4062
`
`J. Org. Chem. 1998, 63, 4062-4068
`
`Optimization and Mechanistic Analysis of Oligonucleotide
`Cleavage from Palladium-Labile Solid-Phase Synthesis Supports1
`
`Marc M. Greenberg,* Tracy J. Matray, Jeffrey D. Kahl, Dong Jin Yoo, and Dustin L. McMinn
`Department of Chemistry, Colorado State University Fort Collins, Colorado 80523
`
`Received January 26, 1998
`
`Pd(0)-labile solid-phase synthesis supports have been used to produce oligonucleotides containing
`3¢-alkyl carboxylic acid and 3 ¢-hydroxy termini in quantitative yields. Optimization of the cleavage
`reaction conditions using tetrabutylammonium formate buffer resulted in quantitative yields of
`oligonucleotides using 4 molar equiv of Pd2(dba)3(cid:226)CHCl3 in 1 h at 55 °C. A proton source facilitates
`cleavage of the oligonucleotide from the supports. Trace amounts of water, acting as a nucleophile
`on the Ł3-complex, presumably preventing back biting by the initially released oligonucleotide, are
`required to obtain reproducibly high yields of cleaved oligonucleotides during a 1 hreaction. The
`previously observed lability of (cid:226)-cyanoethyl groups to the Pd(0) conditions has been examined using
`a mononucleotide substrate. Cleavage of the (cid:226)-cyanoethyl group was shown to proceed to the
`exclusion of other alkyl groups. A mechanism involving initial insertion by Pd(0) into the carbon-
`oxygen bond of the (cid:226)-cyanoethyl group is suggested to account for the cleavage of this group.
`
`The Tsuji-Trost Pd(0) cleavage reaction has proven
`to be very useful in oligonucleotide synthesis. Noyori and
`Hayakawa were the first to utilize this reaction for
`deprotecting the phosphate diesters and exocyclic amines
`in oligonucleotides.2 Their strategy has been used in
`conjunction with photolabile solid-phase supports to
`prepare oligonucleotides containing alkaline labile nucle-
`otides at defined sites.3 More recently, the Tsuji-Trost
`Pd(0) cleavage reaction has been employed to cleave
`oligonucleotides from their solid-phase supports (1).4,5
`The exocyclic amine, 5¢ -hydroxyl, and commercially avail-
`able methyl phosphate protecting groups are unaffected
`by the Pd(0) cleavage reaction conditions.4 Hence, the
`Pd(0)-labile supports can also be used to produce pro-
`tected oligonucleotides in solution, which are useful for
`synthesizing oligonucleotide conjugates in high yield
`under mild conditions.6 Supports 2 and 3 have expanded
`the array of functionalization obtainable from palladium-
`labile solid-phase synthesis supports to include optimal
`production of oligonucleotides containing 3¢ -hydroxy ter-
`mini. The optimization of reaction conditions for oligo-
`nucleotide cleavage from these Pd(0)-labile supports, as
`well as relevant mechanistic observations, is described
`below.
`Our original interest in Pd(0)-labile solid-phase syn-
`thesis supports was an outgrowth of work involving
`orthogonal photolabile solid-phase supports.7 During the
`past decade, we and others have utilized photochemistry
`
`(1) A portion of this manuscript was taken from the Ph.D. disserta-
`tion of T.J.M., Colorado State University, 1997.
`(2) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am.
`Chem. Soc. 1990, 112, 1691.
`(3) Greenberg, M. M.; Barvian, M. R.; Cook, G. P.; Goodman, B. K.;
`Matray, T. J.; Tronche, C.; Venkatesan, V. J. Am. Chem. Soc. 1997,
`119, 1828.
`(4) Matray, T. J.; Yoo, D. J.; McMinn, D. L.; Greenberg, M. M.
`Bioconjugate Chem. 1997, 8, 99.
`(5) (a) Zhang, X.; Gaffney, B. L.; Jones, R. A. Nucleic Acids Res. 1997,
`25, 3980. (b) Lyttle, M. H.; Hudson, D.; Cook, R. M. Nucleic Acids Res.
`1996, 24, 2793. (c) Bergmann, F.; Kueng, E.; Iaiza, P.; Bannwarth, W.
`Tetrahedron 1995, 51, 6971.
`(6) (a) McMinn, D. L.; Matray, T. J.; Greenberg, M. M. J. Org. Chem.
`1997, 62, 7074. (b) McMinn, D. L.; Greenberg, M. M. J. Am. Chem.
`Soc. 1998, 120, 3289.
`
`and, in particular, the o-nitrobenzyl photoredox reaction
`in developing methods for solid-phase synthesis.8-10 In
`our own research, the o-nitrobenzyl photoredox reaction
`has been used to synthesize solid-phase supports that
`release oligonucleotides (protected or unprotected) con-
`taining 3¢-hydroxy, 3¢-alkyl carboxylic acids, or 3¢-alkyl-
`amines (4-6).7 The supports are compatible with com-
`mercially available reagents and automated oligonucleotide
`
`(7) (a) McMinn, D. L.; Greenberg, M. M. Tetrahedron 1996, 52, 3827.
`(b) Venkatesan, H.; Greenberg, M. M. J. Org. Chem. 1996, 61, 525. (c)
`Yoo, D. J.; Greenberg, M. M. J. Org. Chem. 1995, 60, 3358.
`(8) (a) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.;
`Stern, D.; Winkler, J.; Lockhart, D. J.; Moris, M. S.; Fodor, S. P. A.
