J. CHEM. SOC. PERKIN TRANS. I 1994
`
`211
`
`Synthesis of 3' -O-(w-Aminoalkoxymethyl)thymidine 5'-Triphosphates,
`Terminators of DNA Synthesis that Enable 3' -Labelling
`
`Jari Hovinen, *,a,b Elena Azhayeva,a,c Alex Azhayev,a,c Andrei Guzaev 8 and
`Harri Lonnberg a
`a University of Turku, Department of Chemistry, FIN-20 500 Turku, Finland
`6 Turku Centre for Biotechnology, BioCity, FIN-20 520 Turku, Finland
`c Joint Biotechnology Laboratory, BioCity, FIN-20 520 Turku, Finland
`
`Treatment of 5' -0-benzoylthymidine 1 with a mixture of acetic anhydride, acetic acid and dimethyl
`sulfoxide yielded 5'-0-benzoyl-3'-0-methylthiomethylthymidine 2. which was converted via the 3'-
`0-bromomethyl derivative into 3' -0-(ro-aminoalkoxymethyl)thymidines 7 bearing a 6, 8 or 10
`methylene groups long hydrocarbon chain, and finally to their 5' -triphosphates 10. The latter
`compounds were shown to be terminators of DNA synthesis catalysed by thermostable Tet/z DNA(cid:173)
`polymerase. and may be labelled at the aliphatic amino group with fluorescent probes.
`
`Quite m.:ently a rapid solid-phase method, called mini(cid:173)
`sequencing, has been introduced for the detection of point
`mutation of DNA. 1•2 The method involves hybridization of
`immobilized single-stranded DNA with a primer that ends
`immediately before the site of mutation, and elongation of the
`chain with a single labelled deoxyribonucleoside 5' -triphos(cid:173)
`phate. Parallel runs with each of the four possible nucleotides
`enable identification of the mutated base. Thus far only
`radiochemically labelled nucleotides have been used. Applic(cid:173)
`ation of nonradioactive techniques, such as fluorescence
`detection, is hampered by the fact that the nucleoside tri(cid:173)
`phosphates bearing the fluorescent probe should be reasonably
`good substrates of DNA polymerase. As a first step in our
`efforts to find a common labelling strategy for all the nucleotides
`occurring in DNA, we now report on the preparation of some
`thymidine 5' -triphosphates bearing a long chain aminoalkoxy(cid:173)
`methyl side arm at 3' -0. Their ability to serve as specific
`terminators of DNA synthesis catalysed by DNA polymerases
`were studied before and after attachment of fluorescent probes
`to their amino function.
`
`Results and Discussion
`The reaction sequence utilized in the preparation of 3'-O-(co(cid:173)
`aminoalkoxymethyl)thymidine 5'-triphosphates is depicted in
`Scheme 1. The key steps are discussed below.
`
`Formation of the Methylthiomethyl Ether 2.~Methylthio(cid:173)
`methyl ethers are well-known by-products of the oxidation of
`alcohols with a mixture of acetic anhydride and dimethyl
`sulfoxide (DMSO). 3- 8 The amount of these by-products has
`been shown to vary from traces to more than 70% depending on
`the nature of the alcohol. Addition of acetic acid to the reaction
`mixture markedly increases the yield of the methylthiomethyl
`ether. 9 This observation was exploited in the preparation of 3' -
`O-methylthiomethylthymidine. Treatment of 5'-O-benzoyl(cid:173)
`thymidine 10 I with a mixture of DMSO, acetic acid and acetic
`anhydride (54: 11: 35. v /v) for 2 days at ambient temperature
`gave, after purification, an almost 70% yield of the desired
`3'-O-methylthiomethyl derivative 2.
`
`Attachment of the Aliphatic Arm to 03' 3.~The methyl(cid:173)
`thiomethyl ether, 2, was first converted into the more reactive
`bromomethyl ether, 11 14 by
`treating 2 with N-bromo(cid:173)
`succinimide (NBS) or molecular bromine in dry dichloroethane.
`When the 5'-O-benzoyl-3' -O-bromomethylthymidine obtained
`
`was allowed to react in situ with a long-chain co-bromo alcohol
`(6 to 10 carbons), in the presence of 2,6-lutidine (2,6-di(cid:173)
`methylpyridine),
`the corresponding 3' -O-(w-bromoalkoxy(cid:173)
`methyl)thymidines, 3a--c, were formed. The same methodology
`has successfully been used also with appropriately protected
`guanosine, cytidine and adenosine analogues without severe
`side reactions. 9 It has been shown that when N-iodosuccinimide
`(NIS) is used as the halogen source in a large excess (sixfold),
`the main product is the corresponding succinimide derivative,
`3d. 12• 13 In the present synthesis only traces of this kind of
`side product were found, and the formation of the side product
`was completely prevented by using molecular bromide as a
`thiophilic promoter instead of N-halogenosuccinimide. How(cid:173)
`ever, a small amount ( ~ 10%) of another, slow-migrating side
`product was formed (Rr 0.29 compared to 0.37 of 3a; MeOH(cid:173)
`CH2Cl2, 3: 97, v/v). The product had an identical UV spectrum
`with the main product, and its 1 H N MR spectrum exhibited
`double signals for H6 and sugar protons, but only single peaks
`for the aliphatic arm and a singlet (2 H) at i5 5.47 probably
`referring to NCH 2O. After deprotection with methanolic
`ammonia, the 13C NMR showed four signals in the carbonyl
`carbon region of the thymidine residue. Accordingly, the side
`product may tentatively be assigned as a dimer 3e containing a
`methylene bridge between 03' of one thymidine and N3 of the
`other.
`The w-bromo substituent was displaced by azide ion by
`heating 3a--c in a mixture of sodium azide and lithium chloride
`in dimethylformamide (DMF). The reaction mixture was
`'buffered' with ammonium chloride to prevent the cleavage of
`the base labile 5'-O-benzoyl group. The displacement was
`almost quantitative. After conventional work up, the product,
`4a--c, was purified by silica gel column chromatography. The
`overall yield starting from 2 was normally about 50%.
