FULL PAPER
`
`Syntheses of Nucleosides Designed for Combinatorial DNA Sequencing
`
`Mike B. Welch,[a] Carlos I. Martinez,[b] Alex J. Zhang,[a] Song Jin,[a]
`Richard Gibbs,[b] and Kevin Burgess*[a]
`
`I
`Abstract: Nucleoside triphosphates
`with 3’-O-blocking groups that are both
`photolabile and fluorescent were re-
`quired to investigate the viability of a
`strategy for sequencing DNA in a com-
`binatorial fashion (see Figure 1). Four
`compounds were prepared to realize this
`goal. Two of them, 14 a and 14 t, had
`dansyl-functionalized,
`3’-O-(2’’-nitro-
`benzyl) ether groups, while the other
`two, 18 a and 18 t, had similar pendant
`carbonate groups. Tests for incorpora-
`tion of these analogues were performed
`
`by using five different DNA replicating
`enzymes, but the analogues were not
`incorporated. These results were sur-
`prising in view of the fact that previous
`studies had shown that 3’-O-(2’’-nitro-
`benzyl)adenosine triphosphate II was
`incorporated by Bst DNA polymerase
`I. However, molecular simulations with
`the coordinates of a T7 polymerase
`
`Keywords: DNA sequencing · en-
`zyme catalysis · nucleotides
`
`crystal structure as a model demon-
`strates that analogues 14 a, 14 t, 18 a
`and 18 t are too large to fit into the
`enzyme active site, whereas accommo-
`dation of the unsubstituted 2-nitroben-
`zyl compound II is much less demand-
`ing. We conclude that both the nucleo-
`side
`triphosphates
`and the DNA
`polymerase enzyme must be modified
`if
`the proposed DNA sequencing
`scheme is to be viable.
`
`Introduction
`
`Complete DNA sequence analysis of the human genome is a
`costly and time-consuming project. Accelerated methods for
`sequencing large DNA strands are therefore highly desirable.
`Most of
`the current efforts to improve sequencing are
`technological improvements of the Sanger[1, 2] or Maxam –
`Gilbert[3] schemes.[4] These include adaptation of robotic
`systems for processing fluorescent dideoxy-terminated nu-
`cleotides on commercially available DNA sequencing ma-
`chines[5, 6] coupled with ultra thin,[7] or capillary gel,[8–11]
`electrophoresis to improve efficiency. Conventional gel elec-
`trophoresis is not required for some of these approaches,
`nevertheless they are unlikely to reduce the cost and time
`factors to acceptable levels. Other modifications of conven-
`tional sequencing schemes focus on the primer, but still
`require gel electrophoresis.[6, 12–14] For instance, contiguous
`hexamer strings may be used in primer walking methods
`wherein the appropriate primers are drawn from an oligonu-
`cleotide library,[15, 16] but the feasibility of this methodology
`remains to be proven for large-scale projects.
`
`[a] Prof. Dr. K. Burgess, M. B. Welch, A. J. Zhang, S. Jin
`Texas A & M University, Chemistry Department
`P. O. Box 300012, College Station, Texas 77842 (USA)
`[b] C. I. Martinez, R. Gibbs
`Department of Molecular and Human Genetics
`Baylor College of Medicine, One Baylor Plaza
`Houston, TX 77030 (USA)
`
`Other advances in sequencing focus on simultaneous
`processing of data. The simplest and most widely applied
`form of such multiplexing is separations of combinations of
`Sanger sequencing reactions in single gel lanes.[17] Schemes
`involving combination of two sets of Sanger sequencing
`reactions have also been proposed,[17] but are not used
`frequently. Multichannel capillary electrophoresis has also
`been explored, and shows considerable promise.[8, 18–20] Other
`forms of multiplex sequencing involve oligonucleotide probes
`to visualize fragments after they have been transferred to a
`nylon membrane.[21–23] However, efficiency enhancements
`from any one of these methods is unlikely to raise the
`throughput of sequence data by more than one or two orders
`of magnitude.
`There are few fundamentally new approaches to sequenc-
`ing. Novel methodologies include those involving scanning
`tunneling microscopy,[24] single molecule detection,[25, 26] meth-
`ods based on detection of the pyrophosphate liberated in each
`addition step,[27, 28] mass spectrometry,[29] and hybridization
`(SBH-techniques).[30–32] These protocols may offer significant
`increases in efficiency over the established procedures. They
`generally do not require gel electrophoresis, therefore some
`can potentially process larger numbers of samples without
`concomitant increases in equipment, reagents, or time. How-
`ever, at this stage these procedures are largely unproven, and
`some have obvious disadvantages. Scanning tunneling micro-
`scopy and other single molecule detection methods, for
`instance, have not evolved to the level required for reliable
`
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`sequencing, and the techniques based on pyrophosphate
`liberation and hybridization cannot be used to characterize
`repeated sequences. The main applications of SBH-techni-
`ques appear to be for detecting mutations.[33–35] Numerous
`other schemes have been proposed but work on these has
`apparently ceased.[36, 37]
`Our group and others have been exploring another way to
`facilitate parallel analyses of multiple samples in an array
`without gel electrophoresis.[38–43] We call
`this the Base
`Addition Sequencing Scheme or BASS. Central
`to this
`approach is a set of four nucleoside triphosphates I that have
`3’-O-blocking groups that are both labile and fluorescent. The
`
`fluorescence of the protecting group should enable the parent
`base on the ribose skeleton (A, T, G, or C) to be identified. A
`cycle in the proposed sequencing scheme would consist of the
`following steps (Figure 1): 1) incorporation of the appropriate
`
`Figure 1. The base addition sequencing scheme (BASS).
