Protein Prenylation and Their Applications

Abstract

Prenylation is a universal covalent post-translational modification found in all eukaryotic cells, comprising attachment of either a farnesyl or a geranylgeranyl isoprenoid. Prenyl group is important for protein-protein binding through specialized prenyl-binding domains. Farnesylation and geranyl geranylation are very important in C-terminal anchoring of proteins to the cell membrane. These post-translational modification are most often catalyzed by either protein farnesyl transferase (FTase) or protein geranyl geranyl transferase-I (GGTase-I). These enzymes typically recognize a CaaX motif, where “C” is the cysteine to be prenylated and the remainder of the motif leads to recognition by FTase and/or GGTase-I. Prenylation plays vital role in diversification of natural products flavonoids, coumarins, and isoflavonoids. Many prenylated compounds have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-spasmodic, anti-bacterial, anti-fungal, anti-inflammatory, and anti-androgen activity. Due to their beneficial effects on diseases, prenylated compounds are of particular interest as lead compounds for producing drugs and functional foods. In this chapter, we concise the prenylation reactions of aromatic compounds such as indole, ketones, and aldehydes that may results to lead molecules discovery. Prenylation reactions are applied on azoles, anilines, thioles, indole, α-carbonyl bromides, and aryl bromide. There are several drugs that are obtained from prenylation, i.e. (-)-17-hydroxy-citrinalin, (+)-stephacidin, prenylated. In this text there is no referencing, it is a chemical name, so keep as it is.

1. Introduction

Prenylation is class of modification of molecules involving irreversible covalent bonding of isoprenoid unit to chemical compound or protein. Prenylation is known as lipidation or isoprenylation (Figure 1) [1].

Figure 1.

Structure of isoprenyl group.

Prenylation of protein is required to make protein fully functional. In a three-step process, farnesyl (15-carbon) or geranylgeranyl (20-carbon) group is transferred to protein, which occurs on carboxyl terminal of cysteine residues of protein. The RAM mutant, a Saccharomyces cerevisiae mutant, was used to characterize the location of prenylation in proteins changed by these substituents. This mutant was found to be defective in the post-translational processing of both the ras gene product and the yeast a-mating factor. A common carboxy terminal region was discovered when the predicted amino acid sequences of these two proteins, other Ras proteins and mating factors, and the known prenylated proteins lamin B and prelamin A were compared. A CAAX box is a carboxy terminal amino acid sequence in which C is cysteine, A is an aliphatic amino acid, and X is the carboxy terminal amino acid. The three AAX amino acids were removed from the structure of yeast mating hormone a-factor, which was discovered to be farnesylated and carboxymethylated at the carboxy terminal cysteine ("). This discovery anticipated that a carboxy terminal cysteine in mammalian ras protein would be prenylated with farnesyl. The modifications of proteins by prenylation increase lipophilicity of prenylated proteins for efficient anchoring on plasma membranes or organellar membranes. Prenylated proteins plays role in a number of signaling and regulatory pathways that are responsible for basic cell operations [1, 2]. The first prenylated polypeptide to be discovered in late 1970 and early 1980 which is the mating factor from the fungus Rhodosporidium toruloides which is an undecapeptide containing a C-terminal S-farnesyl-cysteine methyl ester. These mating factors are found as farnesyl group attached to cysteine residue of short peptide [3, 4]. Farnesyltransferase and geranylgeranyl transferase type 1 (GGTase-I) are enzymes that catalyze the attachment of a single farnesyl (15 carbon) or geranylgeranyl (20 carbon) isoprenoid group to proteins. The addition of two geranylgeranyl groups to two cysteine residues in sequences such as CXC, CCXX near the C-terminus of Rab proteins is catalyzed by geranylgeranyl transferase type 2 (GGTase-II or Rab geranylgeranyl transferase) (Figures 2 and 3) [5, 6].

Figure 2.

Structures of 1 (farnesyl diphosphate, FPP) and 2 (geranylgeranyl diphosphate, GGPP).

Figure 3.

Reactions catalyzed by prenyl transferase enzymes.

Natural chemicals can be prenylated to add structural variety, change biological activity, and improve medicinal potential. Prenylated natural products are a large class of bioactive molecules with demonstrated medicinal properties such as anti-emetics, anticonvulsants, antidepressants, and analgesics. Examples include prenyl-flavonoids, prenyl-stilbenoids, and cannabinoids. Prenyl transfer of natural products catalyzed by enzymes has high regio- and stereo-specificity, but it requires expensive isoprenyl pyrophosphate substrates [4]. In prenylated natural products, the carbon lengths of the prenyl side chains differ. Based on the size of the carbon, four different forms of prenyl side chains have been identified: C5 (isopentenyl), C10 (geranyl), C15 (farnesyl), and C20 (isopentenyl) (geranylgeranyl) [5]. Prenyltransferases catalyze the fundamental isoprenoid chain-elongation reaction, resulting in prenyl diphosphate with a variety of chain lengths and stereochemistries. All of the compounds are made up of linear isoprenoid diphosphates, which are made up of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, two isomeric 5-carbon unit intermediates (DMAPP). According to the specificities of particular prenyltransferases, the reactions are regulated to run continuously and to cease exactly at certain chain lengths [7].

