Isolation and synthesis of trans- and cis-(−)-clovamides and their deoxy analogues from the bark of Dalbergia melanoxylon

N-(3′,4′-Dihydroxy-trans-cinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine [(?)-clovamide], the major phenolic metabolite (0.1%) in the bark of Dalbergia melanoxylon, is associated with minor proportions of its cis-isomer, and similar pairs of geometrical isomers of their deoxy analogues N-(4′-hydroxycinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine and N-(4′-hydroxycinnamoyl)-3-(4-hydroxyphenyl)-L-alanine. (?)-Trans-clovamide is synthesized by direct condensation of the acid chloride of caffeic acid with L-DOPA. Diagnostic CD spectra of these compounds and ¹³C spectra of (?)-trans- and (?)-cis-clovamides are recorded.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

Model studies pertaining to the hydrazinolysis of glycopeptides and glycoproteins: hydrazinolysis of the 1-N-acetyl and 1-N-(l-β-aspartyl) derivatives of 2-acetamido-2-deoxy-β-d-glucopyranosylamine

The products of hydrazinolysis of the 1-N-acetyl and 1-N-(l-β-aspartyl) derivatives of 2-acetamido-2-deoxy-β-d-glucopyranosylamine could not be converted quantitatively into 2-amino-2-deoxy-d-glucose under mild conditions. Proton and ¹³C-n.m.r. measurements indicated that the hydrazone of 2-amino-2-deoxy-d-glucose was a major product of the hydrazinolysis of 2-acetamido-1-N-acetyl-2-deoxy-β-d-glucopyranosylamine. Control experiments showed that acetohydrazide is slowly converted into 4-amino-3,5-dimethyl-1,2,4-triazole under-the conditions of hydrazinolysis, and that 2-amino-2-deoxy-d-glucose reacts slowly with acetohydrazide in dilute acetic acid. The implications of these results in relation to the hydrazinolysis of glycopeptides and glycoproteins are discussed.     

Basic structure of the oligosaccharide repeating-unit of the Shigella flexneri O-antigens.

The reaction of sodium D-glucuronate with a synthetic peptide, AcTyrLysGlyNH₂ acetate, under physiological conditions, gave as major product the sodium salt of AcTyr-N-(D-arabino-5-carboxy-2,3,4,5-tetrahydroxy-1-pentenyl)-N-(D-arabino- 5-carboxy-3,4,5-trihydroxy-2-oxopentylidene)LysGlyNH₂ (2). The structure was elucidated on the basis of p.m.r., ¹³C-n.m.r., i.r., and u.v. spectra, and pH titration. Compound 2 is the product of oxidation of the sodium salt of AcTyr-N,N-bis(D- arabino-5-carboxy-2,3,4,5-tetrahydroxy-1-pentenyl)LysGlyNH₂, the bis-enol form of the di-D-fructuronic acid peptide obtained through the Amadori rearrangement. A new type of condensation that gives a product having a conjugated enol-keto-immonium group might take place when D-glucuronic acid reacts with peptides or proteins containing a lysine residue.     

Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N′,N′-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N′-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N′-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′-methylurea, which is the product resulting from combined N demethylation and hydroxylation.     

Novel amide alkaloids from the roots of Piper guineense

Two novel amide alkaloids, wisanine and wisanidine, have been isolated from the petroleum-extract of the roots of Piper guineense, and found to be N-piperidyl-5 (2-methoxy-4,5-methylenedioxyphenyl)-trans-2-trans-4-pentadienamide and N-pyrrolidyl-5-(2-methoxy-4, 5-methylenedioxyphenyl)-trans-2-trans-4-pentadienamide respectively. The structure of wisanidine has been confirmed by synthesis. N-Isobutyl)-trans-2-trans-4-eicosadienamide, recently reported to be present in the fruits of the plant as well as Piperine and Δ^α,β-dihydropiperine have also been found to be major constituents of the roots.     

Derivatives of N-amidinoproline and their use in conventional and solid phase peptide synthesis

N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, ¹H NMR, and ¹³C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisothiourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95–98 fragment of fibrinogen α chain.     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Characterization of aminoalkylphosphonyl cerebrosides in muscle tissue of Turbo cornutus

From muscle tissues of the marine snail (Turbo cornutus) aminoalkylphosphonyl cerebrosides, which had been shown to be present in visceral parts, were isolated.Their structure was determined by degradative methods and by characterization of components by gas chromatography-mass spectrometry.The aminoalkylphosphonyl cerebroside fraction consisted of a major portion of 1-O-[6′-O-(N-methylaminoethylphosphonyl)galactosyl] ceramide and a minor portion of a novel lipid, 1-O-[6′-O-(aminoethylphosphonyl)galactosyl] ceramide.The fatty acids of the fraction were mainly palmitic (53.3%) and 2-hydroxy palmitic acid (14.6%). The long chain bases were mainly dihydroxy C_{22 : 2} (36.6%), C_{18 : 1} (14.6%) and C_{18 : 2} (11.3%), and trihydroxy bases were also found as minor components.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (^NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (^NU_m), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of ^NU and ^NU_m substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either ^NU or ^NU_m and of DNA:RNA hybrid duplexes containing ^NU_m. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to ^NU, ^NU_m decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the ^NU_m containing DNA:DNA duplex structure. The conformation of ^NU_m, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of ^NU_m to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.     

Metabolism of 4-N-Hydroxy-Cytidine in Escherichia coli

4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu⁻. Measurement of the incorporation of 4-N-hydroxy-cytidine-2-¹⁴C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu⁻ cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound.     

The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove

The crystal and molecular structure of 2′-O-Me(CGCGCG)₂ has been determined at 1.19 Å resolution, at 100 K, using synchrotron radiation. The structure in space group P3₂12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3′-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2′-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O⁶ and N⁷ of guanosine and N⁴ of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2′-O-Me(CGCGCG)₂ structure in the P3₂12 and P6₁22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2′-O-Me(CGCGCG)₂ is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Cytokinin activity of N-phenyl-N′-(4-pyridyl)urea derivatives

We have synthesized 35 N-phenyl-N′-(4-pyridyl)urea derivatives and tested their cytokinin activity in the tobacco callus bioassay. Among them, N-phenyl-N′- (2-chloro-4-pyridyl)urea is highly active, the optimum concentration of which is lower than 4 × 10^?9 M (0.001 ppm), 3 compounds, i.e. N-(2-methylphenyl)-N′-(2-chloro-4-pyridyl)urea, N-(3-methylphenyl)-N′-(2-chloro-4-pyridyl)urea and N-(3-chlorophenyl)-N′-(2-chloro-4-pyridyl) urea are as active as N⁶-benzyladenine (concentration for optimum yield: 4.4 × 10^?8 M or 0.01 ppm), and N-phenyl-N′-(2-methyl-4-pyridyl)urea and N-(2-chlorophenyl)-N′-(2-chloro-4-pyridyl)urea are as active as N-phenyl-N′-(4-pyridyl)urea (concentration for optimum yield: 4.7 × 10^?7 M or 0.1 ppm), while the activity of the other 29 compounds are not so remarkable and 11 of them are almost or completely inactive.     

Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG^C8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG^C8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG^C8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG^C8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG^C8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG^C8-N-ABA bypass catalyzed by Dpo4.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.