Search

The procedure for 17-hydroxylation of pregnan-20-ones using oxygen, butoxide, and triethyl phosphite was extended to steroid analogues with aromatic C-rings, viz. 18-nor-17α(H)- and -17β(H)-pregna-4,8,11,13-tetraene-3,20-dione (and to 1-acetylindan). At temperatures higher than that we adopted (–50 °C), side-chain cleavage to give the 17-ketone (or equivalent) became prominent. Results of an X-ray crystallographic study on one of the two 17-epimeric C-aromatic products, 17β-hydroxy-18-nor-17β(H)-pregna-4,8,11,13-tetraene-3,20-dione, are presented. The crystals are orthorhombic, space group P212121, with four molecules in a cell of dimensions a= 9.643(3), b= 17.043(4), c= 10.015(3)A. The structure was solved by direct methods and refined by full-matrix least-squares calculations; R= 0.059 for 1 219 observed reflections. Ring A has a 1α,2β-half -chair conformation, ring B is in a 5α,6β-half-chair conformation, aromatic ring C is planar, and ring D is a C(16)β-envelope. Molecules are linked to form infinite chains by intermolecular O–H ⋯ O hydrogen bonds [O ⋯ O 2.801 (4)A].

The identity of eriocarpin with desglucosyrioside (1) has been established by comparison of their triacetates. 13C and 1H N.m.r. spectra show that the conversion of labriformidin into desglucosyrioside involves solely the reduction of a 3-keto-function in the carbohydrate portion (B) which is identical (in structure and stereochemistry) with that in uscharidin (7). Structures are proposed for labriformidin and labriformin. 13C N.m.r. data establish the β-configuration of the 7,8-epoxide in labriformidin, desglucosyrioside, and in the 5β-cardenolide sarverogenin (11).

Polypeptides expressed on the surface of merozoites, the invasive stage of the asexual blood cycle, are good candidates for the development of malaria vaccines. Five synthetic peptides with predetermined specificity deduced from a genomic DNA clone coding for the NH2-terminal portion of the main merozoite surface polypeptide of Plasmodium falciparum were evaluated for their capability to raise antibodies that react with the P. falciparum merozoites. Antibodies induced by two of the peptides (3 and 5) reacted with the membrane surfaces of seven of seven isolates of P. falciparum from different geographic areas. Antibodies against peptide 4, which contains a repeated amino acid sequence (Gly-Gly-Ser and Val-Ala-Ser), reacted with six of seven isolates. Structural analysis of the deduced polypeptides suggests that peptide 3 is exposed at the surface of merozoites. When it was used to immunize monkeys, three of the four animals were partially protected from a challenge infection that induced a fulminant infection in control animals.

The objective of this introduction is to develop concisely the context in which biochemical studies of Epstein—Barr virus (EBV) have been undertaken. Elements of history and biology are relevant to an understanding of the somewhat unusual course of biochemical research with EBV.

Two of the Sal I fragments and all of the internal BamHI fragments (with the exception of BamHI c, a 0.6 x 10(6) dalton fragment) of Epstein-Barr virus (EBV) DNA have been cloned in pBR322. The termini and other parts of the DNA (including the EcoRI fragment which contains BamHI c) have been cloned as EcoRI fragments in bacteriophage Charon 4A. The cloned DNAs have been used to derive a complete map of the BamHI fragments of EBV DNA and to align the BamHI, EcoRI, HindIII, and SalI cleavage sites in EBV DNA.

Epstein-Barr virus (EBV) is the human prototype of a family of closely related herpesviruses of Old World primates. These agents probably evolved and spread among the Old World primates since the divergence of apes from monkeys about 30 million years ago. Although the DNAs of the EBV family have no sequence homology to other herpesviruses, there are some features in common with other herpesviral genomes. EBV DNA is unusual in having five tandem direct repeat elements which divide the genome into five unique sequence domains. The tandem direct repeats at the ends of the linear virion DNA probably mediate circularization of the viral DNA to form the circular episomal viral DNA which is characteristic of EBV-infected cells. In latent transforming infection, messenger RNAs are encoded by three widely separate regions of the EBV genome. The remainder of the viral genome encodes many RNAs and proteins which are expressed in productive infection. Early and late viral genes are intermixed along the full length of EBV DNA.

