.:) 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 21 5815-5820
Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA Andre Guy, Anne-Marie Duplaa, Jacques Ulrich' and Robert Teoule* Service d'Etudes des Systemes Moleculaires, Departement de Recherche Fondamentale de la Matibre Condensee, Centre d'Etudes Nucleaires de Grenoble, BP 85X, 38041 Grenoble Cedex and 'Departement de Biologie Moleculaire et Structurale, Spectrometrie de Masse des Proteines, Centre d'Etudes Nucleaires de Grenoble, BP 85X, 38041 Grenoble Cedex, France Received October 1, 1991; Accepted October 10, 1991
ABSTRACT 2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.
INTRODUCTION The chemical modifications induced in the DNA in cells exposed to the action of ionizing radiation are thought to be responsible
for biological effects such as cellular lethality, mutagenesis and carcinogenesis (1,2). When thymidine is gamma-irradiated in aqueous solutions under aerobic conditions the predominant hydrolysis product is 2-deoxyribosylformylamine (3,4). The nature of modified bases on the DNA chain has been analysed after mild acid hydrolysis of irradiated DNA (5). Pyrimidine radiation products are generated by the attack of OH radicals at the 5,6-double bond of the pyrimidine ring (6). Subsequently, pyrimidine ring opening gives rise to formylamino residue. Ionizing radiation produces a large spectrum of DNA modifications and each individual damage appears in low yield so that irradiation cannot conveniently be used to prepare an oligonucleotide with a single formylamino residue. One of the difficulties is that very few methods are available to insert only one damage into an oligonucleotide (7). Our approach was to prepare first the modified phosphoramidite and then to introduce the alkali labile altered nucleotide during the chemical synthesis of DNA fragments. *
To whom correspondence should be addressed
In contrast to other oxidative DNA damages such as thymine glycols or urea residues containing DNA (8-1 1), the role played by the formylamino residue has not been investigated. It would be of interest to know if this lesion blocks the directed DNA polymerase synthesis on a template bearing this damage and to determine whether or not this defect is processed by DNA repair enzymes. In order to examine the biological consequences of this oxidative DNA damage we have selectively introduced the defect into DNA fragments. In this article, we describe the chemical synthesis of oligodeoxyribonucleotides containing the formylamino nucleotide in a well defined position within the sequence. Results concerning the role played by the lesion in in vitro replication are reported.
EXPERIMENTAL General Solvents were dried or distilled before use. 4-Methoxytriphenylchloromethane (MMT Cl): Fluka purum. Lead tetraacetate (Pb(OAc)4: Fluka purwm) was vacuum-dried at 10 mm Hg over P205 and KOH, room-temperature, overnight. KMnO4: Prolabo RP Normapur. 2-Cyanoethyltetraisopropyl-phosphoro-diamidite was prepared according to (27). Tetrazole was sublimated under reduced pressure. Diisopropylammonium tetrazolide was prepared according to (14). 5'-O-(4,4'-dimethoxytrityl)-Nprotected-2 ' -deoxynucleoside-3 '-O-(2-cyanoethyl-N, Ndiisopropylamino)-phosphoramidites with labile base protection (N6-phenoxyacetyl adenine, N2-phenoxyacetyl guanine, N4-isobutyryl cytosine and thymine) and the 1 Amole nucleoside (Thymidine or isobutyryl cytidine) grafted on a chemical CPG support were synthesized according to (17). Thin layer chromatography (TLC) was performed on precoated silica gel 60 F 254 (Merck), column chromatography: silica gel 60 H (Merck), and analytical HPLC mode using different purification
techniques to acquire pure compounds (Varian 5020 liquid chromatograph, variable wavelenght UV-Vis Cecil detector). UV shadowing or staining with cysteine-ethanolic-sulfuric acid spray was used to visualize UV absorbing or trityl derivative on TLC. UV spectra: Beckman DU-8B spectrophotometer: X max (e) in
5816 Nucleic Acids Research, Vol. 19, No. 21 nm. NMR spectra: Briker AC 200 ('H; 200 MHz) or WM 250 C1P; 101.2 MHz) spectrometers; chemical shift values in ppm rel. to TMS as internal reference (1H) or to 85% H3 P04 as external standard (31p). Pyrolysis Mass Spectrometry and positive or negative ion fast atom bombardment mass spectrometry (FAB-MS) (28): Kratos MS 50 and VGMS (selected peaks: m/z (%)). Oligonucleotide syntheses were performed on an Applied Biosystems 381 A DNA synthesizer using new PAC-phosphoramidite monomers and the improved phosphoramidite procedure introducing N-methylimidazole and phenoxyacetic anhydride capping (29,30) and IM iodine/pyridine/THF/water oxidizing solution (31).
5'"O-(4-methoxytrityl)-thymidine (2) Compound 2 was prepared from 1 according to (12). The oily product was purified on silica gel column (80 g SiO2, 6 x 8 cm) using a step gradient of 0-4% MeOH in CH2Cl2 to give a 79% yield (7.7 g). TLC (CHCl3/MeOH, 90: 10): Rf = 0.74. UV (EtOH): 266.7 (9700). Anal. cal. for C30H30N206 (514.55): C 70.02, H 5.88, N 5.34, 0 18.76; found: C 69.74, H 6.16, N 5.44, 0 18.98. H'-NMR (200 MHz, CD3COCD3): 1.60 (s, 3H, CH3-C(5)); 2.45 (m, 2H, H-C(2'), H-C(2')); 3.50 (m, 2H, HC(5'), H-C(5')); 3.90 (s, 3H, MeO, MMt); 4.15 (q, 1H, H-C (4')); 4.70 (q, 1H, H-C (3')); 6.5 (t, 1H, H-C(l')); 7.0-7.6 (m, 14H, H arom.); 7.75 (s, 1H, H-C(6)); 10.2 (s, 1H, H-N). FAB-MS (neg. ions, glycerol matrix): 513 (37, [M-H]-), 241 (20, [M-H-MMT]-), 125 (100, B-).
2-deoxy-5-0-(4-methoxytrityl)-,B-D-ribofuranosyl-1-formylamine (3) To a solution of 2 (4.12 g, 8 mmol) in acetone (50 ml) and pyridine (10 ml) fine needles of potassium permanganate (2.6 g, 16.4 mmol) were gradually added under stirring. During the reaction period the pH was controlled at 8.2 and the temperature maintained at 25°C. The end of the oxidative reaction was checked by TLC (solvent CHCl3/MeOH, 90: 10). Aqueous NaHSO3 (0.8 M, 60 ml) was added, salts were filtered off, washed with acetone and filtrate was evaporated in vacuo to 20 ml. CH2CI2 (2 x 100 ml) was added and the organic extract was evaporated to dryness. The oily residue was dissolved in dry pyridine (20 ml) and anhydrous powdered Pb (OAc)4 (3.8 g, 8.4 mmol) was added under stirring. After completion (2 h, TLC monitoring) pyridine was removed in vacuo on a rotatory evaporator, the oily residue was dissolved in CH2CI2 (2 x 100 ml) and washed with 5% aqueous NaHCO3 (200 ml). The filtered organic layer was washed with H20 (200 ml) and dried (Na2SO4). Evaporation of the filtrate gave an oily residue which was loaded onto silica gel column (6 x7 cm, Merck silica gel G, 10-40 ttm, CH2Cl2). Elution with CH2CI2 containing 2% MeOH gave 3 Purification by HPLC (Lichroprep silica gel 60, 1 x 30 cm column) using a linear gradient of 0-5 % MeOH in CH2Cl2 gave a 32% yield of pure compound (1.13 g). TLC (Ethyl Acetate/Isopropanol/Water, 98: 4: 2): Rf = 0.58. Anal. cal. for C26H27NO5 (433.48): C 72.04, H 6.28, N 3.23, 0 18.46; found: C 72.33, H 6.30, N 3.26, 0 18.11. IH-NMR (200 MHz, CD30D, 2 conformers): 2.02 (m, 2H, H-C(2), HC (2')); 3.16 (m, 2H, H-C (5), H-C (5')); 3.76 (s, 3H, CH30 (MMT)); 3.95 (m, 1H, H-C (4)); 4.30 (m, 1H, H-C(3)); 5.50 (t, 0.4H, J = 6.5, H-C (1), minor conformer); 5.78 (t, 0.6H, H-C (1), major conformer); 6.80-7.50 (m, 14H, H arom.); 8.03 (s, 0.6H, H-C = 0, major conformer); 8.20 (s, 0.4H, H-C = 0, minor conformer); major conformer: 4J (H-C = 0 and HC (1)) = 0.8 Hz; minor conformer, 4J (H-C = 0 and H-C(l))
< 0.1 Hz. FAB-MS (pos. ions, PEG 200 matrix, Na I): 456 (55, [M + Na]+) FAB-MS (neg. ions, glycerol matrix): 432 (80, [M-H]-), 160 (100, [M-H-MMT]-). HR FAB-MS (neg.ions, PEG 200 matrix): 432.1835 [M-H] -, C26H2605N, calc. 432.1811.