`Science 1996, 274, 610. (b) Cho, C. Y.; Moran, E. J.; Cherry, S. R.;
`Stephans, J. C.; Fodor, S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs,
`J. W.; Schultz, P. G. Science 1993, 261, 1303.
`(9) Pirrung, M. C.; Fallon, L.; Lever, D. C.; Shuey, S. W. J. Org.
`Chem. 1996, 61, 2129.
`(10) (a) Holmes, C. P. J. Org. Chem. 1997, 62, 2370. (b) Burgess,
`K.; Martinez, C. I.; Russell, D. H.; Shin, H.; Zhang, A. J. J. Org. Chem.
`1997, 62, 5662. (c) Holmes, C. P.; Chinn, J. P.; Look, G. C.; Gordon, E.
`M.; Gallop, M. A. J. Org. Chem. 1995, 60, 7328.
`
`S0022-3263(98)00135-2 CCC: $15.00 © 1998 American Chemical Society
`Published on Web 05/27/1998
`
`Illumina Ex. 1105
`IPR Petition - USP 10,435,742
`
`

`

`Oligonucleotide Cleavage from Pd(0)-Labile Solid Supports
`
`J. Org. Chem., Vol. 63, No. 12, 1998 4063
`
`Scheme 1a
`
`Scheme 2a
`
`synthesis protocols. Isolated yields as high as 98% of
`oligonucleotide containing photodamage below detectable
`limits are obtainable. However, decreases in oligonucleo-
`tide yields are observed when the length of the biopoly-
`mer is increased from 20 to 40 nucleotides.7a This
`decrease in yield prompted us to investigate the Tsuji-
`Trost reaction as a method for the cleavage of protected
`oligonucleotides from solid-phase supports, the yields of
`which we assumed would be independent of oligonucleo-
`tide length. Our preliminary experiments demonstrated
`that this was indeed the case.4
`
`consisted of N-acylated product. Following desilylation
`of the coupling product (12), 13 was oxidized to the
`carboxylic acid (14), which was then loaded directly onto
`the LCAA-CPG.12 Using the free carboxylic acid to load
`the LCAA-CPG marks a departure from previous syn-
`theses of orthogonal solid-phase supports prepared in our
`group, which involved prior activation and isolation of
`the carboxylic acid as the respective trichlorophenyl
`ester.4,7 The synthesis of 3 was accomplished using 13
`as a branching point. Sebacic acid was coupled to 13,
`and the resulting carboxylic acid (15) was loaded onto
`the LCAA-CPG.
`
`Results and Discussion
`Synthesis of Pd(0)-Labile Solid-Phase Oligonu-
`cleotide Synthesis Supports. The general approach
`for the synthesis of 1 was presented previously (Scheme
`1).4 However, the experimental details are described in
`the Experimental Section of this paper. Two Pd(0)-labile
`supports (2, 3) that release oligonucleotides containing
`3¢ -hydroxy termini were designed on the basis of the
`successful utilization of 1. When compared to 2 and
`previously described Pd(0)-labile (1) and photolabile (4-
`6) supports, solid-phase support 3 was designed to
`contain a longer tether between the long chain alkyl-
`amine controlled-pore glass support (LCAA-CPG) and the
`reactive center. This longer tether was introduced in
`order to examine whether increasing the distance be-
`tween the support and the reactive functionality (thereby
`increasing the accessibility of reagents to the Alloc group)
`increased the efficiency of the cleavage reaction.
`Solid-phase support 2 was prepared via coupling the
`chloroformate (11) of the previously reported alcohol (8)
`with the dianion of 5¢ -O-dimethoxytrityl thymidine
`(Scheme 2).11 The major impurity of this reaction
`
`(11) Nicolaou, K. C.; Prasad, C. V. C.; Somers, P. K.; Hwang, C.-K.
`J. Am. Chem. Soc. 1989, 111, 5330.
`(12) Kumar, P.; Sharma, A. K.; Sharma, P.; Garg, B. S.; Gupta, K.
`C. Nucleosides & Nucleotides 1996, 15, 879.
`
`The Effect of Reaction Buffer on the Efficiency
`of Pd(0)-Labile Solid-Phase Supports. Excellent
`yields of undamaged oligonucleotides were obtained from
`1 using n-BuNH2/HCO2H as reaction buffer. However,
`the reaction required 5 h to proceed to completion, and
`the workup of the biphasic Pd(0) reaction mixture was
`made difficult by residual reagents. Consequently, tetra-
`butylammonium formate (TBA) was investigated as an
`alternative buffer system.13 TBA offered several poten-
`tial advantages over n-BuNH2/HCO2H, including mono-
`phasic reaction conditions and a more facile workup. In
`addition, we anticipated that cleavage of the oligonucleo-
`tides from the solid-phase supports might proceed more
`quickly using the tetralkylammonium buffer, by elimi-
`nating the possibility for formation of weak (cid:243)-complexes
`between Pd(0) and the alkylamine in the buffer, which
`reduces the amount of Pd(0) available for reaction.
`Indeed, quantitative yields of 16 were obtained from
`O-methyl phosphate protected polythymidylate in 1 h at
`55 °C using 20 molar equiv of Pd2(dba)3(cid:226)CHCl3, 1,2-
`bis(diphenylphosphino)ethane (DIPHOS, 100 molar equiv),
`and 0.12 M TBA (Table 1). Control experiments carried
`
`(13) Bufalini, J.; Stern, K. H. J. Am. Chem. Soc. 1961, 83, 4362.
`
`

`

`4064 J. Org. Chem., Vol. 63, No. 12, 1998
`
`Greenberg et al.
