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1 Journal of the Korean Chemical Society 2005, Vol. 49, No. 4 Printed in the Republic of Korea 대사추적용 P-32 표지시약의화학적합성에관한연구 xá Á Áwx Á x* w yw w ( ) Study on the Novel Synthesis of P-32 Labeled Agents for Tracing Metabolism Kang-Hyuk Choi, Byung-Jae Ahn, Ul-Jae Park, Hyon-Soo Han, and Kook-Hyun Yu* Department of Chemistry, Dongguk University, Seoul , Korea Radioisotope Production and Application Division, Korea Atomic Energy Institute, Daejun , Korea (Received March 24, 2005). [ 32 P] γ-atp [ 32 P] α-dctp t P-32 t yw, y RNAù DNA P-32 t». yw yw w w w. P-32 t yw yw w, O-8-(5-chloroquinolyl)-S-phenyl phosphorothiate w. O-8-(5-chloroquinolyl)-S-phenyl phosphorothiate w 42%. w [ 32 P] γ-atp [ 32 P] α-dctp w t ƒƒ 95%, 40%. : [ 32 P]H 3PO 4, [ 32 P] γ-atp, [ 32 P] α-dctp, yw w, O-8-(5-Chloroquinolyl)-S-Phenyl Phosphorothiate ABSTRACT. [ 32 P] γ-atp (adenosine 5'-[γ- 32 P]triphosphate) and [ 32 P] α-dctp (deocycytidine 5'-[α- 32 P]triphosphate) are representatives of P-32 labeled compounds and used as phosphorylating agents or labeled substrates for several RNA and DNA polymerase reactions. These are usually produced by enzymatic processing. Our studies have been focused mainly on preparing these compounds by chemical processing for mass production. O-8-(5-chloroquinolyl)-S-phenyl phosphorothiate, a coupling agent, is proper for the systhesis of [ 32 P] γ-atp and [ 32 P] α-dctp. Coupling agent has been prepared in a yield of 42%. [ 32 P] γ-atp and [ 32 P] α-dctp were prepared in radiochemical yields of 95% and 40%; respectively. Keywords: [ 32 P] H 3PO 4, [ 32 P] γ-atp, [ 32 P] α-dctp, Chemical Method, O-8-(5-Chloroquinolyl)-S-Phenyl Phosphorothiate P-32 w t yw w y» w w xk š. ƒ t yw [ 32 P] γ-atp(adenosine 5'-[- 32 P]- triphosphate) [ 32 P] α-dctp(deocycytidine 5'-[α- 32 P]- triphosphate)ƒ, w t yw t DNA RNA t». ü P-32 t yw 2002» 3526 mci 1, w wš. w št P-32 ƒ [ 32 P] H 3PO 4 xk (30 Ci/batch) š t yw w ƒ w š. w xk P-32 t yw w α γ e k t w yw 2-6 w. [ 32 P] γ-atp yw w Johnson/walseth 7) Maxam/gilbert 8 381

2 382 xá Á Áwx Á x wš. [ 32 P] α-dctp w nucleoside monophosphate kinase nucleoside diphosphate kinase nucleotide kinase w yw w y mù p (trichloroacetonitrile) w yw w ww w yw w z w x w w. w yw w POCl 3 tri-n-butylammonium pyrophosphate w y(triphosphate) w j y j yw trichloroethyl phosphate, trichloroethyl phosphate quinolyl i»(quinolyl phosphate wš p, group) j p (nucleotide)» yw k k» w» w. w 8- i (8-quinolyl phosphate) r q»(phenylsulfide) w k» sw w (coupling agnet). Fig. 1 2 yw w, k ey ƒ w. r q» Ag + w y y e y, i» Cu 2+ w y y g ey Fig. 1. O-8-(5-chloroquinolyl)-S-phenyl