Leo A. J. M. Sliedregt

Trimethylsilyl triflate mediated chemoselective condensation of arylsulfenyl glycosides

Abstract Condensation of a fully benzoylated phenylsulfenyl glycoside with either benzylated or benzoylated phenylthio glycosyl acceptors under the agency of trimethylsilyl triflate proceeds with a good degree of chemoselectivity in the presence of the scavenger triethylphosphite.

Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the asialoglycoprotein Receptor

Abstract Structurally well defined di- and tri-antennary lysine-based galactose- and N-acetylgalactosamine-containing ligands for the hepatic asialoglycoprotein receptor (ASGP-R) could be assembled on a solid support, using a combined Fmoc/Alloc protecting group strategy for the amino functions of lysine. This methodology allowed easy introduction of spacers, the length of which could be readily accommodated for optimal binding to the ASGP-R.

Cyclic diguanylic acid behaves as a host molecule for planar intercalators

Cyclic ribodiguanylic acid, c‐(GpGp), is the endogenous effector regulator of cellulose synthase. Its three‐dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X‐ray diffraction analysis at 1 Å resolution. In both crystal forms, two independent c‐(GpGp) molecules associate with each other to form a self‐intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32°), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c‐(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage‐like model consisting of a tetrameric c‐(GpGp) aggregate in which a large cavity (‘host’) is generated to afford a binding site for certain planar intercalators (‘guests’).

Design and synthesis of a multivalent homing device for targeting to murine CD22

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail. and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohns disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAc alpha-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper. the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAc alpha-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAc alpha-2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAc alpha-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAc alpha-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.

Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity

The device of new hepatotrophic prodrugs of the antiviral nucleoside 9‐(2‐phosphonylme‐thoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi‐ and trivalent cluster glycosides (K(GN)2 and K2(GN)3, respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10‐fold higher than that of the parent drug (52±6% and 62±3% vs. 4.8±0.7% of the injected dose for PMEA) and could be attributed for 90% to parenchymal cells. Accumulation of the PMEA prodrugs in extrahepatic tissue (e.g., kidney, skin) was substantially reduced. The ratio of parenchymal liver cell‐to‐kidney uptake—a measure of the prodrugs therapeutic window—was increased from 0.058 ± 0.01 for PMEA to 1.86 ± 0.57 for K(GN)2‐PMEA and even 2.69 ± 0.24 for K2(GN)3‐PMEA. Apparently both glycosides have a similar capacity to redirect (antiviral) drugs to the liver. After cellular uptake, both PMEA prodrugs were converted into the parent drug, PMEA, during acidification of the lysosomal milieu (t1/2≈100 min), and the released PMEA was rapidly translocated into the cytosol. The antiviral activity of the prodrugs in vitro was dramatically enhanced as compared to the parent drug (5‐ and 52‐fold for K(GN)2‐PMEA and K2(GN)3‐PMEA, respectively). Given the 15‐fold enhanced liver uptake of the prodrugs, we anticipate that the potency in vivo will be similarly increased. We conclude that PMEA prodrugs have been developed with greatly improved pharmacokinetics and therapeutic activity against viral infections that implicate the liver parenchyma (e.g., HBV). In addition, the significance of the above prodrug concept also extends to drugs that intervene in other liver disorders such as cholestasis and dyslipidemia.—Biessen, E. A. L., Valentijn, A. R. P. M., de Vrueh, R. L. A., van de Bilt, E., Sliedregt, L A. J. M., Prince, P., Bijsterbosch, M. K., van Boom, J. H., van der Marel, G. A., Abrahams, P. J., van Berkel, T. J. C. Novel hepatotrophic prodrugs of the antiviral nucleoside 9‐(2‐phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity. FASEB J. 14, 1784–1792 (2000)

Synthesis of a Lipophilic Prodrug of 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) and Its Incorporation into a Hepatocyte-Specific Lipidic Carrier

