Bioconjugate Chem. 1004, 5, 31-39
31
Thiolate and Phosphorothioate Functionalized Fluoresceins and Their Use as Fluorescent Labels Christopher Bieniarz,; Douglas F. Young, and Michael J. Cornwell Abbott Laboratories, Diagnostics Division, Department of Immunochemistry, Abbott Park, North Chicago, Illinois 60064-3500.Received May 26,1993”
We report the syntheses of two new fluorescein derivatives, 3‘,6’-dihydroxy-3-0~0-2[(phosphonothio)acetyllspiro[isobenzofuran-1(3H),9’-9H-xanthene]-6-carboxylic acid hydrazide, disodium salt, a phosphorothioate fluorescein, and 3’,6’-dihydroxy-3-oxo-2-(mercaptoacety1)spiro[isobenzofuran-1(3H),9’9H-xanthene]-6-carboxylic acid hydrazide, a mercaptoacetyl fluorescein. The latter is derived from the first compound by hydrolysis of the phosphate. Direct nonenzymaticlabeling of the maleimide-derivatized IgG molecule by the novel mercaptoacetyl fluorescein is discussed. We also present a new method of bioconjugating phosphorothioate-functionalizedfluorophores to a maleimide-derivatized protein, based on the alkaline phosphatase-catalyzed hydrolysis of the S-P bond of the phosphorothioate and the concomitant liberation of the fluorophore thiolate. This last species reacts in situ with the maleimide on the protein. A high degree of conjugation control is achieved in that modulation of the stoichiometry of the label and enzyme results in incorporation from seven to eight fluorophores per protein, depending on the ratio of the phosphorothioate fluorescein to alkaline phosphatase. The quantum yield of the mercaptoacetyl fluorescein relative to 6-carboxyfluorescein is 0.22 and A,,, = 494 nm and b,,, = 517 nm.
INTRODUCTION The reaction of thiolate anion with a suitable electrophile is one of the most important methods of bioconjugation chemistry ( I ) . In general, excellent results are obtained by functionalizing the first entity with an electrophilic reagent (Le., maleimide or haloacetyl) and a subsequent reaction with a thiolate functionalityon the second entity. The three reagents considered below are introduced through a nucleophilic attack by the amine of the protein or hapten on the carbonyl of the active ester of the thiolating reagent. The generation of thiolates on proteins is frequently achieved through a reduction of the intrinsic disulfide bonds (2). However, cleaving the cystine bonds has the drawback of affecting the tertiary and quaternary structure of some proteins. Lately, the use of 2-iminothiolane (3) has seen increased use, mainly because of the efficiency and high yield of the thiolations. While 2-iminothiolane generates thiol functionality directly as a consequence of the ring opening of the Q-mercaptobutyrimidate, SPDP’ (3) and SATA (4) introduce thiols in a protected form as 2-pyridyl disulfide and thioacetyl moieties, respectively. Consequently, it is necessary to deprotect the thiols prior to the reaction with the thiolreactive group. In the case of SPDP, this is accomplished by reductive cleavage of the 2-pyridyl disulfide group using DTT as a reducing agent. After reduction, DTT and pyridine-2-thione have to be removed from the reaction medium by chromatography or dialysis. Deprotection of the thiol of the SATA reagent calls for even harsher conditions of 0.2 N NaOH, aqueous NH3 (51, or hydroxylamine ( 4 ) which may be incompatible with base-sensitive haptens or proteins. While the coupling reagents men@
Abstract published in Advance ACS Abstracts, December
15, 1993.
1 Abbreviations (in order of appearance in the text): SPDP, N-succinimidyl3-(2-pyridy1dithio)propionate;SATA, N-succinimidyl S-acetylthioacetate;DTT, dithiothreitol;DMF, dimethylformamide; hCG, human chorionic gonadotropin; TNBS, 2,4,6trinitrobenzenesulfonicacid; SEC, size exclusion chromatography; 6-CF, 6-carboxyfluorescein.
1043-1802/94/2905-0031$04.50/0
tioned above are most frequently used in the modification of proteins, they also may be applied for the modification of small molecules, i.e., haptens, fluorophores, chromophores, chemiluminophores, and drugs for the purpose of conjugating these entities to proteins. We have recently reported an efficient, high-yielding method of converting halides to mercaptans (6). The method consists of reacting an aliphatic or activated aromatic halide with 1 or 2 equiv of the sodium thiophosphate tribasic dodecahydrate (Sigma) in methanol or aqueous DMF. The intermediate alkyl or aryl phosphorothioate is hydrolized in situ over a broad pH range, 4-7, yielding thiolate ion and phosphate. The latter is an innocuous byproduct which in most bioconjugations need not be removed, since it does not interfere in the reaction process or in the next step of conjugation. In this paper we describe the adaptation of this method to the synthesis of a novel thiolated fluorescein derivative and the use of this fluorophore in labeling of an immunoglobulin, goat anti-hCG IgG. We also describe a new method of self-catalyzed conjugation of a novel fluorescein phosphorothioate to alkaline phosphatase, consisting of exposing in a buffered aqueous solution the fluorescein phosphorothioate to the action of bovine intestinal alkaline phosphatase suitably modified with maleimides. The principle is shown in Scheme 1. Alkaline phosphatase is first chemically modified by introduction of maleimide groups. This modification has minimal effect on the enzymatic activity. Alkaline phosphatase catalyzes the hydrolytic cleavage of the sulfurphosphorus bond of the phosphorothioate (7). The deprotected nucleophilic thiolate of the label molecule (in this work, a fluorescein derivative) reacts with the maleimides on the enzyme completing the conjugation. While most fluorescein labels are functionalized with electrophilic groups which may react with nucleophiles on the protein, very few fluorescein derivatives functionalized with nucleophilic groups have been reported. The most important examples are 4’-(aminomethyl)fluorescein (8) and 5- and 6-(aminomethyl)fluorescein (9). In this work we extend the repertoir of the nucleophilic fluorescein 0 1994 American Chemical Soclety
Bienlarz et al.
