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N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [Mock et al. Nat Chem. Biol., 2020, 16, 667−675]. Here, we describe the structure−activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [MockNat Chem. Biol., 2020, 16, 667−67532393901]. Here, we describe the structure–activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus–pituitary–adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Most known β2 selective proteasome inhibitor suffer from relatively poor cell permeability as the result of a net positive charge caused by the basic moiety at P1. Here we describe the synthesis of oligopeptide vinyl sulfones that contain different amino acids bearing amino groups with reduced basicity at P1 and/or P3. For this, we developed the first enantioselective synthesis of lysine(4-ene) and lysine(4-yn). These amino acids, as well as histidine and diaminopropionic acid-glycine, were incorporated at the P1 and/or P3 position of oligopeptide vinyl sulfones. All inhibitors inhibit β2, however, with loss of potency compared to our most potent and selective β2 inhibitor, LU-102. These results notwithstanding, our study described here provides important insights for the future design of β2 selective proteasome inhibitors.

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation ( kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

The interpretation of high-dimensional structure-activity data sets in drug discovery to predict ligand-protein interaction landscapes is a challenging task. Here we present Drug Discovery Maps (DDM), a machine learning model that maps the activity profile of compounds across an entire protein family, as illustrated here for the kinase family. DDM is based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm to generate a visualization of molecular and biological similarity. DDM maps chemical and target space and predicts the activities of novel kinase inhibitors across the kinome. The model was validated using independent data sets and in a prospective experimental setting, where DDM predicted new inhibitors for FMS-like tyrosine kinase 3 (FLT3), a therapeutic target for the treatment of acute myeloid leukemia. Compounds were resynthesized, yielding highly potent, cellularly active FLT3 inhibitors. Biochemical assays confirmed most of the predicted off-targets. DDM is further unique in that it is completely open-source and available as a ready-to-use executable to facilitate broad and easy adoption.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

SummaryThe elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer.This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure.The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised,EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.

The journey towards a detailed mechanistic understanding of the anticoagulant action of heparin has resulted in synthetic mimetics with improved pharmacodynamic profiles. Inspired by the ternary complex formation of heparin with antithrombin III and thrombin, the active pentasaccharide fondaparinux has been succeeded by several clinical candidates, such as SR123781, that have tailor-made factor Xa and thrombin inhibitory activities combined with less aspecific binding (e.g. binding to platelet factor 4 involved in thrombocytopenia). Novel compounds with both antithrombin III-mediated inhibition of factor Xa and direct thrombin inhibition are emerging. Org42675 is one such compound, balancing dual inhibition of factor Xa and thrombin in one anticoagulant drug, with excellent pharmacokinetic properties and strong inhibitory activity toward clot-bound thrombin.

Heparin is a sulfated glycosaminoglycan isolated from animal organs that has been used clinically as an antithrombotic agent since the 1940s. In the early 1980s it was discovered that a unique pentasaccharide domain in some heparin chains activates antithrombin III (AT-III), a serine protease inhibitor that blocks thrombin and factor Xa in the coagulation cascade. Sanofi-Synthélabo and Organon developed a synthetic analogue of this pentasaccharide. The resulting antithrombotic drug arixtra, which went on the market in the USA and Europe in 2002, shows superior antithrombotic activity and brings about AT-III-mediated activity against factor Xa exclusively. Structure-based design has subsequently led to analogues with longer-lasting activity, such as idraparinux, as well as novel conjugates and long oligosaccharides with specific anti-Xa and antithrombin activities. The new drug candidates are more selective in their mode of action than heparin and less likely to induce thrombocytopenia.

Heparin, ein aus tierischen Organen gewonnenes sulfatiertes Glycosaminoglycan, findet seit den 40er Jahren als antithrombotischer Wirkstoff klinische Verwendung. Ein Durchbruch in der Heparinforschung war in den fruhen 80er Jahren die Entdeckung, dass eine charakteristische Pentasacchariddomane in manchen Heparinketten den Serinproteaseinhibitor Antithrombin III (AT-III) aktiviert, der in der Gerinnungskaskade Thrombin und den Faktor Xa inhibiert. Sanofi-Synthelabo und Organon entwickelten das Analogon 2 (Fondaparinux) des Pentasaccharids. Die Verbindung wurde zum antithrombotischen Medikament Arixtra weiterentwickelt, das 2002 in den USA und Europa auf den Markt kam. Arixtra bewirkt ausschlieslich die AT-III-vermittelte Inhibierung des Faktors Xa. Durch strukturbezogenes Design wurden auch Analoga mit langerer Wirkungsdauer wie Idraparinux sowie neue Konjugate und lange Oligosaccharide mit spezifischer Anti-Xa- und Antithrombin-Aktivitat zuganglich. Die neuen Wirkstoffkandidaten wirken selektiver als Heparin, was insbesondere die Wahrscheinlichkeit einer Thrombopenie verringert.