2. History behind protein prenylation

Rhodotorucine A, a mating factor peptide of the yeast Rhodosporidium toruloides, was found in 1979 in Japan as the first example of prenylation of a carboxy-terminal cysteine residue. A S-farnesyl cysteine was discovered near the carboxy-terminus of the mature form. Over the next five years, more fungal peptidyls will be discovered. sex hormones covalently modified by a farnesyl group on a C-terminal cysteine were made public This modification's function was unknown, but it was stoichiometric and stable, indicating that it is a key component of the function. a list of mate-inducing variables Prenylation in mammals was discovered independently for the first time. Compactin, a cholesterol production inhibitor was discovered to have a negative effect on cells in 1984 [1].

Compactin inhibits 3-hydroxy-3-methylglutaryl-CoA-reductase (HMG-CoA reductase), an enzyme that produces mevalonic acid, a key step in the isoprenoid pathway. Compactin treatment of cultured cells resulted in cell cycle arrest and morphological changes. The fact that these effects could be reversed by giving the cells mevalonate but not cholesterol, dolichol, ubiquinone, or isopentenyl adenine, the principal products of isoprenoid biosynthesis, demonstrated that one or more non-sterol isoprenoids play a significant role in cell cycle and shape. Covalent incorporation of an isoprenoid derivative into cellular proteins was seen following the destiny of radiolabeled mevalonate in cultured 3T3 fibroblasts with their mevalonate production suppressed by lovastatin [3].

Isoprenylation was shown to be a common process in mammalian cells, including proteins from several compartments such as the nuclear envelope, plasma membrane, and cytoplasm. In 1986, a novel gene from the yeast Saccharomyces cerevisiae was discovered that was necessary for posttranslational modification of both the RAS proteins and an a-mating factor termed RAM. Except for the C-terminus, which consists of a cysteine followed by two residues with aliphatic side chains and an ultimate C-terminal amino acid residue, there was no evident sequence similarity between the yeast RAS proteins and the precursor polypeptide for a-factor. This so-called CaaX motif was discovered in a number of other proteins as well, although at the time, it was thought to communicate palmitoyl group alteration. New eukaryotic protein carboxyl methylation processes, such as modification of the subunit of cGMP phosphodiesterase and nuclear lamin B, were identified in 1985. These responses did not fall into any recognized activity category. The discovery of the CaaX-motif in the aforementioned proteins, as well as the carboxymethylation of several fungal mating factors at the C-terminus, led to the notion that the CaaX sequence signal related prenylation, proteolytic cleavage, and methylation.

The discovery in 1988 that the yeast a-factor contains a C-terminal farnesylcysteine methyl ester and the discovery in 1989 that all mammalian Ras proteins are isoprenylated on the conserved cysteine residue supported this hypothesis. Upstream cysteine residues in a subset of Ras proteins could be assigned to palmitoylation, which was previously thought to modify this amino acid. At the same time, lamin B was discovered to be the first isoprenylated protein. In 1990, trans, trans-farnesyl, and all-trans-geranylgeranyl were found as the primary isoprenoids bound to mammalian protein. The discovery that nuclear lamin B, mammalian Ras, and yeast RAS are farnesylated was followed by the discovery that a -subunit of a mammalian heterotrimeric G-protein is geranylgeranylated. The finding that Ras farnesylation is essential for oncogenic forms to convert cells spurred research efforts in this sector, resulting to the identification of the farnesyltransferase enzyme and the creation of particular protein prenylation inhibitors for cancer treatment [8].

