N-nitrosation by nitrite ion in neutral and basic medium.

Formaldehyde catalyzed the conversion of various secondary amines to nitrosamines in the pH range 6.4 to 11.0. Chloral was also an effective catalyst. The reaction proceeds easily enough to have potential synthetic applications; the proposed mechanism could explain some reported anomalies regarding the synthesis of carcinogenic N-nitroso compounds in vivo and in vitro.

Proteins in a nonvenomous defensive secretion: biosynthetic significance.

In common with many arthropods, the true bug, Leptoglossus phyllopus, when disturbed, emits a two-phase secretion that consists of an organic phase and an aqueous phase. The organic phase is a mixture of highly reactive low-molecular-weight compounds, analogous to those produced by other arthropods, and is deterrent to many kinds of predators. The aqueous phase, heretofore ignored in most analyses of arthropod defensive secretions, contains proteins. Even though the secretion is not injected, the proteins enzymatically catalyze the derivation of the most reactive components within the impermeable cuticular storage reservoir and, thus, constitute part of the defensive system that appears to be commonly used by arthropods producing irritating chemicals.

Benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide adenosine and deoxyadenosine adducts: structure and stereochemistry.

The structure and absolute stereoconfigurations of four adenosine adducts with (+/-)-7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) and their deoxyadenosine analogs have been determined. They result from both cis and trans addition of the N6 amino group of ademine to the 10 position of both enantiomers of BDPE. This was determined from studies of the nuclear magnetic resonance spectra, mass spectra, and circular dichroism spectra, as well as from their pKa values and chemical reactivities.

Selective induction of intestinal tumors in rats by methyl(acetoxymethyl)nitrosamine, an ester of the presumed reactive metabolite of dimethylnitrosamine.

Methyl(acetoxymethyl)nitrosamine (DMN-OAc) was synthesized and tested for toxicity and carcinogenicity in rats to test the hypothesis that alpha-hydroxylation is required for metabolic activation of dimethylnitrosamine (DMN) to a reactive, proximate carcinogen. The acute median lethal doses (LD50) of DMN-OAc and DMN injected ip into 5-week-old male Sprague-Dawley (Charles River (CD) rats were determined to be 0.19 and 0.59 mmole/kg body weight or 25 mg DMN-OAc/kg and 44 mg DMN/kg body weight, respectively. Single ip injections of one-half the LD50 DMN-OAc (13 mg/kg body weight) in 5-week-old rats of both sexes resulted in a high incidence of epithelial tumors of the intestinal tract. Mean survival times for rats with intestinal tumors were 353 days for males and 433 days for females. Tumors were rarely found at other sites. DMN at equivalent toxic (one-half the LD50, 22 mg/kg) and molar (= one-sixth LD50, 7.0 mg/kg) dose levels, yielded (as expected) tumors of kidneys, lungs, and occasionally other organs, but at a much lower incidence. The finding of the potent carcinogenicity of DMN-OAc supported the postulate that alpha-hydroxylation of DMN in vivo generates a proximate carcinogen.

Use of 3,4-dichlorobenzenethiol as a trapping agent for alkylating intermediates during in vitro metabolism of nitrosamines.

Our studies using 3,4-dichlorobenzenethiol as a probe for methylating agent production during exposure of N-nitrosodimethylamine to rat liver S-9 preparations produced results different from those of an investigation reported in the literature. Methyl-3,4-dichlorophenyl thioether was detected, but the quantities found were not significantly different from the background levels of methylation product detected in the absence of nitrosamine. Only about 10% of the thioether isolated after incubating N-nitrosodi[14C]methylamine as substrate was radioactive. The results indicate that the majority of the methyl groups transferred to the sulfur nucleophile in our experiments came from components of the incubation mixture other than the nitrosamine. Some artifactual methylation was also associated with the analytical procedure. We conclude that 3,4-dichlorobenzenethiol should be used with caution in studies of alkylation during the in vitro metabolism of carcinogenic nitrosamines.

A p21(Waf1/Cip1)carboxyl-terminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.

Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.

Cord factor (trehalose-6-6'-dimycolate) of in vivo-derived Mycobacterium lepraemurium.