`
`Isolated Yields of Fully Deprotected
`Table 1.
`Oligonucleotides Obtained via Pd(0)-Mediated Cleavagea
`solid-phase
`reaction
`isolated
`support
`time (min)
`yield (%)d,e
`oligonucleotide
`99 ( 5
`16b
`1
`60
`81 ( 3
`16c
`1
`30
`102 ( 6
`1
`17b
`60
`98 ( 16
`18c
`2
`60
`103 ( 3
`18c
`3
`45
`80 ( 9
`18c
`3
`30
`a All reactions were carried out at 55 °C using 20 molar equiv
`of Pd2(dba)3(cid:226)CHCl3 and 100 molar equiv of DIPHOS relative to
`DNA. The concentration of TBA buffer was 0.12 M. b O-Methyl-
`protected phosphoramidites were used. c O-(cid:226)-Cyanoethyl-protected
`phosphoramidites were used. d Yields reported with a standard
`deviation are the average of at least three separate reactions.
`e Isolated yields are determined via comparison of the isolated yield
`of oligonucleotide obtained via Pd(0) cleavage and subsequent
`NH4OH treatment, versus that obtained via direct NH4OH treat-
`ment of resin bound oligonucleotide from the same oligonucleo-
`tide synthesis.
`
`Table 2. Optimization of Isolated Yields of 18 Obtained
`from 3 via Pd(0)-Mediated Cleavagea,b
`isolated
`molar equiv
`[TBA]
`reaction
`[H2O]
`yield (%)c,d
`of Pd(0)
`(M)
`(M)
`time (min)
`98 ( 2
`40
`0.12
`0.55
`60
`94 ( 1
`40
`0.06
`0.55
`60
`96 ( 7
`8
`0.12
`0.55
`60
`94 ( 5
`8
`0.06
`0.55
`60
`104 ( 4
`8
`0.06
`2.75
`60
`77 ( 9
`4
`0.12
`0.55
`60
`78 ( 3
`4
`0.06
`2.75
`60
`102 ( 1
`4
`0.12
`0.55
`120
`44 ( 5
`2
`0.12
`0.55
`60
`a All reactions were carried out at 55 °C using 2.5 molar equiv
`of DIPHOS relative to Pd(0). b O-(cid:226)-Cyanoethyl-protected phos-
`phoramidites were used to prepare the oligonucleotides. c Yields
`reported with a standard deviation are the average of at least three
`separate reactions. d Isolated yields are determined via comparison
`of the isolated yield of oligonucleotide obtained via Pd(0) cleavage
`and subsequent NH4OH treatment, versus that obtained via direct
`NH4OH treatment of resin bound oligonucleotide from the same
`oligonucleotide synthesis.
`
`Scheme 3
`
`out in the absence of Pd(0) resulted in no detectable
`cleavage of the oligonucleotide. Isolated yields of oligo-
`nucleotides were independent of sequence (e.g. 17).
`However, slightly lower yields were obtained upon short-
`ening the reaction time 30 min. The reaction conditions
`that were successful for 16 and 17 from 1 gave rise to
`quantitative yields of 18 from 2 (Table 1). Unfortunately,
`the solid-phase support containing a longer tether (3)
`resulted in only marginally greater reactivity (Table 1).
`The integrity of the biopolymers, following aminolysis,
`which would result in cleavage at damaged sites (and
`result in less than the observed quantitative yields), were
`established by electrospray mass spectrometry, and/or
`enzymatic digestion, followed by reverse phase HPLC
`analysis of the nucleoside components.14
`Optimization of Pd(0) Cleavage Conditions. Dur-
`ing the course of these studies, it was determined that a
`slight excess of formic acid ((cid:25)0.5 equiv) relative to TBA
`was required in order to obtain quantitative yields of
`cleaved oligonucleotides. No DNA was cleaved from the
`resin in the absence of formic acid, suggesting that a
`proton source is required for a successful reaction.
`Additional acid results in degradation of the biopolymer,
`presumably due to cleavage of the glycosidic bonds. A
`proton source is available in the n-BuNH2/HCO2H buffer
`system used in previous experiments, but not when using
`TBA in the absence of additional formic acid.2,4 We
`suggest that the proton facilitates the insertion of the
`Pd(0) into the carbon-oxygen bond by increasing the
`electrophilicity of the carboxyl group (Scheme 3). How-
`ever, involvement of acid in a later step cannot be ruled
`out.
`The final optimization of the Pd(0)-mediated cleavage
`conditions was carried out using 3. The choice of support
`is arbitrary, as all three of the Pd(0)-labile supports
`
`(14) See Supporting Information.
`
`exhibited comparable reactivity. Although the cost of
`DNA on a molar basis is greater than that of Pd2(dba)3(cid:226)
`CHCl3, we sought to minimize the number of molar
`equivalents of Pd(0) employed in the cleavage reaction.