32 Bioconjugate Chem., Vol. 5, No. 1, 1994
Scheme 1
J
Fluorescein phosphorothioate
Maleimide functionalized alkaline phosphatase
derivatives by disclosing thiolated and phosphate-protected thiolated fluoresceins and describe methods of conjugating these new fluorophores to biologically important molecules. The alternative methods relying on the reaction of iminothiolane nucleophile and iodoacetyl fluoresceins have the disadvantage of the required prefunctionalizationof the protein with a thiolate which under many experimental conditions undergoes oxidative dimerization to disulfidesand possibly undesirable crosslinking of the protein to be labeled with the fluorophore. The new nucleophilicfluorophoresdescribed in this paper avoid the above complications. A very useful feature of this chemistry is that it allows the quantitation of the maleimide functionalities introduced into a protein at an intermediate stage of a bioconjugation. Since the fluorescein derivatives are highly chromogenic, the measurement of absorbance of the maleimide linker-functionalized protein after the reaction with the mercaptoacetyl fluorescein allows the quantitation of the introduced maleimides. EXPERIMENTAL PROCEDURES Materials. Except as noted, reagents were obtained commercially and used without further purification. All solvents were HPLC grade. Anhydrous DMF, Sephadex G-25 gel filtration packing, hydrazine hydrate, and silica gel 60 Merck 70-230 mesh were purchased from Aldrich Chemical Co. 6-Carboxyfluorescein N-hydroxysuccinimide ester was obtained from Research Organics, Inc. Bromoacetic acid N-hydroxysuccinimide ester, sodium thiophosphate dodecahydrate, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 5,5’-dithiobis(2-nitrobenzoicacid) (Ellman’s reagent), p-nitrophenyl phosphate, and all buffer componentswere from Sigma Chemical Co. The extended heterobifunctional maleimide active ester, succinimidyl 4- [(N-maleimidomethyl)tricaproamido]cyclohexane-1carboxylate (30 atom linker), was prepared as previously described (IO). Bovine intestinal alkaline phosphatase, purchased from Boehringer Mannheim Co. as a 10 mg/ mL solution in triethanolamine, NaC1, MgC12, and ZnC12, was dialyzed against 0.1 M phosphate buffer pH 7.0 containing 0.1 M NaC1,O.l M MgC12, and 0.1 M ZnClz and used as a 1 mg/mL solution. Anti-hCG IgG was from Abbott Laboratories. Determination of protein concentrations was accomplished with Bio-Rad Protein Assay Kit, from Bio-Rad Laboratories. General Procedures. Electronic spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. Nuclear magnetic resonance spectra were obtained on a Varian Gemini-300instrument. Fluorescencespectra were recorded on a Hitachi F-3010 fluorescence spectrophotometer. HPLC analyseswere performed on a SpectraPhysics instrument equipped with an SP8490 dualwavelength detector. Elemental analyses were by Oneida Research Services Inc., Whitesboro, NY. 3’,6’-Dihydroxy-3-oxospiro[isobenzofuran-1(3R),9’9H-xanthenel-6-carboxylicAcid Hydrazide (2). To a stirred solution of 6-carboxyfluorescein N-hydroxysuccinimide ester (1) (2.500 g, 5.29 mmol) in methanol (25 mL) was added slowly dropwise a solution of hydrazine ’
Fluorescein-labeled alkaline phosphatase
hydrate (0.270 g, 5.29 mmol) in methanol (5 mL). The solutionwas stirred for 1h a t room temperature and stored overnight at 2 “C. The precipitated product was filtered and dried a t reduced pressure: yield 1.50 g, 73 % ;lH NMR (300 MHz, DMSO-&) 6 4.59 (br, 2H), 6.57 (dd, 4 H, J = 12 Hz), 6.70 (s,2H), 7.64 (5, 1H), 8.06 (d, 1H, J = 8 Hz), 8.13 (d, 1H, J = 8 Hz), 10.02 (br, 2 H); MS (FAB) m/z 391 (M + H)+. 3’,6’-Dihydroxy-3-oxospiro[isobenzofuran-l(3R),Y9H-xanthenel-6-carboxylic Acid (Bromoacetyl)hydrazide (3). To a solution of bromoacetic acid N-hydroxysuccinimide ester (0.582 g, 2.46 mmol) in dry DMF (20 mL) was added dropwise over 2.5 h a solution of hydrazide fluorescein 2 (1.000 g, 2.46 mmol) in DMF (50 mL). The reaction mixture was stirred for a further 4 h and evaporated on the rotary evaporator. The product was chromatographed on silica gel using a 5-20 % gradient of methanol in methylene chloride as the eluant to yield 0.500 g (40%) of 3. Silica TLC showed a single spot, Rf 0.37 CH2C12/CH30H (4/1): ‘H NMR (300 MHz, DMSO&) 6 3.95 (s, 2H), 6.59 (dd, 4 H, J = 8 Hz), 6.70 (s, 2 H), 7.72 (s, 1H), 8.12 (d, 1H, J = 8 Hz), 8.18 (d, 1 H, J = 8 Hz), 10.16 (s, 2 H); MS (FAB) m/z 513 (M H)+. 