Harvests of Mycobacterium lepraemurium obtained from livers of moribund infected mice yielded M. lepraemurium cell walls that were extracted with solvent to provide crude M. lepraemurium cell wall lipids. By solvent fractionation and chromatography on DEAE cellulose and cellulose, a cord factor-like glycolipids contaminated with mycoside C was obtained. Additional solvent treatment provided the purified glycolipid, which was identified as 6,6'-trehalose dimycolate, by infrared and chromatographic comparison with authentic samples from M. tuberculosis, by identification of trehalose and specific mycolates of M. lepraemurium, and by permethylation analysis. This constitutes the first unequivocal identification of cord factor as a product of in vivo-derived mycobacteria.

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

D-Val22 containing human big endothelin-1 analog, [D-Val22]Big ET-1[16-38], inhibits the endothelin converting enzyme.

Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin-1 (ET-1) from a precursor, big endothelin-1 (Big ET-1). We synthesized four analogs of human Big ET-1[16-38], substituted with single D-amino acids at P1, P2, P1' and P2' positions. ECE activity was determined using an ET-1 specific radioimmunoassay system. None of the D-amino acid containing Big ET-1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [D-Val22]Big ET-1[16-38], strongly inhibited the ECE activity. Furthermore, when this D-Val22 containing peptide was preadministered to rat striatum, it was found to inhibit the dopamine release induced by Big ET-1. This result suggests that the D-Val22 containing peptide inhibits the ECE activity in vivo. The D-Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.

L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.

4'-O-[2-(2-fluoromalonyl)]-L-tyrosine: a phosphotyrosyl mimic for the preparation of signal transduction inhibitory peptides.

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.

Discovery of small-molecule inhibitors of Bcl-2 through structure-based computer screening.

Bcl-2 belongs to a growing family of proteins which regulates programmed cell death (apoptosis). Overexpression of Bcl-2 has been observed in 70% of breast cancer, 30-60% of prostate cancer, 80% of B-cell lymphomas, 90% of colorectal adenocarcinomas, and many other forms of cancer. Thereby, Bcl-2 is an attractive new anti-cancer target. Herein, we describe the discovery of novel classes of small-molecule inhibitors targeted at the BH3 binding pocket in Bcl-2. The three-dimensional (3D) structure of Bcl-2 has been modeled on the basis of a high-resolution NMR solution structure of Bcl-X(L), which shares a high sequence homology with Bcl-2. A structure-based computer screening approach has been employed to search the National Cancer Institute 3D database of 206 876 organic compounds to identify potential Bcl-2 small-molecule inhibitors that bind to the BH3 binding site of Bcl-2. These potential Bcl-2 small-molecule inhibitors were first tested in an in vitro binding assay for their potency in inhibition of the binding of a Bak BH3 peptide to Bcl-2. Thirty-five potential inhibitors were tested in this binding assay, and seven of them were found to have a binding affinity (IC(50) value) from 1.6 to 14.0 microM. The anti-proliferative activity of these seven active compounds has been tested using a human myeloid leukemia cell line, HL-60, which expresses the highest level of Bcl-2 protein among all the cancer cell lines examined. Compound 6 was the most potent compound and had an IC(50) value of 4 microM in inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compounds had moderate activity in inhibition of cell growth. Compound 6 was further evaluated for its ability to induce apoptosis in cancer cells. It was found that 6 induces apoptosis in cancer cells with high Bcl-2 expression and its potency correlates with the Bcl-2 expression level in cancer cells. Furthermore, using NMR methods, we conclusively demonstrated that 6 binds to the BH3 binding site in Bcl-X(L). Our results showed that small-molecule inhibitors of Bcl-2 such as 6 modulate the biological function of Bcl-2, and induce apoptosis in cancer cells with high Bcl-2 expression, while they have little effect on cancer cells with low or undetectable levels of Bcl-2 expression. Therefore, compound 6 can be used as a valuable pharmacological tool to elucidate the function of Bcl-2 and also serves as a novel lead compound for further design and optimization. Our results suggest that the structure-based computer screening strategy employed in the study is effective for identifying novel, structurally diverse, nonpeptide small-molecule inhibitors that target the BH3 binding site of Bcl-2.

Hydrolysis of iodine labelled urodilatin and ANP by recombinant neutral endopeptidase EC. 3.4.24.11.

