47 modified, N, N′-dicyclohexylcarbodiimide (DCCI), represented in Figure 2.3, is arguably the most popular (Han and Kim, 2004), and is the coupling agent of choice in this study. DCCI rapidly facilitates concurrent activation and coupling when introduced into a mixture of the carboxyl- and amine-bearing compounds. Other synthetic routes entail the use of DCCI in the generation of active carboxylic acid esters, to which the amino component is added at a later stage. In general, DCCI-mediated couplings rapidly give high yields especially when used in conjunction with strategies which limit undesired reactions with other functional groups on the starting material. Finally, the urea byproduct of DCCI reactions is insoluble in most organic solvents, and is easily eliminated by filtration following product formation (Bodanszky, 1988).
Figure 2.3: A ball and stick model of DCCI.
48 2.2.2 Methods
2.2.2.1 Synthesis of cholesterylformylhydrazide (SH01)
Cholesterylformylhydrazide was prepared according to the method published by Singh and Ariatti (2006).
Briefly, a solution of hydrazine (240 mg, 7.5 mmol) in CHCl3:MeOH (3:0.6 ml) was added dropwise, with stirring, to an ice-cold solution of cholesterylchloroformate (1.13 g, 2.5 mmol) in CHCl3 (5 ml). The reaction was maintained at room temperature for 2.5 hours, during which it was periodically monitored by TLC on silica gel 60F254 plates developed in CHCl3:MeOH (95:5, v/v). Cholesterol derivatives appeared as purple spots after the plate was treated with 10 % H2SO4 and heated to approximately 60 ˚C. The crude product was
deposited as a white crystalline mass after rotary evaporation (Rotavapor-R, Büchi) of the solvent. This was recrystallised from CHCl3:MeOH (approximately 4:1, v/v). Liquors were collected and subjected to further recrystallisation. Finally 927 mg of product (83.4 % yield) was obtained. Rf (retention factor) = 0.56. mp (melting point), (first crop): 211 – 213 ˚C. mp (second crop): 217 – 219 ˚C.
2.2.2.2 Synthesis of lactobionylcholesterylformylhydrazide (SH02)
Lactobionic acid (107 mg, 0.3 mmol), which was dried in vacuo, and SH01 (111 mg, 0.25 mmol) were dissolved, with heating, in pyridine (2 ml). Thereafter, a solution of DCCI (85 mg, 0.4 mmol) in pyridine (0.5 ml) was introduced. Product formation was monitored by TLC on silica gel 60F254 plates in CHCl3:MeOH:H2O (6:4:1, v/v/v) as recommended by Wang et al. (2006), Rf = 0.39. Components of the reaction mixture were observed after spraying the plate with 10 % H2SO4, followed by heating. After 6 days at room temperature, the solution was filtered to remove dicyclohexylurea (DCU) crystals and concentrated to a foam in vacuo. Residual pyridine and DCCI were eliminated by co-evaporation with toluene and extraction with petroleum ether (60 – 80 ˚C) respectively. Thereafter, the crude product was evaporated to dryness. Water was added to the residue, in order to extract unreacted
49 lactobionic acid, whereupon a gel was formed. The gel was centrifuged at 3000 rpm
(Eppendorf 5702R centrifuge, Merck, Darmstadt, Germany) for 5 minutes. After discarding the supernatant, a slurry remained. This was dried in vacuo, with CHCl3:MeOH
(approximately 1:1, v/v) and CHCl3:EtOH:MeOH (undefined ratios) respectively, to an off- white powder (102 mg, 52 % yield). Approximately 20 mg of product was purified on a silica gel 60 column (7.95 cm3), eluted with CHCl3:MeOH (4:1, v/v), CHCl3:MeOH (6:4, v/v) and CHCl3:MeOH:H2O (6:4:1, v/v/v) respectively. Fractions of approximately 3 ml each were collected and analysed by TLC using the corresponding solvent system. Finally, fractions exhibiting greatest product purity were combined and evaporated to dryness, to afford 17 mg of SH02. mp: 188.5 ˚C (decomp.).
2.2.2.3 Synthesis of urocanylcholesterylformylhydrazide (SH04)
The coupling of urocanic acid to the cholesterol anchor molecule, SH01, was achieved in several steps.
2.2.2.3.1 Preparation of the diethylammonium salt of N-tritylurocanic acid
This protocol is a modified version of that which was employed by Cloninger and Frey (1998) for the synthesis of the dibutylammonium salt of N-tritylurocanic acid.
Urocanic acid (276 mg, 2 mmol) was partially dissolved in dry dimethylformamide (DMF) (7 ml). Upon introduction of triethylamine (1.09 g, 10.8 mmol) and tritylchloride (1.22 g, 4.4 mmol) a homogeneous solution was obtained. This was stirred at room temperature for 24 hours. High Performance Liquid Chromatography (HPLC)-grade methanol (7 ml) was added, and the solution stirred for an additional 24 hours, after which it was dried to a brown oil under vacuum. The oil was diluted with ethylacetate, until the solution separated into two layers. Following in vacuo concentration of the organic layer, diethylamine (146 mg, 2 mmol) was added, and the solution stored overnight at 4 ˚C. The product was obtained as crystals, which were repeatedly washed with ethylacetate and hexane. The washings were collected and dried under vacuum, followed by the addition of hexane, in order to increase product yield. Eventually 954 mg (quantitative yield) of the diethylammonium salt of N- tritylurocanic acid was recovered.
