Anal. Chern. 1987, 5 9 , 1102-1105
1102
N-Substituted I-Cyanobenz[ f Iisoindole: Evaluation of Fluorescence Efficiencies of a New Fluorogenic Label for Primary Amines and Amino Acids Bogdan K. Matuszewski,' Richard S. Givens,* K a s t u r i Srinivasachar,z Robert G. Carlson, and Takeru Higuchi
T h e Center for Bioanalytical Research and Department of Chemistry, The University o f Kansas, Malott Hall, Lawrence, Kansas 66045
We report here the physical and spectral properties of Nsubstituted 1-cyanobenz[f]solndole (CBI) derlvatlves, the product from the reaction of 2,&naphthalenedlcarboxyaldehyde and cyanide wlth a prlmary amlne or amlno acld. The quantum efflclencles (9,'s) were measured as a function of solvent and buffer composition In water and acetonltrlle/ water mixtures and ranged between 0.5 and 0.8 for amlno acld and ollgopeptide derlvatlves and 0.4-0.8 for aliphatic amlne derlvatlves. The 9, values for amino acld and ollgopeptlde derlvatlves Increased wlth an Increase In solvent water content, in contrast wlth an approximate 10-fold decrease for analogous dansylated derlvatlves. Phosphate and Imidazole buffers dld not slgnlflcantly affect the emisslon yields. For dl- and trlpeptkles, a decrease of 20% In 9,was observed In extendhrg the amlno acld chaln length In contrast wlth a 10-fold decrease reported for the o-phthalaldehyde derlvatlves. These features suggest that the CBI derlvatlves are especially well sulted for assaylng prlmary amlno acids by fluorescence detectlon methods when coupled with reverse-phase hlgh-performance llquld chromatography (RPHPLC).
Optimization of the fluorescence efficiency (Q of analytes is a primary concern in devising ultrasensitive fluorescencebased analytical methods. Equally significant considerations are the sensitivity of the fluorescence efficiency to changes in the protic and aprotic makeup of the solvent, to changes in the pH, to the presence of buffer salts, and to minor changes in structural features of a series of closely related analytes. These considerations are particularly important for development of analytical methodology for reverse-phase high-performance liquid chromatography (RP-HPLC) where variations in these parameters are both frequently encountered and often a necessity. Furthermore, these considerations are important whether the analyte itself fluoresces or must be chemically altered by incorporation of a fluorescent ligasd through a derivatization reaction. Recent results in our laboritories have indicated that Nsubstituted 1-cyanobenz[flisoindole (CBI) derivatives are useful fluorogenic reagents for ultrasensitive assay of amino acids and primary amines (eq 1) (1-3). Detection limits for 7N
+
RNHz
NaCN
&\ N-R
\
'
(1)
C HO
Present address: Research Laboratories, W26-372, Merck, Sharp and Dohme, West Point, P A 19486. *Present address: Laboratory of Molecular Biology, Building 36, Room 1B-08, National Institute of Mental Health, Bethesda, MD 20205.
derivatized amino acids including small peptides have been significantly improved (3-5)over other fluorescence methods (6-9) including the o-phthalaldehyde/Z-mercaptoethanol (OPA/2-ME) (6) and the dansyl chloride (7)derivatization methods. We wish to report our quantitative evaluation of the fluorescence efficiencies of a series of N-substituted 1cyanobenz[flisoindoles, the fluorogenic labeled derivatives of amines and amino acids, and to compare these results with other fluorescence-based labeling methods, namely, the OPA-thiol(6) and dansyl-based (7) assay methods that are currently employed. EXPERIMENTAL SECTION Materials. Acetonitrile (Fisher, HPLC grade) and water (distilled twice from a glass apparatus) contained no significant absorbing or fluorescent impurities detectable at the highest sensitivities used for fluorescence determinations. Quinine bisulfate (QB, Aldrich) was used as received. A stock solution in 1.0 N H2S04was used, after appropriate dilution, both as the fluorescence (af)standard (10,11) and for the calculation of the spectral sensitivity factor (S,) (12)of the spectrofluorimeter between 400 and 600 nm (vide infra). Quinine sulfate in 0.1 N H2S04 is also recommended as a fluorescence quantum yield standard in the emission region of 400-600 nm by the IUPAC Photochemistry Commission coordinated by D. F. Eaton, Feb., 1985. The N-substituted 1-cyanobenz[flisoindole (CBI) derivatives