Optimization and Mechanistic Analysis of Oligonucleotide Cleavage from Palladium-Labile Solid-Phase Synthesis Supports

Article abstract of DOI:10.1021/jo980135b

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)₃3·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 η³-complex, presumably preventing back biting by the initially released oligonucleotide, are required to obtain reproducibly high yields of cleaved oligonucleotides during a 1 h reaction. The previously observed lability of β-cyanoethyl groups to the Pd(0) conditions has been examined using a mononucleotide substrate. Cleavage of the β-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 β-cyanoethyl group is suggested to account for the cleavage of this group.

Full text of DOI:10.1021/jo980135b

4062
J . Org. Chem. 1998, 63, 4062-4068
Op tim iza tion a n d Mech a n istic An a lysis of Oligon u cleotid e
Clea va ge fr om P a lla d iu m -La bile Solid -P h a se Syn th esis Su p p or ts1
Marc M. Greenberg,* Tracy J . Matray, J effrey D. Kahl, Dong J in Yoo, and Dustin L. McMinn
Department of Chemistry, Colorado State University Fort Collins, Colorado 80523
Received J anuary 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‚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 η3-complex, presumably preventing back biting by the initially released oligonucleotide, are
required to obtain reproducibly high yields of cleaved oligonucleotides during a 1 h reaction. The
previously observed lability of â-cyanoethyl groups to the Pd(0) conditions has been examined using
a mononucleotide substrate. Cleavage of the â-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 â-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.
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
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.; J ones, 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.
(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.; J acobs,
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.
(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.
S0022-3263(98)00135-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/27/1998

Oligonucleotide Cleavage from Pd(0)-Labile Solid Supports
J . Org. Chem., Vol. 63, No. 12, 1998 4063
Sch em e 1a
Sch em e 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.
Resu lts a n d Discu ssion
Syn th esis of P d (0)-La bile Solid -P h a se Oligon u -
cleotid e Syn th esis Su p p or ts. 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.
Th e Effect of Rea ction Bu ffer on th e Efficien cy
of P d (0)-La bile Solid -P h a se Su p p or ts. 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 σ-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‚CHCl3, 1,2-
bis(diphenylphosphino)ethane (DIPHOS, 100 molar equiv),
and 0.12 M TBA (Table 1). Control experiments carried
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.
(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.
Ta ble 1. Isola ted Yield s of F u lly Dep r otected
Ta ble 2. Op tim iza tion of Isola ted Yield s of 18 Obta in ed
fr om 3 via P d (0)-Med ia ted Clea va gea ,b
Oligon u cleotid es Obta in ed via P d (0)-Med ia ted Clea va gea
solid-phase
support
reaction
time (min)
isolated
molar equiv
of Pd(0)
[TBA]
(M)
[H2O]
(M)
reaction
time (min)
isolated
oligonucleotide
yield (%)d,e
yield (%)c,d
16b
16c
17b
18c
18c
18c
1
1
1
2
3
3
60
30
60
60
45
30
99 ( 5
81 ( 3
102 ( 6
98 ( 16
103 ( 3
80 ( 9
40
40
8
8
8
4
4
4
2
0.12
0.06
0.12
0.06
0.06
0.12
0.06
0.12
0.12
0.55
0.55
0.55
0.55
2.75
0.55
2.75
0.55
0.55
60
60
60
60
60
60
60
120
60
98 ( 2
94 ( 1
96 ( 7
94 ( 5
104 ( 4
77 ( 9
78 ( 3
102 ( 1
44 ( 5
a
All reactions were carried out at 55 °C using 20 molar equiv
of Pd2(dba)3‚CHCl3 and 100 molar equiv of DIPHOS relative to
b
DNA. The concentration of TBA buffer was 0.12 M. O-Methyl-
a
protected phosphoramidites were used. c O-â-Cyanoethyl-protected
All reactions were carried out at 55 °C using 2.5 molar equiv
d
b
phosphoramidites were used. Yields reported with a standard
of DIPHOS relative to Pd(0). O-â-Cyanoethyl-protected phos-
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.
phoramidites were used to prepare the oligonucleotides. c Yields
reported with a standard deviation are the average of at least three
d
separate reactions. 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.