`The original idea was to debenzoylate 4a--c, phosphorylate
`the deblocked 5'-OH (5a--c) and then reduce the azido group
`to an amino function. Unfortunately, the reduction of azido
`to amino with triphenylphosphine in a mixture of aqueous
`ammonia and dioxane failed, when applied to triphosphates,
`although the corresponding azido nucleosides Sa--c were
`rapidly reduced with an excess of triphenylphosphine in
`pyridine. It has also been reported that 3'-azido-3'-deoxythy(cid:173)
`midine 5'-triphosphate can easily be reduced to the 3'-amino
`analogue by the method described above. 15
`Since the azido group was reduced at nucleoside level, the
`resulting amino function has to be protected before phosphoryl(cid:173)
`ation of 5' -OH. This was done by treating the aminoalkoxy-
`
`Illumina Ex. 1060
`IPR Petition - USP 10,435,742
`
`

`

`212
`
`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`Ill
`
`BzO?
`
`OCH20(CH2)n Br
`3a-c
`n=6,8or10
`
`0
`0
`0
`II
`II
`II
`-0-)-0-)-0-)-ovo✓
`o-
`o-
`o- ~
`
`OCH20(CH2ln Na
`
`HO?
`
`v
`
`BzO?
`
`OCH20(CH2ln Na
`Sa-c
`
`OCH20(CH2ln Na
`4a-c
`
`HO?
`
`~
`
`HOvO✓
`~ viii
`
`OCH20(CH2ln NH2
`7a-c
`
`OCH20(CH2ln NHCOCFa
`8a-c
`
`0
`0
`0
`II
`II
`II
`-o-)-0-f-0-)-ovo✓
`o-
`o-
`o- ~
`
`OCH20(CH2ln NHCOCFa
`
`X
`
`0
`0
`0
`II
`II
`II
`-o-P-O-P-0-P-O?
`I
`I
`I
`0
`o-
`o-
`o-
`
`OCH20(CH2)n NH2
`
`10a-c
`
`11
`
`OH
`
`Scheme 1 Reagents and conditions: i, AcOH-Ac2O, DMSO; ii, NBS or Br 2, CICH2CH2CI; iii, Br(CH2).OH, lutidine; iv, N 3 - , DMF;
`v, NH 3-MeOH; vi, (a) Ph3P, pyridine (b) NH 3 (aq.); vii, CF3CO2Me, DMF; viii, (a) PO(triazoleh-MeCN (b) P2O 7-DMF (c) H 2O; ix, NH 3(aq.);
`x, FITC,pH JO
`
`BzO?
`
`0 ~ft
`e,o?)
`
`OCH20(CH2l6Br
`3e
`
`3d
`
`methyl nucleosides 7a--c with an excess of methyl trifluoro(cid:173)
`acetate in dry DMF. When the ester was dry and freshly
`distilled, the reaction was quantitative and the product 8a--c
`was easily isolated on a short silica gel column.
`
`Synthesis of 5'-Triphosphates.-A modification of the
`method of Otvos et al. 16 was applied to the preparation of the
`nucleoside 5' -triphosphates, 9a--c. According to the original
`method, the nucleoside is allowed to react with phosphorus
`oxychloride in dry trimethyl phosphate, and the intermediate
`formed is treated in situ with bis(tributylammonium) pyro-
`
`phosphate. Unfortunately, phosphorylation of the nucleoside
`with POC13 requires a prolonged treatment, and hence the
`hydrogen chloride liberated may cleave the acetal linkage in
`8a--c. To avoid this, a slightly different approach was applied.
`The nucleoside was first treated with phosphoryltris(triazole)
`in dry acetonitrile 1 7 and the intermediate obtained was allowed
`to react overnight with bis(tributylammonium) pyrophosphate.
`The remaining triazole ligand was hydrolysed by addition of
`water to give 9a--c. Treatment of 9a--c with aqueous ammonia
`gave lOa--c as the final products.
`In order to demonstrate the usefulness of 3'-0-(co-amino(cid:173)
`alkoxymethyl)thymidine 5' -triphosphates prepared as sub(cid:173)
`strates of DNA polymerases even when attached to bulky
`fluorescent groups, 10a was labelled with fluorescein isothio(cid:173)
`cyanate. The reaction was quantitative and the product, 11,
`was purified by HPLC.
`
`Termination of DNA Synthesis Catalysed by Thermostable
`Tet/z-DNA Polymerase.-Compounds lOa--cdiffersignificantly
`from the well-known terminators of DNA synthesis, such as
`2',3' -dideoxyribonucleoside 5' -triphosphates 18 and 3' -amino-
`2',3' -dideoxyribonucleoside 5' -triphosphates. 19 Their co-amino(cid:173)
`alkoxymethyl side arms enable attachment of reporter groups,
`either before or after enzymatic incorporation into DNA, and
`hence labelling of the 3'-terminus of DNA. The potential
`advantages of the usage of a long flexible acetal arm were
`
`

`

`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`expected to be (i) the reporter groups may be kept distant from
`the catalytic centre of the polymerase enzymes, and (ii) the
`flexible arm would not severely restrict the conformational
`motion that the sugar ring undergoes on binding to enzyme.
`Indeed, compounds IOa--c, as well as their analogues bearing a
`trifluoroacetyl 9a--c or fluorescein group 11, at the amino
`function were observed to be specific terminators of DNA
`synthesis catalysed by thermostable polymerases.* Fig. I shows
`the results obtained with Tet/z DNA-polymerase, using 2',3'(cid:173)
`dideoxyribonucleoside 5'-triphosphates and compounds 9a,
`toa--c and 11 as terminators. It is clearly seen that 3' -O-(w(cid:173)
`aminoalkoxymethyl)thymidine 5' -triphosphates IOa--c as well
`as their analogues, 9a and 11, terminate DNA synthesis, giving
`patterns very similar to that of 2',3'-dideoxythymidine 5'-tri(cid:173)
`phosphate.