2-deoxy-5-0-(4-methoxytrityl)-,B-D-ribofuranosyl-1-
formylamine-3i--(2-cyanoethyl)-NN'-diiso-propylphosphoramidite (4) To dry 3 (1.08 g, 2.5 mmol) dissolved in anhydrous CH2C12 (10 ml) at room temperature under dry Ar atmosphere were added with stirring 0.6 eq of anhydrous diisopropylammonium tetrazolide (0.25 g, 1.5 mmol) and 1.1 eq of bis-(N, N'-diisopropylamino)-2-cyanoethoxyphosphine (0.9 g, 2.75 mmol). After completion of the reaction (1 h, TLC analysis (CH2Cl2/MeOH, 9: 1)), ethyl acetate (50 ml) was added and the reaction mixture was extracted with 5% aqueous NaHCO3 (2 x 100 ml) then washed with brine. The organic extract was dried (Na2SO4), fitered and evaporated to an oily residue. Attempts to precipitate in cold (-70°C) hexane failed and lyophilisation from benzene gave 4 as white gel stored in sealed amber vials at -20°C. TLC (Ethyl acetate/dichloromethane/ triethylamine 40: 20: 2): Rf = 0.52. FAB-MS (pos. ions, PEG 200 matrix, Na I): 655.9 (24, [M + Na]+). HR FAB-MS (neg. ions, PEG matrix): 579.2647 [M-CNE]-, C32H40N206P, calc. 579.2624. 31P-NMR (101.2 MHz, CD3CN/CH3CN 1:2):148.8. General phophoramidite methodology for DNA synthesis Each synthesis was performed on a DNA synthesizer using a 1 mol protected nucleoside grafted on a chemical modified CPG support in a short teflon column. For optimal coupling efficiency the flow rate of reagents was fitted at 2.3 ml.mn-'. The protected phosphoramidite 4 and the N-blocked-5'-O-DMT-2'-
deoxynucleoside-3'-0-(2-cyanoethyl-N, N'-diisopropylamino)phosphoramidites of (MeO)2 Tr pac6 Ad, (MeO)2 Tr pac2 Gd, (MeG)2 Tr ibu4Cd, (MeO)2Tr Td were used in 0.08 M dry MeCN (or toluene/MeCN, 10: 100) solutions. The syntheses were carried out following the standard cycle protocol but with N-methylimidazole/phenoxyacetic anhydride and IM iodine/ pyridine/ THF/water for the capping and oxidizing steps respectively. During the detritylating step of the synthesis cycle monomethoxytrityl and dimethoxytrityl protecting groups were eliminated in the same mild acid condition (3% trichloroacetic acid in CH2Cl2 for 140 sec.). After removal of the trityl group, cleavage of the oligonucleotides from support and deprotection of the N-blocked deoxynucleosides and the phosphate groups were carried out with a 28% ammonia solution (5 x200 il) during 6 h at room temperature. HPLC purifications were performed on an anion-exchange column (0.75 x 30 cm, 10 jAm of Partisil SAX) using a linear gradient of 0.3 M KH2PO4 buffer (pH 6.7) with 30% Me CN over 50 min. Desalting prior to use by dialysis against distilled water (5 x 600 ml) gave products which were further purified by 20% denaturing polyacrylamide gel electrophoresis. Fast atom bombardment mass spectrometry for DNA sequencing The short model sequence 5' d(C-G-F-A-T)3' was prepared following general phosphoramidite procedure with 42 repetitive chemical cycles of trichloroacetic acid detritylation, phenoxyacetic anhydride/N-methylimidazole capping, I2/pyridine/THF/H20 oxidation, CH3CN washing steps in order to be in the same conditions for chemical preparation of a long polynucleotide. The
Nucleic Acids Research, Vol. 19, No. 21 5817 crude oligomer from ammonia treatment was purified on a reverse-phase column (0.46x25 cm, Hypersil ODS, 10 Am, Societe Francaise de Chromato Colonne) using a linear gradient of 3-10% MeCN in 0.025 M TEAA (pH 6.8) over 30mn. To evaluate the integrity of the formylamino residue we have performed mass spectrometric sequence analysis of this oligonucleotide. Negative ions FAB-MS unambiguously confirmed the complete ammonia deprotection of 5' d(C-G-F-AT)3' and the presence of the formylamino residue. FAB-MS (neg. ions, glycerol matrix, NH40H): 1395.0 ([M-H]-), calc. 1395.3. The complete sequence of the nucleotide (Figure 2) with the main fragmentations can be read independently starting either from the 5' or from the 3'-end: 306 (Cp-), 635 (Cp Gp-), 858 (Cp Gp Fp-), 1171 (Cp Gp Fp Ap-), 1395 (Cp Gp Fp Ap T-) or 321 (pT-), 634 (pA pT-), 857 (pF pA pT-), 1186 (pG pF pA pT-), 1395 (Cp Gp Fp Ap T-). In vitro replication (_y-32P) ATP (5000 Ci/mmol) was purchased from Amersham. T4 polynucleotide kinase and E. coli DNA polymerase I (Klenow fragment) were from Boehringer. Taq DNA polymerase was from Perkin Elmer Cetus. 10 pmol of the 5'-end 32p labeled 19 base long d(ACGACTTAGCGAGGTTAGC) primer were annealed with 10 pmol of the DNA template 9 (figure 1) as described (32).
Klenow fragment assay conditions: Primer elongation by the Klenow fragment was carried out for 15 min at 37°C in a buffer containing 50 mM Tris-HCl (pH = 7.6), 8 mM MgCl2, 1 mM DTT, 5 pmol of the labeled primer-template (about 1 x 106 cpm/pmol), 2 units of the enzyme and 25 ,tM of each of the four dNTPs in a final volume of 10 Al. Taq DNA polymerase assay conditions: Polymerisation by Taq polymerase was carried out by mixing 5 pmol (1. 106 cpm/pmol) of the labeled primer-template, 5 units of the enzyme, 25 utM each dATP, dCTP, dGTP, dTTP, in 10 A1 of 10 mM Tris-HCl (pH = 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01 % gelatin. The incubation time was 5 min at 72°C. The two samples were cooled at 0°C and added to 3,^l of stop buffer containing 40 mM EDTA, 80% (v/v) formamide, 0.05 % (w/v) xylene cyanol and bromophenol blue. After heating at 90°C for 2 min they were quickly loaded on a denaturing polyacrylamide gel (20% (w/v) polyacrylamide, 1/20 crosslinked, 7 M urea). Electrophoresis was carried out at 50 V/cm for 3 hours. The extension products located by autoradiography were excised and 32P radioactivity counted by Cerenkov effect. The upper bands of the gel were extracted in 3 ml of 10 mM Tris-HCl (pH = 7.2), 0.5 M NaCl, 1 mM EDTA, purified on a Nensorb 20 cartridge (Dupont Nen) and dried in a Speed Vac concentrator (Savant) for sequencing according to the Maxam and Gilbert procedure (20).
