An improved synthesis of the dinucleotides pdCpA and pdCpdA

Article abstract of DOI:10.1081/NCN-100002081

An improved route was developed for the preparation of the dinucleotide hybrid 5′-O-phosphoryl-2′-deoxycytidylyl-(3′ → 5′)adenosine (pdCpA) 7. This simple and concise synthesis involves the successive coupling of 2-cyanoethyl N, N, N′, N′-tetra- isopropylphosphorodiamidite with 4-N-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-cytidine 1 and 6-N,6-N,2′-O,3′-O-tetrabenzoyladenosine 2 as the key step. Some dinucleotide derivatives bearing different protecting groups were also synthesized and the selective deprotection conditions were studied in detail. The utility and efficiency of this approach has been further demonstrated by its application to the synthesis of total DNA dinucleotide pdCpdA 17 and total RNA dinucleotide 21.

Full text of DOI:10.1081/NCN-100002081

This article was downloaded by: [University of Chicago Library]
On: 14 November 2014, At: 13:32
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
AN IMPROVED SYNTHESIS OF THE DINUCLEOTIDES
pdCpA AND pdCpdA
Xue-Feng Zhu a & A. Ian Scott b
a Center for Biological NMR, Department of Chemistry , Texas A&M University , P.O. Box
30012, College Station, TX, 77842-3012, U.S.A.
b Center for Biological NMR, Department of Chemistry , Texas A&M University , P.O. Box
30012, College Station, TX, 77842-3012, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Xue-Feng Zhu & A. Ian Scott (2001) AN IMPROVED SYNTHESIS OF THE DINUCLEOTIDES pdCpA AND
pdCpdA, Nucleosides, Nucleotides and Nucleic Acids, 20:3, 197-211, DOI: 10.1081/NCN-100002081
To link to this article: http://dx.doi.org/10.1081/NCN-100002081
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(3), 197–211 (2001)
AN IMPROVED SYNTHESIS OF THE
DINUCLEOTIDES pdCpA AND pdCpdA
Xue-Feng Zhu and A. Ian Scott∗
Center for Biological NMR, Department of Chemistry, Texas A&M
University, P.O. Box 30012, College Station, TX 77842-3012
ABSTRACT
An improved route was developed for the preparation of the dinucleo-
tide hybrid 5ꢀ-O-phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ → 5ꢀ)adenosine (pdCpA)
7. This simple and concise synthesis involves the successive coupling of 2-
cyanoethyl N,N,Nꢀ,Nꢀ-tetra- isopropylphosphorodiamidite with 4-N-benzoyl-
5ꢀ-O-(4, 4ꢀ-dimethoxytrityl)-2ꢀ-deoxy-cytidine 1 and 6-N,6-N,2ꢀ-O,3ꢀ-O-
tetrabenzoyladenosine 2 as the key step. Some dinucleotide derivatives bearing
different protecting groups were also synthesized and the selective deprotec-
tion conditions were studied in detail. The utility and efficiency of this approach
has been further demonstrated by its application to the synthesis of total DNA
dinucleotide pdCpdA 17 and total RNA dinucleotide 21.
INTRODUCTION
Recently, a general biosynthetic method for site-specific incorporation of un-
natural amino acids with novel steric or electronic properties into proteins has
been developed independently by the groups of Schultz (1–7) and Chamberlin
(8–11). This technique is a powerful tool for probing protein structure and function
and has many other important applications (12–15). The crucial step of the tech-
nique involves the construction of a misacylated suppressor tRNA bearing the de-
sired unnatural amino acid (16,17). Hecht and co-workers originally developed one
successful approach to this problem, which depends on the T4 RNA ligase mediated
∗Corresponding author.
197
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
REPRINTS
198
ZHU AND SCOTT
coupling of 2ꢀ(3ꢀ)-O-aminoacylated dinucleotide 5ꢀ-O-phosphorylcytidylyl-
(3ꢀ → 5ꢀ)adenosine (pCpA) to 3ꢀ-truncated run-off tRNA lacking the 3ꢀ-terminal
pCpA [tRNA(-CA)] (18–20). Chamberlin (8) and Schultz (2) simplified this method
by replacing the cytidine unit of pCpA with a 2ꢀ-deoxycytidine unit, resulting
in the synthesis of dinucleotide hybrid 5ꢀ-O-phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ →
5ꢀ)adenosine (pdCpA). It was shown that 2ꢀ(3ꢀ)-O-aminoacylated pdCpA deriva-
tives are still substrates of T4 RNA ligase and therefore can be coupled with a
truncated tRNA as efficiently as pCpA (2). Furthermore, the resulting aminoacy-
lated tRNA analogs conserve good compatibility with other enzymes involved in
protein biosynthesis (10).
Although the substitution of pCpA by pdCpA offers several advantages, such
as synthetic simplicity and product stability, the current synthetic routes to pdCpA
are tedious and rather inefficient (2,10,21). In this paper, we describe an improved
procedure which produces pdCpA in high yield under mild reaction conditions.
Furthermore, 5ꢀ-O-phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ → 5ꢀ)-2ꢀ-deoxy adenosine
(pdCpdA), a potential substitute for pCpA and pdCpA, was also efficiently pre-
pared by this strategy.
RESULTS AND DISCUSSION
The synthetic route to pdCpA is shown in Scheme 1. In order to reduce the
number of synthetic steps and increase the overall yield, we developed a one-pot
reaction to produce the dinucleotide 4-N-benzoyl-5ꢀ-O-(4, 4ꢀ-dimethoxytrityl)-2ꢀ-
deoxycytidylyl-{3ꢀ−OP-2-cyanoethyl)]→ 5ꢀ}-6-N,6-N, 2ꢀ-O, 3ꢀ-O-tetrabenzoyl-
adenosine (structure not shown in Scheme 1), a key intermediate for pdCpA.