`While the molar ratio of DIPHOS to Pd2(dba)3(cid:226)CHCl3 was
`maintained at 5, we found that the Pd(0) could be reduced
`to 8 molar equiv relative to support-bound oligonucleo-
`tide without any sacrifice in isolated yield of 18 (Table
`2). However, to obtain quantitative yields of 18 when
`using 2 molar equiv of Pd2(dba)3(cid:226)CHCl3, the reaction time
`had to be increased to 2 h. No adverse consequences in
`the yield of 18 were observed when the concentration of
`TBA was reduced to 60 mM. It is also relevant to note
`that high yields were unobtainable when using TBA as
`a buffer system under scrupulously dry conditions;
`whereas the addition of small amounts of water to the
`reaction mixture resulted in consistently high yields. We
`believe that when TBA is used as reaction buffer, water
`acts as a nucleophile in competition with the initially
`cleaved oligonucleotide (“biting back”) to release the Pd(0)
`from the Ł3-complex (Scheme 3).15 Biting back by leaving
`groups, in this case the 3¢ -alkoxy oligoncleotide, during
`nucleophilic substitution of allylic substrates mediated
`by Pd(0) is not uncommon.16
`Pd(0)-Mediated Cleavage of (cid:226)-Cyanoethyl Phos-
`phate Protecting Groups. 31P NMR experiments on
`
`(15) Upon initial cleavage of the oligonucleotide from 3, it is expected
`that the biopolymer undegoes rapid decarboxylation to produce a 3¢ -
`terminal alkoxide. In the absence of a proton source, this oligonucleo-
`tide is believed to attack the Ł3-complex, resulting in an oligonucleotide
`which is still bound to the solid-phase support.
`(16) Merzouk, A.; Guibe´, F.; Loffet, A. Tetrahedron Lett. 1992, 33,
`477.
`
`

`

`Oligonucleotide Cleavage from Pd(0)-Labile Solid Supports
`
`J. Org. Chem., Vol. 63, No. 12, 1998 4065
`
`Scheme 4
`
`Figure 1. 31P NMR of the reaction of 19 with Pd(0), DIPHOS,
`(bottom) reaction of 19, (middle) reaction
`and TBA in THF:
`of 19, spiked with 20 and 21, (top) reaction of 19, spiked with
`20--22.
`
`19 using catalytic Pd2(dba)3(cid:226)CHCl3 and DIPHOS in TBA
`buffer confirmed the proposal that the (cid:226)-cyanoethyl group
`was cleaved under these Pd(0) conditions (Figure 1).4 Of
`the four possible products that can be formed from 19
`(20-22 and 5¢ -O-dimethoxytritylthymidine), only 20 is
`observed, indicating that cleavage of the (cid:226)-cyanoethyl
`group occurs to the exclusion of the other two phosphate
`triester alkyl groups. (cid:226)-Cyanoethyl cleavage occurs
`regardless of the buffer system used and requires the
`presence of palladium. On the basis of analogies to Pd(0)
`insertion into acyl halides, as well as halides, two possible
`mechanisms for the cleavage of the (cid:226)-cyanoethyl group
`were considered. Although carbon-oxygen insertions
`into carboxylic acid esters do not occur, acid halides do
`react with Pd(0).17,18 We considered the possibility that
`the lower pKa of (cid:226)-cyanoethanol compared to simple
`alcohols increased the reactivity of the phosphate triester
`such that its reactivity was closer to that of an acyl
`halide. Phosphorus-oxygen bond insertion, followed by
`hydrolysis, would yield the phosphate diester and (cid:226)-cy-
`anoethanol. In contrast, carbon-oxygen bond insertion
`(in analogy to reactions of alkyl halides19), followed by
`(cid:226)-elimination, and subsequent reductive elimination
`would release the phosphate diester and acrylonitrile.
`Observation of acrylonitrile by GC/MS, but not (cid:226)-cyano-
`ethanol, leads us to propose that carbon-oxygen inser-
`tion is the pathway by which Pd(0) gives rise to depro-
`tection of the (cid:226)-cyanoethyl group in phosphate triesters
`(Scheme 4).
`Summary. Oligonucleotides can be cleaved from
`solid-phase supports in quantitative yield in 1 h using a
`
`(17) Hegedus, L. S. Transition Metals in the Synthesis of Complex
`Organic Molecules, University Science Books: Mill Valley, CA, 1994.
`(18) Labadie, J. W.; Stille, J. K. J. Am. Chem. Soc. 1983, 105, 669.
`(19) Canty, A. J. In Comprehensive Organometallic Chemistry II;
`Puddephatt, R. J., Ed.; Pergammon Press: Oxford, 1995; Vol 9.
`
`slight excess of the less costly Pd(0) reagent. When
`carried out in tetrabutylammonium formate buffer, the
`reaction needs a small amount of water and acid in order
`to obtain reproducible quantitative yields of product. In
`addition, the previously observed cleavage of (cid:226)-cyanoethyl
`groups from phosphate triesters by Pd(0) is believed to
`occur via initial carbon-oxygen bond insertion, followed
`by elimination of acrylonitrile. This facile process sug-
`gests that the (cid:226)-cyanoethyl group could be employed as
`an alternative to the Alloc protecting group.
`
`Experimental Section
`General Methods. 1H NMR spectra were obtained at 270
`or 300 MHz. 13C and 31P NMR spectra were obtained at the
`respective frequencies using the same spectrometers; 31P NMR
`spectra were referenced against external phosphoric acid.
`Reverse phase HPLC analysis utilized a Rainin Microsorb-
`MV C18 (5 (cid:237)m) column. GC/MS was carried out on a DB1 fused
`silica capillary column. All reactions were carried out in oven-
`dried glassware, under a nitrogen atmosphere, unless other-
`wise stated. THF was distilled from Na0/benzophenone ketyl.
`Pyridine, DMF, CH3CN, 1,2-dichloroethane, acetic anhydride,
`and CH2Cl2 were distilled from CaH2.
`Oligonucleotides were synthesized using standard cycles,
`commercially available reagents, and the solid-phase supports
`described above. Commercially available DNA synthesis
`reagents were purchased from Glen Research (Sterling, VA).
