ANALYTICAL
BIOCHEMISTRY
194,
77-81
(1991)
Synthesis of a Reagent for Fluorescence-Labeling of Vitamin D and Its Use in Assaying Vitamin D Metabolites Masato
Shimizu,
Shinichi
Kamachi,*
Yasuho
Nishii,*
and Sachiko
Yamadal
Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kano!u, Chiyoda-ku, Tokyo 101, and *Biosensor Laboratories Co., Ltd. 3-41-8 Takada, Toshima-ky Tokyo 171, Japan
Received
June
4, 1990
The fluorogenic dienophile 1,2,4-triazoline-3,5dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25Hydroxyvitamin D, , la,25-dihydroxyvitamin D,, and 24(R),25-dihydroxyvitamin D, reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that la,25-dihydroxyvitamin D, at a concentration as low as lo-* M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using highperformance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQTAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite. o ISPI Academic PWS. IIIC.
know accurately the concentrations of the active and other major metabolites in human plasma in various clinical situations. The competitive binding assay currently in use lacks specificity, so it is troublesome and requires special techniques (2,3). The precision of this assay is poor as recent interlaboratory studies have demonstrated (4,5). Though the gas chromatographymass spectrometry is a definitive method, it has many difficulties when used for routine vitamin D assay. The need for a convenient, precise, and highly sensitive method for assaying vitamin D metabolites has prompted us to develop a novel fluorescence-labeling reagent that targets the s-cis-diene part of vitamin D. We synthesized fluorogenic dienophile 4-[2-(6,7-dimethoxy - 4-methyl-3-oxo-3,4dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD) as a reagent to detect vitamin D in high specificity, and used it in a new fluorometric assay of the major vitamin D metabolites, 25-hydroxyvitamin D, (25-(OH)D,), 1,25(OH),D,, and 24(R),25-dihydroxyvitamin D, (24,25(OH),D,). This is the first practical fluorometric method for assaying vitamin D metabolites. MATERIALS
It is now well recognized that the active vitamin D, metabolite la,25dihydroxyvitamin D, (1,25-(OH),D,)2 is a multifunctional steroid hormone regulating not only calcium metabolism but also proliferation and differentiation of a variety of its target cells (1). To fully understand the biological role of vitamin D, it is important to
1 To whom correspondence should be addressed. ’ Abbreviations used: 1,25-(OH),D,, la,25dihydroxyvitamin D,; DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dhydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,Sdione; 25(OH)D,, 25-hydroxyvitamin D,; 24,25-(OH),D,, 24(R),25dihydroxyvitamin D,; DMF, dimethylformamide; DPPA, diphenylphosphoryl azide; DMSO, dimethyl sulfoxide. 0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
AND METHODS
General Procedure Melting points were obtained on a Yanagimoto MR melting point apparatus and are uncorrected. Mass spectra (at 70 eV) were measured on a Jeol DX-300 spectrometer. Relative intensities are given in parentheses. Infrared spectra (ir) were obtained with KBr disks on a JASCO FT/IR-7000 spectrometer. Proton nuclear magnetic resonance spectra (‘H NMR) were recorded with a Jeol GX-270 spectrometer. Chemical shifts are reported in parts per million downfield from tetramethylsilane, and coupling constants are reported in hertz. Fluorescence spectra were recorded on a Shimazu RF-5000 spectrofluorophotometer. Ultraviolet spectra (uv) were 77
Inc. reserved.
78
SHIMIZU
obtained on a Hitachi U-3200 spectrophotometer. High-performance liquid chromatography (HPLC) was performed with a JASCO 880-PU equipped with a solvent programmer 801-SC and a fluorescence detector 821-FP (flow-cell volume, 16 ~1) or a Tosoh CCPM with a solvent programmer PX-8010 and a fluorescent detector FS-8010 (flow-cell volume, 16 ~1).
Chemicals 25-(OH)D, and 1,25-(OH),D, were purchased from Philips Duphar (Amsterdam). 24,25-(OH),D, was kindly donated by Kureha Chemical Industry (Tokyo Japan). Methanol-free dichloromethane was used for the synthesis and reactions of DMEQ-TAD: A reagent grade dichloromethane was washed successively with concentrated sulfuric acid, water, 5% Na,CO,, and water, dried over calcium chloride, and distilled from CaH,.
Synthesis
of Fluorogenic Dienophile DMEQ-TAD
6,7-Dimethoxy-3-oxo-3,4-dihydroquinoxaline-2-propionic acid (I) (7,s). A solution of dinitroveratrole (0.91 g, 4 mmol) in ethanol (80 ml) was shaken in the presence of PtO, (89 mg) under hydrogen atmosphere until the theoretical amount of hydrogen (540 ml) was absorbed. The reaction mixture was filtered directly into a flask containing 2-ketoglutaric acid (0.58 g, 4 mmol) under nitrogen atmosphere and the mixture was refluxed for 1.5 h. After cooling, the precipitated crystals were filtered to give I (0.80 g, 72%). I: mp 250-252°C (ethanol); mass spectrum, m/z 278 (M+, 36), 260 (loo), 232 (88), 217 (32), 189 (31), 161 (21); ir (KBr) 1715,1644 cm-‘; ‘H NMR (CDCl,) 6 2.80 (2 H, t, J = 7.3 Hz), 3.15 (2 H, t, J = 7.3 Hz), 3.92 (3 H, s), 3.93 (3 H, s), 6.78 (1 H, s), 7.18 (1 H, s), 11.92 (1 H, br). 6,7 -Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxaline-2-propionic acid (II). A solution of I (606 mg, 2.2 mmol) in dimethylformamide (DMF) (20 ml) was added to a stirred suspension of NaH (176 mg, 7.3 mmol) in DMF (3 ml) at 0°C under nitrogen. After 30 min, methyl iodide (455 ~1, 7.3 mmol) was added to the solution and the mixture was stirred at 0°C for 1.5 h. The reaction mixture was poured into ice-water, stirred at room temperature for 30 min, acidified with 0.5 N HCl, and the crystals precipitated were filtered to give the carboxylic acid (II) (570 mg, 89%). II: mp 239-241°C (methanolCHCl,); mass spectrum, m/z 292 (M+, 53), 274 (99), 246 (loo), 231 (53), 203 (41), 175 (27); ir (KBr) 1734, 1628 cm -l; ‘H NMR (DMSO-d& 6 2.70 (2 H, t, J = 6.9 Hz), 3.00 (2 H, t, J = 6.9 Hz), 3.65 (3 H, s), 3.84 (3 H, s), 3.94 (3 H, s), 6.98 (1 H, s), 7.22 (1 H, s).