`
`Experimental
`All the solvents used were of analytical grade and were distilled
`and dried before use. Thymidine was purchased from Sigma
`and w-bromo alcohols and fluorescein isothiocyanate from
`Aldrich. Adsorption column chromatography was performed
`on columns of silica gel 60 (Merck), and triphosphates were
`purified on Fractogel TSK DEAE-640(M) (Merck). TLC was
`conducted on silica-60 F 254 plates (Merck). The melting points
`reported are uncorrected. NMR spectra were recorded on a
`JEOL JNM GX-400 spectrometer operating at 399.8, 100.5 and
`161.9 MHz for 1H, 13C and 31P, respectively. Coupling
`constants are given in Hz. Tetramethylsilane was used as
`internal (1H, 13C) and H 3PO4 as external reference (3 1P). 13C
`NMR data of the compounds prepared are listed in Table I. IR
`spectra were recorded on Perkin-Elmer I 600 FT-IR spectro(cid:173)
`photometer and UV spectra on Perkin-Elmer A-2 spectro(cid:173)
`photometer. Elemental analyses were performed by Analytische
`Laboratorien, Germany, or at Department of Chemistry,
`University of Oulu, Finland. Tet/z DNA-polymerase from
`Thermus thermophilus was generously supplied by Dr. V.
`Kiselev (Research Center of Molecular Diagnostics and
`Therapy, Moscow, Russia). 2'-Deoxyadenosine 5'-[a:- 35S]tri(cid:173)
`phosphate (initial specific activity 1300 Ci mmoJ- 1) was
`obtained from NEN. 2' -Deoxy-and 2' ,3' -dideoxy-ribonucleo(cid:173)
`side 5'-triphosphates were from Pharmacia, and control single
`stranded M 13 mp 18 DNA from USB.
`
`2.- Com-
`5' -0-Benzoyl-3' -0-methylthiomethylthymidine
`pound 1 10 (15.0 g, 43.3 mmol) was dissolved in DMSO (145
`cm 3 ) and acetic acid (29 crn 3 ) and acetic anhydride (93 cm 3 )
`were added . The mixture was stirred at room temperature for
`2 days, i. e. until no starting material was detected by TLC, and
`was then evaporated to give an oily residue. The oil was
`dissolved in methylene chloride ( 150 cm 3 ) and washed with sat.
`aq. NaHCO 3 (3 x 75 cm 3). The organic layer was dried
`(Na2SO4), filtered and evaporated to dryness. The resulting
`solid was purified on a silica gel column, using CH 2Cl 2
`containing from O to 2% MeOH as eluent. Pure fractions were
`combined and concentrated, and the residue was crystallized
`from toluene to give the title compound 2 (12.0 g, 68%) as a
`white powder; m.p. 135 °C; Rr 0.66 (silica gel, CH 2Cl 2- MeOH,
`9 : I, v/v); ).m • .(MeOH)/nm 266; 6H(CDCl 3) 8.68 (l H, br s,
`HJ), 8.04-7.48 (5 H, m, arom), 7.24 (I H, s, H6), 6.30 (l H, dd,
`Jl'2' 7.6,Jl'2'' 6,J,Hl'),4.69(2H,JAB I l.6,OCH2S),4.65(1 H,dd,
`J4._5 . 3.4, J 5 ,_ 5 • 12.2, HS'), 4.58 (l H, m, HJ'), 4.55 (I H, dd,
`J4._ 5 .. 4.0, HS"), 4.37 (l H, m, H4'), 2.16 (I H, ddd, J 2 •• 2 .. 13.8,
`Jr. 3 • 3.4, H2"), 2.16 (3 H, s, SCH 3 ) , 2.13 (I H, m, J 2 , _3 • 5.9,
`
`* These results will be published elsewhere.
`
`213
`
`T
`
`--
`
`-
`
`-
`
`---- - --
`
`-
`
`----
`------
`-----
`--
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`Fig. 1 Gel pattern of the DNA synthesis catalysed by a thermostable
`DNA-polymerase, Tet/z, and
`terminated with 2',3'-dideoxyribo(cid:173)
`nucleoside 5' -triphosphates (ddNTP) and various 3' -O-(ro-amino(cid:173)
`alkoxymethyl)thymidine 5'-triphosphates (9a, lOa-c and 11). Tracks
`1---4 show termination with ddGTP (7 µmol dm-3 ), ddATP (147 µmol
`dm- 3) , ddCTP (56 µmol dm-3 ) and ddTIP (86 µmol dm-3 ), respectively.
`Tracks 5- 9 show termination with 10a (455 µmol dm- 3 ), 10b (350 µmol
`dm-3 ) and Hie (210 µmol dm- 3) , 9a (455 µmol dm-3 ) and 11 (260 µmol
`dm-3), respectively.
