Chemical synthesis of dolichyl phosphates, their analogues and

Several methods for simple and efficient chemical synthesis of dolichyl phosphates and their analogues and derivatives are briefly summarized with a special emphasis on chemical modification of phosphoryl group and preparation of dolichyl phosphates labelled at the omega-end and at the gamma-isoprene unit of the isoprene chain by fluorescent groups, 2-aminopyridine and 1-aminonaphtalene residues. Additionally, data on biochemical assays with application of the compounds mentioned above are presented.

To study any of the biochemical steps of the biosynthetic cycles mentioned above in vitro it is necessary to have phosphates of polyprenols and dolichyl phosphates.It is worth to notice that living cells are a good source of polyprenols or dolichols whereas concentrations of their phosphates are extremely low and these labile compounds (especially polyprenol derivatives) usually decompose during isolation and purification procedures.
The chemical synthesis of polyprenyl phosphates poses problems different from those encountered with derivatives of dolichols (2,3-dihydropolyprenols).
The aim of this paper is to summarize briefly several methods for simple, reproducible and efficient chemical synthesis of dolichyl phosphates and their analogues and derivatives with a special emphasis on chemical modification of phosphoryl L.L. Danilov and T.N.Druzhinina group and preparation of dolichyl phosphates labelled at the ω-end and at the γ-isoprene unit of isoprene chain by fluorescent groups.
Additionally, data on biochemical assays with application of the compounds mentioned above are presented.The main results of investigation in this field were obtained in the N.D. Zelinsky Institute of Organic Chemistry RAS.

Methods for phosphorylAtIon of IsoprenoId AlCohols
Historically the first method for phophorylation of polyprenols was developed in 1972 (Warren & Jeanloz, 1972).It was based on the reaction of the alcohol with o-phenylene phosphorochloridate and 2,6-lutidine followed by elimination of the protective group including treatment with lead tetraacetate and further alkaline treatment.Purification of the phosphates was difficult due to the presence of coloured side-products formed after oxidative deblocking.
Up to 1981 this method was practically the only method for phosphorylation of polyprenolic alcohols in spite of all its inconveniences and deficiencies.
The first synthesis of dolichyl phosphate was achieved in 1974 via the procedure cited above (Wedgwood et al., 1974).Initially this method was used in our group for preparation of a series of prenyl phosphates with different chain length (3-16 isoprene units) in 35-62% yields (Kalinchuk et al., 1985) (Scheme 1).
The multistep procedure, the necessity to remove the protecting group and insufficient method of desired product isolation (usually TLC) retarded investigation of the dolichol pathway.Search for alternative methods of chemical phosphorylation of dolichols and polyprenols turned out a vital task.
In 1981 the first really simple and efficient procedure for preparation of high purity dolichyl phosphate via phosphorus oxychloride as the phosphorylating agent in the presence of base (triethylamine) followed by hydrolysis of intermediate dichloride, isolation and purification of the desired product by anion-exchange chromatography was developed (Danilov & Chojnacki, 1981) (Scheme 2).
Dolichyl phosphates with 11, 18, 19, 20 and 21 isoprene units were obtained in 57-84% yields (Table 1).The reaction gave good results even when performed on a very small scale for the preparation of radiolabelled derivatives (yield was calculated based upon radioactivity incorporation).Furthermore citronellyl phosphate (Cit-P, ωs-P) was prepared through similar way in 67% yield (Kalinchuk et al., 1985).But this efficient simple method of phosphorylation proved not to be applicable to fully unsaturated polyprenols (no phosphorylated polyprenols were isolated from reaction mixtures).
In recent years, the use of protected phosphochloridates for the synthesis of dolichyl phosphates was practically discontinued after introducing POCl 3 as a phosphorylating agent for dolichols.
The propagation of this suitable principle in practice of other researchers was summarized in the following reviews (Danilov & Shibaev, 1991;Shibaev & Danilov, 1992;1997).