3’,6’-Dihydroxy-3-oxospiro[isobenzofuran-1(3R),Y9H-xanthenel-6-carboxylicAcid 2-(Mercaptoacety1)hydrazide (5). To a solution of 3 (0.250 g, 0.490 mmol) in DMF (0.75 mL) was added a solution of sodium thiophosphate (0.182 g, 0.490 mmol) in water (3 mL). The mixture was stirred for 20 h after which the solvents were removed under reduced pressure. The residue was dissolved in water (2 mL), diluted to 150 mL with ethanol, and cooled in the refrigerator. The precipitated solid was removed by filtration, and the filtrate was concentrated to 5 mL. Product was precipitated out by addition of 200 mL of diethyl ether to yield 0.220 g (89%) of 5. TLC analysis revealed a single, fluorescent spot, Rf 0.40, CH1CldCH30H (4/1),1% CH3COOH V/V:‘H NMR (300MHZ, CD30D) 6 3.61 (s, 2H), 6.62 (d, 4H, J = 12 Hz), 6.92 (d, 2 H , J = 9 Hz), 7.75 (s, lH), 8.08 (s, 2H); MS (FAB) m/z 463 (M - H)+. Anal. Calcd for C23H16N207S-2H20: C, 55.20; H, 4.02; N, 5.60. Found: C, 55.39; H, 4.02; N, 5.84. 3’,6’-Dihydroxy-3-oxospiro[isobenzofuran-1(3R),9’9H-xanthenel-6-carboxylicAcid 2-[ (Phosphonothio)acetyllhydrazide, Disodium Salt (4). This compound was prepared like 5 except the mixture of 3 (0.100 g, 0.195 mmol) and sodium thiophosphate dodecahydrate (0.0772 g, 0.195 mmol) was stirred in aqueous DMF for only 20 min. The solvents were removed under reduced pressure, and the residual solid was dissolved in 4 mL of MeOH, diluted to 200 mL with acetone, and cooled in an ice bath. The product was filtered out and dried under vacuum to yield 68 mg (0.063 mmol, 33%) of 4. Repeated recrystallization from a minimal volume of MeOH yielded sample used for elemental microanalysis. Silica TLC done in solvents of increasing polarity revealed a fluorescent spot a t the origin, without any higher Rfcomponents: lH NMR (300 MHz, CD30D) 6 3.44 (d, 2H, J = 15.4 Hz), 6.54 (d, 4H, J = 12 Hz), 7.03 (d, 2H, J = 9 Hz), 7.75 (s, lH), 8.06 (s, 2H); 31PNMR (CD30D) 6 18.12 (s), H3P04as external standard; MS (FAB) m/z 465 (M - PO3)+;IR (KBr) 3240,
+
Bioconjugate Chem., Voi. 5,
Thiolate and Phosphorothioate Fluorescein Labels
1570, 1460, 1385, 1120, 1090, 962 cm-l. Anal. Calcd for C23H15N20&3PNa2.4HpO.4NaBr: C, 25.85; H, 2.17; N, 2.62. Found: C, 25.74; H, 2.20; N, 2.95. Labeling of Anti-hCG IgG with Mercaptoacetyl Fluorescein 5 . ( a ) Functionalization of Anti-hCG IgG with 30-Atom Linker Maleimide. Anti-hCG IgG (1.3 mL of 6.6 mg/mL stock solution) was diluted to 2.0 mL with 0.1 M phosphate buffer, pH 7.0. This solution was concentrated to 200 pL using an Amicon Centricon concentrator equipped witha 30 000 MW cutoff membrane and diluted with 2.0 mL of the buffer, and the procedure was repeated three more times to purify the protein. Protein concentration was 7.05 mg/mL as determined by the Warburg-Christian method (11). To that buffered pH 7.0 solution of IgG (1.13 mL) was added 0.720 mg (20 equiv/protein) of the 30-atom maleimide linker in DMF (150 pL). The solution was incubated for 1 h at room temperature while rotating at 100 rpm, after which the conjugate was chromatographed on a G-25 column using 0.1 M phosphate buffer, pH 7.0. The fractions containing the protein were collected and pooled. ( b ) Conjugation of Mercaptoacetyl Fluorescein 5. To 0.90 mL of pH 7.0 buffered 2.23 mg/mL solutions of the 30-atom linker derivatized IgG were added 20,40, or 100 pL of 2.0 mg/mL (5, 10, or 25 equiv/IgG) solutions of 5. The solutions were incubated overnight at 5 "C while rotating at 100 rpm. The conjugates were chromatographed on a G-25 column using pH 7.0 phosphate buffer as eluant. Collected 25-drop fractions were examined at Apso and A490 for the presence of protein and fluorescein derivative, respectively. The appropriate fractions were pooled and examined by HPLC using a Bio-Rad Bio-Si1 SEC-125 column. ( c ) Determination of the Number of Fluorophore LabelslIgG by UVlvis Spectroscopy. IgG concentrations in the pooled fractions above were determined by BioRad Protein Assay, based on the Bradford dye-binding procedure (12). From the standard curve of protein concentration vs absorbance, the value of IgG concentration of the pooled fractions was 0.43 mg/mL (2.9 X lo4 M) for the 25 equiv of 5/IgG prep. From the plot of fluorophore concentration vs absorbance at X = 490 nm and the measurement of absorbance at that wavelength of the pooled labeled protein fractions, the number of fluorophores/IgG was determined. ( d ) Determination of the Number of Fluorophore LabelslIgG by Fluorescence Spectroscopy. Fluorescence measurements were made at submicromolar concentrations of the fluorophore in order to avoid inner filter effect (13 ) . The