Our strategy is based on employing 2- cyanoethyl N,N,Nꢀ,Nꢀ-tetraisopropyl-
phosphorodiamidite, rather than previously reported 2,2,2-trichloroethyl phospho-
rodichloridite, as a bifunctional phosphitylating reagent (22,23). Although the
2,2,2-trichloroethyl group has also been widely used as a phosphate(phosphite)
protecting group, the major problem associated with its use is difficulty in com-
plete removal (normally by reductive elimination with zinc-copper couple). In ad-
dition, the phosphitylation of 2,2,2-trichloroethyl phosphorodichloridite with nu-
cleosides should be performed at low temperature (−78◦C) (24). On the other hand,
2-cyanoethyl N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodiamidite possesses two reactive
centers (the two iPr2N groups) attached to the same phosphorus atom, allowing
it to react with two different properly protected nucleosides in the presence of
1H-tetrazole thus forming a dinucleotide linked by a phosphite triester under
very mild conditions. Another significant advantage of the use of 2-cyanoethyl
N,N,Nꢀ,Nꢀ-tetraisopropylphosphoro- diamidite is that in the final step of the din-
ucleotide preparation, all protecting groups from the heterocyclic bases (N-ben-
zoyl) and from the phosphates (2-cyanoethyl) can be cleaved at the same time by
treatment with aqueous ammonia.
4-N-benzoyl-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-2ꢀ-deoxycytidine 1 was chosen as
the dC component, and 6-N,6-N,2ꢀ-O,3ꢀ-O-tetrabenzoyladenosine 2 obtained from

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
199
NHBz
N
NHBz
N
DMTrO
N
O
DMTrO
N
O
(i)
O
O
(ii)
NHBz
N
2
O
P
HO
N
O
NC
HO
O
N
O
O
1
3
NBz2
O
P
NBz2
N
NBz2
N
N
P
N
O
N
N
N
N
N
O
NC
O
N
N
NC
O
HO
O
N
N
(i)
(iii)
O
O
1
BzO
(iv)
OBz
BzO
OBz
BzO
OBz
4
2
5
NH2
NHBz
N
NC
N
O
O
O
O
−O
P
O
N
O
P
O
N
O
O
O
−O
NC
NH2
NBz2
N
O
P
O
P
(V)
N
N
N
−O
O
N
O
O
NC
O
O
N
N
N
O
O
BzO
OBz
OH
HO
7 (pdCpA)
6
Scheme 1. Reagents and Conditions: (i) 1H-tetrazole, (iPr2N)2POCH2CH2CN, CH2Cl2, rt, 2 h;
(ii) (a) 1H-tetrazole, 2, CH2Cl2, rt, 4 h, (b) I2/THF/H2O/Pyridine, rt, 10 min, (c) 1% TFA in
CH2Cl2, rt, 5 min; (iii) (a) 1H-tetrazole, 1, CH2Cl2, rt, 4 h, (b) I2/THF/H2O/Pyridine, rt, 10
min, (c) 1% TFA in CH2Cl2, rt, 5 min. (iv) 1H-tetrazole, iPr2NP(OCH2CH2CN)2, CH2Cl2, rt,
3 h, (b) I2/THF/H2O/Pyridine, rt, 10 min; (v) NH4OH/CH3OH/1,4-dioxane, rt, 24 h. DMTr=di(p-
methoxyphenyl)phenylmethyl.
6-N-benzoyl- adenosine in high yield (83%) was employed as the “A” com-
ponent rather than the 6-N,2ꢀ-O,3ꢀ-O-tribenzoyl derivative because of its better
solubility and easy separation from the product by flash chromatography. Thus,
2ꢀ-deoxycytidine 1 was condensed with 2- cyanoethyl N,N,Nꢀ,Nꢀ-tetraisopropyl-
phosphorodiamidite in the presence of 1H-tetrazole to give phosphoramidite 3,
which, without isolation, was subsequently coupled to the protected adenosine 2.
After iodine oxidation of the resulting phosphite triester, selective removal of the
trityl group by 1% TFA in dichloromethane provided the phosphotriester 4 in 67%
overall yield. It was also observed that inverting the addition sequence of 1 and 2
(via intermediate 5) has little effect on the yield of 4 (60%). Conversion of 4 into
the corresponding 5ꢀ-phosphorylated product 6 was readily achieved by condens-
ing 4 with excess di(2-cyanoethyl) N,N-diisopropylphosphoramidite (2 equiv.),

ORDER
REPRINTS
200
ZHU AND SCOTT
NHBz
NHBz
N
NC
NC
N
O
P
O
O
HO
N
O
NHBz
N
O
O
N
O
O
O
N
N
NHBz
N
NHBz
N
O
P
O
P
HO
N
N
(i)
(ii)
O
N
N
O
O
O
NC
NC
N
O
N
O
RO OR
N
O
O
RO
OR
RO OR
8a. R = TBDMS
9a. R = TBDMS
O
b. ORRO =
O
O
TBDMS
O
10a. R =
b. ORRO =
O
b. ORRO =
O
(iv)
(iii)
NH2
NHBz
N
N
NC
O
O
O
−O
O
P
N
O
P
O
N
O
O
O
−O
O
NC
NH2
O
NHBz
N
O
P
N
−O
P
O
N
pdCpA (7)
N
(v)
(iv)
O
O
NC
O
N
N
O
N
N
O
O
RO OR
TBDMS
HO OH
12a. R =
b. ORRO =
11
O
O
Scheme 2. Reagents and Conditions: (i) (a) 3 (prepared in situ as shown in Scheme 1), 1H- tetrazole,
8, CH2Cl2, rt, 3 h, (b) I2/THF/H2O/Pyridine, rt, 10 min, (c) 1% TFA in CH2Cl2, rt, 5 min; (ii) (a)
1H-tetrazole, iPr2NP(OCH2CH2CN)2, CH2Cl2, rt, 3.5 h, (b) I2/THF/H2O/Pyridine, rt, 10 min; (iii)
1N HCl, rt, 3.5 h; (iv) NH4OH/CH3OH/1,4-dioxane, rt; (v) From 12a, TBAF/THF, rt, 2 h, From 12b,
1N HCl, 1.5 h, rt.
followed by iodine oxidation. After the simultaneous removal of benzoyl and
2-cyanoethyl groups with ammonium, pdCpA 7 was obtained in high overall yield
(Scheme 1).
The generality of this method was demonstrated by the preparation of analogs
of 4 bearing different protecting groups removable under neutral or acidic condi-
tions, thus allowing selective deprotection of the amino groups of the nucleotide
bases or of the two hydroxy groups of the ribose moiety, as depicted in Scheme 2.
These deprotection protocols are required to introduce a wide range of functionality
in the “unnatural” substrates to be used in future studies based on a theoretical pre-
diction that the RNA World could sustain formation of the carbon-carbon bond and
carbon-oxygen bond (25). Among them, partially protected dinucleotide 11 is an
especially important one for our current investigation of developing programmable
molecular machines used for organic synthesis. Dinucleotide 11 is a key interme-
diate in the synthesis of misacylated dinucleotides that are crucial precursors of our
polyprenoid and polysaccharide synthesis.
