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

Abstract

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.

Figure 1

Farnesyl diphosphate and related DATFP-containing photoprobes.

Scheme 1

Synthesis of amide-linked photoaffinity analog 3.

Figure 2

Structure of compound 10 determined by X-ray crystallography to establish the E stereochemistry of the C-6 alkene.

Figure 3

Rearrangement of DATFP-amides to triazolones.

Figure 4

Evaluation of amide 3 as an alternative substrate and inhibitor of ScPFTase. (A) Reaction between N-dansyl-GCVIA and FPP or amide 3 catalyzed by ScPFTase monitored by fluorescence spectroscopy. (a) Kinetic data using FPP as a substrate. (b) Kinetic data using amide 3 as a substrate. Fluorescence was monitored at 30°C by excitation at 340 nm and emission at 505 nm. (B) .Inhibition of ScPFTase-catalyzed farnesylation of N-dansyl-GCVIA by amide 3. Reactions contained 2.0 μM FPP, 2.0 μM N-dansyl-GCVIA and 3 at varying concentrations and were monitored using a continuous spectrofluorometric assay. Each point is the average of 2-3 determinations with the error bars indicating the standard error for each measurement.

Scheme 2

Structures of ScPFTase modified N-dansyl-GCVIA peptide products. 11: peptide product from reaction with FPP (1); 12: peptide product from reaction with allylic ester 2; 13: peptide product from reaction with amide 3; 14: peptide product from reaction with alkyl ester 4.

Figure 5

Analysis of photolabeling of ScPFTase with DATFP-amide ([³²P]3) by SDS-PAGE. Lanes 1 and 1′: molecular weight standards. Lanes 2 and 2′: sample containing PFTase and [³²P]3, no UV irradiation. Lanes 3 and 3′: PFTase irradiated in the presence of [³²P]3. Lanes 4 and 4′: PFTase irradiated in the presence of [³²P]3 and FPP (substrate). Lanes 1, 2, 3, and 4 show the silver-stained proteins. Lanes 1′, 2′, 3′, and 4′ show the radiolabeled proteins.

Figure 6

Analysis of photolabeling of HsPGGTase I with DATFP-amide ([³²P]3) by SDS-PAGE. Lanes 1 and 1′ contain samples of PGGTase I and [³²P]3 which were not irradiated. Lanes 2 and 2′ contain samples of HsPGGTase I irradiated at 254 nm in the presence of [³²P]3. Lanes 3 and 3′ contain samples of PGGTase I irradiated in the presence of [[³²P]3 and FPP (substrate). Lanes 1, 2, and 3 show the silver-stained proteins. Lanes 1′, 2′, and 3′ show the radiolabeled proteins.

Figure 7

Analysis of photolabeling of RnPGGTase II with DATFP-amide ([³²P]3) by SDS-PAGE. Lanes 1 and 1′: PGGTase II and [³²P]3, no UV irradiation. Lanes 2 and 2′: RnGGPTase II and [³²P]3, 1 min UV irradiation. Lanes 3 and 3′: RnPGGTase II, [³²P]3, and GGPP, 1 min UV irradiation. Lanes 1, 2, and 3 show the silver-stained proteins. Lanes 1′, 2′, and 3′ show the radiolabeled proteins.

Scheme 3

Synthesis of dihydroester-linked photoaffinity analog 4.

Scheme 4

DATFP-containing allylic- and alkyl-ester model compounds and their putitive hydrolysis products.

Figure 8

¹H-NMR spectra of DATFP-containing model esters obtained under mild acidolytic conditions. Spectra on the left are the compounds before heating and spectra on the right are the compounds after heating at 100°C for 4 h. (A) Compound 18 treated under acidic conditions (CD₃CN/D₂0 10%/TFA-d 0.1%) before heating. (B) Compound 18 treated under acidic conditions (CD₃CN/D₂0 10%/TFA-d 0.1%) after heating to 100°C for 4 h. The appearance of three new doublets of doublets after heating indicates the presence of 21. (C) Compound 19 treated under acidic conditions (CD₃CN/D₂0 10%/TFA-d 0.1%) before heating. (D) Compound 18 treated under acidic conditions (CD₃CN/D₂0 10%/TFA-d 0.1%) after heating to 100°C for 4 h. Compound 19 shows no visible change in the ¹H-NMR spectrum after 4 h. In panels (C) and (D), peaks labeled “S” are from D₂O, p-xylene (internal standard for integration) and CD₃CN.