Subsequently in 1988a simple and universal method for preparation of monophosphates from alcohols (including dolichols and polyprenols) was developed in our group.Trichloroacetonitrile was proposed initially as a condensing reagent for the reaction of isoprenoid alcohols with bis(triethylammonium) hydrogen phosphate (Cramer & Boehm, 1959) but this procedure resulted in of mono-and diphosphates of the alcohols.It was revealed in our experiments that the use of tetran-butylammonium dihydrogen phosphate instead of the bis(triethylammonium) salt in the presence of approximately equimolar amount of trichloracetonitrile resulted in preferable monophosphorylation of alcohols and only small amounts of the diphosphates were detected in reaction mixture.After anion-exchange chromatography pure monophosphates of alcohols were isolated in 80-87% yield (Scheme 3; for more details including reaction conditions and mechanism see Danilov & Shibaev, 1991;Shibaev & Danilov, 1997).
Phosphorylation with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was also successfully applied to a series of polyprenols  with modified isoprenoid chain in yields of 46-87% and dolichol C 95 in 80% yield (Danilov et al., 1989), retinol, cholesterol, nonacosanol, ethanol, tert-butanol and some other alcohols.In all cases the corresponding monophosphates predominated and were isolated via anion-exchange chromatography in high yields.
At present this seems to be the simplest and most efficient procedure for the preparation of polyprenyl phosphates and related derivatives, first of all for dolichyl phosphates.
Dolichyl thiophosphate (6) was obtained by the reaction of Dol-OH with PSCl 3 and Py following saponification and isolation by liquid-liquid extraction as potassium salt.
A modified analogue of dolichyl phosphate, dolichyl phosphorofluoridate (8) was prepared in a 72% yield from dolichyl-H-phosphonate, chlorotri-methylsilane, iodine and triethylamine trihydrofluoride.As an example of a dolichyl phosphate derivative lacking the phosphate group dolichyl sulfate ( 9) was also prepared.The procedure includes reaction of dolichol with Py•SO 3 complex in DMFA followed by isolation the desired product via anion-exchange chromatography (81%) or liquid-liquid extraction (73%).
The possibility of simple and effective preparation of Dol-P stimulated the appearance of a new generation of methods, resulting in the synthesis of more complex dolichyl phospho compounds such as radiolabelled dolichyl [β-33 P]diphosphate via reaction of dolichyl phosphoroimidazolide with trioctylammonium salt of phosphoric acid in 18% yield (Shabalin et al., 1995).Recently, dolichyl diphosphate was prepared in 64% yield by reaction of Dol-P with diphenyl phosphorochloridate and interaction of the activated reaction product with tetra-n-butylammonium dihydrogen phosphate (Maltsev et al., 1995).
For dolichyl diphosphate sugars the pioneer method of synthesis proposed in 1978 included interaction of dolichyl diphenyl diphosphate with salt of fully acetylated glycosyl phosphates in the presence of pyridine (Warren & Jeanloz, 1978).Protecting acetyl groups were eliminated by treatment with sodium methoxide.The results were satisfactory (40-79%) only in the cases of derivatives of 2-acetamido-2-deoxy-glycosyl phosphates or containing their oligosaccharides (for a review see Danilov &  Shibaev, 1991); derivatives of 1,2-cis-glycosyl phosphates could be obtained only in 6-12% yield due to considerable decomposition of the peracetylating products obtained in the process of base deacylation probably due to fast splitting of diphosphate linkages and to intramolecular nucleophilic attack of the alkoxide ion appearing on the neighbouring phosphorus atom.
