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Anal. Chem. 1987, 59, 1096-1101
Naphthalene-2,3-dicarboxaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary Amines Pierre d e Montigny, J o h n F. Stobaugh,* Richard S. Givens, Robert G.Carlson, K a s t u r i Srinivasachar,’ L a r r y A. Sternson,2 a n d T a k e r u Higuchi The Center for Bioanalytical Research, Department of Pharmaceutical Chemistry, and Department of Chemistry, The University of Kansas, Malott Hall, Lawrence, Kansas 66045
On the basls of the isolndoie formatlon mechanism in the o -phthalaMehyde/2-mercaptoethanol (OPA/P-ME) derivatlzation of primary amlnes and the structure-stability reiationships for isolndoies, an Improved fluorogenlc reagent, naphthaiene-2,3-dlcarboxaldehyde (NDA) in the presence of cyanide Ion (CN-), has been developed. Reaction of NDA/ CN- with prlmary amlnes in aqueous medla results in the formatlon of N-substituted I-cyanobenz[l]isoindoie (CBI ) derlvatives which have slgnlfkantly Improved stabliRy compared to the corresponding OPA/2-ME derlvathres (for giyckre greater than 5Mdd hrprovemenl was realized) and have Hgh quantum efflclencies for fluorescence (a, = 0.54 in 60% aqueous acetonitrile for the CBI-n-propyiamine derlvative) In solvent systems commonly used In iiquld chromatography. Parameters In the NDNCN- derlvatlzation of alanine are defined (Le., pH and the reagent component concentrations) and used in the development of a labeling procedure for amlno acid mixtures. Gradient dutlon fractlonatlon of 18 CBI-amino acid derivatives was accomplished In 60 min and permltted detection limits of less than 200 fmol InJected(excitatlon 246 nm) or 3 pmd InJected(excitation 420 nm). The utility of the reagent In assaylng amino acid mixtures resuitlng from the enzymatic hydrolysis of the peptldes Met-enkephalin and glucagon Is demonstrated.
The trace analysis of amino acids is limited when highperformance liquid chromatography (HPLC) is employed due to the low response of conventional liquid chromatography detectors to the transparent analytes. To circumvent this limitation, a variety of successful analytical schemes for amino acids have incorporated a derivatization step. For example, ninhydrin derivatization following ion-exchange fractionation constitutes the classical method as first proposed by Spackman, Stein, and Moore ( I ) for amino acid analysis. With recent advances in column technology new approaches that utilize gradient elution fractionation with either hydrophobic or cation-exchange columns in conjunction with either preor postcolumn derivatization have evolved. Reagents, such as ninhydrin ( Z ) , fluorescamine (Z), dansyl chloride ( 3 ) ,7fluoro-4-nitrobenz-2,1,3-oxadiazole (NBD-F) ( 4 ) ,and the ophthalaldehyde (OPA, 1) reagent (5-8) have commonly been used in amino acid analysis. Recently, the use of the OPA reagent in both pre- and postcolumn modes has become popular. OPA reacts with primary amino acids in the presence of a thiol, 2-mercaptoethanol (2-ME) has been used most frequently, to form intensely fluorescent products (5). These products, in an elegant study (9),were shown to be the N-substituted l-alkylthioisoindoles, 2, depicted in Scheme I. Unfortunately, these Present address: Laboratory of Molecular Biology, Building 36, Room 1B-08,National Institute of Mental Health, Bethesda, MD 20205.
Present address: Smith, Kline and French Laboratories, 1500 Spring Garden St., Philadelphia, PA 19101.
Scheme I. Alternative Mechanism for the Fluorogenic o-Phthalaldehyde Reaction Depicting the Key Intermediates
aCHO It11
R N H 2 , RSH
d\ N
-
R
CHO
/
tRNHz,
- He0
SR
2
. He0
t I
SR
OH
3
isoindole derivatives exhibit a time-dependent degradation and thus place considerable restrictions on the method when used in precolumn applications. Our present objective has been to improve this widely utilized approach by the modification of the OPA reagent to provide for the formation of stable products while retaining and/or improving the high fluorescence quantum efficiencies. We wish to report the results of our research from which the (NDA) and a reaction of naphthalene-2,3-dicarboxaldehyde primary amine in the presence of cyanide ion (CN-) to form intensely fluorescent N-substituted 1-cyanobenzwisoindole (CBI) derivatives has emerged as an improved fluorogenic derivatization method in the analysis of amino acids.