3. Classes of prenylated proteins

The growing realization that the presence of a CAAX motif at a protein’s carboxyl terminus indicated that it was a candidate for prenylation sparked a flurry of effort aimed at detecting the prenylation status of such proteins. Initial searches of protein sequence databases yielded 40 such possibilities; unexpectedly, the majority of these proteins were members of the GTP-binding protein class (sometimes known as “small G proteins”), which is linked to the ras proteins. Furthermore, the majority of the remaining possibilities (e.g., the subunits of heterotrimeric G proteins, cGMP phosphodiesterase) were recognized to have a role in cellular signaling. Almost all of the potential proteins that have been thoroughly examined have been discovered to be prenylated. Only the 15-carbon farnesyl and 20-carbon geranylgeranyl groups have been shown to alter proteins so far, with geranylgeranyl being the most often attached isoprenoid. The COOH-terminal amino acid (“X”) of the CAAX box is now known to dictate which isoprenoid is linked to a candidate protein, which is of great interest to researchers investigating the enzymology of the prenylation processes (see below). The protein includes the farnesyl isoprenoid if this residue is a serine, methionine, or glutamine, whereas a leucine at that location drives geranylgeranyl addition. On proteins with the CAAX-motif, prenylation is not the sole posttranslational modification. The three COOH-terminal amino acids (the “AAX”) are missing from the mature versions of these prenylated proteins. These three amino acids are removed by a cellular peptidase, leaving the prenylated cysteine as the COOH-terminal residue. Furthermore, a significant percentage of prenylated proteins have the carboxyl group of this cysteine residue methylated in all situations where the prenylated proteins have been thoroughly investigated. The end outcome of these three seemingly unrelated processing processes is a mature protein with a highly hydrophobic COOH-terminus, significantly improving the protein's inherent hydrophobic characteristics. It has recently been discovered that prenylation is not confined to proteins with the COOH-terminal C U X - motif, but also occurs on the rabIYPT1 family of GTP-binding proteins. The COOH-terminal sequences of the majority of these proteins finish in Cys-Cys or two cysteines separated by another amino acid (Cys-X-Cys). The rab/YPTl proteins appear to have the ability to reversibly connect with membranes and have been involved in the modulation of intracellular membrane trafficking pathways. Furthermore, both cysteine residues are prenylated with the geranylgeranyl isoprenoid in many (if not all) of these proteins. No proteolytic processing is necessary to expose the carboxylate of the prenylated cysteine residue for methylation because these alterations occur at the extreme COOH-terminus, although this methylation of the carboxyl group has been proven to occur [9].

4. Isoprenoid analogs

To examine various aspects of the prenylation reaction and prenyltransferases, a significant number of isoprenoid analogues with varied functions have been produced. Photo affinity probes like compounds 6 through 4 were employed extensively to investigate the structural properties of yeast and mammalian forms of FTase and GGTase-I before the crystal structure of FTase was discovered. These analogues revealed the role of the subunit of FTase and GGTase-I in isoprenoid substrate recognition and binding, as well as changes in active site layout between mammalian and yeast FTases. Similar tests using GGTase-II were later carried out, leading to the discovery of proteins with which Rab5 interacts via the isoprenoid group. While three was recognized as a substrate by FTase, four, which included one more isoprenoid unit, was found to be a powerful inhibitor of yeast FTase. A novel photoactive isoprenoid probe with a diazirine group was recently published, with a size that is closer to that of FPP. In cross-linking investigations of Icmt, peptides containing that photoactive isoprenoid were employed. Phosphonate 6 and related analogues 66 have been particularly effective in crystallographic investigations that have demonstrated that the isoprenoid binds in an extended conformation and that isoprenoid binding causes many active site residues to rearrange when compared to the unliganded enzyme. Isoprenoid substrate specificity of prenyltransferase enzymes has been investigated recently using isoprenoid analogues. Gibbs and colleagues created a variety of all-trans FPP and GGPP geometric isomers. They discovered that whereas most of the analogues were substrates for mammalian FTase, they were not accepted as substrates by mammalian GGTase-I. Molecule 7 inhibited GGTase-I, showing a more rigorous selectivity for the enzyme's isoprenoid substrates. The binding site of FTase is very plastic, according to Spielmann and colleagues. Analog 8, for example, was an effective FTase substrate because all isoprene units were substituted with aryl groups. The anilinogeranyl-based isoprenoid analogues 9 and 10. The downstream enzymes Rce1 and Icmt processed proteins changed with these analogues, but the resulting modified proteins were not physiologically active, showing the necessity of enhanced hydrophobicity during prenylation. They also discovered that some anilinogeranyl-based analogues, such as 11, were substrates for FTase when a peptide-based on K-Ras C-terminal sequence, dansyl-GCVIM, was used, but that they were potent inhibitors of the enzyme when dansyl-GCVLS (IC50 of 16 was 3.0 nM), a sequence based on H-Ras C-terminal sequence, was used (Figure 4) [10].

Figure 4.

Structures of isoprenoid analogs used to study structure, mechanism and isoprenoid substrate specificity of FTase and GGTase-I.

Gibbs and colleagues synthesized a significant number of analogues with alterations at the 3- or 7-positions of FPP. They concluded that even minor modifications in the functionality incorporated at these points can result in significant and surprising differences in incorporation efficiency. For example, they discovered that the 3-vinyl analogue 12, which is a slow FTase substrate in cells, and the 3-allyl analogue, which is an FTase inhibitor, is both slow FTase substrates. During the screening of analogues against an eight-sequence CaaX library, the 7-allyl analogue 14, could only farnesylate the CVIM sequence, whereas 15 was an exceptionally efficient substrate for practically all of the sequences in their library. These minor differences are likely due to the fact that when bound to FTase, the protein and isoprenoid substrates interact over a large surface area thus, small changes in one of the substrate structures necessitate compensatory changes in the other substrate to achieve optimal complementarity (Figure 5) [11, 12].

Figure 5.

