POLITICA DE PRECIOS Y DESCUENTOS
SEGMENTOS DE MERCADO
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Silica gel 60 F254 plates from Merck (Darmstadt, Gennany) were used for thin layer chromatography. Detection was achieved by using UV -light and/or by dipping the
plate into a solution containing 88 % ethanol, 1 0 % water and 2 % conc. sulphuric
acid followed by heating with a heat gun. Compounds containing a free amine could be developed to a purple colour by dipping the plate into a solution of 200 mg of
ninhydrin in 94 mL n-butanol, 4 mL water and 2 mL glacial acetic acid followed by
heating.
Flash Chromatography
Silica Gel 60, 0.04-0.06 mm (230-400 mesh ASTM) from Scharlau Chemie S .A.
(Spain) was used for flash chromatography.
High Performance Liquid Chromatography (HPLC) :
The purification of peptides and glycopeptides was carried out on a Philips PU 4 1 00 liquid chromatograph connected to a PU 4 1 1 0 UV/VIS detector using a Phenomenex Jupiter C 1 8 column (5).!, 250 x 1 0 mm). Peaks were monitored at A. 2 1 4 nm.
Kinetic investigations were carried out on a Dionex Summit HPLC system connected to a UVD-340S Photo Array Detector and a RF 2000 Fluorescence detector using a Phenomenex Jupiter C I 8 column (5).!, 250 x 4.60 mm). Peaks were monitored at A. 2 1 4 nm on the UV detector. The excitation wavelength for the fluorescence detector was 495 nm and the emission wavelength was 520 nm.
Nuclear Magnetic Resonance (NMR)- Spectroscopy:
All I H-NMR and I JC NMR spectra as well as 2D experiments were recorded on a Bruker Avance 300 spectrometer. The proton frequency is 300. 1 3 MHz and that for DC is 75 .47 MHz . The Avance 300 is equipped with a Quad Nuclei Probe and the sample temperature is normally 3 0 QC. If necessary l H- 1 H-COSY, I H- 13C-HMQC and DEPT - experiments were perfonned to achieve the full assignment of signals. Nuclear Overhauser effect spectroscopy (nO e) was used to detennine the stereochemistry of the C-glycopeptides.
Chapter 8 -Materials and Methods 1 2 1
Electrospray M ass Spectrometry (ES-MS)
Electrospray mass spectrometry was carried out on a Perkin-Elmer API 3 00 ES-IMS.
Solid Phase Peptide Synthesis er:
Solid phase peptide synthesis was carried out on an Applied Biosystems Peptide Synthesiser Model 43 1 A using Rink® resins and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry.
Solvents
All solvents used for chemical reactions were obtained from Aldrich® in Sure/Seal ™
bottles. Solvents for recrystallisations and work-up procedures were reagent grade from various suppliers. Solvents used for column chromatography were distilled in our laboratory prior to usage. HPLC-grade acetonitrile was used for HPLC
chromatography. Water used was distilled and filtered by a MilE Q® system.
Reagents
All chemicals used were reagent grade obtained from various suppliers (mainly Aldrich®, Acros Organics and Lancaster).
Microanalysis
Microanalytical investigations were carried out by the Campbell Microanalytical Laboratory in the Department of Chemistry at the University of Otago, N ew Zealand! Aotearoa.
Chapter 8 - Materials and Methods 1 22
8.2 General Procedures
Solid Phase Peptide Synthesis
For the solid phase peptide synthesis 0.42 g of Rink® resin ( loading 0.6 mmol/g) was reacted with 1 mmo! of each amino acid using Fmoc-chemistry. The peptide chains were assembled from the C-to the N-tenninus. The peptides were cleaved from the resin with a solution of 9.S mL TF A and O.S mL water. After filtration and washing of the resin with chloroform, the solvent was evaporated using a rotary evaporator, the residue taken up in water and the solution washed twice with cold ether. After lyophilisation the peptides were purified on a Phenomenex Jupiter Cl 8 column (S/-!, 2S0 x 1 0 mm) at a flow rate of 3 mL Imin. A linear gradient was employed over l h, with initial conditions 1 00 % water (0. 1 % TF A) and final conditions 20 % water (0. 1
% TFA) and 80 % acetonitrile (0.08 % TFA). Peaks were monitored at Iv 2 1 4 nm . The
peptides were dissolved and injected as solutions in DMF.
