Amide group assisted 3′-dephosphorylation of oligonucleotides synthesized on universal A-supports

Article abstract of DOI:10.1016/S0040-4020(01)00409-4

(±)-3-Amino-1-(4,4′-dimethoxytriphenylmethyl)-2-propanediol was attached to succinylated alkylamino-controlled pore glass via the second amide bond. The resulting solid phase was acylated to give seven new universal solid supports, compatible with the preparation of all common types of oligodeoxyribonucleotides. These resins allow for fast elimination of the 3′-terminal phosphodiester or phosphorothioate function by ammonia in methanol at room temperature.

Full text of DOI:10.1016/S0040-4020(01)00409-4

TETRAHEDRON
Pergamon
Tetrahedron 57 #2001) 4977±4986
Amide group assisted 30-dephosphorylation of oligonucleotides
synthesized on universal A-supports
Alex V. Azhayevp and Maxim L. Antopolsky
Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland
Received 22 February 2001;revised 20 March 2001;accepted 5 April 2001
AbstractÐ#^)-3-Amino-1-#4,40-dimethoxytriphenylmethyl)-2-propanediol was attached to succinylated alkylamino-controlled pore glass
via the second amide bond. The resulting solid phase was acylated to give seven new universal solid supports, compatible with the
preparation of all common types of oligodeoxyribonucleotides. These resins allow for fast elimination of the 30-terminal phosphodiester
or phosphorothioate function by ammonia in methanol at room temperature. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
treatment with basic volatile reagents, or the need to employ
a desalting step with sodium hydroxide, makes this solid
support unattractive in industrial high output multi-well
synthesizers.
A universal solid support, permitting the direct coupling of
any nucleoside residue and the straightforward elimination
of the terminal phosphodiester linkage at the same time as
the deprotection step, would help eliminate the possibility of
errors in parallel synthesis applications where up to 192
wells may contain different supports. Such an approach
would eliminate the need of several supports for oligo-
nucleotide synthesis and simplify the preparation of oligo-
nucleotides having modi®ed nucleosides at the 30-terminus,
or oligonucleotide mixtures with varying nucleosides at the
30-termini.
Azhayev3,4 reported on the new universal solid supports
U-3±U-5 #Scheme 1), which are not only compatible with
the preparation of all common types of oligonucleotides but
also function well for oligomers with unusual base labile
units. These supports allow for a faster elimination of the
30-terminal phosphodiester function #concentrated ammo-
nium hydroxide/808C/2±8 h) when compared with the
earlier reported universal support U-1. In addition, these
phases permit the use of neutral conditions for the terminal
dephosphorylation with aqueous zinc chloride or even
water. Apparently supports U-3±U-5 appear to be more
attractive than supports U-1 and U-2. Nevertheless, they
also require prolonged treatment with basic or neutral
reagents at elevated temperatures. In addition, multistep
preparations with supports U-2±U-5 result in relatively
high production costs. These characteristics also prevent
these solid phases from being ideal for general industrial
application.
McLean and coworkers1 have described the solid support
U-1 #Scheme 1) for universal applications. This solid
support allows for the detritylation, the addition of the
®rst nucleoside monomer and the remainder of the oligomer
preparation to proceed without changes to standard proto-
cols. The elimination of the terminal phosphodiester group
utilizes the same reagents, which are needed for the routine
deprotection of oligonucleotides, but requires very aggres-
sive and lengthy conditions #e.g. concentrated ammonium
hydroxide/808C/17 h, compared to the standard oligomer
deprotection conditions of ammonium hydroxide/558C/5±
6 h). More recently Lyttle and coworkers2 proposed a
uridine-based linker immobilized onto polystyrene particles
via a phosphotriester group #U-2) as an alternative to U-1.
The support U-2 gives oligonucleotide cleavage with
aqueous ammonia in about 8 h at 608C. Although all con-
ditions mentioned above are suitable for the preparation of
unmodi®ed oligonucleotides, they are not compatible with
base-labile nucleoside analogues. Furthermore, prolonged
Thus, the new universal supports, which lack the limitations
of U-1±U-5 remain important. We now report on new
universal solid phases, which allow faster elimination of
the 30-terminal phosphodiester group in methanolic ammo-
nia solutions at room temperature.
2. Results and discussion
Azhayev3,4 demonstrated that aminomethyl- and diamino-
ethyl- substituents are capable of facilitating cleavage of a
phosphodiester group linked to a cis-diol-containing bridge
on a universal support. Thus, the idea of neighboring group
Keywords: oligonucleotides;amide group;solid supports.
Corresponding author. Tel.: 1358-17-162204;fax: 1358-17-162456;
e-mail: Alex.Azhayev@uku.®
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020#01)00409-4

4978
A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
OH
ODMTr
OH
HO
HN
HO
HN
O
HO
H2N
HN
O
O
O
O
O
OCH3
CH3O
R
1-4
R
1a-4a
DMTrO
U-1
O
O
ODMTr
CN
O
O
O
1b-4b
O
HN
O
HO
O
P
O
Ura
O
O
R
O ODMTr
O
O
O
OAc
ODMTr
U-2
HN
O
AQ- 1 - AQ- 4
HN
O
R
O
CF3CONH
Scheme 2. 1, 1a, 1b, AQ-1: RˆCF3± 2, 2a, 2b, AQ-2: R ˆ CH3± 3, 3a, 3b,
AQ-3: RˆC6H5± 4, 4a, 4b, AQ-4: RˆC#CH3)3±.
NH
O
O
O
OBn
DMTrO
2-hydroxy groups of 1a±4a were acylated with hydro-
quinone-O,O0-diacetic acid and N,N0-diisopropylcarbo-
diimide to give compounds 1b±4b. Finally, derivatives
1b±4b were linked to a lcaaCPG, employing a N,N0-diiso-
propylcarbodiimide/N-hydroxybenzotriazole condensation.