ET
AL.
1 - Ethoxycarbonyl-4-[2-(6,7-dimethoxy-4-methyl-30x0-3,4-dihydroquinoxalinyl)ethyl]semicarbazide (III). Triethylamine (0.36 ml, 2.57 mmol) was added at room temperature to a solution of the carboxylic acid (II) (500 mg, 1.71 mmol) in DMF (50 ml). Then diphenylphosphoryl azide (DPPA) (0.55 ml, 2.57 mmol) was added to the solution and the mixture was stirred at room temperature for 2.5 h. The solvent was evaporated in vacua, the residue was dissolved in benzene (20 ml) and the solution was refluxed for 1 h. The reaction mixture was cooled to room temperature and to the solution was added ethyl carbazate (178 mg, 1.71 mmol). The mixture was refluxed for 30 min and then the solvent was evaporated. The residue was chromatographed on silica gel (90 g) to give a by-product (102 mg, 18%) eluting with CHCl, and then III (394 mg, 59%) eluting with 4% methanol/CHCl, (v/v). III: mass spectrum, m/z 393 (M+, 5), 347 (0.8), 330 (l), 289 (92), 246 (47), 234 (loo), 219 (22), 205 (24); ir (KBr) 1731, 1669, 1649 cm-‘; ‘H NMR (DMSO-de)61.18(3H,t,J=6.9Hz),2.95(2H,t,J=5.9 Hz), 3.52 (2 H, m), 3.67 (3 H, s), 3.89 (3 H, s), 3.97 (3 H, s), 4.02 (2 H, q, J = 6.9 Hz), 6.40 (1 H, br), 6.91 (1 H, s), 7.31 (1 H, s), 7.70 (1 H, br), 8.65 (1 H, br). 4 -[2-(6,7-Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazolidine-3,5dione (IV). A suspension of III (272 mg, 0.69 mmol) and K&O, (191 mg, 1.38 mmol) in ethanol (20 ml) was refluxed for 6 h. The solvent was evaporated and the residue was dissolved in water (30 ml). The solution was made acidic by adding 2 N HCl and extracted with 10% methanol/ CHCl,. The extracts were dried (Na,SO,) and evaporated to give IV (222 mg) as a crystalline solid. The crude product was recrystallized from methanol/CHCl, to give IV (194 mg, 81%) as pale yellow prisms. IV: mp 250-253’C; mass spectrum, m/z 347 (M+, 5), 289 (2), 246 (loo), 231 (35), 203 (20), 175 (20), 101 (12); ir (KBr) 1694, 1640 cm-l; ‘H NMR (DMSO-$) 6 3.02 (2 H, t, J = 6.9 Hz), 3.65 (3 H, s), 3.80 (2 H, t, J = 6.9 Hz), 3.84 (3 H, s), 3.95 (3 H, s), 6.96 (1 H, s), 7.26 (1 H, s), 9.95 (2 H, s); fluorescence (ethanol) X,, 370 nm, X,, 440 nm. 4 - [2 - (6,7-Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5dione (DMEQTAD). Iodobenzene diacetate (10.0 mg, 0.031 mmol) was added to a stirred suspension of IV (8.6 mg, 0.025 mmol) in methanol-free dichloromethane (1.5 ml) at room temperature and the mixture was stirred for 3.5 h. The reaction mixture was filtered, the filtrate was stored in a freezer (-20°C) over night, and the precipitated red crystals were filtered with the aid of argon pressure to give DMEQ-TAD (6.7 mg, 79%) as red needles. DMEQ-TAD: mp 200-202°C (decompose); ir (KBr) 1785,1769,1638 cm-‘; ‘H NMR (CDCl,)G 3.29 (2 H, t, J = 6.8 Hz), 3.68 (3 H, s), 3.95 (3 H, s), 4.02 (3 H, s), 4.15 (2 H, t, J = 6.8 Hz), 6.68 (1 H, s), 7.12 (1 H, s); uv
FLUOROMETRIC
(CH,CN) 277,225
X,, nm.
526,366,295,241,212
ASSAY
OF
B. Analytical scale A solution (ethanol) of 1,25(OH),D, (7.2 X 10-l’ mol) was placed in a conical tube, and the solvent was removed completely in vacua. A solution of DMEQ-TAD (7.2 X lo-’ mol) in methanolfree dichloromethane (1 ml) was added to the tube and the mixture was stirred at room temperature. After 30 min, the excess of the reagent was quenched by adding ethanol and the solvent was evaporated. The residue was dissolved in methanol and a portion was analyzed by HPLC (column, LiChrospher RP-18(e) (Cica-Merck, Tokyo) 4 X 250 mm, 35°C; solvent, linearly programmed gradient system from 60% methanol/H,0 (v/ v) to 80% methanol/H,0 (v/v) during 40 min; rate, 1 ml/min; detector ex 370 nm, em 440 nm). The yield of the adduct (Vb) was nearly 100% as calculated on the basis of the area of the two peaks of the C(6) epimers eluted at 26.5 and 30 min. Under similar conditions, 25-(OH)D, and 24,25(OH),D, were converted to the corresponding fluorescent (Va and Vc) adduct quantitatively.
