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ANALYTICALBIOCHEMISTRY 178,1-7 (1989) REVIEW Formation and Instability of o-Phthalaldehyde Derivatives of Amino Acids M. C. Garcia Alvarez-Coque,’ and C. Mongay Fernhdez M. J. Medina Hernhdez, Departamento Facultad de Q&mica Anulitica, de Q&mica, Universidad o-Phthalaldehyde reacts with amino acids in the presence of a thiol to give highly fluorescent l-alkylthio-2-alkyl-substituted isoindoles. However, the instability of the derivatives limits the general utility of the reaction. Mechanistic descriptions of isoindole formation and degradation proposed in the last years, which permit a better understanding of the factors affecting isoindole stability, are presented. The use of alternative thiols and o-phthalaldehyde-like reagents is also reviewed. 0 1989 Academic Press. Inc. One of the most sensitive fluorogenic reagents available for the determination of amino acids is o-phthalaldehyde (OPA)’ when used in conjunction with high-performance liquid chromatography. The reagent has some convenient properties: it rapidly forms fluorescent derivatives (h,, = 340 nm, h,, = 455 nm) at room temperature, is nonfluorescent itself, and when present in excess does not break down or react to form fluorescent by-products. However, the derivatives formed are somewhat unstable and this severely limits the general utility of the reaction. The reaction of OPA with amino acids in the presence of 2-mercaptoethanol at pH N 9.5 was first reported by Roth in 1971 (1). Roth considered the possibility that the same species was produced regardless of the nature of the primary amine, as in the case of the ninhydrin reaction. However, differences in quantum yields of the amine derivatives suggested that the same fluorophore was not obtained in each case. In addition, the thiol compound was originally thought to function simply as a reducing agent, but Si’ To whom correspondence should be sent. ’ Abbreviations used: OPA, o-phthalaldehyde; NAC, N-acetyl-Lcysteine; OAB, acetobenzaldehyde; OBB, o-benzoylbenzaldehyde; NDA, naphthalene-2,3-dicarboxaldehyde. 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form Inc. reserved. R. M. Villanueva de Valencia, Camaiias, 46100-Burjassot, Valencia, Spain mons and Johnson showed that the thiol actually becomes part of the final product. Based on data collected from proton NMR, ir and mass spectral analysis of several crystallized OPA-amine-thiol derivatives, these authors concluded that the fluorescent products were l-alkylthio-2-alkyl-substituted isoindoles (I) (2-5). However, long reaction times (15 min), bulky alkyl thiols, and low temperatures of crystallization (OOC)were used to permit precipitation of the fluorescent derivatives and these conditions are not typical of those normally used in OPA-derivatization of primary amines in HPLC applications. Normally, on-line HPLC procedures are carried out over a much shorter period of time (less than 1 min) and at or above room temperature. Therefore, it is not necessarily valid to assume that the fluorescent products, obtained under such vastly different conditions, are the same. For this reason, Simpson et al. (6) performed gas chromatography-mass spectrometry analysis of methylene chloride extracts of postcolumn OPA-derivatives of various aliphatic amines with 2-mercaptoethanol and ethanethiol. The study demonstrated that the fundamental structure of the fluorophore (I) was consistently present in all derivatives: MECHANISM FOR ISOINDOLE FORMATION Simons and Johnson presented a mechanistic description for isoindole formation (5) in which OPA first reacts with 2-mercaptoethanol and subsequently with the amine (Scheme 1). The reaction sequence proceeds by protonation of the imine intermediate (III), followed by a “partial &l-like intramolecular reaction” to give a protonated isoindole (V). However, isoindole formation GARCfA ALVAREZ-COQUE S-R’ + WA R’-SH ET AL. S-R’ B II 6 d I?-NHz SCHEME nobenzylsulfide 3. Alternate (X) (7). mechanism for formation of the a-alkylami- 1-W a S-R’ I V SCHEME mons 1. Mechanism and Johnson for isoindole formation proposed by Si- (5). is known to proceed preferentially in alkaline solutions, where formation of a protonated hemithioacetal (IV) or isoindole (V) would be unfavorable. Sternson et al. proposed an alternative pathway (Scheme 2) (7), where the amine reacts with free OPA to form a carbinolamine (VIII) which then dehydrates to give the highly reactive protonated imine (IX). This, in turn, is rapidly attacked by the thiol anion to form an a-alkylaminobenzylsulfide (X). Intramolecular nucleophilic attack by the resulting secondary amine on the remaining carbonyl group forms the isoindole ring to give the intermediate XI, which undergoes facile dehydration to yield the fluorescent isoindole (I). Operationally the derivatization reaction is indeed conducted by first combining OPA with the thiol and then adding the primary amine. Aqueous solutions of OPA are in equilibrium with the cyclic hydrate (VI) (a), and in the presence of thiols a reversible pathway to the VIII - 2. Mechanism et al. (7). 