RESULTS AND DISCUSSION Synthesis of the 2-deoxyribosylformylamine building block It is well known that potassium permanganate oxidizes 5,6-pyrimidine double bond to give cis glycol. The primary products of the KMnO4 action on pyrimidine bases in neutral conditions are 5,6-dihydro-5,6-dihydroxy-derivatives which may subsequently be oxidized with the formation of fragmentation products. To obtain the protected 2-deoxyribosylformylamino phosphoramidite which is required to insert the damaged residue
in an oligodeoxyribonucleotide chain we have investigated two approaches. The first one involved the oxidation of thymidine, the tritylation of resulting mixture and a difficult separation of the tritylated compounds before phosphitylation. The second approach starting from tritylated thymidine was much more efficient. Introduction of a tritylated group at the first step of the synthesis allowed an easy detection of the resulting oxidative products. Mono-p-methoxytrityl (MMT) was chosen as protecting group for the 5'-O-nucleoside 1 because it is more stable than the dimethoxytrityl protecting group in many useful organic solvents. This MMT-group is also a suitable marker for following reactions of non UV products on thin layer chromatography and further purifications on column chromatography. Based on the latter approach we have developed a route to the protected deoxyribosylformylamine 3 involving KMnO4 oxidative reaction medium and lead tetraacetate ring opening. The synthesis of the protected phosphoramidite 4 needed for the chemical synthesis of oligodeoxyribonucleotides is outlined in the enclosed scheme 1. Compound 2 was prepared from 1 according to (12) with a 79% yield. Permanganate oxidation at pH 8 of 5'-Omethoxytritylthymidine 2 in acetone-pyridine solvent mixture was followed by lead tetraacetate treatment to give 3. The desired compound was separated from a mixture of oxidative side products by silica gel chromatography and a further high performance liquid chromatography yielded 32% of 5-0methoxytrityl-2-deoxyribosylformylamine 3. The structure of pure isolated product was confirmed by IH-NMR and high resolution FAB mass spectrometry. 1H-NMR showed the presence of two conformers which were due to the energy barrier to amide bond rotation of the formylamino groups (scheme 2). 0
JCH3 MMT Cl
H
MMT-019Z
1. KMnO
HO
HO
NaHSb3
s 3. Pb(OAc),, HCO
MMT-OCH2
NH
Hi=O
N (iPr)?
rO
NH
NCCH20H2O/N( i(Pr)
.~~~4
T
H0
Pr
MMT:Mon,tiri, iPr ls.pwopyl, Ac:Acety Scheme 1. Preparation of the 2-deoxyribosylformylamine phosphoramidite 4. MMT
/H
H\ Q-CH2 O
N-C
HO
tl
MMT OC 2
0
/
N- - C~
HO HO Scheme 2. Cis and trans conformer
H
structure
H of compound 3.
5818 Nucleic Acids Research, Vol. 19, No. 21 d d d
8 9
d Y(O-T-0G0-T-A-T
I
11 Figure
5(A-CG0-A-C-T-T-A-G-C-G-A-G-G-F-T)3
5 6 7
Y(ACG0-A-C-T-T-A-0.C-G-A-O-F-T-T)3 Y(A.C-O-A.C.T.T.A.-.C-O.A-F-.GT-T)3 .-C-A-C-T-T-C---A-A-C-0-T-A-F-T-O.C.--C-T-A-A-C-C-T.-C.C-T-A-A-TeC0-)3
d
5(G-T-O4-.T-A-T-G-G-C-A-C-T-T-C4-0G-A-A-C-G-T-C-F-T-G3-C-0-0-C-T-A-A-C-C-T-C40-C-T-A-A40-T-C4-G-s
d d
-T(C-C-A.T0-F-T-C-G-C)3 5(C0-F.A-T3
1. Sequence of the
olieodeoxyribonucleotides chemically synthesized with the PAC nucleoside phosphoramidites.
The cis-trans conformer ratio (minor: major = 2: 3) was determined by integration of the well separated formyl proton resonances at low field (minor: 8.20 ppm, major: 8.03 ppm). The structures of major and minor conformers could be assigned according to previous results (13) on the basis of several criteria, the must reliable being the NOE method. Irradiation of the minor Ha proton produced an increase of the integrated intensity in the minor Hb band. In contrast, irradiation of the major H. proton band did not result in an increase of the major Hb band. The four bond coupling interactions between the anomeric Ha and the formyl Hb protons are 4Jab: 0.8 Hz, (major) and 4Jab < 0.1 Hz (minor). Their relative values are in agreement with the stereochemical trends for 4J interactions. Phosphitylation (14,15) of 3 to obtain the phosphoramidite 4 was carried out with 1.1 eq of 2-cyanoethyltetraisopropylphosphorodiamidite and 0.6 eq of diisopropylammonium tetrazolide as activator in anhydrous CH2C12, at room temperature for 30 min. (98%). 31P-NMR and high resolution FAB-MS confirmed the structure.
.
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I
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1395 1186
(4,4'-dimethoxytrityl)-N-protected-2'-deoxynucleoside-3 '-O(2-cyanoethyl-N,N-diisopropylamino)-phosphoramidites with
SC
G
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ibk
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S
ldi
r
i
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T
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A
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634 ,'
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ih
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17
857
; ,'
A-
ih
t
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A
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635 r,,
Assembling of the phosphoramidites building blocks on a solid support The phosphoramidite 4 was used, likewise each of the four 5'-Obase protection (N6-phenoxyacetyl adenine, N2-phenoxyacetyl guanine, N4-isobutyryl cytosine and thymine) described previously (16,17), for the synthesis of oligodeoxyribonucleotides on silica gel support. The protected phosphoramidite 4 was inserted using the standard phosphoramidite methodology (15,18,19), with the same high coupling efficiency (> 97%), as measured and calculated by the amount of released trityl cation. During the detritylating step of the synthesis cycle monomethoxytrityl and dimethoxytrityl protecting groups were completely eliminated in the same acidic condition (3% trichloroacetic acid in dichloromethane for 140 sec). A variety of chemically synthesized DNA fragments 5-11 bearing the formylamino residue (Figure 1) and ranging from 5 to 47 mer was synthesized for biological purposes. They were prepared on an automatic synthesizer with reaction time, flow rate of reagents delivery and solvent washing conditions optimized for better coupling efficiency. The overall yields for compounds 5-11 ranged from 54 to 72%. After acidic removal of the trityl group and mild ammonia deprotection at room temperature during 6 hours, the crude oligodeoxyribonucleotides were purified by
pT
_-
F is 2-deoxyribosylformylamine.
;
A
T
!
labile
HPLC on an anion exchange column and desalted by dialysis. Additional purification was performed by preparative polyacrylamide gel electrophoresis. The yield of pure oligomers 8 and 9 was 41% and 44% respectively. Deoxyribosylformyl-
H O
-P0
--O -P-0-4- O
-
D
OH 306
OH
OH
I- 0 OH
_J J _~~~~~-
635
0 *-'
58
1171
~J
1395
FgE 2. Sequencing of dS'(C-G-F-A-T)3' by FAB-MS. a) Negative ion FAB mass spectrum including the glycerol matrix. b) Oligonucleotide structure with the main fragnentations. Dotted lines show the friagentons with corresponding mass.
amine is unstable under heating in basic conditions, so the ammonium hydroxyde used for cleavage from the solid support and room temperature deprotection was carefully removed by evaporation without heating on a Speed Vac concentrator and lyophilized.
Stability of the inserted 2-deoxyribosylformylamine The length and the sequence of oligonucleotides were checked by Maxan and Gilbert chemical degradation (20) after 32p labeling and polyacrylamide gel electrophoresis. For modified oligonucleotides the product band on the autoradiogram gave no
Nucleic Acids Research, Vol. 19, No. 21 5819 a
Gi
G
A
A
C
IIb
G+ A
G
A> C
C
C+ T
_ _ _ _
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Figure 3. Chemical sequencing products obtained after extension of the 5' labeled primer-template by a) Klenow fragment, b) Taq DNA polymerase as described in Experimental. The central letters (G,A,T,C) denote the sequence template 9 (figure 1) where X is the formylamino residue position, and boxed letters are opposite the 3'-end of the primer. The incorporated nucleotides are indicated at the left and right sides for the Klenow fragment and Taq polymerase respectively. Note the misinsertion of the base G opposite X at the position 24 for the Klenow fragment and the 'blank' for the Taq polymerase reaction. The arrows (positions 27, 32, 35) indicate the incorrect incorporations of the base G along the sequence downstream the damage.
