Regular paper on-line at: www.actabp.pl Prenyl sulfates as alkylating reagents for mercapto amino acids

A new methodology for prenylation of thiol compounds has been developed. The approach is based on the use of prenyl sulfates as new reagents for S-prenylation of benzenethiol and cysteamine in aqueous systems. The C(10)-prenols geraniol and nerol that differ in the configuration (E or Z, correspondingly) of the alpha-isoprene unit were efficiently O-sulfated in the presence of a pyridine-SO(3') complex. The obtained geranyl and neryl sulfates were tested as alkylating agents. These compounds were chosen to reveal the influence of the alpha-isoprene unit configuration on their alkylation (prenylation) ability. S-Geranyl cysteine was prepared to demonstrate the applicability of this method for prenylation of peptides containing mercapto amino acids.


INTRODUCTION
Prenylated proteins play important roles in eukaryotic cells (Omer & Gibbs, 1994;Takai et al., 2001).The first prenylated polypeptide, the α-mating factor of yeast, was discovered 25 years ago (Kamiya et al., 1978).Studies during the last decades have shown that the proper functioning of many proteins requires a series of post-translational lipid modifications (Volkert et al., 2001;Pechlivanis & Kuhlmann, 2006).The interest in protein lipidation increased rapidly after recognition that Ras and Rab proteins are subject to post-translational lipidation (Hancock et al., 1989;Farnsworth et al., 1991).Both proteins belong to the Ras superfamily and are key regulators of cell growth in all eukaryotic cells.Gene expression, apoptosis and remodelling of the cytoskeleton are all controlled by Ras proteins.Ras proteins were originally identified as the products of oncogenes capable of inducing cell transformation.Over the last twenty-five years they have been studied in great detail because mutant Ras proteins are associated with many types of human cancer (Bu-day & Downward, 2008).It has been shown that the attached lipid groups function not only as anchors linking the modified protein to the cytoplasmic or vesicular membranes, but could also be involved in signal transduction by facilitating protein-protein and protein-lipid interactions (Wittinghofer & Waldmann, 2000;Kuhn et al., 2001).The post-translational modification of proteins with lipids is a key mechanism in the regulation of protein localization and function.Prenylation is irreversible but it is frequently associated with reversible post-translational modifications, such as palmitoylation and phosphorylation.Both of them act like switches that modulate the dynamics of protein-membrane or protein-protein interactions and target the modified proteins to specific membrane compartments (Perez-Sala, 2007).Recent work (Abankwa et al., 2007) has shown that Ras proteins are laterally segregated into non-overlapping, dynamic domains of the plasma membrane called nanoclusters.This lateral segregation is important in determining Ras interactions with membrane-associated proteins, effectors and scaffolding proteins, and is critical for Ras signal transduction
In vivo S-prenylation of proteins is achieved by enzymatic formation of S-geranylgeranyl or Sfarnesyl cysteine sulfides, the cysteine residues being located at (or close to) the C-terminus.This involves either one or two geranylgeranyl transferases or a farnesyl transferase (Zhang & Casey, 1996;Lane & Beese, 2006.).Prenyltransferases attach the isoprenoid lipids to the C-terminus of several guanosine triphosphate-binding proteins.The prenyl groups are essential for the biological activity of these proteins.The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis and high serum cholesterol-level-associated diseases (Simonen et al., 2008;Baron & Seabra, 2008).Control of Ras GTPase signalling might reduce the aberrant DNA methylation and, accordingly, may reduce the risk of cancer development (Patra, 2008).
A regulatory role of prenylation of plant proteins in developmental processes and hormone signal transduction has been suggested (Rodriguez-Concepcion et al., 1999;Galichet & Gruissem, 2006).In additon to farnesyl and geranylgeranyl derivatives, various polyprenyl and dolichyl as well as phytyl residues were also discovered to covalently modify plant proteins (Swiezewska et al., 1993;Shipton et al., 1995;Gutkowska et al., 2004).
Synthetic strategies that give access to lipidated peptides have been developed in the last ten years both for solution (Silvius & Lheureux, 1994;Shahinian & Silvius, 1995;Kadereit et al., 2001) and solid-support approaches (Ludolph & Waldmann, 2003;Kragol et al., 2004;Lumbierres et al., 2005).For both methods (Brunsveld et al., 2006), two basic techniques are used for introduction of the lipid functionalities.In the first technique, the lipid groups are introduced through coupling of prelipidated amino acids, whereas in the second one, they are introduced through lipidation of a selectively deprotected peptide (i.e., a peptide with a free thiol group).Quite frequently, a combined solution/solid-phase approach is used (Xue et al., 1992;Naider & Becker, 1997;Reents et al., 2005, Brunsveld et al., 2005).In this approach, the majority of the peptide sequence is, first, rapidly synthesized on a solid support, then cleaved off and coupled with lipidated amino acids in solution or lipidated by alkylation of free thiol side chains of cysteines.In addition, the use of prelipidated building blocks has been suggested for the synthesis of lipidated peptides on a solid support (Kragol et al., 2004;Lumbierres et al., 2005).
In thioalkylation reactions, cysteine or cysteine residues of peptides are reacted with pre-nyl bromide or prenyl chloride in organic or mixed organic/aqueous media.On-resin prenylation of the cysteines requires a large excess of prenyl halide in order to achieve complete conversion.Depending on the solubility of the cysteine-containing peptide, prenylation is accomplished either in alkaline, neutral or acidic conditions.Synthesis under alkaline conditions is usually performed in N,N-dimethylformamide (DMF) with aqueous KHCO 3 (Naider & Becker, 1997) or in tetrahydrofuran/liquid ammonia mixtures (Schroeder et al., 1997, Kuhn et al., 2001, Ludolph et al., 2002).Reaction under neutral conditions proceeds in DMF and KF•2H 2 O in the presence of an equimolar amount of N,N-diisopropylethylamine (Silvius & Lheureux, 1994;Shahinian & Silvius, 1995).S-Prenylation procedure under acidic conditions is carried out in a DMF/aqueous trifluoroacetic acid/n-butanol (or acetonitrile) mixture in the presence of Zn(OAc) 2 as a catalyst (Xue et al., 1992).All these procedures provide the required compounds in good yields for short peptides.However, the use of alkyl halides for thioalkylation of natural long-chain peptides and proteins requires a mixed organic/aqueous medium, which is problematic for solubility reasons.The bivalent character of the lipidated peptides, featuring both the highly hydrophobic lipid groups and polar amino acids, makes them difficult to handle.The intrinsic instability of prenyl moieties in acidic conditions limits the number of applicable methods for Sprenylation of cysteine residues.
The successful development of solid-phase synthesis of lipidated Ras and Rab proteins demands new techniques for the incorporation of the required lipid groups.In this paper we demonstrate a new method for the chemical prenylation of mercapto amino acids using prenyl sulfates as alkylating reagents.This approach avoids the use of organic solvents and allows the S-prenylation reaction to be performed in aqueous systems.

