Aldehydes and Dialdehydes

Product List N1138 P2331MP A2333 A2334 A6222 A10192 naphthalene-2,3-dicarboxaldehyde (NDA) o-phthaldialdehyde (OPA) *high purity* ATTO-TAG CBQCA Amine-Derivatization Kit ATTO-TAG FQ Amine-Derivatization Kit ATTO-TAG CBQCA derivatization reagent (CBQCA; 3-(4-carboxybenzoyl)quinoline- 2-carboxaldehyde) ATTO-TAG FQ derivatization reagent (FQ; 3-(2-furoyl)quinoline- 2-carboxaldehyde)

Table 1. Spectral Properties after conjugation Dye ATTO-TAG CBQCA ATTO-TAG FQ NDA OPA Excitation 450 nm 480 nm 419 nm 334 nm Emission ~550 nm ~590 nm 493 nm 455 nm

2) Reactive Group Structure/Reaction: Figure 1. General reaction sequence of aldehyde with primary amine.

Figure 2. Fluorogenic amine-derivatization reaction of naphthalene-2,3-dicarboxaldehyde (NDA).

1-cyano-2- substituted-benz[f]isoindole Figure 3. Fluorogenic amine-derivatization reaction of CBQCA.

Cyanobenz[f]isoindole Figure 4. Fluorogenic amine-derivatization reaction of o-phthaldialdehyde (OPA).

Thioisoindole 3) Mechanism The condensation reaction of an amine with either an aldehyde or ketone proceeds through the intermediate formation of an unstable carbinolamine, formation to imine (Schiff base) with the elimination of water, then reductive amination to a stable amine (Figure 1). The first step is a nucleophilic addition to the carbonyl group followed by a rapid proton transfer. The resulting product, a hemiaminal or carbinolamine, is generally unstable and cannot be isolated. A second reaction occurs in which water is eliminated from the carbinolamine and an imine (Schiff base) is formed. The imine (Schiff base) may be reduced to a stable amide bond by reductive amination with the addition of sodium borohydride or sodium cyanoborohydride.

NDA and CBQCA/FQ reactions requires the presence of cyanide to create the final adduct of N-substituted cyanobenz[f]isoindole (Figure 2 and 3). The optimal reaction pH is near the pKa for the primary amine for deprotonation of the amine. Cyanide degrades NDA, they must be made separately and only mixed shortly before the reaction1. OPA reaction requires the presence of a thiol to create the final adduct of N-substituted thioisoindoles. The thiol component of the reaction, the added beta-mercaptoethanol, functions as a nucleophile toward the intermediate imine1 (Figure 4). Degradation of OPA-adducts is accelerated by the presence of excess aldehyde component of the reagent2-4. The isoindole ring readily undergoes auto-oxidation; unless the final adducts are to be analyzed immediately they should be stored desiccated under nitrogen or argon. OPA comparison with Fluorescamine: 1) OPA exhibits greater quantum yields (~one order of magnitude greater); OPA is 5 to 10-fold more sensitive than fluorescamine5. 2) OPA is soluble and stable in aqueous buffers. 3) OPA does not form a fluorophore with secondary amines5. NDA comparison with OPA: 1) NDA final product more stable than OPA final products, e.g., OPA-glycine t1/2 = 1.5 hours; NDA-glycine <10% loss after 10 hours1. 2) OPA-adducts decompose readily in the presence of excess OPA whereas NDA-adducts are unaffected by 100- to 1000-fold excess of NDA. Both OPA and NDA react with lysine, ornithine and cadaverine to form bis-NDA-analyte or bis-OPA-analyte products which exhibit diminished fluorescence. Bis-NDA shows reduced fluorescence; bis-OPA quenching can be alleviated by addition of surfactant, such as Brij; the addition of surfactant does not work to eliminate quenching of bis-NDA. Mono-lysine-OPA adducts yields low fluorescence unless Brij is present6. Detection Limits: femtomolar range (10-15 M). 3 picomoles (420 nm, tungsten lamp; 200 femtomoles for most amino acids, down to attomoles of NDA-arginine (246 nm, deuterium lamp)6. ATTO-TAG CBQCA: subattomole range (<10-18 moles)* ATTO-TAG FQ: subfemtomole range (<10-15 moles)* OPA: NDA: * using capillary electrophoresis with laser excitation source. Fluorescent adducts are stable in acetonitrile with minimal changes in fluorescence efficiencies and thus, may be used with RP-HPLC6. 4) Reaction Conditions OPA Stock solution: 500 mg to 800 mg OPA dissolved into 10 mL 95% ethanol; add 200 uL mercaptoethanol, methanethiol or ethanethiol. Mix this solution into 1 L of 3% boric acid solution, pH 9.7 to 10 (adjust pH with KOH or NaOH). Degas solution with vacuum aspirator and add 7.5 mL of a 30% Brij 35 solution. Filter sterilize through 0.2 um filter. Store in covered bottles (protect from light) under nitrogen; this solution is stable at room temperature for ~2 weeks5,7.