evidence of the chemical structure of formylamino residue. This lesion has no UV absorption above 220 nm and the detection of the deoxyribosylformylamine by reversed phase HPLC analysis of the enzymatic nucleotide digestion was unsuccessful. The stability of the glycosylformylamine inserted in the DNA chain was investigated under the chemical conditions required for the synthesis and the final deprotection step. The presence of this deoxyribosylformylamine (F) was confirmed on a short sequence model 5'd(C-G-F-A-T)3' by fast atom bombardment mass spectrometry (FAB-MS) (21). This instrumental method can analyse the molecular weight (< 3000) of oligonucleotides using the negative ion fragmentation pattern. To evaluate the stability and the authenticity of this defect for longer oligomers the same base sequence oligonucleotide was submitted to 42 repetitive cycles of acidic detritylation, solvent washing, phenoxyacetic anhydride capping, iodine oxidation, final alkali cleavage from the solid support and base deprotection. Molecular weight measurement of the oligodeoxynucleotide by FAB-MS clearly identified the presence of 2-deoxyribosylformylamine. Figure 2a shows the negative ion FAB mass spectrum of the 5' d(C-G-F-A-T)3' DNA fragment containing the formylamino residue (F). The highest mass m/z = 1395 is the [M-H]- ion. The upper part of the spectrum is representative of the 3' to 5' sequence ions (5'-phosphate ends) and the lower part is representative of the 5' to 3' sequence (3'-phosphate ends). The complete base sequence can be read independently starting from the 5' or from the 3'-end. Figure 2b shows the oligonucleotide structure with the main fragmentation ions. The dotted lines
indicate the main fragments with their mass, those above corresponding to the 5'-P sequence ions and those below to the 3'-P sequence ions. The mass difference between two markers is representative of a nucleotide. Oligodeoxynucleotide bearing the Formylamino defect as template for the Klenow fragment and Taq DNA polymerase The analysis of replication products synthesized on the single stranded DNA template 9 bearing the formylamino residue at position 24 has been performed with the Klenow fragment and the Taq DNA polymerase, in the presence of 2'-deoxynucleoside triphosphates and Mg2+ as divalent cation. The 19 base long d(ACGACTTAGCGAGGTTAGC) primer, 32P-labeled at its 5'-end, has been used to initiate the copy. Bypass frequency at the formylamino site was estimated to be 33 % and 11 % for the Klenow fragment and Taq DNA polymerase respectively. The full length products were sequenced by the Maxam and Gilbert method. Autoradiography of the sequencing polyacrylamide gel electrophoresis which is given in figure 3 reveals that the Klenow fragment mainly directs the misinsertion of the base G opposite the formylamino lesion at position 24. With the Taq DNA polymerase a deletion is observed opposite the defect. After the lesion the copy of the template is correct with Taq DNA polymerase but not perfect with the Klenow fragment. When the Klenow fragment is used additional G can be inserted with the correct bases in different positions 27, 32 and 35. The results obtained with the formylamino residue can be compared with those reported for the apurinic sites. Experiments performed with single stranded DNA templates carrying an apurinic site and DNA polymerase III holoenzyme have been reported and the value of the bypass frequency has been estimated to be 10-15% (22). It has been found that DNA polymerase I of E. coli as well as avian myeboblastosis virus reverse transcriptase and DNA polymerase a insert a base adenine opposite AP sites in vitro (23-26). Abasic sites are incapable of normal hydrogen bonding and the nature of the opposite base inserted during replication can be determined by other factors such as stacking interactions of dNTPs with the previous added base, polymerase imposed bias in productive nucleoside triphosphate binding, nucleotide hydrophobicity. To amplify copies of DNA fragments the polymerase chain reaction is widely used, for example, in forensic samples, archeological remains and museum specimens. In the latter cases most of the DNA is damaged and the lesions can contribute to the population of molecules that makes up the resulting amplified DNA. In the same way, when amplification is initiated from a single template molecule the damage can have a great influence on the result. DNA degradation can take place by oxidative pathways and the formylamino residue is one of the major lesions which can occur in this way. Formylamino residue seems to be a highly mutagenic lesion.
CONCLUSION In our experimental conditions, the alkali labile formylamino residue was stable enough to be processed during all the chemical steps of phosphoramidite synthesis, chain elongation, cleavage from silica gel support and base deprotection. We have shown that the modified phosphoramidite 4, in conjunction with new labile base protected phosphoramidites which allow milder ammonia deprotection can be selectively used in the automated
5820 Nucleic Acids Research, Vol. 19, No. 21 synthesis of oigodeoxyribonucleotides. This chemistry opens new prospects for inserting alkali labile chemical or radiation induced DNA base damages into oligonucleotides. The resulting modified oligonucleotides bearing these damages are very useful to analyse the function of DNA repair enzymes and DNA polymerases.
ACKNOWLEDGEMENT Dr D. Molko for IH-NMR spectra is gratefully acknowledged. REFERENCES 1. Ward, J.F. (1985) Radiation Res., 104, S.103. 2. Thacker, J. (1986) Int. J. Radiat. Biol., 50, 1. 3. Teoule, R. and Cadet, J. (1978) in 'Effects of Ionizing Radiation on DNA', ed. by Huttermann, J., Kohnlein, W., Teoule, R., and Bertinchamps, A., (Berlin: Springer-Verlag) 171. 4. Cadet, J. and Teoule R. (1975) Bull. Soc. Chim. Fr., 891. 5. Teoule, R., Bert, C. and Bonicel A. (1977) Radiat. Res., 72, 190. 6. Teoule, R. (1987) Int. J. Radiat. Biol., 51, 573. 7. Ide, H., Melamede, R.J. and Wallace, S.S. (1987) Biochem., 26, 964. 8. Ide, H., Kow, Y.W. and Wallace, S.S. (1985) Nucleic Acids Res.13, 8035. 9. Demple, B. and Linn, S. (1980) Nature, 287, 203. 10. Kow, Y.W. and Wallace, S.S. (1987) Biochem., 26, 8200. 11. Bailly, V. and Verly, W.G. (1987) J. Biochem., 242, 565. 12. Schaller, H., Weimann, G., Lerch. B. and Khorana, H.G. (1963) J. Am. Chem. Soc., 85, 3821. 13. Cadet, J., Nardin, R., Voituriez, L., Remin, M. and Hruska, F.E. (1981) Can. J. Chem., 59, 3313. 14. Barone, A.D., Tang. J.Y. and Carudiers, M.H. (1984) Nucleic Acids Res., 12, 4051. 15. Sinha, N.D., Biernat, J., MacManus, J. and Koster, H. (1984) Nucleic Acids Res., 12, 4539. 16. Schulhof, J.C., Molko, D. and Teoule, R. (1987) Tetrahedron Len., 38, 51. 17. Schulhof, J.C., Molko, D. and Teoule, R. (1987) Nucleic Acids Res., 15, 397. 18. Beaucage, S.L. and Caruthers, M.H. (1981) Tetrahedron Lett., 22, 1859. 19. Matteucci, M.D. and Carudhers, M.H. (1981) J. Anm Chemn. Soc., 103, 3185. 20. Maxam, A.M. and Gilbert, W. (1985) in 'Methods in Enzymology', Eds Grossman, L. and Moldave, K. Academic Press, New-York, 65, 499. 21. Grotjahn, L., Frank, R. and Blocker, H. (1982) Nucleic Acids Res.,10, 4671. 22. Hevroni, D. and Livneh, Z. (1988) Proc. Natl. Acad. Sci. USA, 85, 5046. 23. Kunkel, T.A., Schaaper, R.M. and Loeb, L.A. (1983) Biochemistry, 22, 2378. 24. Sagher, D. and Strauss, B. (1983) Biochemistry, 22, 4518. 25. Randall, S.K., Eritja, R., Kaplan, B.E., Petruska, J. and Goodman, M.F.J. (1987) J.Biol. Chem., 262, 6864. 26. Takeshita, M., Chang, C.N., Johnson, F., Will, S. and GroUlman, A.P. (1987) J. Biol. Chem., 262, 10171. 27. Nielsen, J. and Dahl, 0. (1987) Nucleic Acids Res., 15, 3626. 28. Ulrich, J., Guy, A., Molko, D. and Teoule, R. (1984) Org. Mass. Spectrom., 19, 585. 29. Eadie, J.S. and Davidson, D.S. (1987) Nucleic Acids Res., 15, 8333. 30. Chaix, C., Molko, D. and Teoule, R. (1989) Tetrahedron Lett., 30, 1, 71. 31. Pon, R.T. (1987) Nucleic Acids Res., 15, 7203. 32. Guschlbauer, W., Duplaa, A.M., Guy A., Teoule, R. and Fazakerley, G.V. (1991) Nucleic Acids Res., 19, 1753.