`Deprotection of standard oligonucleotide ((cid:226)-cyanoethyl or
`methyl protected) was carried out in concentrated NH4OH at
`55 °C for 12 h. All oligonucleotides were purified via 20%
`polyacrylamide denaturing gels [(20 (cid:2) 40 (cid:2) 0.1 cm), 5% cross-
`link, 45% urea (by weight)]. Oligonucleotides were visualized
`using 254 nm light. Bands were excised and eluted with a
`solution of NaCl (0.2 M) and EDTA (1 mM), filtered through
`Quick Sep filters desalted on C18 Sep-Pak cartridges. Oligo-
`nucleotides were quantitated by UV absorption at 260 nm.
`Molar extinction coefficients were calculated using the nearest
`neighbor method.20
`Preparation of 7. 4,4¢ -Dimethoxytrityl chloride (5.0 g,
`14.76 mmol) and 1,4-butanediol (6.41 g, 71.1 mmol) were
`stirred in pyridine (50 mL) at 0 °C overnight. After removal
`of the pyridine and excess alcohol in vacuo, the residue was
`taken up in diethyl ether (100 mL), washed with H2O (25 mL)
`and brine (25 mL), and then dried over MgSO4. Flash
`chromatography (hexanes:EtOAc 2:1 to 1:3) yielded 4.9 g (84%)
`of the dimethoxytritylated alcohol as a colorless oil: 1H NMR
`(CDCl3) (cid:228) 7.41 (d, 2H, J ) 8 Hz), 7.24 (m, 6H), 7.18 (m, 1H),
`6.80 (d, 4H, J ) 9 Hz), 3.76 (s, 6H), 3.61 (t, 2H, J ) 6 Hz),
`3.09 (t, 2H, J ) 6 Hz), 2.99 (bd s, 1H), 1.66 (m, 4H); IR (film)
`3357, 2934, 2868, 2835, 1607, 1508, 1463, 1445, 1301, 1249,
`1176, 1034 cm-1.
`Pyridinium dichromate (14.2 g, 37.71 mmol) and the above
`alcohol (3.7 g, 9.43 mmol) were stirred in DMF (50 mL) for 18
`
`(20) Borer, P. N.; In Handbook of Biochemistry and Molecular
`Biology; Fasman, G. D.; CRC Press: Boca Raton, FL, 1975; p 589.
`
`

`

`4066 J. Org. Chem., Vol. 63, No. 12, 1998
`
`Greenberg et al.
`
`h at room temperature. The mixture was poured into H2O
`(350 mL) and extracted with diethyl ether (5 (cid:2) 100 mL). The
`combined organic layers were washed with brine (75 mL) and
`dried over Na2SO4. Flash chromatography (EtOAc:hexanes
`1:2) yielded 1.62 g (42%) of 7 as a light yellow oil: 1H NMR
`(CDCl3) (cid:228) 7.45 (d, 2H, J ) 9 Hz), 7.31 (m, 6H), 7.22 (m, 1H),
`6.82 (d, 4H, J ) 9 Hz), 3.78 (s, 6H), 3.14 (t, 2H, J ) 6 Hz),
`2.50 (t, 2H, J ) 7.5 Hz), 1.94 (tt, 2H, J ) 6, 7.5 Hz); 13C NMR
`(CDCl3) (cid:228) 179.8, 158.3, 145.1, 136.3, 130.0, 128.1, 127.7, 126.6,
`113.0, 85.9, 62.1, 55.1, 31.2, 25.1; IR (film) 3600, 2932, 2835,
`1707, 1608, 1509, 1445, 1300, 1250, 1176, 1075, 1035 cm-1.
`Preparation of 9. Silyl alcohol 811 (530 mg, 2.17 mmol)
`and dimethoxytrityl carboxylic acid 7 (1.10 g, 2.71 mmol) were
`combined with DCC (582 mg, 2.82 mmol) and DMAP (26 mg,
`0.22 mmol) in CH2Cl2 (17 mL) at 0 °C. The solution was
`filtered after allowing the reaction to stir and warm to room
`temperature over 3 h. The filter cake was washed with
`CH2Cl2. After removal of the solvents in vacuo, flash chro-
`matography (Et2O:hexanes 1:4) yielded 1.27 g (92.5%) of 9 as
`a colorless oil: 1H NMR (CDCl3) (cid:228) 7.42 (d, 2H, J ) 9 Hz), 7.24
`(m, 7H), 6.81 (d, 4H, J ) 9 Hz), 5.73 (dt, 1H, J ) 15, 7 Hz),
`5.53 (dt, 1H, J ) 15, 6 Hz), 4.48 (dd, 2H, J ) 1, 6 Hz), 3.77 (s,
`6H), 3.58 (dd, 2H, J ) 3, 6 Hz), 3.09 (t, 2H, J ) 6 Hz), 2.44 (t,
`2H, J ) 7.5 Hz), 2.04 (m, 2H), 1.91 (m, 2H), 1.47 (m, 4H), 0.89
`(s, 9H), 0.04 (s, 6H); 13C NMR (CDCl3) (cid:228) 173.3, 158.3, 145.2.
`136.4, 136.2, 130.0, 128.1, 127.7, 126.6, 124.0, 113.0, 85.8, 65.1,
`62.9, 62.3, 55.1, 32.3, 32.0, 31.5, 25.9, 25.4, 25.1, 18.3, -5.3;
`IR (film) 2930, 2857, 1735, 1608, 1509, 1463, 1445, 1301, 1251,
`1175, 10907, 1037 cm-1.