RESULTS
of the Fluorescence
The highly fluorescent synthesized in essentially
Labeling
79
D METABOLITES
nm, Xmin 468,317,
Reaction of vitamin D metabolites with the fluorescence labeling reagent (DMEQ-TAD). A. Preparative scale To a solution of vitamin D (5 pmol) in dichloromethane (0.2 ml) was added a solution of DMEQ-TAD (6 pmol) in dichloromethane (1 ml) at room temperature. The solvent was evaporated and the residue was purified on silica gel TLC (8% methanol/chloroform) or HPLC (LiChrosorb Si 60 (Cica-Merck, Tokyo), 10 X 250 mm, 8% methanol/dichloromethane) to give the adduct (V) as a mixture of two C(6) epimers (quantitative total yield). DMEQ-TAD adduct of 25(OH)D, (Va): ‘H NMR (CDCl,) 6 0.52 and0.51 (3 H (2:1), s), 1.20 and 1.21 (6H (2:1), s), 3.69 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.69 (1 H, d, J = 9.6 Hz), 4.88 (1 H, d, J = 9.6 Hz), 6.68 (1 H, s), 7.23 (1 H, s); fluorescence (ethanol) X,, 370 nm, X,, 440 nm. DMEQ-TAD adduct of 1,25-(OH),D, (Vb): ‘H NMR (CDCl,) 6 0.50 and0.51 (3 H (5:4), s), 1.21 and 1.20 (6 H (5:4), s), 3.69 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.30 (1 H, m), 4.70 (1 H, d, J = 9.9 Hz), 4.93 (1 H, d, J = 9.9 Hz), 6.68 (1 H, s), 7.23 (1 H, s). DMEQ-TAD adduct of 24,25-(OH),D, (Vc): ‘H NMR (CDCl,) F 0.54 and0.52 (3 H (2:1), s), 1.16 (3 H, s), 1.21 (3 H, s), 3.70 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.69 (1 H, d, J = 9.6 Hz), 4.88 (1 H, d, J = 9.6 Hz), 6.68 (1 H, s), 7.23 (1 H, s).
Synthesis
VITAMIN
Reagent
dienophile DMEQ-TAD was five steps in 24% overall yield
0 I : R=H II : R=Me
DMEQ-TAD
FIG. 1. Synthesis pathway of the fluorogenic dienophile (DMEQTAD). Reagents (yields): a, HJPtOJethanol then 2-ketoglutaric acid/reflux (72%); b, NaH/CH,I/DMF then H,O (89%); c, DPPA/ triethylamine/DMF, refluxlbenzene, ethyl carbazate/retlux (59%); d, K,CO,/ethanol/reAux (81%); e, iodobenzene diacetate/dichloromethane (79%).
from dmitroveratrole via the pathway shown in Fig. 1. Catalytic reduction of dinitroveratrole followed by the reaction with 2-ketoglutaric acid gave quinoxalinone propionic acid (I). After N-methylation, the carboxylic acid was converted to the semicarbazide (III) by Curtius rearrangement and then it was cyclized to give dihydro derivative (IV) of the desired reagent DMEQ-TAD. The triazolidine (IV) was oxidized with iodobenzene diacetate (9) to give the desired fluorogenic triazoline (DMEQ-TAD) as a red crystalline solid. The reagent is reasonably stable at room temperature and is expected to be stable when stored in a freezer under an inert-gas atmosphere for at least several years.
Reaction of Vitamin D Metabolites Labeling Reagent (DMEQ-TAD)
with the Fluorescence
The fluorescence-labeling reagent (DMEQ-TAD) reacted quantitatively with the three major vitamin D metabolites, 25-(OH)D,, 1,25-(OH),D,, and 24,25(OH),D,, by simply mixing a solution (dichloromethane) of the two compounds at room temperature to give the corresponding 6,19-adducts (V) (Fig. 2). The reaction was very fast: at room temperature at a concentration of more than lop4 M, all three vitamin D metabolites reacted instantaneously with an equimolar amount of DMEQ-TAD. At a concentration as low as 7.2 x lo-’ M (about 3 pg/pl, dichloromethane) with 1000 eq of DMEQ-TAD, nearly 100% of 1,25-(OH),D, was converted to the fluorescent adduct (Vb) within 30 min. 25-(OH)D, and 24,25-(OH),D, reacted similarly under
80
SHIMIZU
V 25-OH-D3 1,25-(OH&D,
: R’zR3-OH,
24,25-(OH),D,
: R’rH,
FIG.
2.
a : Rq=R2=H,
: R’=R2=H,R3x0H
Reaction
R2-H R2=R3z0H
of vitamin
b : R’:R3=OH, c:
R’=H
D metabolites
R3=0H R2=H
R%R3zOH
with
DMEQ-TAD.
similar high dilution conditions. Thus it is clear in a model system that the three major vitamin D, metabolites present in 1 ml of plasma can be converted to the corresponding fluorescent adducts in nearly quantitative yield within 30 min. Each vitamin D yields the corresponding adduct (V) as a mixture of C(6) epimers in a ratio characteristic of each vitamin D. The ratio of the two C(6) epimers of the adducts were approximately 2:l for 25(OH)D, adduct (Va), 54 for 1,25-(OH),D, adduct (Vb), and 2:l for 24,25-(OH),D, adduct (Vc) (see Materials and Methods and Fig. 3). The stereochemistry of each epimer has not been determined. A pair of the retention times as well as the ratio of the two epimers, however, were very useful to confirm the identity of the particular metabolite when they are analyzed by HPLC (see below). The adducts are all very stable, more stable than the parent vitamin D, since the light- and oxygen-sensitive conjugated triene function of vitamin D was masked in the adducts.
