Identifier: TDX:801
Authors: Benito Alifonso, David
Abstract:
During the past decades, much progress has been made in the knowlledge of calcium signalling and how cells employ calcium in order to regulate their processes. 1D-myo-Inositol 1,4,5-trisphosphate (IP3) is a second messenger that plays an important role in intracellular calcium stores activity as well as in extracellular calcium entry. Extracellular stimuli such as hormones, neurotransmitters or growing factors (first messengers) are capable to bind to specific receptors located a the outer face of cell membrane. This bind results in an activation of Phospholipase C located on the cell membrane, which in turn catalyses the hydrolysis of phospholipids, releasing diacyl-glycerol (DAG) and IP3 (second messenger). In 1993, Takahashi et al. isolated from a Penicillium brevicompactum, culture, two potent glyconucleotides trisphosphate: Adenophostin A and B. These compounds are the most potent IP3 agonists ever reported until now, being from 10 to 100 fold times more active than IP3 itself.From a chemical point of view, Adenophostins share with IP3 a trans-diequatorial bis-phosphate moiety flanked by an hydroxyl group at C-2'.(see Figure) Moreover, Adenophostins are resistent to enzymes that metabolize IP3 such as IP3-phosphatase and IP3-kinase. Many Adenophostin analogues have been synthesized in order to elucidate strucural features responsible of Adenophostin activity, and to obtain more active compounds. However, until now, only few analogues has overcome IP3 activity and none of them has reach Adenophostine activity. Structure-activity-relationship studies has allowed to design a pharmacophore model for Adenophostin A. Main features of this model are: The trans-diequatorial bis-phosphate moiety flanked by 2''-OH, which is a key point for Adenophostin biological activity, and mimics 4,5-bisphosphate-6-hydroxyl groups in IP3.The presence of adenine (or any equivalent structure) increases its activity respect to IP3. In that direction to possible adenine roles have been proposed. Adenine would allow to position 2'-phosphate in an optimal disposition through a C2'-endo conformation (indirect role). On the other hand, adenine could be directly involved in complementary interactions with a region located near the binding site (direct role) allowing to avoid the effect of C-terminal inhibitory domain. In particular, it is proposed the existence of a cation- interaction between adenine ring and an arginine residue (Arg504).A recent study carried out in collaboration with Dr. Morère, has allow to show that that mannose-6-phosphate receptors are capable of recognize mannose analogues incorporating carboxylate isoster groups. In particular, it has been showed that carboxyl analogues have same affinity for the receptor as mannose-6-phosphate. Moreover, with this substitution (phosphate-carboxylate) lability of phosphate groups can be avoided.As has been mencioned before, Adenophostins are resistent to IP3 metabolizing enzymes. In that direction, it is well known that the presence of a fluorine atom at 2' position of glycosides increases glycosidic bond stability specially towards acidic hydrolysis.With this background in mind, it was proposed to study whether IP3R are capable of recongnising effectively Adenophostin analogues in which one or more phosphate groups have been replaced by methylene carboxylate moieties. On the other hand, it was also proposed to increase Adenophostin stability by means of fluorine introduction at position C-2''.Present work has been focused in two points:The first one was focused in confirming the interactions of adenine with the receptor and the role of 2'-phosphate in Adenophostin activity. In this sense, biological avaluation of IP3, Adenophostin A, inositol 4,5-bisphosphate (IP2), and 2'-dephospho-Adenophostin A has been carried out. First assays were made with using full lenght receptor, binding domain fragment and binding domain fragment incorporating a mutation at position 568 (from Arg to Gln). This residue interacts with phosphate 1 of IP3 and it is supposed to interact with Adenophostin 2'-phosphate as well. Second assays were made using binding domain receptor fragment incorporating a mutation at position 504 (from Arg to Gln). This residue it is supposed to form a cation- stacking with adenine moiety. With this biological avaluations, it can be deduced that Adenophostin A activity is mainly due to the adenine presence, and moreover, the supposed optimal 2'-phosphate disposition is not determinant in Adenophostin high affinity. Furthermore, it has been confirmed the presence of cation- stacking interaction at the binding core.The second point of the present work has been the design and synthesis of new Adenophostin analogues based in biological study results and antecendents mentioned above.Thus, it has been synthesized the precursor of an Adenophostin analogue incorporatin a fluorine atom at C-2'' in order to confer more stability to the glycosydic bond and avaluate the role of 2''-OH in hydrogen bonding. The introduction of fluorine into Adenophostin structure was carried out by means of electrophilic fluorination with Selectfluor® of 3,4,6-tri-O-acetyl-D-glucal, which lately was transformed into the corresponding glycosyl bromide. On the other hand, adenosine was conveniently protected and used as glycosyl acceptor in the glycosylation with fluorinated building block. Next steps after glycosylation were protecting group manipulation in order to afford a suitable substrate for phosphate group introduction at desired positions (2', 3', 4''). Furthermore, considering the secundary role of 2'-phosphate in Adenophostin activity, we focused in the synthesis of two Adenophostin analogues in which phosphate groups at position 3'' and 4'' were replaced by a methylenecarboxylate moiety. Thus, a part from incorporate non metabolizable groups, alternate substitution would allow to know the independent role of each phosphate in receptor binding. Basic structure for two analogues was afforded from the glycosylation of conveniently protected adenosine with a thioglycoside derivative incorporating the methylenecarboxylate moiety in its structure. Methyl (4,6-O-benzylidene)-1-O--glucoside was used as starting material for both carbohydrate fragments, which were afforded depending on the protecting groups sequence used. Introduction of methylencarboxylate precursor into the scaffolds was made via radical allylation, affording allylderivatives with desired stereochemistry. The acid was obtained upon oxidative cleavage of allyl group. Lasts steps of the carbohydrate fragment synthesis involved the hydrolysis of anomeric position and thioglycoside synthesis. Finally, thioglycoside obtained was reacted with adenosine derivative affording the basic structure for both analogues. After several deprotection steps, apropiate substrates for phosphorilation at desired positions were afforded. In summary, the present work has allowed to:1. Stablish the structural features that confere to Adenophostins a activity higher than IP3.2. Synthesize three precursors of Adenophostin analogues presenting new structural modifications that would allow to a) know the independet role of each phosphate in the natural product and b) the role of 2''-OH in receptor interaction.
Repositori URV