Sch em e 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
Op tim iza tion of P d (0) Clea va ge Con d ition s. Dur-
ing the course of these studies, it was determined that a
slight excess of formic acid (≈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.
exhibited comparable reactivity. Although the cost of
DNA on a molar basis is greater than that of Pd2(dba)3‚
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‚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‚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 η3-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
P d (0)-Med ia ted Clea va ge of â-Cya n oeth yl P h os-
p h a te P r otectin g Gr ou p s. 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 η3-complex, resulting in an oligonucleotide
which is still bound to the solid-phase support.
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
(16) Merzouk, A.; Guibe´, F.; Loffet, A. Tetrahedron Lett. 1992, 33,
477.
(14) See Supporting Information.

Oligonucleotide Cleavage from Pd(0)-Labile Solid Supports
J . Org. Chem., Vol. 63, No. 12, 1998 4065
Sch em e 4
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 â-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 â-cyanoethyl group could be employed as
an alternative to the Alloc protecting group.
F igu r e 1. 31P NMR of the reaction of 19 with Pd(0), DIPHOS,
and TBA in THF: (bottom) reaction of 19, (middle) reaction
of 19, spiked with 20 and 21, (top) reaction of 19, spiked with
20--22.
Exp er im en ta l Section
Gen er a l Meth od s. 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 µm) 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 (â-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 × 40 × 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
19 using catalytic Pd2(dba)3‚CHCl3 and DIPHOS in TBA
buffer confirmed the proposal that the â-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 â-cyanoethyl
group occurs to the exclusion of the other two phosphate
triester alkyl groups. â-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 â-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 â-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 â-cy-
anoethanol. In contrast, carbon-oxygen bond insertion
(in analogy to reactions of alkyl halides19), followed by
â-elimination, and subsequent reductive elimination
would release the phosphate diester and acrylonitrile.
Observation of acrylonitrile by GC/MS, but not â-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 â-cyanoethyl group in phosphate triesters
(Scheme 4).
P r ep a r a tion 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) δ 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,
Su m m a r y. Oligonucleotides can be cleaved from
solid-phase supports in quantitative yield in 1 h using a
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
(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.
(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 × 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) δ 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) δ 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,
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 µmol),
LCAA-CPG (100 mg, ≈5 µmol amine), and HOBt‚hydrate (9.5
mg, 70 µmol) 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 µL), 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 (λmax ) 498
nm, ꢀ ) 7 × 104 M-1 cm-1).
1707, 1608, 1509, 1445, 1300, 1250, 1176, 1075, 1035 cm-1
.
P r ep a r a tion 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) δ 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) δ 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,
P r ep a r a tion 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
1
(11) was analyzed by IR and H 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 × 40 mL). The combined organic layers were washed with
brine (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) δ 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,
1175, 10907, 1037 cm-1
.
P r ep a r a tion 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 × 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) δ 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) δ 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
.
1176, 1103, 972 cm-1
. Anal. Calcd for C45H58N2O10Si: C,
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 × 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) δ 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) δ 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,
66.31; H, 7.17; N, 3.49. Found: C, 66.55; H, 6.97; N, 3.42.
P r ep a r a tion 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) δ 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.
P r ep a r a tion 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 × 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
1733, 1707, 1608, 1509, 1445, 1301, 1250, 1175, 1034 cm-1
.
P r ep a r a tion 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 × 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) δ 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) δ
(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) δ 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
portion of 1.0 N NaOH solution (10 µL) was added. TLC
analysis after an additional 30 min indicated that starting
material had been completely consumed. The reaction mixture
was then poured into a separatory funnel containing CH2Cl2
(5 mL) and H2O (10 mL). The layers were separated, and the
H2O layer was extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were then washed with brine, dried
over MgSO4, and concentrated in vacuo. Flash chromatogra-
phy (MeOH:EtOAc 1:4) yielded 65.1 mg (99%) of the 20: 1H
NMR (CD3OD) δ 7.65 (s, 1H), 7.42 (d, 2H, J ) 7.2 Hz), 7.30
(m, 7H), 6.86 (d, 4H, J ) 8.7 Hz), 6.33 (dd, 1H, J ) 8.1, 5.7
Hz), 4.99 (d, 1H, J ) 6.3 Hz), 4.24 (m, 1H), 3.76 (s, 6H), 3.72
(m, 2H), 3.42 (m, 2H), 2.50 (m, 2H), 1.52 (m, 2H), 1.31 (s, 3H),
0.87 (t, 3H, J ) 7.4 Hz); 13C NMR (CD3OD) δ 169.0, 160.5,
154.0, 146.2, 137.6, 137.0, 131.7, 129.7, 129.1, 128.3, 114.0,
112.1, 88.4, 86.9, 86.2, 77.4, 69.0, 68.4, 65.3, 55.9, 40.6, 26.7,
25.3, 12.5, 10.9; 31P NMR (CD3OD) δ -0.86; IR (film) 3410,
2962, 1690, 1607, 1508, 1464, 1250, 1176, 1074, 1033, 830
cm-1; HRMS (FAB) calcd 666.2342 (M + H), found 666.2369.
