Amino acids 9-Fluorenylmethyl chloroformate

A derivatization with acid chlorides is also possible. Amino acids can be derivatized with 9-fluorenylmethyl chloroformate (FMOC) and separated on a CSP with X-cyclodextrin (ChiraDex gamma ), a cyclic oligosaccharide which consists of eight glucose units. 

Bartok, T., Szalai, G., Lorincz, Zs., Borcsok, G., and Sagi, F., High-speed RP-HPLC/FL analysis of amino acids after automated two-step derivatization with o-phthaldialdehyde/3-mercaptoproprionic acid and 9-fluorenylmethyl chloroformate, /. Liq. Chromatogr., 17, 4391, 1994. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Amino acids derivatized with 9-fluorenylmethyl chloroformate (90) were separated by CE and determined by LIF with a pulsed laser LOD 0.5 nM (SNR 2)322. A sensitive technique for amino acids is capillary zone electrophoresis (CZE) combined with LIF of their fluorescein isothiocyanate (133) derivatives. Not all amino acids give good resolution. LOD for proline and arginine were 0.3 and 0.5 nM, respectively323. 

Bank RA, Jansen EJ, Beekman B and Te Koppele JM (1996) Amino acid analysis by reverse-phase high-performance liquid chromatography improved derivatization and detection conditions with 9-fluorenylmethyl chloroformate. Anal Biochem 240, 167-176. 

C. Clapp, J. Swan, andj. Poechmann,/. Cbem. Educ. 69, A122-A126 (1992). Using 9-fluorenylmethyl chloroformate and HPLC to identify amino acids. 

PA Haynes, D Sheumack, LG Grieg, J Kibby, JW Redmond. Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate. J Chromatogr 588 107-114, 1991. 

J Kirschbaum, B Luckas, WD Beinert. Pre-column derivatization of biogenic amines and amino acids with 9-fluorenylmethyl chloroformate and heptylamine. J Chromatogr A 661 193-199, 1994. 

K Ou, MR Wilkins, JX Yan, AA Gooley, Y Fung, D Sheumack, KL Williams. Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate. J Chromatogr A 723 219-225, 1996. 

DT Blankenship, MA Krivanek, BL Ackerman, AD Cardin. High-sensitivity amino acid analysis by derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection applications in protein structure determination. Anal Biochem 178 227-232, 1989. 

B Carratu, C Boniglia, G Bellomonte. Optimization of the determination of amino acids in parenteral solutions by high-performance liquid chromatography with precolumn derivatization using 9-fluorenylmethyl chloroformate. J Chromatogr A 708 203-208, 1995. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

A second amino protecting group, the 9-fluorenylmeth xycarbonyl protecting group, abbreviated as Fmoc, is formed by reacting the amino acid with 9-fluorenylmethyl chloroformate in a nucleophilic acyl substitution reaction. 

It was soon realized that major side products in this reaction are the corresponding Fmoc dipeptides (and even tripeptides) at extents of 1-5%, occasionally up to 14%. This side reaction occurs via a multistep mechanism involving, (1) Fmoc protection (2) formation of a noixed anhydride with excess 9-fluorenylmethyl chloroformate (17) and (3) peptide coupling with unreacted amino add.f " The formation of Fmoc dipeptides is particularly favored with nonhindered amino acids, e.g. in the case of glycine 14.4% dipeptide and 0.2% tri-... 

Scheme 2 Fluorenylmethyl Chloroformate and the Fmoc/Amino Acid Mixed Anhydride...

P. A. Haynes, D. Shenmack, L. G. Greig, J. Kibby, and J. W. Redwood, Applications of antomated amino acid analysis using 9-fluorenylmethyl chloroformate, J. Chromatogr. 588 107 (1991). 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

Pre-column derivatization—RPC analysis of phenylisothiocyanate (PITC), o-phthaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), or other derivatives of amino acids with UV or fluorescence detection is most common. This is the preferred methodology for life science research because of its higher sensitivity. More examples of precolumn derivatization of amino acids are shown in the life science section of this chapter. 

The solubility of amino acids in CO2 modified with methanol, water and methylamine was increased sufficiently for SFC on columns packed with silica by deri-vatizing with 9-fluorenylmethyl chloroformate (FMOC) [16]. Valine, alanine, phenylalanine, lysine and stTine were separated (Figure 4) within 40 minutes. Derivatiza-tion with (-b)-l-(9-fluorenyl)ethyl chloroformate (FLEC) allowed separation of enantiomers more rapidly than by HPLC but with similar selectivity. 

Koros, A., Varga, Z., Molnar-Perl, I. (2008). Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese hy high-performance hquid chromatography. Journal of Chromatography A, 1203,146-152. http //dx.doi.Org/10.1016/j.chroma.2008.07.035. 

Derivatization of sarcosine with 9-fluorenylmethyl chloroformate (FMOC) facilitates detection via fluorescence, but unfortunately the sarcosine derivatives are among the least stable of the amino acid derivatives formed this way, and the instability can lead to peak broadening. 

An alternative protecting group is known as Fmoc it is formed by treating an amino acid with 9-fluorenylmethyl chloroformate. 

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