The side reaction discussed above strongly limited application of the diphenyl diphosphate method for the synthesis of dolichyl-and polyprenyl diphosphate sugars.A practical preparation of the compounds became possible after our group developed a phosphoroimidazolide method based on interaction of polyprenyl phosphoroimidazolides with unprotected glycosyl phosphates (Danilov et al., 1981; for a review see Danilov & Shibaev, 1991).The yields of desired compounds were 22-84% for polyprenyl diphosphate sugars, containing from monoto tetrasaccharide residues; this method was applicable to this class of compounds containing a dolichol residue (Lee & Coward, 1992).The synthesis of P 1dolichyl, P 2 -2-acetamido-2-deoxy-α-d-glucopyranosyl diphosphate using dolichyl phosphorodichloridate as activated reagent was performed in a 60% yield (Imperiali & Zimmerman, 1990).It proved that at present the phosphoroimidazolide method for the synthesis of lipid diphosphate sugars is the method of choice for dolichyl and polyprenyl glycosyl diphosphates thanks to its efficiency and universality.

synthetIC derIvAtIves wIth fluoresCent lAbel At IsoprenoId ChAIn
This group of fluorescent dolichyl phosphate derivatives is the largest one, comprising compounds with different number of isoprene units and modified at different positions of the isoprenoid chain.
Derivatives of dolichyl phosphate containing 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the ω-or γ-isoprene unit of isoprenoid chain were synthesized in our group (Scheme 5) according to the principle of phosphorylation with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile (see above, Scheme 3) from corresponding aminoalcohols, specially synthesized (Grigorieva et al., 2000;Shibaev et al., 2000;Xing et al., 2000;Veselovsky et al., 2001;Lamani et al., 2006).The phosphorylation of the aminoalcohols was found to proceed more slowly and to require about double excess of the reagents than for the reaction of nonmodified polyprenols and dolichols, which could be explained by the influence of the amino function in the molecule.The predominant procedure for purification of the desired phosphates was anion-exchange column chromatography, in some cases liquid-liquid extraction or adsorption chromatography were used.The derivatives showed UV-absorption characteristic for the incorporated chromophores and intensive fluorescence with excitation/emission maxima at 315/360 nm for compounds 10a,b and 340/410 nm for 11a-e and 12a,b (in n-heptane/ 2propanol, 4:1, v/v).Considerable variations in yields (18-93%) of the compounds could be dependent on structural differences, properties of amino groups in the molecule and the type of isolation and purification procedures.
As a result of these synthetic approaches a new instrument for studies of enzyme reactions was successfully developed.Such collection of dolichyl phosphate derivatives with a fluorescent label in isoprenoid chains should prove to be useful for investigation of intra-and inter-molecular events during enzyme catalysis.Results of such kind of research with recombinant yeast Dol-P-Man synthase are summarized in the final article of this series (see Lamani et al., 2006).
In conclusion of this part we could say that the phosphoroimidazolide method developed in our group for the synthesis of polyprenyl, dolichyl (and Scheme 5. Dolichyl phosphate derivatives containing fluorescent label at ω-and γ-isoprene unit. other lipid) diphosphate sugars in the course of 30 years remains the most general and convenient procedure for preparation of these biologically important derivatives.As it is evident from the data cited above this method has been used successfully up to now and the most recent publication on this subject appeared in the current year.
We have a great pleasure in stressing that at the very beginning of the cascade of syntheses and biochemical investigations there was the idea of Professor Tadeusz Chojnacki to develop a simple and efficient method for dolichol phosphorylation that was first realized in 1981.That synthesis gave the impetus to acceleration and expansion of new chemical and biochemical research.

bIoCheMICAl reseArCh wIth synthetIC derIvAtIves of dolIChols
Examples of application of synthetic derivatives of dolichols as lipid acceptors in enzyme cycles could be divided into two groups.In the first group, experiments were performed in the last century with partially purified enzymes, evaluation of substrate characteristics was not always complete and analysis of product structure was preliminary in some experiments.
Research in the second group started in 1999 and final discussion was published in 2006.This series of experiments with homogeneous recombinant enzyme, dolichyl-phosphate-mannose synthase and syntetic Dol-P analogs containing fluorescent probes allowed the intramolecular distances within the protein molecule to be establish as well as the localization of the active and catalytic sites and of the substrates on the enzyme surface, resulting in the construction of a three-dimensional model and proposing a molecular mechanism of catalysis.
