Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes.

Elucidation of the cyclization mechanism catalyzed by terpene synthases is important for the rational engineering of terpene cyclases. We developed a chemoenzymatic method for the synthesis of systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specifically deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases. We examined the cyclization mechanism of tetracyclic diterpene phomopsene with phomopsene synthase. A detailed EI-MS analysis of phomopsene labeled at various positions allowed us to propose the structures corresponding to the most intense peaks, and thus elucidate a cyclization mechanism involving double 1,2-alkyl shifts and a 1,2-hydride shift via a dolabelladien-15-yl cation. Our study demonstrated that this newly developed method is highly sensitive and provides sufficient information for a reliable assignment of the structures of fragmented ions.

Exploration of GGTase-I substrate requirements. Part 1: Synthesis and biochemical evaluation of novel aryl-modified geranylgeranyl diphosphate analogs.

Protein geranylgeranylation is a type of post-translational modification that aids in the localization of proteins to the plasma member where they elicit cellular signals. To better understand the isoprenoid requirements of GGTase-I, a series of aryl-modified geranylgeranyl diphosphate analogs were synthesized and screened against mammalian GGTase-I. Of our seven-member library of compounds, six analogs proved to be substrates of GGTase-I, with 6d having a krel=1.93 when compared to GGPP (krel=1.0).

Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

Protein prenylation is a type of post-translational modification that aids certain proteins in localizing to the plasma member where they activate cell signaling. To better understand the isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of GGTase-I, with 11d having a krel=0.95 when compared to GGPP (krel=1.0).

Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.

Design and synthesis of non-hydrolyzable homoisoprenoid α-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases.

An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.

Syntheses of deuterium labeled prenyldiphosphate and prenylcysteine analogues for in vivo mass spectrometric quantification.

A Wittig reaction employing Li(CD3)2CP(C6H5)3 was used to prepare d6-farnesol and d6-geranylgeraniol. Reductive amination of aniline-2,3,4,5,6-d5 was used to prepare the unnatural isoprenoid analogues d5-anilinogeraniol and d5-anilinofarnesol. All of these deuterated isoprenols were elaborated into their diphosphate and cysteine thioether derivatives suitable for use as stable-isotope labeled standards for quantitative mass spectrometric analysis.

Simultaneous dual protein labeling using a triorthogonal reagent.

Construction of heterofunctional proteins is a rapidly emerging area of biotherapeutics. Combining a protein with other moieties, such as a targeting element, a toxic protein or small molecule, and a fluorophore or polyethylene glycol (PEG) group, can improve the specificity, functionality, potency, and pharmacokinetic profile of a protein. Protein farnesyl transferase (PFTase) is able to site-specifically and quantitatively prenylate proteins containing a C-terminal CaaX-box amino acid sequence with various modified isoprenoids. Here, we describe the design, synthesis, and application of a triorthogonal reagent, 1, that can be used to site-specifically incorporate an alkyne and aldehyde group simultaneously into a protein. To illustrate the capabilities of this approach, a protein was enzymatically modified with compound 1 followed by oxime ligation and click reaction to simultaneously incorporate an azido-tetramethylrhodamine (TAMRA) fluorophore and an aminooxy-PEG moiety. This was performed with both a model protein [green fluorescent protein (GFP)] as well as a therapeutically useful protein [ciliary neurotrophic factor (CNTF)]. Next, a protein was enzymatically modified with compound 1 followed by coupling to an azido-bis-methotrexate dimerizer and aminooxy-TAMRA. Incubation of that construct with a dihydrofolate reductase (DHFR)-DHFR-anti-CD3 fusion protein resulted in the self-assembly of nanoring structures that were endocytosed into T-leukemia cells and visualized therein. These results highlight how complex multifunctional protein assemblies can be prepared using this facile triorthogonal approach.

Isoprenyl-thiourea and urea derivatives as new farnesyl diphosphate analogues: synthesis and in vitro antimicrobial and cytotoxic activities.

A series of new isoprenyl-thiourea and urea derivatives were synthesized by the reaction of alkyl or aryl isothiocyanate or isocyanate and primary amines. The structures of the compounds were established by (1)H NMR, (13)C NMR, MS, HRMS and elemental analysis. The new compounds were screened for in vitro antimicrobial activity against seven strains representing different types of gram-positive and gram-negative bacteria. More than a third of the synthesized compounds showed variable inhibition activities against the tested strains. Best antimicrobial activities were found for those thiourea analogues with 3-methyl-2-butenyl, isobutyl or isopentyl groups and aromatic rings possessing electron withdrawing substituents. The new compounds were also subjected to a preliminary screening for antitumoral activity. The presence of a highly lipophilic group and an electron withdrawing group in the aromatic rings enhanced anticancer activity of the synthesized compounds, showing in most cases more activity than that of the controls.

Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis.

Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.

Synthesis of frame-shifted farnesyl diphosphate analogs.

A set of synthetic approaches were developed and applied to the synthesis of eight frame-shifted farnesyl diphosphate (FPP) analogs. These analogs bear increased or decreased methylene units between the double bonds and/or diphosphate moieties of the isoprenoid structure. Evaluation versus mammalian FTase revealed that small structural changes can lead to dramatic changes in substrate ability.

Flexible and general synthesis of functionalized phosphoisoprenoids for the study of prenylation in vivo and in vitro.

