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BIOTINYLATEDPSORALEN
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odontal pathogens, 38 and Leishmania. 39 Biotinylated probes have also been used for in situ cytohybridization assays for detection of viral infections (e.g., human papillomavirus4°,4~ and bovine herpesvirus 42) and to determine levels of mRNA expression in cells o r t i s s u e s . 43.44 New applications for biotinylated probes in molecular biology and clinical diagnostics will continue to emerge as nonisotopic detection techniques acquire the speed, sensitivity, and reproducibility to compete effectively with radioisotopes. Acknowledgments We thank Dr. DietmarRabussayfor continuedsupport and Dr. ReynaldoPless and Dr. Susan Bromleyfor critical readingof the manuscript. We also thank KathleenBlairfor help in typing the manuscript. 37 H. Bialkowska-Hobrzanska, J. Clin. Mk'robiol. 25, 338 (1987). 3~ C. K. French, E. D. Savitt, S. L. Simon, S. M. Eklund, M. C. Chen, L. C. Klotz, and K. K. Vaccaro, Oral Microbiol. hnmunol. 1, 58 (1986). 39 p. G. Trejo, Focus (Bethesda Res. Lab.~Life Technol.) 9~ 11 (1987). 40 A. M. Beckmann, D. Myerson, J. R. Daling, N. B. Kiviat, C. M. Fenoglio, and J. K. McDougall, J. Med. Virol. 16, 265 (1985). 41 K. Milde and T. Loning, J. Oral Pathol. 15, 292 (1986). 42 D. C. Dunn, C. D. Blair, D. C. Ward, and B. J. Beaty, Am. J. Vet. Res. 47, 740 (19861. 43 R. H. Singer, J. B. Lawrence, and C. Villnave, BioTechniques 4, 230 (1986). 44 p. Liesi, J.-P. Julien, P. Vilja, F. Grosveld, and L. Rechardt, J. Histochem. Cytochem. 34, 923 (1986).
[67] B i o t i n y l a t e d P s o r a l e n D e r i v a t i v e for L a b e l i n g N u c l e i c Acid Hybridization Probes B y COREY L E V E N S O N , ROBERT WATSON, and EDWARD L. SHELDON
Nucleic acid hybridization probes are widely used biochemical reagents with applications in research and diagnostics. Such probes require at least two structural features: a single-stranded region of nucleic acid capable of annealing to a complementary "target" sequence and one or more detectable moieties (labels). The label(s) may be incorporated in the single-stranded hybridizing region as long as its presence does not compromise the ability of the probe to anneal to the target sequence. Nonradioactive labels are preferable owing to their ease of handling and disposal and their long shelf life, One of the preferred labels for nonMETHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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IC) A
8
[6 7]
¢
FIG. 1. Probe constructs that are suitable substrates for labeling using biotinylated psoralen: (A) two complementary single-stranded oligonucleotides, (B) a partially self-complementary oligonucleotide, and (C) an M 13 phage-derived "gapped circle" [V. Courage-Tebbe and B. Kernper, Biochim. Biophys. Acta 697, 1 (1982)]. Note: These representations are not drawn to scale.
isotopically labeled probes is biotin. 1,2 Conjugates of various enzymes with avidin or streptavidin are employed for colorimetric detection. 3,4 Nonisotopic labels can be introduced into probes using appropriately modified derivatives of psoralens. Psoralens are planar compounds that are capable of intercalating into double-stranded nucleic acids. The intercalated complexes are photoactivated by long-wavelength (360 nm) ultraviolet light and form interstrand cross-links. Figure 1 depicts three types of hybridization probes that are suitable substrates for reaction with psoralen labeling reagents. Each probe consists of a single-stranded hybridizing region linked to a double-stranded region containing preferred psoralen-binding sites (5 '-TpA-3 '). Once these probes have been labeled with a psoralen derivative, the double-stranded region is cross-linked and therefore becomes incapable of being denatured; the duplex region serves only as a carrier for the label(s). The psoralen-derived labeling reagents have three domains: the trimethylpsoralen ring system, a long, flexible, hydrophilic "spacer arm," and the nonisotopic label. For the reagent described herein, the psoralen ring system is derived from trimethylpsoralen (trioxsalen), the spacer arm is derived from tetraethylene glycol, and the label is biotin. The synthesis of the biotinylated psoralen labeling reagent is shown in Fig. 2. Reaction of tetraethylene glycol with p-toluenesulfonyl chloride in pyridine yields I j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975). 2 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 3 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983). 4 E. L. Sheldon, D. E. Kellogg, R. Watson, C. Levenson, and H. Erich, Proc. Natl. Acad. Sci. U.S.A. 83, 9085 (1985).