MATERIALS AND METHODS
Geraniol and nerol were purchased from Lancaster (UK); cysteamine and l-cysteine from Aldrich (USA); pyridine-SO 3 complex, triethylamine and benzenethiol from Fluka (Germany).The equilibrium mixture of n-butanol and water was prepared by shaking equal volumes of both components.The upper layer of the equilibrium mixture contained n-butanol and water in a ratio of 86:14 (v/v) and the bottom layer of the mixture contained n-butanol and water in a ratio of 14:86 (v/v).Flash column chromatography (FCC) was performed on a column (1.4 × 18 cm) with Kieselgel 60 (0.040-0.063 mm) (Merck).TLC was performed on Kieselgel 60 F 254 (Merck) developed with chloroform/methanol/ Prenyl sulfates as alkylating reagents for mercapto amino acids water (60:25:4, by vol., solvent A) and light petroleum (40-60 o C)/ethyl acetate (95:5, v/v, solvent B).Unsaturated compounds were detected on TLCplates with iodine vapor with subsequent charring by spraying with 4% sulfuric acid in methanol and heating.Compounds with free amino group were detected by spraying with 1% ninhydrin in ethanol and heating to 100 o C. UV spectra were measured on a Specord UV-VIS. 1 H and 13 C NMR spectra ( 1 H at 500 MHz, 13 C at 125 MHz) were recorded on a Bruker AMX-500 spectrometer for solutions in CDCl 3 , unless otherwise indicated.Chemical shifts (δ in ppm) are given relative to those for tetramethylsilane, J values are given in Hz.ES-MS spectra were obtained with a Finnigan LCQ spectrometer, EI-MS spectra were measured with a Jeol DX-300 apparatus.