In this paper, a glycosylation-based approach towards novel aminoglycoside analogues is presented. Three different cyclitol building blocks were condensed with different thioglycosides to give, after removal of the protective groups, several neamine-type analogs varying in the positioning and the stereochemistry of the amino functions. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Enantiomerically pure 4,6-diaminocyclohexenols are obtained from carbohydrate derived 1,7-dienes by ring-closing metathesis and palladium catalyzed allylic amination using o-nitrobenzenesulfonylamides as nucleophiles. In the latter reaction the use of a cyclic carbonate as a leaving group proved to be essential to facilitate a smooth substitution. The obtained compounds were converted into orthogonally protected diaminocyclitols, which are stereoisomers of the naturally occurring 2-deoxystreptamine, a constituent of aminoglycoside antibiotics.

A combinatorial library of 32 tricomponent bisubstrate fucosyltransferase analogs has been generated using solid-phase peptide synthesis with orthogonally protected lysine as a scaffold.

A novel chemoselective glycosylation sequence is described that employs the recently developed BSP/Tf2O and DPS/Tf2O reagent systems to activate thioglycosides. In the first glycosylation event a relatively armed thioglycoside is activated with the BSP/Tf2O activator system and condensed with an acceptor thioglycoside to yield the thiodisaccharide, which is activated with the more potent DPS/Tf2O activator in the next glycosylation event. Quenching of (N-piperidino)phenyl(S-thiophenyl)sulfide triflate, which is formed upon activation of the first thioglycoside, with triethyl phosphite is crucial for a productive glycosylation.

Historically, pharmaceutical industries have focussed on the discovery of compounds that target the protein products of genes. The intermediary product between gene and protein, consisting of RNA, has remained largely unexplored. Several drugs targeting the rRNA of bacteria have been, however, in clinical use for over half a century. One of these drug classes, the aminoglycoside antibiotics, also targets human rRNA, and have been developed as therapeutics for genetic disorders. Targeting at the RNA level is an economical approach to address non-drugable proteins and targets that have failed to give leads by hits in HTS, as it can build on biological knowledge gathered over years. RNA also offers entirely new opportunities for drug development, such as targeting of non-coding RNA sequences.

Four heparin pentasaccharides bearing either one (i.e. 3b and 4b) or two (i.e. 3c and 4c) positively charged amino groups at the reducing end have been synthesized and evaluated for their antithrombin III mediated anti-Xa activity. The positively charged groups were introduced to interact specifically with the negatively charged amino acid residues Glu113 and Asp117 of antithrombin III, which are located in the heparin binding site in close proximity to the reducing end of the saccharide. It turned out that the target compounds 3b,c and 4b,c exhibited relatively low anti-Xa activities, indicating unfavorable interactions between the new pentasaccharides and antithrombin III rather than the anticipated enhancement of association. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

A flexible synthetic route towards chirally pure 4,6-diaminocyclohexene derivatives based on palladium catalyzed allylic amination of carbohydrate derived cyclohexenes is presented.

Despite intense research over the last 10 years, aided by the availability of X-ray structures of enzyme-inhibitor complexes, only very few truly orally active thrombin inhibitors have been found. We conducted a comprehensive study starting with peptide transition state analogues (TSA). Both hydrophobic nonpeptide analogues as well as hydrophilic peptidic analogues were synthesized. The bioavailability in rats and dogs could be drastically altered depending on the overall charge distribution in the molecule. Compound 27, a tripeptide TSA inhibitor of thrombin, showed an oral bioavailability of 32% in rats and 71% in dogs, elimination half-lives being 58 and 108 min, respectively. The thrombin inhibition constant of compound 27 was 1.1 nM, and in an in vivo arterial flow model, the ED(50) was 5.4 nmol/kg.min, comparable to known non-TSA inhibitors. A molecular design was found that combines antithrombotic efficiency with oral bioavailability at low dosages.