[67]
BIOTINYLATEDPSORALEN
579
HO~O~O~/0~0H 1) Tosyl chloride/pyridine 2) Lithium a z i d e / D M F
3)
H2NI~ O~
0 " ~ / 0 ~ , ~ ~ NH 2
I
H2N~
O~
Triphenylphosphine/OH-
Di-t-butyl dicarbonate
0 l 0 / ~ O . ~ NHJJ~o-~ I 4'-Formyl
HN ~
0,~
0
(I)
0~
0,~
(If)
trioxsalen/NaCNBH 3
NH..LLO,l~ ---
(III)
1) HCI
0
2) BNHS H N/~JO~ 0" "0" "~
"O" \
0~/0.~
H ~
H
NH
(Iv)
FIG. 2. Synthesis of the biotinylated psoralen labeling reagent.
bistosylate. Reaction of the tosylate with lithium azide in DMF yields bisazide. The azide is reduced to the bisamine (I) by triphenylphosphineammonium hydroxide. The bisamine is converted to the mono-tert-butyloxycarbonyl (BOC) derivative (II), which is purified by silica gel column chromatography. The mono-BOC-protected psoralen derivative (lid is produced via a reductive amination reaction employing compound II and 4 '-formyltrioxsalen. Treatment of compound II! with HC1 in ethyl acetate removes the BOC group, and subsequent reaction with biotin-N-hydroxysuccinimide ester (BNHS) yields the biotinylated psoralenamine (IV). For the purpose of quantifying levels of incorporation, compound IV, may also be synthesized using tritiated BNHS. Synthesis of Reagents
Materials. The following chemicals are obtained from Aldrich (Milwaukee, WI): tetraethylene glycol, p-toluenesulfonylchloride, triphenylphosphine, di-tert-butyl dicarbonate, and dimethylaminocinnamaldehyde. Lithium azide is obtained from Kodak (Rochester, NY). 4'-Formyltrioxsalen is obtained from HRI Associates (Berkeley, CA). N-Hydroxysuccinimide ester of biotin (BNHS) is obtained from Pierce
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Chemical Co. (Rockford, IL). Tritiated BNHS is obtained from Amersham (Arlington Heights, IL). Analytical thin-layer chromatograms are run on Bakerflex IB2-F plates (J. T. Baker, PhiUipsburg, NJ). Preparative TLC is performed using uniplate silica gel GF tapered layer plates (Analtech, Newark, DE). Biotinylated compounds are detected on TLC plates using a spray reagent consisting of equal volumes of 2% (v/v) sulfuric acid in ethanol and 0.2% (w/v)p-dimethylaminocinnamaldehyde in absolute ethanol. 5 NMR spectra are obtained using a Varian FT80, and shifts are reported as parts per million (ppm) downfield from tetramethylsilane as an internal standard. Mass spectral data are provided by the mass spectrometry laboratory at the University of California at Berkeley, Department of Chemistry. Bistosyl-3,6,9-trioxaundecane-1, l l - d i o l . To a chilled solution of tetraethylene glycol (42 g, 216 mmol) in 500 ml of dry pyridine is added ptoluenesulfonyl chloride (100 g, 525 mmol). The solution is stirred at 4° for 18 hr. To the solution is added 100 ml of methanol, and stirring is continued for an additional hour. The solvent is removed under reduced pressure and the residue partitioned between 300 ml of ethyl acetate and 300 ml of 0.5 M citric acid. The organic layer is washed with saturated NaC1 (2 times, 300 ml each). The organic extract is dried over magnesium sulfate, filtered, and concentrated to a syrupy residue (92 g) which may be used directly for the next reaction. If desired, the material may be purified by silica gel column chromatography using dichloromethane-methanol (97:3) as eluant (TLC, Rf = 0.54, detect with iodine vapor). NMR (DMSO-d6): 2.41(s), 3.46(s), 3.55(m), 4.14(m), 7.46(d, J = 8.3 Hz), 7.82 (d, J = 8.3 Hz). 1 , 1 1 - D i a z i d o - 3, 6, 9 - t r i o x a u n d e c a n e . To a solution of bistosyl-3,63trioxaundecane-l,ll-diol (50.3 g, 100 mmol) in 250 ml of dry dimethylformamide (DMF) is added lithium azide (30 g, 613 mmol). The mixture is heated to 75 ° with stirring until TLC on silica gel (ethyl acetate) reveals no starting material remaining. The solvent is removed under vacuum and the residue partitioned between 500 ml ethyl acetate and 250 ml of saturated NaC1. The organic layer is dried over magnesium sulfate, filtered, and concentrated to an oil (21 g). This material can be either reduced directly in the following reaction or purified by column chromatography on silica gel using ethyl acetate as eluant (TLC, R f -- 0.71, iodine vapor detection). NMR (DMSO-d6): 3.34(m), 3.57(s), 3.65(m). IR (neat): 2100 cm-1. 1 , 1 1 - D i a m i n o - 3, 6, 9 - t r i o x a u n d e c a n e (I). Triphenylphosphine (89 g, 339 mmol) is added to a chilled, stirred solution of 1,11-diazido-3,6,9-