`
`K. Burgess et al.
`
`nucleotide triphosphate analogue I by a DNA replicating
`enzyme(s); 2) spectroscopic identification of the base ana-
`logue incorporated; and, 3) removal of the blocking group P*
`to regenerate a 3’-hydroxy terminus on the (now elongated)
`polynucleotide chain.
`If realized, base addition sequencing would have several
`significant advantages over the methodologies currently used
`for sequencing DNA. First, results from each DNA sample
`will be distinguished by direct analysis of the array, so addition
`of more samples would not proportionately increase the
`amount of effort or materials required. This compares
`favorably with procedures wherein each DNA template must
`be handled separately, and for which every additional sample
`requires a new gel lane. Consequently, the proposed scheme
`potentially has a much greater capacity than conventional
`sequencing methods. Second,
`if the experiment could be
`arranged in such a way that millions of primed DNA
`fragments were analyzed simultaneously, then it would not
`be necessary to characterize each primer. Instead the primers
`could be generated by a combinatorial method, and the
`arrangement of sequences would be deduced from overlaps in
`the data. It is possible that the extent of multiplexing would be
`such that the method would be viable even if a relatively short
`read of DNA sequence was obtained from each individual
`experiment (for example 10 – 50 bases). Moreover, the ease of
`primer synthesis would represent a highly significant cost/time
`saving advantage. Finally, the method could have incidental
`benefits like circumvention of artifacts due to gel compres-
`sions (frequently associated with G,C-rich strings in the
`sequence).
`In preliminary work we found that 3’-O-(2’’-nitrobenzyl)-
`adenosine triphosphate II could be incorporated by a DNA
`polymerase, and that photodeprotection of the 3’-hydroxy was
`possible facilitating DNA replication.[38] This encouraging
`result indicated that 3’-modifications could be tolerated by
`DNA replicating enzymes. Herein we describe syntheses of
`nucleosides protected with photolabile 3’-O-blocking groups
`that are also fluorescent, and report preliminary tests for
`incorporation by DNA polymerase enzymes.
`
`Results and Discussion
`
`Syntheses of nucleoside triphosphates with 3’’-ether linkages:
`Initial attempts to prepare 3’-O-protected nucleosides fo-
`cused on the use of nitrobenzoic acid derivatives as illus-
`trated in Scheme 1. The readily available starting material
`1[44] was coupled with (N-allyloxycarbonyl)pentan-5-ol amine,
`via the acid chloride, to give the ester 2. A phase transfer
`catalyst was used to form the critical ether linkage; devel-
`opment of conditions for this step required considerable
`experimentation. Removal of the 5’-silyl protecting group,
`triphosphorylation,[45] and removal of the allyloxycarbonyl
`group then gave the nucleotide amine 6. A potentially
`attractive feature of this route was that addition of fluorescent
`labels at the very end of the synthesis would allow one ad-
`vanced intermediate to be transformed into several com-
`pounds. Unfortunately,
`labeling of the triphosphate with
`BODIPY-SE 503/512 was unsuccessful, due to the small
`
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`

`Nucleosides Designed for Combinatorial Sequencing
`
`951 – 960
`
`Coupling of derivatives III (R(cid:136) H) to nucleosides was
`problematic, but eventually, suitable conditions were devel-
`oped. Scheme 2 outlines syntheses of derivatives of adenosine
`
`Scheme 1. Synthesis of nucleoside amine 4.
`
`amounts of material and difficulty in handling triphosphates.
`A disadvantage of the design 6 is that 2-nitrobenzyl groups
`substituted with carboxy functionalities cleave less readily
`under photolytic conditions than comparatively electron-rich
`systems.[46] This factor, combined with the experimental
`difficulties associated with the triphosphorylation and label-
`ing steps, led us to investigate alternative routes featuring
`more photosensitive molecules.
`The generic structure III represents the photolabile con-
`nection sought in the next phase of this work. Photodecom-
`position of the methyl substituted compounds III, R(cid:136) Me,
`gives nitroso ketones, whereas the corresponding compounds
`without this methyl substituent (R(cid:136) H) give nitroso alde-
`hydes.[47] Nitroso ketones are less reactive by-products hence
`initial efforts focused on the methyl-substituted compounds.
`However, the secondary benzylic alcohol III where R(cid:136) Me
`did not undergo coupling with the 3’-hydroxy of the nucleo-
`side under a variety of conditions.
`
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`Scheme 2. Preparation of 14 a and 14 t.
