“One-Pot” Syntheses of Malondialdehyde Adducts of Nucleosides

Short, “one-pot” syntheses of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine are described. These syntheses proceed in improved yield and easier purification than previous syntheses and are well suited for the preparation of isotopically labeled nucleoside adducts for biomarker and metabolic studies.     

Synthesis of novel ammonium and selenonium ions and their evaluation as inhibitors of UDP-galactopyranose mutase

The syntheses of two ammonium salts of 1,4-dideoxy-1,4-imino-d-galactitol containing erythritol sulfate side chains are described. The parent compound is a known inhibitor of the enzyme UDP-galactopyranose mutase (UDP-galactopyranose furanomutase, E.C. 5.4.99.9), which is responsible for the conversion of UDP-galactopyranose into UDP-galactofuranose and is presumably protonated in its active form. The side chain was chosen because it is present in a known sulfonium ion alpha-glucosidase inhibitor, salacinol. The syntheses of the selenonium analogues derived from 1,4-dideoxy-1,4-seleno-d-galactitol are also described. The synthetic strategy in the syntheses of all four salts involved the nucleophilic attack of a protected derivative of the alditol at the least hindered carbon of 2,4-O-benzylidene d- or l-erythritol-1,3-cyclic sulfate to give adducts that were subsequently deprotected. The importance of different protecting groups used in the various syntheses is also highlighted. Enzyme inhibition assays carried out on these compounds, and the corresponding sulfonium ions synthesized previously, show that they are poor inhibitors of UDP-galactopyranose mutase.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

The synthesis and in vitro testing of structurally novel antibiotics derived from acylnitroso Diels-Alder adducts

The structural similarity between beta-lactam antibiotics, such as penicillin, and isoxazolidine-3,5-dicarboxylic acids led to the hypothesis that isoxazolidine-3,5-dicarboxylic acids could be effective analogs of beta-lactam antibiotics. The syntheses of relevant isoxazolidine-3,5-dicarboxylic acids from acylnitroso Diels-Alder adducts and subsequent biological testing have shown that these first examples are inhibitors of Escherichia coli X580.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Biosynthesis of "drosopterins" by an enzyme system from Drosophila melanogaster.

The red eye pigment of Drosophila melanogaster consists of six complex pteridines known as neodrosopterin, drosopterin, isodrosopterin, fraction e, and aurodrosopterins (2); these pigments are greatly reduced in the purple mutant. Conditions for biosynthesis of these "drosopterins" are described and compared with those for the synthesis of sepiapterin. The enzymes are contained in a soluble, pteridine-free extract obtained between 40 and 60% saturated ammonium sulfate. The results indicate that sepiapterin synthase consists of two enzymes, the first of which provides a precursor for "drosopterin" biosynthesis. The evidence is (1) the purple mutant, low in accumulated sepiapterin and "drosopterins", is known to have approximately 10% of the sepiapterin synthase activity of wild type; (2) unlabeled sepiapterin does not cause isotope dilution of "drosopterin" synthesis; (3) the 600g pellet prepared from a wild-type head homogenate contains "drosopterin" synthesizing activity and no sepiapterin synthase, yet a heat-labile factor in this fraction stimulates sepiapterin synthesis in the 100000g supernatant of wild-type or pr flies; (4) sepiapterin and "drosopterin" syntheses require Mg2+; (5) sepiapterin synthesis is stimulated by NADPH; "drosopterin" synthesis responds to either NADPH or NADH. Although "drosopterins" are complex pteridine-type pigments, we have demonstrated their biosynthesis by soluble enzymes. This allows us to consider investigation into the mechanism by which the amounts of these pigments are regulated.     

Lipid-phenolic radical adducts as a plausible mechanism of "plant ageing" pigment formation

Co-oxidation of chlorogenic acid, caffeic acid, aesculetin and lucigenin with linoleic acid and egg phosphatidyl choline leads to the formation of fluorescent polymer materials. The fluorescent products are more lipophylic, they have lower elution volumes on Sephadex LH-20 column than related phenols and they differ by their fluorescence and chromatographic properties considerably from polymer lipid peroxidation products. From the presence in the excitation fluorescence spectra of a band corresponding to the phenols it was concluded that the fluorophoric groups were similar in both cases. The data are discussed in terms of liquid phase peroxidation and the appearance of the fluorescent species are attributed to the production of molecular adducts as a result of lipid and phenoxyl radical recombination. The characteristics of products obtained are compared with properties of fluorescent "plant ageing" pigments accumulated in aged and damaged plant cells.     