5 D. B. McCormick and J. A. Roth, this series, Vol. 18, p. 383.
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BIOTINYLATEDPSORALEN
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trioxaundecane (24.4 g, 100 mmol) in 250 ml pyridine. Nitrogen bubbles evolve. The solution is stirred with cooling on ice for 45 min and then allowed to warm to room temperature. After 45 rain at room temperature, 100 ml of concentrated ammonium hydroxide is added and the mixture stirred overnight. The solvent is removed under reduced pressure, and the mixture is partitioned between 500 ml of 0.5 M citric acid and 250 ml of ethyl acetate. The aqueous phase is washed with ethyl acetate (2 times 250 ml each) to remove triphenylphosphine oxide. The aqueous phase is saturated with sodium chloride and exhaustively extracted with n-butanol. The alcohol extract is concentrated under reduced pressure and the residue taken up in ! 00 ml of absolute ethanol. Concentrated hydrochloric acid (15 ml) is added, and the solvent is removed under reduced pressure. The residue is crystallized from ethanol-ether. (Note: the product will not crystallize unless it is anhydrous.) The hygroscopic crystals (21 g) are dried under vacuum. TLC, Rf = 0.28 [silica gel, dichloromethane-methanol-acetic acid (70 : 30 : 5), ninhydrin detection]. NMR (DMSO-d6): 3.39(s), 3.57(s), 3.64(s), 8.10(br). 1 - A m i n o - 12 - t e r t - b u t y l o x y c a r b o n y l a m i n o - 3 , 6 , 9 , - t r i o x a u n d e c a n e
(I1). To a solution of I (13.26 g, 50 retool) and triethylamine (8 ml, 5.8 g, 57.4 mmol) in 100 ml of methanol is added a solution of di-tert-butyl dicarbonate (12 g, 55 retool) in 10 ml of methanol. Carbon dioxide bubbles evolve, and the reaction is slightly exothermic. Stirring is continued until gas evolution ceases. The solvent is removed under reduced pressure, and the residue is adsorbed onto 20 g of silica gel and fractionated on a column using dichloromethane-methanol-acetic acid (70:30:5) as eluant. The fractions containing product are pooled and concentrated to yield the mono-BOC (butyloxycarbonyl) derivative as a syrup. TLC, Rf = 0.67 [dichloromethane-methanol-acetic acid (70 : 30 : 5), ninhydrin positive]. 12 - tert - B u t y l o x y c a r b o n y l a m i n o - 1 - [ ( 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n - 4 'm e t h y l e n y l ) a m i n o ] - 3 , 6 , 9 - t r i o x a u n d e c a n e (III). 4 '-Formyltrioxsalen (1 g,
3.9 mmol) and 1 g of 3 /k molecular sieves are added to a solution of compound |I (2.28 g, 7.80 retool) and sodium cyanoborohydride (246 rag, 3.9 mmol) in 50 ml of anhydrous methanol. The suspension is adjusted to pH 7 by addition of triethylamine and is stirred in the dark at room temperature until TLC [silica gel, dichloromethane-methanol (8: 1)] shows no formyltrioxsalen remaining. The mixture is filtered and evaporated to a residue which is purified by silica gel column chromatography using the TLC solvent system as eluant. Fractions containing the fluorescent product are pooled and evaporated to dryness to yield 1.8 g. TLC, Rf = 0.41 [dichloromethane-methanol (8:1); fluorescent under longwavelength UV; ninhydrin positive after spraying the plate with 3 M HCI
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in ethyl acetate]. NMR (DMSO-d6): 1.36(s), 2.39(s), 2.44(s), 2.65(t, J = 5.5 Hz), 3.07(t, J = 5.5 Hz), 3.48(s), 3.81(s), 6.25(s), 6.73 (br t), 7.73(s).