The synthetic biochemistry platform for the production of prenyl natural products.

First, glucose is broken down into pyruvate through a glycolysis pathway modified to regulate NADPH levels (12 enzymatic steps). Then, either PDH or the PDH bypass converts pyruvate into acetyl CoA. Acetyl-CoA is converted into GPP via the mevalonate pathway (eight enzymatic steps). By varying the aromatic prenyltransferase (aPT) and aromatic substrate we can produce various prenyl-flavonoids and prenyl-stilbenoids using the same central pathway. We developed the prenyltransferase NphB (dNphB) variants to produce CBGA or CBGVA. CBGA is converted to cannabidiol and CBGVA is converted to cannabidivarin acid via cannabidiolic acid synthase (CBDAS). It is possible to produce other cannabinoids by using different cannabinoid synthases (THCAS and CBCAS) [13].

5. Mechanism of protein prenylation

5.1 Binding mechanism

There is currently a substantial body of knowledge about the role of the active site Zn²⁺ in PFTase. Mutagenesis of the protein-derived Zn²⁺ ligands resulted in significantly reduced Zn²⁺ binding and activity [14]. The replacement of Co²⁺ for Zn²⁺ provided more direct proof of Zn²⁺ in catalysis. Comparisons of the Co²⁺ –PFTase absorbance spectrum to that of model compounds revealed the presence of one thiolate ligand, possibly Cys299β, in the metal's inner coordination sphere. When peptide substrate was added, the spectrum resembled that of two thiolate ligands. This means that the cysteine in the peptide substrate is directly thiolate-coordinated to the metal. Cysteine ligation was later crystallographically verified with a Zn^2+–sulfur bond length of 2.5.52. Although the spectra of the Co²⁺ –PFTase–product complex differed from that of the ternary complex containing substrates, the Co²⁺ –PFTase–product complex showed more sulfur–cobalt coordination than the free enzyme. The farnesylated peptide interpreted the UV spectrum in terms of thioether coordination to Co²⁺. Co²⁺–PFTase has similar activity to the conventional Zn²⁺ enzyme. Single turnover tests utilizing absorbance stopped-flow spectrophotometry produced a two-exponential trace whose rates and extinction coefficient changes corresponded to the development of a Co²⁺ –PFTase–FPP–peptide ternary complex and subsequent conversion to the Co²⁺ –PFTase–product complex. On a time scale consistent with catalysis, the creation and disappearance of Co²⁺ –thiolate species were established [15].

5.2 Chemical mechanism

The molecular mechanism of the prenyltransferase reaction is currently being researched, and a better knowledge of the reaction's transition state should aid in the development of inhibitors for these enzymes. The diphosphate moiety in the prenyl substrate is displaced by the sulfur in the peptide/protein substrate in this reaction. It could happen through an associative mechanism in which the peptide thiolate attacks the C1 site of FPP directly, causing the diphosphate to be displaced (Figure 6).

Figure 6.

Possible reaction mechanisms for protein prenyltransferases.

The reaction would continue through a single transition state in this situation. An associative mechanism can take a variety of forms, ranging from an early transition state with little positive charge growth in the allylic moiety of the prenyl substrate to a late transition state with significant positive charge development. The nucleophilicity of the substrate thiolate provides much of the “driving power” for an associative mechanism with an early transition state. The nucleophilicity of the thiolate and the capacity of the isoprene moiety to stabilize the growing positive charge are critical for a mechanism with a later transition state. An allylic carbocationic intermediate would be used in a dissociative process (Figure 6). The production of the pyrophosphate anion-allylic carbocation ion pair in the active site would result in significant charge separation. There would be two distinct transition states in this reaction. The stabilization of the allylic carbocation by resonance provides a significant portion of the “driving power” in this mechanism. L-b-farnesylaminoalanine (faA), a transition state analogue, was designed to mimic the charge and shape of a putative alkylated thiol intermediate formed by adding the prenyl group to a protonated sulfur [16]. The faA (IC50 = 51 mM) and tetrapeptide faA-VIA (IC50 = 14 mM) derivatives showed modest inhibitory activity. The IC50 of faA-VIA was reduced to 370 nM in the presence of inorganic pyrophosphate. This class of books has minimal binding. The fact that the thiolate form of cysteine is the species that assaults FPP is easily explained by subsequent research. Several fluorine derivatives of FPP were used to study the evolution of positive charge in the transition state. As the quantity of fluorines in the C3 methyl grows, a cationic intermediate becomes increasingly unstable. This approach produced an excellent Hammett plot for the rates of solvolysis Ksolv (a dissociative reaction) vs the rates of prenyl transfer in the reaction catalyzed by FPP synthase where the nucleophile is a carbon-carbon double bond, with a slope of 1 [17]. The enzyme-catalyzed reaction's response to substitution was thus very similar to a dissociative solvolysis reaction involving allylic carbocationic intermediates. Increased fluorine substitution revealed a high Hammett connection with the solvolysis hypothesis in the instance of PFTase. The slope, on the other hand, was significantly smaller than unity. Hammett plots of kcat vs the rate of associative displacement between allylic reactants and azide showed a considerably better correlation. The most obvious explanation of these findings is that the PFTase-catalyzed reaction's transition state is electrophilic, but the mechanism is associative with a late transition state with significant electrophilic character. The difference between PFTase and FPP synthase could be due to the thiolate prenyl acceptor's higher nucleophilicity when compared to a carbon-carbon double bond.