`
`

`

`214
`
`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`Table I
`
`13C NMR chemical shifts of the thymidine analogues prepared"
`
`Chemical shift
`
`Compound C2
`
`C4
`
`C5
`
`C6
`
`5-CH 3 Cl' C2' C3' C4' CS' Others
`
`2
`3a
`
`Sa
`Sb
`5c
`7a"
`7b"
`
`7c"
`8a
`
`8b
`8c
`
`150.1 163.4 111.4 134.8 12.3
`150.4 163.9 111.3 134.8 12.2
`
`150.5 164.0 111.1 136.8 12.6
`150.0 162.3 111.3 137.0 12.7
`150.3 163.6 111.2 136.7 12.7
`150.4 164.0 109.4 135.9 12.5
`150.7 163.7 109.5 135.9 12.2
`
`150.4 163.6 109.4 135.9 12.2
`150.6 164.2 111.2 136.9 12.5
`
`150.3 164.2 111.2 136.8 12.6
`150.3 163.3 111.2 136.8 12.6
`
`85.3 37.8 78.0 82.3 64.2 166.2 (C=O), 139.5-134.6 (arom.), 74.1 (OCH 2S), 13.9 (SCH 3 )
`85.0 38.4 76.4 82.5 64.0 166.0 (C---0), 134.8-128.6 (arom), 94.6 (OCH,0),
`68.4, 33.8, 32.6, 29.3, 27.8, 25.3 (6 x CH,)
`86.4 38.1 76.6 85.3 62.4 94.8 (OCH 20), 68.4, 51.4, 36.6, 35.9, 33.7, 32.9 (6 x CH 2 )
`76.7 85.2 62.6 94.9 (OCH 20), 68.6, 51.7, 24.0-20.5 (8 x CH 2)
`86.7 38.1
`86.6 38.1 76.6 85.3 62.6 94.9 (OCH 20), 68.7, 51.6, 29.7-26.3 (10 x CH 2)
`85.0 36.8 76.3 83.5 61.2 93.5 (OCH 20), 67.3, 41.5, 33.2, 29.1, 26.2, 25.6 (6 x CH 2)
`85.0 36.9 76.4 83.8 61.3 93.6 (OCH 20), 67.4, 41.6, 33.2, 29.1, 28.9, 28.8, 26.4, 25.6
`(8 x CH,)
`85.0 36.9 76.4 83.8 61.2 93.6 (OCH 20), 67.3, 41.3, 32.9, 28.9-25.6 (10 x CH 2 )
`86.4 38.2 77.2c 85.4 62.4 157.6 (C---0), 94.8 (OCH 20), 68.3, 39.9, 29.4, 28.8, 26.3, 25.7
`(6 x CH,)
`86.6 38.0 77.2' 85.4 62.5 157.6 (C-0), 94.8 (OCH 20), 68.4, 40.0, 29.5-26.4 (8 x CH 2 )
`86.6 38.0 77 .2 C 85.3 62.5 157.6
`(C=O), 94.8
`(OCH 20), 68.6, 40.0. 29.6---26.6
`(10 x CH,)
`
`• In CDC13 ; ppm from internal tetramethylsilane. • In [ 2H 6]DMSO.' Partial overlapping with the solvent signal.
`
`H2') and 1.67 (3 H, s, 5-CH 3) (Found: C, 56.0; H, 5.4; N, 6.8.
`C 19H 22N 2O 6S 1 requires: C, 56.2; H, 5.46; N, 6.90%).
`
`3a-
`3' -0-( w-Bromoalkoxymethyl)-5' -O-benzoylthymidines
`c. -Compound 2 (2.0 g, 5.0 mmol) was dissolved in dry 1,2-
`dichloroethane (25 cm 3) and NBS (I.I equiv., 1.0 g) or Br2 (282
`mm 3 ) was added to it. After 10 min the appropriate bromo
`alcohol (1.5 equiv.; 6-10 carbons) and lutidine (1.2 cm 3) were
`added. The mixture was stirred overnight at room temperature.
`The reaction was quenched by addition of aq. NaHSO 3 (20
`cm 3). The organic layer was separated, dried over Na2SO4 and
`then concentrated. The oil obtained was normally used for the
`next reaction without further purification. In one case the oil
`was purified on a silica gel column eluting with CH 2Cl 2
`containing from 0 to 3.5% MeOH to give the title compound
`3a; Rr 0.37 (silica gel, CH2Cl 2-MeOH, 97: 3, v /v); Amax(cid:173)
`(MeOH)/nm 266; ..5tt(CDCl3 ) 9.73 (l H, s, H3), 8.03-7.47 (5 H,
`arom), 7.24 (I H, s, H6), 6.32 (l H, dd,J1 ,, 2 , 6.811 _2 .. 6.4, HI'),
`4.76 (2 H, s, OCH 2O), 4.66 (1 H, m, 1 4 ,. 5, 3.8, 1 5 ,. 5 ,, 12.0, H5'),
`4.55 (1 H, m, 14 -y- 4.8, HS'), 4.44 (l H, m, H3'), 4.35 (I H. m,
`H4'), 3.56 (2 H, m, OCH 2 ), 3.39 (2 H, t, l 6.8, CH 2Br), 2.52 (1
`H, m, 1 2 •• 2 ,, 14.2, H2"), 2.17 (1 H, m, 1 2 .. , 3 , 2.8, 1 2 ,, 3 , 5.1, H2'),
`1.83 (2 H, m, OCH2CH2 ), 1.66 (3 H, s, 5CH 3 ), 1.59 (2 H, m,
`CH2CH2Br), 1.44 (2 H, m, OCH 2CH2CH2 ) and 1.38 (2 H, m,
`CH2CH 2CH 2Br).
`
`3' -0-( w-Azidoalkoxymethyl)-5' -O-benzoylthymidines 4a--c.(cid:173)
`LiCI (0.33 g, 7.8 mmol) and NaN3 (1.66 g, 25.5 mmol) were
`suspended in DMF (25 cm3). The mixture was kept at 120 °C
`for 30 min and then allowed to cool to 90 °C. NH 4Cl (0.56 g)
`was added and the whole mixture was poured onto crude 3a--c
`(5 mmol). The suspension was stirred at 50 °C overnight, i.e.
`until HPLC analysis showed that all the starting material was
`consumed, cooled and concentrated under reduced pressure.
`The residue was suspended in methylene chloride (50 cm3) and
`washed with water (50 cm3). The organic layer was dried over
`Na 2SO4 , filtered and then concentrated. The resulting oil was
`dissolved in a small amount of methylene chloride and added
`dropwise into a mixture of diethyl ether-hexane (1: 3, v/v, 250
`cm 3) to remove lutidine and unchanged long chain alcohol. The
`solution was allowed to precipitate and was then decanted. The
`remaining oil was purified by silica gel column chromatography.