Measurement of steroid synthesis in zona glomerulosa cells by liquid chromatography-electrospray ionization-mass spectrometry: inhibition by nitric oxide

A liquid chromatography-electrospray ionization-mass spectrometry method was developed to simultaneously determine the concentrations of aldosterone, corticosterone, cortisol, deoxycorticosterone, pregnenolone, and progesterone in bovine adrenal zona glomerulosa (ZG) cells. Steroids were extracted by liquid-liquid extraction, separated on a reverse-phase C18 column, ionized by electrospray, and detected by single-quadrupole mass spectrometry in a positive ion mode. All steroids formed sodium adducts at high abundance. Factors affecting the formation and signal of sodium adducts were investigated. The limits of detection (S/N=3) using selected ion monitoring are 2 pg for these steroids and 10 pg for pregnenolone. DETA NONOate, a nitric oxide donor, inhibited the basal, angiotensin-II-stimulated, and 25-hydroxycholesterol-stimulated syntheses of these steroids in ZG cells in a concentration-dependent manner. The technique demonstrates the ability to determine the individual steroid in each enzymatic step of aldosterone synthesis and the activity of steroidogenic enzymes in adrenal ZG cells.     

Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2'-protecting group for large-scale syntheses. Unlike 2'-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2'-deprotection. The syntheses of 2'-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2'-Cpep monomers were transformed into 3'-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.     

A-DNA accommodates adducts derived from diol epoxides of polycyclic aromatic hydrocarbons bound in a "side-stacking" mode

The minor groove of undistorted A-DNA provides a good binding site for planar, hydrophobic moieties such as unmetabolized polycyclic aromatic hydrocarbons (PAHs), and the base pairs at the ends of short oligodeoxynucleotide helices. It also accommodates the chief adduct derived from the metabolically activated form of the carcinogen benzo[a]pyrene. B-DNA lacks such a site. Computerized models have been generated for the major (N2-guanine-linked) adducts formed at this site by both + and - enantiomers of anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) with poly(dG).poly(dC) in the A-DNA conformation. The BPDE adducts lie in the shallow, relatively hydrophobic minor groove of the A-DNA after empirical potential energy minimization using the program AMBER. We term this binding mode "side-stacking." The side-stacked + anti-BPDE may constitute the chief carcinogenic lesion derived from benzo[a]pyrene.     

Syntheses of fluorinated amino acids: from the classical to the modern concept

Summary A survey of the synthetic pathways leading to the fluorine-containing analogues of amino acids is given. From the great number of syntheses the typical examples are selected and divided into two groups: classical syntheses and the modern ones. The classical ammonolysis of halogeno acids and equivalent reactions are discussed as first, followed by a few examples of oxo amino group transformation. Conversion of the oxo compounds into amino acids richer by one carbon atom is realized by the Strecker and hydantoin syntheses. For the prolongation by two carbons, the Erlenmeyer azlactone method and alkylation of CH-acidic esters are applied. The modern syntheses are represented by direct fluorination by elemental fluorine and other electrophilic fluorinating reagents. Further examples include the applications of the Yarovenko reagent, sulphur tetrafluoride and its derivative DAST. The use of trifluoropyruvates as the fluoro synthons is mentioned briefly. Finally, the examples of the amidocarboxylation method and the syntheses of diverse-fluorinated methionines are shown.     

Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ-deoxyguanosine (dG) and two BPQ-deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827-838). However, the identification of BPQ-DNA adducts by 32P postlabeling methods from in vitro and in vivo exposures required 3'-monophosphate derivatives of BPQ-dG, BPQ-dA, and BPQ-deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3'), dAMP(3'), and dCMP(3') were prepared. The syntheses of the BPQ-3'-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ-nucleotide adducts to the corresponding known BPQ-nucleoside adduct standards. The BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adduct standards were used in 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ-DNA adducts, the following were identified: 1 BPQ-dGMP adduct, 2 BPQ-dAMP adducts, and 3 BPQ-dCMP adducts. This study represents the first reported example of the characterization of stable BPQ-DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ-DNA adduct studies in vivo and thereby to the contribution of BPQ in B[a]P carcinogenesis.     

Base excision repair system targeting DNA adducts of trioxacarcin/LL-D49194 antibiotics for self-resistance

Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases—TxnU2, TxnU4, LldU1 and LldU5—important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-{"type":"entrez-nucleotide","attrs":{"text":"D49194","term_id":"702903"}}D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.     

Structural basis for the sequence-dependent effects of platinum–DNA adducts

The differences in efficacy and molecular mechanisms of platinum based anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) have been hypothesized to be in part due to the differential binding affinity of cellular and damage recognition proteins to CP and OX adducts formed on adjacent guanines in genomic DNA. HMGB1a in particular exhibits higher binding affinity to CP-GG adducts, and the extent of discrimination between CP- and OX-GG adducts is dependent on the bases flanking the adducts. However, the structural basis for this differential binding is not known. Here, we show that the conformational dynamics of CP- and OX-GG adducts are distinct and depend on the sequence context of the adduct. Molecular dynamics simulations of the Pt-GG adducts in the TGGA sequence context revealed that even though the major conformations of CP- and OX-GG adducts were similar, the minor conformations were distinct. Using the pattern of hydrogen bond formation between the Pt–ammines and the adjacent DNA bases, we identified the major and minor conformations sampled by Pt–DNA. We found that the minor conformations sampled exclusively by the CP-GG adduct exhibit structural properties that favor binding by HMGB1a, which may explain its higher binding affinity to CP-GG adducts, while these conformations are not sampled by OX-GG adducts because of the constraints imposed by its cyclohexane ring, which may explain the negligible binding affinity of HMGB1a for OX-GG adducts in the TGGA sequence context. Based on these results, we postulate that the constraints imposed by the cyclohexane ring of OX affect the DNA conformations explored by OX-GG adduct compared to those of CP-GG adduct, which may influence the binding affinities of HMG-domain proteins for Pt-GG adducts, and that these conformations are further influenced by the DNA sequence context of the Pt-GG adduct.     