`Preparation of 10. An equimolar mixture of TBAF and
`HOAc (0.5 M; 24.4 mL) in THF was added to 9 (1.0 g, 1.58
`mmol) in THF (10 mL) at 0 °C. After the mixture was allowed
`to warm to room temperature and stir overnight, the reaction
`was poured into saturated NaHCO3 (25 mL) and extracted
`with Et2O (2 (cid:2) 75 mL). The combined organic layers were
`washed with brine (25 mL) and dried over MgSO4. Flash
`chromatography (EtOAc:hexanes 1:2) yielded 817 mg (100%)
`of the desilylated product: 1H NMR (CDCl3) (cid:228) 7.40 (d, 2H, J
`) 9 Hz), 7.24 (m, 7H), 6.79 (d, 4H, J ) 9 Hz), 5.72 (dt, 1H, J
`) 15, 6.5 Hz), 5.53 (dt, 1H, J ) 15, 6 Hz), 4.48 (dd, 2H, J ) 1,
`6 Hz), 3.76 (s, 6H), 3.61 (t, 2H, J ) 6 Hz), 3.08 (t, 2H, J ) 6
`Hz), 2.44 (t, 2H, J ) 7.5 Hz), 2.06 (m, 2H), 1.91 (m, 2H), 1.51
`(m, 5H); 13C NMR (CDCl3) (cid:228) 173.4, 158.3, 145.1, 136.4, 135.8,
`130.0, 128.1, 127.7, 126.6, 124.2, 112.9, 85.8, 65.0, 62.6, 62.3,
`55.1, 32.1, 31.9, 31.5, 25.4, 25.0; IR (film) 3400, 2933, 1733,
`1608, 1509, 1446, 1301, 1250, 1175, 1073, 1034 cm-1.
`PDC (2.03 g, 5.41 mmol) and the primary alcohol obtained
`above (800 mg, 1.55 mmol) were stirred at room temperature
`in DMF (12 mL) for 12 h. The solution was poured into H2O
`(100 mL) and extracted with Et2O (3 (cid:2) 100 mL). The
`combined organics were washed with brine (50 mL) and dried
`over MgSO4. Flash chromatography (EtOAc:hexanes 1:2)
`yielded 592 mg (72%) of 10: 1H NMR (CDCl3) (cid:228) 7.40 (d, 2H, J
`) 9 Hz), 7.23 (m, 7H), 6.80 (d, 4H, J ) 9 Hz), 5.69 (dt, 1H, J
`) 6.5, 15 Hz), 5.55 (dt, 1H, J ) 6, 15 Hz), 4.49 (d, 2H, J ) 6
`Hz), 3.77 (s, 6H), 3.08 (t, 2H, J ) 6 Hz), 2.44 (t, 2H, J ) 7.5
`Hz), 2.33 (t, 2H, J ) 7.5 Hz), 2.08 (m, 2H), 1.91 (m, 2H), 1.71
`(m, 2H); 13C NMR (CDCl3) (cid:228) 179.1, 173.4, 158.3, 145.1, 136.4,
`134.6, 130.0, 128.1, 127.7, 126.6, 125.1, 113.0, 85.8, 64.9, 62.3,
`55.2, 33.2, 31.5, 31.4, 25.4, 23.7; IR (film) 3340, 2934, 2836,
`1733, 1707, 1608, 1509, 1445, 1301, 1250, 1175, 1034 cm-1.
`Preparation of 1. 2,4,5-Trichlorophenol (260 mg, 1.32
`mmol), DCC (272 mg, 1.32 mmol), 10 (560 mg, 1.05 mmol),
`and DMAP (13 mg, 0.11 mmol) were combined in CH2Cl2 (13
`mL) at 0 °C. After warming to room temperature and stirring
`for 12 h, the reaction mixture was poured into H2O (20 mL)
`and extracted with CH2Cl2 (3 (cid:2) 20 mL). The combined organic
`layers were dried over Na2SO4 and concentrated in vacuo.
`Flash chromatography (EtOAc:hexanes 1:8) yielded 624 mg
`(83%) of the requisite trichlorophenyl ester as a colorless oil:
`1H NMR (CDCl3) (cid:228) 7.52 (s, 1H), 7.42 (d, 2H, J ) 9 Hz), 7.24
`(m, 8H), 6.81 (d, 4H, J ) 9 Hz), 5.72 (dt, 1H, J ) 6, 15 Hz),
`5.58 (dt, 1H, J ) 6, 15 Hz), 4.50 (d, 2H, J ) 6 Hz), 3.77 (s,
`6H), 3.08 (t, 2H, J ) 6 Hz), 2.59 (t, 2H, J ) 7 Hz), 2.45 (t, 2H,
`J ) 7.5 Hz), 2.16 (m, 2H), 1.87 (m, 4H); 13C NMR (CDCl3) (cid:228)
`
`173.3, 170.4, 158.3, 145.8, 145.1, 136.4, 134.2, 131.4, 131.0,
`130.5, 129.9, 128.1, 127.7, 126.6, 126.1, 125.5, 125.3, 113.0,
`85.8, 64.8, 62.3, 55.2, 33.0, 31.5, 31.3, 25.4, 23.8; IR (film) 2934,
`2836, 1774, 1732, 1608, 1582, 1510, 1462, 1350, 1302, 1250,
`1175, 1105, 1081, 1035 cm-1; HRMS FAB (M+) calcd 710.1605,
`found 710.1595.