`
`

`

`FULL PAPER
`
`and thymidine. The synthesis began with alkylation of vanillin
`to install a linker for the fluorescent reporter. Nitration
`followed by reduction provided the primary benzylic alcohol
`9. The critical coupling step involved conversion of the
`benzylic alcohol 9 into the corresponding benzyl bromide,
`then phase transfer catalyzed reaction with a 5’-protected
`nucleoside under biphasic conditions. Numerous other ap-
`proaches were attempted, but only phase transfer based
`methods gave positive results. In preparation for the con-
`version to the nucleotide, the amine was deprotected then
`dansylated; 5’-desilation then afforded nucleosides 13 a and
`13 t.
`Triphosphorylation of nucleosides 13 and 17, and of many
`other unnatural nucleosides prepared in our laboratories, has
`proved to be experimentally difficult and tends to give poor
`yields. The protocol developed by Eckstein et al. was the best
`out of several approaches attempted,[45] although none of
`those were entirely satisfactory. Fortunately, only small
`amounts of the product are required for feasibility tests in
`bioassays. Debenzoylation of the adenosine derivative 13 a
`and 17 a was performed after the triphosphorylation sequence
`(NH4OH, 60 8C, 3 h). Both pairs of final products, 14 a/14 t and
`18 a/18 t, were purified by chromatography, first on diethyl-
`aminoethyl (DEAE) cellulose, then by RP HPLC.
`
`Syntheses of nucleoside triphosphates with 3’’-carbonate link-
`ages: Difficulties encountered in the syntheses of the ether
`linked derivatives, as outlined above,
`led us to explore
`preparations of structurally similar, but hopefully more
`accessible compounds. Syntheses of structural variants would
`also help probe the tolerance of DNA replicating enzymes to
`unnatural nucleosides. Consequently, two carbonate-linked
`compounds were constructed as described in Scheme 3. Alter-
`native routes to the same compounds were attempted, for
`instance, by forming a chlorocarbonate functionality from the
`nucleoside 3’-hydroxy, but none worked as well as that shown.
`
`Tests for incorporation of 3’’-blocked nucleoside triphos-
`phates: Analogues 14 a, 14 t, 18 a, and 18 t were tested as
`substrates for a series of commercially available DNA
`replicating enzymes. The protocol used for these experiments
`was based on a procedure we have reported previously.[38]
`Briefly, 5’-fluorescein-labeled universal primer was annealed
`to
`a
`synthetic
`oligo
`template,
`5’-TACGGAGGTG-
`GACTGGCCGTCGTTTTACA (italic sequence indicates
`the replication region). The reactions were carried out in
`the presence of a mixture containing the corresponding
`enzyme, some dNTPs, and no other added nucleotides
`(control), a ddNTP (positive control), or a sample of analogue
`
`K. Burgess et al.
`
`Scheme 3. Preparation of carbonate-linked compounds.
`
`14 a, 14 t, 18 a, or 18 t. After incubation the reactions were
`stopped and loaded on a 20 % acrylamide gel, subjected to gel
`electrophoresis, the gel was scanned, and the results were
`visualized with a fragment analysis software.
`Table 1 shows the results for the incorporation assays. The
`enzymes tested were unable to recognize the nucleotide
`
`Table 1. Tests of analogues 14 a, 14 t, 18 a, 18 t as substrates for DNA polymerases.
`
`Analogue
`
`Klenow
`
`rTth DNA Pol.
`
`Polymerase
`Vent (exo-) DNA Pol.
`
`inhibition
`(100 mm)
`no incorporation
`
`no incorporation
`no incorporation
`
`inhibition
`(10 mm)
`nonselective inhibition
`(6.5 mm)
`no incorporation
`no incorporation
`
`inhibition
`(100 mm)
`no incorporation
`
`no incorporation
`no incorporation
`
`14 a
`
`14 t
`
`18 a
`18 t
`
`954
`
`Ampli Taq DNA Pol.
`
`Ampli Taq FS
`
`nonselective inhibition
`(100 mm)
`no incorporation
`
`nonselective inhibition
`(100 mm)
`no incorporation
`
`no incorporation
`inhibition (1.8 mm)
`
`no incorporation
`no incorporation
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`

`

`Nucleosides Designed for Combinatorial Sequencing
`
`951 – 960
`
`analogues as substrates for the termination of the DNA
`amplification under the conditions studied. No incorporation
`was observed in most of the experiments, but in some there
`was evidence that the analogue being tested inhibited the
`polymerase under study, that is no incorporation was ob-
`served and only a band corresponding to the unreacted primer
`could be seen. This was the case for Ampli Taq DNA
`polymerase for which a 1.18 mm final concentration of the
`thymidine carbonate 18 t caused complete inhibition. Klenow
`Fragment, rTth, and Vent (exo(cid:255) ) DNA polymerases ex-
`hibited the same behavior but at 100 mm of the adenosine ether
`14 a. Nonspecific inhibition (termination of the amplification
`reaction at different positions along the template with no
`specificity) was observed for Ampli Taq DNA polymerase, FS
`and Ampli Taq DNA polymerase when a 100 mm of 14 a was
`used (Figure 2).