phosphorothiate which contains two leaving group is use as coupling agent. ww. w w s j p w» w. ù x ¾ yw w w P-32 t yw w x w k p, w P-32 t yw w š. w [ 32 P] γ-atp [ 32 P] α-dctp w w w x twš w. š w Scheme 1 2 (3) w w. yw (2) w 0, x w. w Cl v w v (xk e ). v w š w y yw (2) w. w yw (2) w xk k w š HMPA DMSO w. yw (3) Tsujiaki Hata 19 šw 10 w w r q (phenyldisulfide) w yw (3) 10%. v mù p (acetonitrile) yw w 2.5 r q yw (3) w w TLC w y w. w r q ƒw, (yw (2)) w y wš p ps v(tributylphosphine) ƒw 60% yw (3). ATP w [ 32 P] γ-atp(5) w Scheme 2 P-32 t Scheme 1. Reaction step in the preparation of O-8-(5-chloroquinolyl)-S-phenyl phosphorothiate. Journal of the Korean Chemical Society

3 P-32t yw w w 383 Scheme 2. Reaction step in the preparation of [ 32 P] γ-atp using chemical processing. yw w» w w ( 31 P) ATP w w. ATP w 2 w» (One-spot Reaction) ww. yw (4) y Ag + r q» w AgSPh e y ƒ w TLC y w. e ep v l(nylon, pore size 0.45 µl) w w. yw (4) i» w» w Cu 2+ w j»ƒ w 2+ Zn w. w w ATP qw ATP TLC w y w. y n-buoh :EtOH:H 2O:NH 3(1:1:1:1) w, ATP 0.53, AMP 0.70, yw (3) 0.85 ùkü. Scheme 2 w mw P-32 t yw w w y w. Scheme 3. Reaction step in the preparation of [ 32 P] α-dctp using chemical processing. 2005, Vol. 49, No. 4

4 384 xá Á Áwx Á x Fig. 2. Partially protected phosphoric acid with Triethylamine. dctp w [ 32 P] α-dctp (8) w Scheme 3 t yw w w» w w dctp w y w. dcmp (6) w H 3PO 4 e»ƒ CCl 3CN 11 ú. wƒ ù y» v w. w w» w p p (triethylamine) w y» w. H 3PO 4 pka 1, pka 2, pka 3 ƒƒ 2.12, 7.21, p p (pka = 11.1) ƒw H 3PO 4» k y w. y H 3PO 4 Fig. 2 k, k y ƒ w. wr, v pka 5.25 H 3PO 4 pka 1 pka 2 ƒ, k w» y w. w dcmp y w y C 3H 7OH : H 2O : NH 4OH(11 : 4 : 5) w dcmp 0.55 ùkü. dctp w ATP w w w. TLC y w C 3H 7OH : H 2O:NH 4OH(55 : 20 : 25) 0.35, n-buoh : H 2O : MeOH : NH 4OH (1:1:1:1) dcmp dctp w y. [ 32 P] H 3PO 4 [ 32 P] H 3PO 4 w t, t» ƒ yw w ùkú, t yw w w w. t yw w [ 32 P] H 3PO 4 yw y w. n [ 32 P] H 3PO 4 w Table 1. Analytical data of 10-fold diluted [ 32 P] γ-atp solution. Element-analysis is carried out with ICP-AES and anions are analyzed with spot test Elements and Ions Contents (ppm) Elements and Ions Contents (ppm) Fe 0.8 Al 4 Na 10 Ba 0.4 Mg 1.6 Si 4 Ca 4 2 CO 3 N.D K N.D 2 SO 4 N.D Cu N.D CN N.D Mn ClO 4 N.D Zn 0.4 SCN N.D Cd 0.4 Cl Detection w ICP-AES w w. Cl, CN 2, ClO 4 ƒ w ƒ sww Cl y. y ƒ [ 32 P] H 3PO 4 1ml 20 x w. Na 10 ppm, Ca 4 ppm, Al 4 ppm š Si 4 ppm ùkû. ùkù w Cl Na + Cl ewey ww k» w t yw w ƒ. [ 32 P] H 3PO 4 ƒ w y. t yw [ 32 P] γ-atp(5) w w [ 32 P] H 3PO 4 2 mci/1 ml HCl k, Scheme 2 x w y Table 2 ùkü. 3 w 5 z x w 40% [ 32 P] H 3PO 4 š w [ 32 P] γ-atp 60%. w HCl w w w 95%. ww [ 32 P] H 3PO 4 w yƒ ùkû w y ƒ w š w. w k [ 32 P] H 3PO 4 wš ATP, 2, 5 y w x 4. w [ 32 P] Journal of the Korean Chemical Society