The resulting solid supports AQ-1±AQ-4, contained 40±
100 mmol of DMTr-groups per gram of CPG.7
U-3
CF3CONH
CF3CONH
O
HN
In order to investigate the usefulness of AQ solid supports in
the preparation of oligodeoxynucleotides, the short
sequence oligo-T6 was assembled on a synthesizer, using
standard phosphoramidite chemistry and recommended
protocols. No differences in coupling ef®ciency #.98%
as determined by trityl assay) were detected between
AQ-supports and the commercial T-derivatized support,
which is consistent with the compatibility of AQ-supports
with standard assembly steps. After completion of the syn-
thesis, the addition of ammonia in methanol rapidly cleaved
the hydroquinone-O,O0-diacetate linker and released a
hexamer having a cyanoethyl-protected phosphotriester
function linked to the 1-hydroxyl of #^)-3-acylamido-1,2-
propanediol #Scheme 3, Structure A). During subsequent
treatment with ammonia, the acylamido fragment #as the
acylamide anion) assists the vicinal 2-hydroxyl group in
attacking the phosphorus and ®nally gives the desired
oligo-T6. At the same time, a fraction of phosphodiester
#Scheme 3, Structure B) was formed due to b-elimination
of the cyanoethylene prior to formation of the deacylated
phosphotriester #Scheme 3, Structure A). This side reaction
®nally leads to a stable 30-tethered oligomer t-oligo-T6
#Scheme 3). Investigation of the properties of AQ-supports
under various reaction conditions is reported hereafter.
O
O
O
X
DMTrO
U-4,5
Scheme 1. U-4: Xˆ±OBn; U-5: XˆH;DMTr ˆ4,40-dimethoxytriphenyl-
methyl;
ˆlcaaCPG;
ˆpolystyrene beads.
assistance for elimination of the terminal phosphodiester to
generate termini with 30-hydroxyls seems to be worthwhile.
Amides of carboxylic acids may be regarded as weak N±H
acids.5 Amide groups will give rise to amide anions in, for
example methanolic ammonia or ammonium hydroxide.
These amide anions, in turn, display basic properties. There-
fore, it seemed interesting to see if a vicinal amide group of
3-acylamido-1,2-propanediol tether would enhance the
elimination of a terminal phosphodiester in a basic solution,
along with a linker originally on the solid support.
We report the preparation of four solid supports, AQ-1±
AQ-4 #Scheme 2) in which a #^)-3-acylamido-1-#4,40-
dimethoxytriphenylmethyl)-2-propanediol is attached to a
long chain alkylamino controlled pore glass #lcaaCPG) via
a labile hydroquinone-O,O0-diacetyl linker.6 The starting
#^)-3-amino-1,2-propanediol was ®rst acylated to give the
3-acylamido derivatives 1±4, and then converted to their
dimethoxytrityl derivatives 1a±4a in good yield. The
The assembled sequences were ®rst cleaved from AQ
supports with ammonia in organic solvents and the extent
of dephosphorylation and cleavage of the terminal tether

A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
O ODMTr
4979
O
O
O
AQ
HN
O
HN
O
N
C
R
i
O
P
O-N
O O
O
O
O
O
HN
O
HN
O
R
ii
_
O
H
O
R
N
O
O
P
C N
O-N
O
A
N-OH = oligo-T6
+
_O
O-N
P
O O
O
O
O
O
O
HN
O
B
HN
R
ii
H
O
R
N
O
O_
P
_
t-Oligo-T6
O-N
O
O
Scheme 3. #i) oligonucleotide synthesis;#ii) NH 3/MeOH.
was investigated, using ion exchange HPLC #Fig. 1, panel
A). The effect of ammonium hydroxide at 208C for 2 h on
the cleavage from T-bound support is shown in Fig. 1, panel
B for comparison. In each of the traces shown in Fig. 1, the
product, with a retention time of 13 min, corresponds to
oligo-T6, as demonstrated by an authentic standard. The
later eluting product in Fig. 1, panel A is the hexamer having
a terminal phosphodiester linked to the 3-acylamido-1,2-
propanediol tetherÐt-oligo-T6. derived from supports
AQ-1±AQ-4 #Scheme 3;Fig. 1). All four t-oligo-T6 oligo-
mers derived from supports AQ-1±AQ-4 were isolated by
HPLC and their structure was con®rmed by electrospray
ionization mass spectrometry8 #data not shown).
Figure 1. Ion exchange HPLC traces of oligo-T6 synthesized on T-bound
support and AQ-supports. A: oligo-T6 synthesized on AQ-3-support and
treated with 9 M NH3/MeOH±dioxane #1:1), 1 h at 208C;B: oligo-T
synthesized on T-bound support and treated with 32% ammonium hydrox-
ide, 2 h at 208C. Chromatograms of oligo-T6 synthesized on AQ-1-, AQ-2-
and AQ-4-supports were analogous to that shown for the AQ-3- support
#data not shown). Time is in minutes.
6
vage/dephosphorylation. It is noteworthy to mention that
with ammonium hydroxide cleavage/dephosphorylation,
only 43% of the oligo-T6 was obtained with support AQ-1.
Apparently, the presence of water facilitates the competing
reaction #Scheme 3, Structure B), making the use of aqueous
ammonia for oligonucleotide cleavage/deprotection less
ef®cient.
The results of these studies under different conditions and at
different temperatures are summarized in Table 1. The best
yields of oligo-T6 #75±88%) were achieved with AQ-1 solid
support, by incorporating tri¯uoroacetamido function and,
more importantly, using 2±9 M ammonia in methanol±
dioxane #or methanol±toluene, data not shown) for clea-
As follows from the experiments described above, the struc-
ture of the acylamido function incorporated onto the AQ
support did not signi®cantly in¯uence the yield of
the desired oligo-T6. Apparently, reactions with all of the

4980
A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
Table 1. Yield of oligo-T6 derived from the universal solid supports AQ
under various cleavage/elimination conditions
O
H2N
NH
HO
Support
Dephosphorylation/cleavage
procedurea
Purity #%)b
6
(lcaaCPG)
O
O
AQ-1
AQ-1
AQ-1
AQ-1
AQ-1
AQ-2
AQ-2
AQ-3
AQ-3
AQ-4
I
II
43
82
85
87
88
75
79
79
83
79
F3C
N
H
ODMTr
III
IV
V
III
IV
III
IV
IV
OH
2a
H2N
ODMTr
5
OH
6
O
OH
H
N
a
Procedure I: 33% NH3/H2O, 208C, 2h;procedure II: 9 M NH /MeOH±
3
ODMTr
HN
HN
dioxane #1:1), 208C, 12 h;procedure III: 9 M NH 3/MeOH±dioxane #1:1),
48C, 1 h or 208C, 1 h;procedure IV: 9 M NH 3/MeOH±dioxane #1:1),
2208C, 1 h or 208C, 1 h;procedure V: 2 M NH /MeOH±dioxane #1:1),
7
O
R
3
48C, 1 h or 208C, 1 h.