12-Biotinylamino-l-[(4,5 ',8-trimethylpsoralen-4 '-methylenyl)amino]3,6, 9-trioxaundecane (IV). To a solution of III (71.2 mg, 133.7 ~mol) in 1 ml of dry dioxane is added 0.5 ml of a saturated solution of HC1 in dioxane ( - 1 2 M). The solution becomes cloudy, and a precipitate is formed which begins to crystallize. The mixture is checked by TLC [silica gel, 2-butanone-acetic acid-water (70 : 30 : 25)]. When the BOC-protected material is no longer evident ( - 3 0 min), the solvent is removed under a stream of air. The crystalline residue is taken up in 0.5 ml of methanol and taken to dryness. The residue is reconstituted in 0.4 ml of dry DMF, and 120/~1 (689/.Lmol) of diisopropylethylamine is added. This solution is added to a suspension of BNHS (50 mg, 146.5 /xmol) in 0.2 ml of dry DMF. All material dissolves. After 2 hr, the mixture is taken to dryness and reconstituted in 1 ml of methanol. The solution is applied to two 20 x 20 cm preparative TLC plates, and the plates are developed with 2-butanoneacetic acid-water (70 : 30 : 25). The product band (Rf = 0.77, fluorescent and biotin positive) is scraped and eluted from the silica gel with 20 ml of methanol. The extract is filtered, and the concentration of product is determined spectrophotometrically using an extinction coefficient of 25,000 at 250 nm. Yield, 87.6/zmol of IV (66%). Fast atom bombardment high-resolution mass spectroscopy: M + H = 659.3109 (C33H47N4OsS = 659.829). Probe Preparation and Biotinylation Partially double-stranded probe constructs (as depicted in Fig. 1) are biotinylated by mixing the labeling reagent with the probe DNA at a molar ratio (reagent to base pairs) of 2 : 1 and a DNA concentration of 100/zg/ml in 100 mM NaC1, 10 m M Tris (pH 7.5), and 1 mM EDTA. The mixture is irradiated with 360-nm UV light (B-100A Black-Ray Lamp, UV Products, San Gabriel, CA) at a flux of 30 mW/cm 2 for 10 min, and the biotinylated gapped circles are purified from the reaction mixture by chromatography on a 0.7 × 30 cm Sephacryl S-200 column in 10 mM NaCI, 10 mM Tris, 1 mM EDTA. These conditions result in the incorporation of one biotinylated psoralen labeling reagent for every 10 base pairs (10%). Levels of label incorporation are assayed by scintillation counting of probes that had been photoreacted with tritiated biotinylated psoralen. Comments
Biotinylated nucleic acid probe constructs have previously been made by enzymatic incorporation of biotinylated nucleotides into doublestranded DNA, 2 by chemical synthesis of oligonucleotides using biotin
[67]
BIOTINYLATEDPSORALEN
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derivatives,6,7 o r by reaction of the probe with photoreactive biotin derivatives. 8 The disadvantages of the enzymatic method of biotinylation include the expense of reagents and difficulties involved with control and scale-up of the reaction. Biotinylated oligonucleotides are difficult to prepare and have the disadvantage of delivering very few biotins to the site of hybridization. Photoreactive biotin derivatives previously reported are based on arylazides that generate arylnitrenes upon photoactivation. These nitrenes do not discriminate between single- or double-stranded nucleic acids or between nucleic acids and other molecules. The attribute of biotinylated psoralen that makes it attractive as a labeling reagent is its ability to discriminate between single- and doublestranded nucleic acids; psoralens are known to interact preferentially with the duplex form. It was demonstrated previously9 that M13 probes could be treated (cross-linked) with trioxsalen without compromising the ability of the probe to hybridize to target nucleic acids. We have demonstrated that partial duplex probes are also suitable substrates for reaction with psoralen derivatives. 4 The presence of a large substituent at the 4' position of the psoralen ring system does not prevent the molecule from intercalating and subsequently photoreacting with the duplex region. The level of incorporation (psoralens per 100 base pairs) is comparable for both 4'-aminomethyltrioxsalen and the biotinylated psoralen described herein. It is possible to bind one psoralen per 4-5 base pairs for either compound.~° We have found that this level of incorporation is unnecessary for optimal signal detection. Because of the size of the enzyme conjugates used for detection, it is probably unnecessary to have more than approximately one biotinylated psoralen per 10 base pairs. Higher levels of incorporation lead to higher levels of background, presumably owing to aggregation of the heavily biotinylated probe or increased nonspecific adherence to the membrane. The extent of incorporation of biotinylated psoralen with nucleic acids is easily controlled by adjusting the base pair to psoralen stoichiometry and has been found to be insensitive to scale-up. Milligram quantities of probe DNA can be labeled in the single reaction. Acknowledgments The authors would like to acknowledgethe creative input of Kary Mullis and Henry Rapoport, and the technical assistance of Diana Ho, David Kellogg, Todd Smith, Rick Snead, and Dragan Spasic. 6j. Kempe, W. I. Sundquist, F. Chow, and S. L. Ho, Nucleic Acids Res. 13, 45 (1985). 7A. Cholletand E. H. Kawashima,Nucleic Acids Res. 13, 1529(1985). s A. C. Forster, J. L. Mclnnes, D. C. Skingle, and R. H. Symons,Nucleic Acids Res. 13, 745 (1985). 9 D. M. Brown, J. Frampton, P. Goelet, and J. Karn, Gene 20, 139 (1982). ~0S. T. Isaacs, C. J. Shen, J. E. Hearst, and H. Rapoport, Biochemistry 16, 1058(1977).