`
`Figure 2. Illustrative data from incorporation assay. Attempted incorpo-
`ration of 14 a by Ampli Taq DNA polymerase, FS. Arrow marks bands
`corresponding to no incorporation. Template amplification sequence is
`shown on the right of the gel. Fluorescein-labeled universal primer was
`annealed to a complementary oligo template (5’-TACGGAGGTG-
`GACTGGCCGTCGTTTTACA). Lane 1 contained no dNTPs or ddNTPs.
`Lanes 2 – 8 contained 0.1 mm dCTP, in addition lanes 3 and 4 contained
`0.5 mm ddATP and 0.1 mm dATP, 0.1 mm dTTP, respectively. Lanes 5 to 8
`contained 0.1 mm, 2 mm, 10 mm and 100 mm D*ATP, respectively.
`
`Molecular simulations of 3’’-blocked nucleoside triphosphates
`in the active sites of DNA replicating enzymes: Molecular
`simulations were performed to rationalize the lack of
`incorporation of the analogues prepared in the course of the
`work described above. It was perplexing that compound 3’-O-
`(2’’-nitrobenzyl)adenosine triphosphate II was previously
`incorporated by a DNA polymerase, whereas similar ana-
`logues prepared in the current study were not.
`Coordinates for a crystal structure of T7 DNA polymerase
`encapsulating a primed template and ddGTP were down-
`loaded from the Protein Data Bank and used as a model for
`this study. This particular set of coordinates was used since
`they include all the components (enzyme, primed template,
`and nucleoside triphosphate) and because the data set was
`recorded at high resolution (2.2 (cid:138)). An expansion of the
`active site of this enzyme complex is shown in Figure 3 top.
`Removal of the ddGTP entity gave a vacant active site, and
`several conformers of 3’-O-(2’’-nitrobenzyl)adenosine tri-
`phosphate II were fitted in this void by visually docking to
`form reasonable contacts and avoid unfavorable interactions.
`A few orientations seemed reasonable, and one illustrative
`representation is shown in Figure 3 middle. Conversely,
`
`Figure 3. a) Active site of T7 polymerase highlighting two magnesium
`atoms coordinated to dideoxyguanosine triphosphate with critical side-
`chains of the protein (grey) and terminus of an encapsulated primer (blue)
`highlighted; b) as above but with 3’-O-(2’’-nitrobenzyl)adenosine triphos-
`phate encapsulated; c) as in a) but with the nucleoside triphosphate 18 a
`encapsulated.
`
`attempts to fit the analogues with fluorescent groups and a
`methoxy-substituent attached to the aromatic ring were less
`successful. Figure 3 bottom shows a representation of 18 a in
`the active site; several interactions were involved that would
`not be permissible in reality. These docking experiments are
`too crude to allow detailed conclusions to be formulated, but
`it does seem clear that 3’-O-(2’’-nitrobenzyl)adenosine tri-
`phosphate II is much more easily accommodated in this
`particular enzyme than any of the analogues prepared in this
`study.
`
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`Conclusions
`
`3’-O-Blocked nucleoside triphosphates, wherein the 3’-protect-
`ing group is both photolabile and fluorescent, are syntheti-
`cally accessible. However, they tend to be too big to fit into
`the active site of DNA polymerases as evidenced by the data
`from the activity screens and the molecular-simulation experi-
`ments. It seems clear that modifications to the polymerase
`enzyme as well as to the nucleoside triphosphate are required
`if BASS is to be developed into a viable sequencing scheme.
`There are indications that modern methods for combinatorial
`mutagenesis of proteins, gene shuffling,[48] have the potential
`to overcome this obstacle, but those methods must be refined
`further before this goal can be realized.
`
`Experimental Section
`
`General procedures: High-field NMR spectra were recorded on a Unity(cid:135)
`300 spectrometer (1H at 300 MHz, 13C at 75 MHz), 1H chemical shifts are
`reported in d relative to CHCl3 (7.23 ppm) as internal standard, and 13C
`chemical shifts are reported in ppm relative to CDCl3 (77.0 ppm) unless
`otherwise specified. Multiplicities in 1H NMR are reported as (br) broad,
`(s) singlet, (d) doublet, (t) triplet, (q) quartet, (p) quintet and (m) mul-
`tiplet. Thin-layer chromatography was performed on silica gel 60 F254
`plates from Whatman. Flash chromatography was performed on SP silica
`gel 60 (230 – 600-mesh ASTM). BODIPY 503/512 was purchased from
`Molecular Probes. Other chemicals were purchased from commercial
`suppliers and used as received.
`4-bromomethyl-3-nitrobenzoate
`(N-Allyloxycarbonyl)-5-aminopent-1-yl
`(2):
`4-Bromomethyl-3-nitrobenzoic
`acid (2.0 g,
`7.7 mmol), CH2Cl2
`(38 mL), and DMF (0.1 mL) were cooled to 0 8C under a nitrogen
`atmosphere. Oxalyl chloride (1.95 g, 15.4 mmol, 1.3 mL) was added,
`resulting in a vigorous evolution of gas. The reaction mixture was stirred
`for 3 h, concentrated, and taken up in CH2Cl2 (38 mL). A solution of
`triethylamine (1.56 g, 15.4 mmol, 2.15 mL), catalytic DMAP, and (N-
`allyloxycarbonyl)-5-aminopentanol (1.44 g, 7.7 mmol) in CH2Cl2 (10 mL)
`was added to the above acid chloride, and the mixture was stirred for 3 h.