6 - Hz0 I SCHEME Sternson x IX XI for isoindole formation proposed by cyclic thiol addition product (VII) exists (5)) but neither addition products VI or VII would be expected to be reactive toward a primary amine. In addition, if, as proposed by Simons and Johnson (5), the formation of X proceeds by initial reaction of the hemithioacetal (II) with the primary amine to form the protonated imine (XII) (Scheme 3), this would be followed by attack of the thiol anion to provide the hemithioacetal (XIII), which will rapidly decompose in basic media (9,10), generating X. Further reaction would proceed as shown in Scheme 2. The plausibility of the mechanism was further substantiated by substituting methyl-(o-formyl)benzoate for OPA in the amine-thiol condensation (7). Formation of 1-alkylthio-2-alkylphthalimidine supported the feasibility of thiocarbinolamine (X) intermediacy and the likelihood that cyclization is initiated by attack of the amino moiety of X on an o-carbonyl. In the case of OPA, such an attack would occur at an aldehydic carbon and would be expected to proceed with ease. The final dehydration step (XI -W I) has been previously observed in the synthesis of 1-aryl isoindoles from o-aminomethyl benzophenones (11). FACTORS AFFECTING ISOINDOLE STABILITY Early applications of the fluorogenic OPA reaction included the postcolumn derivatization of amino acids separated by ion-exchange chromatography (12,13). However, this approach must scrupulously avoid impurities in the reagents and mobile phase buffers, which can contribute to high background fluorescence (14). Postcolumn schemes also result in some loss of both resolution and sensitivity due to mixing of the mobile phase with diluent (1516). More recently, precolumn derivatization of primary amines with the OPA-2-mercaptoethanol reagent, followed by reverse-phase HPLC separation, has become popular (14-18). The precolumn approach offers the advantages of improved detection limits, simplification of the chromatographic system, and reduced analysis time. Unfortunately, precolumn OPA approaches present the problem of derivative instability, with glycine, alanine, lysine, and ornithine derivatives being particularly unstable (2,4,5,19-25). Thus, careful timing of the reaction or even instrumental automation is required to ensure acceptable analytical precision. o-PHTHALALDEHYDE DERIVATIVES When isoindoles are generated using equimolar concentrations of all reactants (amine, OPA, and thiol), or when the amine is present in excess, degradation slows drastically. In contrast, addition of excess OPA subsequent to isoindole formation results in rapid degradation (26,27). Cooper et al. (28) indicated that when the OPA-2-mercaptoethanol amino acid derivatives were injected into the HPLC column immediately after the reaction was carried out, they were observed to be stable, including the glycine, ornithine, and lysine isoindoles. Only the diacidic amino acids, aspartic and glutamic, were shown to undergo a significant loss of fluorescence. The reason for the enhanced stability may be the removal of excess OPA from the derivatives during the chromatographic process, or that immobilization of the OPA-2-mercaptoethanol amino acid derivatives on the reverse phase of the HPLC column retards degradation. Several studies on the degradation kinetics of isoindoles suggested that the process occurs via at least two parallel routes, described by a linear dependence of the observed rate constant, kobs,vs OPA concentration (27): hbs = h + MOPAl, where b and kl are the apparent rate constants for the uncatalyzed and OPA-catalyzed processes, respectively. The OPA independent degradation pathway appears unimportant in the presence of a moderate excess of OPA. The destabilization of OPA depends strongly on thiol structure (26). Because $ is much less variable than kl , stability differences related to structural variations may almost solely be due to inhibition of the OPA dependent process (27). In the presence of an excess of OPA, a nonlinear decrease in decay rate with increasing thiol concentration was observed (27,29). This observation is contrary to previous studies in which excess 2-mercaptoethanol was reported to have no effect on derivative stability. In one of these studies (30), however, no excess OPA was present when thiol concentration was varied, so that the primary destabilizing influence was absent. In the second (26), the maximum thiol concentration examined was too low. The observed supression of the