Weller and Distefano have mentioned measurement of the secondary kinetic isotope effect for prenylation of dansyl- GCVIA with [1-2H2] FPP [9]. They discovered kH/kD 1. If fully expressed, a dissociative mechanism should have a kH/kD of 1.2, and an associative mechanism with an early transition state should have a small inverse impact. The partial suppression of a kinetic isotope effect is predicted by commitment factors estimated from yeast PFTase rate constants. The lack of a detectable isotope effect supports an associative process, albeit it is unclear whether the experiments were done precisely enough to detect a minor isotope effect. R- and S-[1-2H] FPP were used to investigate the stereochemistry of the PFTase reaction [18]. The reaction preceded with inversion of configuration at C1 of FPP, according to the 1H NMR spectra of the products. While the stereochemical conclusion is compatible with an associative reaction, it could simply reflect the substrate's orientation in the active site prior to catalysis [19] Inversion of configuration has also been found for FPP synthase and dimethylallyl tryptophan synthase, both of which have strong evidence for a dissociative prenyl transfer mechanism. As a result, the stereochemical findings are compatible with either process. There is no precedent for an associative enzyme-catalyzed prenylation process other than PFTase. Dissociative alkylations appear to be catalyzed by FPP synthase and dimethylallyl tryptophan synthase, which prenylate weakly nucleophilic carbon-carbon double bonds and aromatic rings, respectively. Two copies of the highly conserved DDXXD or DDXXXXD motifs are found in FPP synthase and closely related chain elongation prenyltransferases [20].

The magnesium complexes of the allylic and homoallylic diphosphate substrates are recognized by these patterns. The aspartate-rich motif in FPP synthase has been subjected to site-directed mutagenesis, which suggests that these residues are critical for both catalysis and substrate binding. Although it is possible that a distinct structural pattern evolved for a dissociative alkylation, the absence of related aspartate-rich motifs in the active site of protein prenyltransferases could indicate that the mechanism for sulfur alkylation is different. A shift from the dissociative process reported in FPP synthase to an associative reaction for protein prenylation is consistent with a cysteine thiolate's high nucleophilicity [21].

6. Reagents for prenylation reaction and their applications

Prenylating reagent plays a significant function in bioactive and synthetic substances because it has a high binding affinity for proteins and increases membrane permeability, enhancing bioactivity and bioavailability of prenylated drugs. Through a specific prenyl-binding domain, the prenyl group plays an important role in protein-protein binding [22, 23].

6.1 Prenyl ester

Prenyl ester is used for the synthesis of prenyl alcohol. Prenyl ester is synthesis from alkanoic acid and isoprene. When alkanoic acid (it is a mixture of carboxylic acid RCO₂H) Where R is a (C1-C2) alkyl group reacted with 3-Methyl but-1-3-ene (isoprene) in the presence of an inorganic acid catalyst to a mixture at room temperature or applying gentle heat to the reaction mixture will formation of Prenyl ester (Figure 7) [24].

Figure 7.

Preparation of prenyl ester.

When strong acidic condition giving to the reaction then reaction will occur however it will yield prenyl ester (Ex. Sulfuric acid or p-toluene sulfonic acid).

6.2 Prenyl acetate

Prenyl acetate is a derivative of prenyl ester. Prenyl ester is insoluble in water and soluble in heptanes and octane. Prenyl acetate is synthesized from alkanoic acid and isoprene.

When alkanoic acid (it is a mixture of carboxylic acid RCO₂H) Where R is a (C1-C2) alkyl group preferably acetic acid in molar excess is used for alkanoic acid, reacted with 2-methyl-1, 3- butadiene (isoprene) in the presence of phosphoric acid as the catalyst and reaction occurs at room temperature heating is not essential. However, absent heating, the reaction is slow.

Hence, gentle heating is giving to the reaction (about 40°C to about 100°C) of the reaction mixture under pressure (due to the volatility of isoprene). Then yield is form Prenyl Acetate (Figure 8) [25].

Figure 8.

Preparation of prenyl acetate.

Formation of prenyl alcohol is one of the applications of Prenyl acetate. Prenyl acetate is saponified using Sodium hydroxide or Sodium carbonate in the presence of aqueous methanol to yield prenyl alcohol (Figure 9) [26].

Figure 9.

Formation of prenyl alcohol.