AbstractPurpose. 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of Hepatitis B virus replication, is in vivo hardly taken up by parenchymal liver cells (the site of infection). Our aim is to examine whether lactosylated reconstituted HDL (LacNeoHDL), a lipidic particle that is specifically internalized by parenchymal liver cells, is a suitable carrier for the selective delivery of PMEA to this cell type. Methods. To incorporate PMEA into LacNeoHDL, we synthesized a lipophilic prodrug (PMEA-LO) by coupling PMEA via an acid-labile phosphonamidate bond to lithocholic acid-3α-oleate. Results. The yield of the synthesis was 52% ([3H]PMEA-LO: 24%). [3H]PMEA-LO readily incorporated into LacNeoHDL (13 molecules/ particle) without affecting the size and net negative charge of the carrier. Further, incubation studies at lysosomal pH showed [3H]PMEA was completely released from the carrier whereas, at neutral pH or in plasma, appreciable release was not observed. Conclusions. The conjugation of PMEA with lithocholic acid-3α-oleate results in a lipophilic prodrug that readily associates with LacNeoHDL. The association of the prodrug does not affect the physico-chemical properties of the particle, and PMEA is released from the carrier at lysosomal pH. These findings indicate that by using the prodrug approach, LacNeoHDL is a suitable carrier to deliver PMEA to parenchymal liver cells.

Stimulation of Liver-Directed Cholesterol Flux in Mice by Novel N-Acetylgalactosamine–Terminated Glycolipids With High Affinity for the Asialoglycoprotein Receptor

Objective—Interventions that promote liver-directed cholesterol flux can suppress atherosclerosis, as demonstrated for scavenger receptor-BI overexpression in hypercholesterolemic mice. In analogy, we speculate that increasing lipoprotein flux to the liver via the asialoglycoprotein receptor (ASGPr) may be of therapeutic value in hypercholesterolemia. Methods and Results—A bifunctional glycolipid (LCO-Tyr-GalNAc3) with a high-nanomolar affinity for the ASGPr (inhibition constant 2.1±0.2 nmol/L) was synthesized that showed rapid association with lipoproteins on incubation with serum. Prior incubation of LCO-Tyr-GalNAc3 with radiolabeled low-density lipoprotein or high-density lipoprotein (0.5 &mgr;g/&mgr;g of protein) resulted in a dramatic induction of the liver uptake of these lipoproteins when injected intravenously into mice (70±3% and 78±1%, respectively, of the injected dose at 10 minutes of low-density lipoprotein and high-density lipoprotein), as mediated by the ASGPr on hepatocytes. Intravenously injected LCO-Tyr-GalNAc3 quantitatively incorporated into serum lipoproteins and evoked a strong and persistent (≥48 hour) cholesterol-lowering effect in normolipidemic mice (37±2% at 6 hours) and hyperlipidemic apoE−/− mice (32±2% at 6 hours). The glycolipid was also effective on subcutaneous administration. Conclusions—LCO-Tyr-GalNAc3 is very effective in promoting cholesterol uptake by hepatocytes and, thus, may be a promising alternative for the treatment of those hyperlipidemic patients who do not respond sufficiently to conventional cholesterol-lowering therapies.

Approaches for the Design of Novel Anti-Atherogenic Compounds

Atherosclerosis, a chronic disease related to the vascular system, is the most common cause of morbidity and mortality in Western society. Epidemiological studies have established that the serum cholesterol/triglyceride level is one of the major causative factors for its occurrence. At present, patients with persistent high levels of serum cholesterol and/or triglyceride are therapeutically treated with various types of drugs. Frequently applied drugs are inhibitors of a rate-limiting enzyme in the cholesterol synthesis pathway, i.e., 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, or bile acid sequestrants (fibrates). These drugs lead to an enhanced expression level of low-density lipoprotein (LDL) receptors, thereby stimulating the catabolism of LDL, the main carrier of cholesterol in human blood. Despite the general therapeutic efficacy of these drugs, a significant number of patients, including those suffering from familial hypercholesterolemia and dysbetalipoproteinemia, do not respond sufficiently to treatment with these drugs. In addition, HMG-CoA reductase inhibitors may induce unwanted side effects, since they only interfere with cholesterol synthesis but also lower the synthesis of other physiologically active isoprenoids. These considerations have prompted efforts to explore new frontiers in anti-atherosclerotic theraphy in order to expand the therapeutic scope for treating hyperlipidemia and hypercholesterolemia.

Potential usefulness of 4-nitrophenylthio- β-D-glycosides in the chemoselective synthesis of oligosaccharides

Condensation of 4-nitrophenylthioD-glycosyl acceptors with ethylthioD-glycosyl donors in the presence of thiophilic promoters IDCP and NIS/TfOH, according to the “latent-active” principle proposed by Royet al, did not proceed in all cases as expected. On the other hand, replacement of the ethylthio function by an n-pentenyloxy group shows promise for the future chemoselective assembly of oligosaccharides.