rate of OPA decomposition at high thiol concentration is probably due to the formation of one or more OPA-thiol derivatives which decreases the free OPA concentration. Several avenues are therefore available for maximizing isoindole stability when using precolumn OPA-derivatization; the control of thiol or OPA concentration, and the use of an appropriate thiol. Use of a minimum OPA concentration is the most obvious route for in situ stabilization, Very large excesses should indeed be avoided, but the need for rapid and quantitative deriva- OF AMINO 3 ACIDS tive formation imposes some limits, particularly in samples where widely varying substrate concentrations are encountered. Thus, finally, the use of an appropriate thiol seemsto be the best way to achieve stabilization. EFFECT OF THIOL STRUCTURE ISOINDOLE STABILITY ON Jacobs et al. (27) studied a series of 4-aminobutyric acid derivatives and diverse thiols and observed that stability clearly increases with increased branching of the side-chain (Y to the thiol function (-C(CH& > -CH(CH& > -CH2C!HB > -CHB). This trend lends credence to the assumption that steric bulk of the thiol is a dominant factor in determining derivative stability. Mercaptoethanol has become more extensively utilized than other thiols for the OPA-derivatization of amino acids. Several researchers have investigated the possibility of enhancing isoindole stability by varying the structure of the thiol compound, but for some thiols (dithiothreitol, ethanethiol, and 2-methyl-2-propanethiol) the fluorescence intensity achieved is weaker than that obtained with 2-mercaptoethanol, and others result in nonfluorescent compounds (methylmercaptoacetate and mercaptosuccinic acid) (20,22,31). Simons and Johnson proposed ethanethiol as a substitute for 2-mercaptoethanol, due to the improved stability of ethanethiol-amino acid-derived isoindoles (4,22). Recently Stobaugh et al. (32) checked the higher stability of these derivatives. However, ethanethiol is more volatile than 2-mercaptoethanol and one of the major disadvantages of the latter, its pervasive odor, is increased in ethanethiol. Arylthiols (benzylmercaptan, thiophenol, and triphenylmethylmercaptan) (33), which are less malodorous, have also been investigated. However, benzylmercaptan does not produce fluorescent products, thiophenol seems to form a different derivative, and the formation of fluorescent isoindoles from the latter is too slow. Other thi01s proposed are 3-mercaptopropionic acid (34,35) and thioglycerol(36), but neither has received extensive use. Recently, N-acetyl-L-cysteine (NAC) has been substituted for 2-mercaptoethanol in the pre- and postcolumn OPA-derivatization of primary amines and amino acids with good results. Its optical activity enables the separation of enantiomers (37-40). The specific fluorescence of the corresponding OPA-NAC derivatives is nearly identical to that of the OPA-2-mercaptoethanol products, the reaction is rapid, and the NAC derivatives are more stable than either 2-mercaptoethanol or ethanethiol derivatives (including the glycine isoindole). The sensitivity achieved in the analysis of secondary amines is also improved, presumably because the fluorophore is more stable to hypochlorite, which is used for the oxidative cleavage of the imino linkage (41). NAC is commercially 4 available, inexpensive, odor. GARCiA ALVAREZ-COQUE ET AL. and presents no objectionable EFFECT OF AMINE STRUCTURE The stability of OPA-derived isoindoles is known to vary depending on the nature of the amine moiety. Nakamura et ~2. (30) reported qualitative data on the influence of primary amine structure on the stability of the OPA-2mercaptoethanol derivatives. Later, Stobaugh et al. (26) evaluated quantitatively the kinetic stability of several isoindoles. Examination of the isoindole structures and the degradation rate constants revealed slight increases in stability as the N-substituents of the isoindole became larger and when the N-substituent was additionally substituted at C-10. Still greater stability was afforded to the isoindole by the presence of a carboxylate in the amine substrate, with the degree of stabilization increasing as the carboxylate approaches C-10. Thus, (Yamino acids tend to form more stable isoindoles than other amines. Lindroth and Mopper (18) claimed that the stabilizing effect of the carboxylate group of the a-amino acids was due to an electron-donating effect on the isoindole at the C-10 position. However, the results of Stobaugh et al. (26), together with those of other workers (42,43) indicate that increased isoindole stability is predominantly due to steric factors. Jacobs et at. (27) examined the stability of isoindoles where amine structure was varied systematically. The introduction of branch points into the amine side chain