Solution Phase Peptide Synthesis
Solution phase peptide synthesis was carried out using N-t-butoxycarbonyl (Boc) chemistry with dicyclohexyl carbodiimidel l -hydroxybenzotriazole activation in DMF. The general protocol was as follows: the carboxyl-protected amino acid component ( 1 .0 equiv.) and the Boc-protected carboxyl component ( 1 .S equiv.) were coupled in DMF at 0 QC using HOBt ( 1 .S equiv.) and DCC ( 1 .6 equiv.). After 1 2 h of stirring at room temperature the DCU (dicycIohexylurea) was filtered off and the solvent evaporated. The residue was taken up in chloroform and washed with sat. (saturated) sodium hydrogen carbonate solution, brine and water. After drying over sodium sui fate, the residue was subjected to column chromatography using silica gel.
Boc-Group Cleavage
For the solution phase peptide synthesis Boc-groups were cleaved using 4 M
hydrochloric acid in l ,4-dioxane at 0 QC following a procedure previously described by Wang et al. [S] .
Chapter 8 - M aterials and Methods
Synthesis of ,B-Glycopyranosylamines
,B-Configured glycopyranosylamines were synthesised following the procedure of Likhosherstov et al. [ 1 1 9] .
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The deacetylated glycoside was dissolved in sat. ammonium hydrogen carbonate solution and the solution left standing at r.t. (room temperature) for 24 d. If the reaction flask is sealed, the reaction does not go to completion. A practical method was to close the flask with a rubber septum into which was then inserted a Luer-lock needle. The progress of the reaction was monitored by TLC (CHCh :MeOH, 1 : 1 , ninhydrin stain) and upon completion (very minor traces of starting material might still be detectable) the solution was repeatedly lyophilised to remove the ammonium hydrogen carbonate. The resulting compound was used without further purification.
Synthesis of Glycopeptide Mimetics
The coupling reaction between the free amino-group of the glycoside building block and the carboxy group of the side chain in aspartic or glutamic acid in pre-assembled peptides was carried out using a method based on that of Anisfeld and L ansbury [ 1 1 7]
and S .Y.c. Wong et al. [ 1 1 8 ] : 1 . 5 eq. (equivalent) of the glycoside and l eq. of the
peptide component were coupled in DMSO/DMF at r.t. using 3 eq. HOBt, I eq. 2- ( I H-benzotriazol- l -yl)- 1 , I ,3,3-tetramethyluronium hexafluorophosphate (HBTU) and
1 eq. N,N-diisopropylethylamine. The reaction was monitored by HPLC (for
conditions see below). After completion (typically 4 h) the glycopeptide was isolated
from the reaction mixture by RP HPLC (Phenomenex Jupiter e I 8, 5j.t, 250 x 1 0 mm)
at a flow rate of3 mL *min- I . The following method was used to elute the product
(solvent A: I 00 % water, 0. 1 % TFA; solvent B : MeCN, 0.08 % TFA): 1 ) isocratic
flow of 1 00 % A for 5 minutes, 2) linear gradient: 1 00 % A � 65 % A and 35 % B
over 3 0 min.
Method for the HPLC-based Discontinuous Assay using FITC-Ova
Kinetic investigations using FITC-ova as the substrate were carried out on a Dionex Summit HPLC system connected to a RF 2000 Fluorescence detector using a
Phenomenex Jupiter C 1 8 column (5j.t, 250 x 4.60 mm). The excitation wavelength was
Chapter 8 - Materials and Methods 1 24
used (solvent A : 1 00 % water, 0. 1 % TFA; solvent B: MeCN, 0.08 % TFA): 1 ) 80 % A and 20 % B � 60 % A and 40 % B over 1 5 min. 2 ) 60 % A and 40 % B � 30 % A and 70 % B over 1 0 min. 3) 30 % A and 70 % B � 80 % A and 20 % B over 1 0 min. The flowrate was 1 mL *min-l .
Method for the HPLC-based Discontinuous Assay using Synthetic N
Glycopeptides
Kinetic investigations using synthetic N-glycopeptides as the substrate were carried out on a Dionex Summit HPLC system connected to a UVD-340S Photo Array Detector using a Phenomenex Jupiter C l 8 column (51-!, 250 x 4.60 mm). Peaks were
monitored at ').. 2 1 4 nm. The following linear gradient was used (solvent A: l OO %
water, 0. 1 % TFA; solvent B : MeCN, 0.08 % TFA): 1 ) isocratic flow of 1 00 % A for 2 min., 2) 1 00 % A � 60 % A and 40 % B over 20 min., 3) 60 % A and 40 % B �
Chapter 8 -Materials and Methods