HPLC purification

Another major advantage of using HH.C is that solvent additives can be used which help to suppress the formation of cation adducts. Apffel and co-woikers reported the use of hexafluoro-2-propanol (HFIP) for ESI-MS analysis of oUgonucleotides up to 74 bases long [17], Addition of HFIP to a water/methanol gradient allows good HPLC separation of oUgonucleotides and efficient electrospray ionisation with a minimum of cation adducts formed. 

HPLC solvents were prepared from a stock solution of aqueous 0.8M HFIP, pH 6.8 (adjusted with triethylamine), diluted to 0.4M with water for solvent A and methanol for solvent B [10 17], The gradient generally used for SOMA analysis was 60% A 40% B programmed to 40% A 60% B in 5 mins, returning to the initial conditions in OA mins. 

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This group, used for 5 -phosphate protection, has hydrophobicity similar to that of the dimethoxytrityl group and thus was expected to assist in reverse-phase HPLC purification of product from failure sequences in oligonucleotide synthesis. The group is cleaved with Bu4N F in DMSO at 70°. ... 

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

Another set of studies on allergy and inflammation consists of a direct determination of PAF-acether after HPLC purification of human blood extracts. PAF-acether... 

In the current era many medicinal chemists are unaware of the very important role of compound soUd state properties on aqueous solubility and therefore to oral absorption. In many organizations compound purification by crystallization has disappeared being replaced by automated reverse-phase HPLC purification. If medicinal chemists isolate a compound as a white powder from evaporation of... 

HPLC purification generated the final compounds represented by 18 (mixture of diastereomers) and 19. 

Reaction of hydroxyketone 308 with Lawesson s reagent afforded dithiaphos-pholane 309 as a single P-diastereomer in 52% yield after HPLC purification (Scheme 73) [110]. 

In order to investigate whether the above glycerides are derived from the animal s diet or by biosynthesis, [2-14C]RS-mevalonic add DBED salt was injected into the digestive gland of A. montereyensis and A. odhneri spedmens, which were placed in a running sea-water aquarium for 24 h and then extracted [79]. Incorporation of the label was checked in the major metabolites of the two nudibranchs, namely 23 and 51 in A. montereyensis and 47 in A. odhneri, after HPLC purification and conversion to derivatives that were also purified via... 

The tritium-labelled 9-cri-retinoic acid [11,12-3H2], 96, the natural ligand for retinoid X receptor (RXR)83, has been produced84 by small-scale photoisomerization of all-trans-retinoic acid [11,12-3H2(N)], 97, followed by HPLC purification (equation 37). 

The two major approaches for HPLC purification are fast gradient separation and parallel purification. Yan et al.178 utilized the former (Figure 1.51). The purification lab received a 96-well plate containing synthesized products at 0.1 to 0.2 mmol/well. A Hydra 96-probe liquid handler prepared QC plates for all samples that were analyzed with a MUX-LCT eight-channel parallel LCMS instrument at a throughput of 2000 samples/day. Only samples with purities above 10% were purified on a... 

Preparative reversed-phase HPLC purification was carried out using Gilson liquid handlers and HPLC equipment controlled by Unipoint Version 3.2 software. Initial and final HPLC gradient... 

After removal of the protein by ultrafiltration, the reaction mixture was subjected to semi-preparative HPLC. Purification was performed using a /iBondapak NH2 column (4.0 mm X 150 mm) with the following conditions CH3CN/H20,5 mL min , 25 °C injection volume 10 pL, 4 mg. 

Cheney et al. (1995) analyzed steroids by coupling an HPLC purification step with GC/MS. The steroids were initially characterized by their HPLC retention times compared with the retention times of tritium-labeled recovery standards. Next, the nemosteroids were characterized by their GC retention times. Finally, they were identified by their unique fragmentation spectra following derivatization with heptafluorobutyric anhydride or methoxyamine hydrochloride. For structmal identification, the mass spectra were compared to appropriate reference standards. This approach is highly specific, and its sensitivity is increased by the use of SIM. The detection limit for measuring allopregnanolone achieved in the 1995 study was 0.63 pmol (0.2 ng) starting from 100-300 mg of brain tissue. 

The synthetic route should aim at incorporating the label as late as possible in the sequence. This requires the development of rapid syntheses (generally not more than 3 h for compounds) including HPLC purification and formulation of the radiopharmaceutical for intravenous injection. The large amounts of reagents compared to those of the labelled substrate [20] usually lead to rapid reactions. However, unexpected labelled compounds can also arise from side reactions of reagents in excess or from reactive impurities present in the reaction medium [21]. 