HPLC Analysis of the Adducts of Vitamin Fluorogenic Reagent DMEQ-TAD
ET
AL.
The specificity of most of the known fluorescence-labeling reagents is poor, since they target common functional groups, such as amino, hydroxyl, and carboxyl groups (10). It is very difficult to derivatize trace amounts of substrate with high efficiency, since most of the reagents require rather vigorous conditions to be applied to sensitive compounds and the rate of the reaction is too slow under extremely high dilution. We synthesized fluorogenic dienophile, DMEQ-TAD, in which the most reactive dienophilic group, 1,2,4-triazoline3,Sdione (11,12), is connected to the extremely sensitive fluorescent group, 6,7dimethoxyquinoxalinone (8). DMEQ-TAD is ideal as the reagent to assay vitamin D metabolites in that (i) it targets the uncommon conjugated diene group making the reagent highly selective; (ii) the reagent is extremely reactive so that the derivatization is highly efficient (quantitative) even at a concentration as low as 7 X lo-’ M (3 pg/pl); (iii) since the sensitive conjugated triene group of vitamin D is protected in the labeled products, they are much more
6
D with
We analyzed the fluorescent adduct of vitamin D metabolites by HPLC equipped with a fluorescence detector. As Fig. 3 shows, all pairs of the DMEQ-TAD adducts of three vitamin D metabolites were cleanly separated on a single column of HPLC. Under the HPLC conditions shown in the figure, the adduct of each vitamin D metabolite was linearly quantified down to 10 fmol, and detected down to 1 fmol. DISCUSSION
This paper reports the first practical fluorometric method for assaying vitamin D metabolites. Fluorescence labeling is versatile and one of the most sensitive methods for detecting small amounts of compounds. However that method has some defects when used to assay trace amounts of biological compounds in plasma.
0
FIG. 3.
10 Retention
20 time (mln)
30
40
HPLC profile of the fluorescent adducts of the three major vitamin Da metabolites. A solution of DMEQ-TAD (5.9 X 10-r mol) in dichloromethane (500 ~1) was added to a conical tube containing 25-(OH)D3 (2.2 x 10-a mol), 1,25-(OH)aDa (1.2 X lo-’ mol), and 24,25-(OH),D, (2.1 x 10e9 mol) at room temperature and the mixture was stirred for 30 min. Ethanol was added, the solvent was evaporated, the residue was dissolved in 50 ml of methanol, and 10 pl of the solution was injected to an HPLC (column, LiChrospher RP-18 (e) 4 X 250 mm, 35’C; solvent, linear gradient system from 60% methanol/ water (v/v) to 80% methanol/water (v/v) during 40 min; flow rate, 1 ml/min; detector, ex 370 nm, em 440 nm). Peaks: 1 and 2, 24,25-(OH)xD3 DMEQ-TAD adduct (Vc); 3 and 4, 1,25-(OH)zD, DMEQ-TAD adduct (Vb); 5 and 6,25-(OH)D, DMEQ-TAD adduct Wa).
FLUOROMETRIC
ASSAY
OF
stable than the parent vitamin D’s; (iv) the sensitivity of the labeled compounds is extremely high, more than 10 times higher than that of the radio receptor assay. We demonstrated in a model system that the fluorescent dienophile (DMEQ-TAD) is a very useful reagent for assaying vitamin D metabolites. We are currently applying the fiuorometric method to the assay of vitamin D metabolites in plasma samples and the results will be reported elsewhere.
and Herrath, and Clinical
D. v. Endo-
2. Shepard, R. M., Horst, R. L., Hamstra, A. J., and DeLuca, H. F. (1979) Biochem. J. 182, X-69. 3. Porteous, C. E. Coldwell, R. D., Trafford, D. J. H., and Makin, H. L. J. (1987) J. Steroid Biochem. 28, 785-801.
81
D METABOLITES
4. Mayer, 1204.
E., and Schmidt-Gayk,
5. Jongen, Kuiper, 399-403.
M. J. M., Van Ginkel, F. C., van der Vijgh, W. J. F., S., Netelenbos, J. C., and Lips, P. (1984) Clin. Chem. 30,
H. (1984)
Clin.
6. Coldwell, R. D., Porteous, C. E., Trafford, H. L. J. (1987) Steroti 49,155-196. 7. Budesinsky, Z., and Valenta, A. (1971) Collect. mun. 36,2527-2539. 8. Iwata, T., Yamaguchi, M., Hara, S., Nakamura, Y. (1986) J. Chromatogr. 362, 209-216. 9. Moriarty, Commun.
REFERENCES 1. Norman, A. W., Schaefer, K., Grigoleit, H.-G., (Eds.) (1988) Vitamin D, Molecular, Cellular crinology, de Gruyter, Berlin/New York.
VITAMIN
R. M., Prakash, 17,409-413.
10. Seiler, N., and Demisch, Chromatography (Blau, Heyden, London.
I., and
Penmasta,
Chcm.
30,
1199-
D. J. H., and Makin, Czech.
Chem.
Com-
M., and Ohkura, R. (1987)
Synth.
L. (1978) in Handbook of Derivatives for K., and King, G. S., Eds.), pp. 346-390,
11. Barton, D. H. R., Shioiri, T., and Widdowson, D. A. (1971) J. Chem. Sot. C 1968-19’74. 12. Burrage, M. E., Cookson, R. C., Gupte, S. S., and Stevens, I. D. R. (1975) J. Chem. SOL, Perkin Trans. 2 1325-1334.