P r ep a r a tion of 21. To thymidine â-cyanoethyl phosphor-
amidite (100 mg, 0.13 mmol) stirring in CH3CN (3 mL) was
added 0.1 M tetrazole solution (1.5 mL, 0.15 mmol). The
reaction was stirred at room temperature for 30 min when TLC
analysis (MeOH:CH2Cl2 1:4) indicated that the starting amid-
ite 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 consumed by dropwise addition of Na2S2O3
solution (5 mol %) until the solution became clear. The
contents of the flask were then poured into a separatory funnel
containing CH2Cl2 (3 × 15 mL), and the combined organic
layers were dried over MgSO4 and concentrated in vacuo.
Flash chromatography (MeOH:EtOAc 3:7) afforded 72.1 mg
(79%) of 21 as a mixture of salts. The mixture of salts was
passed through a cation exchange column (Dowex, Na+ cation-
exchange resin), affording predominantly the sodium salt of
21: 1H NMR (CD3OD) δ 7.68 (s, 1H), 7.42 (d, 2H, J ) 7.2 Hz),
7.25 (m, 7H), 6.86 (d, 4H, J ) 8.4 Hz), 6.39 (dd, 1H, J ) 7.8,
5.7 Hz), 5.05 (t, 1H, J ) 6.3 Hz), 4.26 (m, 1H), 3.95 (m, 2H),
3.76 (s, 6H), 3.46 (m, 2H), 2.70 (t, 2H, J ) 6.3 Hz), 2.55 (m,
2H), 1.32 (s, 3H); 13C NMR (CD3OD) δ 166.4, 160.5, 152.4,
146.1, 137.8, 136.9, 131.6, 129.7, 129.1, 128.3, 119.4, 114.4,
112.0, 88.4, 86.8, 86.1, 77.6, 65.2, 61.8, 55.9, 40.5, 20.5, 12.1;
31P NMR (CD3OD) δ -1.16; IR (film) 2956, 2835, 2359, 2341,
C
39H42N2O11: C, 65.54; H, 5.92; N, 3.92. Found: C, 65.38; H,
6.10; N, 3.70.
P r ep a r a tion 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 × 50 mL).
After drying over Na2SO4, flash chromatography (CH2Cl2:
MeOH; 19:1) gave 15 (115 mg, 93%) as a white foam: 1H NMR
(CDCl3) δ 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.
.
:
Exa m p le of Loa d in g of F r ee Ca r boxylic Acid s on to
LCAA-CP G Su p p or ts.12 To a small glass vial containing 14
(5 mg, 7 µmol) was added DMAP (1 mg, 8.1 µmol) in CH3CN
(200 µmol), 2,2′-dipyridyl disulfide (2.3 mg, 8.1 µmol) in a 1:1
1,2-dichloroethane:CH3CN (100 µL) solution, PPh3 (2.1 mg, 8.1
µmol) in CH3CN (100 µL), and LCAA-CPG (50 mg, 2.5 µmol
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 µmol/g. Typical loadings ranged from 40 to 50
µmol/g.