were synthesized, isolated, and purified in the laboratories of the Center for Bioanalytical Research, according to previously described procedures (1). Instrumentation. The fluorescence spectra were recorded with an Aminco-Bowman spectrofluorimeter (Model SPF 4-8940 SP) equipped with an IP 28 photomultiplier. The emission spectra (excitation at 366 nm) were corrected according to the procedure described by Parker (13). The spectral sensitivity factors were calculated at 5-nm intervals between 400 and 600 nm by dividing the ordinates of the experimentally normalized quinine bisulfate spectrum obtained with our instrument by the corresponding ordinates of the corrected quinine bisulfate spectrum reported by Berlman (14). The SK values were then employed for the correction for all subsequent CBI derivative spectra. The emission maxima and full width at half-height for all of the CBI derivatives were nearly identical and were not appreciably affected by the changes in the solvent. Thus, the calculated fluorescence quantum efficiencies of the other CBI derivatives are relative to those of the CBI-Gly derivative which served as the secondary standard, necessitating correction of the CBI-GLY emission spectrum only. Because of the very similar spectral wavelength distribution and maxima for the emission bands of the CBI derivatives when compared with quinine bisulfate, the use of uncorrected spectra However, separate emission introduced less than a 5% error in spectra corrections were necessary for the N-tert-butyl(tert-butylthio)benz[flisoindole (TBBI-t-Bu) and the dimethylaminosubstituted derivative (DMA-CBI-t-Bu, see Table I). The absorption spectra were recorded on a Hewlett-Packard 8450A diode array spectrometer in a 1-cm fluorescence cell vs. the appropriate solvent mixture used for the afdetermination. Fluorescence Quantum Efficiencies (af).The fluorescence quantum efficiencies (Of)were determined relative to that of
0003-2700/87/0359-1102$01.50/00 1987 American Chemical
Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8 , APRIL 15, 1987
Where Gfand G[ are the fluorescence quantum efficiencies for the CBI derivative and QB, respectively, and A and A'are the absorbances of the CBI and QB solutions, respectively. The absorbance values A and A' were obtained immediately before recording the fluorescence spectrum. In order to minimize errors due to an inner filter effect, the concentrations of all samples and standards were adjusted so that the absorbances were 0.100 f 0.010 (13). The errors due to small refractive index changes of the solvent did not exceed 2 %, which is well within the estimated overall error (&15%),and, therefore, are not included in the calculations.
Table I. Structures of N-Substituted 1-Cyanobenz[f]isoindoles and Related Derivatives Employed in This Study
&
N-R
\
\
R
amino acid GlY Gly-Gly Gly-Gly-Gly Ala CBI-Lys
-CH2COONa -CHzCONHCHzCOONa -CH2CONHCH2CONHCOONa
RESULTS The structures of the CBI derivatives of a representative series of primary amines and amino acids (1) that were examined for this study are given in Table I and their fluorescence efficiencies (@is) are listed in Tables I1 and 111. The solvent effects on the fluorescence efficiencies were evaluated for a series of acetonitrile-H20 mixtures that were representative of the range used in many RP-HPLC separaM tions. Two common buffers were also investigated, imidazole and 0.05 M phosphate (1-4). All reported afvalues are for aerated solutions since deaeration with argon did not significantly change in the fluorescence yield. The A,, values of the two visible absorption bands, their molar absorptivities (A), the fluorescence maxima, and the full width at half-height ( Wll2) for the representative series of CBI derivatives are presented in Table IV. With these data and the fluorescence efficiencies (in Tables I1 and 111), the intrinsic fluorescence sensitivity (IFS) factor was determined according to eq 3. According to Lloyd (12),the IFS
-CH(CH3)CONHCH(CH3)COONa -(CH,),CH(CBI)COONa -(CHz),CH(NH2)COONa
LYS
amine
R
t-Bu n-Bu
-C(CHB)B -C(CHz)&H3 -(CHz)zCH3 -(CHZ)&BI
n-pr
(CBI)z(CHz), other
S-t-Bu
N-I-Bu TBBI - I
W
N
-
t
- Bu
-
B
u
IFS = @fAA(max)/WI/2
NEH&
O M A - C B I - t -Bu
= Zf(1 - lo-A')/[If(l
- 10-91
(3)
values serve as an empirical comparative indicator of a derivatives performance as a fluorescing analyte.
