A similar outcome was observed for the deprotection of 1-O-phenoxyacetyl 89. Partial cleavage of the ester group was observed, even when reaction time was minimised. Presumably the sodium bicarbonate used to generate the sodium salts of the phosphates must play a role in this unwanted side-reaction. Potentially, this problem
A
may be overcome simply by omitting the sodium bicarbonate for the duration of the debenzylation, then adding it to rapidly form the desired salt before work-up. This way, the length of time that the compound is exposed to the bicarbonate is minimised. However, given the apparent tendency of the 1-phosphate of this class of compounds to migrate over time, further efforts to optimise conditions for this step could not be justified.
3.6 Summary
Following the problems encountered in the first synthetic approach towards novel PtdIns(3,4,5)P3 analogues, a second approach was devised. This involved the introduction of appropriate functionality to the 4-position as the penultimate step in the synthesis. A racemic synthesis of the key 4-hydroxy intermediate 66, bearing protected phosphates at the 3- and 5-positions and a phosphodiester at the 1-position, was achieved in high overall yield. Despite steric hindrance from the neighbouring phosphate groups, the 4-position of alcohol 66 was successfully functionalised to synthesise analogue precursors with either phosphinate, sulfonate, carbonate or ester groups at this position. Several attempts had been made to synthesise further analogue precursors, but the lack of reactivity of alcohol66 and purification difficulties curtailed the success of such efforts.
Unfortunately, final debenzylation of the penultimate compounds was problematic. Prolonged exposure to aqueous conditions facilitated the migration of the 1-position phosphodiester to a neighbouring alcohol of the inositol ring. This was observed to occur during reaction in basic conditions, as with the synthesis of the 4-dimethlphosphinate33, or with the isolated material over time as with the 4-mesylate 100. Furthermore, the carbonate and ester groups of compounds 89 and 90 were partially cleaved under the deprotection conditions employed.
In order to reduce the chances of migration of the 1-position phosphate, both during the final step, in storage and in conditions for biological testing, it seemed that modification of the target compound was required. Also, alteration of debenzylation conditions
appears necessary in order to isolate carbonate and ester-bearing analogues such as 89 and 90. The introduction of less base-labile groups at the 4-position may well be required if analogues bearing an aromatic group are to be synthesised.
Chapter 4. Results and Discussion Part 3
4.1 The rationale for InsP4analogue synthesis
The second route towards the rationally designed PtdIns(3,4,5)P3analogues (chapter 3) enabled the synthesis of several precursor compounds. However, the fact that the 1- phosphodiester was prone to migration either during the final deprotection, or over time with the stored product, suggested that the design of the target compounds should be modified. One option was to increase the size or length of the diester group to provide the more commonly reported dibutanoyl or dihexanoyl phosphodiester, to give analogues of the type 103 (Figure 4.1). The added steric bulk of the diester should prevent migration. However, one potential problem with this approach is that additional lipophilicity, and the resulting disordered nature, of the phosphodiester is likely to reduce the chances of achieving co-crystallisation of the analogues with PKB PH. This is evidenced by the fact that biologists have failed to obtain crystal structures of short chain PtdIns(3,4,5)P3 compounds bound to PKB PH, and have only achieved co- crystallisation with the inositol head group InsP4.54 Another option would be to replace the phosphodiester altogether with a simple phosphate, to give trisphosphates of the type 104 (Figure 4.1). If anything, this modification should increase the chances of obtaining ligand-PKB PH crystal structure complexes, as compared to the original design. Given the potential utility of such crystal structures for active compounds, it was decided that this latter approach would be adopted. Such a modification should also significantly reduce the likelihood of phosphate migration, as there would be no phosphate ester in the molecule.