BIOTINYLATEDPSORALEN
577
odontal pathogens, 38 and Leishmania. 39 Biotinylated probes have also been used for in situ cytohybridization assays for detection of viral infections (e.g., human papillomavirus4°,4~ and bovine herpesvirus 42) and to determine levels of mRNA expression in cells o r t i s s u e s . 43.44 New applications for biotinylated probes in molecular biology and clinical diagnostics will continue to emerge as nonisotopic detection techniques acquire the speed, sensitivity, and reproducibility to compete effectively with radioisotopes. Acknowledgments We thank Dr. DietmarRabussayfor continuedsupport and Dr. ReynaldoPless and Dr. Susan Bromleyfor critical readingof the manuscript. We also thank KathleenBlairfor help in typing the manuscript. 37 H. Bialkowska-Hobrzanska, J. Clin. Mk'robiol. 25, 338 (1987). 3~ C. K. French, E. D. Savitt, S. L. Simon, S. M. Eklund, M. C. Chen, L. C. Klotz, and K. K. Vaccaro, Oral Microbiol. hnmunol. 1, 58 (1986). 39 p. G. Trejo, Focus (Bethesda Res. Lab.~Life Technol.) 9~ 11 (1987). 40 A. M. Beckmann, D. Myerson, J. R. Daling, N. B. Kiviat, C. M. Fenoglio, and J. K. McDougall, J. Med. Virol. 16, 265 (1985). 41 K. Milde and T. Loning, J. Oral Pathol. 15, 292 (1986). 42 D. C. Dunn, C. D. Blair, D. C. Ward, and B. J. Beaty, Am. J. Vet. Res. 47, 740 (19861. 43 R. H. Singer, J. B. Lawrence, and C. Villnave, BioTechniques 4, 230 (1986). 44 p. Liesi, J.-P. Julien, P. Vilja, F. Grosveld, and L. Rechardt, J. Histochem. Cytochem. 34, 923 (1986).
[67] B i o t i n y l a t e d P s o r a l e n D e r i v a t i v e for L a b e l i n g N u c l e i c Acid Hybridization Probes B y COREY L E V E N S O N , ROBERT WATSON, and EDWARD L. SHELDON
Nucleic acid hybridization probes are widely used biochemical reagents with applications in research and diagnostics. Such probes require at least two structural features: a single-stranded region of nucleic acid capable of annealing to a complementary "target" sequence and one or more detectable moieties (labels). The label(s) may be incorporated in the single-stranded hybridizing region as long as its presence does not compromise the ability of the probe to anneal to the target sequence. Nonradioactive labels are preferable owing to their ease of handling and disposal and their long shelf life, One of the preferred labels for nonMETHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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IC) A
8
[6 7]
¢
FIG. 1. Probe constructs that are suitable substrates for labeling using biotinylated psoralen: (A) two complementary single-stranded oligonucleotides, (B) a partially self-complementary oligonucleotide, and (C) an M 13 phage-derived "gapped circle" [V. Courage-Tebbe and B. Kernper, Biochim. Biophys. Acta 697, 1 (1982)]. Note: These representations are not drawn to scale.
isotopically labeled probes is biotin. 1,2 Conjugates of various enzymes with avidin or streptavidin are employed for colorimetric detection. 3,4 Nonisotopic labels can be introduced into probes using appropriately modified derivatives of psoralens. Psoralens are planar compounds that are capable of intercalating into double-stranded nucleic acids. The intercalated complexes are photoactivated by long-wavelength (360 nm) ultraviolet light and form interstrand cross-links. Figure 1 depicts three types of hybridization probes that are suitable substrates for reaction with psoralen labeling reagents. Each probe consists of a single-stranded hybridizing region linked to a double-stranded region containing preferred psoralen-binding sites (5 '-TpA-3 '). Once these probes have been labeled with a psoralen derivative, the double-stranded region is cross-linked and therefore becomes incapable of being denatured; the duplex region serves only as a carrier for the label(s). The psoralen-derived labeling reagents have three domains: the trimethylpsoralen ring system, a long, flexible, hydrophilic "spacer arm," and the nonisotopic label. For the reagent described herein, the psoralen ring system is derived from trimethylpsoralen (trioxsalen), the spacer arm is derived from tetraethylene glycol, and the label is biotin. The synthesis of the biotinylated psoralen labeling reagent is shown in Fig. 2. Reaction of tetraethylene glycol with p-toluenesulfonyl chloride in pyridine yields I j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975). 2 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 3 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983). 4 E. L. Sheldon, D. E. Kellogg, R. Watson, C. Levenson, and H. Erich, Proc. Natl. Acad. Sci. U.S.A. 83, 9085 (1985).