spectra were acquired at A,, = 490 nm and A,, = 517 nm. From the standard plot of the fluorescence vs concentration of mercaptoacetyl fluorescein 5 and the measurements of the fluorescence of the pooled antibody fractions, the number of fluorophores/IgGwas measured. Labeling of Calf-Intestinal Alkaline Phosphatase with Phosphorothioate Fluorescein 4. (a) Functionalization of Calf-Intestinal Alkaline Phosphatase with 30-Atom Maleimide Linker. Calf-intestinal alkaline phosphatase (1.5 mL of 10 mg/mL solution) was diluted to 2.0 mL with pH 7.0 phosphate buffer containing 0.1 M NaC1, MgCl2, and ZnClp and concentrated down to 200 pL using an Amicon Centricon concentrator equipped with a 30 000 MW cutoff membrane, and rediluted to 2.0 mL with phosphate buffer, and the procedure was repeated two more times to purify the protein. Protein concentration was 9.00 mg/mL by the Warburg-Christian method (11). To 1.33 mL of the buffered solution of that protein, 2.70 mg (50equiv/enzyme)of the 30-atom maleimide linker
No. 1, 1994 33
in 300 pL of DMF was added, and the solution was incubated for 1 h at room temperature while rotating at 100 rpm. The conjugate was then chromatographed on a G-25 column, and fractions containing protein were collected and pooled. Quantitation of TNBS reactive amines revealed that only seven amines were titratable in the linker-functionalized enzyme while 20 could be titrated in the native enzyme. ( b )Conjugation of Phosphorothioate Fluorescein 4. To three aliquots of 30-atom maleimide linker functionalized alkaline phosphatase (1.40 mL of 2.5 mg/mL solutions) was added 0.686,1.37, or 2.06 mg of 4 in 900 pL of pH 7.0 phosphate buffer (27, 55, or 82 equiv/protein). The solutions were incubated at 5 "C while rotating at 100 rpm. After 48 h the material was fractionated on a G-25 column. The fractions which showed absorbance at both 280 and 490 nm were collected, pooled, and examined on a size exclusion Bio-Rad Bio-Si1 SEC 400 HPLC column. ( c ) Determination of the Number of Fluorophore LabelslAlkaline Phosphatase by U V / v i s Spectroscopy. Protein concentrations of the pooled fractions above were determined as for the case of IgG. From the standard curve of protein concentration vs absorbance, the values of alkaline phosphatase concentrations of the pooled fractions were 7.73 X lo4, 8.20 X lo4, and 6.93 X lo4 M for preps run with 27, 55, and 82 equiv of 4lalkaline phosphatase, respectively. From a standard plot of fluorophore concentration vs absorbance at ,A, = 490 nm and the determination of the absorbance of the pooled labeled protein fractions at that wavelength, the number of fluorophores/alkaline phosphatase was determined. ( d ) Determination of the Number of Fluorophore LabelslAlkaline Phosphatase by Fluorescence Spectroscopy. This was done following the same technique as described above for IgG labeling. Measurement of the Relative Quantum Yield of 5. The quantum yield of this fluorophore relative to 6-carboxyfluorescein (6-CF) was determined by the quotient of the integrated emission intensities over all wavelengths of mercaptoacetyl fluorescein 5 and 6-CF (14) QFS/QF&cF= 15/16-CF(oD6-CF/oD5) where 15 and I6-CF refer to the integrated emission intensities of the sample and standard, respectively. All solutions were 0.10 M phosphate buffer, pH 7.0. RESULTS
The synthesis of the novel fluorophores 4 and 5 is depicted in Scheme 2. Hydrazinolysis of 6-carboxyfluorescein N-hydroxysuccinimide ester 1in methanol yielded hydrazide fluorescein 2, which was subsequently reacted with bromoacetic acid N-hydroxysuccinimide ester in dry DMF. The resulting (bromoacety1)hydrazidefluorescein 3 was reacted with 1 equiv of sodium thiophosphate in aqueous DMF yielding phosphorothioate fluorescein 4. The cleavageof the phosphate was done by aqueous dilute acid at pH 4-5 (6,15,16),by prolonged stirring in a neutral aqueous solution, or enzymatically by alkaline phosphatase-catalyzed hydrolysis at pH 7.0. The resulting mercaptoacetyl fluorescein 5 was completely free of the difluorescein disulfide as demonstrated by the following experiments. When a 1:l aqueous methanolic solution of an aliquot of 5 was incubated with a 20-fold excess of N-ethylmaleimide for 1 h at room temperature, TLC of the reaction solution revealed complete disappearance of the original single spot at Rf 0.40, CHpClp/CH30H (4/1), 1%CH3COOH v/v, and concomitant appearance of a new fluorescent spot at Rf 0.72. In a second experiment,
34
Bioconjugate Chem., Vol. 5, No. 1, 1994
Blenlarz et ai.