`A mixture of the trichlorophenyl ester (50 mg, 70 (cid:237)mol),
`LCAA-CPG (100 mg, (cid:25)5 (cid:237)mol amine), and HOBt(cid:226)hydrate (9.5
`mg, 70 (cid:237)mol) was shaken overnight in the dark at 25 °C using
`a vortexer. The resin was filtered, washed well with dry
`EtOAc, and dried under vacuum. Unreacted amine was
`capped by treatment with acetic anhydride (250 (cid:237)L), pyridine
`(2 mL), and DMAP (25 mg) for 1 h. The resin was filtered,
`washed, and dried as described above. Free amine was
`measured on 1 mg of resin (1) via quantitative ninhydrin
`analysis.21 Resin loading was measured by treatment with
`p-toluenesulfonic acid in CH3CN and quantitation of the
`dimethoxytrityl cation by absorption spectroscopy ((cid:236)max ) 498
`nm, (cid:15) ) 7 (cid:2) 104 M-1 cm-1).
`Preparation of 12. Phosgene (1.9 M in toluene, 3.1 mL)
`was added via syringe to 8 (250 mg, 1.0 mmol) in THF (2 mL).
`After the reaction mixture was stirred for 3 h, N2 was bubbled
`through the solution for 1 h to remove excess phosgene. After
`removal of the solvent in vacuo, an aliquot of the crude product
`(11) was analyzed by IR and 1H NMR (CDCl3); IR showed one
`carbonyl stretch at 1778 cm-1. 1H NMR showed a shift of the
`allylic alcohol methylene protons from 4.07 to 4.69 ppm. The
`above analytical methods indicated that the reaction had gone
`to completion. The sodium alkoxide salt of 5¢ -O-(4,4¢ -dimethoxy-
`trityl)thymidine [prepared by addition of sodium hydride (150
`mg, 3.75 mmol) to 5¢ -O-(4,4¢ -dimethoxytrityl)thymidine dis-
`solved in THF (6 mL) (820 mg, 1.5 mmol)] in THF (6 mL) was
`added, and the mixture was stirred under N2 for 2 h at 25 °C.
`The reaction was diluted with EtOAc (40 mL) and poured into
`H2O (100 mL). The aqueous layer was extracted with EtOAc
`(3 (cid:2) 40 mL). The combined organic layers were washed with
`brine (2 (cid:2) 100 mL) and dried over Na2SO4. Flash chroma-
`tography (EtOAc:hexanes 1:2) yielded 12 as a white foam (391
`mg, 48%): 1H NMR (CDCl3) (cid:228) 8.01 (s, 1H), 7.58 (s, 1H), 7.37-
`7.21 (m, 9H), 6.82 (d, 4H, J ) 9 Hz), 6.44-6.40 (m, 1H), 5.81
`(dt, 1H, J ) 6.6, 15 Hz), 5.55 (dt, 1H, J ) 6.3, 15 Hz), 5.32 (d,
`1H, J ) 6 Hz), 4.55 (d, 2H, J ) 6.6 Hz), 4.20 (s, 1H), 4.75 (s,
`6H), 3.58 (t, 2H, J ) 6 Hz), 3.55-3.40 (m, 2H), 2.57-2.37 (m,
`2H), 2.10-2.03 (m, 2H), 1.58-1.38 (m, 4H), 0.86 (s, 9H), 0.01
`(s, 6H); IR (film) 2930, 1744, 1696, 1607, 1508, 1458, 1253,
`1176, 1103, 972 cm-1. Anal. Calcd for C45H58N2O10Si: C,
`66.31; H, 7.17; N, 3.49. Found: C, 66.55; H, 6.97; N, 3.42.
`Preparation of 13. An equimolar solution of glacial acetic
`acid and TBAF in THF (0.5 M, 1.5 mL) was added to a solution
`of 12 (150 mg, 0.18 mmol) in THF (5 mL). After 24 h an
`additional 0.3 mL of the buffered TBAF solution was added.
`After 12 additional hours, the reaction was diluted with EtOAc
`(50 mL), washed with brine (100 mL), and dried over Na2SO4.
`Flash chromatography (EtOAc:hexanes 1:1) yielded 13 as a
`white foam (135 mg, 92%): 1H NMR (CDCl3) (cid:228) 8.05 (s, 1H),
`7.56 (s, 1H), 7.35-7.19 (m, 9H), 6.80 (d, 4H, J ) 9 Hz), 6.41-
`6.36 (m, 1H), 5.79 (dt, 1H, J ) 6, 14.5 Hz), 5.54 (dt, 1H, J )
`7, 14 Hz), 4.53 (d, 2H, J ) 7.5 Hz), 4.18 (s, 1H), 3.74 (s, 6H),
`3.64-3.56 (m, 2H), 3.50-3.35 (m, 2H), 2.55-2.34 (m, 2H),
`2.10-2.02 (m, 2H), 1.50-1.38 (m, 4H), 1.33 (s, 3H); IR (film)
`3464, 3060, 2932, 1743, 1692, 1607, 1582, 1509, 1252, 1202,
`1177, 1153, 1066, 973 cm-1. Anal. Calcd for C39H44N2O10: C,
`66.84; H, 6.33; N, 4.00. Found: C, 66.72; H, 6.32; N, 3.81.
`Preparation of 14. PDC (210 mg, 0.56 mmol) was added
`to a solution of 13 (100 mg, 0.14 mmol) in DMF (1.3 mL). The
`reaction was allowed to stir for 15 h at 25 °C, after which the
`solution was poured into H2O (50 mL) and extracted with Et2O
`(3 (cid:2) 30 mL). The combined organic layers were washed with
`saturated NaHCO3 (50 mL) and then concentrated in vacuo.
