2.3 Results and discussion
2.3.4 Urocanylcholesterylformylhydrazide (SH04)
64 It is noteworthy that SH02 did not melt but decomposed, as evidenced by a change in the birefingent crystals from colourless to dark brown, due to charring of the sugar residues. The fact that no additional change occurred even when SH02 was subjected to temperature in excess of 230 ˚C, which corresponds to the melting point of DCU, confirms that DCU was not present as a contaminant. This was further corroborated by the absence of a peak corresponding to the molecular weight of DCU in the mass spectrum of SH02. A disadvantage associated with DCCI coupling protocols is the possibility of O to N
transacylation within some of the O-acylisourea intermediate species, resulting in stable N- acylisourea, due to competition between the amine-bearing compound and the nucleophilic center of O-acylisourea for the acyl group (Bodanszky, 1988; Bodanszky et al., 1976).
However the mass spectrum confirmed that SH02 was free of N-acylisourea molecules, as a peak corresponding to a molecular weight of this species was not evident (refer to Appendix 2).
65 N
HN
C OH
O
Urocanic acid
Cl
i) tritylchloride NH ii) diethylamine
N N
C O
O N
H H
i) NaOH ii) AcOH iii) NaCl
N N
C OH O N-tritylurocanic diethylammonium salt
N-tritylurocanic acid DCCI
(dry DMF)
N OH O
O NHS
N N
C O O
N O
O
N-hydroxysuccinimide ester of N-tritylurocanic acid
O C NH H2N
O
cholesterylformylhydrazide
N N
C O
NH NH C O O
N-tritylurocanylcholesterylformylhydrazide (SH05)
N HN
C O
NH NH C O O
urocanylcholesterylformylhydrazide (SH04) i) CHCl3
ii) AcOH 37 oC, 8.5 hours
(SH01)
Figure 2.12: Scheme outlining the synthesis of urocanylcholesterylformylhydrazide (SH04) from urocanic acid and SH01.
66 An urocanic acid molecule contains both amino and carboxyl functionalities, from which amide bonds may be derived via DCCI coupling reactions. Therefore, the introduction of a coupling reagent to a mixture of urocanic acid and SH01 may generate homopolymers and SH01-linked higher homologues of urocanic acid, in addition to the desired novel lipid. This would impact negatively on the yield of the desired product; and presents difficulties with respect to product purification. In an attempt to prevent the formation of amide bonds between urocanic acid molecules, its amino group was rendered inert by appending a trityl group. The trityl moiety is well documented as a protecting group for amines. It is relatively simple to introduce and can be removed under conditions which preserve the integrity of the newly synthesised molecule (Divakaran, 2008).
The reaction of urocanic acid with tritylchloride generates hydrochloric acid as a byproduct, which is likely to react with the basic nitrogen atom of the imidazole ring, to yield a
hydrochloride salt. This was prevented by performing the reaction in the presence of a 5.4 molar excess of the organic base, triethylamine that was introduced into the reaction mixture prior to tritylchloride. Triethylamine served to sequester hydrochloric acid, forming its hydrochloride salt, which is insoluble in DMF. Solubilisation of triethylamine hydrochloride was achieved by the addition of methanol. In fact, methanol serves a dual purpose in this reaction as it also eliminates unreacted tritylchloride as methyl ethers. For the purposes of product recovery the tritylated urocanic acid was obtained in high yield as a
diethylammonium salt. The unreactive nature of this compound necessitated its conversion to the free acid. However, due to the low reactivity of the carboxyl functionality, and with reference to the preparation of urocanic acid-modified carriers reported in the literature (Bailey, 1990; Yang et al., 2010), this group required activation to merit its participation in subsequent synthesis. For this reason NHS was coupled to the free acid, to afford an active ester.
Apart from the procedure for product visualisation outlined in 2.2.2.3.3, two other methods also confirmed the formation of the active ester via TLC, both during its synthesis and after product recovery. The phenolic ring system of the trityl group and heterocyclic ring structures of urocanic acid and NHS are able to absorb ultraviolet (UV) light. Therefore, UV
illumination of the fluorescent silica gel plate enabled visualisation of the product and unreacted starting material as dark spots. An alternative method entailed spraying the plate
67 with sulphuric acid, whereupon the product assumed an intense yellow colour (Figure 2.13).