quinine bisulfate (QB) in 0.1 N H2S04(af= 0.55) (IO, 11). The measured fluorescence intensity (If)of the CBI derivatives, obtained by integration of the corrected emission spectra, were compared with those of either the primary (QB) or secondary (CBI-Gly)standards. The value for a given derivative was then calculated according to the following expression (15): @f/@[
1103
DISCUSSION Fluorescence Quantum Efficiencies and Solvent Effects. The data presented here clearly indicate that N-substituted 1-cyanobenz[flisoindole derivatives of amines, amino acids, and small peptides exhibit excellent fluorescence emission properties, especially when compared with other
(2)
Table 11. Fluorescence Quantum Efficiencies" of N-Substituted 1-Cyanobenz[f]isoindole Derivatives of Amino Acids and Small Peptides as a Function of Solvent Composition solvent
Gly
Gly-Gly
Gly-Gly-Gly
Ala
Ala-Ala1
Lys
CBI-Lys
HZO H20/MeCN (70/30) HzO/MeCN (40/60) MeCN
0.80 0.76 0.73
0.58 0.56 0.48
0.68 0.59 0.59
0.02c
0.7OC 0.55c
0.02c 0.03'
b
b
0.54 0.50 0.50 0.11
0.44c
b
0.75 0.67 0.58 0.15
b
b
imidazole buffer (0.01 M, pH 7.2)/MeCN (70/30) phosphate buffer (0.05 M, pH 7.0)/MeCN (40/60)
0.79
0.55 0.46
0.59 0.49
0.70
0.54
0.57
0.50
0.70 0.30
0.02 0.04
0.66
" Determined from corrected fluorescence spectra, with an estimated error of 15%. *Not measured due to poor solubility. Instead of H,O. 0.01 N NaOH was used here. Table 111. Fluorescence Quantum Efficiencies" of Various N-Substituted 1-Cyanobenz[f]isoindole Derivaives of Amines as a Function of Solvent Composition
solvent
t-Bu
n-Bu
n-Pr
(CBI)z(CH2)S TBBI-t-Bu
H20/MeCN (60/40) HzO/MeCN (40/60) MeCN
0.38 0.38 0.33
0.60 0.58 0.41
0.64 0.54 0.42
0.01 0.02 0.04
imidazole buffer (0.01 M, pH 7.2)/MeCN (20/80) phosphate buffer (0.05 M, pH 7.0)/MeCN (40/60)/MeCN
0.30 0.26
0.44 0.38
0.45 0.41
0.03 0.02
b 0.06 0.12 0.04
0.02
DMACBI-tBuc 0.02 0.03 0.08 0.04 0.02
a Determined from the corrected fluorescence spectra and corrected for refractive index changes. An estimated error was 15%. *Not measured due to poor solubility. cEstimated value due to large red shift of the emission spectrum.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8. APRIL 15. 1987
-- --- - - - - - - -Table IV. Absorption" and Fluorescence Data of I-Cyano-N-Substituted and Related Benz[f]isoindole Derivatives of Amines, Amino Acids, and Small Peptides in Phosphate Buffer ( p H 7.0, 0.05 M)/MeCN (40/60) I -
derivative
absorption niax rim (A,)
CBI-Gly CBI-Gly-Gly CBI-Gly-Gly-Gly CBI-Ala CBI-Ala-Ala (CBI),-Lys CBI-LYS CBI-t-Bu CBI-n-Bu CBI-n-Pr (CBI)2-(CH2)5 TBBI-t-Bu CMA-CBI-t-Bu
419 (9400), 442 (8900) 420 (6000), 443 (5500) 420 (6700), 442 (6100) 421 (7000), 443 (6500) 422 (52001,444 (4700) 419 (WOO), 443 (8700) 417 (6800), 440 (6500) 422 (8300). 447 (7500) 417 (8000),440 (7700) 417 (8500), 440 (8100) 418 (108001, 441 (10100) 446,' 465' 429 (lOZOO), 453 (9500)
~~~
*f
emission mdx, nm
(Wl,2, cm
~
Table V. Comparison of Quantum Efficiencies for OPA, Dansyl-, and CBI-Labeled Amino Acids and Oligopeptides in Aqueous Media
')
493 (2700) 494 (2700) 494 (2800) 496 (2600) 496 (2800) 488 (2700) 486 (2800) 492 (2700) 486 (2700) 486 (2700) 490 (2750) 534 (2900) 598 (3200)
IFSb 2.3
1.o
1.2 1.5 0.9 0.1 0.7 0.8 1.1 1.3
0.08 c
0.06
OReported data are for visible region only. bCalculated from the equation IFS = QfAh(,,,,)/Wl,2; Ah(mar)is the absorptivity at 420 nm. 'Not determined due to significant instability of this derivative.
derivatives currently available for the determination of these classes of compounds. The af value of each of the CBI derivatives in aqueous solvents is uniformly high (0.3-0.8, Tables I1 and 111) and shows remarkably little variation with solvent change from water to acetonitrile or with the buffer composition of the solvent. The relatively small response to changes in the analytical conditions makes these CBI derivatives especially well suited for RP-HPLC analysis. Our results should be viewed in the context of those reported earlier by Chen (7) for dansyl derivatives of amino acids, for which the af values are quite sensitive to solvent variations, decreasing by nearly an order of magnitude as the solvent composition approaches 100% H20. No detailed determination of the fluorescence efficiencies as a function of solvent composition was reported for the OPAIB-ME derivatives, possibly due to the instability of the thio-substituted isoindole and the attendant difficulties with isolation of the amino acid derivatives. The only data available are limited to studies with aqueous borate buffer (pH 9.0) solution in which the derivatives were generated in situ (6). Even here, the efficiencies are considerably lower than we have found for the corresponding CBI analogues (Table V). Fluorescence efficiencies of 0.5-0.8 were also obtained for the CBI derivatized di- and tripeptides, Gly-Gly, Ala-Ala and Gly-Gly-Gly, thus displaying little diminution (