`The reaction mixture was then diluted with CH2Cl2 (200 mL), washed with
`HCl (0.5 m 2(cid:2) 250 mL), and the aqueous layer was back extracted with
`CH2Cl2 (50 mL). Purification of the crude product by flash chromatography
`with a gradient of 25 % to 30 % EtOAc/hexanes as the eluant gave 2 as a
`thick liquid with an orange tinge (2.9 g, 87 % yield). Rf(cid:136) 0.66 (50 % EtOAc/
`hexanes); 1H NMR (CDCl3, 300 MHz): d(cid:136) 8.57 (d, J(cid:136) 1.5 Hz, 1 H), 8.21
`(dd, J (cid:136) 8.1, 1.5 Hz, 1 H), 7.74 (d, J(cid:136) 8.1 Hz, 1 H), 5.90 – 5.77 (m, 1 H), 5.20
`(d, J(cid:136) 17.1 Hz, 1 H), 5.12 (d, J(cid:136) 10.2 Hz, 1 H), 4.94 (s, 2 H), 4.88 (br s, 1 H),
`4.47 (d, J(cid:136) 5.1 Hz, 2 H), 4.31 (t, J(cid:136) 6.6 Hz, 2 H), 3.14 (q, J (cid:136) 6.6 Hz, 2 H),
`1.76 (p, J(cid:136) 7.5 Hz, 2 H), 1.58 – 1.36 (m, 4 H); 13C NMR (CDCl3, 75 MHz)
`d(cid:136) 163.9, 156.2, 147.8, 136.5, 134.0, 132.7, 131.7, 126.0, 117.4, 65.7, 65.3, 42.2,
`40.6, 29.5, 28.1, 27.9, 23.0.
`3’’-O-[(4’’’’-(N-Allyloxycarbonyl)-5’’’’’’-aminopent-1-yl-oxycarbonyl)-2’’’’-nitro-
`phenylmethyl]-5’’-O-(tert-butyldiphenylsilyl)thymidine (3): A solution of 2
`(676 mg, 1.57 mmol) in CH2Cl2 (4.0 mL, 0.39 m) was added dropwise over
`10 min to a stirred mixture of 5’-O-(tert-butyldiphenylsilyl)thymidine
`(771 mg, 1.60 mmol), aqueous Bu4NOH (70 %, 0.5 mL), NaI (cat), CH2Cl2
`(5 mL), H2O (5 mL), NaOH (1m, 5 mL), and stirred for 17 h at 25 8C. The
`reaction mixture was diluted with CH2Cl2 (100 mL) and extracted with HCl
`(0.5 m, 2(cid:2) 75 mL). The combined aqueous layers were then back extracted
`with CH2Cl2 (50 mL). Purification of the crude product was by flash
`chromatography with a gradient of 4:1:5 EtOAc/hexanes/CH2Cl2 increased
`to 100 % EtOAc as the eluting solvent. Appropriate fractions were
`combined and concentrated to yield 3 as a yellow foam (717 mg, 54 %
`yield). Rf (cid:136) 0.28 (50 % EtOAc/hexanes); IR (neat): n˜ (cid:136) 3448, 2934, 2859,
`1715, 1668 cm(cid:255)1; 1H NMR (CDCl3, 300 MHz): d(cid:136) 8.59 (d, J(cid:136) 1.8 Hz, 1 H),
`8.10 (dd, J(cid:136) 8.1, 1.5 Hz, 1 H), 7.64 – 7.61 (m, 4 H), 7.54 (d, J(cid:136) 1.2 Hz, 1 H),
`7.45 – 7.37 (m, 6 H), 7.25 (d, J(cid:136) 8.1 Hz, 1 H), 6.34 (dd, J(cid:136) 7.8, 5.4 Hz, 1 H),
`5.93 – 5.80 (m, 1 H), 5.49 (s, 2 H), 5.24 (dd, J (cid:136) 17.1, 1.5 Hz, 1 H), 5.16 (dd,
`
`K. Burgess et al.
`
`J(cid:136) 10.5, 1.5 Hz, 1 H), 4.78 (br s, 1 H), 4.55 – 4.50 (m, 3 H), 4.30 (t, J(cid:136)
`6.3 Hz, 2 H), 4.00 – 3.80 (m, 3 H), 3.16 (q, J(cid:136) 6.6 Hz, 2 H), 2.35 (ddd, J(cid:136)
`13.2, 5.7, 2.4 Hz, 1 H), 2.22 – 2.12 (m, 1 H), 1.75 (p, J(cid:136) 7.2 Hz, 2 H), 1.63 (s,
`3 H), 1.59 – 1.38 (m, 4 H), 1.06 (s, 9 H); 13C NMR (CDCl3, 75 MHz): d(cid:136)
`164.3, 163.2, 156.3, 150.7, 148.8, 136.9, 135.5, 135.2, 134.1, 133.8, 132.8, 132.2,
`130.5, 130.2, 130.1, 128.4, 128.0, 127.9, 125.9, 117.6, 110.3, 87.0, 85.4, 72.0,
`65.5, 64.0, 41.6, 41.1, 40.8, 29.6, 28.2, 27.0, 23.1, 19.3, 12.8; HRMS (positive-
`ion FAB, nitrobenzyl alcohol (NBA)): calcd for C43H52N4O11SiNa 851.3299,
`found 851.3327.