`Elution with 0 to 4% MeOH in CH 2Cl 2 gave the title
`compounds 4a--c as colourless oils. 4a: (1.60 g, 57% from 2) Rr
`0.37 (silica gel, CH 2Cl2-MeOH, 97: 3, v/v); AmaiMeOH)/nm
`268; 611(CDCl 3) 8.20 (I H, br, H3), 8.04-7.46 (5 H, arom.), 7.23
`(1 H, s, H6), 6.30 (I H, dd,11 ,, 2 , 7.2,11 .. 2 6.5, HI'), 4.75 (2 H, s,
`
`OCH 2O); 4.66 (1 H, dd, 1 5 ._ 5 .. 12.0, H5'), 4.54 (I H, dd, H5"),
`4.42 (I H, m, H3'), 4.34 (1 H, m, H4'), 3.54 (2 H, m, OCH 2), 3.25
`(2H, t,16.8, CH 2N 3),2.51 (l H,m,12 .z" 13.8, H2"),2.15(1 H,m,
`H2'), 1.66 (3 H, s, 5-CH3), 1.60 (4 H, m, 2 x CH 2) and 1.43 (4 H,
`m, 2 x CH 2 ). 4b: (1.20 g, 45% from 2) Rr 0.39 (silica gel,
`CH 2Cl,-MeOH, 97:3, v/v); Ama.(MeOH)/nm 269;
`..5tt(cid:173)
`(CDCl3) 8.23 (1 H, br s, H3), 8.03-7.47 (5 H, arom), 7.23 (1 H, s,
`H6),6.30(l H,dd,Jl',2' 7.3,Jl',2"6.4,Hl '),4.75(2H, s, OCH2O),
`4.67 (1 H, dd, 14 -, 5 , 2.9, l 5 ._ 5 ,. 12.2, H5'), 4.54 (l H, dd, 14 ,, 5 ,. 3.9,
`H5"), 4.43 (I H, m, H3'), 4.35 (l H, m, H4'), 3.55 (2 H, m,
`OCH 2 ), 3.25 (2 H, t, l 6.8, CH 2N 3), 2.52 (1 H, m, 1 2 ,_ 2 ,. 13.7,
`1 2 ,., 3 , 2.9, H2"), 2.15 (I H, m, 1 2 ,, 3 , 6.8, H2'), 1.66 (3 H, s, 5-
`CH3) and 1.60--1.31 (12 H, m, 6 x CH 2). 4c: (1.28 g, 40% from
`2) Rr 0.40 (silica gel, CH 2Cl 2-MeOH, 97: 3, v/v); Am • .(MeOH)/
`nm 269; ..5tt(CDCl 3) 8.20 (I H, br s, H3), 8.04-7.47 (5 H,
`arom), 7.20 (I H, s, H6), 6.30 (I H, dd, 1 1 ., 2 . 7.3, 1 1 ,, 2 .. 6.3,
`HI'), 4.75 (2 H, s, OCH 2O), 4.66 (1 H, dd, 1 5 ,_ 5 ,. 12.1, H5'), 4.45
`(I H, dd, H5"), 4.37 (l H, m, H3'), 4.37 (1 H, m, H4'), 3.57 (2 H,
`m, OCH 2), 3.25 (2 H, t, l 6.8, CH 2N 3), 2.54 (1 H, m, 1 2 ._ 2 ,. 13.6,
`H2"), 2.15 (1 H, m, H2'), 1.69 (3 H, s, 5-CH 3) and 1.62-1.27 (I 6
`H, m, 8 x CH 2).
`
`3' -O-(ro-Azidoalkoxymethyl)thymidines 5a--c.-Compound
`4a--c was dissolved in sat. methanolic ammonia and stirred
`overnight at room temperature. When the reaction was
`complete, solvent was evaporated under reduced pressure and
`the residue was purified on a silica gel column eluting with
`CH 2Cl2 containing from Oto 5% MeOH. The title compounds
`5a--c were obtained as colourless oils (95% yield). 5a: Rr 0.52
`(silica gel, CH 2Cl2-MeOH, 9:1, v/v); Amax(MeOH)/nm 267;
`..5tt(CDC1 3 ) 9.30 (1 H, br s, H3), 7.43 (I H, s, H6), 6.18 (1 H, dd,
`1 1-. 2 , 7.0,1 1,. 2 ,6.7, Hl'),4.73(2H,s, OCH 2O),4.43(1 H,m,H3'),
`4.07 (1 H, m, H4'), 3.94 (I H, dd, h.s· 2.7,J5 ._ 5 ,. 11.9, H5'), 3.81
`(I H,dd,14.5 3.1, H5"),3.55(2H, t,16.7, OCH 2),3.27(2H, t,J
`6.7, CH 2N 3 ), 2.96(1 H, br 5'-OH), 2.36(2 H, m, H-2', H2"), 1.94
`(3 H, s, 5-CH 3), 1.60 (4 H, m, 2 x CH 2 ) and 1.40 (4 H, m,
`2 X CH2); Vmax/cm- 1 2096s (N3). 5b: Rr 0.54 (silica gel,
`v/v); Am • .(MeOH)/nm 267;
`Jtt(cid:173)
`CH 2Cl,-MeOH, 9: 1,
`(CDCl3)8.30(1 H, brs, H3), 7.38(1 H,s, H6), 6.15(1 H,dd,11 .,2,.
`6.8, HI'), 4. 73 (2 H, s, OCH 2O), 4.43 ( I H, m, H3'), 4.08 ( 1 H, m,
`H4'),3.95(1 H,dd,15 ., 5 ,. 11.8, H5'),3.80(1 H,dd,H5"),3.54(2H,
`t,J 6.8, OCH2 ), 3.28 (2 H, t,J 6.8, CH2N 3), 2.96 (l H, br, 5' -OH),
`2.36 (2 H, m, H2', H2"), 1.92 (3 H, s, 5-CH 3 ) and 1.61-1.33 (12 H,
`m, 6 X CH2); Vmax/cm- 1 2096s (N 3), 5c: Rr 0.56 (silica gel,
`CH 2Cl 2-MeOH, 9:1, v/v); lma/MeOH)/nm 267; ..5tt(CDCl3)
`8.40(1 H, br s, H3), 7.38 (l H, s, H6), 6.16 (1 H, dd,11 ,, 2 , 7.0,11 ,,2 ,.
`6.8, H 1 '), 4. 73 (2 H, s, OCH2O), 4.41 (1 H, m, H3'), 4.07 (I H, m,
`
`

`

`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`H4'),3.94(1 H,dd,15 ., 5 .. l l.9,H5'),3.81 (1 H,dd,H5"),3.54(2H,
`t, 16.8, OCH 2), 3.26 (2 H, t, 16.8, CH2N 3), 2.90 (1 H, hr, 5' -OH),
`2.36 (2 H, m, H2', H2H), 1.92 (3 H, s, 5-CH3) and 1.64-1.29 (16 H,
`m, 8 X CH2); Vmax/cm-1 2096 (N3).