`The residue was purified by flash chromatography (EtOAc:
`hexanes 3:2) to give 14 (58 mg, 57%) as a foam:
`1H NMR
`
`(21) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal.
`Biochem. 1981, 117, 147.
`
`

`

`Oligonucleotide Cleavage from Pd(0)-Labile Solid Supports
`
`J. Org. Chem., Vol. 63, No. 12, 1998 4067
`
`(CDCl3) (cid:228) 9.19 (bd s, 1H), 7.57 (s, 1H), 7.35-7.18 (m, 9H),
`6.82-6.77 (d, 4H, J ) 9 Hz), 6.42-6.37 (m, 1H), 5.81-5.71
`(m, 1H), 5.61-5.52 (m, 1H), 5.29 (d, 1H, J ) 3.3 Hz), 4.59-
`4.46 (m, 2H), 4.19 (bd s, 1H), 3.74 (s, 6H), 3.51-3.36 (m, 2H),
`2.56-2.29 (m, 4H), 2.14-2.05 (m, 2H), 1.76-1.66 (m, 2H), 1.33
`(s, 3H); IR (film) 3172, 3035, 2930, 1736, 1702, 1552, 1509,
`1462, 1252, 1202, 1070, 1033, 912, 703 cm-1. Anal. Calcd for
`C39H42N2O11: C, 65.54; H, 5.92; N, 3.92. Found: C, 65.38; H,
`6.10; N, 3.70.
`Preparation of 15. DCC (30 mg, 0.14 mmol) was added
`to a solution of 13 (100 mg, 0.14 mmol), sebacic acid (113 mg,
`0.56 mmol), and DMAP (9 mg, 0.07 mmol), in THF (1 mL).
`The solution was allowed to stir at room temperature for 6 h,
`at which time the reaction mixture was concentrated in vacuo,
`to give a white solid. The solid was resuspended in EtOAc
`(50 mL) and washed with H2O (50 mL) and brine (3 (cid:2) 50 mL).
`After drying over Na2SO4, flash chromatography (CH2Cl2:
`MeOH; 19:1) gave 15 (115 mg, 93%) as a white foam: 1H NMR
`(CDCl3) (cid:228) 9.12 (s, 1H), 7.57 (s, 1H), 7.35-7.17 (m, 9H), 6.79
`(d, 4H, J ) 9 Hz), 6.42-6.37 (m, 1H), 5.78 (dt, 1H, J ) 6.6, 15
`Hz), 5.55 (dt, 1H, J ) 6.5, 15 Hz), 5.28 (d, 1H, J ) 5.7 Hz),
`4.60-4.46 (m, 2H), 4.18 (s, 1H), 4.01 (t, 2H, J ) 6.3 Hz), 3.75
`(s, 6H), 3.50-3.37 (m, 2H), 2.55-2.33 (m, 2H), 2.30 (t, 2H, J
`) 7.2 Hz), 2.24 (t, 2H, J ) 7.5 Hz), 2.10-2.00 (m, 2H), 1.63-
`1.51 (m, 6H), 1.47-1.38 (m, 2H), 1.33 (s, 3H), 1.32-1.19 (m,
`8H); IR (film) 3182, 3036, 2932, 1738, 1731, 1713, 1704, 1674,
`1607, 1557, 1510, 1455, 1252, 1177, 1068, 1034, 973 cm-1.
`Anal. Calcd for C49H61N2O11: C, 65.32; H, 6.82; N, 3.11.
`Found: C, 65.37; H, 6.84; N, 3.05.
`Example of Loading of Free Carboxylic Acids onto
`LCAA-CPG Supports.12 To a small glass vial containing 14
`(5 mg, 7 (cid:237)mol) was added DMAP (1 mg, 8.1 (cid:237)mol) in CH3CN
`(200 (cid:237)mol), 2,2¢ -dipyridyl disulfide (2.3 mg, 8.1 (cid:237)mol) in a 1:1
`1,2-dichloroethane:CH3CN (100 (cid:237)L) solution, PPh3 (2.1 mg, 8.1
`(cid:237)mol) in CH3CN (100 (cid:237)L), and LCAA-CPG (50 mg, 2.5 (cid:237)mol
`of amines). The vial was shaken for 1 h, after which the resin
`was filtered and washed with EtOAc (10 mL), CH3CN (10 mL),
`and Et2O (10 mL). After air-drying, the resin was added to
`DMAP (9 mg, 0.073 mmol) and acetic anhydride (215 mg, 2.1
`mmol) dissolved in dry pyridine (2 mL). After filtration and
`drying, it was determined (via trityl assay) that the resin was
`loaded to 44 (cid:237)mol/g. Typical loadings ranged from 40 to 50
`(cid:237)mol/g.
`Preparation of 19. To thymidine (cid:226)-cyanoethyl phosphor-
`amidite (100 mg, 0.13 mmol) stirring in CH3CN (3 mL) were
`added a 0.1 M tetrazole solution (5 mL, 0.5 mmol) and n-propyl
`alcohol (0.5 mL, 8.3 mmol). The reaction was allowed to stir
`at room temperature for 30 min when TLC analysis (MeOH:
`CH2Cl2; 1:9) indicated that the starting amidite was completely
`consumed. To the reaction mixture was then added dropwise
`a 1.0 M solution of I2 in THF:2,6-lutidine:H2O (2:2:1), until an
`orange color persisted. The excess I2 in solution was then
`treated with Na2S2O3 solution (5 mol %), until