The 2ꢀ,3ꢀ-di-O-TBDMSoxy analogue 10a was initially chosen as a promis-
ing candidate. Following a procedure similar to the one described for synthesis of

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
201
O
P
CBz
C
RO
O
−O
O
O
O
−O
CBz
A(Bz)2
O
P
O
P
DMTrO
HO
(iii)
(i)
O
O
−O
+
O
O
NC
A(Bz)2
A
O
O
HO
OBz
O
OBz
O
13
14
HO
17 (pdCpdA)
15 R = H
(ii)
16 R = (NCCH2CH2)2P(O)
U
RO
O
O
P
OTBDMS
U
UBz
20 R = H
21 R = (NCCH2CH2)2P(O)
DMTrO
HO
(ii)
(i)
O
O
O
O
+
NC
UBz
O
HO OTBDMS
BzO OBz
O
18
19
BzO OBz
Scheme 3. Reagents and Conditions: (i) (a) 1H-tetrazole, 13, (iPr2N)2POCH2CH2CN, 14 or 1H-
tetrazole, 18, (iPr2N)2POCH2CH2CN, 19, CH2Cl2, rt, 3 h, (b) I2/THF/H2O/Pyridine, rt, 10 min,
(c) 1% TFA in CH2Cl2, rt, 5 min; (ii) (a) 1H-tetrazole, iPr2NP(OCH2CH2CN)2, CH2Cl2, rt, 3.5 h,
(b) I2/THF/H2O/Pyridine, rt, 10 min; (iii) NH4OH/CH3OH/1,4-dioxane, rt. CBz = 4-N-
benzoylcytosin-1-yl; A(Bz)2 =6-N,6-N-dibenzoyladenin-9-yl;
U
=
uracil-1-yl; UBz
=
3-N-
benzoyluracil-1-yl.
6, dinucleotide derivative 10a was obtained in 75% overall yield from 8a. How-
ever, the selective deprotection of 2ꢀ,3ꢀ-di-O-TBDMS groups from 10a proved
unexpectedly difficult. Many attempts were made to transform 10a into diol 11 by
using a variety of deprotection conditions (TBAF/THF, TBAF-HOAc/THF, HF-
Pyridine/THF), but were all unsuccessful. One possible explanation for this result
is that nucleophilic fluoride ions attack cyanoethyl groups of phosphate ester and
cleave them as a result of β-elimination since, when studied in model reactions, 8a
can be completely converted into 6-N-benzoyl adenosine under exactly the same
conditions as mentioned above. On the basis of these observations, we now re-
placed 2ꢀ,3ꢀ-di-O-TBDMS groups with an isopropylidene group, which is also an
acid-labile protecting group, to allow the synthesis of isopropylidene acetal 10b
in 69% overall yield from 8b. Several methods were also tried for removing the
acetonide unit from 10b. Both ferric chloride hexahydrate (26) and 95% TFA gave
a complex mixture of products, while aqueous acetic acid and conc.HCl-MeOH
(1:70) were ineffective. Finally, it was found that stirring 10b in 1N HCl for 3.5 h
at rt accomplished complete conversion (TLC monitoring), providing diol 11 in
90% yield after flash chromatography separation. As expected, dinucleotides 10a
and 10b were smoothly converted into 2ꢀ,3ꢀ-O protected pdCpA 12a and 12b by
ammonium hydroxide treatment. Both 11 and 12 can be further transformed into
pdCpA 7 in good yield under the conditions shown in Scheme 2.

ORDER
REPRINTS
202
ZHU AND SCOTT
In order to further show the utility of this useful one-pot procedure, we suc-
cessfully applied it to the synthesis of the fully protected pdCpdA 16 and the
total RNA dinucleotide 21 in high yields (Scheme 3). Upon treatment with aque-
ous ammonia, dinucleotide 16 can be transformed into pdCpdA 17. The synthesis
of pdCpdA as a substitute of pdCpA is especially interesting. As we know, both
2ꢀ-and 3ꢀ-O-aminoacylated pdCpA derivatives are substrates of T4 RNA ligase,
therefore we can infer that 3ꢀ-O-aminoacylated pdCpdA derivatives should also
be substrates. If this assumption is true, pCpA can be further replaced by pdCpdA
rather than pdCpA, from which 2ꢀ- and 3ꢀ-O-aminoacylated mixtures are always
obtained. In addition, pdCpdA derivatives are useful in determining the function-
ality of the free 3ꢀ(2ꢀ)-OH group of 2ꢀ( 3ꢀ)-O-aminoacylated pdCpA in the in vitro
protein synthesizing system. These investigations are currently under way in our
laboratory.
In conclusion, we have developed a novel and general approach to the syn-
thesis of the fully-protected phosphotriester 4 by using 2-cyanoethyl N,N,Nꢀ,Nꢀ-
tetraisopropyl- phosphorodiamidite as the coupling reagent. With good overall
yields and very mild reaction conditions, it allows the synthesis of analogs of
4, as illustrated by the preparation of DNA/RNA dinucleotides hybrids 10, 11 and
12, all of which are key precursors of pdCpA. In addition, the synthetic approach
described herein is versatile, as shown by the synthesis of total DNA dinucleotides
15, 16 and total RNA dinucleotides 20 and 21.
EXPERIMENTAL
1H (500 MHz), 13C (125 MHz) and 31P (202.5 MHz) NMR spectra were
recorded in CDCl3, DMSO-d6 or D2O on a Bruker ARX-500 spectrometer. All
compounds, except 7, 12a, 12b and 17, are diastereoisomers at 3ꢀ → 5ꢀ phosphorus
atom and instrumentally analyzed without separation, therefore, the number of pro-
tons and carbons are doubled in NMR spectra. Coupling constants are expressed in
Hz. LSI mass spectra were obtained on a VG analytical 70s high-resolution double-
focussing magnetic-sector mass spectrometer; MALDI mass spectra were obtained
on a Perseptive Biosystems Voyager XL-ELITE mass spectrometer. UV spectra
were recorded on a Varian DMS 90 UV- Visible spectrometer. λ are quoted in nm
and band intensities in parenthesis, as ε. High-performance liquid chromatogra-
phy (HPLC) was performed on a Hewlett Packard HP 1100 series HPLC system
equipped with a HP-3390A integrator. The column employed was a 250 mm × 22
mm reverse phase C-18 preparative (C-18 10U, Alltech) using a solvent system
consisting of 10 mM aqueous AcOH : CH3CN (linear gradient from 90:10 to 10:90
in 60 min) at a flow rate of 5 ml/min.