Despite currently available antithrombotic therapies, venous thromboembolism (VTE) remains a major cause of morbidity and mortality. Fondaparinux sodium (pentasaccharide), the first in a new class of antithrombotic agents developed for the prevention and treatment of VTE, inhibits thrombin generation by selectively inhibiting factor Xa. Fondaparinux exhibits complete bioavailability by the subcutaneous route and is rapidly absorbed, reaching its maximum concentration approximately 2 h post dosing. It has a terminal half-life of 13 to 21 h, permitting once-daily dosing. Fondaparinux's reproducible linear pharmacokinetic profile exhibits minimal intrasubject and intersubject variability, suggesting that individual dose adjustments will not be required for the vast majority of the population and that there will be no need for routine hemostatic monitoring. At therapeutic concentrations (2 mg/L), fondaparinux exhibits94% binding to its target protein, antithrombin. Within this same concentration range there is no specific binding by fondaparinux to plasma proteins commonly involved in drug binding, indicating a low potential for drug-drug interactions by protein displacement. Unlike antithrombotic agents prepared from animal extracts (heparins), fondaparinux is chemically synthesized; this leads to batch-to-batch consistency and the absence of potential risk of contamination problems. In recently completed phase III clinical trials in VTE prevention in major orthopedic surgery, fondaparinux showed significant superiority over the low-molecular-weight heparin enoxaparin, providing an overall50% (P0.001) reduction in VTE risk without increasing clinically important bleeding. Additional clinical data support its potential benefits in other venous and arterial thrombotic disorders. In view of these collective findings, fondaparinux is expected to play a major role in the prevention and treatment of venous and arterial thrombotic disease.

Heparin and heparan sulfate are key players in a plethora of physiological processes. Organic synthesis is the method of choice for the production of these oligosaccharides and their derivatives and analogues. The highly complex structure of these polysaccharides presents a formidable synthetic challenge and the incorporation of the full array of variations in oligosaccharides of significant length is a daunting task. This review records the development of strategies to access these exciting biomolecules.

The carbocyclisation of sugar derived spiroketals and ketosides under the agency of triisobutylaluminium is presented. In addition, the synthetic usefulness of cyclooctenic product 21, derived from the corresponding exo-glycal 20, was shown by its transformation into conformationally locked l-idose analogues 22 and 23.

A single disaccharide building block is required to obtain synthetic carbohydrates that reproduce the anticoagulant activity of heparin and inhibit thrombin (n>6) and/or factor Xa (n≥2; see reaction scheme). Thus, there is evidence that heparin fragments with at least 15 saccharide units are required for thrombin inhibition. Lev=levulinoyl.

Ausgehend von einem einzigen Disaccharidbaustein wurden synthetische Kohlenhydrate erhalten, die die gerinnungshemmende Aktivitat von Heparin aufweisen und Thrombin (n>6) und/oder Faktor Xa (n≥2) inhibieren (siehe Schema). Damit ist auch ein Hinweis darauf gegeben, das fur die Inhibierung von Thrombin Heparinfragmente aus mindestens 15 Zuckereinheiten erforderlich sind. Lev = Lavulinoyl.

A three-step approach to chiral pyranose [5,4], [5,5], [5,6] and [5,7] unsaturated spiroacetal derivatives from perbenzylated glucopyranolactone 1 is presented. The strategy involves Grignard addition of vinyl or allyl magnesium bromide to 1 to give 2 and 12 , respectively, K-10 mediated glucosidation of different terminal alkenols with 2 and 12 followed by ring-closing metathesis.

A synthesis is presented of the cyclic trimeric d-oligonucleotide 3′-isopropylphosphate I, comprising one formacetal and two (3′ → 5′)-internucleosidic phosphodiester bonds. The ester linkages connect d-guanosine with the 3′ and 5′ ends of thymidine and 5-hydroxymethyl-2′-deoxyuridine-3′-isopropylphosphate (HMDUpiPr), respectively. The 5′-end of the thymidine unit is anchored via the formacetal bond to the allylic hydroxyl group of HMDUpiPr. The cyclic arrangement of the three d-nucleosides in I mimics, as based on molecular modeling, the key structural features of the conformationally constrained T7pG8pT9p-domain of the thrombin-binding DNA aptamer d(G1G2T3T4G5G6 T 7 G 8 T 9 G10G11T12T13G14G15). Biological evaluation showed that compound I did not exhibit anti-thrombin activity.