Prenyl alcohol is known as prenol, 3-Methyl-2-butenyl alcohol, 3, 3-Dimethylallyl alcohol, 3-Methyl-2-buten-1-ol, 3-Methyl-2-butenol, butenol methyl, Methyl-3-but-2-en-1-ol. Prenol is used in the manufacturing industries as an intermediate in pharmaceutical companies and aroma compounds it is also used in the manufacture of Vitamins A and E, the anti-acne drugs tretinoin and isotretinoin [27, 28, 29].

Formation of citral is the application of prenol. Citral is an acyclic monoterpene aldehyde with two isoprene units, making it a monoterpene. Citral is a generic word for two geometric isomers, each of which has its own name; the E-isomer is known as Geranial (trans-Citral) or Citral A, or Neral (cis-Citral) is the name given to the Z-isomer [8, 9]. Citral is used for to hide the smell of smoking; Citral is utilized in the manufacture of vitamin A, lycopene, ionone, and methylionone [30].

Synthesis of Citral from prenol by one pot process: One pot process means it is a method of increasing the effectiveness of a chemical reaction by exposing a reactant to many chemical reactions in a single reactor. Chemists prefer this because it saves time and money while enhancing chemical yield by eliminating a long separation procedure and purification of intermediate chemical compounds (Figure 10) [31].

Figure 10.

Synthesis of Citral from prenol by one pot process.

Formation of prenylated indole derivatives: Prenylated indole alkaloids are a vast class of fungal secondary metabolites that include cytotoxic, insecticidal, and antifungal properties. Antibacterial and anthelmintic properties make them a growing subject of study for synthetic and biological researchers [32].

6.3 Prenyl sulfate

Geranyl sulfate, triethylammonium salt and Neryl sulfate, triethylammonium salt these two-reagent system was created for the S-Prenylation of thiols. As alkylating reagents, prenyl sulfate was utilized, allowing the S-Prenylation processes to be carried out in aqueous solutions at room temperature [33].

Synthesis of Geranyl sulfate and Neryl sulfate: Geraniol and Nerol (C10-Prenol) treated with the molar excess of pyridine-SO₃ complex with triethylamine the intended prenyl sulfate precipitated together with the non-consumed prenols when the reaction mixtures were treated with water. The latter were subsequently separated from the residue using cold n-heptane to get yield of triethylammonium salts of pure geranyl sulfate (75 %) and neryl sulfate (72 %) (Figure 11) [34].

Figure 11.

Synthesis of prenylated sulfates.

6.4 Application of Geranyl sulfate and neryl sulfate

Synthesis of benzenethiol derivative: Benzene thiol is known as thiophenol. The thiophenolate is very nucleophilic, implying a high rate of alkylation [35]. Thiophenol are used to make medications such as sulfonamides. Butoconazole and Merthiolate, both antifungal medicines, are thiophenol derivatives [36].

Synthesis of Geranyl cysteine: Reaction proceeds through S-Prenylation. When L-cysteine is treated with sulfate in the presence of aq. KOH then reaction mixture was extracted with diethyl ether to remove triethylamine and geraniol (which was generated, most likely, due to incomplete hydrolysis of sulfate), as per usual S-prenylation technique. It was simple to purify and isolate the required geranyl cysteine as a potassium salt 43% yield (Figure 12) [37].

Figure 12.

Synthesis of Geranyl cysteine.

6.5 Prenyl benzaldehyde

The IUPAC name of prenyl benzaldehyde is 4-Chloro-5-(1, 1-dimethylallyl)-2-methoxybenzaldehyde. The synthesized benzaldehydes are being utilized to make a variety of new analogues of Licochalcone A, a well-known antibacterial molecule, as well as to investigate the pharmacophoric components required for antibacterial action (Figure 13) [38].

Figure 13.

Structure of prenyl Benzaldehyde.

Application of prenyl benzaldehyde: The prenylated benzaldehydes were employed to discover the pharmacophoric components responsible for Licochalcone A as an antibacterial action. It was discovered that the hydroxyl group at position 4′ of the A ring was important for action [39].

6.6 Prenyl bromide

Prenyl bromide is also known as 3, 3-Dimethylallyl bromide, 1-Bromo-3-methyl-2-butene, and Prenyl bromide (Figure 14).

Figure 14.

Structure of Prenyl Benzaldehyde.

Prenyl bromide is soluble water and other organic solvents like ether and acetone. Propyl bromide (n-PB) is used as an intermediate in organic synthesis and in the manufacture of Agrochemicals and Pharmaceuticals. N-PB and n-PB formulations are also used as organic cleaning solvent for precision cleaning, degreasing, electronics and metal cleaning applications [40].