BIOCHEMISTRY
194,
77-81
(1991)
Synthesis of a Reagent for Fluorescence-Labeling of Vitamin D and Its Use in Assaying Vitamin D Metabolites Masato
Shimizu,
Shinichi
Kamachi,*
Yasuho
Nishii,*
and Sachiko
Yamadal
Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kano!u, Chiyoda-ku, Tokyo 101, and *Biosensor Laboratories Co., Ltd. 3-41-8 Takada, Toshima-ky Tokyo 171, Japan
Received
June
4, 1990
The fluorogenic dienophile 1,2,4-triazoline-3,5dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25Hydroxyvitamin D, , la,25-dihydroxyvitamin D,, and 24(R),25-dihydroxyvitamin D, reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that la,25-dihydroxyvitamin D, at a concentration as low as lo-* M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using highperformance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQTAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite. o ISPI Academic PWS. IIIC.
know accurately the concentrations of the active and other major metabolites in human plasma in various clinical situations. The competitive binding assay currently in use lacks specificity, so it is troublesome and requires special techniques (2,3). The precision of this assay is poor as recent interlaboratory studies have demonstrated (4,5). Though the gas chromatographymass spectrometry is a definitive method, it has many difficulties when used for routine vitamin D assay. The need for a convenient, precise, and highly sensitive method for assaying vitamin D metabolites has prompted us to develop a novel fluorescence-labeling reagent that targets the s-cis-diene part of vitamin D. We synthesized fluorogenic dienophile 4-[2-(6,7-dimethoxy - 4-methyl-3-oxo-3,4dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD) as a reagent to detect vitamin D in high specificity, and used it in a new fluorometric assay of the major vitamin D metabolites, 25-hydroxyvitamin D, (25-(OH)D,), 1,25(OH),D,, and 24(R),25-dihydroxyvitamin D, (24,25(OH),D,). This is the first practical fluorometric method for assaying vitamin D metabolites. MATERIALS
It is now well recognized that the active vitamin D, metabolite la,25dihydroxyvitamin D, (1,25-(OH),D,)2 is a multifunctional steroid hormone regulating not only calcium metabolism but also proliferation and differentiation of a variety of its target cells (1). To fully understand the biological role of vitamin D, it is important to
1 To whom correspondence should be addressed. ’ Abbreviations used: 1,25-(OH),D,, la,25dihydroxyvitamin D,; DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dhydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,Sdione; 25(OH)D,, 25-hydroxyvitamin D,; 24,25-(OH),D,, 24(R),25dihydroxyvitamin D,; DMF, dimethylformamide; DPPA, diphenylphosphoryl azide; DMSO, dimethyl sulfoxide. 0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
AND METHODS
General Procedure Melting points were obtained on a Yanagimoto MR melting point apparatus and are uncorrected. Mass spectra (at 70 eV) were measured on a Jeol DX-300 spectrometer. Relative intensities are given in parentheses. Infrared spectra (ir) were obtained with KBr disks on a JASCO FT/IR-7000 spectrometer. Proton nuclear magnetic resonance spectra (‘H NMR) were recorded with a Jeol GX-270 spectrometer. Chemical shifts are reported in parts per million downfield from tetramethylsilane, and coupling constants are reported in hertz. Fluorescence spectra were recorded on a Shimazu RF-5000 spectrofluorophotometer. Ultraviolet spectra (uv) were 77
Inc. reserved.
78
SHIMIZU
obtained on a Hitachi U-3200 spectrophotometer. High-performance liquid chromatography (HPLC) was performed with a JASCO 880-PU equipped with a solvent programmer 801-SC and a fluorescence detector 821-FP (flow-cell volume, 16 ~1) or a Tosoh CCPM with a solvent programmer PX-8010 and a fluorescent detector FS-8010 (flow-cell volume, 16 ~1).
Chemicals 25-(OH)D, and 1,25-(OH),D, were purchased from Philips Duphar (Amsterdam). 24,25-(OH),D, was kindly donated by Kureha Chemical Industry (Tokyo Japan). Methanol-free dichloromethane was used for the synthesis and reactions of DMEQ-TAD: A reagent grade dichloromethane was washed successively with concentrated sulfuric acid, water, 5% Na,CO,, and water, dried over calcium chloride, and distilled from CaH,.
Synthesis
of Fluorogenic Dienophile DMEQ-TAD
6,7-Dimethoxy-3-oxo-3,4-dihydroquinoxaline-2-propionic acid (I) (7,s). A solution of dinitroveratrole (0.91 g, 4 mmol) in ethanol (80 ml) was shaken in the presence of PtO, (89 mg) under hydrogen atmosphere until the theoretical amount of hydrogen (540 ml) was absorbed. The reaction mixture was filtered directly into a flask containing 2-ketoglutaric acid (0.58 g, 4 mmol) under nitrogen atmosphere and the mixture was refluxed for 1.5 h. After cooling, the precipitated crystals were filtered to give I (0.80 g, 72%). I: mp 250-252°C (ethanol); mass spectrum, m/z 278 (M+, 36), 260 (loo), 232 (88), 217 (32), 189 (31), 161 (21); ir (KBr) 1715,1644 cm-‘; ‘H NMR (CDCl,) 6 2.80 (2 H, t, J = 7.3 Hz), 3.15 (2 H, t, J = 7.3 Hz), 3.92 (3 H, s), 3.93 (3 H, s), 6.78 (1 H, s), 7.18 (1 H, s), 11.92 (1 H, br). 6,7 -Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxaline-2-propionic acid (II). A solution of I (606 mg, 2.2 mmol) in dimethylformamide (DMF) (20 ml) was added to a stirred suspension of NaH (176 mg, 7.3 mmol) in DMF (3 ml) at 0°C under nitrogen. After 30 min, methyl iodide (455 ~1, 7.3 mmol) was added to the solution and the mixture was stirred at 0°C for 1.5 h. The reaction mixture was poured into ice-water, stirred at room temperature for 30 min, acidified with 0.5 N HCl, and the crystals precipitated were filtered to give the carboxylic acid (II) (570 mg, 89%). II: mp 239-241°C (methanolCHCl,); mass spectrum, m/z 292 (M+, 53), 274 (99), 246 (loo), 231 (53), 203 (41), 175 (27); ir (KBr) 1734, 1628 cm -l; ‘H NMR (DMSO-d& 6 2.70 (2 H, t, J = 6.9 Hz), 3.00 (2 H, t, J = 6.9 Hz), 3.65 (3 H, s), 3.84 (3 H, s), 3.94 (3 H, s), 6.98 (1 H, s), 7.22 (1 H, s).