This is because the acid releases the trityl group as a stable carbocation, which is yellow in colour. In other TLC analyses, the active ester was detected as a dark purple spot on the plate after applying basic hydroxylamine and ferric chloride. Excess hydroxylamine in alkaline medium is known to convert N-hydroxysuccinimide and its esters to their corresponding hydroxamic acids. In the presence of Fe(III) cations, hydroxamic acid forms a ferric
hydroxamate complex that is purple in colour (Maguire and Dudley, 1977; Ong and Brady, 1972).
Figure 2.13: Formation of the active ester of N-tritylurocanic acid after 24 hours of reaction time, as observed on silica gel 60F254 plates developed in CHCl3:MeOH (95:5, v/v). The plate was viewed under ultraviolet light and thereafter, sprayed with 10 % H2SO4 and heated.
The N-hydroxysuccinimide ester of N-tritylurocanic acid prepared exactly as outlined in 2.2.2.3.3 was used as a precursor to SH04 in this study. At a later stage however, a second batch of the active ester was prepared, and ethanol was employed as the solvent for
recrystallisation. In this instance, the product was obtained in high yield (80.8 %) as white, birefringent crystals, which melted at a higher temperature (127-130 ˚C). A comparison on
Lane 1: N-tritylurocanic acid
Lane 2: Reaction mixture (N-tritylurocanic acid, NHS, DCCI in DMF)
a: Product (N-hydroxysuccinimide ester of N-tritylurocanic acid)*
b: N-tritylurocanic acid c: NHS
denotes a UV-absorbent compound
*Note that the product was purified by recrystallisation prior to use.
68 TLC revealed that the Rf values of the amorphous and crystalline products were identical. It appears, therefore, that ethanol is a more suitable solvent for the recovery of the active ester of N-tritylurocanic acid in crystalline form.
The next reaction entailed the formation of the amide linkage between the activated, N- protected urocanic acid and the cholesterol anchor molecules (SH01). Progress of this reaction was evidenced by the release of UV-absorbent NHS by TLC (Figure 2.14).
Eventually the product, SH05, was identified on the chromatogram due to its UV-absorbent, trityl-positive and cholesterol-positive properties. Finally, the trityl group was removed under mildly acidic conditions to afford the imidazolylated lipid, SH04, with a 9.771 Å spacer between the cholesterol and imidazole rings (Figures 2.4c and 2.5c).
Figure 2.14: Coupling of carboxyl activated N-tritylurocanic acid to SH01 as observed on silica gel 60F254 TLC plates in CHCl3:MeOH (98:2, v/v). Reaction components were visualised under UV illumination, and after applying 10 % H2SO4.
Lane 1: N-hydroxysuccinimide ester of N-
tritylurocanic acid, SH01 and NHS, each applied successively.
Lane 2: Reaction mixture (N-hydroxysuccinimide ester of N-tritylurocanic acid and SH01 in CHCl3) a: N-hydroxysuccinimide ester of N-tritylurocanic acid b: SH01
c: Product (SH05)*
d: N-tritylurocanic acid e: NHS
denotes a UV-absorbent compound
*Note that the product was purified by preparative TLC before use.
69 In summary, two novel cholesterol derivatives, lactobionylcholesterylformylhydrazide
(SH02) and urocanylcholesterylformylhydrazide (SH04) were successfully synthesised in reactions mediated by the coupling reagent dicyclohexylcarbodiimide.
Cholesterylformylhydrazide (SH01) was prepared from cholesterylchloroformate and hydrazine as a precursor to both SH02 and SH04. A direct coupling reaction between SH01 and lactobionic acid gave the glycolipid, SH02. Observations made as per TLC suggested that this reaction proceeded largely via a lactone intermediate. The synthesis of the imidazolylated lipid, SH04, from urocanic acid and SH01 was achieved in several steps:
urocanic acid was N-tritylated, recovered as a diethylammonium salt, and converted to the free acid; the amide bond was formed between the N-protected urocanic acid and SH01, and the trityl group was removed. NMR and high resolution mass spectrometry were used to validate the structures of both cholesterol derivatives.
70 CHAPTER THREE
LIPOSOME PREPARATION AND CHARACTERISATION