P r ep a r a tion of 19. To thymidine â-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 the solution
became clear. The contents of the flask were then poured into
a separatory funnel containing CH2Cl2 (15 mL) and saturated
NaHCO3 (15 mL). The aqueous layer was extracted with
CH2Cl2 (3 × 15 mL), and the combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo to afford
crude product as a white foam. Flash chromatography (EtOAc:
hexanes 3:1) afforded 19 as a white foam (76 mg, 76%): 1H
NMR (CD3OD) δ 7.63 (s, 1H), 7.41 (d, 2H, J ) 7.2 Hz), 7.27
(m, 7H), 6.87 (d, 4H, J ) 8.4 Hz), 6.33 (t, 1H, J ) 6.6 Hz),
5.17 (m, 1H), 4.27 (m, 1H), 4.21 (m, 2H), 4.03 (m, 2H), 3.77 (s,
6H), 3.46 (m, 2H), 2.82 (p, 2H, J ) 5.7 Hz), 2.59 (m, 2H), 1.66
(m, 2H), 1.40 (s, 3H), 0.94 (m, 3H); 13C NMR (CD3OD) δ 166.3,
160.5, 152.4, 146.0, 137.5, 136.8, 131.6, 129.6, 129.2, 128.4,
118.6, 114.5, 112.2, 88.6, 86.1, 85.9, 80.4, 71.9, 64.5, 64.2, 55.9,
39.9, 24.8, 20.3, 12.2, 10.5; 31P NMR (CD3OD) δ -1.17, -1.46;
IR (film) 3177, 3057, 2965, 2360, 1690, 1607, 1509, 1464, 1251,
1177, 1029, 828 cm-1; HRMS (FAB) calcd 720.2686 (M + H),
found 720.2664.
1692, 1607, 1508, 1467, 1250, 1176, 1075, 1032, 829 cm-1
HRMS (FAB) calcd 677.2138 (M), found 677.2196.
;
P r ep a r a tion of 22. To 21 (67 mg, 0.1 mmol) stirring in
MeOH (2 mL) at 55 °C was added 1.0 N NaOH (0.1 mL). The
reaction was stirred for 1 h, with monitoring by TLC (MeOH:
CH2Cl2 1:1) every 15 min. After 1 h, an additional portion of
1.0 N NaOH solution (10 µL) was added. After an additional
2 h, TLC indicated that the starting material had been
completely consumed. The reaction mixture was concentrated
in vacuo to afford 64 mg (106%) of a white film as the desired
product in >95% purity according to 1H NMR: 1H NMR
(CD3OD) δ 7.65 (s, 1H), 7.42 (d, 2H, J ) 7.2 Hz), 7.25 (m, 7H),
6.84 (d, 4H, J ) 8.4 Hz), 6.54 (dd, 1H, J ) 9.0, 5.4 Hz), 4.97 (t,
1H, J ) 6.3 Hz), 4.33 (m, 1H), 3.72 (s, 6H), 3.51 (dd, 1H, J )
10.2, 3.0 Hz), 3.41 (dd, 1H, J ) 10.2, 2.4 Hz), 2.66 (m, 1H),
2.39 (m, 1H), 1.23 (s, 3H); 31P NMR (CD3OD) δ 0.24; IR (film)
3365, 2935, 1692, 1659, 1606, 1510, 1463, 1439, 1300, 1252,
1118, 984 cm-1; ESMS (M) calcd 622.6, found 623.0.
Gen er a l P r oced u r e for Clea va ge of Oligon u cleotid es
F r om P d (0)-La bile Resin s. Initial preparation for this
reaction entailed the preparation of several reagent solutions.
In an oven-dried scintillation vial was placed THF (15 mL)
which was sparged with argon for 30 min. In an oven-dried 1
dram vial equipped with a septum was weighed out Pd2(dba)3‚
CHCl3 and DIPHOS in a 1:2 mass ratio. The reagent mixture
was diluted with the appropriate amount of sparged THF. In
an oven-dried scintillation vial, tetrabutylammonium formate
(TBA) was weighed out under an argon blanket and then
diluted with the appropriate amount of sparged THF. In an
oven-dried reaction vessel purged with argon was placed ≈1.0
mg of resin containing protected oligonucleotide. The vessel
P r ep a r a tion of 20. To 19 (65.9 mg, 0.09 mmol) stirring
in MeOH (2 mL) was added 1.0 N NaOH solution (90 µL). The
reaction was allowed to stir for 1 h while being monitored by
TLC (MeOH:CH2Cl2 1:4) every 15 min. After 1 h, an additional

4068 J . Org. Chem., Vol. 63, No. 12, 1998
Greenberg et al.
was purged a second time with argon, followed by the addition
of the Pd(0) solution (100 µL) and the TBA solution (100 µL).