Purity was assessed using IE HPLC by integrating peaks at 260 nm.
b
O
O
O
H
N
ODMTr
A-support
O
acylamides reported here resulted in anions capable of
assisting the vicinal 2-hydroxyl group in attacking the
phosphorus #Scheme 3, Structure A). On the other hand,
the structure of the linker at the 2-hydroxy group appears
to be critical. The cleavage of the hydroquinone-O,
O0-diacetate group proceeds faster than b-elimination of
Scheme 5. Af: RˆH±; Ap: RˆC6H5OCH2±; Aac: RˆCH3±; Amac
:
:
RˆCH3OCH2±; Ampac
:
Rˆp-CH3OC6H4±; Adcac
:
RˆCl2CH±, Atcac
RˆCl3C±.
the protecting group on the phosphorous. Therefore, attack
on the phosphotriester function by the 2-hydroxyl group of
the 3-acylamido-1,2-propanediol tether takes place, and the
desired oligonucleotide is 30-dephosphorylated.
R
O
O
O
H
N
ODMTr
In a basic solution, the diamide of dicarboxylic acid #e.g.
succinic acid), linking lcaaCPG and the 3-amino-1,2-pro-
panediol tether, should also be capable of assisting a similar
attack of the 2-hydroxyl group on the phosphorous #Scheme
4, Structure A), provided that this 2-hydroxy function of
the tether is instantly deproptected with ammonia prior to
phosphate deprotection. This attack should lead to the elimi-
nation of the desired oligonucleotide oligo-T6. Interestingly,
the competing reaction should lead to the formation of a
lcaaCPG bounded oligonucleotide with the phosphodiester
group at the 30-terminus #Scheme 4, Structure B). This inter-
mediate should ultimately lead to a stable phosphodiester
linked to the solid-phase #Scheme 4, Structure C). Taking
this kind of reasoning into consideration, we designed and
synthesized several new universal A-supports #Scheme 5).
In these structures the succinyl group bridged the lcaaCPG
and 3-amino-1-#4,40-dimethoxytriphenylmethyl)-2-propane-
diol via two amide bonds and the 2-hydroxyl function was
acylated.
HN
A-support
O
i
R
O
O
O
O
O
O
H
N
O
HN
P
C N
O-N
O
ii
_
O
H
N
_
N
O
O
O
P
C N
A
O
ON
N-OH = oligo-T6
+
R
_
O
O
O
The preparation of A supports appeared to be straight-
forward #Scheme 5). Initially lcaaCPG was succinylated
and capped to give succinylamido-CPG #6). #^)-3-Tri-
¯uoroacetamido-1-#4,40-dimethoxytriphenylmethyl)-2-pro-
panediol #2a) was converted to the corresponding amine 5,
which, in turn, was attached to 6 in the presence of N,N0-di-
isopropylcarbodiimide and N-hydroxybenzotriazole to give
solid phase 7.
_
N
_
N
O
O
O
P
O
O
ON
B
ii
_
O
OH
_
N
_
N
O
O
O
P
C
ON
Finally the free hydroxyl group on compound 7 was either
,
acetylated to give Aac, phenoxyacetylated to give Apac
Scheme 4. #i) oligonucleotide synthesis;#ii): NH 3/MeOH.

A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
4981
Table 2. Relative yield of oligo-T6 derived from the universal solid
supports A under various cleavage/elimination conditions
Support
Dephosphorylation/cleavage
procedurea
Relative yield
#%)b
Purity
#%)c
T-CPG
Af
Aac
Apac
Amac
Ampac
Adcac
Atcac
Af
I
I
100
42
4
48
24
30
68
72
76
97
82d
74
95
94
95
96
96
96
II
II
II
II
II
II
II
a
Procedure I: 33% NH3/H2O, 208C, 2 h;procedure II: 2 M NH 3/MeOH,
208C, 1 h or 33% NH3/H2O, 208C, 1 h.
Yield of oligo-T6 resulting from the commercial T-support was taken as
100%. Relative yield of oligomer, resulting from A-support, was calcu-
lated relative to the yield from the T support.
b
c
Purity was assessed using IE HPLC by integrating peaks at 260 nm.
Along with 7.8% of an unidenti®ed product.
d
various reaction conditions. The assembled oligo-T6
sequence was cleaved from A-support with ammonia in
methanol, and the extent of dephosphorylation/cleavage
from the controlled pore glass was investigated by ion
exchange HPLC #Fig. 2, panel A). The effect of ammonium
hydroxide at 208C for 2 h on cleavage from T-CPG is shown
in Fig. 2, panel B, for comparison. In each of these chro-
matograms the product, with a retention time of about
13 min, corresponds to oligo-T6, as demonstrated by an
authentic standard.
The optimal conditions for recovering oligo-T6 from
A-supports was assessed from IE HPLC by peak integration,
corresponding to oligo-T6 #260 nm) derived from 1 mmol of
initial solid support. Typically, 1/50 of the total amount of
obtained oligomer was injected and the oligonucleotide
peak area was compared to the corresponding area of
oligo-T6, resulting from commercial T-CPG. The results
of these are summarized in Table 2.
It can be seen that after completion of the synthesis, addition
of ammonia in methanol to all A-supports released the
oligo-T6 in solution #Scheme 4;Fig. 2;Table 2). As
expected, the competing reaction most probably led to the
formation of some lcaaCPG bounded oligomer with the
phosphodiester group at the 30-terminus #Scheme 4, Struc-
ture C). This assumption is supported by the relative yield
data given in Table 2. Thus, the only oligonucleotide
product in the solution appeared to be the desired oligomer,
along with small amounts of truncated sequences resulting
from the non-quantitative coupling steps in the process of
oligonucleotide assembly.