Nucleic Acids Research, Vol. 19, No. 21 5815-5820
Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA Andre Guy, Anne-Marie Duplaa, Jacques Ulrich' and Robert Teoule* Service d'Etudes des Systemes Moleculaires, Departement de Recherche Fondamentale de la Matibre Condensee, Centre d'Etudes Nucleaires de Grenoble, BP 85X, 38041 Grenoble Cedex and 'Departement de Biologie Moleculaire et Structurale, Spectrometrie de Masse des Proteines, Centre d'Etudes Nucleaires de Grenoble, BP 85X, 38041 Grenoble Cedex, France Received October 1, 1991; Accepted October 10, 1991
ABSTRACT 2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.
INTRODUCTION The chemical modifications induced in the DNA in cells exposed to the action of ionizing radiation are thought to be responsible
for biological effects such as cellular lethality, mutagenesis and carcinogenesis (1,2). When thymidine is gamma-irradiated in aqueous solutions under aerobic conditions the predominant hydrolysis product is 2-deoxyribosylformylamine (3,4). The nature of modified bases on the DNA chain has been analysed after mild acid hydrolysis of irradiated DNA (5). Pyrimidine radiation products are generated by the attack of OH radicals at the 5,6-double bond of the pyrimidine ring (6). Subsequently, pyrimidine ring opening gives rise to formylamino residue. Ionizing radiation produces a large spectrum of DNA modifications and each individual damage appears in low yield so that irradiation cannot conveniently be used to prepare an oligonucleotide with a single formylamino residue. One of the difficulties is that very few methods are available to insert only one damage into an oligonucleotide (7). Our approach was to prepare first the modified phosphoramidite and then to introduce the alkali labile altered nucleotide during the chemical synthesis of DNA fragments. *
To whom correspondence should be addressed
In contrast to other oxidative DNA damages such as thymine glycols or urea residues containing DNA (8-1 1), the role played by the formylamino residue has not been investigated. It would be of interest to know if this lesion blocks the directed DNA polymerase synthesis on a template bearing this damage and to determine whether or not this defect is processed by DNA repair enzymes. In order to examine the biological consequences of this oxidative DNA damage we have selectively introduced the defect into DNA fragments. In this article, we describe the chemical synthesis of oligodeoxyribonucleotides containing the formylamino nucleotide in a well defined position within the sequence. Results concerning the role played by the lesion in in vitro replication are reported.
EXPERIMENTAL General Solvents were dried or distilled before use. 4-Methoxytriphenylchloromethane (MMT Cl): Fluka purum. Lead tetraacetate (Pb(OAc)4: Fluka purwm) was vacuum-dried at 10 mm Hg over P205 and KOH, room-temperature, overnight. KMnO4: Prolabo RP Normapur. 2-Cyanoethyltetraisopropyl-phosphoro-diamidite was prepared according to (27). Tetrazole was sublimated under reduced pressure. Diisopropylammonium tetrazolide was prepared according to (14). 5'-O-(4,4'-dimethoxytrityl)-Nprotected-2 ' -deoxynucleoside-3 '-O-(2-cyanoethyl-N, Ndiisopropylamino)-phosphoramidites with labile base protection (N6-phenoxyacetyl adenine, N2-phenoxyacetyl guanine, N4-isobutyryl cytosine and thymine) and the 1 Amole nucleoside (Thymidine or isobutyryl cytidine) grafted on a chemical CPG support were synthesized according to (17). Thin layer chromatography (TLC) was performed on precoated silica gel 60 F 254 (Merck), column chromatography: silica gel 60 H (Merck), and analytical HPLC mode using different purification
techniques to acquire pure compounds (Varian 5020 liquid chromatograph, variable wavelenght UV-Vis Cecil detector). UV shadowing or staining with cysteine-ethanolic-sulfuric acid spray was used to visualize UV absorbing or trityl derivative on TLC. UV spectra: Beckman DU-8B spectrophotometer: X max (e) in
5816 Nucleic Acids Research, Vol. 19, No. 21 nm. NMR spectra: Briker AC 200 ('H; 200 MHz) or WM 250 C1P; 101.2 MHz) spectrometers; chemical shift values in ppm rel. to TMS as internal reference (1H) or to 85% H3 P04 as external standard (31p). Pyrolysis Mass Spectrometry and positive or negative ion fast atom bombardment mass spectrometry (FAB-MS) (28): Kratos MS 50 and VGMS (selected peaks: m/z (%)). Oligonucleotide syntheses were performed on an Applied Biosystems 381 A DNA synthesizer using new PAC-phosphoramidite monomers and the improved phosphoramidite procedure introducing N-methylimidazole and phenoxyacetic anhydride capping (29,30) and IM iodine/pyridine/THF/water oxidizing solution (31).
5'"O-(4-methoxytrityl)-thymidine (2) Compound 2 was prepared from 1 according to (12). The oily product was purified on silica gel column (80 g SiO2, 6 x 8 cm) using a step gradient of 0-4% MeOH in CH2Cl2 to give a 79% yield (7.7 g). TLC (CHCl3/MeOH, 90: 10): Rf = 0.74. UV (EtOH): 266.7 (9700). Anal. cal. for C30H30N206 (514.55): C 70.02, H 5.88, N 5.34, 0 18.76; found: C 69.74, H 6.16, N 5.44, 0 18.98. H'-NMR (200 MHz, CD3COCD3): 1.60 (s, 3H, CH3-C(5)); 2.45 (m, 2H, H-C(2'), H-C(2')); 3.50 (m, 2H, HC(5'), H-C(5')); 3.90 (s, 3H, MeO, MMt); 4.15 (q, 1H, H-C (4')); 4.70 (q, 1H, H-C (3')); 6.5 (t, 1H, H-C(l')); 7.0-7.6 (m, 14H, H arom.); 7.75 (s, 1H, H-C(6)); 10.2 (s, 1H, H-N). FAB-MS (neg. ions, glycerol matrix): 513 (37, [M-H]-), 241 (20, [M-H-MMT]-), 125 (100, B-).
2-deoxy-5-0-(4-methoxytrityl)-,B-D-ribofuranosyl-1-formylamine (3) To a solution of 2 (4.12 g, 8 mmol) in acetone (50 ml) and pyridine (10 ml) fine needles of potassium permanganate (2.6 g, 16.4 mmol) were gradually added under stirring. During the reaction period the pH was controlled at 8.2 and the temperature maintained at 25°C. The end of the oxidative reaction was checked by TLC (solvent CHCl3/MeOH, 90: 10). Aqueous NaHSO3 (0.8 M, 60 ml) was added, salts were filtered off, washed with acetone and filtrate was evaporated in vacuo to 20 ml. CH2CI2 (2 x 100 ml) was added and the organic extract was evaporated to dryness. The oily residue was dissolved in dry pyridine (20 ml) and anhydrous powdered Pb (OAc)4 (3.8 g, 8.4 mmol) was added under stirring. After completion (2 h, TLC monitoring) pyridine was removed in vacuo on a rotatory evaporator, the oily residue was dissolved in CH2CI2 (2 x 100 ml) and washed with 5% aqueous NaHCO3 (200 ml). The filtered organic layer was washed with H20 (200 ml) and dried (Na2SO4). Evaporation of the filtrate gave an oily residue which was loaded onto silica gel column (6 x7 cm, Merck silica gel G, 10-40 ttm, CH2Cl2). Elution with CH2CI2 containing 2% MeOH gave 3 Purification by HPLC (Lichroprep silica gel 60, 1 x 30 cm column) using a linear gradient of 0-5 % MeOH in CH2Cl2 gave a 32% yield of pure compound (1.13 g). TLC (Ethyl Acetate/Isopropanol/Water, 98: 4: 2): Rf = 0.58. Anal. cal. for C26H27NO5 (433.48): C 72.04, H 6.28, N 3.23, 0 18.46; found: C 72.33, H 6.30, N 3.26, 0 18.11. IH-NMR (200 MHz, CD30D, 2 conformers): 2.02 (m, 2H, H-C(2), HC (2')); 3.16 (m, 2H, H-C (5), H-C (5')); 3.76 (s, 3H, CH30 (MMT)); 3.95 (m, 1H, H-C (4)); 4.30 (m, 1H, H-C(3)); 5.50 (t, 0.4H, J = 6.5, H-C (1), minor conformer); 5.78 (t, 0.6H, H-C (1), major conformer); 6.80-7.50 (m, 14H, H arom.); 8.03 (s, 0.6H, H-C = 0, major conformer); 8.20 (s, 0.4H, H-C = 0, minor conformer); major conformer: 4J (H-C = 0 and HC (1)) = 0.8 Hz; minor conformer, 4J (H-C = 0 and H-C(l))
< 0.1 Hz. FAB-MS (pos. ions, PEG 200 matrix, Na I): 456 (55, [M + Na]+) FAB-MS (neg. ions, glycerol matrix): 432 (80, [M-H]-), 160 (100, [M-H-MMT]-). HR FAB-MS (neg.ions, PEG 200 matrix): 432.1835 [M-H] -, C26H2605N, calc. 432.1811.
