Fmoc loading determination

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... 

Four aminomethyl MicroTubes (note 1) immersed in DCM (4mL) were treated with Fmoc-Cl (0.104 g, 400 pmol note 2) and DIEA (0.14 mL, 800 pmol). The reaction mixture was shaken (note 3) at room temperature for 2h. After the supernatant was removed by aspiration, the MicroTubes were washed with MeOH, DCM, and ethyl ether (note 4) and dried under vacuum for 24 h. Each MicroTube was then treated with 2 mL of 20% piperidine in DMF at room temperature for 2h. An aliquot (20 pL) of the solution was diluted to 1 mL with 20% piperidine in DMF. The loading was determined by measuring UV absorption of the... 

Pathway C seemed to be especially attractive, because it should enable addition of acyl anion equivalents to a large number of readily accessible activated carboxylic acids (Figure 3.6.10). Thus diversity in all relevant positions should be readily attainable. High-loaded triphenyl phosphine resin 12 (1.6 mmol g-1) was alkylated with bromoacetonitrile under the action of microwave irradiation yielding phos-phonium salt 13 quantitatively. 13 was converted into stable ylide 14 by treatment with tertiary amine. Carboxylic acids were activated in the presence of N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC) and reacted with 14 yielding acyl cyanophosphoranes 15. The reaction was monitored by ATR-IR coupling yields could be determined by spectrophotometric Fmoc-determination and were 90% for Fmoc-phenylalanine as reference amino acid. 

Fmoc determination (1) Weigh duplicate samples of 5 to 10 mg loaded resin in an Eppendorf tube and add 1.0 mL 20% piperidine/DMF, stir for 20 min, and centrifuge down the resin. (2) Transfer 100 pL of the above solution to a tube containing 10 mL DMF and mix. (3) Set the spectrophotometer at 301 nm. Transfer 2 mL DMF into each of the two cells of the spectrophotometer (reference and sample cells), set the spectrophotometer to zero. Empty the sample cell, transfer 2 mL of the solution to measure, and check absorbance. (4) Substitution of the resin = [101 X (Absorbance)]/[7.8 x (weight in mg)] Check absorbance three times at 301 nm calculate average substitution. 

Fmoc-D-cycloserine (4-aminoisoxazolidine-3-one) and its enantiomer were immobilized on SASRIN resin or 2-chlorotrityl linker resins using Mitsunobu-type reaction or direct tritylation, respectively. The loading of the resulting resins (0.59-0.69 mmol g) was determined by spectrophotometry of the in rr/ -generated piperidine-dibenzofulvene... 

The resin was then filtered off and washed vtith CH2CI2 (2x7 mb), DMF (3x7 mb), and CH2CI2 (3x7 mb) to give the desired product 85. The loading level was determined by Fmoc reading (UV-active piperidine-dibenzofulvene adduct) after Fmoc deprotection -with 20% piperidine in DMF (2 + 20 min). In the next step, coupling of Fmoc-amino acids and 2-fluoro-5-nitrobenzoic acid to the previous amino acid was performed under the same reaction conditions, except for the shorter reaction time (24 h). 

HEM A functionalized crowns are esterified with Fmoc-jS-Ala as follows. All equipment and solid reagents are dried in a desiccator over silica under vacuum for at least 3 h before use. Fmoc- -Ala is activated with 1 equiv. of DIPCDI at a concentration of 0.05 M and 0.01 M DMAP in DMF-DCM (1 3). After activation for 3 min, the solution is added to HEMA crowns (0.15 mL/crown) and reacted for 25 min at 2TC in a constant-temperature bath. The crowns are washed with DMF and DCM, and any excess HEMA hydroxyl groups are capped by acetylation with acetic anhydride-DIEA-DMF (5 1 50), 0.15 mL/crown, for 90 min at room temperature. The average loading per crown of Fmoc-/8-Ala is determined in triplicate using quantitative Fmoc analysis and is found to be typically 1.5 jumol, with a variation of -0.1 /ixmol. 

The Fmoc group, when present on the resin or introduced via a derivatized amino acid (usually Fmoc-Nle or Fmoc-Gly) esterified to the resin, can be quantitated to determine the resin loading. Removal of the Fmoc group by... 

The linker was synthesized from Merrifield resin or carboxypolystyrene and a DMT-protected diol (Scheme 2). The cleavage of the resulting DMT-protected Unker allowed for UV quantitation of the resin loading [22]. The linker can also be installed directly from the diol [23]. The loading can be determined by Fmoc quantitation [24]. 

Fmoc quantification is a technique used to determine the amount of Fmoc present on the resin using UV absorptions see Chapter 2 for more details). This will give a good estimate of the resin loading. 

In order to determine the quality of the dipeptidyl resin, an Fmoc quantification is carried out to determine the loading of the resin see Chapter 2). 

Determine the loading by Fmoc UV test with the use of Fmoc deprotecting solution A (r Note 20, w Subheading 3.5 and Chapter 2). 

Determine the loading of the resin with the UV Fmoc test (see Subheading 3.5). The average loading for this step is... 

The resin is dried under vacuum and an Fmoc quantification is performed to determine loading. 

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