At least three separate regions of the Epstein-Barr virus (EBV) genome encode RNA in a cell line that is growth transformed and nonpermissively infected with EBV. Six polyadenylylated cytoplasmic RNAs have been identified from these three regions. An abundant RNA 3.0-3.1 kilobases (kb) long is encoded by DNA of the internal reiteration, IR, and DNA that maps at 25.7-30 megadaltons. A second, abundant, 2.9-kb RNA is primarily encoded by DNA at 110-03 megadaltons but probably has a 3' end to the left of 110 megadaltons. A third, abundant, 3.7-kb RNA is largely encoded by DNA at 63-66 megadaltons and has a 5' end to the left of 63 megadaltons. A less-abundant 1.5-kb RNA is also encoded by IR. The least-abundant polyadenylylated RNAs identified are 2.3 and 2.0 kb. These RNAs have 3' ends mapping of 5-7 megadaltons and 5' ends mapping to the right of 7 megadaltons. The data suggest that there may be two additional polyadenylylated cytoplasmic RNAs, a 3-kb RNA mapping at 26.2-30 megadaltons and a minor RNA mapping at 102-110 megadaltons. An abundant 0.16-kb nonpolyadenylylated RNA is also present in the cytoplasm of IB-4 cells. This RNA precipitates from the cytoplasm in the presence of high concentrations of magnesium, indicating that it is complexed with protein or polyribosomes.

The ring-C aromatic analogue of progesterone, 18-norpregna-4,8,11,13-tetraene-3,20-dione (2), was formed together with its 17-epimer (3) when 9α,11α-epoxy-20ξ-hydroxy-3-oxopregn-4-eno-18,20-lactone (14) was subjected to oxidative decarboxylation, and the resulting products treated with boron trifluoride. The precursor (14) was synthesised as follows. 11α-Hydroxyprogesterone (4) was dehydrated, and the product then selectively reduced (by two routes) to 20β-hydroxypregna-4,9(11)-dien-3-one (9). The latter was functionalised at C-18 to give 3-oxopregna-4,9(11)-dieno-18,20-lactone (10) which was oxidised, after opening of the lactone ring, to the corresponding lactol (12). The latter was converted into the required lactol epoxide (14). Internal displacement on the 9α,11α-epoxide occurred when the lactol epoxide (14) was treated with acid, yielding 9α-hydroxy-3,20-dioxopregn-4-eno-18,11β-lactone (16).

Reduction of gomphoside with sodium cyanoborohydride occurred with opening of the 2′-hemiacetal to yield the 3-(4,6-dideoxy-β-D-ribo-hexopyranoside) and the 3-(4,6-dideoxy-β-D-arabino- hexopyranoside) of 2α-hydroxyuzarigenin. Sodium borohydride reduction of 3′-didehydrogomphoside and 3′-epi-gomphoside 3′-acetate led instead to 2α-hydroxyuzarigenin 3-(4,6-dideoxy-β-D-lyxo-hexopyranoside). The structures of these stereoisomeric products are shown by 1H and 13C n.m.r. spectra. Borohydride reduction of uscharidin likewise yielded 2α,19-dihydroxyuzarigenin 3-(4,6-dideoxy-β-D-lyxo-hexopyranoside). In contrast, a product with an intact 2′-hemiacetal, viz. 19-hydroxy-3′-epi- gomphoside, was obtained by Reichstein et al. under related conditions.

Eight cardenolide glycosides and one genin, including two new glucoside conjugates, have been isolated from adult monarch butterflies (Danaus plexippus L.) the larvae of which were reared on Asclepias fruticosa L. leaves. The glycosides calactin, calotropin (major components), calotoxin, and afroside were identified by comparison with authentic samples. The remaining compounds, after conversion into peracetates, have been shown by 400 MHz 1H n.m.r. and mass spectra (and for the first two compounds by direct comparison) to be the genin calotropagenin and the glycosides frugoside, gofruside, gomphogenin 3-β-D-glucopyranoside, and calotropagenin 3-β-D-glucopyranoside. The relationship between these compounds from monarch butterflies and those previously isolated from the food plant A. fruticosa is discussed.