ET
AL.
1 - Ethoxycarbonyl-4-[2-(6,7-dimethoxy-4-methyl-30x0-3,4-dihydroquinoxalinyl)ethyl]semicarbazide (III). Triethylamine (0.36 ml, 2.57 mmol) was added at room temperature to a solution of the carboxylic acid (II) (500 mg, 1.71 mmol) in DMF (50 ml). Then diphenylphosphoryl azide (DPPA) (0.55 ml, 2.57 mmol) was added to the solution and the mixture was stirred at room temperature for 2.5 h. The solvent was evaporated in vacua, the residue was dissolved in benzene (20 ml) and the solution was refluxed for 1 h. The reaction mixture was cooled to room temperature and to the solution was added ethyl carbazate (178 mg, 1.71 mmol). The mixture was refluxed for 30 min and then the solvent was evaporated. The residue was chromatographed on silica gel (90 g) to give a by-product (102 mg, 18%) eluting with CHCl, and then III (394 mg, 59%) eluting with 4% methanol/CHCl, (v/v). III: mass spectrum, m/z 393 (M+, 5), 347 (0.8), 330 (l), 289 (92), 246 (47), 234 (loo), 219 (22), 205 (24); ir (KBr) 1731, 1669, 1649 cm-‘; ‘H NMR (DMSO-de)61.18(3H,t,J=6.9Hz),2.95(2H,t,J=5.9 Hz), 3.52 (2 H, m), 3.67 (3 H, s), 3.89 (3 H, s), 3.97 (3 H, s), 4.02 (2 H, q, J = 6.9 Hz), 6.40 (1 H, br), 6.91 (1 H, s), 7.31 (1 H, s), 7.70 (1 H, br), 8.65 (1 H, br). 4 -[2-(6,7-Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazolidine-3,5dione (IV). A suspension of III (272 mg, 0.69 mmol) and K&O, (191 mg, 1.38 mmol) in ethanol (20 ml) was refluxed for 6 h. The solvent was evaporated and the residue was dissolved in water (30 ml). The solution was made acidic by adding 2 N HCl and extracted with 10% methanol/ CHCl,. The extracts were dried (Na,SO,) and evaporated to give IV (222 mg) as a crystalline solid. The crude product was recrystallized from methanol/CHCl, to give IV (194 mg, 81%) as pale yellow prisms. IV: mp 250-253’C; mass spectrum, m/z 347 (M+, 5), 289 (2), 246 (loo), 231 (35), 203 (20), 175 (20), 101 (12); ir (KBr) 1694, 1640 cm-l; ‘H NMR (DMSO-$) 6 3.02 (2 H, t, J = 6.9 Hz), 3.65 (3 H, s), 3.80 (2 H, t, J = 6.9 Hz), 3.84 (3 H, s), 3.95 (3 H, s), 6.96 (1 H, s), 7.26 (1 H, s), 9.95 (2 H, s); fluorescence (ethanol) X,, 370 nm, X,, 440 nm. 4 - [2 - (6,7-Dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5dione (DMEQTAD). Iodobenzene diacetate (10.0 mg, 0.031 mmol) was added to a stirred suspension of IV (8.6 mg, 0.025 mmol) in methanol-free dichloromethane (1.5 ml) at room temperature and the mixture was stirred for 3.5 h. The reaction mixture was filtered, the filtrate was stored in a freezer (-20°C) over night, and the precipitated red crystals were filtered with the aid of argon pressure to give DMEQ-TAD (6.7 mg, 79%) as red needles. DMEQ-TAD: mp 200-202°C (decompose); ir (KBr) 1785,1769,1638 cm-‘; ‘H NMR (CDCl,)G 3.29 (2 H, t, J = 6.8 Hz), 3.68 (3 H, s), 3.95 (3 H, s), 4.02 (3 H, s), 4.15 (2 H, t, J = 6.8 Hz), 6.68 (1 H, s), 7.12 (1 H, s); uv
FLUOROMETRIC
(CH,CN) 277,225
X,, nm.
526,366,295,241,212
ASSAY
OF
B. Analytical scale A solution (ethanol) of 1,25(OH),D, (7.2 X 10-l’ mol) was placed in a conical tube, and the solvent was removed completely in vacua. A solution of DMEQ-TAD (7.2 X lo-’ mol) in methanolfree dichloromethane (1 ml) was added to the tube and the mixture was stirred at room temperature. After 30 min, the excess of the reagent was quenched by adding ethanol and the solvent was evaporated. The residue was dissolved in methanol and a portion was analyzed by HPLC (column, LiChrospher RP-18(e) (Cica-Merck, Tokyo) 4 X 250 mm, 35°C; solvent, linearly programmed gradient system from 60% methanol/H,0 (v/ v) to 80% methanol/H,0 (v/v) during 40 min; rate, 1 ml/min; detector ex 370 nm, em 440 nm). The yield of the adduct (Vb) was nearly 100% as calculated on the basis of the area of the two peaks of the C(6) epimers eluted at 26.5 and 30 min. Under similar conditions, 25-(OH)D, and 24,25(OH),D, were converted to the corresponding fluorescent (Va and Vc) adduct quantitatively.