1. Urodilatin is a 32 amino-acid peptide of similar sequence to atrial natriuretic peptide (ANP), with four additional amino-acids at the N-terminus. Although ANP and urodilatin bind to the same receptors with similar affinities, urodilatin is more active than ANP as a natriuretic agent. Previous studies, using neutral endopeptidase EC 3.4.24.11 (NEP) derived from crude membrane preparations, were inconclusive, but suggested that urodilatin was more resistant than ANP to degradation by this enzyme. In the present study, we compared the degradation rates of [125I]-urodilatin and [125I]-ANP by pure recombinant NEP (rNEP). 2. Incubation of radioactively labelled ANP with rNEP resulted in a much more rapid degradation of the peptide than that for labelled urodilatin. 3. Both phosphoramidon and SQ-28,603, potent inhibitors of NEP, completely protected both peptides from metabolism by rNEP. 4. The circular dichroism spectra of the two peptides indicate that they are very similar and exist largely in unordered or flexible conformations. 5. These results support the relative resistance of urodilatin to NEP, and indicate that urodilatin may be of use as a therapeutic agent, in conditions in which ANP is ineffective.

Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz[a]anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects.

The leukemogens 7,12-dimethylbenz[a]anthracene (DMBA) and 7,8,12-trimethylbenz[a]anthracene (TMBA) bind covalently in vivo to DNA of Long-Evans rats in the hematopoietic organs, spleen and bone marrow, and in the liver, a non-target organ. Both TMBA and DMBA depleted bone marrow cells and both agents bound persistently to the DNA of bone marrow and of liver, and less to that of spleen. The three main DMBA:deoxyribonucleoside adducts in spleen, bone marrow and liver were the same as those found previously in the liver (Dipple et al. (1983) Cancer Res., 43, 4132). There were no organ-specific or age-dependent differences in the relative amounts of adducts formed. There appears to be no direct correlation between the susceptibility of an organ to carcinogenesis and the nature and relative amount of the specific adducts formed, at least for the three organs studied here.

Mutagenicity of N-nitroso pyridinol carbamate with the Salmonella/mammalian microsome test.

Pyridinol carbamate was nitrosated to the N,N1-dinitroso derivative and the structure was proved by spectroscopic methods, including chemical ionization mass spectroscopy. By the Ames test, the dinitroso derivative showed a significant dose-dependent mutagenic response with Salmonella typhimurium strains TA 1535 and TA 100; the response became more pronounced in the presence of microsomes. As the dosage of N-nitroso pyridinol carbamate increased, the number of revertant colonies also increased. Pyridinol carbamate was not mutagenic.

Physico-chemical and microbiological comparison of nystatin, amphotericin A and amphotericin B, and structure of amphotericin A.

Two polyene antibiotics, nystatin and amphotericin A, were compared by physico-chemical and microbiological methods. The two antibiotics were found to have the same molecular weight, 926, by plasma desorption and electron-impact MS. However, 13C NMR spectrometry and HPLC studies indicated that the two molecules are different. The 200 MHz NMR studies indicated a chemical environment of 24 carbons of amphotericin A identical with that of the carbons of amphotericin B and nystatin. The structure of amphotericin A is identical with that of amphotericin B, except that there is a single bond between carbons 28 and 29 instead of a double bond, as shown by two-dimensional NMR studies.

Structure of fredericamycin A, an antitumor antibiotic of a novel skeletal type; spectroscopic and mass spectral characterization.

IR, UV-visible spectroscopy, circular dichroism, 1H and 13C NMR studies, high resolution electron impact, field desorption, and fast atom bombardment mass spectral studies are reported for fredericamycin A (NSC-305263), a novel antitumor antibiotic of acid-base indicator type produced by Streptomyces griseus (FCRC-48). The spectral data are correlated with the structure obtained by X-ray crystallography as (E,E)-6',7'-dihydro-4,9,9'-trihydroxy-6-methoxy-3'-(1,3-pentadienyl++ +)-spiro- [2H-benz[f]indene-2,8'-[8H]-cyclopent-[g]-isoquinoline]-1,1', 3,5,8(2'H)-pentone. The novel spiro ring antibiotic exhibits unusual 1H and 13C NMR spectroscopic and chemical behavior, not previously observed in other antibiotic structures.