2-Cyanoethyl N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodiamidite (24), di(2-
cyanoethyl) N,N- biisopropylphosphoramidite (27) and nucleosides (1, 8b, 13,
14, 18, 19) were prepared following published procedures. Nucleoside 2 was
prepared by Schultz protocol. However, we modified this procedure by using

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
203
N-benzoyladenosine, rather than adenosine, as the starting material, resulting in
higher yield of 2. The synthesis of 8a was reported in our previous paper (28).
Thin-layer chromatographies (TLC) were performed on pre-coated 0.25 mm silica
gel 60F254 polyester plates (20 × 20 cm, Aldrich). Flash column chromatographies
were carried out on Mallinckrodt Silica Gel 150 (60-200 mesh). All solvent and
reagents were purified when necessary by standard literature methods. All air- and
water-sensitive reactions were conducted in anhydrous solvent and under nitrogen
or argon atmosphere. In the work-up stage, organic solutions were dried over an-
hydrous sodium sulfate. All evaporations were carried out under reduced pressure
using a rotary evaporator, unless stated otherwise.
4-N-benzoyl-2ꢀ-deoxycytidylyl-{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N,6-N,2ꢀ-O,
3ꢀ-O-tetra-benzoyl adenosine (4) To a solution of 1 (180 mg, 0.284 mmol) in
dichloromethane (1 ml) at rt was added 1H-tetrazole (25 mg, 0.357 mmol) and 2-
cyanoethyl N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodiamidite (107 mg, 0.355 mmol).
After stirring for 2 h at rt, 1H-tetrazole (25 mg, 0.357 mmol) and 2 (243 mg, 0.355
mmol) were added. The resulting mixture was stirred at rt for 4 h and then iodine
(270 mg, 1.063 mmol) in THF-H2O-pyridine (66:33:1, 2.7 ml) was added. After
10 min, the reaction mixture was poured into ethyl acetate (50 ml), washed with
0.2 M sodium bisulfite (10 ml × 2) and brine (10 ml), dried, filtered and concen-
trated. The residue was then stirred with 1% TFA solution in dichloromethane (25
ml) at rt for 5 min. The reaction was quenched by adding saturated aqueous sodium
hydrogen carbonate (5 ml). After separation, the organic phase was washed with
brine (5 ml), dried, filtered and evaporated. The crude product was purified by flash
column chromatography using dichloromethane-methanol (100:0 to 100:4) as the
eluent to give 4 (215 mg, 67%, 2 diastereoisomers, 1:1 ratio) as a white solid. UV
(CH2Cl2) λ 252 nm (ε 41 900), λmax 232 nm (ε 47 600). δH(CDCl3) 1.96 (2H, br
s), 2.34 (1H, m), 2.43 (1H, m), 2.71-2.80 (6H, m), 3.72-3.83 (4H, m), 4.17-4.31
(6H, m), 4.51-4.58 (4H, m), 4.70 (2H, m), 5.05 (1H, m), 5.14 (1H, m), 6.13 (2H,
m), 6.18 (1H, m), 6.21 (1H, m), 6.25 (1H, m), 6.29 (1H, m), 6.53 (1H, d, J 4.9),
6.55 (1H, d, J 4.9), 7.33-7.38 (16H, m), 7.45-7.48 (8H, m), 7.51-7.58 (8H, m),
7.82 (8H, d, J 7.6), 7.89-7.91 (8H, m), 7.93 (4H, d, J 7.4), 8.22 (1H, d, J 7.5),
8.26 (1H, d, J 7.5), 8.49 (1H, s), 8.50 (1H, s), 8.67 (1H, s), 8.69 (1H, s) and 9.08
(2H, br s); δC(CDCl3) 19.6 (2C), 39.6 (1C), 39.7 (1C), 61.3 (1C), 61.5 (1C), 62.5
(1C), 62.6 (1C), 66.7 (1C), 66.8 (1C), 70.6 (1C), 70.7 (1C), 73.9 (1C), 74.1 (1C),
78.7 (1C), 78.9 (1C), 81.2 (1C), 81.3 (1C), 86.3 (1C), 86.5 (1C), 86.8 (2C), 88.0
(1C), 88.2 (1C), 96.6 (2C), 113.1 (2C), 116.5 (1C), 116.6 (1C), 127.6 (arom.C, 2C),
127.8 (arom.C, 2C), 128.2 (arom.C, 2C), 128.4 (arom.C, 2C), 128.6 (arom.C, 8C),
128.8 (arom.C, 8C), 129.0 (arom.C, 4C), 129.4 (arom.C, 8C), 129.8 (arom.C, 4C),
129.9 (arom.C, 4C), 132.8 (arom.C, 2C), 133.2 (arom.C, 4C), 133.3 (arom.C, 2C),
133.8 (arom.C, 4C), 133.9 (arom.C, 2C), 134.0 (arom.C, 2C), 143.6 (1C), 143.8
(1C), 146.1 (2C), 151.9 (1C), 152.0 (1C), 152.5 (2C), 152.6 (2C), 152.7 (1C),
152.8 (1C), 162.0 (1C), 162.1 (1C), 165.2 (4C), 165.3 (1C), 165.4 (1C), 172.4 (2C)
and 172.5 (2C); HRMS [M+1]+, Found: 1130.3085. C57H49N9O15P requires m/z,
1130.3086.

