Fmoc determination

The method described below provides a quick and easy means of obtaining the substitution of any Fmoc-functionalized resin. The theoretical substitution of a peptidyl resin can be calculated from the substitution of the base resin using Equation 1. 

M = molecular weight of target peptide, plus all protecting groups  

Protocol 14. Estimation of level of first residue attachment 

Weigh dry Fmoc-amino acid resin (approx. 1 xmol with respect to Fmoc) into two of the cells. 

Dispense freshly prepared 20% piperidine in DMF (3.00 ml) into each of the three UV cells. 

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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. 

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... 

When AMPA or gluphosinate are determined alone, the sensitivity is higher because a higher dilution is not required. For glyphosate, when the transfer volume is precisely adjusted to 280 p.1 for the FMOC-glyphosate-containing fraction, a limit of detection of about 0.2 p.g 1 can be reached (28). 

Aurora Biomolecules dedicates to peptide synthesis (and polyclonal antibody production) for any small quantity purpose. FMOC chemistry (on Perceptive Biosystems Pioneer instruments) is used for peptides synthesis Online monitoring of the coupling efficiencies and HATU activation helps insure that the major component of the synthesis is the correct oligopeptide. Purification is firstly carried out by size exclusion chromatography, and then by HPLC on a PE vision purification workstation. Typically, 20 mg of pure peptide are obtained. The molecular weight of the purified peptide is determined as a final confirmation of quality. 

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... 

The resin substitution level is based on spectrophotometric determination of the Fmoc-derived chromophore liberated upon treatment with 20% piperidine/DMF using e29o nm = 5253 M 1 cm-1, which was used to calculate the percent efficiency. 

To accurately determine anchoring, coupling, and cleavage yields, resins are extended further with an internal reference amino acid2 (IRAA lie is used), introduced as its Fmoc derivative by standard coupling methods, at a point before introduction of the handle. 

FIGURE 5.23 Synthesis of cyclic peptides by head-to-tail cyclization of resin-bound peptides using Boc/Bzl chemistry59 and Fmoc/tBu chemistry.60 The carboxy-terminal protectors are orthogonal to the other protectors. The nature of the linker determines the nature of the product. Both chemistries are compatible with the two types of linkers. All = allyl. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

An alternative approach is the cleavage of a UV-active protecting group from the resin, such as the widely used Fmoc Test. The quantitation of the 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group for amines is used in SPPS as an indirect method to determine the extent of a peptide coupling reaction. Similar approaches have also been recently reported for the quantitation of supported thiols [151, 154] and have also been the subject of an excellent review [148]. 

Figure 1 Proportion of the Xaa(P)-Deleted Peptide, ASQGLEDPA-NH2, Determined in Crude Products Obtained from the PyBroP/DIPEA, BOP/HOBt/NMM, BOP/HOBt/DIPEA, HBTU/HOBt/DIPEA, HATU/ HOAt/DIPEA, and HATU/DIPEA Coupling of Fmoc-iyr[PO(OBzl)(OH)]-OH (Y), Fmoc-Ser[PO-(OBzl)-(OH)]-OH (S), and Fmoc-Thr[PO(OBzl)(OH)]-OH (T) (3 equiv) for 1 hJ4Sl b...

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