5 P-32t yw w w 385 Table 2. Preparative conditions of [ 32 P] γ-atp and its yield. [ 32 P] γ-atp is identified by Radio TLC scanning Exp. Precursor Cpds (3) (mg) AMP (mg) Solvent MPD/HMPA AgNO 3 [ 32 P]-H 3PO 4 CuCl 2 Yield Exp mg 10 mg 0.9 ml /0.3 ml 1.1 eq 5-fold Dilution 250 µl 5 eq 40% Exp mg 10 mg 0.9 ml /0.3 ml 1.1 eq 100 µl 5 eq 60% Exp mg 11 mg 0.9 ml /0.3 ml 1.5 eq 100 µl 5 eq 73% Exp mg 11 mg 0.9 ml /0.3 ml 2 eq 100 µl 5 eq 95% Exp mg 11 mg 0.9 ml /0.3 m 5 eq 100 µl 5 eq 88% Fig. 3. Radio TLC Scanning chromatogram of [ 32 P] γ-atp with n-buoh : EtOH : H 2O : NH 3 (1:1:1:1) as eluents Free Phosphoric acid([ 32 P]GH 3PO 4) and [ 32 P] γ-atp are identified in R f = 0.1 and γ-atp j m Fig. 3 t 97%. t yw [ 32 P] α-dctp(8) w Scheme 3 [ 32 P] α-dctp w [ 32 P] dcmp w w y w. w [ 32 P] H 3PO 4 3 mci/1 ml HCl Fig. 4. TLC Scanning chromatogram of prepared [ 32 P] dcmp. (Distance from solvent front : 80 mm, Eluents; C 3H 7OH : H 2O : NH 4OH=55 : 20 : 25, Radio chemical yield: 55%) : Free Phosphoric acid([ 32 P]GH 3PO 4) and [ 32 P] dcmp are identified in R f = 0.1 and k w v w» 14/20 jointƒ x w w. [ 32 P] dcmp w j w. [ 32 P] H 3PO 4 1z 10~30%, 3z 40~55% [ 32 P] dcmp ùkû (Table 3. Fig. 4). p, p p w û y w. w 3z k [ 32 P] H 3PO 4 wš w p p w. w w [ 32 P] Table 3. Preparative conditions of [ 32 P] α-dcmp and its yield. [ 32 P] α-dcmp is identified by Radio TLC scanning Exp. Deoxycytidine CCl 3CN DMSO [ 32 P]H 3PO 4 Yield š Exp. 1 20Gmg 8.2 µl 0.2Gml 0.1GmlG(3GmCi) - Exp. 2 20Gmg 8.2 µl 0.2Gml 10 µlg(0.03gmci) - Exp. 3 20Gmg 8.2 µl 0.2Gml 0.1GmlG(0.25GmCi) 30% dried [ 32 P] H 3PO 4 Exp. 4 20Gmg 8.2 µl 0.2Gml 0.1GmlG(0.3GmCi) 11% dried [ 32 P] H 3PO 4 Exp. 5 20Gmg 3.0 µl 0.5Gml 0.1GmlG(0.3GmCi) 42% dried [ 32 P] H 3PO 4 Exp. 6 20Gmg 3.0 µl 0.2Gml 0.1GmlG(0.3GmCi) 55% dried [ 32 P] H 3PO , Vol. 49, No. 4

6 386 xá Á Áwx Á x š w» w k š j m v/»(fab) w. j m Bioscan System 200 imaging scanner AC-3000 w. w GBC Scientific Equipment Pty Ltd (GBC Integra XMZ-Australia) ICP-AES w. Fig. 5. TLC Scanning chromatogram of prepared [ 32 P] α-dctp. (Distance from solvent front : 90 mm, Eluents; n-buoh : H 2O : MeOH : NH 4OH=1:1:1:1, Radio chemical yield: 65%) : [ 32 P] α-dctp was prepared using sep-pak filtered [ 32 P] dcmp. Free Phosphoric acid([ 32 P]GH 3PO 4) and [ 32 P] α-dctp are identified in R f = 0.1 and dcmp sep-pak ep w w w [ 32 P] α-dctp w ATP w w ww. j m y w [ 32 P] α-dctp(8) t 65% (Fig. 5), w t 40%. [ 32 P] α-dctp(8) w [ 32 P] dcmp w ƒ j w š y w.»» P-32 w [ 32 P] H 3PO 4 w, 1-chloro-8-hydroxyquinoline, phenyl disulfide adenosine 5'-monophosphate