O-Phthalaldehyde with

Phthalimidines (isoindolin-l-ones) can be valuable intermediates for the synthesis of isoindoles and some natural products, and there has been recent interest in the development of simple methods for the direct conversion of o-phthalaldehyde into /V-substituted phthalimidines. Condensation of o-phthalaldehyde with primary aliphatic amines using acetonitrile as solvent gives disappointing yields with a-methylbenzylamine, for example, the yield of the phthalimidine 1 is only 21%. By contrast, treatment of o-phthalaldehyde with a-amino acids in hot acetonitrile gives generally excellent yields of the corresponding phthalimidines. With L-valine, for example, 2 is formed in 87% yield. 

The formation of dialkyl benzo[f]furan-l-yl phosphonates by Lewis acid-promoted reaction of o-phthalaldehyde with trialkyl phosphites has been described (Equation 127) <2006S4124>. 

The precolumn derivatisation is based on the formation of substituted iso-indoles (Fig. 5) on reaction of o-phthalaldehyde with primary amines... 

Scheme 12.37 Divergent reactions of o-phthalaldehyde with N-substituted enaminones and 4-hydroxy-6-methyl-2H-pyran-2-one.

Li has described the preparation of complex pentacyclic systems containing an indeno[2,l-c]quinoline core (compounds 125) by treatment of o-phthalaldehyde with 4-hydroxy-6-methyl-2/f-pyran-2-one and cyclic enaminones [85]. The domino process summarized in Scheme 3.37 was proposed to explain this transformation. 

Dipping solution Make 0.1 g o-phthalaldehyde (phthaldialdehyde, OPA) and 0.1 ml 2-mercaptoethanol (2-hydroxy-l-ethanethiol) up to 100 ml with acetone. 

In the presence of 2-mercaptoethanol o-phthalaldehyde reacts with primary amines to yield fluorescent isoindole derivatives [20] ... 

Another reagent that readily forms fluorescent derivatives with primary amines is o-phthalaldehyde (trade name "Fluoropa"). The reaction proceeds in aqueous solution in the presence of a mercaptan at a pH of 9-11 producing an isoindole. 

A versatile route to 3-benzoheteropines has been reported starting from o-phthalaldehyde, including the first preparations of 3-benzarsepines and the parent 3-benzothiepin and 3-benzoselenepins <96CC2183>. l,7-Dihydro-l//-dibenzo[c,c]tellurepin has been prepared from 2,2 -bis(bromomethyl)biphenyl and potassium tellurocyanate and its complexes with palladium and ruthenium species have been studied, a number of mono- and binuclear complexes are formed <96RTC427>. 

Figure 4.4 Release of amino acids from cortical slices exposed to 50 mM K+. Measurements by HPEC and fluorescence detection after reaction of amino acids with o-phthalaldehyde 1, aspartate 2, glutamate 3, asparagine 4, serine 5, glutamine 6, histidine 7, homoserine (internal standard) 8, glycine 9, threonine 10, arginine 11, taurine 12, alanine 13, GABA 14, tyrosine. Glutamate concentration is almost 1 pmol/gl which represents a release rate of 30 pmol/min/mg tissue...

ATPase with pyridoxal-5 -phosphate [341-344], adenosine-5 -triphosphopyridoxal [99], 2,4,6-trinitrobenzene sulfonate [344-346], fluorescamine [347,348], methylbenzi-midate [349], acetic anhydride and maleic anhydride [344], and o-phthalaldehyde [350],... 

A method to determine nabam by HPLC after acidic hydrolysis to ethylenediamine and post-column derivatization with o-phthalaldehyde-mercaptoethanol has also been reported. " ... 

Benson, J. R. and Hare, P. E., o-Phthalaldehyde fluorogenic detection of primary amines in the picomole range comparison with fluorescamine and ninhydrin, Proc. Natl. Acad. Sci. U.S.A., 72, 619, 1975. 

The reaction of or o-phthalaldehyde and a thiol compound with an amino acid to form an isoindole derivative can be used to enhance the detection sensitivity for the normally only weakly UV-detectable amino acid compounds, and to introduce an... 

Mopper [265] has described developments in the reverse phase performance liquid chromatographic determination of amino acids in seawater. He describes the development of a simple, highly sensitive procedure based on the conversion of dissolved free amino acids to highly fluorescent, moderately hydrophobic isoindoles by a derivatisation reaction with excess o-phthalaldehyde and a thiol, directly in seawater. Reacted seawater samples were injected without further treatment into a reverse-phase high-performance liquid chromato-... 

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

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. 

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. 

A study of residual analysis of thirty pesticides and their transformation products was based on SPE on-line with HPLC-UVD or post-column derivatization with o-phthalaldehyde (73) and fluorescence detection (FLD), according to EPA method 531.1 and others. The method allowed determination of many pesticides in river and well waters at 0.01 to 0.5 -ig/L levels195. An automatized procedure was proposed for determination... 

Latent fingerprints on paper have been revealed by combining the amino acids present with reagents such as ninhydrin (see 37), dansyl chloride (92), fluorescamine (154), 4-chloro-7-nitrobenzofurazan (127a) and o-phthalaldehyde (see reaction 7). To avoid some problems encountered with these reagents it was proposed to use 1,8-diazafluorenone (155), leading to the formation of highly fluorescent ylides (156)349. 

A method for analysis of A-nitroso-A-alkylureas (288b) has been developed by forming fluorescent derivatives with sodium sulfide, taurine (77) and o-phthalaldehyde (73)... 

After extraction, the urethanated films were subjected to alkaline hydrolysis of urethanes to liberate the corresponding amines, while the adipoylated films were hydrolyzed after having reacted with 7-hydroxycoumarin. Amounts of the released amines and coumarin were determined by fluorescence spectroscopy as described in the Experimental section. Since aniline as well as butylamine has no appreciable fluorescence by themselves, their fluorescence assay was made after reacting with o-phthalaldehyde in the presence of mercaptoethanol. In Figure 3, where relative fluorescence intensities are plotted as a function of concentrations of amines and hydroxycoumarin, one can see that the fluorescence intensities vary linearly with their concentration to permit us the quantitative determination of extremely small amounts of amines and hydroxycoumarin. 

Figure 3. Relative fluorescence intensities of n-butylamine and aniline after reaction with o-phthalaldehyde, a-naphthylamine, and 7-hydroxycoumarin (O) aniline (O) n-butylaminej(A) a-naphthylamine (9) 7-hydroxycoumarin...

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Piepponen TP, Skujins A. 2001. Rapid and sensitive step gradient assays of glutamate, glycine, taurine and y-amino-butyric acid by high-performance liquid chromatography-fluorescence detection with o-phthalaldehyde-mercap-toethanol derivatization with emphasis on microdialysis samples. J Chromatogr B 757 277-283. 

Oguri, S., Maeda, Y., and Mizusawa, A. (2004). On-column derivatization-capillary electro-chromatography with o-phthalaldehyde/alkylthiol for assay of biogenic amines. /. Chromatogr. A 1044, 271-276. 

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

---