RESULTS AND DISCUSSION
In prenylated peptides (Omer & Gibbs, 1994), geranylgeraniol (E,E,E -C 20 isoprenoid alcohol) and farnesol (E,E -C 15 isoprenoid alcohol) are the major prenols that are covalently attached to the thiol groups of cysteine residues at (or near) the C-terminus (see Fig. 1).Some proteins from the Ras superfamily are post-translationally converted into C-terminal methyl esters.
In this work, we used short C 10 -prenols geraniol 1 and nerol 2 (Scheme 1) that differ in the configuration (E or Z, correspondingly) of the α-isoprene unit.These compounds were chosen to reveal the influence of the α-isoprene unit configuration on the alkylation (prenylation) reaction.Geranyl sulfate 3 and neryl sulfate 4 were tested as alkylating agents.They were synthesized by the method that we had developed previously for long-chain polyprenyl sulfates (Maltsev et al., 2001).The method was modified for the synthesis of short-chain prenyl sulfates 3 and 4 because of the different solubility of long and short prenols and their sulfates in organic solvents.Prenols 1 and 2 were transformed into their sulfates 3 and 4 using triethylamine (instead of DMF, as in Maltsev et al., 2001) as a solvent (see Scheme 1).
We discovered that prenols 1 and 2 could be efficiently O-sulfated in the presence of a two-fold molar excess of pyridine-SO 3 complex.After treatment of the reaction mixtures with water, the de- sired prenyl sulfates precipitated together with the non-consumed prenols.The latter were then extracted from the residue with cold n-heptane to provide pure geranyl sulfate 3 (75%) and neryl sulfates 4 (72%) as triethylammonium salts.The products were characterized by NMR. 1 H-NMR spectra of 3 and 4 virtually coincided with those of prenols 1 and 2 (Crombie et al., 1975) 1).
Triethylammonium salts of prenyl sulfates 3 and 4 were used for S-prenylation of benzenethiol, cysteamine and l-cysteine in aqueous solutions in the presence of KOH.For the preparation of S-prenyl benzenethiols 6 and 7, a solution of geranyl sulfate 3 (or neryl sulfate 4) in the lower layer of the equilibrium mixture n-butanol/water was treated with benzenethiol 5 and aq.KOH (Scheme 2).The prenylation products 6 and 7 were extracted with light petroleum and isolated in 48 and 54% yields, respectively.The products were characterized by NMR, UV and EI-MS.We observed a large characteristic upfield shift of the signals for H-1 (δ 3.48 for 6 and 3.53 for 7) as compared with the signals for the sulfates 3 and 4 (δ 4.53 and 4.57, respectively) in 1 H-NMR spectra (see Table 1). 13C-NMR spectra showed a large upfield shift of the C1 signal.Signals of pseudo-molecular ions (m/z 246.42 for 6 [M + ] and 246.15 for 7 [M + ]) for both thioethers in mass spectra were close to the calculated value of m, 246.41.
Cysteamine 8 was S-prenylated under similar conditions (Scheme 3), thus providing S-geranyl 9 (54%) and S-neryl 10 (76%) derivatives of cysteamine.Their structures were confirmed by the characteristic chemical shifts for H-1 signal in the 1 H-NMR spectra (see Table 1) and by MS data.The main signals in the mass spectra corresponded to the pseudomolecular ions for compounds 9 (m/z 214.16 [M+H] + ) and 10 (m/z 214.23 [M+H] + ).
Worth noting is the observation that the yields of S-neryl derivatives of benzenethiol (7) and

2008
S. Maltsev and others cysteamine (10) were higher than those for the corresponding S-geranyl derivatives (6 and 9), thus indicating that prenyl sulfate 4 containing (Z)-α-isoprene unit seems to be a more efficient prenylating reagent than the (E)-isomer 3.However, since prenyl residues in natural prenylated proteins contain (E)-isoprene units, only geranyl sulfate 3 was used for S-alkylation of l-cysteine 11 (Scheme 4).
After the standard S-prenylation procedure, the reaction mixture was extracted with diethyl ether to remove triethylamine and geraniol (which was formed, probably, due to partial hydrolysis of 3).The desired geranyl cysteine 12 was easily purified and isolated as potassium salt in 43% yield.Structure of the product was confirmed by NMR (see Table 1) and MS.The high-field position (compared to 3) of the H-1 signal in the 1 H-NMR spectrum and the presence of the signal of the C=O group at δ C 180.0 in the 13 C-NMR spectrum are characteristic of S-geranyl cysteine 12.The main signal in the mass spectrum corresponded to the pseudo-molecular ion of 12 (m/z 255.30[M−H] − ).
In conclusion, a rather simple and convenient method has been developed for S-prenylation of thiols.Prenyl sulfates were used as alkylating reagents, which allowed the S-prenylation reactions to be carried out in aqueous solutions at room temperature.This provides a new chemical tool for introduction of lipid functionalities into cysteine-containing peptides.

Figure 1 .
Figure 1.Farnesyl (A) and geranylgeranyl (B) sulfide linkages found in peptides and proteins.Note that double bonds are all in trans (E) configuration.