50 2.2.2.3.2 Preparation of N-tritylurocanic acid
The diethylammonium salt of N-tritylurocanic acid (408.3 mg, 0.9 mmol) was dissolved, with heating, in ethanol (15 ml), and mixed with distilled water (15 ml). A pellet of NaOH (52.1 mg, 1.3 mmol) was introduced to displace diethylamine, which was subsequently expelled under mild vacuum. The solution was acidified to a pH of approximately 5, by the dropwise addition of glacial acetic acid. Thereafter, excess NaCl was added to induce precipitation of the free acid. The product, which was recovered as a white powder (270 mg, 79 % yield) after vacuum filtration, was rinsed with distilled water and dried for
approximately 12 hours in a drying pistol. mp: 218 – 220 ˚C. TLC: Rf = 0.1 (silica gel 60F254 plates in CHCl3:MeOH, 95:5, v/v).
2.2.2.3.3 Preparation of the N-hydroxysuccinimide ester of N-tritylurocanic acid
N-tritylurocanic acid (250 mg, 0.66 mmol), DCCI (135.8 mg, 0.66 mmol) and NHS (75.7 mg, 0.66 mmol) were dissolved in dry DMF (3.5 ml) and maintained at room temperature. TLC was performed on silica gel 60F254 plates in CHCl3:MeOH (95:5, v/v), in order to monitor product formation (Rf = 0.84). The active ester was observed as a purple spot after spraying the plate with a mixture (2.3:1, v/v) of 14 % (w/v) hydroxylamine hydrochloride and 3.5 N NaOH; and subsequently, FeCl3 in 1.2 N NaOH (5 % w/v). After 5 days, the solution was filtered to remove DCU crystals, and concentrated in vacuo. The residue was then dissolved in warm isopropanol, and stored at 4 ˚C overnight, whereupon the product was deposited as a film. The supernatant was discarded and the film dried under vacuum, first using a rotary evaporator and subsequently a drying pistol, to afford 261 mg of product as white, amorphous solids (83 % yield). mp: 99 – 103 ˚C.
2.2.2.3.4 Preparation of N-tritylurocanylcholesterylformylhydrazide (SH05)
SH01 (46.7 mg, 0.1 mmol) and the N-hydroxysuccinimide ester of N-tritylurocanic acid (50 mg, 0.1 mmol) were each dissolved in 0.5 ml CHCl3. The solutions were combined and stored in the dark, at room temperature. The reaction was monitored by TLC on silica gel 60F254 plates developed in CHCl3:MeOH (98:2, v/v) and, after 7 days, product was recovered
51 via preparative TLC. Briefly, the reaction mixture was applied to four 10 cm × 20 cm TLC plates which were developed in the above-mentioned solvent system. The TLC plates were viewed under ultraviolet light, so as to locate the position of the product. Silica gel at this position was scraped off, the product extracted into EtOH with heating, and the eluent evaporated to dryness under vacuum. This afforded 45.9 mg (54 % yield) of product as a white powder, with mp of 124 – 127 ˚C, and Rf of 0.39.
2.2.2.3.5 Detritylation of SH05
SH05 (approximately 40 mg) was dissolved in 500 µl each of CHCl3 and AcOH, and maintained at 37 ˚C for 8.5 hours. Samples were periodically applied to silica gel 60F254 plates which were developed in CHCl3:MeOH (95:5, v/v), in order to monitor the removal of the trityl group, whereupon the solution was evaporated to dryness. The released tritanol was extracted twice into 5 ml petroleum ether (60 – 80 ˚C), and the crude product deposited as a white powder after rotary evaporation. This was dissolved, with heating, in a mixture of CHCl3 and MeOH (undefined ratios); and purified on two 10 × 20 cm silica gel 60F254 TLC plates, which were developed in CHCl3:MeOH (85:15, v/v), to afford 16 mg (57 % yield) of SH04. mp: 232 – 235 ˚C; 227 – 228 ˚C (decomp.). Rf = 0.41.
2.2.2.4 Spectral analyses
1H and 13C NMR spectra were obtained on a Varian Gemini 300 instrument (Varian Inc., Palo Alto, CA.) at 300 MHz and 75 MHz respectively. 1H chemical shifts were recorded relative to C5H5N (8.57 ppm) or CHCl3 (7.24 ppm) and 13C chemical shifts relative to C5H5N (135.5 ppm). Abbreviations for signal multiplicities are as follows: s (singlet), d (doublet), t (triplet), m (multiplet). Chemical shifts are reported for 1H spectra as: shifts (multiplicity, integration, coupling constant, coupling assignment). Mass spectra were obtained on a Bruker ESI-(Q)TOF instrument operating in positive mode. Infrared (IR) spectra were obtained on a Nicolet Impact 420 spectrophotometer using a KBr disc technique (0.5 % dilution).
NMR and mass spectra are given in Appendices 1 and 2, respectively. Note that naming of both cholesterol derivatives and intermediates in their synthesis, according to IUPAC
52 nomenclature, is listed in Appendix 4. This was performed using ChemAxon software
accessed via http://www.chemicalize.org.