The substitution of a hydrogen for a fluorine-18 has been widely used. Direct radiofluorination of l-DOPA [68] (3,4-dihydroxyphenyl-L-alanine) yields the three possible stereoisomers [2- F], [5- F] and [6- F]fluoro-L-DOPA in 12,1.7 and 21 % yields respectively (Scheme 6). A complex HPLC purification leads to the preferred [6- F]fluoro-L-DOPA in 3 % radiochemical yield. 

Three-component treatment of ketosulfones and related CH-acids with aldehydes and 5-aminopyrazoles was also patented by Han and Hu [74]. They used stirring of the starting materials in THF at 70°C and HPLC purification to synthesize biologically active pyrazolopyrimidines containing sulfonic group. 

This general procedure was applied to a variety of scaffolds to produce a small library of compounds. After the work-up and preparative HPLC, the final yields were in the range of 40-63%, which is comparable to the previously reported batch procedures. A typical 35 min run finally gave 0.5 mmol of the product that afforded 40-80 mg after the HPLC purification this accounts to approximately 80-160 mg/h productivity. 

N02-dG was achieved by the selective photodissociation of persulfate anions that generate 003 and N02 by one-electron oxidation of bicarbonate and nitrite anions in solution. " The 03 site-selectively generates G(—H) in the 11-mer that combines with N02 to yield the 8-N02-dG-adducted strand in 3% following HPLC purification. The nitrated 11-mer was stable at 4°C for 4 days, but depuri-nated at room temperature at pH 7 with a half-life of 20 h. " ... 

F. Fuchtner, P. Angelberger, H. Kvaternik, F. Hammerschmidt, B. Peric-Simovc, J. Steinbach, Aspects of 6-[ F]fluoro-L-DOPA preparation Precursor synthesis, preparative HPLC purification and determination of radiochemical purity, Nucl. Med. Biol. 29 (2002) 477-481. 

O-Acylated Hmb is stable to acidolysis by TFA giving a useful additional feature to Hmb-containing peptides. Side-chain protection and the V -protection, if it is first replaced with Boc before Hmb O-acetylation, can be removed and backbone protection retained. After deacetylation of Hmb with aqueous hydrazine the peptide can be further purified before removal of the Hmb groups, useful when the product is poorly soluble. This feature is also useful for the purification of large polypeptides as the presence of backbone protection on the side-chain deprotected peptide prevents the formation of relatively stable folded structures that can complicate HPLC purification.1 11... 

The monomeric peptides [Cys-I], [Cys,Cys(Acm-II], [Cys(Acm),Cys-III], and [Cys-IV] were synthesized by Fmoc chemistry on Rink-amide resin as 5-Mob derivatives and cleaved/deprotected with 1M TMSBr/thioanisole in TFA in the presence of m-cresol and 1,2-ethanedithiol as scavengers. Following gel filtration on Sephadex G-10 with 1M AcOH as solvent and HPLC purification the peptides were obtained in 30—40% yield. Each product was characterized by LSIMS, HPLC, and amino acid analysis. 

Since this postsynthetic, chemoselective lipidation procedure bypasses all the difficulties encountered during preparative HPLC purification of lipopeptides, the a-hydrazinoacetyl peptides were further developed into a strategy of lipidation by chemoselective hydrazone formation between lipophilic glyoxaldehyde vectors and prepurified hydrazino peptides (Scheme 10).11241 The hydrazino handle can be located at the N-termini or even at preselected lysine side-chain positions. Various lipophilic vectors were proposed containing one or more fatty acids coupled via amides to spacers which in turn are linked to glyoxylic acid via an amide bond.1124-1271... 

To a soln of the linear peptide in DMF (1.5 x 10 mol-L-1), DPPA (3 equiv), and NaHC03 (5 equiv) were added and the soln was stirred for 1 h at rt. After completion of the reaction (monitored by HPLC), NaHC03 was filtered off. A few drops of H20 were added to the soln to hydrolyze excess DPPA and the mixture was concentrated, the residue dissolved in tBuOH/H20 (or dioxane/H20) and lyophilized. The protecting groups were removed after optional HPLC purification and the cyclic peptide purified and characterized. 

The preparation of conjugated dienes from pyridines is exemplified by the transformation of 2-picoline into the sex pheromone (669) of Lobesia botrana, a major pest of vineyards (Scheme 154) (80TL67). Thus, the lithio salt of 2-picoline was alkylated by 2-(5-chloropentyl-oxy)tetrahydropyran, the resulting pyridine (665) N-methylated, and the pyridinium salt reduced by sodium borohydride. Quaternization of the 1,2,3,6-tetrahydropyridine (666) and Hofmann elimination gave the (7 , 9Z)-undecadien-l-ol (667) as the sole isomer. Protection of the alcohol and treatment of the corresponding ammonium salt (668) of the amine with lithium dimethylcuprate gave pure (669) after hydrolysis, acetylation and HPLC purification. 

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