CHARACTERISATION PROCEDURES AND INSTRUMENTATION

n.m.r. spectra were recorded on the Varian VXR 400 (400 MHz), AMX 400, Bruker AC 300 (300 MHz) and AVANCE 500 instruments, using CDCI3, CD3OD

or (CD3)2S0 2 as solvents. The chemical shift data for each signal is given in units

o f 6 relative to tetramethylsilane (TMS); 6 (TMS) = 0. The multiplicity o f the

signals is denoted as : s - singlet, d - doublet, t - triplet, dd - doublet o f doublets, dt - doublet o f triplets, m - multiplet, br s - broad singlet, etc..

*^C n.m.r. spectra were recorded on the Varian VXR 300 and 400 (75 MHz, 100 MHz) instruments using CDCI3 as solvent. The chemical shift data for each signal is given in units o f 5 relative to TMS; Ô (TMS) = 0.

Mass spectra were recorded either on a VG ZAB SE machine (E.I. and F.A.B. ionisation) or a Micromass LC Quattro machine (A.P.C.I.+ and E.SP.+ ionisation.). High resolution mass spectrometry was recorded using F.A.B. on a VG Analytical ZAB SE machine.

Melting points were determined using a Gallenkamp instrument.

Thin layer chromatography (tic) was carried out on pre-coated 0.25 mm thick Merck 60 F2 5 4 silica plates. Visualisation was by spraying with basic potassium

permanganate solution, or basic bromocresol green (to reveal acids), or with ninhydrin solution (to reveal amines), or absorption o f u.v. light. Column chromatography was performed using commercially available silica 60 BDH, and the column diameter is given in cm.

Infra red spectra were recorded on a Perkin-Elmer 1600 FT-IR spectrometer and are in CHCI3 solutions (with KBr cell), unless otherwise stated.

Optical rotations were measured using an Optical Activity:polAAR2000 polarimeter and concentration is expressed in g/100ml. In the experimental section [a]^ values are given in 1 0"^ deg cm^ g"^.

Microanalyses o f all products were determined wherever possible; however, many compounds prepared in this work were viscous oils and their purity was determined by chromatographic and spectroscopic means. Accurate mass spectrometry was also undertaken.

The target test peptide was synthesised on an automated Milligen 9050 Plus

PepSynthesizer using the Fmoc strategy. Amino acids, coupling reagents and resins were obtained from Novabiochem and Perceptive Biosystems. The control peptide was synthesised on a ABI 430A automated synthesiser using the Fmoc strategy

Analytical reverse phase HPLC (flow rate Iml/min) was carried out using a Waters 600E quaternary gradient system pump, Rheodyne 7125 valve injector, Gilson 115 variable wavelength u.v. detector and Hewlett-Packard HP3396 A integrator with Vydac™ C^g column, 4.6 mm x 25 cm. Preparative reverse phase HPLC (flow rate 18ml/min) was carried out using a Waters 600E quaternary gradient system pump, Rheodyne 7125 valve injector, Gilson Holochrome variable wavelength u.v. detector and Waters 745B Data Module with Vydac™ Cjg column, 21.4 mm x 25 cm. Liquid Chromatography-Mass Spectrometry was carried using a Hewlett-Packard HP 1100 for HPLC and Micromass Quattro LC for mass spectrometry. LC-MS was carried out on the Vydac™ C^g column analytical HPLC column. Retention times are given in minutes.

CD spectra were recorded with a Jasco J720 and J600 spectropolarimeters, flushed with nitrogen to improve performance below 200 nm. Solution concentration (test or control peptide in a particular solvent) o f 0.300 mg/ml were prepared by weighing the peptide using a microbalance Mettler Toledo MT5 (sensitivity ± Ipg), and 0,05 cm cell pathlength was used in the far-UV CD region (185-250 nm). The pH meter fitted in the spectropolarimeter determined the pH o f the test peptide in water as 6 and the pH o f the control peptide in water as 5.2.

All CD spectra were reported in terms o f A£=8l-Sr (M"^ cm"^) using a mean

molecular weight (MW=113) p er amino acid residue. The contents o f secondary structure were estimated with the Malik method^®^ using a principle component regression analysis (Plsplus o f Grams/32 suite program, Galactic Industries Corporation) with a calibration data set o f 16 proteins obtained from Hennessey and Johnson.^

The attempted ^H n.m.r. o f the test peptide in CD3OH was carried out on a Varian VMX 600 (600 MHz) instrument.

The qualitative Kaiser test^®^ was carried out as follows: taking a very small sample o f peptidyl-resin into a test tube and adding 4 drops o f bottle A (20 g o f phenol in 5 ml o f absolute ethanol), followed by 8 drops o f bottle B (2 ml o f 1

mM KCN in 98 ml o f pyridine) and 4 drops o f bottle C (1 g o f ninhydrin in 20 ml o f absolute ethanol). The final colour o f the suspension was indicative o f the outcome o f the reaction after the mixture was heated at 100°C for 2 minutes.

Distillations o f solvents and reagents were all done under a dry N2 atmosphere.

THF solvent was dried by distillation from a THF/sodium/benzophenone suspension. DCM was dried by distillation from DCM/calcium hydride or DCM/phosphorus pentoxide suspensions. TEA was distilled from a TEA/calcium hydride suspension. Allyl alcohol was distilled over magnesium turnings activated

by iodine. Lithium bromide was dried at 140°C at 0 .1mm Hg for 14 hours. For the peptide synthesis, either distilled DMF (from a DMF/calcium hydride suspension) or commercially supplied DMF (HPLC grade) from BDH was used. Piperidine, dioxane, acetic anhydride and diisopropylethylamine were all distilled over calcium hydride and collected at their respective boiling points under a dry inert atmosphere. Unless specified all other reagents were commercially purchased and used without further purification. Any required purification was carried out by literature methods.^ ^^

Titration o f «-BuLi was carried out as follows^^^: 0.075 mg (0,394 mmol) o f the titre ( 1 -methyl-2-amido-rgrr-butyIcarbonyl benzene, FW 191.26 g/mol) (Figure 105) was dissolved in 2 ml o f dry THF under nitrogen, and a clear solution was obtained. This was then titrated with a certain volume o f «-BuLi until a final orange colour change o f the solution is observed. The accurate concentration o f the M-BuLi can be determined from the added volume which is equimolar to that o f the titre.

O

Figure 105. The titre reagent used in the titration o f «-BuLi.