EXPERIMENTAL SECTION Apparatus. Isocratic liquid chromatographyexperiments were conducted on a modular HPLC system consisting of an Altex Model llOA pump, a Rheodyne Model 7120 fixed loop injector, and a Schoeffel FS 970 LC fluorometer. Fluoresence detection in the HPLC experiments was accomplished with either a deutrium source for excitation at 246 nm or a tungsten-halogen source for excitation at 420 nm in conjunction with a 470-nm emission cutoff filter. Gradient elution experiments utilized two Altex Model llOA pumps interfaced to a Systec SLIC 1400 system controller and an Altex Ultrasphere-ODS 5-pm (250 X 4.6 mm) column. Isocratic elution studies were performed with Shandon Hypersil ODS 5-pm (150 X 4.6 mm) columns packed in our laboratories using an established high-pressure upward slurry technique (IO). Chromatograms were recorded with a Houston Instruments Omniscribe strip chart recorder with quantitation accomplished by manual peak height measurements. The buffer-independent pH-rate profile for CBI-alanine derivative formation was obtained with the use of a Metrohm pH-stat device consisting of a 632 pH meter, a 614 Impulsomat, a 655 MultiDosimat, and a 649 Swing-Out magnetic stirrer. Temperature was controlled in kinetic experiments with a Haake Model D2L circulating bath. Fluorescence studies were performed with a Perkin-Elmer Model 650-40 spectrofluorometer interfaced to a Model 3600 data station. ‘H NMR, IR, and mass spectra were obtained on Varian XL 300, Beckman Acculab 3, and Ribermag 10-10instruments, respectively. A Hewlett-Packard Model 185-B CHN analyzer was used for elemental analysis determinations and melting points (uncorrected)were taken on an Electrothermal melting point apparatus. Reagents. Amino acids, Met-enkephalin, glucagon, and aminopeptidase M were obtained from Sigma and used as received.
0003-2700/87/0359-1096$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
The Met-enkephalin and glucagon, while not contaminated with 80% or greater) due other peptides, were of undefined purity (a. to residual salts and water remaining after isolation. OPA (Sigma) was recrystallized from hexane and NDA was synthesized by a literature procedure (11,21)and recrystallized from petroleum ether. Water used throughout was deionized in mixed bed ionexchange columns followed by double distillation from an all-glass still. All other chemicals were of the highest purity available and used as received. Solutions. Buffers. Sodium borate buffer (pH 9.5,O.l M) was prepared by dissolving boric acid in water, adding the required amount of lithium perchlorate to control ionic strength, and titrating to the required pH with standard sodium hydroxide (0.5 N). Sodium borate buffer (pH 9.1, 0.01 M) was prepared by dissolving sodium tetraborate decahydrate in water. Sodium phosphate buffer (pH 7.0, 0.2 M) was obtained by preparing separate solutions of monobasic sodium phosphate and dibasic sodium phosphate, respectively. These solutions were then blended to obtain a pH 7.0 buffer. Potassium phosphate buffer (pH 6.8, 0.05 M) was similarly prepared from the individual phosphate salts. These four buffers will hereafter be referred to as pH 9.5 borate buffer, pH 9.1 borate buffer, pH 7.0 phosphate buffer, and pH 6.8 phosphate buffer, respectively. N D A and 0PA4/2-ME. Methanolic solutions of NDA and OPA/2-ME were freshly prepared on a weekly basis in actinic glassware. Sodium Cyanide. Stock solutions were prepared by dissolving the required amount of sodium cyanide in water and adjusting the pH to the desired value with either perchloric acid (0.1 N) or sodium hydroxide (0.1 N). The solutions were discarded after one week. Amino Acids. Stock solutions of the amino acids were prepared by accurately weighing and dissolving with water. The required working solutions were obtained by further dilution with water. Standard solutions were prepared fresh weekly and stored protected from light during their use period. Peptides. Met-enkephalin standard solutions were prepared by accurately weighing and dissolving in pH 7.0 phosphate buffer. Glucagon solutions, due to solubility considerations, were prepared in pH 9.1 borate buffer. Aminopeptidase M. Stock solutions of the enzyme (0.2 mg/mL) were prepared in pH 7.0 phosphate buffer and then diluted to the desired concentration with additional pH 7.0 phosphate buffer. Fresh solutions were prepared at the time of each peptide hydrolysis experiment. Mobile Phases. HPLC mobile phases were prepared by fdtering pH 6.8 phosphate buffer through a 0.45-pm cellulose acetate membrane filter and then mixing with the required prefiltered HPLC-grade organic modifier and degassing prior to use. Isocratic elution studies were all performed with a 30% acetonitrile/pH 6.8 phosphate buffer mobile phase. Kinetic Investigations. Alanine pH-Rate Profile. The rate of formation of the CBI-alanine derivative was characterized as a function of pH at constant ionic strength (0.1 M) and temperature (30 "C). As in actual analytical applications of the reagent system, NDA and CN- were present in excess quantities with respect to the alanine. The pH was maintained constant during the reaction with a pH-stat device and the reaction was M), initiated by the addition of sodium cyanide (0.1 mL, 2 X M) to alanine (0.1 mL, 1 X 10"' M) and NDA (0.1 mL, 2 X an aqueous lithium perchlorate solution (9.7 mL, 0.1 M) preequilibrated at 30 "C. The formation of the CBI-alanine derivative was quantified by removing aliquota of the reaction solution at timed intervals and analyzing by RP-HPLC using the isocratic mobile phase. Kinetic Dependency. The kinetic order of NDA and CN- in the derivatization of alanine was investigated at pH 9.5 and 25 "C via spectrofluorometry. Under conditions with excess NDA and CN- the reaction was initiated by the addition of an alanine solution (0.1 mL, 6 X lob M) and then pH 9.5 borate buffer (2.7 mL) to a quartz cuvette. To this solution was added sodium cyanide (0.1 mL) and NDA (0.1mL). The concentrations of the NDA and sodium cyanide solutions were such that final con. to 4 X 10 M and 5 X 10 to centrations ranged from 1 X 1.6 X M, respectively.