OH O HO O O O R P P O-Na+ +Na-O O +Na-O O-Na+ O P O O-Na+ O-Na+ 1 2 3 4 5 6 103 OH O HO O O O R P P O-Na+ +Na-O O +Na-O O-Na+ O P O O-Na+ O 1 2 3 4 5 6 O O C5H11 O C5H11 O 104
Compounds such as trisphosphate 104 can be seen as 4-position-modified InsP4 analogues. InsP4 itself is synthesised from Ins(1,4,5)P3 by Ins(1,4,5)P3-3-kinase.166 Its characteristic short life-time has led to the proposition that this inositol may also act as a second messenger.167 As noted earlier, InsP4 binds PKBαPH with similar affinity to PtdIns(3,4,5)P3 in vitro.34 This is not the case for all the PH domain-containing proteins that bind to PtdIns(3,4,5)P3, where InsP4 affinity is sometimes significantly less than for the phospholipid. Proteins that are selective for InsP4 are rare. Also, the ability of proteins such as PKB to distinguish between InsP4and PtdIns(3,4,5)P3in vivo remains unclear. Clearly, the crucial difference between these inositols involves the presence of the fatty glyceryl unit at the PtdIns(3,4,5)P31-position phosphate. As it is the phosphate interactions which are seen as most significant for binding, it is possible that the glyceryl unit acts more as a recognition module, the extent to which may vary between the protein substrate. The lipid chains themselves serve only to anchor the molecule in the membrane. Such debate further underlines the complexity of inositol signalling.
It is, therefore, conceivable that omitting the glyceryl unit in the proposed analogues may improve selectivity against some PtdIns(3,4,5)P3-binding proteins that display reduced affinity for InsP4. A long-term aim for the project would involve the initial discovery of an active InsP4 analogue, followed by the synthesis of a related PtdIns(3,4,5)P3 analogue which bears a long-chain phosphodiester at the 1-position. Comparison of activity could reveal useful information concerning the nature of PtdIns(3,4,5)P3/InsP4 selectivity for PH domain binding.
4.2. The racemic synthesis of 4-position InsP4analogues
4.2.1 Synthesis of the 4-dimethylphosphinate InsP4analogue 108
The synthesis of the racemic bisphosphate 67 was outlined in chapter 3. This material was used to synthesise the key 1-hydroxy intermediate 106 in two steps. Thus, phosphitylation of the 1-position of67was achieved by reaction with bis(benzyloxy)-N,N- diisopropylamino phosphine and 1H-tetrazole followed by oxidation by mCPBA, giving
intermediate 105 in 87% yield (Scheme 4.1). Efficient PMB-deprotection of compound 105using ceric ammonium nitrate afforded the 4-hydroxy-trisphosphate106.
OBn O BnO O OPMB O P P OBn BnO O O P O OBn OBn OBn OBn OBn O BnO O OH O P P OBn BnO O O P O OBn OBn OBn OBn OBn O BnO O OPMB OH P P OBn BnO O BnO OBn O i ii 67 105 106
Scheme 4.1.Synthesis of key intermediate106.Reagent and conditions:i. a.bis(Benzyloxy)-N,N-diisopropylamino phosphine, 1H-tetrazole, CH2Cl2,b.mCPBA -78 °C → RT, 87% yield; ii.CAN, CH3CN/H2O (4:1), 85% yield.
The synthesis of the first 4-position modified InsP4analogue was achieved as outlined in Scheme 4.2. The previously optimised conditions for phosphinylation were employed, in
which alcohol 106 was reacted with dimethylchlorophosphine in
pyridine/dichloromethane to give compound 107in 62% yield. Global debenzylation was then performed under the same conditions employed previously, using 20 equivalents of Pd black in the presence of sodium bicarbonate (6 equivalents), with t-butanol/water (6:1) as the solvent under an atmosphere of hydrogen. Clean product 108 was obtained in quantitative yield after 5 hours. As anticipated, no phosphate migration was observed by1H and31P NMR analysis. However, obtaining useful mass spectrometric data proved difficult. It was found that the addition of diethylamine to a dilute solution of the sample in methanol/water was required to obtain interpretable spectra. This promoted the association of the phosphate groups with H+ as opposed to Na+ to form the phosphate acid. Thus, in ES- mode, the (M-6Na+5H)- ion for the resulting acid of compound 108 was identified, as well as the ions corresponding to the mono- and di-sodiated material (see Figure 4.2). The fully sodiated material, however, was not observed by this method. Also, due to the hygroscopic nature of polyphosphorylated compounds such as analogue 108, as well as the likely preference of mixed sodium salts of the compound, accurate elemental analysis could not be obtained.