[67]
BIOTINYLATEDPSORALEN
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HO~O~O~/0~0H 1) Tosyl chloride/pyridine 2) Lithium a z i d e / D M F
3)
H2NI~ O~
0 " ~ / 0 ~ , ~ ~ NH 2
I
H2N~
O~
Triphenylphosphine/OH-
Di-t-butyl dicarbonate
0 l 0 / ~ O . ~ NHJJ~o-~ I 4'-Formyl
HN ~
0,~
0
(I)
0~
0,~
(If)
trioxsalen/NaCNBH 3
NH..LLO,l~ ---
(III)
1) HCI
0
2) BNHS H N/~JO~ 0" "0" "~
"O" \
0~/0.~
H ~
H
NH
(Iv)
FIG. 2. Synthesis of the biotinylated psoralen labeling reagent.
bistosylate. Reaction of the tosylate with lithium azide in DMF yields bisazide. The azide is reduced to the bisamine (I) by triphenylphosphineammonium hydroxide. The bisamine is converted to the mono-tert-butyloxycarbonyl (BOC) derivative (II), which is purified by silica gel column chromatography. The mono-BOC-protected psoralen derivative (lid is produced via a reductive amination reaction employing compound II and 4 '-formyltrioxsalen. Treatment of compound II! with HC1 in ethyl acetate removes the BOC group, and subsequent reaction with biotin-N-hydroxysuccinimide ester (BNHS) yields the biotinylated psoralenamine (IV). For the purpose of quantifying levels of incorporation, compound IV, may also be synthesized using tritiated BNHS. Synthesis of Reagents
Materials. The following chemicals are obtained from Aldrich (Milwaukee, WI): tetraethylene glycol, p-toluenesulfonylchloride, triphenylphosphine, di-tert-butyl dicarbonate, and dimethylaminocinnamaldehyde. Lithium azide is obtained from Kodak (Rochester, NY). 4'-Formyltrioxsalen is obtained from HRI Associates (Berkeley, CA). N-Hydroxysuccinimide ester of biotin (BNHS) is obtained from Pierce
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Chemical Co. (Rockford, IL). Tritiated BNHS is obtained from Amersham (Arlington Heights, IL). Analytical thin-layer chromatograms are run on Bakerflex IB2-F plates (J. T. Baker, PhiUipsburg, NJ). Preparative TLC is performed using uniplate silica gel GF tapered layer plates (Analtech, Newark, DE). Biotinylated compounds are detected on TLC plates using a spray reagent consisting of equal volumes of 2% (v/v) sulfuric acid in ethanol and 0.2% (w/v)p-dimethylaminocinnamaldehyde in absolute ethanol. 5 NMR spectra are obtained using a Varian FT80, and shifts are reported as parts per million (ppm) downfield from tetramethylsilane as an internal standard. Mass spectral data are provided by the mass spectrometry laboratory at the University of California at Berkeley, Department of Chemistry. Bistosyl-3,6,9-trioxaundecane-1, l l - d i o l . To a chilled solution of tetraethylene glycol (42 g, 216 mmol) in 500 ml of dry pyridine is added ptoluenesulfonyl chloride (100 g, 525 mmol). The solution is stirred at 4° for 18 hr. To the solution is added 100 ml of methanol, and stirring is continued for an additional hour. The solvent is removed under reduced pressure and the residue partitioned between 300 ml of ethyl acetate and 300 ml of 0.5 M citric acid. The organic layer is washed with saturated NaC1 (2 times, 300 ml each). The organic extract is dried over magnesium sulfate, filtered, and concentrated to a syrupy residue (92 g) which may be used directly for the next reaction. If desired, the material may be purified by silica gel column chromatography using dichloromethane-methanol (97:3) as eluant (TLC, Rf = 0.54, detect with iodine vapor). NMR (DMSO-d6): 2.41(s), 3.46(s), 3.55(m), 4.14(m), 7.46(d, J = 8.3 Hz), 7.82 (d, J = 8.3 Hz). 1 , 1 1 - D i a z i d o - 3, 6, 9 - t r i o x a u n d e c a n e . To a solution of bistosyl-3,63trioxaundecane-l,ll-diol (50.3 g, 100 mmol) in 250 ml of dry dimethylformamide (DMF) is added lithium azide (30 g, 613 mmol). The mixture is heated to 75 ° with stirring until TLC on silica gel (ethyl acetate) reveals no starting material remaining. The solvent is removed under vacuum and the residue partitioned between 500 ml ethyl acetate and 250 ml of saturated NaC1. The organic layer is dried over magnesium sulfate, filtered, and concentrated to an oil (21 g). This material can be either reduced directly in the following reaction or purified by column chromatography on silica gel using ethyl acetate as eluant (TLC, R f -- 0.71, iodine vapor detection). NMR (DMSO-d6): 3.34(m), 3.57(s), 3.65(m). IR (neat): 2100 cm-1. 1 , 1 1 - D i a m i n o - 3, 6, 9 - t r i o x a u n d e c a n e (I). Triphenylphosphine (89 g, 339 mmol) is added to a chilled, stirred solution of 1,11-diazido-3,6,9-