2-deoxy-5-0-(4-methoxytrityl)-,B-D-ribofuranosyl-1-
formylamine-3i--(2-cyanoethyl)-NN'-diiso-propylphosphoramidite (4) To dry 3 (1.08 g, 2.5 mmol) dissolved in anhydrous CH2C12 (10 ml) at room temperature under dry Ar atmosphere were added with stirring 0.6 eq of anhydrous diisopropylammonium tetrazolide (0.25 g, 1.5 mmol) and 1.1 eq of bis-(N, N'-diisopropylamino)-2-cyanoethoxyphosphine (0.9 g, 2.75 mmol). After completion of the reaction (1 h, TLC analysis (CH2Cl2/MeOH, 9: 1)), ethyl acetate (50 ml) was added and the reaction mixture was extracted with 5% aqueous NaHCO3 (2 x 100 ml) then washed with brine. The organic extract was dried (Na2SO4), fitered and evaporated to an oily residue. Attempts to precipitate in cold (-70°C) hexane failed and lyophilisation from benzene gave 4 as white gel stored in sealed amber vials at -20°C. TLC (Ethyl acetate/dichloromethane/ triethylamine 40: 20: 2): Rf = 0.52. FAB-MS (pos. ions, PEG 200 matrix, Na I): 655.9 (24, [M + Na]+). HR FAB-MS (neg. ions, PEG matrix): 579.2647 [M-CNE]-, C32H40N206P, calc. 579.2624. 31P-NMR (101.2 MHz, CD3CN/CH3CN 1:2):148.8. General phophoramidite methodology for DNA synthesis Each synthesis was performed on a DNA synthesizer using a 1 mol protected nucleoside grafted on a chemical modified CPG support in a short teflon column. For optimal coupling efficiency the flow rate of reagents was fitted at 2.3 ml.mn-'. The protected phosphoramidite 4 and the N-blocked-5'-O-DMT-2'-
deoxynucleoside-3'-0-(2-cyanoethyl-N, N'-diisopropylamino)phosphoramidites of (MeO)2 Tr pac6 Ad, (MeO)2 Tr pac2 Gd, (MeG)2 Tr ibu4Cd, (MeO)2Tr Td were used in 0.08 M dry MeCN (or toluene/MeCN, 10: 100) solutions. The syntheses were carried out following the standard cycle protocol but with N-methylimidazole/phenoxyacetic anhydride and IM iodine/ pyridine/ THF/water for the capping and oxidizing steps respectively. During the detritylating step of the synthesis cycle monomethoxytrityl and dimethoxytrityl protecting groups were eliminated in the same mild acid condition (3% trichloroacetic acid in CH2Cl2 for 140 sec.). After removal of the trityl group, cleavage of the oligonucleotides from support and deprotection of the N-blocked deoxynucleosides and the phosphate groups were carried out with a 28% ammonia solution (5 x200 il) during 6 h at room temperature. HPLC purifications were performed on an anion-exchange column (0.75 x 30 cm, 10 jAm of Partisil SAX) using a linear gradient of 0.3 M KH2PO4 buffer (pH 6.7) with 30% Me CN over 50 min. Desalting prior to use by dialysis against distilled water (5 x 600 ml) gave products which were further purified by 20% denaturing polyacrylamide gel electrophoresis. Fast atom bombardment mass spectrometry for DNA sequencing The short model sequence 5' d(C-G-F-A-T)3' was prepared following general phosphoramidite procedure with 42 repetitive chemical cycles of trichloroacetic acid detritylation, phenoxyacetic anhydride/N-methylimidazole capping, I2/pyridine/THF/H20 oxidation, CH3CN washing steps in order to be in the same conditions for chemical preparation of a long polynucleotide. The
Nucleic Acids Research, Vol. 19, No. 21 5817 crude oligomer from ammonia treatment was purified on a reverse-phase column (0.46x25 cm, Hypersil ODS, 10 Am, Societe Francaise de Chromato Colonne) using a linear gradient of 3-10% MeCN in 0.025 M TEAA (pH 6.8) over 30mn. To evaluate the integrity of the formylamino residue we have performed mass spectrometric sequence analysis of this oligonucleotide. Negative ions FAB-MS unambiguously confirmed the complete ammonia deprotection of 5' d(C-G-F-AT)3' and the presence of the formylamino residue. FAB-MS (neg. ions, glycerol matrix, NH40H): 1395.0 ([M-H]-), calc. 1395.3. The complete sequence of the nucleotide (Figure 2) with the main fragmentations can be read independently starting either from the 5' or from the 3'-end: 306 (Cp-), 635 (Cp Gp-), 858 (Cp Gp Fp-), 1171 (Cp Gp Fp Ap-), 1395 (Cp Gp Fp Ap T-) or 321 (pT-), 634 (pA pT-), 857 (pF pA pT-), 1186 (pG pF pA pT-), 1395 (Cp Gp Fp Ap T-). In vitro replication (_y-32P) ATP (5000 Ci/mmol) was purchased from Amersham. T4 polynucleotide kinase and E. coli DNA polymerase I (Klenow fragment) were from Boehringer. Taq DNA polymerase was from Perkin Elmer Cetus. 10 pmol of the 5'-end 32p labeled 19 base long d(ACGACTTAGCGAGGTTAGC) primer were annealed with 10 pmol of the DNA template 9 (figure 1) as described (32).
Klenow fragment assay conditions: Primer elongation by the Klenow fragment was carried out for 15 min at 37°C in a buffer containing 50 mM Tris-HCl (pH = 7.6), 8 mM MgCl2, 1 mM DTT, 5 pmol of the labeled primer-template (about 1 x 106 cpm/pmol), 2 units of the enzyme and 25 ,tM of each of the four dNTPs in a final volume of 10 Al. Taq DNA polymerase assay conditions: Polymerisation by Taq polymerase was carried out by mixing 5 pmol (1. 106 cpm/pmol) of the labeled primer-template, 5 units of the enzyme, 25 utM each dATP, dCTP, dGTP, dTTP, in 10 A1 of 10 mM Tris-HCl (pH = 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01 % gelatin. The incubation time was 5 min at 72°C. The two samples were cooled at 0°C and added to 3,^l of stop buffer containing 40 mM EDTA, 80% (v/v) formamide, 0.05 % (w/v) xylene cyanol and bromophenol blue. After heating at 90°C for 2 min they were quickly loaded on a denaturing polyacrylamide gel (20% (w/v) polyacrylamide, 1/20 crosslinked, 7 M urea). Electrophoresis was carried out at 50 V/cm for 3 hours. The extension products located by autoradiography were excised and 32P radioactivity counted by Cerenkov effect. The upper bands of the gel were extracted in 3 ml of 10 mM Tris-HCl (pH = 7.2), 0.5 M NaCl, 1 mM EDTA, purified on a Nensorb 20 cartridge (Dupont Nen) and dried in a Speed Vac concentrator (Savant) for sequencing according to the Maxam and Gilbert procedure (20).