Scheme 2
H2N-NH2
H2N- HN
0
0
0
1
2
3
alkallne phosphatase * or dilute acid
0
5
4
Scheme 3
YNH2 +
30 atom linker 20 equiv/lgG
Mercaptoacetyl fluorescein labeled IgG
aqueous solution of 5 was oxygenated by bubbling air into the solution through a micropipette. After 24 h the starting material was almost completely oxidized; the new spot, Rf 0.00 showed greatly diminished fluorescence. The compounds 4 and 5 were stored lyophilized at room temperature for periods of several months without any decomposition, We have first exploited the nucleophilicity of the novel mercaptoacetyl fluorescein derivative 5 for labeling of a model IgG molecule, anti-hCG, prefunctionalized with extended length heterobifunctional maleimide active esters (30-atom linkers). In an earlier work we demonstrated that this extended length coupling agent, succinimidyl4-[(N-maleimidomethyl)tricaproamido]cyclohexane-1-carboxylate, offers several advantages as a
coupling agent over the shorter, more hydrophobic heterobifunctional reagents used in the past (IO). Scheme 3 shows the construction of the conjugate. IgG was first derivatized with a 20-fold molar excess of the 30-atom heterobifunctional maleimide succinimide active ester. The maleimide ring is known to be unstable at neutral or higher pH (I7).Consequently,the maleimidefunctionalized protein was never stored in buffered solution for longer than 24 h. Instead, it was chromatographed on a size-exclusion column in order to remove the unreacted heterobifunctional reagent and was used immediately in the thiolation step. In preliminary experiments (data not shown), we determined that a 25-fold molar excess of 5 over IgG resulted in optimal labeling.
Thlolate and Phosphorothloate Fluorescein Labels
Bioconjugate Chem., Vol. 5, No. 1, 1994 35
QPn.(I
i n
-1
0.9
s
9 g c
P n
280nm
0.3 0.2
U
0.1 nn "."
300
400
500
600
Wavelength (nm)
Figure 1. Dependence of the absorption spectra of 1.0 X 10J M mercaptoacetyl fluorescein 5 upon the pH. The sigmoidal fit of the absorbances vs pH yielded a pK, value of 6.4 for the formation of the dianion of 5.
This stoichiometry allowed a controlled labeling of protein by the fluorophores a t the optimal 2:l ratio of the label per IgG, thus avoiding ambiguities associated with fluorophore self-quenching and impairment of the antibodybinding capacity observed with overmodification. Sizeexclusion HPLC chromatogram of unlabeled and fluorophore-labeled anti-hCG IgG revealed two peaks, at 7.74 and 6.76 min, respectively. Integration of the peak areas at AZWshowed 71% fluoresceinated IgG and 29% unmodified IgG. In a 0.1 M phosphate buffer at pH 7.0 and at X 490 nm, 5 has an apparent extinction coefficient e490 = 23 064 M-l cm-l. The dependence of the absorbance spectra of 1 X M 5 upon the pH is shown in Figure 1. These spectral changes reflect the equilibria between the dianion and monoanion of the fluorescein derivative 5 and are consistent with the data reported in the literature for other fluorescein derivatives (18,19). From the above data we determined the pK, for the formation of the most intensely chromophoric dianionic species of 5 to be 6.4, as compared to 6.5 of the carboxyfluorescein (19). This reagent is a strong fluorophore, with A,, = 494 nm and A,, = 517 nm. The quantum yield of fluorescence of 5 relative to 6-CF measured as described in the Experimental Procedures is QB/Q~-cF = 0.22. In order to study the effect of the mercaptan substituent of 5 on its fluorescence and extinction coefficient, 5 X 10-5 M pH 7.0 solution of 5 was incubated with 5 X 10-3 M N-ethylmaleimide. The solution was examined by periodic absorbance and fluorescence scans over a period of 24 h. There was no change in fluorescenceor absorbance profiles of 5. Figure 2 depicts the plot of absorbances at and A490 of G-25 eluted fractions. Since the molar absorptivity of this fluorophore at 490 nm is four times larger than at 280 nm, only 6% of the absorbance at 280 nm should be due to the conjugated fluorophore, the remaining 94% being attributable to the IgG in the fraction. The unconjugated fluorophore elutes at fraction number above 20 (data not shown). Since our interest centers around the use of these novel mercaptofluoresceins as protein labels, we required a controlled conjugation of these fluorophores to the protein. In order to ascertain the fluorophore/IgG ratio, the quantitation based on absorbance measurements was compared with fluorescence determination. The measurements of the IgG concentrations using Bio-Rad Protein Assay were done by constructing a standard curve of A594 us anti-hCG IgG which gave a linear fit (R2= 0.996) over a 10-fold range of IgG concentrations and reading the unknown concentrations off the curve. On the basis of
Fraction Number
Figure 2. Plots of absorbances at 280 and 490 nm of G-25eluted fractions of 5/anti-hCG conjugates. IgG was prefunctionalized with 20 equiv of the 30-atom maleimide heterobifunctional linker and incubated at 5 OC with 4.0 X le7M (25 equiv) of 5. Fractions 10-12 were collected,pooled, and examined by HPLC. Integration for the peak areas at Am determined 71 % fluoresceinated IgG and 29 % unmodified IgG. Table 1. Number of Fluorophore Labels Introduced into Two Different Proteins, Anti-hCG (Experiment 1) and Bovine Calf Intestinal Alkaline Phosphatase (Experiment 2) as a Function of the Stoichiometry of 5 and 4 with Respect to Anti-hCG and Alkaline Phosphatase, Respectively, As Determined by Absorbance (A) and Fluorescence (F) Measurements experiment 1 equiv of 5/IaG - used 5 10 25