ORDER
REPRINTS
204
ZHU AND SCOTT
4-N-benzoyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-2ꢀ-deoxycytidylyl-3ꢀ
-OP-2-cyanoethyl)]→ 5ꢀ}-6-N,6-N,2ꢀ-O,3ꢀ-O-tetrabenzoyladenosine (6) To a
solution of 4 (330 mg, 0.292 mmol) in dichloromethane (2 ml) at rt was added
1H-tetrazole (41 mg, 0.584 mmol) and di(2-cyanoethyl) N,N-diisopropylphos-
phoramidite (158 mg, 0.584 mmol). The resulting mixture was stirred at rt for 4
h, then iodine (297 mg, 1.169 mmol) in THF-H2O-pyridine (66:33:1, 3 ml) was
added. After 10 min, the reaction mixture was diluted with ethyl acetate (60 ml),
washed with 0.2 M sodium bisulfite (10 ml × 2) and brine (10 ml), dried, filtered and
concentrated. Flash column chromatography of the residue using dichloromethane-
methanol (100:2 to 100:4) as the eluent yielded 6 (377 mg, 98%, 2 diastereoiso-
mers, 1:1 ratio) as a white solid. UV (CH2Cl2) λmax 257 nm (ε 40 000), λ 236 nm
(ε 36 200). δH(CDCl3) 2.26 (1H, m), 2.31 (1H, m), 2.67-2.86 (14H, m), 4.18-
4.43 (18H, m), 4.55-4.57 (4H, m), 4.70 (2H, m), 5.14 (2H, m), 6.13-6.16 (2H,
m), 6.18 (1H, m), 6.21 (1H, m), 6.26 (1H, m), 6.29 (1H, m), 6.54 (1H, d, J
4.8), 6.56 (1H, d, J 4.9), 7.33-7.39 (16H, m), 7.45-7.59 (16H, m), 7.82 (8H,
m), 7.86 (4H, m), 7.88-7.95 (8H, m), 7.98 (2H, m), 8.50 (1H, s), 8.52 (1H, s)
and 8.68 (2H, s); δC(CDCl3) 19.6 (3C), 19.7 (3C), 39.1 (2C), 62.7 (6C), 66.7
(2C), 66.9 (1C), 67.1 (1C), 70.6 (1C), 70.7 (1C), 74.0 (2C), 77.5 (1C), 77.7 (1C),
81.2 (1C), 81.3 (1C), 83.5 (2C), 86.8 (1C), 86.9 (1C), 87.4 (1C), 87.5 (1C), 96.8
(2C), 116.5 (2C), 116.6 (3C), 116.7 (3C), 127.6 (arom.C, 2C), 127.7 (arom.C,
2C), 128.2 (arom.C, 2C), 128.4 (arom.C, 2C), 128.5 (arom.C, 4C), 128.6 (arom.C,
4C), 128.8 (arom.C, 8C), 129.0 (arom.C, 4C), 129.4 (arom.C, 8C), 129.8 (arom.C,
4C), 129.9 (arom.C, 4C), 133.2 (arom.C, 8C), 133.8 (arom.C, 4C), 133.9 (arom.C,
4C), 143.8 (1C), 143.9 (1C), 144.5 (2C), 152.0 (4C), 152.5 (1C), 152.6 (1C),
152.7 (1C), 152.8 (1C), 162.3 (1C), 162.4 (1C), 165.1 (4C), 165.3 (2C) and 172.3
(4C); HRMS [M+Na]+, Found: 1338.3084. C63H55N11NaO18P2 requires m/z,
1338.3099.
5ꢀ-O-Phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ→5ꢀ)adenosine (7) To a solution
of 6 (300 mg, 0.228 mmol) in dioxane (0.45 ml) and methanol (3 ml) was added
concentrated ammonium hydroxide (3.45 ml). The flask was capped and allowed to
stir for 24 h at rt. Then the solvent was removed under vacuum and the residue was
further lyophilized. The crude product was purified by HPLC. After the appropriate
fractions were collected and lyophilized, pdCpA 7 (130 mg, 90%) was obtained as a
white solid. (On a gram-scale preparation, the crude product should be pre-purified
on a Dowex anion exchange column using 0.025 M to 1 M aqueous ammonium
acetate as gradient eluent before the HPLC separation). UV (H2O) λmax 260 nm
(ε 21 000). δP(D2O) 2.32 and 3.46; δH(D2O)1.85 (1H, m), 2.29-2.33 (1H, m), 3.85
(2H, m), 3.97 (2H, m), 4.14 (1H, m), 4.20 (1H, m), 4.38 (1H, m), 4.69 (1H, m),
4.74 (1H, m), 5.92-5.96 (2H, m), 6.01 (1H, d, J 7.9), 7.83 (1H, d, J 7.8), 8.16
(1H, s) and 8.40 (1H, s); δC(D2O) 38.0 (1C), 63.9 (1C), 64.2 (1C), 69.5 (1C), 73.2
(1C), 75.2 (1C), 83.1 (1C), 84.5 (1C), 85.6 (1C), 86.6 (1C), 94.8 (1C), 117.7 (1C),
140.1 (1C), 142.7 (1C), 148.2 (1C), 149.2 (2C), 152.5 (1C) and 159.8 (1C); HRMS
[M+1]+, Found: 637.1190 C19H27N8O13P2 requires m/z, 637.1173.

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
205
4-N-benzoyl-2ꢀ-deoxycytidylyl-{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N
-benzoyl-2ꢀ,3ꢀ-di-O-(tert-butyl-dimethylsilyl)adenosine (9a) Following a simi-
lar procedure described for 4, 9a (515 mg, 82%, 2 diastereoisomers, 1:1 ratio) was
obtained from 8a (0.60 mmol scale) as a white solid. UV (CH2Cl2) λmax 260 nm
(ε 21 200), λ 234 nm (sh ε 11 900). δH(CDCl3) -0.11 (3H, s), -0.01 (3H, s), 0.04
(3H, s), 0.08 (3H, s), 0.12 (3H, s), 0.13 (6H, s), 0.15 (3H, s), 0.86 (9H, s), 0.89
(9H, s), 0.94 (9H, s), 0.95 (9H, s), 2.31 (1H, m), 2.45 (1H, m), 2.70-2.82 (6H, m),
3.79-3.87 (4H, m), 4.22-4.36 (10H, m), 4.41-4.56 (6H, m), 4.83 (1H, m), 4.90 (1H,
m), 5.10 (1H, m), 5.21 (1H, m), 6.01 (2H, m), 6.23 (2H, m), 7.46-7.52 (10H, m),
7.56-7.59 (4H, m), 7.89 (4H, d, J 7.1), 8.06 (4H, m), 8.29-8.34 (4H, m), 8.76 (1H,
s), 8.77 (1H, s), 9.23 (2H, br s) and 9.45 (2H, br s); δC(CDCl3) -5.0 (1C), -4.9
(1C), -4.8 (1C), -4.7 (1C), -4.6 (2C), -4.4 (1C), -4.3 (1C), 17.7 (1C), 17.8 (1C),
17.9 (2C), 19.6 (1C), 19.7 (1C), 25.5 (3C), 25.6 (3C), 25.7 (6C), 39.8 (2C), 61.2
(1C), 61.4 (1C), 62.4 (2C), 66.1 (1C), 66.7 (1C), 70.7 (1C), 71.2 (1C), 74.6 (1C),
74.9 (1C), 78.5 (1C), 78.7 (1C), 81.8 (1C), 82.3 (1C), 86.5 (2C), 87.6 (2C), 89.8
(1C), 89.9 (1C), 96.8 (2C), 116.4 (1C), 116.5 (1C), 123.8 (1C), 124.1 (1C), 127.7
(arom.C, 4C), 127.9 (arom.C, 2C), 128.0 (arom.C, 2C), 128.7 (arom.C, 2C), 128.8
(arom.C, 2C), 128.9 (arom.C, 4C), 132.8 (arom.C, 2C), 132.9 (arom.C, 2C), 133.1
(arom.C, 2C), 133.3 (arom.C, 1C), 133.4 (arom.C, 1C), 142.0 (1C), 142.4 (1C),
145.3 (2C), 149.6 (1C), 149.7 (1C), 151.3 (1C), 151.5 (1C), 152.4 (2C), 154.8 (2C),
162.3 (1C), 162.4 (1C), 165.1 (1C), 165.3 (1C) and 166.7 (2C); HRMS [M+Na]+,
Found: 1068.3842. C48H64N9NaO12PSi2 requires m/z, 1068.3848.