Applications:

Synthesis of N-Prenylation of (-)-indolactam-V: N-prenylation with an electrophilic group. Deprotonation of N-unsubstituted indoles with NaH or, more rarely, KH in DMF, DMSO, THF, or acetone, followed by reaction with prenyl bromide or chloride, is a common method for making N-prenyl indole derivatives. Yields are typically greater than 80%. If electron withdrawing groups (Ac, CHO) were present at C3, alkali hydroxides in the presence of crown ethers, phase transfer catalysts, or K₂CO₃ were utilized as bases. When dealing with NaH/DMF, N-unsubstituted diketopiperazines and phthalimide protected tryptophan derivatives underwent partial epimerization. Alternatively, the disodium salt of tryptophan could be used to selectively N-prenylate unprotected tryptophan by treating it with prenyl bromide/Na in liquid ammonia (Figure 15) [41].

Figure 15.

N-Prenylation of (-)-indolactam.

A series of 5-arylidene-2, 4-thiazolidinediones and its geranyloxy or prenyloxy derivative were synthesized by using Prenyl bromide.

Flavonoids synthesize with prenyl bromide chains, in the presence of anhydrous potassium carbonate using two different methodologies, one based on conventional thermal heating and other by MAOS.

Total Synthesis of (–) Rosiridol and (–)-Bifurcadiol by Catalytic Asymmetric Aldehyde Prenylation:

Synthesis of (-) Rosiridol: Primary alcohol is treated with trityl group as protecting group and triethlamine in the presence of DMAP (acyl transfer reagent) yield compound. The compound undergoes standard allylic oxidation to form alcohol. The obtained compound treated with MnO₂ gives substrate. The catalytic asymmetric prenylation of compound treated with prenyl boronic ester was undergoes the optimized conditions to form homoprenyl alcohol. Removing the protecting group of reaction gives the (-) Rosiridol (Figure 16) [42, 43].

Figure 16.

Synthesis of (-) rosiridol.

Synthetic route of (–) Bifurcadiol: Primary alcohol is treated with trityl group as protecting group and triethlamine in the presence of DMAP (acyl transfer reagent) yield compound. The compound undergoes standard allylic oxidation to form alcohol. The obtained compound treated with MnO₂ gives substrate. The catalytic asymmetric prenylation of compound treated with prenyl boronic ester was undergoes the optimized conditions to form homoprenyl alcohol. Removing the protecting group of reaction gives the (-) Bifurcadiol (Figure 17) [44].

Figure 17.

Synthetic route of (–) Bifurcadiol.

Biogenetic type synthesis of chromanochalcones from prenylated chalcones: By regioselective cyclization using BF₃–Et₂O, we have established a simple and convenient process for the synthesis of chromanochalcones from prenylated chalcones in high yields. Use of BF₃–Et₂O to form a complex with the chelated hydroxyl group (C-2) and α, b-unsaturated carbonyl group of the prenylated chalcone for regioselective cyclization. The production of a flavanone is prevented by this complexation. The chromanochalcone is formed by cyclization of the prenyl group by the second mole of the reagent (Figure 18) [45].

Figure 18.

Biogenetic type synthesis of chromanochalcones from prenylated chalcones using BF₃-Et₂O.

Synthesis of lupiwighteone: The presence of a prenylated side chain (i.e. prenyl, geranyl) on the flavonoid skeleton separates prenylated flavonoids from other naturally occurring flavonoids. Claisen rearrangements are a highly effective approach for regioselective prenylation of isoflavonones compounds.

The 5-hydroxy group in genistein was treated with the two equivalents of acetic anhydride in pyridine gives 7, 4 -diacetoxy-5-hydroxyisoflavone, when condensation of 7, 4 -diacetoxy-5-hydroxyisoflavone with 3-methyl-2-buten-1-ol in the presence of triphenylphosphine and diethyl azodicarboxylate in dry THF will gives the desired ether 7, 4 -diacetoxy-5-(3-methyl-but-2-enyloxy)isoflavone. The compound 7, 4 -diacetoxy-5-(3-methyl-but-2-enyloxy) isoflavone was dissolved in minimum dry CHCl₃ solvent and heated at 60°C. Will form the para-rearrangement product, 7, and 4 -diacetoxy-5-hydroxy-8 prenyl isoflavone, this compound get hydrolysis with 10% aqueous NaHCO₃ at 60°C gives the lupiwighteone (Figure 19) [46].

Figure 19.

Synthesis of lupiwighteone.

7. Biotechnology application

The ability of FTase to change a single cysteine residue in the C-terminal CaaX motif and incorporate isoprenoid analogs with bioorthogonal functions has been utilized for site-specific protein modifications in recent years. This is feasible because the presence of a CaaX-box at the C-terminus of practically any protein is enough to make it an effective FTase substrate. Functionalization of the resultant proteins by bioorthogonal processes provides a simple way for producing a large range of site-specific protein conjugates.