no. of 5/IgG by A by F 0 1 2
1 2
3
experiment 2 no. of 4Jdk phos bv A bv F
equiv of 4/alkDhos used 21 55 82
7 7 8
4
4 5
our determined value of the apparent extinction coefficient of 5 and the IgG concentrations, the number of fluorophores introduced per mole of IgG was calculated at three different stoichiometries of 5/IgG. Similarly, a fluorescence standard curve of 5 allowed the determination of the number of fluorophores per IgG. The results of the labeling of IgG by thiolated fluorophore are shown in Table 1. Clearly, the level of the fluorescein incorporation into the protein increases as the number of equivalents of the fluorophore per mole of IgG is increases. When 5 equiv of 5 per IgG is used, only very low levels of fluoresceination is achieved. Increasing the ratio of 5/IgG to 10or 25 results in introduction of one to three fluorescent labels per IgG. In the following text we show that the precursor of 5 , 3',6'-dihydroxy-3-oxo-2- [(phosphonothio)acetyllspiro [isobenzofuran-l(3H),9'-9H-xanthenel-6-carboxylic acid hydrazide, disodium salt, the phosphorothioate fluorescein 4, is also a very useful fluorophore marker of proteins. Scheme 1 summarizes the process of selfcatalyzed labeling of a maleimide prefunctionalized alkaline phosphatase by 4. We derivatized bovine alkaline phosphatase with extended length heterobifunctional maleimide active ester (30-atom linker) as described in the Experimental Procedures. The residual activity of the derivatized enzyme was 81% of the native alkaline phosphatase activity. When an aqueous solution of 4 is exposed at pH 7-9 to the action of the maleimidederivatized alkaline phosphatase, the latter catalyzes very efficiently the hydrolysis of the sulfur-phosphorus bond of the phosphorothioate, generating mercaptoacetyl fluorescein 5 described above. In preliminary experiments with several alkyl phosphorothioates we determined that
36 Bloconjugate Chem., Vol. 5, No. 1, 1994
Bieniarz et
181
14i
I
\
l
Fraction Number
Figure 3. Plots of absorbances at 280 and 490 nm of G-25eluted fractions of the fluorophore-labeled alkaline phosphatase. Alkaline phosphatase was prefunctionalized with 50 equiv of the 30-atom maleimide heterobifunctional linker and incubated at 5 OC for 48 h with 1.9 X 10-6mol (82 equiv) of 4 according to the method depicted in Scheme 1. Since the molar absorptivity of this fluorophore at 490 nm is four times larger than at 280 nm, 17%of the absorbance at 280 nm should be due to the conjugated fluorophore,the remaining 83% being attributable to the alkaline phosphatase in the fraction. The unconjugated fluorophoreelutes at fraction number above 20. Fractions 8-11 were collected, pooled, and analyzed by HPLC. at pH 7.0 the half-life of the alkaline phosphatase-catalyzed release of thiolate is approximately 15 min while the controls in absence of enzyme revealed virtually no free thiols even after 1-2-h incubation. The resulting mercaptoacetyl fluorescein nucleophile reacts with the maleimide electrophile on the alkaline phosphatase, completing the conjugation. We were interested in ascertaining the number of fluorophores which may be conjugated to alkaline phosphatase by this method without significantly compromising the catalytic activity of the enzyme, while simultaneously eliciting the highest possible fluorescent signal from the labels. We show in Table 1that increasing the stoichiometric ratio of 4 to alkaline phosphatase resulted in higher levels of incorporation of the fluorophores. Interestingly, the conjugation of the fluorophore to the maleimide-derivatized enzyme caused more pronounced loss of enzymatic activity than the loss resulting from the derivatization of the enzyme with the 30-atom linker alone. Approximately 70% of the native enzyme activity was lost after 27, 55, or 82 equiv of 4 per alkaline phosphatase was used as compared to only 20 % loss upon derivatization with 30-atom linker. The extinction coefficient of 4 in 0.1 M phosphate buffer at pH 7.0 is €490 = 53 636 M-l cm-l, 2.3 times higher than the value of the apparent extinction coefficient of 5. Under these conditions, A,, = 496 nm and A,, = 517 nm. Figure 3 shows the plot of absorbances at and A490 of G-25-eluted fractions of the labeled alkaline phosphatase. Since the molar absorptivity of this fluorophore at 490 nm is four times larger than at 280 nm, comparison of the values of the maxima in the plots of Figure 3 shows that 17% of the absorbance at 280 nm should be due to the conjugated fluorophore, the remaining 83 % being attributable to the alkaline phosphatase in the fraction. The unconjugated fluorophore elutes at fraction number above 20 (data not shown). Fluorescence spectra of the compounds 4 and 5 at pH 7.0 are shown in Figure 4. The relative fluorescence intensity 1 4 / 1 5 is 5.0. We interpret this markedly lower fluorescence of the mercaptoacetyl fluorescein 5 as compared to its phosphorothioate precursor 4 as well as the
al.