4-N-benzoyl-2ꢀ-deoxycytidylyl-{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N-
benzoyl-2ꢀ,3ꢀ-O- iso-propylideneadenosine (9b) Following a similar procedure
described for 4, 9b (720 mg, 70%, 2 diastereoisomers, 1:1 ratio) was obtained from
8a (1.20 mmol scale) as a white solid. UV (CH2Cl2) λmax 260 nm (ε 20 800), λ 232
nm (sh ε 10 400). δH(CDCl3) 1.42 (6H, s), 1.63 (6H, s), 2.26 (1H, m), 2.37 (1H, m),
2.63-2.81 (6H, m), 3.81 (4H, m), 4.17- 4.34 (12H, m), 4.51 (2H, m), 5.04-5.16 (4H,
m), 5.49 (2H, m), 6.17 (2H, m), 6.25 (2H, s), 7.46-7.49 (10H, m), 7.55-7.60 (4H,
m), 7.89 (4H, d, J 7.5), 8.03 (4H, d, J 7.5), 8.25-8.29 (4H, m), 8.76 (1H, s), 8.77
(1H, s), 9.29 (2H, br s) and 9.51 (2H, br s); δC(CDCl3) 19.5 (1C), 19.6 (1C), 25.3
(2C), 27.0 (2C), 39.7 (1C), 39.8 (1C), 61.4 (2C), 62.4 (2C), 67.6 (1C), 67.7 (1C),
78.7 (1C), 78.8 (1C), 80.9 (1C), 81.0 (1C), 84.0 (1C), 84.2 (1C), 85.4 (2C), 86.4
(1C), 86.5 (1C), 87.7 (1C), 87.8 (1C), 90.7 (1C), 90.8 (1C), 96.9 (2C), 114.8 (1C),
114.9 (1C), 116.5 (1C), 116.6 (1C), 123.8 (1C), 124.0 (1C), 127.8 (arom.C, 4C),
128.0 (arom.C, 2C), 128.1 (arom.C, 2C), 128.6 (arom.C, 2C), 128.7 (arom.C, 2C),
128.8 (arom.C, 4C), 132.8 (arom.C, 2C), 132.9 (arom.C, 2C), 133.1 (arom.C, 2C),
133.2 (arom.C, 1C), 133.3 (arom.C, 1C), 142.4 (1C), 142.6 (1C), 145.3 (2C), 149.8
(2C), 151.2 (1C), 151.4 (1C), 152.6 (2C), 154.9 (2C), 162.4 (2C), 165.2 (1C), 165.4
(1C) and 166.9 (2C); HRMS [M+1]+, Found: 858.2605. C39H41N9O12P requires
m/z, 858.2612.
4-N-benzoyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-2ꢀ-deoxycytidylyl-
{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N-benzoyl-2ꢀ,3ꢀ-di-O-(tert-butyldimethylsilyl)

ORDER
REPRINTS
206
ZHU AND SCOTT
adenosine (10a) Following a similar procedure described for 6, 10a (650 mg,
92%, 2 diastereoisomers, 1:1 ratio) was obtained from 9a (0.57 mmol scale) as a
white solid. UV (CH2Cl2) λmax 260 nm (ε 20 400), λ 232 nm (ε 14 700). δH(CDCl3)
-0.15 (3H, s), -0.11 (3H, s), 0.01 (3H, s), 0.02 (3H, s), 0.10 (6H, s), 0.12 (6H, s),
0.82 (9H, s), 0.84 (9H, s), 0.92 (18, s), 2.19 (1H, m), 2.25 (1H, m), 2.70-2.84 (12H,
m), 4.13-4.48 (28H, m), 4.82 (1H, m), 4.87 (1H, m), 5.05 (1H, m), 5.10 (1H, m),
5.99 (2H, m), 6.17 (1H, m), 6.21 (1H, m), 7.47-7.50 (10H, m), 7.54-7.56 (4H, m),
7.88 (4H, m), 7.95-8.03 (6H, m), 8.27 (1H, s), 8.30 (1H, s), 8.76 (1H, s), 8.77 (1H,
s), 8.88 (2H, br s) and 9.26 (2H, br s); δC(CDCl3) -4.8 (2C), -4.7 (2C), -4.6 (2C),
-4.4 (2C), 17.9 (2C), 18.0 (2C), 19.6 (3C), 19.7 (3C), 25.7 (6C), 25.8 (6C), 39.3
(2C), 62.5 (2C), 62.7 (6C), 66.8 (1C), 67.0 (1C), 71.1 (1C), 71.3 (1C), 74.6 (1C),
74.8 (1C), 77.3 (2C), 82.0 (1C), 82.3 (1C), 83.6 (2C), 87.5 (2C), 89.9 (2C), 96.8
(2C), 116.5 (3C), 116.6 (3C), 123.9 (1C), 124.1 (1C), 127.7 (arom.C, 4C), 128.0
(arom.C, 4C), 128.8 (arom.C, 4C), 129.0 (arom.C, 4C), 132.8 (arom.C, 4C), 133.3
(arom.C, 2C), 133.5 (arom.C, 2C), 142.3 (1C), 143.4 (1C), 144.4 (2C), 149.8 (2C),
151.4 (1C), 151.5 (1C), 152.5 (2C), 153.9 (2C), 162.3 (2C), 164.9 (2C), and 166.1
(2C); HRMS [M+Na]+, Found: 1254.4039. C54H71N11NaO15P2Si2 requires m/z,
1254.4043.