RESULTS
of the Fluorescence
The highly fluorescent synthesized in essentially
Labeling
79
D METABOLITES
nm, Xmin 468,317,
Reaction of vitamin D metabolites with the fluorescence labeling reagent (DMEQ-TAD). A. Preparative scale To a solution of vitamin D (5 pmol) in dichloromethane (0.2 ml) was added a solution of DMEQ-TAD (6 pmol) in dichloromethane (1 ml) at room temperature. The solvent was evaporated and the residue was purified on silica gel TLC (8% methanol/chloroform) or HPLC (LiChrosorb Si 60 (Cica-Merck, Tokyo), 10 X 250 mm, 8% methanol/dichloromethane) to give the adduct (V) as a mixture of two C(6) epimers (quantitative total yield). DMEQ-TAD adduct of 25(OH)D, (Va): ‘H NMR (CDCl,) 6 0.52 and0.51 (3 H (2:1), s), 1.20 and 1.21 (6H (2:1), s), 3.69 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.69 (1 H, d, J = 9.6 Hz), 4.88 (1 H, d, J = 9.6 Hz), 6.68 (1 H, s), 7.23 (1 H, s); fluorescence (ethanol) X,, 370 nm, X,, 440 nm. DMEQ-TAD adduct of 1,25-(OH),D, (Vb): ‘H NMR (CDCl,) 6 0.50 and0.51 (3 H (5:4), s), 1.21 and 1.20 (6 H (5:4), s), 3.69 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.30 (1 H, m), 4.70 (1 H, d, J = 9.9 Hz), 4.93 (1 H, d, J = 9.9 Hz), 6.68 (1 H, s), 7.23 (1 H, s). DMEQ-TAD adduct of 24,25-(OH),D, (Vc): ‘H NMR (CDCl,) F 0.54 and0.52 (3 H (2:1), s), 1.16 (3 H, s), 1.21 (3 H, s), 3.70 (3 H, s), 3.94 (3 H, s), 4.01 (3 H, s), 4.69 (1 H, d, J = 9.6 Hz), 4.88 (1 H, d, J = 9.6 Hz), 6.68 (1 H, s), 7.23 (1 H, s).
Synthesis
VITAMIN
Reagent
dienophile DMEQ-TAD was five steps in 24% overall yield
0 I : R=H II : R=Me
DMEQ-TAD
FIG. 1. Synthesis pathway of the fluorogenic dienophile (DMEQTAD). Reagents (yields): a, HJPtOJethanol then 2-ketoglutaric acid/reflux (72%); b, NaH/CH,I/DMF then H,O (89%); c, DPPA/ triethylamine/DMF, refluxlbenzene, ethyl carbazate/retlux (59%); d, K,CO,/ethanol/reAux (81%); e, iodobenzene diacetate/dichloromethane (79%).
from dmitroveratrole via the pathway shown in Fig. 1. Catalytic reduction of dinitroveratrole followed by the reaction with 2-ketoglutaric acid gave quinoxalinone propionic acid (I). After N-methylation, the carboxylic acid was converted to the semicarbazide (III) by Curtius rearrangement and then it was cyclized to give dihydro derivative (IV) of the desired reagent DMEQ-TAD. The triazolidine (IV) was oxidized with iodobenzene diacetate (9) to give the desired fluorogenic triazoline (DMEQ-TAD) as a red crystalline solid. The reagent is reasonably stable at room temperature and is expected to be stable when stored in a freezer under an inert-gas atmosphere for at least several years.
Reaction of Vitamin D Metabolites Labeling Reagent (DMEQ-TAD)
with the Fluorescence
The fluorescence-labeling reagent (DMEQ-TAD) reacted quantitatively with the three major vitamin D metabolites, 25-(OH)D,, 1,25-(OH),D,, and 24,25(OH),D,, by simply mixing a solution (dichloromethane) of the two compounds at room temperature to give the corresponding 6,19-adducts (V) (Fig. 2). The reaction was very fast: at room temperature at a concentration of more than lop4 M, all three vitamin D metabolites reacted instantaneously with an equimolar amount of DMEQ-TAD. At a concentration as low as 7.2 x lo-’ M (about 3 pg/pl, dichloromethane) with 1000 eq of DMEQ-TAD, nearly 100% of 1,25-(OH),D, was converted to the fluorescent adduct (Vb) within 30 min. 25-(OH)D, and 24,25-(OH),D, reacted similarly under
80
SHIMIZU
V 25-OH-D3 1,25-(OH&D,
: R’zR3-OH,
24,25-(OH),D,
: R’rH,
FIG.
2.
a : Rq=R2=H,
: R’=R2=H,R3x0H
Reaction
R2-H R2=R3z0H
of vitamin
b : R’:R3=OH, c:
R’=H
D metabolites
R3=0H R2=H
R%R3zOH
with
DMEQ-TAD.
similar high dilution conditions. Thus it is clear in a model system that the three major vitamin D, metabolites present in 1 ml of plasma can be converted to the corresponding fluorescent adducts in nearly quantitative yield within 30 min. Each vitamin D yields the corresponding adduct (V) as a mixture of C(6) epimers in a ratio characteristic of each vitamin D. The ratio of the two C(6) epimers of the adducts were approximately 2:l for 25(OH)D, adduct (Va), 54 for 1,25-(OH),D, adduct (Vb), and 2:l for 24,25-(OH),D, adduct (Vc) (see Materials and Methods and Fig. 3). The stereochemistry of each epimer has not been determined. A pair of the retention times as well as the ratio of the two epimers, however, were very useful to confirm the identity of the particular metabolite when they are analyzed by HPLC (see below). The adducts are all very stable, more stable than the parent vitamin D, since the light- and oxygen-sensitive conjugated triene function of vitamin D was masked in the adducts.