Derivatization of the octadecathymidylate derivative 18 (T18 ODN) containing a free amine function with sulfo-SIAB® gave the corresponding iodoacetyl ODN 21. Conjugation of the latter with the thiol-containing pentasaccharide 17c gave pentasaccharide–ODN conjugate III, which exhibited anti-Xa and antithrombin activities of 173 U mg-1 and 5 U mg-1, respectively.

N-Alkylation of the alpha-glucosidase inhibitor 1-deoxynojirimycin (dNM) dramatically increases its inhibitory potency (Tan et al., J. Biol. Chem., 266, 14504-14510, 1991). However, the possibility of extending the alkyl chain to N-decyl-dNM is limited by an increase of detergent-like (amphiphilic) properties of long-chain alkylated dNM derivatives. Substitution of methylene groups in the N-decyl chain by oxygen reduced the amphiphilicity of N-decyl-dNM derivatives, while retaining their superior inhibitory properties. In intact HepG2 cells, the compound N-7-oxadecyl-dNM was found to result in the most pronounced retention of glucose residues on N-linked glycans. Permeabilization of the plasma membrane with the bacterial toxin Streptolysin O improves the inhibitory properties of the derivatives N-3,6,9-trioxadecyl-, N-7,10,13-trioxatetradecyl-, N-3-oxadecyl- and N-7-oxadecyl-dNM, but not those of dNM. These observations suggest differences in the mode of entry of the oxygen-substituted dNM derivatives in comparison with dNM. We observed that the dNM derivative N-3,6,9-trioxadecyl-dNM, devoid of inhibitory activity in intact cells, was inhibitory in Streptolysin O-permeabilized cells. Thus, the permeability barriers posed by plasma membrane and endoplasmic reticulum membrane are not equivalent. The use of a permeabilized cell system thus allows the elaboration of inhibitory principles for novel bioactive compounds where study of the isolated enzymes may not be possible, and where intact cells are not a suitable target due to permeability barriers.

The syntheses of several analogues of disaccharide Manot(1→6)Mana-OCH3 (1) and of trisaccharide Mana(1→6)[Mana(1→3)]Mana-OCH3 (2) are reported. The syntheses are described of the diastereomeric 6-methyl derivatives 9a and 9b, which are representatives of fixed conformations of disaccharide 1. The syntheses of the 2-amino-2-deoxy analogues 15 and 17 and the synthesis of the 2-fluoro-2-deoxy analogue 28 are also reported.

Heparin, ein sulfatiertes Glycosaminglycan, ist ein bekanntes Anticoagulans, dessen Wirkung uber den Serin-Protease-Inhibitor Antithrombin III (AT III) vermittelt wird. Heparin ist seit mehr als einem halben Jahrhundert zur Prophylaxe und Behandlung venoser Thrombosen und Thromboembolien in klinischer Anwendung. Noch bis in die 80er Jahre hinein nahm man an, das die biologische Aktivitat von Heparin hauptsachlich auf seinen polyanionischen Charakter zuruckzufuhren sei. Als man jedoch entdeckte, das ein Teil der Heparin-Polysaccharide eine wohldefinierte Pentasacchariddomane enthalt, die spezifisch AT III bindet und aktiviert, geriet dieses Paradigma ins Wanken. Biologische Untersuchungen an mehreren synthetischen Heparin-Analoga erhellten die Spezifitat der Wechselwirkung zwischen diesem charakteristischen Pentasaccharid und AT III. Dieser Aufsatz gibt eine Ubersicht uber die Synthese des Heparin-Pentasaccharids, einiger nahe verwandter Strukturen und einiger stark modifizierter Analoga. Mit Molecular Modeling und „masgeschneiderten” strukturellen Modifizierungen an diesem Pentasaccharid konnten zahlreiche Erkenntnisse uber Struktur-Aktivitats-Beziehungen gewonnen werden. Dies gab nicht nur den Anstos fur die Entwicklung wirksamerer und einfacherer Derivate, sondern ermoglichte auch ein detailliertes Verstandnis der Erkennung von Heparin und AT III auf molekularer Ebene.