has a mashed stabilizing effect on the resulting isoindoles, the magnitude increasing as the branch point approaches the isoindole ring (-C(CH,), > -CH(CH& > -CH&HB > -CH3). Simple linear extension of the side chain without branching results in a steady twofold factor per methylene decrease in decay rate (-CH2CH2CH&H3 > -CH&H&H3 > -CH&Ha > -CHB). The presence of the bulky groups seemed to stabilize isoindole fluorophores, but in some cases it decreased the rate of reaction (t-butylamine or Ly-methyl-L-tyrosine with 2-mercaptoethanol) or the fluorescence response (t-butylamine or cyclooctylamine with 2-mercaptoethanol) (30). Cysteine and lysine form derivatives much less fluorescent than most of the other amino acids. The low fluorescence yield with cysteine is due to its sulfhydryl group, which competes intramolecularly with P-mercaptoethanol for position 1 in the isoindole (44). Cysteine can best be determined if previously converted to cysteic acid, S-3-sulfopropylcysteine or S-carboxymethylcysteine. The quenching with lysine derivatives is apparently due to an interaction between the two isoindole groups since fluorescence enhancement can be achieved in the presence of a detergent such as sodium dodecylsulfate, which favors separation of the isoindole groups (20,45). I ‘I N-R+ kCH,ui~-S-~ cl2 XVI SCHEME 4. Degradation mechanism for isoindoles obtained from P-mercaptoethanol, suggested by Simons and Johnson (5). MECHANISM FOR ISOINDOLE DEGRADATION Simons and Johnson characterized the degradation products of isoindoles derived from 2-mercaptoethanol as N-alkylphthalimidines (XVI) (2) and pointed out two possibilities for their formation: acid hydrolysis (4,22) (decay of fluorophores is accelerated in aqueous solutions at low pH) and intramolecular nucleophilic attack by the hydroxyl group of 2-mercaptoethanol (Scheme 4) (5). The reduced rate of decay of the 2-mercaptoethanol derivatives in borate buffer, which is assumed to complex with hydroxyl groups, compared with phosphate buffer, supports the involvement of the hydroxyl group in the isoindole decay (22). Furthermore, ethanethiol forms more stable derivatives than 2-mercaptoethanol. The identity of the degradation product is consistent with the scheme proposed by Stobaugh et al. (26). However, the degradation mechanism of Simons and Johnson does not account for the acceleration of isoindole degradation by increasing OPA concentration. Nakamura et al. (30) suggested that OPA destabilizes the isoindole ring by acting either as a dienophile or a nucleophile, but based on schemes supposing the formation of degradation products that differ from those previously identified. Furthermore, Stobaugh et al. (26) indicated that neither of these roles is likely: for OPA to function as a dienophile toward the isoindole ring it would be necessary to form a product in which the aromaticity of OPA is lost, an energetically unfavorable process. On the other hand, the aldehyde groups of OPA should exhibit electrophilic character and would not undergo nucleophilic attack on the isoindole ring system. Unlike many other aromatic aldehydes, OPA undergoes extensive hydration to a mixture of cis and trans cyclic 1,3-phthalandiols (VI), approximately 80% of OPA existing in the hydrated form in aqueous solution (8). The occurrence of such facile equilibria and the wellknown ability of thioethers to lend anchimeric assistance in displacement reactions (46), together with some kinetic observations, led Stobaugh et al. to postulate an alternative degradation mechanism (Scheme 5) (26). o-PHTHALALDEHYDE DERIVATIVES OF AMINO ACIDS OH S-R’ + // la 1 S-R’ .N-R via -Hz0 I + 0;. -R 4-R ‘+a S-R’ / I Hz0 xxv XXIV SCHEME proposed 6. Autoxidation mechanism by Stobaugh et al. (48). for isoindole N-R d degradation XIX XVI SCHEME 6. 2-mercaptoethanol, Degradation mechanism for isoindoles suggested by Stobaugh et al. (26). obtained from In the absence of excess OPA, the isoindole (I) forms a sulfonium ion (XVII) via path 1. Attack by water and/ or hydroxide may occur at the methylene positions of the sulfonium ion to regenerate I or at C-l of the isoindole ring system to yield XVI. In the presence of excess OPA, the isoindole (I) may rapidly equilibrate with the hemithioacetal (XVIII) via path 2. The enhanced leavinggroup capability offered by the phthalandiol group in XVIII (over that of the bare hydroxyl group in I) facilitates the formation of sulfonium ion (XVII), resulting in a higher steady-state concentration of this species and therefore an enhanced rate of loss of isoindole (I). The use of 3-mercapto-1-propanol in place of 2-mercaptoethanol in the derivatization reaction provides an isoindole derivative of improved kinetic stability because its degradation would require the formation of a four-membered ring sulfonium ion