comparatively low apparent extinction coefficient of 5 in the Discussion below. Table 1shows that the self-catalyzed functionalization of alkaline phosphatase (experiment 2) is a very efficient process. Between seven and eight fluorophores were introduced into the alkaline phosphatase, as determined by absorbance measurements. TNBS titration of the accessible amines of the native alkaline phosphatase evidenced 20 amines, while after the derivatization of the enzyme with the 30-atom heterobifunctional maleimide linker, only seven amines could be titrated. This implies that an average of 13maleimide linkers were incorporated into alkaline phosphatase and up to 60% of these were labeled with the fluorophore 4. Fluorescence-based measurements were consistently lower than the absorbance readings, as opposed to experiment 1,where the absorbance readings were consistently lower than the fluorescence readings. This seeming discrepancy is explainable and will be addressed in the Discussion. DISCUSSION
The two new fluorescein derivatives, phosphorothioate fluorescein 4 and mercaptoacetyl fluorescein 5 , allow nucleophilic attachment of the fluorescein molecules to the electrophile-functionalizedproteins, thus enriching the repertoire of methods available to the chemist for fluorescence labeling of biologically relevant molecules. The labeling of proteins by thiolation of their lysines with iminothiolane followed by the reaction with iodoacetylated fluorescein has been used frequently in the past (20,21). However, that method does not allow spacial separation between the iminothiolane-derivatized protein and the fluorophore label. By functionalizing the protein with the extended arm 30-atom maleimide linker and building phosphate-protected thiolate into the fluorophore we achieved greater control of the distance between the label and the protein as well as improved control of the bioconjugation process. Often it is desirable to detect and quantitate the number of maleimide or haloacetyl linkers introduced into a protein in the first stage of the conjugation process. In the concluding paragraph of the Results we have demonstrated the utility of these fluorescein derivatives for that purpose. The derivative 4, a phosphate-protected mercapto fluorescein, is particularly useful in alkaline phosphatase-catalyzed deprotection and labeling, depicted in Scheme 1. We have been inspired in the design of this methodology by the reported excellence of alkyl and aryl phosphorothioates as substrates for alkaline phosphatase (7). Although thiols are classically protected as S-acetyl, S-benzoyl (221, unsymmetrical disulfides (23))or thiosulfates (24),these methods, which originate from organic synthesis, require harsh deprotection conditions and yield byproducts which have to be removed before reaction with the protein. In contrast, the phosphorothioates treated by buffered solutions of alkaline phosphatase yield thiolates very efficiently, as demonstrated by experiments summarized in Table 1. Moreover, the byproduct is an innocuous phosphate ion which need not be removed since it does not affect the conjugation process. We were interested in establishing the limits of conjugation stoichiometry for the two fluorophores. Table 1 summarizes the results of the direct labeling of the maleimide derivatized anti-hCG IgG with 5 (experiment 1)and self-catalyzed labeling of alkaline phosphatase with 4 (experiment 2). In experiment 1 we aimed at labeling the IgG molecule with very few fluorophores so as not to impair the antibody binding capabilities. We found that
Thiolate and Phosphorothioate Fluorescein Labels
Bioconjugate Chem., Vol. 5, No. 1, 1994 37
r
350
100
400
450
500
550
600
650
350
400
450
500
550
600
650
Wavelength (nm) Figure 4. Excitation and emission spectra of 2.8 X lo4 M phosphorothioate fluorescein 4 (left) and 1 X lV M mercaptoacetyl fluorescein 5 (right). Both spectra were acquired in a 0.1 M phosphate buffer at pH 7.00. Relative fluorescence intensity Z,/Zs is 5.0.
functionalization of IgG with 20 equiv of the 30-atom heterobifunctional maleimide active ester followed by conjugation with fluorophore 5 allows incorporation of one to three fluorophores per IgG depending on the ratio of fluorophore to IgG employed in each conjugation. Under the conditions of our experiments, absorbancebased determinations of the number of fluorophores per IgG yielded lower values than the fluorimetric determination. This most likely is due to the error in the Bradford dye-binding procedure (12) because of reading very low absorbance values of the fluoresceinated antibody at 490 nm and also possibly to the overestimate of the IgG concentration after its derivatization with the 30-atom heterobifunctional linker reagent. We showed that mercaptoacetyl fluorescein 5 was completely free from dimeric disulfide oxidation impurities as demonstrated by spot to spot TLC conversion of the compound 5 to its N-ethylmaleimide adduct of higher Rf, the oxidation of the compound 5 to a new compound of Rf 0.00, and very low fluorescence. Indeed, dimers of fluorescein linked through coupling arms possessing disulfides have been reported to have very low quantum yields, 0.11 and 0.17, for difluorescein disulfide and N,N’-difluorescein thiocarbamylcystamine, respectively, presumably due to internal quenching through aromatic stacking interactions ( 2 5 ) . Micromolarconcentrations of fluorophoresmay be reliably determined fluorimetrically provided no inner filter effect or other forms of fluorescence quenching are present (26). Thus, we believe that in case of low levels of fluorophore functionalization the fluorimetric determination is more reliable. In experiment 2, summarized in Table 1,we aimed at maximal functionalization of the protein calf intestinal alkaline phosphatase with phosphorothioate fluorescein 4. We found that the self-catalyzed labeling of alkaline phosphatase is an efficient process, seven to eight fluorophores being incorporated into the protein. We have previously determined that derivatization of this enzyme with 50 equiv of 30-atom maleimide heterobifunctional reagent causes only about 20% loss of the enzymatic activity. Since 30% of the native unlabeled alkaline phosphatase activity remained regardless of whether 27, 55, or 82 equiv of 4 was employed, this invariance of the
residual enzymatic activity on the stoichiometry of 4 used in the labeling suggests that the additional loss of the enzymatic activity most likely occurred as the result of covalent attachment of fluorophore to the enzyme. Unlike in experiment 1, in experiment 2 the fluorimetric determination of the number of incorporated fluorophores yielded about 40% lower values as compared to the determinations done by absorbance. The number of incorporated fluorophores as measured fluorimetrically stays approximately constant yielding values between four and five fluorophores per protein. However, the determinations performed by absorbance reading yielded values of seven to eight fluorophores per protein. We attribute the discrepancy between the fluorescence and absorbance measurements to the concentration quenching of the fluorescein labels on the surface of the enzyme (26). Since the molecular radius of the alkaline phosphatase is approximately 30A ( 2 3 ,eight molecules of 4 on the surface of the enzyme correspond to an effective molarity of the fluorophore of approximately 1.3 X lo4 M, aconcentration at which the inner filter effect indeed should be expected to introduce significant errors in the measurements (28). Thus, at higher concentrations of the fluorophores, we believe that the values determined by absorbance readings are more reliable. An intriguing feature of mercaptoacetyl fluorescein 5 is its markedly lower extinction coefficient as compared to the parent carboxyfluorescein at pH 7.00. Thus, we measured the extinction coefficient of mercaptoacetyl fluorescein 5 to be 35% and 44% of the extinction coefficients of 6-CF and phosphorothioate fluorescein 4, respectively, at pH 7.00. Since our measurement of the pK, = 6.4 for the dianionic 5 is so close to the reported value of pKa = 6.5 for 6-CF, the differences in the values of the extinction coefficients cannot be explained in terms of differences in ionization of the xanthene phenols. It is also unlikely that this discrepancy is due to the formation of the spiro lactone of the phthalate carboxylate at C9’ in the compound 5 because the extinction coefficient of 4 at 53 636 M-l cm-l is 2.3 times higher than the value of the apparent extinction coefficient of 5. We explain these
Bieniarr et al.