Eight new cardenolide glycosides, 19-deoxyuscharin (1b), 4′β-hydroxygomphoside (1k), 3′-didehydrogomphoside (1d), 3′-epigomphoside (1e), 3′-epiafroside (2e), 3′-epigomphoside 3′-acetate (1f), 3′-didehydroafroside (2d), and 3′-epiafroside 3′-acetate (2f), have been isolated from Asclepias fruticosa R. Br. The structures of all new compounds were established by spectral comparisons with known compounds. The chirality at C-3′ of uscharin (3b), voruscharin, and 19-deoxyuscharin (1b) is proposed to be S. By comparison with 4′β-hydroxygomphoside (1k), the β configuration of the 4′-hydroxy group of calotoxin (3k)(from Calotropis procera) has been established.

Irradiation at 365 nm of an oxygenated solution of 7-methylbenz[c]acridine in methanol yields a mixture of photoproducts of which two were shown by 1H and 13C n.m.r. to be trans-6-hydroxy-5-methoxy-7-methyl-5,6-dihydrobenz[c]acridine and trans-5-hydroxy-6-methoxy-7-methyl-5,6-dihydrobenz[c]-acridine.

Synthese de N 2 -alanyl- et N 2 -leucyl methotrexates et des N 4 -t-butoxycarbonylaminoacyl- et N 2 ,N 4 -bis-t-butoxycarbonylaminoacyl methotrexates de di-t-butyle correspondants

Analogues of methotrexate (amethopterin)(1) with α- or γ-monoamide functions [viz. the α- and γ-primary amides (16a) and (26a); the following N-substituted amides: α-methyl-, α-ethyl-, α- and γ-propyl-, α-isopropyl-, α-butyl-, α-isobutyl-, α-sec-butyl-, α-t-butyl, α-benzyl-, and α- and γ-cyclohexylamide (16b–d), (26d), (16e–k), (26k) respectively; and the NN-disubstituted amides: γ-piperidide (26l) and γ-morpholide (26m)] were synthesized starting with t-butyl L-isoglutamine (12a), t-butyl L-glutamine (22a), or the appropriateN′-alkyl or N′N′-dialkyl analogues (12b–k), (22d), (22k), (22l), and (22m). The corresponding N-benzyloxycarbonyl compounds (11) and/or (21) from which the above L-glutamic acid derivatives were obtained were generally synthesized by mixed-anhydride coupling of N-benzyloxycarbonyl-L-glutamic acid (9) with the appropriate amine, conversion into the t-butyl ester, and chromatographic separation. The resulting α-monoamide γ-t-butyl ester (11) and γ-monoamide α-t-butyl ester (21) are unambiguously distinguished by mass spectrometry and 13C n.m.r. spectroscopy. Factors which affect the γ-amide/α-amide product ratio are discussed. The N-deprotected L-glutamic acid monoamide t-butyl esters (12) or (22) were individually coupled to N-trifluoracetyl-p-methylaminobenzoic acid, and the resulting α- or γ-monoamide t-butyl esters (13) or (23) of N-(p-methyl (trifluoroacetyl)aminobenzoyl)-L-glutamic acid was hydrolysed. The N-deprotected product, viz. t-butyl N-(p-methylaminobenzoyl)-L-glutamate α- or γ-monoamide (14) or (24) was converted into the appropriate methotrexate-monoamide t-butyl ester (15) or (25), and thence the desired methotrexate-monoamide (16) or (26), by reaction with 2,4-diamino-6-bromomethylpteridine (17) or by the Taylor procedure. Features of the mass and 13C n.m.r. spectra of the intermediates are discussed.