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Stability Comparisons. The stability of glycine derivatized with NDA/CN- compared to OPA/2-ME was quantitatively evaluated by generating each derivative in situ in a solvent system composed of 20/80 methanol/pH 9.5 borate buffer at 25 "C (the methanol was necessary due to solubility limitations). In each experiment, to a solution of glycine was added sodium cyanide and NDA or 2-ME and OPA. These additions were such that the final concentration ratios were NDA/CN-/glycine 5 X M/5 X MI1 X lo4 M and OPA/2-ME/glycine 5 X M/1.42 X M/1 X lo4 M. The degradation rate of each derivative was determined by taking aliquots of the derivatization solution, maintained at 25 "C, at timed intervals and analyzing by RP-HPLC using the isocratic mobile phase. Procedures. Amino Acid Derivatization. For trace analysis of amino acids the derivatization consisted of adding an aliquot of the amino acid mixture (50 pL) to pH 9.1 borate buffer (200 pL) followed by the addition of sodium cyanide (50 pL, 1 X M) and NDA (200 pL, 1 X M). After a reaction time of 15 min at ambient temperature, a 50-pL aliquot of the reaction mixture was analyzed by gradient elution HPLC. Peptide Hydrolysis. Met-enkephalin (25 pL, 2.5 nmol) was added to an aminopeptidase M solution (25 p L , 2.5 nmol). The reaction was conducted at ambient temperature and aliquots were removed at predetermined intervals and derivatized with NDA/CN-. The resulting derivatives were then assayed by gradient elution HPLC. Glucagon (25 pL, 2.5 nmol) was hydrolyzed at 37 "C in a similar fashion and derivatized, and the resulting mixture was assayed by the same procedure. Due to the undefined purity of each peptide the amino acid composition of the enzymatic hydrolysates was determined on a relative basis, Le., an amino acid of known quantity in each peptide served as the reference. Thus, MET and VAL served as the reference amino acids for Met-enkephalin and glucagon, respectively. Synthesis of 1-Cyano-2-ethylisoindole.This compound was prepared by the method of D'Amico et al. (12) with minor nodifications. To a methanolic solution (10 mL) of OPA (134.1 mg, 1mmol) was added sodium cyanide (49 mg, 1 mmol) and an aqueous solution (10 mL) of ethylamine hydrochloride (81.55mg, 1mmol). The reaction mixture was shielded from light and stirred for 2 h. Upon completion, the purple reaction mixture was chilled on ice to promote complete precipitation of the isoindole. The solid was collected by filtration, washed several times with water, and dried under vacuum overnight to give a white crystalline solid in 45% yield: mp 59-61 "C; IR (KBr) 2200,1510,1415,1330,1230, 1150,1095,780,750,cm-'; lH NMR (CDC13,300 MHz), 7.63 (d of d, 2 H), 7.36 (s, 1 H), 7.23 and 7.10 (2 t, 2 H), 4.39 (9, 2 H), 1.61 (t, 3 H); mass spectrum in CI mode (CH,) m / e 171 (M + 1). Anal. Calcd for Cl1Hl,N2: C, 77.65; H, 5.88; N, 16.47. Found: C, 77.38; H, 6.08; N, 16.29.
RESULTS AND DISCUSSION Background and Rationale. The mechanism initially proposed for the formation of N-substituted 1-alkylthioisoindoles from the reaction of OPA with primary amines in the presence of a thiol was presented in the literature several years ago (13). Recently, investigations in our laboratories with a modified OPA-like reagent has led to the formulation of an alternative mechanism (14)of which the key elements are presented in Scheme I. Subsequent kinetic studies have supported this mechanism and have ruled out the originally proposed sequence (15). It is well-known that, in general, the isoindole ring system readily undergoes autoxidation (16)and/or electrophilic attack a t positions 1 and 3 (17) by species such as the aldehyde component of the OPA reagent. However, significantly enhanced stability to autoxidation (18)and electrophilic attack (19)has been observed when the isoindole ring bears strong electron-withdrawing substituents. On the basis of the mechanism of Scheme I a rational modification of the OPA reagent was undertaken. Key to the approach was the reahation that the thiol component of the reagent functions ab a nucleophile toward the intermediate imine 3 % e reasoned that uther nudeophiiic bpec~ebwuid seive this role JudiLious Lhoice of the nucleophile incorpo
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
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Table I. Spectroscopic Properties of in Situ Generated Isoindole and Benz[f]isoindole Derivatives of Alanine"
dialdehyde OPA
nucleophile 2-ME HS04CN-
NDA
2-ME HSOBCN-
excitation max, nm
emission max, nm
450 400 380
1.00 0.08 0.22
440 420 420
570 500 490