Acrose t,, y (CuCl 2), H 3PO 4 Aldrich t, Aldrich Merck p w. w x w» Aldrich KONTES MICROSCALE GLASSWARE KIT w w e w w. w y w TLC Merck Silica gel 60 F 254 w» UV- v I 2 w. IR Bomen 100 FT-IR Spectrophotometer, NMR Varian Gemini 200 w x 5-Chloro-8-quinolyl phosphate (2) w v 1,4- yw (30 ml) POCl ml z 1,4-10 ml w k 5-chloro-8-hydroxy quinoline 0 o C ƒw. 5 k z vl w z wš v (1:1) yw 10 ml 0 C o ƒw. w z mù p (1:1) yw w 1 e mù p w 2 w 63%(1.8 g) y w (2)(5-chloro-8-quinolyl phosphate). TLC (n-buoh : MeOH : H 2O : NH 4OH=1:1:1:0.5): R f = mp= H NMR (DMSO): δ=9.02(dd; 1 H, Ar-H), 8.55(dd; 1 H, Ar-H), 7.73(m; 3 H, 3Ar-H), 5.32 (broad; 2 H, 2 P-OH) MS(FAB, M + +1): C 9H 7ClNO 4P cald. for found O-8-(5-chloroquinolyl) S-phenyl phosphorothiate (3) w» 1 g(3.86 mmol) yw (2) 3 ml v 15 ml mù p ƒ w. 2.1 g(9.65 mmol) r q (phenyldisulfide) š 4.8 ml(19.30 mmol) ps v(tributylphosphine) ƒw. 1 w z j s w» w. yw j k z mù p 0.88 ml (7.72 mmol) j x ƒw -20 C o g w w w, 68%(1.18 g). TLC(MC : MeOH, 7 : 3): R f= H NMR(CD 3OD): δ=8.86(dd; 1 H, quinoline-h), 8.67(dd; 1 H, quinoline- H), 7.89(dd; 1 H, quinoline-h), 7.69(q; 1 H, quinoline- H), 7.65(d; 1 H, quinoline-h), 7.40(m; 2 H, 2Ar-H), 7.20(m; 1 H, Ar-H), 7.13(m; 1 2H, 2Ar-H), 5.32(broad; Journal of the Korean Chemical Society

7 2 H, 2 P-OH). MS(FAB, M + +1): C 21H 24ClN 2O 3PS cald. for found ATP(5) w 1- pv (MPD) x ps s (HMPA) yw 6 ml 100 mg(0.22 mmol) yw (3) 77 mg(0.22 mmol) AMP(Adenosine monophosphate) ƒw 10 w z 45 mg (0.26 mmol) 30 ww. w yw (4) w š w 38 µl (0.66 mmol) 150 mg(1.1 mmol) y (CuCl 2) ƒ z 12 w g. w ATP TLC w y w. TLC (n-buoh : MeOH : H 2O:NH 4OH=1:1:1:1): R f =0.54. dcmp dctp w»w 30(0.5 mmol) m ù p ƒwš 2z ww. 1 ml (7 mmol) p p ƒw w 200 µl mù p ƒw.» 20 mg(0.08 mmol) p (deoxycytidine monohydrate) z mù p w w. 200 µl DMSO 100 µl(1 mmol) y mù p (CCl 3CN) ƒwš z» 40 C 30 o ew. w yw (6) qw dcmp TLC w. yw (6) w w 1- pv x ps s 1 : 1 yw 4 ml ƒw z 45 mg(0.26 mmol) 100 mg(0.22 mmol) yw (3) ƒ wš 30 k. w 30 µl(0.5 mmol) 100 mg(0.73 mmol) y ƒw z 12 ww. w dctp t TLC w y w. TLC (C 3H 7OH : H 2O:NH 4OH=11 : 4 : 5): dcmp (R f= 0.55), dctp (R f =0.35) TLC (n-buoh : MeOH : H 2O : NH 4OH=1:1:1:1): dctp (R f =0.50) t yw [ 32 P] γ-atp(5) w β s 10 mm j q w 2005, Vol. 49, No. 4 P-32t yw w w 387 Microscale vial w w ww. 0.9 ml 1-MPD 0.3 ml HMPA 13 mg(0.029 mmol) yw (3) 10 mg(0.029 mmol) AMP ƒwš 10 k z 10 mg(0.058 mmol) ƒw 30 k. mù p w yw mw 2z k 0.2 mci [ 32 P] H 3PO 4 yw z 19 mg(0.14 mmol) y ƒwš 3 w [ 32 P] γ-atp(5) w w. w y t j m w. TLC (n-buoh : MeOH : H 2O:NH 4OH=1:1:1:1): R f =0.54. Radiochemical yield: 95%. t yw [ 32 P] dcmp(6) w 10 mm j q β sw 14/20 š ƒ x 0.3 mci (100 µl) [ 32 P] H 3PO 4 w. 