Heparin, a sulfated glycosaminoglycan, is well known for its anticoagulant effect mediated by the serine protease inhibitor antithrombin III (AT III). Heparin has been used clinically for more than half a century for the prophylaxis and treatment of venous thrombosis and thromboembolism. Up until the 1980s it was assumed that the biological activity of heparin was mainly caused by its polyanionic character. However, this paradigm was contradicted when it was discovered that part of the heparin polysaccharides contains a well-defined pentasaccharide domain that specifically binds and activates AT III. The specificity of the interaction between the characteristic pentasaccharide and AT III has become more obvious after the synthesis and biological testing of various heparin analogues. This article reviews the synthesis of the heparin pentasaccharide, some closely related counterparts, and some highly modified analogues. With the aid of molecular modeling and through “tailored” molecular modifications of the pentasaccharide, much knowledge has been gained concerning structure-activity relationships. On this basis not only have more potent and simplified derivatives been developed, but also the recognition between heparin and AT III can now be understood in greater detail at the molecular level.

For the synthesis of Gals(1 4)IdoA disaccharides 1, several glycosylations of L-iduronic acid derivatives 5 and 13 and of L-idopyranose derivative 21 were performed with differently protected D-galactopyranosyl donors under various conditions. It was found that these coupling reactions mainly led to the formation of the undesired α-interglycosidic linkage, even when the donor contained a participating group at C-2. On the other hand, coupling of the corresponding D-glucopyranosyl donor 19 with 13 led to a much higher β/α ratio (2/1). By changing the conformation of the acceptor into the 1,6-anhydro-L-idopyranose acceptor 23, 90% of the desired β-coupled disaccharide was obtained in the glycosylation with galactopyranosyl donor 12. Finally it was found that the α/β ratio of the condensation with 25, promoted by an insoluble silver salt, was influenced in an unusual way by the substituents at the galactosyl bromide.

A novel procedure is described for the introduction of a masked thiol function at the 5′-terminus of oligodeoxynucleotides. An acetyl-protected thiol linker phosphoramidite has been prepared which can be coupled efficiently to fully protected oligonucleotides using standard solid-phase techniques. Our strategy is elaborated for oligothymidylates and is further illustrated for oligodeoxynucleotides which are base-protected with the 2-(acetoxymethyl)benzoyl group. Rapid base-deprotection can be accomplished in methanolic potassium carbonate which also effects saponification of the thioacetyl ester. The resultant free thiol is reacted in situ with dithiodipyridine to afford pyridyldisulfide-derivatized oligodeoxynucleotides.

A single disaccharide building block is required to obtain synthetic carbohydrates that reproduce the anticoagulant activity of heparin and inhibit thrombin (n>6) and/or factor Xa (n≥2; see reaction scheme). Thus, there is evidence that heparin fragments with at least 15 saccharide units are required for thrombin inhibition. Lev=levulinoyl.

The syntheses of pentasaccharide I and heptasaccharide II , which correspond to an ovarian O-glycoprotein, are presented. The protected pentasaccharide 1 was prepared by condensing a trisaccharide donor 18c with a disaccharide acceptor ( 8b , while condensation of the same trisaccharide donor ( 18c ) with a tetrasaccharide acceptor ( 25b ) provided the protected heptasaccharide 2 . Special attention was paid to the deblocking of the protected derivatives 1 and 2 to give I and II , respectively. The β-bonds in 8b and 25b were prepared by using glycosyl donors with non -participating groups at C-2, which were earlier applied for the synthesis of α-glycosides. For the synthesis of the trisaccharide donor ( 18c ), condensations of several activated Galα(1->3)Gal disaccharides with protected GlcNAc residues were compared. It was found that the α/β ratio and the yield of this glycosylation are influenced by the leaving group at the anomeric centre, the character of the participating acyl group and, in particular, by the unfavourable steric interaction between donor and acceptor. In order to reduce this steric interaction between donor and acceptor, the conformation of the GlcNAc acceptor was changed to the 1,6-anhydro derivative 16 , which afforded in the glycosylation with glycosyl bromide 13c a high yield of the desired trisaccharide 17 .

The synthesis of an analogue (i.e. compound 7 ) of the AT-III binding sequence of heparin is described in which an essential 6-O-SO 3 group at the non-reducing glucosamine is replaced by a phosphate group. This compound appears to be biologically inactive.