`
`3'-0-(ro-Aminoa/koxymethyl)thymidines 7a-c.-Compound
`Sa (1.12 g, 2.4 mmol) was dissolved in pyridine (30 cm3) and
`Ph3P (2 equiv., 4.0 mmol, 1.05 g) was added. The mixture was
`stirred at room temperature until no starting material could be
`detected by TLC. The resulting ylide was hydrolysed with aq.
`ammonia, evaporated to dryness under reduced pressure,
`suspended in water (50 cm3) and then extracted with diethyl
`ether (3 x 25 cm3). The aqueous layer was evaporated to
`dryness to give compound 7a as a white solid (0.93 g, 93%), Rr
`0.0 (silica gel, CH2Cl2-MeOH 9:1, v/v). The test for primary
`amines was positive.*
`lmaxCH 2O)/nm 267;
`JH([2H 6](cid:173)
`DMSO) 7.70(1 H, s, H6), 6.12(1 H, dd,J1 •• 2• 7.8,11 ., 2 .. 5.9, HI'),
`4.68 (2 H, s, OCH 2O), 4.29 (1 H, m, H3'), 3.91 (l H, m, H4'), 3.61
`(1 H,m,J 4 -, 5-3.9,J5., 5 .. 12.2,H5'),3.56(1 H,m,J4 ., 5 .. 3.9,H5"),3.47
`(2 H, t, J 6.4, OCH2), 2.50 (2 H, m, CH2NH 2 partially
`overlapping with the solvent signal; 2.23 (1 H, m,J2 .,2 .. 13.2, 1 2 .. ,3 ,
`3.6, H2"), 2.14 (1 H, m, 1 2 ,, 3 , 6.2, H2'), 1.77 (3 H, s, 5-CH 3) and
`1.50-1.29 (8 H, m, 4 x CH 2). With the analogues bearing a
`longer aliphatic arm (8 and 10 carbons) at 03' the product (7b
`and c) was not soluble enough in water to be extracted as
`described. Accordingly, after hydrolysis and the first evapor(cid:173)
`ation, the remaining triphenylphosphine was removed by
`stirring the solid material with diethyl ether. The ethereal layer
`was carefully decanted and the remaining solid material was
`dried under reduced pressure. HPLC analysis showed that 7b
`was homogenous, but 7c contained 10% of impurities. 7b: Rr
`0.0 (silica gel, CH2Cl2-MeOH, 9: 1, v/v). The test for primary
`amines was positive. A.maxCMeOH)/nm 266; t58 ([2H 6]DMSO)
`7.70 (l H, s, H6), 6.12 (1 H, dd, J 1., 2, 7.8, 1 1 ., 2 .. 5.9, HI'), 4.68
`(2 H, s, OCH 2O), 4.29 (I H, m, H3'), 3.90 (l H, m, H4'), 3.61 (I
`H, m, 14 ,. 5 .. 3.9, 1 5 •• 5 .. 12.2, H5'), 3.56 (1 H, m, 14 ,_ 5 .. 3.9, H5"),
`3.47 (2 H, t, 1 6.8, OCH 2 ), 2.50 (2 H, m, CH2NH 2 , partially
`overlapping with the solvent signal), 2.24 (I H, m, 12 •• 2 .. 13.7,
`J 2 ... 3, 2.4, H2"), 2.15 (1 H, m, J 2 ,, 3 • < 1, H2'), 1.77 (3 H, s, 5-
`CH3) and 1.50-1.25 (12 H, m, 6 x CH2 ). 7c: Rr 0.0 (silica gel,
`CH 2Cl 2-MeOH, 9:1, v/v). The test for primary amines was
`positive. Amax(MeOH)/nm 266; t5tt([2H 6]DMSO) 7.70 (1 H, s,
`H6), 6.13 (I H, dd, 1 1,.2 , 7.8, 1 1,_ 2 .. 6.0, HI'), 4.68 (2 H, s,
`OCH 2O), 4.29 (1 H, m, H3'), 3.91 (l H, m, H4'), 3.61 (I H, m,
`J 5., 5 .. 12.2, H5'), 3.56 (I H, m, H5"), 3.46 (2 H, t, 16.8, OCH2),
`2.50 (2 H, m, CH2NH 2, partially overlapping with the signal of
`solvent), 2.22 (2 H, m, H2', H2"), 1. 77 (3 H, s, 5-CH3) and
`l.50-1.29 (16 H, m, 8 x CH 2).
`
`3 '-0-(N-Trifluoroacetyl-ro-aminoa/koxymethyl)thymidines
`8a-c.-Compound 7a-c (1.0 mmol) was dissolved in dry DMF
`(2.5 cm3) freshly distilled methyl trifluoroacetate (l.3 cm 3) was
`added. The reaction mixture was stirred overnight at room
`temperature. Solvent and the unchanged ester were evaporated
`under reduced pressure and the resulting oil was purified on a
`silica gel column eluting with 3% MeOH in CH2Cl 2 • The title
`compounds 8a-c were obtained as colourless oils. Sa: (0.44 g,
`95%); Amax(MeOH)/nm 266; JH(CDCl 3) 9.48 (1 H, hr s, H3),
`7.51 (] H,s, H6), 7.09(1 H, brt, NHCOCF3),6.18(1 H,dd,J1 •• 2 •
`6.8, 1 1, •2 .. 6.8, HI'), 4. 73 (2 H, s, OCH2O); 4.44 (l H, m, H3'), 4.09
`(1 H,m, H4'), 3.93(1 H,m,J5 ,. 5 .. 10, HS'), 3.81 (l H,m, H5"), 3.55
`(2 H, t,16.8, OCH 2), 3.36(2 H, q, J6.8, CH2NHCOCF3), 3.26
`(1 H, hr, 5'-OH); 2.39 (1 H, ddd,J2 •• 2 .. 13.7,12 ... 3 . 3.7, H2"), 2.31
`(I H, m, 12 ._3. 6.8, H2'), l.89 (3 H, s, 5-CH3), 1.60 (4 H, m,
`
`• 'Fluram', Roche Spray Reagent for assay of primary amines, F.