For immobilization of proteins (GFP or GST) onto solid surfaces such as glass slides or agarose resin, both the Poulter and Distefano groups employed azide- and alkyne-functionalized FPP analogs in FTase-catalyzed reactions followed by click reactions or Staudinger ligations. Maynard and colleagues used a similar technique to immobilize mCherry protein tagged with 25 onto a patterned azide-functionalized surface produced by microcontact printing. Waldmann and colleagues used a photochemical thiol-ene reaction between farnesylated recombinant proteins and surface-exposed thiols from functionalized surfaces to immobilize functional proteins in an orientated and selective manner (mCherry and Ypt1) Poulter and colleagues have immobilized the glutathione S-transferase enzyme and antibody-binding protein G to self-assembled monolayers on gold surfaces in a highly organized, regioselective manner. They also developed immobilized recombinant antibody-binding protein L sandwich antibody arrays for trapping antibodies for direct and sandwich-type immunofluorescent detection of ligands in a microarray manner. In general, where oriented protein immobilization is required, the prenylation-based immobilization strategy has several potential biomedical and biotechnology applications, such as protein arrays and diagnostic applications based on immunoassays, Surface Plasmon Resonance (SPR), or electrochemical methods.

Alexandrov and colleagues used a fluorescent analog of FPP and phase partitioning to establish a simple and effective approach for the derivatization and purification of recombinant proteins (such as YPT7, Rab7, and GST). Distefano and colleagues recently reported using an aldehyde functionalized FPP analog in combination with a hydrazide resin-based catch-and-release technique to purify and functionalize proteins containing groups such as a fluorophore or PEG moiety.

The production of site-specific protein modifications such as protein-DNA conjugates, PEGylated proteins, and dually labeled proteins is one key use of the prenylation-based labeling method. A nanoscale-sized defined tetrahedron architecture composed of four oligonucleotides and four GFP molecules, therapeutically relevant proteins GIP and HIV NC attached to oligonucleotides, and DNA-protein cross-links as DNA lesions to study DNA repair and replication are just a few of the protein-DNA conjugates that have been synthesized using this method.

Prenylation of a recombinant protein called ciliary neurotrophic factor (CNTF) with an isoprenoid analog modified with an aldehyde group, followed by oxime ligation-based catch-and-release, resulted in PEGylated CNTF, with the PEG group potentially increasing the serum half-life of this biomedically important protein. Rashidian et al. describe multifunctional macromolecular protein self-assembly made up of an antibody nanoring structure with a single chain anti-CD3 antibody as the targeting element, as well as a model cargo protein and a fluorophore, in a separate paper. The essential multifunctional fragment comprising of a cargo protein, fluorophore, and protein dimerizer was created using a trifunctional FPP analog including both aldehyde and alkyne activity. This high-avidity “effector-antibody-fluorophore” combination was endocytosed by T-leukemia cells, indicating that it might be useful in the development of protein-drug conjugates for therapeutic protein administration and monitoring [47].

8. Conclusions

In conclusion, we have described the prenylated reagent as important for enhancing the protein binding and membrane permeability of compounds. Prenyl alcohol, prenyl ester, prenyl acetate, prenyl bromide, prenyl benzaldehyde and prenyl sulfate are act as a prenyl side chain in the reaction. Prenyl bromide is applicable in synthesis (-) indolactum for their pharmacological studies. (−)-Rosiridol inhibits monoamine oxidase B (MAO B), which is included in neurodegenerative diseases and (−)bifurcadiol displays anti-ulcer and anti-tumor activities. Prenyl esters are utilized to make prenyl alcohol both are used in synthesis of citral by one-pot process and prenylated indole derivatives. Prenylated chalcone is used for the synthesis of chromochalcone because it is an easy and appropriate method for high yield of the compound and catalyzed para-Claisen-Cope rearrangement represents an excellent method for the synthesis of lupiwighteone with the help of a prenylated side chain and gives a simple and high yielding procedure. In the further studies, lot of prenylated molecules developed with the help of a prenylated reagent for giving a better therapeutic effect.

Acknowledgments

The authors thank the Principal and Department of Pharmaceutical Chemistry Mahatma Gandhi Vidyamandir Pharmacy College, Panchvati, Nashik. The authors would also like to thank the Principal and Secretary of Shreeshakti Shaikshanik Sanstha’s Divine College of Pharmacy, Satana, Nashik.

Conflict of interest

The authors have no conflicts of interest.

Authors’ contributions

All the authors have equally contributed to the article. All authors read and approved the final manuscript.

Acronyms and abbreviations

DMF	Dimethyl formamide
DMSO	Dimethyl sulfoxide
DMAP	4-Dimethylaminopyridine
FPP	Farnesyl diphosphate
FTase	Farnesyl transferase
GGTase-I	Geranyl geranyl transferase-I
GGTase-II or Rab geranylgeranyl transferase	Geranylgeranyl transferase type 2
GGPP	Geranylgeranyl diphosphate
MAOS	Microwave assisted organic synthesis
KH	Potassium hydride
NaH	Sodium hydride
RAM	Ribosomal ambiguity
KOH	Potassium hydroxide
THF	Tetrahydrofuran