Protein modification with isoprenoid lipids affects hundreds of signaling proteins in eukaryotic cells. Modification of isoprenoids with reporter groups is the main approach for the creation of probes for the analysis of protein prenylation in vitro and in vivo. Here, we describe a new strategy for the synthesis of functionalized phosphoisoprenoids that uses an aminederivatized isoprenoid scaffold as a starting point for the synthesis of functionalized phosphoisoprenoid libraries. This overcomes a long-standing problem in the field, where multistep synthesis had to be carried out for each individual isoprenoid analogue. The described approach enabled us to synthesize a range of new compounds, including two novel fluorescent isoprenoids that previously could not be generated by conventional means. The fluorescent probes that were developed using the described approach possess significant spectroscopic advantages to all previously generated fluorescent isoprenoid analogue. Using these analogues for flow cytometry and cell imaging, we analyzed the uptake of isoprenoids by mammalian cells and zebrafish embryos. Furthermore, we demonstrate that derivatization of the scaffold can be coupled in a one-pot reaction to enzymatic incorporation of the resulting isoprenoid group into proteins. This enables rapid evaluation of functional groups for compatibility with individual prenyltransferases and identification of the prenyltransferase specific substrates.

Chemical synthesis of polyprenyl sialyl phosphate, a probable biosynthetic intermediate of bacterial polysialic acid.

Using reaction of moraprenyl phosphate with the known N-acetylsialyl chloride and the novel N,N-diacetylsialyl (Neu5Ac(2)) chloride α- and ß-anomers of polyprenyl sialyl phosphate were synthesized for the first time. The α-selectivity dramatically increased when Neu5Ac(2) chloride was used as the glycosyl donor.

Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time.

Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K(m) of 1.5 µM and a k(cat) of 1.2s(-1) based on [(14)C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K(m) of 1.1 µM and a k(cat) of 1.0 s(-1), consistent with those measured using radiolabeled substrate. Using this assay, the IC(50) of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.

New synthetic methodology for the construction of 7-substituted farnesyl diphosphate analogs.

Through the use of a 1,2-metalate rearrangement, six 7-substituted farnesol analogs were generated in a concise manner. This new synthetic route allowed us to quickly prepare several diverse farnesyl diphosphate analogs with interesting biological activities against mammalian protein-farnesyl transferase.

Synthesis and evaluation of a new fluorescent transglycosylase substrate: lipid II-based molecule possessing a dansyl-C20 polyprenyl moiety.

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.

Towards the synthesis of bisubstrate inhibitors of protein farnesyltransferase: Synthesis and biological evaluation of new farnesylpyrophosphate analogues.

Protein farnesyltransferase (FTase) has recently appeared as a new target of parasitic diseases, a field poor in drugs in development. With the aim of creating new bisubstrate inhibitors of FTase, new farnesyl pyrophosphate analogues have been studied. Farnesyl analogues with a malonic acid function exhibited the best inhibitory activity on FTase. This group was introduced into our imidazole-containing model leading to new compounds with submicromolar activities. Kinetic experiments have been realized to determine their binding mode to the enzyme.

Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

Characterization of the Rv3377c gene product, a type-B diterpene cyclase, from the Mycobacterium tuberculosis H37 genome.

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 degrees C, Mg(II) (0.1 mM), and Triton X-100 (0.1 %). Under these conditions, the kinetic values of K(M) and k(cat) were determined to be 11.7+/-1.9 microM for GGPP and 12.7+/-0.7 min(-1), respectively, whereas the specific activity was 186 nmol min(-1) mg(-1). The enzyme activity was inhibited at substrate concentrations higher than 50 microM. The catalytic activity was strongly inhibited by 15-aza-dihydrogeranylgeraniol and 5-isopropyl-N,N,N,2-tetramethyl-4-(piperidine-1-carbonyloxy)benzenaminium chloride (Amo-1618). The DXDTT(293-297) motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen-bonding networks. The Rv3377c enzyme could also react with (14R/S)-14,15-oxidoGGPP to generate 3alpha- and 3beta-hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium-labeled GGPP and oxidoGGPP. We found that GGPP and (14R)-oxidoGGPP adopted a chair/chair conformation, but (14S)-oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A-ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)-Oxidosqualene is folded in a boat conformation, whereas (3S)-2,3-oxidosqualene folds into a chair conformation, for the formation of the A-rings of the hopene and tetrahymanol skeletons, respectively.

Synthesis of imidazole-containing analogues of farnesyl pyrophosphate and evaluation of their biological activity on protein farnesyltransferase.

With the aim of creating new bisubstrate inhibitors of protein farnesyltransferase (FTase), new carboxylic farnesyl pyrophosphate analogues have been designed and synthesized. The original structures are built around three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring. All the compounds were evaluated for their ability to inhibit FTase and compared with the corresponding derivatives lacking the imidazole ring, synthesized for that purpose. These new compounds are not bisubstrate inhibitors probably because the imidazole ring is not in the right position to interact with the zinc atom. However these derivatives display FPP competitive inhibition with a good activity in the carboxylic farnesyl pyrophosphate analogues series.

Synthesis and application of photoaffinity probe containing an intact isoprenoid chain.

Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins, and proteins with molecular weight of approximately 70 KDa appeared as a major band upon Streptavidin blot analysis.