One of the first examples of synthetic Dol-P assay in an enzyme system was the use of these compounds as monosaccharide acceptor with glycosyltransferases from red clover (Trifolium pratense L.) seedlings (Druzhinina et al., 1981).Incubation of semi-synthetic 2,3-dihydroundecaprenyl phosphate with GDP[ 14 C]Man and partially purified preparation of glycosyl transferases resulted in a considerable incorporation of [ 14 C]mannose into the fraction of lipid-oligosaccharides.In the case of C 55 -polyprenyl phosphate the yield of lipid-[ 14 C]oligosaccharides was much lover.This result demonstrates that for enzymes from all types of eukaryotic cells, including plants, derivatives of the Dol series but not prenols with fully unsaturated chain are preferable.Stimulation of the biosynthesis of Dol 11 -P and Dol 21 -P-oligosaccharide derivatives was slightly higher in the presence of exogenic dolichyl phosphates with long-er isoprenoid chain.For structural analysis of lipidlinked oligosaccharides TLC, column ion exchange on DE-52 and paper chromatographies as well as gel-filtration were used.
Synthetic phosphates of citronellol and dolichols C 55 and C 105 were assayed later with enzymes of O-antigen biosynthesis from Salmonella anatum (Kalinchuk et al., 1985).For phosphorylation of Dol 11 OH and Dol 21 OH the procedure with o-phenylenechlorophosphate was used as described earlier (Vergunova et al., 1977).Citronellyl phosphate was prepared following the procedure of Danilov and Chojnacki (1981).
The acceptor ability of polyprenyl phosphates were tested in consecutive reactions of O-antigen biosynthesis: three steps of repeating unit assembly, and in reaction of polymerization.Phosphates of dolichol series could serve as monosaccharide acceptors in reactions of trisaccharide repeating units assembly only in the case of Dol 11 -P with efficiency of 39%; the chain length of the most effective derivative coincides with that of native lipid acceptor (11 isoprene units).Dol-P with shorter or longer isoprenoid chain (phosphates of Dol 21 OH and citronellol) are active only with Gal-P transferase, which initiates the process of repeating unit assembly, catalyzing reaction: UDPGal + Pre n -P → Gal(α)ppPre n + UDP.
The final step of O-specific polysaccharide biosynthesis is enzyme polymerization of trisaccharide repeating unit connected via PP-bridge with Dol 11 (Danilov et al., 1989).Biochemically obtained trisaccharide derivatives of Dol 11 -PP manifest their substrate ability up to the reaction of trisaccharide repeating unit polymerization with high efficiency (92%).
Two synthetic analogues of dolichyl phosphate were tested with glycosyl transferases from mammalian microsomes.Dolichyl H-phosphonate could serve as an acceptor for mannosyl-, glucosyland N-acetylglucosaminyl phosphate from GDP-[ 14 C]Man, UDP-[ 14 C]Glc and UDP-[ 14 C]GlcNAc, respectively.The reaction products were characterized by TCL and additional analysis seems to be desirable.Dolichyl sulfate was not a substrate in the enzyme reactions mentioned and demonstrated weak inhibitory properties (Sizova et al., 1998).
The most impressive results were obtained in a series of biochemical research with homogeneous recombinant enzyme, Dol-P-mannose synthase from Saccharomyces cerevisiae (Lamani et al., 2006).In this research fluorescence energy transfer (FRET) methodology was utilized.FRET analysis allows measurements of distances between specific amino-acid residues in the enzyme and fluorophore groups in L.L. Danilov and T.N.Druzhinina specially designed substrate analogues.A series of 1-aminonaphthalene-labelled dolichyl phosphates having different distances between the fluorophore and the phosphate group of Dol-P were used in these experiments.The FRET experiments allowed for the measurement of distances between selected residues of the enzyme as well as those between the enzyme and chromophoric synthetic Dol-P substrate analogues, thus revealing localization of the active site and the hydrophobic substrate on the enzyme surface.A three-dimensional model of the enzyme was produced with bound substrates, Dol-P, GDP-Man and divalent cations.The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.