`3’’-O-[(4’’’’-(N-Allyloxycarbonyl)-5’’’’’’-aminopent-1-yl-oxycarbonyl)-2’’’’-nitro-
`phenylmethyl]thymidine (4): Compound 3 (700 mg, 0.84 mmol), THF
`(8 mL), and terabutylammonium fluoride (TBAF) (1.26 mL, 1.26 mmol)
`were stirred at 25 8C for 15 min. The reaction was concentrated and purified
`directly by flash chromatography with a gradient of 100 % EtOAc, then 1 to
`5 % MeOH in EtOAc as the eluant to give 4 as a foam (589 mg, 88 % yield).
`IR (neat): n˜ (cid:136) 3479, 2947, 1720,
`Rf(cid:136) 0.11 (75 % EtOAc/hexanes);
`1642 cm(cid:255)1; 1H NMR (CDCl3, 300 MHz): d(cid:136) 8.54 (d, J(cid:136) 1.8 Hz, 1 H),
`8.06 (dd, J(cid:136) 8.1, 1.5 Hz, 1 H), 7.60 (s, 1 H), 7.24 (d, J(cid:136) 8.1 Hz, 1 H), 6.17 (t,
`J(cid:136) 6.6 Hz, 1 H), 5.89 – 5.76 (m, 1 H), 5.44 (s, 2 H), 5.21 (d, J(cid:136) 17.4 Hz, 1 H),
`5.12 (d, J(cid:136) 10.5 Hz, 1 H), 4.97 (br, 1 H), 4.47 – 4.41 (m, 3 H), 4.28 (t, J(cid:136)
`6.3 Hz, 2 H), 4.00 (t, J(cid:136) 6.6 Hz, 2 H), 3.89 (br, 1 H), 3.81 – 3.69 (m, 2 H), 3.12
`(q, J(cid:136) 6.6 Hz, 2 H), 3.95 (br, 1 H), 2.25 – 2.19 (m, 2 H), 1.99 (s, 3 H), 1.74 (p,
`J(cid:136) 7.5 Hz, 2 H), 1.58 – 1.35 (m, 4 H); 13C NMR (CDCl3, 75 MHz): d(cid:136) 164.3,
`163.2, 156.4, 150.7, 148.7, 136.8, 135.5, 133.9, 132.8, 130.5, 128.4, 125.8, 117.5,
`110.1, 87.0, 86.5, 71.1, 65.6, 65.4, 62.1, 41.5, 40.7, 40.3, 29.5, 28.2, 23.0, 13.1;
`HRMS (positive-ion FAB, NBA): calcd for C27H34N4O11Na 613.2122, found
`613.2122.
`3’’-O-[(4’’’’-(N-Allyloxycarbonyl)-5’’’’’’-aminopent-1-yl-oxycarbonyl)-2’’’’-nitro-
`phenylmethyl]thymidine-5’’-O-triphosphate (5): Compound 4 was azeo-
`troped with PhH (3(cid:2) 5 mL) and placed in a high vacuum dessicator
`overnight. Compound 4 (369 mg, 0.46 mmol), 1,8-bis(dimethylamino)naph-
`thalene (199 mg, 0.93 mmol), and trimethyl phosphate (4.6 mL) were
`cooled to 0 8C under a nitrogen atmosphere, and phosphorus oxychloride
`(142 mg, 0.93 mmol) was added in one portion. This mixture was stirred at
`0 8C for 140 min. A premixed solution of tributylammonium pyrophosphate
`(894 mg, 1.6 mmol) and tributylamine (296 mg, 1.60 mmol) in DMF
`(2.0 mL) was then added. The reaction was stirred for 5 min and then
`quenched at 0 8C with triethylammonium bicarbonate (1m, pH(cid:136) 5.7). The
`reaction mixture was allowed to warm to 25 8C, stirred for an additional 1 h,
`diluted with deionized H2O (10 mL), lyophilized, taken up in EtOH (3(cid:2)
`15 mL), and concentrated at 25 8C on a high vacuum rotary evaporator.
`Purification by ion exchange chromatography with DEAE-sephadex A-25
`resin, eluting with a 0 to 1.0 m triethylammonium bicarbonate (500 mL
`each) gradient gave the product 5. This was detected by monitoring the
`absorbance at 302 nm. Appropriate fractions were then combined,
`concentrated, redissolved in deionized water (5 mL), and concentrated
`again to yield compound 5 (34 mg, 8.9 % yield). 31P NMR (D2O, 121 MHz):
`d(cid:136) 3.82 (d, J(cid:136) 24.3 Hz), (cid:255) 5.40 (d, J(cid:136) 21.4 Hz), (cid:255) 19.0 (t, J(cid:136) 20.8 Hz);
`MS (MALDI-TOF): calcd for C27H35N4O20P3 828, found 828.