4-N-benzoyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-2ꢀ-deoxycytidylyl-
{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N-benzoyl-2ꢀ,3ꢀ-O-isopropylideneadenosine
(10b) Following a similar procedure described for 6, 10b (548 mg, 99%, 2 di-
astereoisomers, 1:1 ratio) was obtained from 9b (0.53 mmol scale) as a white solid.
UV (CH2Cl2) λmax 260 nm (ε 21 700), λ 234 nm (sh ε 8 600). δH(CDCl3) 1.38
(6H,s), 1.59 (3H, s), 1.60 (3H, s), 2.16 (1H, m), 2.22 (1H, m), 2.67-2.81 (14 H,
m), 4.15-4.49 (24 H, m), 4.99-5.05 (2H, m), 5.11-5.13 (2H, m), 5.45-5.48 (2H,
m), 6.09-6.13 (2H, m), 6.23-6.24 (2H, m), 7.45-7.48 (10H, m), 7.53-7.58 (4H, m),
7.87 (4H, d, J 7.4), 7.95 (1H, d, J 7.5), 7.96 (1H, d, J 7.5), 8.00 (4H, m), 8.26
(1H, s), 8.28 (1H, s), 8.78 (1H, s), 8.79 (1H, s), 8.92 (2H, s) and 9.45 (2H, br s);
δC(CDCl3) 19.6 (3C), 19.7 (3C), 25.3 (2C), 27.0 (2C), 39.2 (2C), 62.5 (2C), 62.8
(6C), 66.8 (1C), 67.8 (1C), 77.5 (2C), 81.0 (2C), 83.5 (2C), 84.1 (2C), 85.5 (2C),
87.6 (2C), 90.8 (2C), 97.0 (2C), 114.87 (1C), 114.9 (1C), 116.6 (3C), 116.7 (3C),
123.8 (1C), 123.9 (1C), 127.8 (arom.C, 4C), 128.0 (arom.C, 2C), 128.1 (arom.C,
2C), 128.8 (arom.C, 4C), 128.9 (arom.C, 4C), 132.9 (arom.C, 4C), 133.2 (arom.C,
4C), 142.6 (1C), 142.7 (1C), 144.4 (2C), 149.8 (2C), 151.3 (2C), 152.5 (2C), 154.6
(2C), 162.6 (2C), 165.1 (2C) and 166.8 (2C); HRMS [M+Na]+, Found: 1066.2639.
C45H47N11NaO15P2 requires m/z, 1066.2626.
4-N-benzoyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-2ꢀ-deoxycytidylyl-
{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N-benzoyladenosine (11) Dinucleotide 10b (35
mg, 0.033 mmol) was stirred with 1M HCl (2 ml) at rt. After 3.5 h, the starting
material was completely disappeared (TLC monitoring), then the reaction mix-
ture was neutralized by slowly adding saturated aqueous sodium hydrogen car-
bonate. The resulting solution was extracted with ethyl acetate (15 ml × 4). The
combined extracts were washed with H2O (5 ml) and brine (5 ml), dried , fil-
tered and concentrated. The residue was subjected to flash chromatography using

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
207
dichloromethane-methanol (100:4) as eluent to afford 11 (30 mg, 90%, 2 di-
astereoisomers, 1:1 ratio) as a white solid. UV (CH3CN) λmax 258 nm (ε 20 100), λ
232 nm (sh ε 15 300). δH(DMSO-d6) 2.40 (2H, m), 2.66 (2H, m), 2.87-2.94 (12H,
m), 4.16-4.39 (28H, m), 4.73 (2H, m), 5.04 (2H, m), 5.74 (2H, br s), 6.08 (2H, d,
J 5.2), 6.15-6.19 (2H, m), 7.36 (2H, br s), 7.52 (8H, m), 7.62 (4H, m), 7.99 (4H,
d, J 7.7), 8.04 (4H, d, J 7.8), 8.13 (2H, d, J 6.8), 8.66 (2H, s), 8.76 (2H, s), 11.18
(2H, br s) and 11.23 (2H, br s); δC(DMSO-d6) 19.0 (3C), 19.1 (3C), 38.0 (2C),
62.8 (6C), 66.5 (2C), 67.6 (2C), 69.9 (2C), 72.8 (1C), 72.9 (1C), 77.2 (1C), 77.3
(1C), 82.3 (1C), 82.4 (1C), 83.0 (1C), 83.1 (1C), 86.9 (2C), 87.8 (2C), 96.5 (2C),
118.4 (6C), 125.8 (2C), 128.5 (arom.C, 16C), 132.5 (arom.C, 2C), 132.8 (arom.C,
2C), 133.1 (arom.C, 2C), 133.3 (arom.C, 2C), 143.2 (2C), 145.1 (2C), 150.4 (2C),
151.7 (2C), 152.2 (2C), 154.2 (2C), 163.3 (2C), 165.7 (2C) and 167.3 (2C); HRMS
[M+1]+, Found: 1004.2513. C42H44N11O15P2 requires m/z, 1004.2494.
5ꢀ-O-Phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ → 5ꢀ)-2ꢀ,3ꢀ-O-(tert-butyldime-
thylsilyl)adenosine (12a) Following a similar procedure described for 7, 12a
(68 mg, 97%) was obtained from 10a (0.081 mmol scale) as a white solid, m/z
(MALDITOF) 865.25 ([M+1]+). The structure of 12a was further confirmed by
transforming it into pdCpA 7 upon treatment with 1M TBAF in THF.
5ꢀ-O-Phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ→5ꢀ)-2ꢀ,3ꢀ-O-isopropylideneaden-
osine (12b) Following a similar procedure described for 7, 12b (62 mg, 95%)
was obtained from 10b (0.096 mmol scale) as a white solid. UV (H2O) λmax 260
nm (ε 20 000). δP(D2O) -0.13 and 1.21; δH(D2O) 1.30 (3H, s), 1.51 (3H, s), 1.87
(1H, m), 2.23 (1H, m), 3.85 (2H, m), 3.95 (2H, m), 4.08 (1H, m), 4.50 (1H, m),
4.61 (1H, m), 5.05 (1H, dd, J 2.3 and 6.1), 5.31 (1H, dd, J 2.9 and 6.1), 5.87
(1H, m), 6.04 (1H, d, J 7.9), 6.08 (1H, d, J 2.9), 7.85 (1H, d, J 7.9), 8.12 (1H, s)
and 8.28 (1H, s); δC(D2O) 23.7 (1C), 25.5 (1C), 38.1 (1C), 64.0 (1C), 65.0 (1C),
75.6 (1C), 80.4 (1C), 83.2 (1C), 84.6 (1C), 84.7 (1C), 85.7 (1C), 90.0 (1C), 94.7
(1C), 114.4 (1C), 117.8 (1C), 140.7 (1C), 143.0 (1C), 147.7 (1C), 148.2 (1C), 148.8
(1C), 152.0 (1C), 159.5 (1C); HRMS [M+1]+, Found: 677.1459. C22H31N8O13P2
requires m/z, 677.1486.