`Hoffman-La Roche Co. Limited, Diacnostica, Basie, Switzerland.
`
`215
`
`2 x CH2) and 1.38 (4 H, m, 2 x CH2) (Found: C, 49.2; H, 6.2;
`N, 8.8. C 19H 28F 3N 3O 7 requiresC,48.82; H, 6.04; N, 8.99%). Sb:
`(0.44 g, 90%); ,l,m0,.(MeOH)/nm 266; dtt(CDCl3) 8.94 (1 H,
`hrs, H3), 7.44 (I H, s, H6), 6.67 (1 H, br t, NHCOCF 3), 6.16 (1
`H, dd, 1 1 ,,2 • 6.8, 1 1 .,2 .. 6.8, HI'), 4.73 (2 H, s, OCH 2O), 4.42 (I
`H, m, H3'), 4.08 (1 H, m, H4'), 3.93 (1 H, m, 15 .,5 .. 12.0, H5'),
`3.81 (l H, m, HS"), 3.55 (2 H, t, 16.8, OCH2), 3.36(2 H, q,16.8,
`CH2NHCOCF 3), 2.85 (1 H, hr, 5'-OH), 2.36 (2 H, m, H2',
`H2"), 1.89 (3 H, s, 5-CH 3) and 1.58-1.33 (12 H, m, 6 x CH 2)
`(Found: C, 50.95; H, 6.4; N, 8.4. C 21 H 32F 3N 3O 7 requires C,
`50.90; H, 6.51; N, 8.48%). Sc: (0.46 g, 87%); J.maxCMeOH)/nm
`268;t58 (CDC13) 8.63 (1 H, hrs, H3), 7.41 (l H, s, H6), 6.48 (1 H,
`brt, NHCOCF3 ), 6.16(1 H, dd,11,, 2 , 6.8, 1 1 ,,2 .. 6.8, HI'), 4.73(2
`H, s, OCH2O), 4.42 (1 H, m, H3'), 4.08 (1 H, m, H4'), 3.94 (1 H,
`m, J 5 ., 5 .. 10.0, H5'), 3.81 (l H, m, H5"), 3.54 (2 H, t, 1 6.8,
`OCH 2), 3.36 (2 H, q, 16.8, CH2NHCOCF3), 2.64 (1 H, hr, 5'(cid:173)
`OH), 2.37 (2 H, m, H2', H2"), 1.89 (3 H, s, 5-CH 3) and 1.29 (16
`(Found: C, 52.7; H, 6.8; N, 8.0.
`H, m, 8 x CH2)
`C 23H 36F 3N 3O 7 requires C, 52.77; H, 6.93; N, 8.03%).
`
`3' -0-(N-Trifluoroacetyl-ro-aminoalkoxymethyl)thymidine 5' -
`Triphosphates 9a-c as Triethylammonium Salts.-Dry, recrys(cid:173)
`tallized triazole (4.5 equiv. to nucleoside, 31 mg, 0.45 mmol) was
`dissolved in a mixture of dry acetonitrile (0.5 cm3) and dry
`triethylamine (4.5 equiv., 63 mm3). The mixture was cooled on
`an ice bath and phosphorus oxychloride (1.5 equiv, 14 mm3)
`was added. The mixture was allowed to warm to room
`temperature and stirred for 30 min. The whole mixture was
`quickly filtered and the filtrate was poured on dried nucleoside
`Sa~ (0.1 mmol) and stirred at room temperature until all
`starting material had disappeared (normally 10 to 20 min).
`Then bis(butylammonium)pyrophosphate in DMF (0.2 mo!
`dm-3 ; 1.5 equiv., 2.5 cm3) was added and the reaction was
`stirred overnight at room temperature. The remaining triazole
`ligand was hydrolysed by water (5 cm3). The reaction mixture
`was diluted with water (100 cm 3) containing 50% (v/v)
`acetonitrile. The crude material was applied onto an ion
`exchange column and eluted with a linear gradient [0.0-0.3
`mol dm-3 TEAB (triethylammonium hydrogen carbonate) in
`50%, v/v, MeCN]. The fraction eluted at 0.2 mo! dm-3 TEAB
`was collected and concentrated. The oily residue was further
`purified on a preparative RP-18 column (Reliasil 300 A, 5 µm,
`250 mm x 9 mm) and eluted with water containing 40% (v/v)
`acetonitrile. 9a: JH(D 2O) 7.59 (1 H, s, H6), 6.15 (1 H, dd, 1 1 ,. 2 ,
`8.2, 1 1 •• 2 .. 6.0, HI'), 4.69 (2 H, s, OCH2O, partial overlapping
`with the signal of solvent), 4.40 (1 H, m, H3'), 4.17 (l H, m,
`H4'), 4.05 (2 H, m, H5', H5"), 3.49 (2 H, m, OCH 2), 3.14 (2 H, t,
`16.8, CH2NHCOCF3), 2.31 (I H, m, J2 ,, 2 .. 14.0, H2"), 2.21 (1
`H, m, H2'), 1.76 (3 H, s, 5-CH3) and 1.43-1.30 (8 H, m,
`4 x CH 2). 9b:t58 (D2O)7.60(1 H,s, H6), 6.15 (l H, d,J1 •• 2 , 6.0,
`HI'), 4. 70 (2 H, s, OCH 2O, partial overlapping with the solvent
`signal), 4.42 (l H, m, H3'), 4.15 (1 H, m, H4'), 4.07 (2 H, m, H5',
`H5"), 3.50(2 H,m, OCH2), 3.17 (2H, t,16.8, CH2NHCOCF3),
`2.31 (1 H, m, 1 2 •• 2 .. 14, H2"), 2.21 (1 H, m, H2'), 1.77 (3 H, s, 5-
`CH3) and 1.41-1.29 (12 H, m, 6 x CH 2). 9c: JH(D2O) 7.61 (1
`H, s, H6), 6.17 (l H, dd, 1 1 ,,2 .. 8.3, HI'), 4.70 (2 H, s, OCH2O,
`partial overlapping with the solvent signal), 4.41 (1 H, m, H3'),
`4.18 (l H, m, H4'), 4.06 (2 H, m, H5', H5"), 3.46 (2 H, m,
`OCH2 ), 3.15 (2H, t,J6.8, CH2NHCOCF3 ), 2.32(1 H, m,12 ,,2 ..