HPLC Analysis of the Adducts of Vitamin Fluorogenic Reagent DMEQ-TAD
ET
AL.
The specificity of most of the known fluorescence-labeling reagents is poor, since they target common functional groups, such as amino, hydroxyl, and carboxyl groups (10). It is very difficult to derivatize trace amounts of substrate with high efficiency, since most of the reagents require rather vigorous conditions to be applied to sensitive compounds and the rate of the reaction is too slow under extremely high dilution. We synthesized fluorogenic dienophile, DMEQ-TAD, in which the most reactive dienophilic group, 1,2,4-triazoline3,Sdione (11,12), is connected to the extremely sensitive fluorescent group, 6,7dimethoxyquinoxalinone (8). DMEQ-TAD is ideal as the reagent to assay vitamin D metabolites in that (i) it targets the uncommon conjugated diene group making the reagent highly selective; (ii) the reagent is extremely reactive so that the derivatization is highly efficient (quantitative) even at a concentration as low as 7 X lo-’ M (3 pg/pl); (iii) since the sensitive conjugated triene group of vitamin D is protected in the labeled products, they are much more
6
D with
We analyzed the fluorescent adduct of vitamin D metabolites by HPLC equipped with a fluorescence detector. As Fig. 3 shows, all pairs of the DMEQ-TAD adducts of three vitamin D metabolites were cleanly separated on a single column of HPLC. Under the HPLC conditions shown in the figure, the adduct of each vitamin D metabolite was linearly quantified down to 10 fmol, and detected down to 1 fmol. DISCUSSION
This paper reports the first practical fluorometric method for assaying vitamin D metabolites. Fluorescence labeling is versatile and one of the most sensitive methods for detecting small amounts of compounds. However that method has some defects when used to assay trace amounts of biological compounds in plasma.
0
FIG. 3.
10 Retention
20 time (mln)
30
40
HPLC profile of the fluorescent adducts of the three major vitamin Da metabolites. A solution of DMEQ-TAD (5.9 X 10-r mol) in dichloromethane (500 ~1) was added to a conical tube containing 25-(OH)D3 (2.2 x 10-a mol), 1,25-(OH)aDa (1.2 X lo-’ mol), and 24,25-(OH),D, (2.1 x 10e9 mol) at room temperature and the mixture was stirred for 30 min. Ethanol was added, the solvent was evaporated, the residue was dissolved in 50 ml of methanol, and 10 pl of the solution was injected to an HPLC (column, LiChrospher RP-18 (e) 4 X 250 mm, 35’C; solvent, linear gradient system from 60% methanol/ water (v/v) to 80% methanol/water (v/v) during 40 min; flow rate, 1 ml/min; detector, ex 370 nm, em 440 nm). Peaks: 1 and 2, 24,25-(OH)xD3 DMEQ-TAD adduct (Vc); 3 and 4, 1,25-(OH)zD, DMEQ-TAD adduct (Vb); 5 and 6,25-(OH)D, DMEQ-TAD adduct Wa).
FLUOROMETRIC
ASSAY
OF
stable than the parent vitamin D’s; (iv) the sensitivity of the labeled compounds is extremely high, more than 10 times higher than that of the radio receptor assay. We demonstrated in a model system that the fluorescent dienophile (DMEQ-TAD) is a very useful reagent for assaying vitamin D metabolites. We are currently applying the fiuorometric method to the assay of vitamin D metabolites in plasma samples and the results will be reported elsewhere.
and Herrath, and Clinical
D. v. Endo-
2. Shepard, R. M., Horst, R. L., Hamstra, A. J., and DeLuca, H. F. (1979) Biochem. J. 182, X-69. 3. Porteous, C. E. Coldwell, R. D., Trafford, D. J. H., and Makin, H. L. J. (1987) J. Steroid Biochem. 28, 785-801.
81
D METABOLITES
4. Mayer, 1204.
E., and Schmidt-Gayk,
5. Jongen, Kuiper, 399-403.
M. J. M., Van Ginkel, F. C., van der Vijgh, W. J. F., S., Netelenbos, J. C., and Lips, P. (1984) Clin. Chem. 30,
H. (1984)
Clin.
6. Coldwell, R. D., Porteous, C. E., Trafford, H. L. J. (1987) Steroti 49,155-196. 7. Budesinsky, Z., and Valenta, A. (1971) Collect. mun. 36,2527-2539. 8. Iwata, T., Yamaguchi, M., Hara, S., Nakamura, Y. (1986) J. Chromatogr. 362, 209-216. 9. Moriarty, Commun.
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VITAMIN
R. M., Prakash, 17,409-413.
10. Seiler, N., and Demisch, Chromatography (Blau, Heyden, London.
I., and
Penmasta,
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1199-
D. J. H., and Makin, Czech.
Chem.
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M., and Ohkura, R. (1987)
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11. Barton, D. H. R., Shioiri, T., and Widdowson, D. A. (1971) J. Chem. Sot. C 1968-19’74. 12. Burrage, M. E., Cookson, R. C., Gupte, S. S., and Stevens, I. D. R. (1975) J. Chem. SOL, Perkin Trans. 2 1325-1334.