`3’’-O-[4’’’’-(5’’’’’’-Aminopent-1-yl-oxycarbonyl)-2’’’’-nitrophenylmethyl]thymi-
`dine-5’’-O-triphosphate (6): Compound 5 (81 mg, 0.10 mmol), degassed
`HPLC grade H2O (1.0 mL, 0.1m), [Pd(PPh3)4] (11 mg, 0.01 mmol), and
`degassed morpholine (85 mg, 0.98 mmol) were stirred for 19 h at 25 8C. The
`reaction was then filtered through a 45 mm HPLC filter. Purification was
`performed by C18 RP-HPLC (analytical) with a gradient of 0 % B (time t(cid:136)
`0 min); 50 % B (t(cid:136) 20 min); 70 % B (t(cid:136) 30 min) where A is 0.1m
`triethylammonium acetate, and B is 70 % MeCN/A. This procedure was
`repeated 15 times for small portions of the reaction mixture on an
`analytical instrument. Fractions containing the product 6 were assayed by
`UV absorption at 302 nm, combined, and lyophilized to yield a white
`hygroscopic powder (5.1 mg, 7 % yield). MS (MALDI-TOF): calcd for
`C23H32N4O18P3 745, found 745.
`4-[(N-Allyloxycarbonyl)-5-aminopent-1-yl-oxy]-3-methoxybenzaldehyde (8):
`Compound 7 (3.35 g, 13 mmol), MeCN (40 mL), vanillin (1.98 g,
`0.01 mmol), KI (cat), and K2CO3 (5.6 g, 0.04 mol) were refluxed for 12 h.
`The crude reaction mixture was filtered through a plug of silica gel on
`Celite and diluted with EtOAc (100 mL). The organic layer was extracted
`with water (2(cid:2) 50 mL), HCl (0.5 m, 2(cid:2) 50 mL), and brine (25 mL), dried
`over Na2SO4, filtered, and concentrated to yield 8 as a thick orange liquid
`(4.2 g, 100 % yield). Rf(cid:136) 0.38 (50 % EtOAc/hexanes); IR (neat): n˜ (cid:136) 3454,
`2945, 2866, 1700, 1266 cm(cid:255)1; 1H NMR (CDCl3, 300 MHz): d(cid:136) 9.75 (s, 1 H),
`
`956
`
`(cid:23) WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
`
`0947-6539/99/0503-0956 $ 17.50+.50/0
`
`Chem. Eur. J. 1999, 5, No. 3
`
`

`

`Nucleosides Designed for Combinatorial Sequencing
`
`951 – 960
`
`7.35 (dd, J(cid:136) 8.1, 1.8 Hz, 1 H), 7.32 (s, 1 H), 6.87 (d, J (cid:136) 1.8 Hz, 1 H), 5.89 –
`5.76 (m, 1 H), 5.20 (dd, J(cid:136) 17.1, 1.2 Hz, 1 H), 5.11 (dd, J(cid:136) 10.2, 0.9 Hz,
`1 H), 4.94 (br s, 1 H), 4,46 (d, J(cid:136) 5.4 Hz, 2 H), 4.01 (t, J(cid:136) 6.6 Hz, 2 H), 3.83
`(s, 3 H), 3.13 (q, J (cid:136) 6.3 Hz, 2 H), 1.82 (p, J(cid:136) 6.9, 2 H), 1.57 – 1.41 (m, 4 H);
`13C NMR (CDCl3, 75 MHz): d(cid:136) 190.8, 156.2, 153.9, 149.6, 132.8, 129.7,
`126.6, 117.3, 111.2, 109.0, 68.6, 65.2, 55.8, 40.6, 29.5, 28.3, 23.0; HRMS
`(positive-ion FAB, NBA): calcd for C17H24N1O5 322.1654, found 322.1646.
`1-[(N-Allyloxycarbonyl)-5’’-aminopent-1-yl-oxy]-4-(hydroxymethyl)-2-meth-
`oxy-5-nitrobenzene (9): Compound 8 (4.23 g, 13.2 mmol) in Ac2O (17 mL)
`was slowly added to a stirred mixture of HNO3 (66 mL) and Ac2O (17 mL)
`at 0 8C. After 2 h the reaction was allowed to warm to 25 8C and stirred for
`an additional 2 h. The resulting solution was diluted with EtOAc (200 mL),
`washed with brine (4(cid:2) 100 mL), NaHCO3 (6(cid:2) 100 mL), brine (100 mL),
`dried over Na2SO4, filtered through a plug of silica gel on Celite, and
`concentrated yielding a thick yellow oil. This crude material was dissolved
`in absolute EtOH (66 mL) and cooled to 0 8C. Solid NaBH4 (1.0 g,
`26 mmol) was added in about 50 mg portions; the reduction was monitored
`by TLC until starting material was completely consumed (approximately
`4 h). The reaction was then quenched with saturated aqueous NH4Cl,
`allowed to warm to room temperature, and diluted with EtOAc (200 mL).