`14.3, H2"), 2.22 (1 H, m, H2'), 1.77 (3 H, s, 5-CH 3), 1.44 (4 H,
`m, 2 x CH2) and 1.15 (16 H, m, 8 x CH2). All 1H NMR
`spectra also exhibited signals from the triethylammonium
`group (q 3.03 and t 1.13), which disturbed integration.
`
`5' -Triphosphates
`3 '-O-( ro-Aminoa/koxymethyl)thymidine
`lOa-c.-Compounds 9a--c were dissolved in aq. ammonia,
`stirred overnight at room temperature and then evaporated to
`dryness under reduced pressure to give the title compounds.
`
`

`

`216
`
`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`Table 2 Properties of 3'-O-modified thymidine 5'-triphosphates prepared
`
`31 P NMR chemical shifts'
`
`Compound
`
`la/min"
`
`tR/min b
`
`9a
`9b
`9c
`IOa
`IOb
`IOc
`
`24.2
`24.6
`25.0
`22.1
`22.5
`23.0
`
`23.7
`26.7
`32.9
`14.6
`17.1
`22.3
`
`/,ffil:IX
`
`267
`268
`270
`268
`269
`267
`
`A.min
`
`235
`234
`235
`235
`233
`234
`
`o[P(IX)]
`
`o[P(~)]
`
`o[P(y)]
`
`-11.71
`-11.65
`-11.59
`-8.39
`-8.36
`-8.34
`
`-23.96
`-23.95
`-23.91
`-23.46
`-23.44
`-23.43
`
`- 12.34
`-12.32
`-12.17
`-12.23
`-12.19
`-12.18
`
`• Ion exchangecotumn (SynchropakAX-300, 6.5 µm,4.6 x 250 mm), flow rate 1.0 cm 3 min- 1 , A = 0.03 motdm-3 KH 2 PO4 , 50%formamide(pH =
`5.2), B = 0.03 mot dm-3 KH 2PO4 , 1.0 mol drn 3 (NH 4 ),SO4 , 50% formamide (pH = 5.6), from A to B in 30 min. b Reversed-phase column
`(Nucleosil 300-5 C4, 5 µm, 250 mm x 9 mm; Macherey-Nagel, A = 0.05 mot dm- 1 TEAA, pH 5.5; B = A + 50% MeCN, from 40 to 70% Bin 45
`min, flow rate 0.8 cm 3 min- 1).' In D 2O, external reference H 3PO4 (0.00 ppm). Assignments of P(ix) and P(y) are tentative.
`
`Purification was performed on reversed-phase HPLC as
`described above for 9a-c. IOa: oH(D2O) 7.61 (I H, s, H6), 6.17
`(1 H, dd, J 1 •.i· 6.8, J 1 ,_ 2 .. 5.4, HI'), 4.70 (2 H, s, OCH 2O, partial
`overlapping with the solvent signal), 4.40 (l H, m, H3'), 4.24 (I
`H, m, H4'), 4.05 (2 H, m, H5', HS"), 3.45 (2 H, m, OCH 2), 2.88
`(2 H, t, CH2NH2), 2.32 (I H, m, H2"), 2.21 (I H, m, H2'), 1.68
`(3 H, s, 5-CH 3) and 1.50-1.25 (8 H, m, 4 x CH 2). 10b: oH(D 2O)
`7.61 (I H, s, H6), 6.17 (I H, dd,Jl ',2" 6.8J1·.2" 5.9, HI'), 4.70 (2
`H, s, OCH 2O, partial overlapping with the solvent signal), 4.41
`(I H, m, H3'), 4.18 (1 H, m, H4'), 4.06 (2 H, m, H5', H5''), 3.46
`(2 H, m, OCH2), 2.88 (2 H, t, CH2NH 2 ), 2.32 (I H, m, H2"),
`2.23 (1 H, m, H2'}, 1.79 (3 H, s, 5-CH 3) and 1.45-1.15 (12 H, m,
`6 x CH 2 ). toe: oH(D 2O) 7.61 (I H, s, H6), 6.19 (I H, dd, 1 1 ,, 2 ,
`6.7, J 1 .,2 .. 5.7, HI'), 4.70 (2 H, s, OCH2O, partial overlapping
`with the solvent signal), 4.41 (I H, m, H3'), 4.18 (I H, m, H4'),
`4.06 (2 H, m, H5', H5"), 3.46 (2 H, m, OCH 2 ), 2.89 (2 H, t,
`CH2NH 2), 2.32 (I H, m, H2"), 2.22 ( I H, m, H2'), 1.77 (3 H, s,
`5-CH 3 ) and 1.45-1.29 (16 H, m, 8 x CH 2 ); Table 2 records the
`HPLC retention times, UV absorption maxima and 31 P NMR
`shifts of 9a-c and lOa-c.
`
`Labelling of CompoundlOa with Fluorescein Isothiocyanate.-(cid:173)
`Compound 10a (10 OD) (OD= optical density unit) was
`dissolved in carbonate buffer (0.1 mo! dm-3 ; 2.5 cm3, pH =
`10.3). Fluorescein isothiocyanate (50 mg) in DMF (2.5 cm3 )
`was added and the pH was readjusted to 10 with NaOH (0.1
`mo! dm-3 ). The mixture was kept overnight in the dark at room
`temperature. The pH was adjusted to 3 with HCI (1.0 mol
`dm 3), after which the mixture was extracted with ethyl acetate
`(4 x 10 cm 3). The organic layer was discarded and the aqueous
`layer was neutralized with sat. NaHCO 3 . NaHCO 3 • The crude
`product was purified by ion exchange HPLC [Synchropak AX-
`300, 6.5 µm, 4.6 x 250 mm; A = 0.05 mol dm- 3 KH 2 PO4 , 50%
`formamide, pH 5.2; B = 0.05 mo! dm- 3 K