Fmoc Assay

This is a very important and well tested method for the quantitative determination of loading of Fmoc protected compounds particularly that of Fmoc (fluorenylmethoxycarbonyl) amino acids on solid support. Fmoc groups can 

Commonly used ionic liquids are A(-alkylpyridinium, (V,A( -dialkylimidazolium, alkylammonium and alkylphosphonium salts. 

To date a number of reactions have been carried out in ionic liquids [for examples, see Dell Anna et al. J Chem Soc, Chem Commun 434 2002 Nara, Harjani and Salunkhe Tetrahedron Lett 43 1127 2002 Semeril et al. J Chem Soc Chem Commun 146 2002 Buijsman, van Vuuren and Sterrenburg Org Lett 3 3785 2001]. These include Diels-Alder reactions, transition-metal mediated catalysis, e.g. Heck and Suzuki coupling reactions, and olefin metathesis reactions. An example of ionic liquid acceleration of reactions carried out on solid phase is given by Revell and Ganesan [Org Lett 4 3071 2002], 

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The poor accessibility of the a-nitrogen and its reduced nucleophilicity due to Np-protection requires strong N -acylating conditions. Coupling assays of Boc-Pro-OH or Fmoc-Pro-OH with H-(ZNH)Gly-OEt under various conditions show that the best yield is obtained with a symmetric anhydride or an acyl chloride.[93 The symmetric anhydride method affords Z-Protp[CO-N(NH2)]Ala-NHiPr (78) in 55% yield (Scheme 22)P1 ... 

A procedure to determine PIR residues in bovine milk using HPLC with the derivatization step for UV detection has also been published (211). The PIR was extracted from milk after protein precipitation and a two-step LLE procedure. The extract was evaporated to dryness, dissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (FMOC). The de-rivatized extract was analyzed by reversed-phase HPLC. Overall recovery was 89%, with 4% for coefficient of variation. A linear regression analysis of HPLC/UV results was compared with the HPLC/MS assay (209,210). The procedure takes about 2.5 hours to complete six or eight samples. Pirlimycin is stable in milk frozen to —60°C or below for at least 3 months. 

Figure 9.140 HPLC elution profile of a sample taken from a discontinuous assay of crude mouse spleen extract, using iV,-fluorenylmethoxycarbonyl-EEY(P)AA [Fmoc-EEY(P)AA] as substrate. Chromatography was performed on a C g Novapak column (10 cm X 8 mm) eluted isocratically with 36% acetonitrile-water (0.1% TFA) (v/v) at a flow rate of 2 mL/min. Peaks are due to (A) methanol and methanol-soluble compounds derived from the sample of crude homogenate, (B) Fmoc-EEY(P)AA (1238 pmol), and (C) Fmoc-EEYAA (195 pmol). Appropriate controls showed that no interfering compounds eluted in the position of the peptides. Inset Fluorescence monitoring of HPLC of Fmoc-EEYAA (75 fmol) eluted isocratically with 36% acetonitrile-water (0.1% TFA). Excitation and emission wavelengths, 268 and 307 nm, respectively, with gain X 100 and 10 mV chart scale. (From Nash et al., 1993.)...

Chan et developed an assay for the simultaneous determination of catecholamines and metanephrines using FMOC derivatization. The assay is convenient for the simultaneous analysis of NM, MN, E and DA in human urine sample without prior extraction procedures. In this study, urine was directly derivatized and subjected to a simple extraction step with... 

A simple assay based on potent and specific inhibition of jack bean a-mannosidase has been devised for determining low concentrations of 162 (up to 0.5 cm ) in M anisopliae cultures (110). The new assay was used to demonstrate that the addition of L-lysine (163) to the culture medium stimulated production of the alkaloid by approximately fourfold. Other early metabolic precursors of 162 in this fungus, including a-aminoadipic acid, saccharopine (164), L-pipecolic acid (165), and L-lysine itself, were quantified by reverse-phase HPLC analysis of mycelial extracts derivatised with 9-fluorenylmethyl chloroformate (FMOC) (111). 

A second technique that accomplishes peptide synthesis on polyethylene rods was developed by Geyson et al.l95 A typical array of rods (called pins) consists of 96 pins (typically 4 mm in diameter and 40 mm in length) 194 arranged in 8 rows of 12 rods (see Figure 10.28b). The pins fit into the wells of plates (see Figure 10.28)194 that were developed for the enzyme-linked immunosorbent assay (ELISA). The polyethylene rods are functionalized with acrylic acid and NP-Fmoc-(3-alanyl-l,6-diaminohexane is attached as a spacer (see Figure 10.28a). The idea is to attached an amino acid to each of the pins, and place the solution containing the reactants into the wells. The pins are dipped into the wells and allowed to react. To deprotect the terminal... 

FMOC-Cl has been used for the determination of amino acids [182,184,185], natural -lactams and their intermediates [186] and certain herbicides [187], among others. Perliminary work has been published on a polyamine assay [183]. 

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