Figure 2. Ion exchange HPLC traces of oligo-T6 synthesized on Af-support
and T-bound support. A: oligo-T6 synthesized on Af-support and treated
with 2 M NH3/MeOH, 1 h at 208C followed by 32% ammonium hydroxide,
1 h at 208C;B: oligo-T 6 synthesized on T-bound support and treated with
32% ammonium hydroxide, 2 h at 208C. Time is in minutes.
methoxyacetylated to give Amac, 4-methoxyphenoxyacetyl-
ated to give Ampac, dichloroacetylated to give Adcac, tri-
chloroacetylated to give Atcac or formylated to give solid
support Af. The resulting solid supports contained 70±
100 mmol of DMTr-groups per gram of CPG.7
As in the case of AQ-supports, the short sequence oligo-T6
was assembled on all ®ve A-supports using standard phos-
phoramidite chemistry and recommended protocols. No
differences in coupling ef®ciencies #.98% as determined
from trityl assay) were detected between A-supports and the
commercial T-bound solid support #T-CPG), which is
consistent with the compatibility of A-supports and standard
assembly steps.
When comparing different A-supports, the Aac-support
leads to only traces of oligo-T6. This fact makes Aac-support
not so useful for oligonucleotide synthesis. Also, Af-, Atcac
and Adcac-supports appear to be superior to the Apac-, Amac
-
-
and Ampac-, leading to about two times higher overall yield
of the desired oligomer #65±75%). As in the case of AQ-sup-
ports, the ammonium hydroxide cleavage/dephosphoryl-
ation led to a signi®cantly lower yield of oligo-T6,
compared to the methanolic ammonia treatment #Table 2).
We also investigated the properties of A-supports under

4982
A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
Figure 3. Ion exchange HPLC traces of oligo-T50 synthesized on Af-
support and treated with 2 M NH3/MeOH, 1 h at 208C, followed by 32%
ammonium hydroxide, 1 h at 208C. Time is in minutes.
Similar experiments were carried out with a longer sequence
oligo-T50, which was assembled on Af- and Apac-supports
and commercial T-CPG. Fig. 3 shows traces of a crude
oligo-T50 sample, synthesized on Af-support, after treatment
with 2 M NH3/MeOH #208C, 1 h);followed by the addition
Figure 4. IE HPLC traces of 50-GCGAAGCTTTGGAGAGTGGCAT-
GAAGAAA-30 synthesized on the Af-support and treated with 2 M NH3/
MeOH, 1 h at 208C, followed by 32% ammonium hydroxide, 4 h at 558C.
Time is in minutes.
Table 3. Relative yield of oligo-T50, synthesized on different solid supports.
The yield of oligo-T50 obtained from the T bound support was taken as
100%
of an equal volume of 32% ammonium hydroxide #208C,
1 h). The relative yields of oligo-T50, synthesized on differ-
ent solid supports, are summarized in Table 3.
Solid support
Relative yielda of oligo-T50 #%)
Purity #%)b
The preliminary experiments with A-supports clearly
demonstrated that they may be used as a universal solid
phase, allowing for the fast and easy cleavage of suf®cient
amounts of oligothymidilates.
Af
Ap
T
48
27
100
62
56
61
a
Yield of oligo-T50 resulting from the commercial T-CPG support was
taken as 100%. Yield of oligomer, resulting from A-support, was calcu-
lated relative to the yield from T-CPG.
In order to investigate the applicability of A-supports for
the preparation of oligodeoxyribonucleotides of mixed
b
Purity was assessed using IE HPLC by integrating peaks at 260 nm.

A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
4983
Fig. 4 shows the typical analysis of crude 29 mer
50-GCGAAGCTTTGGAGAGTGGCATGAAGAAA-30
assembled on the Af-support and cleaved with 2 M ammo-
nia in methanol #208C, 1 h), and ®nally deprotected by
adding an equal amount of 32% ammonium hydroxide
and heating at 558C for an additional 4 h. The structures
of long oligomers were con®rmed by electrospray ioni-
zation mass spectrometry7 #data not shown) after chromato-
graphic puri®cation #compounds were assembled on a
1 mmol scale;yields were 50±75 OD 260) All oligonucleo-
tides appeared to be good primers, and performed well in
PCR experiments #data not shown).
The oligonucleotide phosphorothioate 50-TGGCGTCTTC-
CATTT-30 was synthesized on commercial a T-CPG
support and support Af, following the recommended proto-
col. No differences in coupling ef®ciencies #.98% as deter-
mined from trityl assay) were detected between support Af
and the T-CPG. The support-bound oligonucleotide phos-
phorothioate was cleaved and deprotected as described
above, and then analyzed by IE HPLC. Fig. 5 shows an
analysis of crude oligonucleotide phosphorothioate
prepared on Af-support. This oligomer was identical to the
phosphoprothioate prepared on the T-bound solid phase.
3. Conclusion
In summary, we have developed new universal solid
supports that are compatible for preparing common types
of oligodeoxyribonucleotides. These resins allow for fast
elimination of the 30-terminal phosphodiester or phosphoro-
thioate function with ammonia in methanol at room
temperature. We believe that these inexpensive universal
solid phases may become extremely useful for the prepara-
tion of oligonucleotides from both conventional and parallel
industrial 96- or 192-well synthesizers.
4. Experimental
4.1. General
Figure 5. Ion exchange HPLC traces of 50-TGGCGTCTTCCATTT-30
phosphorothioate synthesized on the Af-support and treated with 2 M
NH3/MeOH, 1 h at 208C, followed by 32% ammonium hydroxide, 4 h at
558C. Time is in minutes.