Reaction at pH >7 is complete within minutes at room temperature; going up to 70oC improves derivatization of highly substituted carbon adjacent to the amino group7. NDA Stock Solution: NDA in 100% methanol (1 mM); store protected from light at 4oC; stored in the refrigerator, the stock solution is stable for 1 week. KCN Stock Solution: KCN in ddH2O (10 mM) with 10 uL of 0.1 N NaOH solution added. The reaction of amino acids/peptides may be done at room temperature in borate buffers (~10 to 100 mM) at pH >7 to <9.5; reaction rate increases with ionic strength. The yield of undesirable fluorescent side products increases with a large excess of NDA; the useful concentration range for NDA is in the range of 50-400 uM with 1.0-2.0 mM cyanide, optimal final working concentrations are dependent upon the concentration of total amines in the sample. Higher cyanide concentrations or pH >9.5 lower the reaction rate. NDA should be added last, after cyanide addition6. General guideline for a recommended NDA reaction mixture sequence8: 1) Dissolve analyte into buffer: 50 uL of analyte at 2 to 40 ug/mL concentration in 50% ethanol or methanol/water solution 2) Add 200 uL of 10 mM borate buffer, pH 9-9.4, 2) Add 50 uL of 10 mM stock cyanide solution. 3) Add 200 uL of 1 mM NDA/methanol stock solution. 4) Incubate at room temperature for ~30 minutes. Final NDA adducts stable for ~12 hours at room temperature; up to 48 hours at -5 to -20oC8. CBQCA/FQ with Amino acids and Peptides: CBQCA is soluble in 100% methanol or DMSO (10 mM); FQ reagent is soluble in 100% methanol (10 mM); solubility in methanol is improved by addition of base (e.g., 15% (v/v) 0.2 M KOH). At a minimum, use a 6-fold molar excess of reagent and 5-fold molar excess of cyanide per mole of amino acids or peptides. For amino acids and smaller peptides, pH 8.5 to 9.5 is optimal for ~1 hour reaction at room temperature. Peptides of >9 residues show no distinct pH optimum for reaction. CBQCA/FQ with Amino Sugars: 2-fold molar excess of reagent and 3-fold excess of cyanide per mole of amino sugars. A pH of 7 is optimal for a ~1 hour reaction at room temperature. 5) Hydrolysis OPA is stable in buffered aqueous solutions; stock solutions can be stored under dry N2 for ~2 weeks at room temperature, protected from light. NDA should be stored in anhydrous solutions and is stable for ~1 week. CBQCA and FQ: The stock solutions of ATTO-TAG CBQCA or ATTO-TAG FQ in anhydrous solvents (either methanol or DMSO) should be stored at -20C in a capped vial. The stock solution must be thoroughly

thawed and mixed before use. A working supply of the stock solution may be kept at room temperature and replaced daily. 6) Stability of Reagents All reagents are stable in powder form, kept frozen, desiccated and protected from light. Stock solutions are stable from 1 to 2 weeks, dependent upon the reagent and the quality of the solvents. Anhydrous stock solutions (for NDA and CBQCQ/FQ) should also be stored desiccated to minimize the gradual accumulation of water from condensation. Post-reaction: The cyanoisoindole derivatives of amino acids and peptides are stable in solution for about 24 hours. Those from amino sugars are stable for at least 10 hours. When evaporated to dryness and frozen, the derivatized products are stable for at least two weeks. OPA-glycine t1/2 = 1.5 hours; NDA-glycine <10% loss after 10 hours1.

7) Storage of Reagent
Reagents are stable for at least 6 months when stored in solid form, kept desiccated, <-20oC, protected from light. References: [1] Naphthalene-2,3-dicarboxaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary Amines." de Montigny P, et al. Anal Chem 59, 1096 (1987) [2] Nakamura, H., Matsumoto, A., Tamura, Z. Anal. Lett. 1982 15:1393-1410. [3] Stobaugh, J.F., Repta, A.J., Sternson, L.A., Garren, K.W. Anal Biochem, 1983 135:495-504. [4] Stobaugh, J.F., Repta, A.J., Sternson, L.A. J. Pharm Biomed Anal. 1986, 4:341-351. [5] o-Phthalaldehyde: Fluorogenic Detection of Primary Amines in the Picomole Range. Comparison with Fluorescamine and Ninhydrin." Benson JR, Hare PE. Proc Natl Acad Sci U S A 72, 619-622 (1975) [6] Naphthalenedialdehyde-Cyanide: A Versatile Fluorogenic Reagent for the LC Analysis of Peptides and Other Primary Amines." Lunte S, Wong OS. LC-GC 7, 908 (1989) [7] Amino Acid Analysis with o-Phthalaldehyde Detection: Effects of Reaction Temperature and Thiol on Fluorescence Yields." Cronin JR, Pizzarello S, Gandy WE. Anal Biochem 93, 174-179 (1979) [8] Fluorescence Detection and Precolumn Derivatization." Yang SS, Smetena I. Chromatographia 37, 593 (1993)