RESULTS AND DISCUSSION Synthesis of the 2-deoxyribosylformylamine building block It is well known that potassium permanganate oxidizes 5,6-pyrimidine double bond to give cis glycol. The primary products of the KMnO4 action on pyrimidine bases in neutral conditions are 5,6-dihydro-5,6-dihydroxy-derivatives which may subsequently be oxidized with the formation of fragmentation products. To obtain the protected 2-deoxyribosylformylamino phosphoramidite which is required to insert the damaged residue
in an oligodeoxyribonucleotide chain we have investigated two approaches. The first one involved the oxidation of thymidine, the tritylation of resulting mixture and a difficult separation of the tritylated compounds before phosphitylation. The second approach starting from tritylated thymidine was much more efficient. Introduction of a tritylated group at the first step of the synthesis allowed an easy detection of the resulting oxidative products. Mono-p-methoxytrityl (MMT) was chosen as protecting group for the 5'-O-nucleoside 1 because it is more stable than the dimethoxytrityl protecting group in many useful organic solvents. This MMT-group is also a suitable marker for following reactions of non UV products on thin layer chromatography and further purifications on column chromatography. Based on the latter approach we have developed a route to the protected deoxyribosylformylamine 3 involving KMnO4 oxidative reaction medium and lead tetraacetate ring opening. The synthesis of the protected phosphoramidite 4 needed for the chemical synthesis of oligodeoxyribonucleotides is outlined in the enclosed scheme 1. Compound 2 was prepared from 1 according to (12) with a 79% yield. Permanganate oxidation at pH 8 of 5'-Omethoxytritylthymidine 2 in acetone-pyridine solvent mixture was followed by lead tetraacetate treatment to give 3. The desired compound was separated from a mixture of oxidative side products by silica gel chromatography and a further high performance liquid chromatography yielded 32% of 5-0methoxytrityl-2-deoxyribosylformylamine 3. The structure of pure isolated product was confirmed by IH-NMR and high resolution FAB mass spectrometry. 1H-NMR showed the presence of two conformers which were due to the energy barrier to amide bond rotation of the formylamino groups (scheme 2). 0
JCH3 MMT Cl
H
MMT-019Z
1. KMnO
HO
HO
NaHSb3
s 3. Pb(OAc),, HCO
MMT-OCH2
NH
Hi=O
N (iPr)?
rO
NH
NCCH20H2O/N( i(Pr)
.~~~4
T
H0
Pr
MMT:Mon,tiri, iPr ls.pwopyl, Ac:Acety Scheme 1. Preparation of the 2-deoxyribosylformylamine phosphoramidite 4. MMT
/H
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N-C
HO
tl
MMT OC 2
0
/
N- - C~
HO HO Scheme 2. Cis and trans conformer
H
structure
H of compound 3.
5818 Nucleic Acids Research, Vol. 19, No. 21 d d d
8 9
d Y(O-T-0G0-T-A-T
I
11 Figure
5(A-CG0-A-C-T-T-A-G-C-G-A-G-G-F-T)3
5 6 7
Y(ACG0-A-C-T-T-A-0.C-G-A-O-F-T-T)3 Y(A.C-O-A.C.T.T.A.-.C-O.A-F-.GT-T)3 .-C-A-C-T-T-C---A-A-C-0-T-A-F-T-O.C.--C-T-A-A-C-C-T.-C.C-T-A-A-TeC0-)3
d
5(G-T-O4-.T-A-T-G-G-C-A-C-T-T-C4-0G-A-A-C-G-T-C-F-T-G3-C-0-0-C-T-A-A-C-C-T-C40-C-T-A-A40-T-C4-G-s
d d
-T(C-C-A.T0-F-T-C-G-C)3 5(C0-F.A-T3
1. Sequence of the
olieodeoxyribonucleotides chemically synthesized with the PAC nucleoside phosphoramidites.
The cis-trans conformer ratio (minor: major = 2: 3) was determined by integration of the well separated formyl proton resonances at low field (minor: 8.20 ppm, major: 8.03 ppm). The structures of major and minor conformers could be assigned according to previous results (13) on the basis of several criteria, the must reliable being the NOE method. Irradiation of the minor Ha proton produced an increase of the integrated intensity in the minor Hb band. In contrast, irradiation of the major H. proton band did not result in an increase of the major Hb band. The four bond coupling interactions between the anomeric Ha and the formyl Hb protons are 4Jab: 0.8 Hz, (major) and 4Jab < 0.1 Hz (minor). Their relative values are in agreement with the stereochemical trends for 4J interactions. Phosphitylation (14,15) of 3 to obtain the phosphoramidite 4 was carried out with 1.1 eq of 2-cyanoethyltetraisopropylphosphorodiamidite and 0.6 eq of diisopropylammonium tetrazolide as activator in anhydrous CH2C12, at room temperature for 30 min. (98%). 31P-NMR and high resolution FAB-MS confirmed the structure.
.
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.
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(4,4'-dimethoxytrityl)-N-protected-2'-deoxynucleoside-3 '-O(2-cyanoethyl-N,N-diisopropylamino)-phosphoramidites with
SC
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.'
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r
i
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Assembling of the phosphoramidites building blocks on a solid support The phosphoramidite 4 was used, likewise each of the four 5'-Obase protection (N6-phenoxyacetyl adenine, N2-phenoxyacetyl guanine, N4-isobutyryl cytosine and thymine) described previously (16,17), for the synthesis of oligodeoxyribonucleotides on silica gel support. The protected phosphoramidite 4 was inserted using the standard phosphoramidite methodology (15,18,19), with the same high coupling efficiency (> 97%), as measured and calculated by the amount of released trityl cation. During the detritylating step of the synthesis cycle monomethoxytrityl and dimethoxytrityl protecting groups were completely eliminated in the same acidic condition (3% trichloroacetic acid in dichloromethane for 140 sec). A variety of chemically synthesized DNA fragments 5-11 bearing the formylamino residue (Figure 1) and ranging from 5 to 47 mer was synthesized for biological purposes. They were prepared on an automatic synthesizer with reaction time, flow rate of reagents delivery and solvent washing conditions optimized for better coupling efficiency. The overall yields for compounds 5-11 ranged from 54 to 72%. After acidic removal of the trityl group and mild ammonia deprotection at room temperature during 6 hours, the crude oligodeoxyribonucleotides were purified by
pT
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;
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HPLC on an anion exchange column and desalted by dialysis. Additional purification was performed by preparative polyacrylamide gel electrophoresis. The yield of pure oligomers 8 and 9 was 41% and 44% respectively. Deoxyribosylformyl-
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FgE 2. Sequencing of dS'(C-G-F-A-T)3' by FAB-MS. a) Negative ion FAB mass spectrum including the glycerol matrix. b) Oligonucleotide structure with the main fragnentations. Dotted lines show the friagentons with corresponding mass.
amine is unstable under heating in basic conditions, so the ammonium hydroxyde used for cleavage from the solid support and room temperature deprotection was carefully removed by evaporation without heating on a Speed Vac concentrator and lyophilized.
Stability of the inserted 2-deoxyribosylformylamine The length and the sequence of oligonucleotides were checked by Maxan and Gilbert chemical degradation (20) after 32p labeling and polyacrylamide gel electrophoresis. For modified oligonucleotides the product band on the autoradiogram gave no
Nucleic Acids Research, Vol. 19, No. 21 5819 a
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Figure 3. Chemical sequencing products obtained after extension of the 5' labeled primer-template by a) Klenow fragment, b) Taq DNA polymerase as described in Experimental. The central letters (G,A,T,C) denote the sequence template 9 (figure 1) where X is the formylamino residue position, and boxed letters are opposite the 3'-end of the primer. The incorporated nucleotides are indicated at the left and right sides for the Klenow fragment and Taq polymerase respectively. Note the misinsertion of the base G opposite X at the position 24 for the Klenow fragment and the 'blank' for the Taq polymerase reaction. The arrows (positions 27, 32, 35) indicate the incorrect incorporations of the base G along the sequence downstream the damage.