#^)-3-Amino-1,2-propanediol was purchased from Fluka,
acetic anhydride, benzoyl chloride, pivaloyl chloride, 2,6-
lutidine, succinic anhydride, tri¯uoroacetic acid, and
solvents were from Aldrich, 4,40-dimethoxytrityl chloride
was purchased from ChemGenes, lcaaCPG was purchased
from Sigma or Glen Research and hydroquinone-O,O0-
diacetic acid was purchased from Lancaster. Reagents
#solvents, activators, etc.) for oligonucleotide synthesis
were purchased from Glen Research. Column ¯ash chroma-
tography was performed on Silica gel 60 #available from
Merck). NMR spectra were recorded on a Bruker Avance
DRX 500 spectrometer with tetramethylsilane as an internal
standard #sˆsinglet;d ˆdoublet;dd ˆdoublet of doublets;
mˆmultiplet;br.s ˆbroad signal). All compounds bearing
free hydroxy- and amino-groups were co-evaporated with
CD3OD before measurements. Electrospray ionization mass
spectra #ESI MS) were aquired using a LCQ quadrupole ion
trap mass spectrometer equipped with an electrospray ioni-
zation source #Finnigan MAT). IR spectra were taken with a
Perkin±Elmer 983 spectrometer.
sequences, seven longer oligonucleotides were synthesized
on a 1 mmol scale:
50-TGGCGTCTTCCATTT-30 #15 mer),
50-GTGGAATTCCAGCAGCAGAAAGAGCTCATC-30
#30 mer),
50-TAT GGATCC TCAGCTGCAAATGAGGG-30 #26
mer),
50-GTGGAATTCATGAAGAAAGAGATGATCATG-30
#30 mer),
50-TATGGTACCTCAGCCGTCCGTGCTGCTT-30 #28
mer),
50-GCGAAGCTTTGGAGAGTGGCATGAAGAAA-30
#29 mer),
50-TATGGATCCAACCATTCAACATGGTGGAC-30
#29 mer).

4984
A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
4.2. Oligodeoxyribonucleotide synthesis
CHaHbNH);ESI MS: found 195,1, C 10H13NO3 requires
195.1;anal. calcd for C 10H13NO3: C, 61.53;H, 6.71;N,
7.18%. Found: C, 61.39;H, 6.83;N, 7.02.
The protected oligonucleotides were assembled on an
Applied Biosystems 392 DNA Synthesizer using phos-
phoroamidite chemistry and recommended DMTr-off proto-
cols for 40 nmol, 0.2 mmol and 1 mmol scales.
4.3.4. 0^)-3-Pivaloylamido-1,2-propanediol 04). Pivaloyl
chloride #0.74 ml, 6 mmol) dissolved in 5 ml of 1,4-dioxane
was added dropwise to a cold stirred solution #08C) of #^)-
3-amino-1,2-propanediol #0.55 g, 6 mmol) in 1,4-dioxane±
water #5:1;20 ml). The reaction mixture warmed to 20 8C
within 2 h, solvents were removed under vacuum and
4.3. HPLC techniques
Oligonucleotides containing phosphodiester functions were
analyzed by ion exchange HPLC #column: Dionex DNAPac
PA-100, 4£250 mm;buffer A: 0.1 M NaAc in 20% MeCN
pH 8, buffer B: 0.1 M NaAc and 0.4 M NaClO4 in 20%
MeCN pH 8;¯ow rate 1 ml min 21;on a linear gradient
from 0±15% B over 20 min for oligo-T6 and modi®ed
oligo-T6 oligonucleotides, and from 5 to 45% B over
30 min for longer oligomers). Phosphorothioate oligo-
nucleotides were analyzed by ion exchange HPLC #column:
derivative
4 was isolated by ¯ash chromatography
#isocratic, 7% of methanol in dichloromethane) as a color-
less oil #0.8 g, yield 77%): nmax #KBr) 3360±3080 #OH and
NH), 2960±2880 #CH aliph.), 1640 #CvO), 1540 #NH),
1480±1430 #CH2), 1400 #C±N), 1370 #CH3), 1220±1050
#C±O); dH #500 MHz CDCl3) 6.96 #br.s, 1H, NH), 3.78
#m, 1H, CHOH), 3.67 #m, 1H, CHaHbOH), 3.47 #m, 1H,
CHaHbNH), 3.40 #m, 1H, CHaHbOH), 3.40 #m, 1H,
CHaHbNH), 1.20 #s, 9H, Me3C);ESI MS: found 175.2,
Ê
Poly LP PolyWax 4.6£100 mm, 5 mm, 300 A;buffer A:
C8H17NO3 requires 175.1;anal. calcd for C H17NO3: C,
8
54.84;H, 9.78;N, 7.99%. Found: C, 54.71;H, 9,91;N, 7.83.
0.05 M KH2PO4 in 50% formamide, pH 6.7;buffer B:
0.05 M KH2PO4 and 1.5 M NaBr in 50% formamide, pH
6.7;¯ow rate 1 ml min 21;on a linear gradient from 5 to
50% B over 30 min).
4.4. 0^)-3-Tri¯uoroacetamido-1-04,40-dimethoxytriphenyl-
methyl)-2-propanediol 01a), 0^)-3-acetamido-104,40-
dimethoxytriphenylmethyl)-2-propanediol 02a), 0^)-3-
benzoylamido-1-04,40-dimethoxytriphenylmethyl)-2-pro-
panediol 03a) and 0^)-3-pivaloylamido-1-04,40-dimethoxy-
triphenylmethyl)-2-propanediol 04a). General procedure
4.3.1. 0^)-3-Tri¯uoroacetamido-1,2-propanediol 01). #^)-
3-Amino-1,2-propanediol #2.73 g, 30 mmol) was dissolved
in methyl tri¯uoroacetate #30 ml, 300 mmol) and left over-
night at 208C. The reaction mixture was evaporated and
dried by coevaporation with toluene #3£100 ml) to give
5.44 g #97%) of 1 as a colorless oil: nmax #KBr) 3360±
3120 #OH and NH), 2960 #CH aliph.), 1720 #CvO), 1570
#NH), 1440 #CH2), 1350 #C±N), 1220±1060 #C±F and
C±O); dH #500 MHz CDCl3) 6.82 #br.s, 1H, NH), 3.92 #m,
1H, CHOH), 3.73 #m, 1H, CHaHbOH), 3.62 #m, 1H,
CHaHbNH), 3.57 #m, 1H, CHaHbOH), 3.42 #m, 1H,
4,40-Dimethoxytrityl chloride #1.7 g, 5 mmol) was added to
a 5 mmol stirred solution of propanediol #1, 2, 3 or 4) in dry
pyridine #20 ml). The mixture was agitated overnight at
208C and the reaction was stopped by the addition of 2 ml
methanol. Solvents were removed by evaporation, and the
residue was dissolved in 50 ml of ethylacetate, then washed
with saturated sodium hydrocarbonate solution #3£30 ml)
and water #2£30 ml). The organic solution was dried over
Na2SO4 and evaporated to dryness. #^)-3-Acylamido-1-
#4,40-dimethoxytriphenylmethyl)-2-propanediols #1a, 2a,
3a and 4a) were isolated by ¯ash chromatography #step
gradient from 0 to 2% methanol in dichloromethane in
the presence of 0.1% of pyridine, for acetamido-1#4,40-
dimethoxytriphenylmethyl)-2-propanediol #2a) and from 0
to 5% methanol in dichloromethane in the presence of 0.1%
pyridine) as pale yellow oils.