`The crude reaction mixture was extracted with water (2(cid:2) 100 mL), brine
`(100 mL), dried over Na2SO4, filtered through a plug of silica gel on Celite,
`and concentrated. The crude product was purified by flash chromatography
`with a gradient of 50 to 60 % EtOAc/hexanes as the eluting solvent yielding
`9 (0.73 g, 53 % yield over two steps) as a yellow oil which crystallized upon
`standing. Rf(cid:136) 0.36 (3(cid:2) 30 % EtOAc/hexanes); IR (neat): n˜ (cid:136) 3439, 2943,
`2862, 1711, 1576, 1323, 1064 cm(cid:255)1; 1H NMR (CDCl3, 300 MHz): d(cid:136) 7.65 (s,
`1 H), 7.15 (s, 1 H), 5.93 – 5.80 (m, 1 H), 5.24 (dd, J(cid:136) 17.4, 1.5 Hz, 1 H), 5.14
`(dd, J(cid:136) 10.2, 1.2 Hz, 1 H), 4.91 (s, 2 H), 4.85 (br, 1 H), 4.49 (d, J(cid:136) 5.4 Hz,
`2 H), 4.02 (t, J(cid:136) 6.6 Hz, 2 H), 3.93 (s, 3 H), 3.16 (q, J(cid:136) 6.3 Hz, 2 H), 3.10 –
`2.85 (br, 1 H), 1.84 (p, J(cid:136) 6.9 Hz, 2 H), 1.60 – 1.42 (m, 4 H); 13C NMR
`(CDCl3, 75 MHz): d(cid:136) 156.3, 154.1, 147.2, 139.4, 132.8, 132.0, 117.6, 110.8,
`109.1, 69.1, 65.4, 62.6, 56.3, 40.7, 29.6, 28.4, 23.0; HRMS (positive-ion FAB,
`NBA): calcd for C17H25N2O7 369.1661, found 369.1666.
`1-[(N-Allyloxycarbonyl)-5’’-aminopent-1-yl-oxy]-4-bromomethyl-2-meth-
`oxy-5-nitrobenzene (10): Compound 9 (114 mg, 0.31 mmol), in an oven-
`dried flask, was azeotroped with PhH (2 mL), charged with distilled EtOAc
`(1.5 mL),
`triphenylphosphine (122 mg, 0.46 mmol), and stirred until
`homogeneous at 25 8C. Carbon tetrabromide (154 mg, 0.46 mmol) was
`added and the resulting orange-red solution was stirred for 1 h. The
`reaction was exothermic and a gummy material formed. The crude reaction
`mixture was filtered through a plug of silica gel on Celite and washed with
`EtOAc (50 mL). Purification of the crude product by flash chromatography
`with a gradient of 30 to 40 % EtOAc/hexanes as the eluting solvent gave
`compound 10 (117 mg, 88 % yield), as an off-white solid. M.p. 107 – 108 8C;
`Rf(cid:136) 0.36 (40 % EtOAc/hexanes); IR (CDCl3): n˜ (cid:136) 3452, 2942, 1718, 1526,
`1282 cm(cid:255)1; 1H NMR (CDCl3, 300 MHz): d(cid:136) 7.60 (s, 1 H), 6.88 (s, 1 H),
`5.93 – 5.81 (m, 1 H), 5.25 (ddd, J(cid:136) 17.4, 3.0, 1.2 Hz, 1 H), 5.16 (ddd, J(cid:136) 10.2,
`2.7, 1.5 Hz, 1 H), 4.82 (s, 2 H), 4.83 – 4.76 (br s, 1 H), 4.50 (d, 5.7 Hz, 2 H), 4.03
`(t, J(cid:136) 6.3 Hz, 2 H), 3.93 (s, 3 H), 3.17 (q, J(cid:136) 6.3 Hz, 2 H), 1.85 (dq, J(cid:136)
`6.6 Hz, 2 H), 1.61 – 1.41 (m, 4 H); 13C NMR (CDCl3, 75 MHz): d(cid:136) 156.2,
`153.4, 148.3, 140.0, 132.9, 127.2, 117.5, 113.7, 109.4, 69.1, 65.4, 56.4, 40.7, 30.2,
`29.6, 28.3, 23.0; HRMS (positive-ion FAB, NBA): calcd for C17H24N2O6Br
`431.0817, found 431.0830.
`3’’-O-[4’’’’-((N-Allyloxycarbonyl)-5’’’’’’-aminopent-1-yl-oxy)-5’’’’-methoxy-2’’’’-
`nitrophenylmethyl]-5’’-O-(tert-butyldiphenylsilyl)thymidine (11 t): A solu-
`tion of 10 (800 mg, 1.85 mmol) in CHCl3 (5.0 mL, 0.37 m) was added over
`10 min to a vigorously stirred solution of 5’-O-(tert-butyldi