Borane phosphonate deoxyoligonucleotides are synthesized from 5′-O-benzhydroxybis(trimethylsilyloxy)silyl-2′-deoxynucleoside-3′-phosphoramidites. The exocyclic amines of adenine and cytosine are protected with dimethoxytrityl and trimethoxytrityl, respectively, whereas guanine protection is with N2-(9-fluorenylmethoxycarbonyl) or N2-trimethoxytrityl. Thymine is protected with N3-anisoyl. Using these synthons and under standard conditions via activation with tetrazole, condensations in excess of 99% are observed. Oxidation with either THF·BH3 or a peroxyanion solution followed by cleavage of the silyl ether with fluoride completes a cycle. Following synthesis of an appropriate oligomer, protecting groups are removed using sequentially acetic acid, a dithiolate and ammonium hydroxide. Oligodeoxynucleotide 10 mers and 12 mers having any combination of borane phosphonate and phosphate internucleotide linkages as well as all four 2′-deoxynucleotides are synthesized in isolated yields of 70–80% and characterized by phosphorus NMR and mass spectrometry.

A novel solid-phase phosphoramidite-based method has been developed for the synthesis of borane phosphonate DNA. Keys to this new approach are replacement of the common 5'-dimethoxytrityl blocking group with a 5'-silyl ether and the use of new protecting groups on the bases (adenine, N6-dimethoxytrityl; cytosine, N4-trimethoxytrityl; guanine, N2-[9-fluorenylmethoxycarbonyl]; thymine, N3-anisoyl). Because of these developments, it is now possible for the first time to synthesize oligodeoxynucleotides having any combination of the four 2'-deoxynucleosides and both phosphate and borane phosphonate internucleotide linkages (including oligomers having exclusively borane phosphonate linkages).

The phosphorylated branched heptasaccharides 7 and 8, the octasaccharide 9 and the phosphorylated trisaccharides 5 and 6, which are fragments of the phosphoglycan portion of the surface lipophosphoglycans from Leishmania mexicana (5) or L. major (6-9), were synthesised by using the glycosyl hydrogenphosphonate method for the preparation of phosphodiester bridges. The compounds were tested as acceptor substrates/putative inhibitors for the Leishmania elongating alpha-D-mannosylphosphate transferase.

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10beta-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most potent compound assayed was phosphate dimer 14a, which was greater than 50 times more potent than the parent drug artemisinin and about 15 times more potent than the clinically used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI(50)) values versus a range of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers 9c and 9d, antimalarially active in the low nanomolar region versus P. falciparum, are inactive as anticancer agents even at concentrations in the millimolar region. This observation emphasizes the importance of two trioxane units for high antiproliferative activity, and we propose that the nature of the linker in dimers of this type plays a crucial role in imparting potent anticancer activity.

Signal transduction cascades maintain control over important cellular processes such as cell growth and differentiation by orchestrating protein phosphorylation and dephosphorylation. Specific control of these processes in vivo and in vitro can be achieved with peptide analogues that mimic the binding properties of phosphoproteins. We present here the solid-phase synthesis of two novel classes of phosphopeptide mimetics, O-boranophosphopeptides and O-dithiophosphopeptides, derivatized on tyrosine, serine, and threonine. The use of H-phosphonate and H-phosphonothioate monoesters containing the base labile 9-fluorenemethyl protecting group was key to the synthesis of both phosphopeptide mimetics. O-Boranophosphopeptides were synthesized by condensing O-(9-fluorenemethyl)-H-phosphonate to the peptide hydroxylic component (tyr, ser, or thr) followed by oxidation with borane complexes. Similarly, the synthesis of O-dithiophosphopeptides used the O-(9-fluorenemethyl)-H-phosphonothioate synthon and oxidation with elemental sulfur. Base elimination of the Fmol protecting group and cleavage from the solid support with concentrated ammonium hydroxide afforded the boranophosphopeptide and dithiophosphopeptide target compounds. Ac-YIIPLPG-NH2, having either dithiophosphoryl tyrosine or boranophosphoryltyrosine but no sequence specificity for Yersinia protein tyrosine phosphatase (PTP), was found to competitively inhibit this enzyme with KI values of 430 +/- 50 and 670 +/- 50 microM, respectively. In addition, both phosphopeptide analogues were resistant toward Yersinia PTP enzymatic hydrolysis. Under conditions (pH 8.0) where the phosphopeptide was rapidly dephosphorylated, the boranophosphopeptide hydrolyzed slowly (t1/2 = 15 h) and the dithiophosphopeptide was completely stable over 24 h.

A tetrasaccharide fragment of Leishmania major lipophosphoglycan (which seems to be involved in a biological mechanism for the parasite transmission) has been synthesised using the thioglycoside, trichloroacetimidate and halide-exchange glycosylation procedures and step-wise chain elongation strategy.

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Gal beta 1-4Man alpha 1-P phosphosaccharide repeat structures assembled by the sequential addition of Man alpha 1-P and beta Gal. The synthetic acceptor substrate Gal beta 1-4Man alpha 1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Gal beta 1-4Man alpha 1-P-decenyl is the elongating cx-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K-m values for the donor and acceptor substrates were found using L, major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Gal beta 1-4Man alpha 1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alpha Man residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the beta Gal residue is required for substrate recognition as well as for catalysis, The rate of Man alpha 1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal beta Gal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Galbeta1-4Manalpha1-P-decenyl is the elongating alpha-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K(m) values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.

A polymer (MPEG) supported synthesis of the phosphorylated tetra- and hexa-saccharide fragments of the lipophosphoglycan from Leishmania has been developed using mono- and di-saccharide H-phosphonates for construction of the phosphodiester bridges.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

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The three structural analogues of dec-9-enyl β-D-galactosyl-(1→4)-α-D-mannosyl phosphate, comprising thiophosphate, boranophosphate and methylphosphonate derivatives, were prepared via disaccharide H-phosphonate or trichloroacetimidate (for the methylphosphonate synthesis) intermediates.

6-N-Biotinylaminohexyl isopropyl phosphorofluoridate 2 was prepared by treatment of the corresponding hydrogen phosphonic diester with triethylamine trihydrofluoride and iodine. The activity of the phosphorofluoridate was tested by the inhibition of a-chymotrypsin.

The triglycosyl monophosphate 1, heptaglycosyl triphosphates 2 and 3, and octaglycosyl triphosphate 4, which are fragments of the phosphoglycan part of Leishmania major lipo- and proteo-phosphoglycans have been prepared using di- and tri-saccharide H-phosphonates for construction of the phosphodiester bridges.

The phosphorylated branched heptasaccharide beta-D-Galp-(1-->4)-alpha-D-Manp-(1-PO3H-6)-[beta-D-Glcp-(1-->3)]-beta-D-Galp-(1-->4)-alpha-D-Manp-(1-PO3H-6)-beta-D-Galp-(1-->4)-alpha-D-Manp-1-PO3H-O[CH2](8)CH=CH2, which is a fragment of the phosphoglycan portion of Leishmania mexicana phosphoglycan and proteophosphoglycan, has been synthesized using the thioglycoside and Helferich methods for the glycosylations and the glycosyl hydrogenphosphonate method for the successive introduction of the disaccharide phosphate and trisaccharide phosphate blocks.

An oligothmidylate containing boranophosphates was synthesized via silylation of H-phosphonate linkages followed by boranation. These compounds formed duplexes having reduced stability with complementary RNA/DNA and stimulated RNase H activity. (C) 1998 Elsevier Science Ltd. All rights reserved.