CHaHbNH);ESI MS: found 187.0. C H8F3NO3 requires
5
187.0;anal. calcd for C H8F3NO3: C, 32.09;H, 4.31;N,
5
7.49%. Found: C, 31.89;H, 4.47;N, 7.40.
4.3.2. 0^)-3-Acetamido-1,2-propanediol 02). #^)-3-
Amino-1,2-propanediol #0.91 g, 10 mmol) was dissolved
in 20 ml of dry pyridine, and acetic anhydride #4.7 ml,
50 mmol) was added. The mixture was left overnight at
208C and evaporated. The oily residue was treated with
25% aqueous ammonia #30 ml) for 1 h at 208C. After
evaporation and drying by coevaporation with toluene
#3£50 ml), compound 2 was used for 4,40-dimethoxytrityl-
ation without further puri®cation.
The yield of 1a was 1.98 g #81%): nmax #KBr) 3510 #OH),
3060±2840 #NH and CH aliph.), 1730 #CvO), 1610 #CvC
arom.), 1580 #NH), 1460 and 1440 #CH2), 1420 #C±N),
1310±1040 #C±O methoxy, C±F and C0±O), 950±710
#CH arom.); dH #500 MHz CDCl3)8.52 #br.s, 1H, NH),
7.43±6.81 #m, 13H, arom.), 3.92 #m, CHOH), 3.79 #s, 6H,
2£CH3OC6H4), 3.61 #m, 1H, CHaHbNH), 3.31 #dd, 1H,
CHaHbNH), 3.25#dd, 1H, Jˆ7.6, 9.7 Hz, CHaHbODMTr),
3.15 #dd, Jˆ7.6, 9.7 Hz, CHaHbODMTr);ESI MS: found
489.3, C26H26F3NO5 requires 489.2;anal. calcd for
C26H26F3NO5: C, 63.80;H, 5.35;N, 2.86%. Found: C,
64.06;H, 5.39;N, 2.70.
4.3.3. 0^)-3-Benzoylamido-1,2-propanediol 03). Benzoyl
chloride #0.7 ml, 6 mmol) in 1,4-dioxane #5 ml) was added
dropwise to a cold #08C) stirred solution of #^)-3-amino-
1,2-propanediol #0.55 g, 6 mmol) in 1,4-dioxane±water
#5:1;20 ml). The reaction mixture was warmed to 20 8C
within 2 h, solvents were removed under vacuum and deri-
vative 3 was isolated by ¯ash chromatography #isocratic,
7% methanol in dichloromethane) as a colorless oil
#0.83 g, 71%): nmax #KBr) 3800±3010 #OH, NH and CH
arom.), 2960 #CH aliph.), 1650 #CvO), 1580 #NH),
1540±1490 #CH2 and CvC arom.), 1400 #C±N), 1260±
1050 #C±O), 930±710 #CH arom.); dH #500 MHz CDCl3)
10.05 #br.s, 1H, NH), 8.06±7.13 #m, 5H, Ph), 3.93 #m, 1H,
CHOH), 3.60 #m, 4H, CHaHbOH, CHaHbOH, CHaHbNH,
The yield of 2a was 1.7 g #78%): nmax #KBr) 3390 #OH, NH
and CH arom.), 2990 #CH aliph.), 1660 #CvO), 1620
#CvC arom.), 1590 #NH), 1530±1520 #CH2 and CvC
arom.), 1470 and 1450 #CH2), 1400 #C±N), 1370 #CH3),

A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
4985
1260±1040 #C±O methoxy and C±O), 990±710 #CH
arom.); dH #500 MHz CDCl3) 7.40±6.80 #m, 13H, arom.),
6.12 #br.s, 1H, NH), 3.88 #m, CHOH), 3.79 #s, 6H, 2£
CH3OC6H4), 3.52 #m, 1H, CHaHbNH), 3.38 #m, 1H,
CHaHbNH), 3.28 #m, 1H, CHaHbODMTr), 3.19 #m, 1H,
CHaHbODMTr), 2.01 #s, 3H, CH3CONH). ESI MS: found
435.1, C26H29NO5 requires 435.2;anal. calcd for
C26H29NO5: C, 71.70;H, 6.71;N, 3.22%. Found: C,
71.51;H, 6.92;N, 3.01.
diol #1 mmol, 1a, 2a, 3a or 4a) and 4-N,N-dimethylamino-
pyridine #10 mg) in dry pyridine #3 ml) and the reaction
mixture was stirred overnight at 208C. Water #1 ml) was
then added and the resulting mixture was evaporated to
dryness. Compounds 1b, 3b and 4b were isolated by ¯ash
chromatography #step gradient from 0 to 5% methanol in
dichloromethane in the presence of 0.1% pyridine for 1b, 3b
and 4b) as pale yellow oils. Crude 2b was dried under
high vacuum to give 0.37 g of a pale yellow oil, which
was used for AQ-2-support preparation without additional
puri®cation.
The yield of 3a was 2.1 g #85%): nmax #KBr) 3800±3020
#OH, NH and CH arom.), 2970 #CH aliph.), 1670 #CvO),
1580 #NH), 1540±1490 #CH2 and CvC arom.), 1460 and
1440 #CH2), 1400 #C±N), 1370 #CH3), 1260±1050 #C±O
methoxy C±O), 980±710 #CH arom.); dH #500 MHz
CDCl3) 7.64±6.80 #m, 18H, arom.) 6.48 #br.s, 1H, NH);
3.86 #m, 1H, CHOH), 3.77 #s, 6H, 2£CH3OC6H4), 3.60
#m, 3H, CHaHbNH, CHaHbNH, CHaHbODMTr), 3.46 #m,
1H, CHaHbODMTr);ESI MS: found 487.3, C 31H31NO5
requires 497.2;anal. calcd for C 31H31NO5: C, 74.83;H,
6.28;N, 2.81%. Found: C, 75.06;H, 6.30;N, 2.69.