Synthesis of analogues of salacinol containing a carboxylate inner salt and their inhibitory activities against human maltase glucoamylase

The syntheses of analogues of the naturally occurring glycosidase inhibitor, salacinol, containing a carboxylate inner salt are described. Salacinol is a sulfonium ion with an internal sulfate counterion. The synthetic strategy relies on the nucleophilic attack of 1,4-anhydro-2,3,5-tri-O-benzyl-4-thio-D- or L-arabinitol at the least hindered carbon of 4,5-anhydro-2,3-O-isopropylidene-D-ribonic acid benzyl ester to yield coupled adducts. Deprotection of the coupled products gives the target compounds. The compound derived from D-arabinitol inhibits recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose oligosaccharides in the small intestine, with a Ki value of 10+/-1 microM.     

High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts

The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens.     

Specific strand loss in N-2-acetylaminofluorene-modified DNA

N-2-Acetylaminofluorene (AAF), a well-known chemical carcinogen, when covalently linked to guanine residues constitutes a premutagenic lesion that is converted in vivo into frameshift mutations. In Escherichia coli, it is thought that -AAF adducts block the replication fork and that the mutagenic processing of the -AAF adducts is mediated by the SOS response. The construction in vitro of plasmids containing -AAF adducts in one strand only of a double-stranded DNA molecule enabled us to investigate the segregation of the strands and the mutagenicity of the lesions in vivo. The two DNA strands were "genetically labelled" by means of a single base-pair mismatch in the tetracycline-resistance gene, one strand carrying the wild-type allele and the other strand a mutant tetracycline-sensitive allele. The two strands contained either no -AAF adducts, -AAF adducts in one strand or -AAF adducts in both strands. When such constructions are used to transform bacterial cells the following are found. When no -AAF adducts are present on either strand of the DNA, a mixture of plasmids having information from both parent strands is found in 80% of the transformed bacterial clones. With -AAF adducts present in one strand only, in 90% of the transformants there is a consistent loss of the parent strand information that contained the -AAF adducts. In the constructions having -AAF adducts in both strands, the transformed bacteria carry either one or the other allele in a pure form. Our results suggest that when blocking lesions (-AAF adducts) are present in one strand only, they trigger the specific loss of that strand. The forward mutation frequency (i.e. the tetracycline-resistance gene inactivation frequency) was found to be more than ten times lower when the -AAF adducts are bound to one strand only compared with the situation where both strands carry the premutagenic lesions. Moreover, when the isolated mutants were sequenced, the mutations were found to consist of a mixture of true -AAF-induced mutations (i.e. -1 or -2 frameshift mutation at previously determined mutation hot spots) and of mutations that are not targeted at -AAF adducts. We suggest that these "background" mutants arose from the mutagenic processing of cryptic lesions present in our DNA. The low mutagenic efficiency of -AAF adducts, when present in one strand only of a duplex DNA, most probably results from the above-described loss of the damaged strand.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of adducts by bisphenol A, an endocrine disruptor, in DNA in vitro and in liver and mammary tissue of mice

Endocrine disruptors (EDs) represent a major toxicological and public health issue, and the xenoestrogen bisphenol A (BPA) has received much attention due to its high production volume and widespread human exposure. Also, due to its similarity to diethylstilbestrol, a known human carcinogen, BPA has been investigated for its genotoxic and carcinogenic properties, but the results have been either inconclusive or controversial. Metabolically activated BPA has previously been shown to form DNA adducts both in vitro and in rat liver. The present study was designed (a) to assess the sensitivity threshold of DNA-adduct detection by ³²P-postlabelling in an acellular system and (b) to evaluate the formation of DNA adducts in both liver and mammary cells of female CD-1 mice receiving BPA in their drinking water (200 mg/kg body weight) for eight consecutive days. The reaction of BPA with calf thymus DNA, in the presence of S9 mix, resulted in a dose-dependent formation of multiple DNA adducts, with a detection limit of 10 ng of this ED under our experimental conditions. Administration of BPA to mice confirmed that DNA adducts are formed in liver (3.4-fold higher levels than in controls). In addition, new evidence is provided that DNA adducts are formed in target mammary cells (4.7-fold higher than in controls). Although DNA adducts do not necessarily evolve into tumours or other chronic degenerative diseases, the formation of these molecular lesions in target mammary cells may bear relevance for the potential involvement of BPA in breast carcinogenesis.     

Biosynthesis of <Emphasis Type="Italic">Veratrum californicum</Emphasis> specialty chemicals in <Emphasis Type="Italic">Camelina sativa</Emphasis> seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.