38 Bioconlugate Chem., Vol. 5, No. 1, 1994
Scheme 4
0
J+&-
\ 2
”
ZWITTERIONIC DIANION MONOMER
discrepancies in terms of the intermolecular dimerization of the compound 5 as depicted in the Scheme 4. The intermolecular nucleophilic attack of the side chain thiolate on C9’ tertiary carbonium ion is likely in view of the known and analogous reactivity of the nucleophiles at C9’ in the acridinium series and the equilibrium between acridinium esters and their colorless, non-chemiluminescent pseudobases (29, 30). The extent to which the equilibrium is shifted to the dimer pseudobase form should manifest itself in the correspondingly lower apparent extinction coefficient a t 490 nm and also proportional diminution of the fluorescence quantum yield of this fluorophore. The intramolecular thiolate addition to C9’ position can be discounted on steric and thermodynamic grounds. We obtained support for this interpretation by reexamining the MS FAB(-) spectrum of 5 in nitrobenzyl alcohol matrix, which showed weak ions at mlz 925 and 947 corresponding to MW of 926 and 948 for the dimer of 5 and its sodium salt. That these did not originate from the difluorescein disulfide was demonstrated in our TLC experiments which clearly indicated presence of free thiol. It is likely that during the isolation of the compound 5 from methanolic ether the less soluble dimeric or even trimeric form of 5 crystallized out. On silica gel or in MS FAB only the monomeric 5 would be observed. The incubation of pH 7.0 solution of 5 with 100-fold molar excess of N-ethylmaleimide led to no change in the fluorescence intensity of 5. This suggests multimeric structure of 5 in which the inner filter effect quenching of the fluorescence would be responsible for the markedly depressed fluorescence of 5. Although several sulfurcontaining fluorescein derivatives have been shown to have strongly reduced fluorescence due to collisional intramolecular quenching between the xanthene moiety and thiolate ion on the substituent chain-apparent quantum yields between 0.01 and 0.11 (25, 26)-this last factor probably plays a much smaller role in the reduced fluorescence of 5 as compared to the proximity effects of the fluorophores in the dimeric or trimeric form of 5. Since our structural data of this compound could not discern between monomeric,dimeric, or higher aggregates, we report our results in terms of the apparent extinction coefficient of 5. Thus, if the real structure of 5 is dimeric, the values in experiment 1of Scheme 1would be half of those reported. Although the present work describes a self-catalytic conjugation of a phosphorothioate derivative
\
PSEUDOBASE DIMER
of a fluorophore to alkaline phosphatase, we have exploited the use of alkaline phosphatase as catalyst in unmasking thiolate for subsequent conjugation of the thiolate to suitably derivatized biological compounds, i.e., antibodies, haptens, and other enzymes. This work is in progress and will be reported in due course. ACKNOWLEDGMENT We are grateful to Dr. Susan J. Tomazic-Allen for critical reading of the manuscript. We also thank Dr. Jeffrey Huff of the Department of Immunochemistry, Abbott Diagnostics Division, and Professor Richard G. Lawton of the University of Michigan Department of Chemistry for stimulating discussions. Supplementary Material Available: Absorbance at 490 nm vs concentration plots of 6-carboxyfluorescein, mercaptoacetyl fluorescein 5, and phosphorothioate fluorescein 4 in 0.1 M phosphate buffer pH 7.0 (1page). Ordering information is given on any current masthead page. LITERATURE CITED (1) (a) Means, G. E., and Feeney, R. E. (1990) Chemical modification of proteins: history and applications. Bioconjugate Chem. 1, 2-12. (b) Kitagawa, T., Shimozono, T., Aikawa,T., Yoshida,T.,and Nishimura, H. (1981)Preparation
and characterization of heterobofunctional crosslinking reagentsfor protein modification. Chem.Pharm. Bull. 29,11301135. (c) Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T. (1983)Enzyme-labeling of antibodies and their fragments for enzymeimmunoassaysand immunohistochemical staining. J. Immunoass. 4 , 209-327. (2)Cleland, W. W. (1964)Biochemistry 3,480-482. (3) (a) Jue, R., Lambert, J. M., Pierce, L. R., and Traut, R. R. (1978) Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemistry 17,53995406. (b) Hillel, S.and Wu, C. W. (1977)Subunit topography of RNA polymerase from Escherichia coli. A cross-linking studywith bifunctionalreagents. Biochemistry 16,3334-3342. (c) Carlsson, J., Drevin, H., and Axen, R. (1978)Protein thiolation and reversible protein-protein conjugation N-succinimidyl3-(2-pyridildithio)propionate,a new heterobifunctional reagent. Biochem. J. 173,723-737. (4)Duncan, R.J. S.,Weston,P.D., and Wrigglesworth,R. (1983) A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132,68-73.
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