The yield of 1b was 0.45 g #65%); nmax #KBr) 3460 #OH),
2950 #NH), 1720 #CvO), 1670 #CvO), 1610±1590 #CvC
arom., NH, COO2), 1510 #CvC arom.), 1470 and 1450
#CH2), 1400 #C±N and COO2), 1300±1040 #C±F, C±O
methoxy and C±O), 900±710 #CH arom.); dH #500 MHz
CDCl3±CD3OD, 4:1) 7.45±6.82 #m, 17H, arom.), 5.17
#br.s, 1H, NH), 4.58 #br.s, 4H, 2£OCOCH2O), 3.79 #s,
6H, 2£CH3OC6H4), 3.77 #m, 1H, CHOH), 3.48 #m, 2H,
CHaHbNH, CHaHbNH), 3.26 #m, 2H, CHaHbODMTr,
CHaHbODMTr). ESI MS: found 697.3, C36H34 F3NO10
requires 697.2;anal. calcd for C 36H34 F3NO10: C, 61.98;
H, 4.91;N, 2.01%. Found: C, 62.21;H, 4.99;N, 1.89.
The yield of 4a was 1.76 g #74%): nmax #KBr) 3660±3010
#OH, NH and CH arom.), 2960±2880 #CH aliph.), 1650
#CvO), 1620 #CvC arom.), 1540 #NH), 1520±1430 #CH2
and CvC arom.), 1450 #CH2), 1400 #C±N), 1370 #CH3),
1270±1020 #C±O methoxy and C±O), 980±710 #CH
arom.); dH #500 MHz CDCl3) 7.41±6.81 #m, 13H, arom.),
5.94 #br.s, 1H, NH), 3.93 #m, 1H, CHOH), 3.77 #s, 6H,
2£CH3OC6H4), 3.51 #m, 1H, CHaHbNH), 3.42 #m, 1H,
CHaHbNH), 3.28 #m, 1H, CHaHbODMTr), 3.11 #m, 1H,
CHaHbODMTr), 1.11 #s, Me3CCONH). ESI MS: found
477.2, C29H35NO5 requires 477.3;anal. calcd for
C29H35NO5: C, 72.93;H, 7.48;N, 2.93%. Found: C,
73.12;H, 7.70;N, 2.79.
The yield of 3b was 0.48 g #69%); nmax #KBr) 3800±3020
#OH, NH and CH arom.), 2980 #CH aliph.), 1720 #CvO),
1670 #CvO), 1620±1580 #CvC arom., NH, COO2),
1540±1490 #CH2 and CvC arom.), 1400 #C±N and
COO2), 1460 and 1440 #CH2), 1400 #C±N), 1370 #CH3),
1260±1050 #C±O methoxy C±O), 980±710 #CH arom.); dH
#500 MHz CDCl3±CD3OD, 4:1) 7.88±6.81 #m, 22H,
arom.), 4.56 #br.s, 4H, 2£OCOCH2O), 3.97 #m, 1H,
CHOH), 3.79 #s, 6H, 2£CH3OC6H4), 3.68 #m, 3H,
CHaHbNH, CHaHbNH, CHaHbODMTr), 3.37 #m, 1H,
CHaHbODMTr). ESI MS: found 705.2, C41H39NO10 requires
705.2;anal. calcd for C 41H39NO10: C, 74.83;H, 6.28;N,
2.81%. Found: C, 74.58;H, 6.33;N, 2.63.
4.4.1. 0^)-3-Amino-1-04,40-dimethoxytriphenylmethyl)-
2-propanediol 05). Compound 1a #2.5 g) was dissolved in
a 9 M solution of ammonia in methanol #75 ml), left over-
night at 208C and ®nally evaporated to dryness to give a
colorless oil. Crude 5 was used for the preparation of
A-supports without additional puri®cation, nmax #KBr)
3480±3020 #OH, NH and CH arom.), 2970±2840 #CH
aliph.), 1620±1520 #CH2 and CvC arom.), 1490±1420
#CH2), 1300 #C±N), 1260±1040 #C±O methoxy and
C±O), 900±710 #CH arom.); dH #500 MHz CDCl3) 7.88±
6.75 #m, 13H, arom.), 3.97 #m, 1H, CHOH), 3.78 #s,
6H, 2£CH3OC6H4), 3.14 #m, 2H, CHaHbODMTr,
CHaHbODMTr), 3.03 #dd, 1H, J 3.1, 12.9 Hz, CHaHbNH2),
2.91 #dd, 1H, Jˆ9.8, 12.9 Hz, CHaHbNH2). ESI MS: found
393.2, C24H27NO4 requires 393.2.
The yield of 4b was 0.43 g #62%); nmax #KBr) 3650±3010
#OH, NH and CH arom.), 1720 #CvO), 1650 #CvO),
1620±1540 #CvC arom., NH, COO2), 1520±1430 #CH2
and CvC arom.), 1400 #C±N and COO2), 1370 #CH3),
1270±1020 #C±O methoxy and C±O), 980±710 #CH
arom.); dH #500 MHz CDCl3±CD3OD, 4:1) 7.37±6.82 #m,
17H, arom.), 6.37 #br.s, 1H, NH), 4.55 #br.s, 4H, 2£
OCOCH2O), 3.91 #m, 1H, CHOH), 3.79 #s, 6H, 2£
CH3OC6H4), 3.53 #m, 1H, CHaHbNH), 3.44 #m, 1H,
CHaHbNH), 3.23 #m, 2H, CHaHbODMTr, CHaHbODMTr),
1.07 #s, 9H, Me3CCONH). ESI MS: found 685.1,
C39H43NO10 requires 685.3;anal. calcd for C 39H43NO10:
C, 68.31;H, 6.32;N, 2.04%. Found: C, 68.53;H, 6.49;N,
1.89.
4.5. Coupling of hydroquinone-O,O0-diacetic acid to 0^)-
3-acylamido-1-04,40-dimethoxytriphenylmethyl)-2-pro-
panediols 1a±4a to give compounds 1b, 2b, 3b and 4b.