100 µl p p ƒ wš 1ml mù p w 30 o C 3z wš š k.» 20 mg(0.08 mmol) p (deoxycytidine monohydrate) z 100 µl DMSO 3 µl y mù p (CCl 3CN) ƒwš z» 40 µl 30 ew. w y t j m w. TLC (C 3H 7OH:H 2O:NH 4OH=11:4:5): R f = Radiochemical yield: 55%. t yw [ 32 P] α-dctp(8) w yw (6) sep-pak z 1- pv x ps s 4 ml ƒw. 13 mg yw (3) ƒw z 10 mg ƒ z 30 k. w 30 µl(0.5 mmol) 100 mg(0.73 mmol) y ƒw z 3 w [ 32 P] α-dctp w w. TLC (n-buoh : MeOH : H 2O:NH 4OH=1:1:1:1): dctp (R f = 0.50). Radiochemical yield: 65%. O-8-(5-chloroquinolyl) S-phenyl phosphorothiate w 42% w y NMR, IR, TLC

8 388 xá Á Áwx Á x w. yw (3) w [ 32 P] γ-atp w w 95% t w 4. [ 32 P] α-dctp w w 35-40%. w 6. yw w mw P-32 t yw [ 32 P] γ-atp( e t ), [ 32 P] α-dctp (α e t ) w [ 32 P] dcmp w j w Sep-pak. w» z w. p, [ 32 P] γ-atp z š q ƒ ƒ w š. wù w w, w w j p. x 1. w z, 2003 Nuclear Energy Yearbook, Korea, 2003, Fumio Hayashi, Osamu Hatano and Takao Shinozawa, Radioisotopes, 1987, 36, Barai, V.N.;G Zinchenko, A.I.; Zalashko, L.M.; Eroshevskaya, L.A. and Mikhailopulo, I.A. Biotechnology letters, 1995, 17(6), Inwon Park and Tae-Neung Johng, Purification of Homemade 32 P-H 3PO 4 and its Application to the Synthesis of [α- 32 P]ATP, Korean Biochem. J., 1984, 17(1), ª, š, [α- 32 P]dCTP, Jouranl of Nuclear and Radiochemistry, 1991, 13(1), Anna Janecka, Methods of preparation of nucleosidetriphosphate labeled with phosphorus-32 in Alfa and gamma position, Post. Biochem., 1983, 29, Johnson, R.A and Walseth, T.F (BrookeG., Greengard. P. and Robinson, G.A., Ed.): Advances in Cyclic Nucleotide Research, 1979, 10, Grossman, I. and Moldave K., (Maxam, A.M and Gilbert, W), Methods in Enzymology, 1980, 65, Symons, R.H. Methods in enzymology, 1974, 29, Symons, R.H. Nucleic acids Reserch, 1977, 4(12), Friedrich Cramer and Gunter Weimann, Chem. Ber., 1961, 94, Donald E. Hoard and Donald G. Ott, Journal of the American Chemical Society, 1965, 87(8), J. Zimmet, L. Jarlebark, R. Hammarberg, P.J.M. von Gslrn, K.S. Jsvobdon, and E. Heilbronn, Nucleosides & Nucleotides, 1993, 12(1), Hiroshi Takaku, Ryuichi Yamaguchi, and Tadaaki Nomoto, Tetrahedron Letters, 1979, 40, Hiroshi Takaku, Chem. Pharm. Bull, 1977, 25(8), Hiroshi Takaku, Toshimi Konishi, and Tsujiaki Hata, Chemistry Letters, 1977, Koichiro Fukuoka, Fuminori Suda, Masahide Ishikawa, and Tsujiaki Hata, Nucleosides &G Nucleotides, 1995, 14, Hiroshi Matsuo, Tomohisa Morguchi, Toshimitsu TaKabe, Stephen Buratowski, Mitsuo Sekine, Yoshimasa Kyogoku, and Gerhard Wagner, J. Am. Chem. Soc., 2000, 122, Tsujiaki HATA and Mitsuo SEKINE, The synthesis of S-phenyl nucleoside phosphorothioates, Chemistry Letters, 1974, 837. Journal of the Korean Chemical Society