5 D. B. McCormick and J. A. Roth, this series, Vol. 18, p. 383.
[67]
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trioxaundecane (24.4 g, 100 mmol) in 250 ml pyridine. Nitrogen bubbles evolve. The solution is stirred with cooling on ice for 45 min and then allowed to warm to room temperature. After 45 rain at room temperature, 100 ml of concentrated ammonium hydroxide is added and the mixture stirred overnight. The solvent is removed under reduced pressure, and the mixture is partitioned between 500 ml of 0.5 M citric acid and 250 ml of ethyl acetate. The aqueous phase is washed with ethyl acetate (2 times 250 ml each) to remove triphenylphosphine oxide. The aqueous phase is saturated with sodium chloride and exhaustively extracted with n-butanol. The alcohol extract is concentrated under reduced pressure and the residue taken up in ! 00 ml of absolute ethanol. Concentrated hydrochloric acid (15 ml) is added, and the solvent is removed under reduced pressure. The residue is crystallized from ethanol-ether. (Note: the product will not crystallize unless it is anhydrous.) The hygroscopic crystals (21 g) are dried under vacuum. TLC, Rf = 0.28 [silica gel, dichloromethane-methanol-acetic acid (70 : 30 : 5), ninhydrin detection]. NMR (DMSO-d6): 3.39(s), 3.57(s), 3.64(s), 8.10(br). 1 - A m i n o - 12 - t e r t - b u t y l o x y c a r b o n y l a m i n o - 3 , 6 , 9 , - t r i o x a u n d e c a n e
(I1). To a solution of I (13.26 g, 50 retool) and triethylamine (8 ml, 5.8 g, 57.4 mmol) in 100 ml of methanol is added a solution of di-tert-butyl dicarbonate (12 g, 55 retool) in 10 ml of methanol. Carbon dioxide bubbles evolve, and the reaction is slightly exothermic. Stirring is continued until gas evolution ceases. The solvent is removed under reduced pressure, and the residue is adsorbed onto 20 g of silica gel and fractionated on a column using dichloromethane-methanol-acetic acid (70:30:5) as eluant. The fractions containing product are pooled and concentrated to yield the mono-BOC (butyloxycarbonyl) derivative as a syrup. TLC, Rf = 0.67 [dichloromethane-methanol-acetic acid (70 : 30 : 5), ninhydrin positive]. 12 - tert - B u t y l o x y c a r b o n y l a m i n o - 1 - [ ( 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n - 4 'm e t h y l e n y l ) a m i n o ] - 3 , 6 , 9 - t r i o x a u n d e c a n e (III). 4 '-Formyltrioxsalen (1 g,
3.9 mmol) and 1 g of 3 /k molecular sieves are added to a solution of compound |I (2.28 g, 7.80 retool) and sodium cyanoborohydride (246 rag, 3.9 mmol) in 50 ml of anhydrous methanol. The suspension is adjusted to pH 7 by addition of triethylamine and is stirred in the dark at room temperature until TLC [silica gel, dichloromethane-methanol (8: 1)] shows no formyltrioxsalen remaining. The mixture is filtered and evaporated to a residue which is purified by silica gel column chromatography using the TLC solvent system as eluant. Fractions containing the fluorescent product are pooled and evaporated to dryness to yield 1.8 g. TLC, Rf = 0.41 [dichloromethane-methanol (8:1); fluorescent under longwavelength UV; ninhydrin positive after spraying the plate with 3 M HCI
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in ethyl acetate]. NMR (DMSO-d6): 1.36(s), 2.39(s), 2.44(s), 2.65(t, J = 5.5 Hz), 3.07(t, J = 5.5 Hz), 3.48(s), 3.81(s), 6.25(s), 6.73 (br t), 7.73(s).