evidence of the chemical structure of formylamino residue. This lesion has no UV absorption above 220 nm and the detection of the deoxyribosylformylamine by reversed phase HPLC analysis of the enzymatic nucleotide digestion was unsuccessful. The stability of the glycosylformylamine inserted in the DNA chain was investigated under the chemical conditions required for the synthesis and the final deprotection step. The presence of this deoxyribosylformylamine (F) was confirmed on a short sequence model 5'd(C-G-F-A-T)3' by fast atom bombardment mass spectrometry (FAB-MS) (21). This instrumental method can analyse the molecular weight (< 3000) of oligonucleotides using the negative ion fragmentation pattern. To evaluate the stability and the authenticity of this defect for longer oligomers the same base sequence oligonucleotide was submitted to 42 repetitive cycles of acidic detritylation, solvent washing, phenoxyacetic anhydride capping, iodine oxidation, final alkali cleavage from the solid support and base deprotection. Molecular weight measurement of the oligodeoxynucleotide by FAB-MS clearly identified the presence of 2-deoxyribosylformylamine. Figure 2a shows the negative ion FAB mass spectrum of the 5' d(C-G-F-A-T)3' DNA fragment containing the formylamino residue (F). The highest mass m/z = 1395 is the [M-H]- ion. The upper part of the spectrum is representative of the 3' to 5' sequence ions (5'-phosphate ends) and the lower part is representative of the 5' to 3' sequence (3'-phosphate ends). The complete base sequence can be read independently starting from the 5' or from the 3'-end. Figure 2b shows the oligonucleotide structure with the main fragmentation ions. The dotted lines
indicate the main fragments with their mass, those above corresponding to the 5'-P sequence ions and those below to the 3'-P sequence ions. The mass difference between two markers is representative of a nucleotide. Oligodeoxynucleotide bearing the Formylamino defect as template for the Klenow fragment and Taq DNA polymerase The analysis of replication products synthesized on the single stranded DNA template 9 bearing the formylamino residue at position 24 has been performed with the Klenow fragment and the Taq DNA polymerase, in the presence of 2'-deoxynucleoside triphosphates and Mg2+ as divalent cation. The 19 base long d(ACGACTTAGCGAGGTTAGC) primer, 32P-labeled at its 5'-end, has been used to initiate the copy. Bypass frequency at the formylamino site was estimated to be 33 % and 11 % for the Klenow fragment and Taq DNA polymerase respectively. The full length products were sequenced by the Maxam and Gilbert method. Autoradiography of the sequencing polyacrylamide gel electrophoresis which is given in figure 3 reveals that the Klenow fragment mainly directs the misinsertion of the base G opposite the formylamino lesion at position 24. With the Taq DNA polymerase a deletion is observed opposite the defect. After the lesion the copy of the template is correct with Taq DNA polymerase but not perfect with the Klenow fragment. When the Klenow fragment is used additional G can be inserted with the correct bases in different positions 27, 32 and 35. The results obtained with the formylamino residue can be compared with those reported for the apurinic sites. Experiments performed with single stranded DNA templates carrying an apurinic site and DNA polymerase III holoenzyme have been reported and the value of the bypass frequency has been estimated to be 10-15% (22). It has been found that DNA polymerase I of E. coli as well as avian myeboblastosis virus reverse transcriptase and DNA polymerase a insert a base adenine opposite AP sites in vitro (23-26). Abasic sites are incapable of normal hydrogen bonding and the nature of the opposite base inserted during replication can be determined by other factors such as stacking interactions of dNTPs with the previous added base, polymerase imposed bias in productive nucleoside triphosphate binding, nucleotide hydrophobicity. To amplify copies of DNA fragments the polymerase chain reaction is widely used, for example, in forensic samples, archeological remains and museum specimens. In the latter cases most of the DNA is damaged and the lesions can contribute to the population of molecules that makes up the resulting amplified DNA. In the same way, when amplification is initiated from a single template molecule the damage can have a great influence on the result. DNA degradation can take place by oxidative pathways and the formylamino residue is one of the major lesions which can occur in this way. Formylamino residue seems to be a highly mutagenic lesion.
CONCLUSION In our experimental conditions, the alkali labile formylamino residue was stable enough to be processed during all the chemical steps of phosphoramidite synthesis, chain elongation, cleavage from silica gel support and base deprotection. We have shown that the modified phosphoramidite 4, in conjunction with new labile base protected phosphoramidites which allow milder ammonia deprotection can be selectively used in the automated
5820 Nucleic Acids Research, Vol. 19, No. 21 synthesis of oigodeoxyribonucleotides. This chemistry opens new prospects for inserting alkali labile chemical or radiation induced DNA base damages into oligonucleotides. The resulting modified oligonucleotides bearing these damages are very useful to analyse the function of DNA repair enzymes and DNA polymerases.
ACKNOWLEDGEMENT Dr D. Molko for IH-NMR spectra is gratefully acknowledged. REFERENCES 1. Ward, J.F. (1985) Radiation Res., 104, S.103. 2. Thacker, J. (1986) Int. J. Radiat. Biol., 50, 1. 3. Teoule, R. and Cadet, J. (1978) in 'Effects of Ionizing Radiation on DNA', ed. by Huttermann, J., Kohnlein, W., Teoule, R., and Bertinchamps, A., (Berlin: Springer-Verlag) 171. 4. Cadet, J. and Teoule R. (1975) Bull. Soc. Chim. Fr., 891. 5. Teoule, R., Bert, C. and Bonicel A. (1977) Radiat. Res., 72, 190. 6. Teoule, R. (1987) Int. J. Radiat. Biol., 51, 573. 7. Ide, H., Melamede, R.J. and Wallace, S.S. (1987) Biochem., 26, 964. 8. Ide, H., Kow, Y.W. and Wallace, S.S. (1985) Nucleic Acids Res.13, 8035. 9. Demple, B. and Linn, S. (1980) Nature, 287, 203. 10. Kow, Y.W. and Wallace, S.S. (1987) Biochem., 26, 8200. 11. Bailly, V. and Verly, W.G. (1987) J. Biochem., 242, 565. 12. Schaller, H., Weimann, G., Lerch. B. and Khorana, H.G. (1963) J. Am. Chem. Soc., 85, 3821. 13. Cadet, J., Nardin, R., Voituriez, L., Remin, M. and Hruska, F.E. (1981) Can. J. Chem., 59, 3313. 14. Barone, A.D., Tang. J.Y. and Carudiers, M.H. (1984) Nucleic Acids Res., 12, 4051. 15. Sinha, N.D., Biernat, J., MacManus, J. and Koster, H. (1984) Nucleic Acids Res., 12, 4539. 16. Schulhof, J.C., Molko, D. and Teoule, R. (1987) Tetrahedron Len., 38, 51. 17. Schulhof, J.C., Molko, D. and Teoule, R. (1987) Nucleic Acids Res., 15, 397. 18. Beaucage, S.L. and Caruthers, M.H. (1981) Tetrahedron Lett., 22, 1859. 19. Matteucci, M.D. and Carudhers, M.H. (1981) J. Anm Chemn. Soc., 103, 3185. 20. Maxam, A.M. and Gilbert, W. (1985) in 'Methods in Enzymology', Eds Grossman, L. and Moldave, K. Academic Press, New-York, 65, 499. 21. Grotjahn, L., Frank, R. and Blocker, H. (1982) Nucleic Acids Res.,10, 4671. 22. Hevroni, D. and Livneh, Z. (1988) Proc. Natl. Acad. Sci. USA, 85, 5046. 23. Kunkel, T.A., Schaaper, R.M. and Loeb, L.A. (1983) Biochemistry, 22, 2378. 24. Sagher, D. and Strauss, B. (1983) Biochemistry, 22, 4518. 25. Randall, S.K., Eritja, R., Kaplan, B.E., Petruska, J. and Goodman, M.F.J. (1987) J.Biol. Chem., 262, 6864. 26. Takeshita, M., Chang, C.N., Johnson, F., Will, S. and GroUlman, A.P. (1987) J. Biol. Chem., 262, 10171. 27. Nielsen, J. and Dahl, 0. (1987) Nucleic Acids Res., 15, 3626. 28. Ulrich, J., Guy, A., Molko, D. and Teoule, R. (1984) Org. Mass. Spectrom., 19, 585. 29. Eadie, J.S. and Davidson, D.S. (1987) Nucleic Acids Res., 15, 8333. 30. Chaix, C., Molko, D. and Teoule, R. (1989) Tetrahedron Lett., 30, 1, 71. 31. Pon, R.T. (1987) Nucleic Acids Res., 15, 7203. 32. Guschlbauer, W., Duplaa, A.M., Guy A., Teoule, R. and Fazakerley, G.V. (1991) Nucleic Acids Res., 19, 1753.