Figure 9

Reversed-phase HPLC analysis with fluorescence detection of reactions between N-dansyl-GCVIA and FPP (1) or dihydroester 4 catalyzed by ScPFTase. (A) N-dansyl-GCVIA (2.4 μM) incubated with FPP (1, 10 μM) and ScPFTase (24 nM). Chromatogram 1 (bottom): Reaction before the addition of the enzyme with the peptide eluting at t_R=25.7 min. Chromatogram 2 (top): Reaction after 5 min with the prenylated peptide (11) appearing at t_R=41.5 min. (B) N-dansyl-GCVIA (2.4 μM) incubated with dihydroester (4, 10 μM) and ScPFTase (24 nM). Chromatogram 1 (bottom): Reaction before the addition of the enzyme with the peptide substrate at t_R=25.7 min. Chromatogram 2 (top): Reaction after 23 h with the prenylated peptide (14) appearing at t_R=36.0 min and some starting material still present.

Figure 10

Inhibition of ScPFTase-catalyzed farnesylation of N-dansyl-GCVIA by dihydroester 4. Reactions contained 2.0 μM FPP, 2.0 μM N-dansyl-GCVIA and 4 at varying concentrations and were monitored using a continuous spectrofluorometric assay. Each point is the average of 3 determinations with the error bars indicating the standard error for each measurement.

Figure 11

Analysis of photolabeling of purified FPP-utilizing enzymes with dihydroester [³²P]4. For all samples, photolysis reactions were fractionated by SDS PAGE followed by phosphorimaging analysis to allow visualization of the radiolabeled proteins. In each case, Lane 1 contains enzyme irradiated in the presence of [³²P]4 while Lane 2 contains enzyme irradiated in the presence of [³²P]4 and FPP (substrate). Where shown, Lane 3 contains [³²P]4 incubated in the presence of protein without irradiation. Panel A: Analysis of photolabeling of purified PFTase. Panel B: Analysis of photolabeling of purified EcFPPSase. Panel C: Analysis of photolabeling of purified NoSTSase.

Figure 12

Selective photolabeling of ScyPFTase in crude E. coli extract using dihydroester 4. Analysis of photolabeling of ScPFTase with [³²P]4 by SDS-PAGE. Lanes 1 and 1′: Molecular weight standards. Lanes 2 and 2′: Crude ScPFTase irradiated in the presence of [³²P]4. Lanes 3 and 3′: Crude ScPFTase irradiated in the presence of [³²P]4 and FPP (substrate). Lanes 4 and 4′: Sample containing crude yPFTase and [³²P]4, no UV irradiation. Panel A: Sypro Orange-stained proteins. Panel B: Radiolabeled proteins visualized via phosphorimaging analysis.

Figure 13

Results of docking experiments with dihydroester 4 and RnPFTase. Top Left (A): Top 3 poses for the R enantiomer of 4 (line representations) docked into RnPFTase. The position of FPP (stick representation) bound to RnPFTase determined via crystallography (pdb code: 1JCR) is shown for comparison. Top Right (B): Top 3 poses for the S enantiomer of 4 docked into RnPFTase. Bottom Left (C): Comparison of highest-scoring docked pose for the R enantiomer of 4 (line representation) with the structure of the protein-bound R enantiomer determined via crystallography (stick representation, green carbons) and FPP (stick representation, yellow carbons). Bottom Right (D): Comparison of highest-scoring docked pose for the S enantiomer of 4 (line representation) with the structure of the protein-bound S enantiomer determined via crystallography (stick representation) and FPP (stick representation, yellow carbons). Colors: C (line representations: grey, stick representations: yellow [FPP], green [crystallographic 4), F (line representations: green, stick representations: white), N (blue), O (red), P (line representations: purple, stick representations: orange). As noted in the text, the term “docked” pose used here refers to the docked poses that have been subjected to energy minimization.

Figure 14

Structure of dihydroester 4 bound to RnPFTase determined by X-ray crystallography. (A) Electron density for dihydroester 4 (mesh) contoured at 1.0 σ. The structures of the two enantiomers of 4 (shown in stick representations) are fit within the electron density. (B) Structures of the two enantiomers of 4 superimposed with the structure of FPP (green) bound to RnPFTase. (C) Active site of RnPFTase showing structures of the two enantiomers of 4 superimposed with the structure of FPP (green). The insert shows the difference in conformation for Y166α and H210α between the structures of 4 and FPP. Colors: C (yellow), N (blue), O (red), α-subunit (grey), β-subunit (light blue), Zn (orange).

Figure 15

CPK models and molecular electrostatic potential (MEP) maps calculated for FPP and dihydroester 4. Above: CPK model (A) and MEP (B) for FPP. Below: CPK model (C) and MEP (D) for 4. For MEP maps, potentials were plotted on a surface of constant density (density = 0.02). Electronegative areas are shown in red and electropositive areas are shown in blue.