General procedure
4.6. AQ-Supports. General procedure
A mixture of lcaa-CPG #0.3 g), hydroxybenzotriazole
#HOBT) #30 mg, 0.2 mmol), N,N0-di-isopropylcarbodiimide
#35 ml, 0.2 mmol), conjugate of #^)-3-acylamido-1-#4,40-
dimethoxytriphenylmethyl)-2-propanediol and hydroqui-
none-O,O0-diacetic acid #1b, 2b, 3b or 4b, 0.15 mmol) in
pyridine #5 ml) was agitated overnight at 208C. Solids were
®ltered off, washed with pyridine #5 ml), tetrahydrofuran
Hydroquinone-O,O0-diacetic acid #0.452 g, 2 mmol) was
dissolved in 5 ml of dry pyridine±dimethylformamide
#4:1), N,N0-di-isopropylcarbodiimide #0.16 ml, 1 mmol)
was added and the mixture was stirred for 1 h at 208C.
The resulting solution was added to a solution of #^)-3-
acylamido-1-#4,40-dimethoxytriphenylmethyl)-2-propane-

4986
A. V. Azhayev, M. L. Antopolsky / Tetrahedron 57 02001) 4977±4986
#5 ml), diethyl ether #10 ml) and dried. The resulting support
was suspended in a mixture of 1-methylimodazole±tetra-
hydrofuran #4:21;5 ml) and acetic anhydride:2,6-lutidine±
tetrahydrofuran #1:1:8;5 ml) and agitated for 45 min at
208C. The ®ltrate was washed with tetrahydrofuran #5 ml),
acetonitrile #10 ml), diethyl ether #10 ml) and dried. The
supports contained the following amount of DMTr-groups
of per gram of CPG: AQ-1Ð100 mmol, AQ-2Ð50 mmol,
AQ-3Ð40 mmol and AQ-4Ð40 mmol.
#5 ml)
and
4-N,N-dimethyaminopyridine
#0.05 g,
0.04 mmol) and agitated overnight at 208C. Solids were
®ltered off, washed with acetonitrile #10 ml) and ®nally
dried. Support Ap contained 100 mmol of DMTr-groups
per gram of CPG; AmacÐ82 mmol of DMTr-groups per
gram of CPG; AmpacÐ86 mmol of DMTr-groups per gram
of CPG; AacÐ97 mmol of DMTr-groups per gram of CPG;
AdcacÐ90 mmol of DMTr-groups per gram of CPG; AtcacÐ
73mmol of DMTr-groups per gram of CPG.
4.6.1. A-Supports. A mixture of lcaa-CPG #1 g), succinic
anhydride #0.75 g, 7.5 mmol), pyridine #5 ml) and 1-methyl-
imidazole±tetrahydrofuran #4:21;5 ml) was agitated over-
night at 208C. Solids were ®ltered off, washed with pyridine
#5 ml) and then tetrahydrofuran #5 ml). The resulting
support 6 was suspended in a mixture of 1-methylimoda-
zole±tetrahydrofuran #4:21;5 ml) and acetic anhydride:2,6-
lutidine±tetrahydrofuran #1:1:8;5 ml) and agitated for
45 min at 208C. The ®ltrate was washed with tetrahydro-
furan #5 ml), 10% aqueous pyridine #10 ml), pyridine
#5 ml), acetonitrile#10 ml) and ®nally dried. A mixture of
derivatized CPG, compound 5 #1.2 g, 3 mmol), 1-hydroxy-
benzotriazole #0.46 g, 3 mmol), pyridine #7.5 ml) and N,N0-
diisopropylcarbodiimide #0.47 ml, 3 mmol) was agitated
overnight at 208C. Solids were ®ltered off, washed with
acetonitrile #5 ml), re-suspended in a mixture of triethyl-
amine±water±acetonitrile #1:1:3;5 ml) and agitated for
45 min at 208C. The ®ltrate was washed with acetonitrile
#10 ml) and ®nally dried.
Acknowledgements
We thank Dr Hugh Mackie #Glen Research Corporation) for
inspiring this work, Dr John Randolf #Glen Research
Corporation) for independent testing of solid supports,
È È
Professor Jouko Vepsalainen #University of Kuopio) for
NMR experiments, Mrs Unni Tengval #University of
Kuopio) for ESI MS measurements and Dr James Callaway
#University of Kuopio) for reading the manuscript.
References
1. Scott, S.;Hardy, P.;Sheppard, R. C.;McLean, M. J. In
Perspectives in Solid Phase Synthesis. Third International
Symposium, Epton, R., Ed.;May¯ower Worldwide: Kings-
winford, West Midlands, 1994;pp 115±124.
2. Lyttle, M. H.;Dick, D. J.;Hudson, D.;Cook, R. M. Nucleosides
Nucleotides 1999, 18, 1809±1824.
3. Azhayev, A. Tetrahedron 1999, 55, 787±800 and references
therein.
The resulting support 7 #0.42 g) was suspended in a mixture
of 2,6-lutidine #3 ml), tetrahydrofuran #8 ml) and acylated
by a mixture #3£0.5 ml over 3 h period) prepared from
formic acid #1.13 ml, 30 mmol) and acetic anhydride
#1.9 ml, 20 mmol). The resulting suspension was agitated
overnight at 208C. The resulting ®ltrate was washed with
acetonitrile #10 ml) and ®nally dried. Support Af contained
100 mmol of DMTr-groups per gram of CPG.
4. Azhayev, A. Collection Symp. Ser. 1999, 2, 129±134.
5. Morrison, T. M.;Boyd, R. M. In Organic Chemistry, 3rd ed.;
Allyn and Bacon: Boston, 1975;pp 758.
6. Pon, R. T.;Yu, S. Y. Tetrahedron Lett. 1997, 38, 3327±3330.
7. Atkinson, T.;Smith, M. In Oligonucleotide Synthesis. A Prac-
tical Approach, Gait, M. J., Ed.;IRL Press: Oxford, 1984;pp
47.
8. Azhayev, A.;Auriola, S.;Hovinen, J. Nucleosides Nucleotides
1998, 17, 1527±1537.
Alternatively, the resulting support 7 #0.5 g) was suspended
in a mixture of the corresponding carboxylic acid #4 mmol),
N,N0-diisopropylcarbodiimide #0.32 ml, 2 mmol), pyridine