Samples of 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (Trimethoprim) were synthesized, enriched with 13C at the following positions: 2 and 5; 4 and 6; α and 4-methoxy. For the first two series (having 13C-enrichment at the pyrimidine), appropriately labelled cyanoacetic acid was converted into labelled methyl 2-(3,4,5-trimethoxybenzyl)cyanoacetate and thence to the labelled 6-hydroxy derivative of Trimethoprim. For the last series (having 13C-enrichment at the trimethoxybenzyl moiety), the key reaction was the nucleophilic attack of C–5 of 2,4-diamino-6-hydroxypyrimidine on labelled 3,4,5-trimethoxybenzyl bromide, again yielding the labelled 6-hydroxy derivative of Trimethoprim. The conversion of the 6-hydroxy derivatives into 13C-enriched Trimethoprim was via the 6-chloride. Also synthesized was [6-13C]-2,4-diaminopyrimidine by cyclization of guanidine and [3-13C]-3-anilinoacrylonitrile. The identity, and the site(s) of the 13C label(s) of each final product and intermediate, were established on the basis of 1H and 13C n.m.r. spectral evidence.

13 C N.m.r. chemical shifts of a number of semi-synthetic cardenolide analogues are presented and the singal assignments justified. Compounds studied include digitoxigenin analogues having αβ-unsaturated 17βside-chains; the α-L-rhamnopyranoside, β-D-glucopyranoside, and β-D-galactopyranoisde of digitoxigenin and/or uzarigenin, and some peracetylated dervatives thereof. The effects of both structural changes in the side-chain and glycoside formation on the chemical shifts of certain carbons on the steroid skeleton are analysed.

The 13C n.m.r. data of the Digitalis and the Cerbera cardiac glycosides, and of ouabain and neriifolin, have been analysed. Both 13C and 1H n.m.r. results show that the 11,12-epoxide in cerbertin has an α-configuration.

Asclepias vestita from Commatti Canyon in California has yielded nine new 19-oxo cardenolide glycosides and one genin, all with the 5(6) double-bond, viz 5,6-dehydrocalactin, 5,6-dehydrocalotropin, 5,6-dehydroasclepin, 5,6-dehydrouscharidin, 5,6-dehydrocalatoxin, 16α-hydroxy-5,6-dehydrocalotropin, 16α-acetoxy-5,6-dehydrocalotropin, 16α-hydroxy-5,6-dehydroasclepin, 16α-acetoxy-5,6-dehydroasclepin, and the genin, 5,6-dehydrocalotropagenin. Most of these Δ5-cardenolide glycosides were accompanied by the known 5α(H) analogues. Also found were two 5α-cardenolides with a 19-hydroxy group, viz. the glycoside 19-dihydrocalotropin and the known genin coroglaucigenin. The position of the unsaturation in the Δ5-cardenolides was located by comparison of the 400 MHz n.m.r. data with those of the 5α-analogues, with consideration of the anisotropy effect of the Δ5 double-bond. A pair of model Δ5-19-oxo and 5α(H)-19-oxo steroids have also been studied for comparison.

The 4,6-dideoxyhexosulose moiety of gomphoside (1) and afroside (2) has been shown to have the 1′S,2′S,3′R,5′R configuration, Formation of 2′,3′-OO-isopropylidene derivatives establishes that the hydroxy-group at C-3′, which is axial, is cis to that at C-2′. The latter group is cis to the anomeric C-1′ hydrogen, as shown by nuclear Overhauser enhancement measurements. Chemical and 1H and 13C n.m.r. data have been used to show that the same chirality at carbons 1′, 2′, and 5′ is present in the following glycosides from plants of the Ascepiadaceae family: calactin (3), syriobioside (10), desglucosyrioside (11), syrioside (12), uscharidin (5), calotoxin (9), uscharin (7), and voruscharin (8).

Aspecioside, a 7β,8β-epoxycardenolide glycoside isolated from Asclepias speciosa(Asclepiadaceae), has been shown by 400 MHz 1H n.m.r., 13C n.m.r., mass spectrometry, and biogenetic considerations to be 12β-hydroxy-5α-tanghinigenin-3-(6-deoxy-β-D-allopyranoside). It is also found in A. syriaca.