12-Biotinylamino-l-[(4,5 ',8-trimethylpsoralen-4 '-methylenyl)amino]3,6, 9-trioxaundecane (IV). To a solution of III (71.2 mg, 133.7 ~mol) in 1 ml of dry dioxane is added 0.5 ml of a saturated solution of HC1 in dioxane ( - 1 2 M). The solution becomes cloudy, and a precipitate is formed which begins to crystallize. The mixture is checked by TLC [silica gel, 2-butanone-acetic acid-water (70 : 30 : 25)]. When the BOC-protected material is no longer evident ( - 3 0 min), the solvent is removed under a stream of air. The crystalline residue is taken up in 0.5 ml of methanol and taken to dryness. The residue is reconstituted in 0.4 ml of dry DMF, and 120/~1 (689/.Lmol) of diisopropylethylamine is added. This solution is added to a suspension of BNHS (50 mg, 146.5 /xmol) in 0.2 ml of dry DMF. All material dissolves. After 2 hr, the mixture is taken to dryness and reconstituted in 1 ml of methanol. The solution is applied to two 20 x 20 cm preparative TLC plates, and the plates are developed with 2-butanoneacetic acid-water (70 : 30 : 25). The product band (Rf = 0.77, fluorescent and biotin positive) is scraped and eluted from the silica gel with 20 ml of methanol. The extract is filtered, and the concentration of product is determined spectrophotometrically using an extinction coefficient of 25,000 at 250 nm. Yield, 87.6/zmol of IV (66%). Fast atom bombardment high-resolution mass spectroscopy: M + H = 659.3109 (C33H47N4OsS = 659.829). Probe Preparation and Biotinylation Partially double-stranded probe constructs (as depicted in Fig. 1) are biotinylated by mixing the labeling reagent with the probe DNA at a molar ratio (reagent to base pairs) of 2 : 1 and a DNA concentration of 100/zg/ml in 100 mM NaC1, 10 m M Tris (pH 7.5), and 1 mM EDTA. The mixture is irradiated with 360-nm UV light (B-100A Black-Ray Lamp, UV Products, San Gabriel, CA) at a flux of 30 mW/cm 2 for 10 min, and the biotinylated gapped circles are purified from the reaction mixture by chromatography on a 0.7 × 30 cm Sephacryl S-200 column in 10 mM NaCI, 10 mM Tris, 1 mM EDTA. These conditions result in the incorporation of one biotinylated psoralen labeling reagent for every 10 base pairs (10%). Levels of label incorporation are assayed by scintillation counting of probes that had been photoreacted with tritiated biotinylated psoralen. Comments
Biotinylated nucleic acid probe constructs have previously been made by enzymatic incorporation of biotinylated nucleotides into doublestranded DNA, 2 by chemical synthesis of oligonucleotides using biotin
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derivatives,6,7 o r by reaction of the probe with photoreactive biotin derivatives. 8 The disadvantages of the enzymatic method of biotinylation include the expense of reagents and difficulties involved with control and scale-up of the reaction. Biotinylated oligonucleotides are difficult to prepare and have the disadvantage of delivering very few biotins to the site of hybridization. Photoreactive biotin derivatives previously reported are based on arylazides that generate arylnitrenes upon photoactivation. These nitrenes do not discriminate between single- or double-stranded nucleic acids or between nucleic acids and other molecules. The attribute of biotinylated psoralen that makes it attractive as a labeling reagent is its ability to discriminate between single- and doublestranded nucleic acids; psoralens are known to interact preferentially with the duplex form. It was demonstrated previously9 that M13 probes could be treated (cross-linked) with trioxsalen without compromising the ability of the probe to hybridize to target nucleic acids. We have demonstrated that partial duplex probes are also suitable substrates for reaction with psoralen derivatives. 4 The presence of a large substituent at the 4' position of the psoralen ring system does not prevent the molecule from intercalating and subsequently photoreacting with the duplex region. The level of incorporation (psoralens per 100 base pairs) is comparable for both 4'-aminomethyltrioxsalen and the biotinylated psoralen described herein. It is possible to bind one psoralen per 4-5 base pairs for either compound.~° We have found that this level of incorporation is unnecessary for optimal signal detection. Because of the size of the enzyme conjugates used for detection, it is probably unnecessary to have more than approximately one biotinylated psoralen per 10 base pairs. Higher levels of incorporation lead to higher levels of background, presumably owing to aggregation of the heavily biotinylated probe or increased nonspecific adherence to the membrane. The extent of incorporation of biotinylated psoralen with nucleic acids is easily controlled by adjusting the base pair to psoralen stoichiometry and has been found to be insensitive to scale-up. Milligram quantities of probe DNA can be labeled in the single reaction. Acknowledgments The authors would like to acknowledgethe creative input of Kary Mullis and Henry Rapoport, and the technical assistance of Diana Ho, David Kellogg, Todd Smith, Rick Snead, and Dragan Spasic. 6j. Kempe, W. I. Sundquist, F. Chow, and S. L. Ho, Nucleic Acids Res. 13, 45 (1985). 7A. Cholletand E. H. Kawashima,Nucleic Acids Res. 13, 1529(1985). s A. C. Forster, J. L. Mclnnes, D. C. Skingle, and R. H. Symons,Nucleic Acids Res. 13, 745 (1985). 9 D. M. Brown, J. Frampton, P. Goelet, and J. Karn, Gene 20, 139 (1982). ~0S. T. Isaacs, C. J. Shen, J. E. Hearst, and H. Rapoport, Biochemistry 16, 1058(1977).