The reaction vessel was then capped and placed in a heating
block at 55 °C for the appropriate reaction time. The mixture
was vortexed every 15 min. After the prescribed reaction time,
the vessel was cooled to room temperature, and the solution
was removed via pipet. The resin was washed with THF (1
mL), H2O (1 mL), and THF (1 mL) again. The washings were
collected into a single sample vial. The washings were then
concentrated in vacuo and resuspended in CH3CN (0.5 mL),
followed by filtration through a 0.2 µm filter, which was
washed with (3 × 0.7 mL) CH3CN and CH3CN:H2O (1:1, 3 ×
0.7 mL). The filtrate was collected into a sample vial and
concentrated in vacuo. The residue was taken up into CH3CN:
H2O (1:1, 3 × 0.3 mL) and transferred to an Eppendorf tube.
The solution was concentrated in vacuo, treated with aqueous
ammonia, analyzed by denaturing gel electrophoresis, isolated,
and quantitated as described in the General Methods section.
P r ep a r a tion of Oligon u cleotid es for ESMS. To the gel-
purified oligonucleotide (6-10 nmol) were added H2O (150 µL)
and NH4OAc (50 µL, 5 M, pH 5.2). The solution was vortexed
and allowed to stand at 25 °C for 5 min. Cold ethanol (0.6
mL) was added and the mixture was inverted several times.
After freezing at -78 °C for 15 min, the solution was
centrifuged for 15 min. The supernatants were removed and
saved. The procedure was repeated a second time. Before
being submitted for ESMS analysis, the oligonucleotide was
requantitated by UV absorption at 260 nm as described in the
General Methods section.
reverse phase HPLC. Precipitation was achieved by addition
of NaOAc (5 µL, 3 M, pH 5), followed by addition of EtOH (200
µL). After freezing (12 min), the supernatants were repre-
cipitated by addition of NaOAc (50 µL, 0.3 M, pH 5), followed
by addition of EtOH (200 µL). After freezing and centrifuga-
tion as described above, the supernatants were removed and
dried in vacuo. Following resuspension of the nucleosides in
water, analysis was carried out using reverse phase HPLC.
Reverse phase HPLC analysis of enzyme digests of oligonucleo-
tides utilized a Rainin Microsorb-MV C18 5 µm column.
Gradient conditions: eluent A, 10 mM KH2PO4 (pH 7.0), 2.5%
MeOH (v:v); eluent B, 10 mM KH2PO4 (pH 7.0), 20% MeOH
(v:v); 0-100% B linearly over 15 min; maintain 100% B for 20
min; flow rate 1.0 mL/min.
GC/MS An a lysis of Rea ction of 19 w ith P d (0). Phos-
phate triester 19 was reacted with Pd(0) as described above
for oligonucleotides, with the exception that Pd(0) was used
in 20 mol %. â-Cyanoethanol and acrylonitrile were analyzed
for by GC/MS. Conditions were as follows: injector, 250 °C;
detector, 270 °C; column oven, Ti ) 35 °C, ti ) 4 min, R ) 50
°C/min, Tf ) 200 °C.
Ack n ow led gm en t. We thank Professor Louis S.
Hegedus for helpful discussions. We are grateful for
support of this research by the National Science Foun-
dation (CHE-9424040). D.L.M. thanks Boehringer-
Ingelheim for partial financial support in the form of a
graduate fellowship. M.M.G. thanks the Alfred P. Sloan
Foundation for support.
Sn a k e Ven om P h osp h od iester a se (SVP D) Digestion of
DNA. SVPD digests were carried out at 37 °C in a solution
(50 µL) containing oligonucleotide (5-10 nmol), SVPD (0.006
units), and reaction buffer [5 µL; MgCl2 (15 mM), Tris acetate
(0.1 M, pH 8.8)]. The reaction was initiated by addition of
enzyme and was allowed to react for 1 h at 37 °C, after which
calf intestinal alkaline phosphatase (10 units) was added.
Digestion was allowed to proceed for an additional 12 h at 37
°C. Upon completion of the digestion, the enzymatic compo-
nents were removed by precipitation prior to analysis by
Su p p or tin g In for m a tion Ava ila ble: Electrospray mass
spectrum and HPLC chromatogram of enzyme digest of 18 (2
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
J O980135B