intermediate and this is a slower process than the formation of the threemembered sulfonium. This mechanism, like that of Simons and Johnson (5) is applicable only to derivatives based on hydroxythiols (like 2-mercaptoethanol). However, the capability of OPA to accelerate isoindole degradation is also present in other thiols. Jacobs et al. (27) suggested that a common rate limiting process should exist, regardless of parent thiol, and since the chemistry of isoindoles is dominated by electrophilic substitutions (47), instability must depend on nucleophilic addition of the isoindole to unreacted OPA. However, the authors did not assign a definite mechanism. Stobaugh et al. observed that 1-alkylthio-2-alkyl-substituted isoindoles apparently react similarly to other non-thio-substituted isoindoles with respect to autoxidation (32,48), with isoindoles derived from hydroxythi01s (where degradation occurs by nonoxidative processes) being a specific case. The authors isolated and identified four degradation products (XXIII, XXIV, XXV, and XXVII), where the thio-substituted N-alkylphthalimidine (XXV) is the major product. An autoxidation mechanism that accounts for three of the observed degradation products was also proposed (Scheme 6). The extensively delocalized cation radical (XX) is formed by electron transfer to oxygen or other radical species present, thus initiating a chain process. Subsequently, the radical XX reacts with oxygen to form a key intermediate, the isoindoyl peroxy radical (XXI), which can further react by pathways previously postulated for isoindoles, including endoperoxide formation (XXII) (49). The nonoxidative degradation product (XXVII) may be derived from reaction of thiyl radicals (produced during the oxidative process described in Scheme 5) with intact parent isoindole, via a homolytic substitution process through intermediate XXVI (Scheme 7). NEW OPA-LIKE REAGENTS Other reagents have been proposed as substitutes for OPA. The minimal structural requirement necessary for SCHEME from 7. isoindoles Formation (49). of a nonoxidative degradation product 6 GARCiA ALVAREZ-COQUE a reagent to condense with a primary amine and a thiol to form an isoindole is the presence of an o-diacylbenzene grouping in which at least one of the carbonyl groups is aldehydic. o-Ketobenzaldehydes such as acetobenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB) undergo such condensation reactions to yield 1,2,3-trisubstituted isoindoles, which are more resistant to autoxidation than OPA isoindoles (7). Isoindoles derived from OBB are significantly more stable than those from OAB, apparently due to a combination of steric, electronic and resonance effects exerted by the 3-phenyl-substituent (in OBB). The isoindole formation rate observed for reaction of OBB was much slower than that exhibited for OPA or OAB under the same conditions, however. This may be explained by the steric effect of the phenyl group, which reduces the opportunity for nucleophilic attack by the secondary amine of the a-alkyl-aminobenzyl sulfide (X, Scheme 2), and by resonance stabilization of the reactant. Thus, although the isoindoles produced from OBB are considerably more stable than from OAB, their slow rate of isoindole formation from OBB makes this reagent less desirable than OAB. Since, in general, the isoindole ring system readily undergoes autoxidation (50) and/or electrophilic attack at positions 1 and 3 (51) by species such as the aldehyde component of the OPA reagent, substitution of thiol by other stronger nucleophilic species may produce a significant stability enhancement. For this purpose, NY, SCN-, HSO,, and CN- were studied (52), but with alanine serving as the primary amine, only HSO; and CNwere observed to give a fluorescent product. Compared to 2-mercaptoethanol, cyanide provided greater stability, but gave a somewhat reduced fluorescence with OPA. Substitution of naphthalene-2,3dicarboxaldehyde (NDA) in the derivatization of amino acids with CN- gave N-substituted l-cyanobenz[flisoindole derivatives, which were much more fluorescent than the OPA2-mercaptoethanol derivatives (52). In contrast, NDA and 2-mercaptoethanol produced unstable and weakly fluorescent derivatives. ET AL. 5. Simons, S. S., Jr., and Johnson, 6. Simpson, R. C., Spriggle, M. J. Medina Hernindez thanks the “Conselleria de Cultura, cacio i Ciincia de la Generalitat Valenciana” for the grant which possible her collaboration in this work. Edumade J. E., and Veening, J. Chroma- H. (1983) togr. 261,407-414. 7. Sternson, L. A., Stobaugh, J. F., and Repta, A. J. (1985) Anal. Biockem. 144,233-246. 8. McDonald, 506516. R. S., and Martin, E. V. (1979) Can& J. Ckem. 67, W. P. (1967) J. Amer. Chem. Sot. 89, 10. Barnett, R., and Jencks, W. P. 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