4-N-benzoyl-2ꢀ-deoxycytidylyl-{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N,6-N, 3ꢀ-
O-tribenzoyl-2ꢀ-deoxy-adenosine (15) Following a similar procedure described
for 4, 15 (187 mg, 62%, 2 diastereoisomers, 1:1 ratio) was obtained from 13 (0.30
mmol scale) as a white solid. δH(CDCl3) 2.28 (1H, m), 2.44 (1H, m), 2.64-2.86 (8H,
m), 3.15 (1H, m), 3.17 (1H, m), 3.76 (4H, m), 4.17-4.53 (14H, m), 5.02 (1H, m), 5.16
(1H, m), 5.75 (2H, m), 6.18 (1H, m), 6.22 (1H, m), 6.62 (2H, m), 7.24-7.34 (8H, m),
7.45-7.48 (14H, m), 7.59-7.61 (4H, m), 7.81-7.86 (12H, m), 8.03 (4H, m), 8.18 (1H,
d, J 7.4), 8.23 (1H, d, J 7.4 Hz), 8.45 (1H, s), 8.50 (1H, s), 8.66 (1H, s), 8.67 (1H,
s) and 8.86 (2H, br s); δC(CDCl3) 19.5 (2C), 37.0 (1C), 37.2 (1C), 39.6 (1C), 39.7
(1C), 53.4 (2C), 61.2 (1C), 61.4 (1C), 62.5 (2C), 67.4 (2C), 74.5 (1C), 74.6 (1C),
78.5 (1C), 79.0 (1C), 83.2 (1C), 83.4 (1C), 84.5 (1C), 84.7 (1C), 86.2 (1C), 86.3
(1C), 87.6 (1C), 87.7 (1C), 96.8 (2C), 116.6 (1C), 116.7 (1C), 127.7 (arom.C, 4C),
128.6 (arom.C, 4C), 128.7 (arom.C, 8C), 128.8 (arom.C, 8C), 129.4 (arom.C, 8C),
129.7 (arom.C, 4C), 133.0 (arom.C, 2C), 133.1 (arom.C, 4C), 133.7 (arom.C, 2C),

ORDER
REPRINTS
208
ZHU AND SCOTT
133.8 (arom.C, 4C), 143.6 (1C), 143.7 (1C), 145.3 (2C), 151.7 (1C), 151.8 (1C),
152.3 (4C), 152.6 (1C), 152.7 (1C), 154.9 (2C), 162.4 (2C), 165.9 (2C), 172.3 (2C)
and 172.4 (2C); HRMS [M+Na]+, Found: 1032.2721. C50H44N9NaO13P requires
m/z, 1032.2694.
4-N-benzoyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-2ꢀ-deoxycytidylyl-
{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-6-N,6-N,3ꢀ-O-tribenzoyl-2ꢀ-deoxyadenosine
(16) Following a similar procedure described for 6, 16 (562 mg, 95%, 2 di-
astereoisomers, 1:1 ratio) was obtained from 15 (0.495 mmol scale) as a white
solid. UV (CH2Cl2) λmax 257 nm (ε 35 000), λ 234 nm (sh ε 27 500). δH(CDCl3)
2.22 (1H, m), 2.33 (1H, m), 2.66-2.87 (16H, m), 3.17 (1H, m), 3.21 (1H, m),
4.12-4.50 (24H, m), 5.10 (1H, m), 5.15 (1H, m), 5.77 (2H, m), 6.20 (2H, m), 6.61
(2H, m), 7.30 (2H, m), 7.34 (8H, m), 7.42-7.50 (12H, m), 7.59 (4H, m), 7.80-7.82
(8H, m), 7.88 (4H, m), 7.99 (1H, d, J 7.4), 8.00 (1H, d, J 7.2), 8.03-8.05 (4H, m),
8.47 (1H, s), 8.49 (1H, s) 8.67 (2H, s) and 9.30 (2H, br s); δC(CDCl3) 19.6 (3C),
19.7 (3C), 37.0 (1C), 37.1 (1C), 39.2 (2C), 46.4 (2C), 62.7 (6C), 66.6 (1C), 66.7
(1C), 67.6 (1C), 67.7 (1C), 74.4 (1C), 74.5 (1C), 77.6 (2C), 83.5 (2C), 84.7 (2C),
87.4 (1C), 87.5 (1C), 96.9 (2C), 116.6 (3C), 116.7 (3C), 127.7 (4C), 127.75 (1C),
127.8 (1C), 128.6 (2C), 128.7 (2C), 128.8 (8C), 128.9 (4C), 129.0 (4C), 129.4 (8C),
129.8 (4C), 132.9 (2C), 133.1 (4C), 133.2 (2C), 133.8 (1C), 133.9 (1C), 133.95
(1C), 134.0 (1C), 143.6 (2C), 143.5 (2C), 151.8 (1C), 151.9 (1C), 152.4 (4C), 152.7
(2C), 154.1 (2C), 162.6 (2C), 166.0 (2C) and 172.3 (4C); HRMS [M+Na]+, Found:
1218.2978. C56H51N11NaO16P2 requires m/z, 1218.2888.
5ꢀ-O-Phosphoryl-2ꢀ-deoxycytidylyl-(3ꢀ→5ꢀ)-2ꢀ-deoxyadenosine(17) Fo-
llowing a similar procedure described for 7, 17 (95 mg, 92%) was obtained from 16
(0.167 mmol scale) as a white solid. UV (H2O) λmax 260 nm (ε 21 700). δP(D2O)
0.17 and 1.59; δH(D2O) 1.47 (1H, m), 2.03 (1H, m), 2.41 (1H, m), 2.69 (1H, m),
3.78 (2H, m), 3.88 (2H, m), 4.02 (1H, m), 4.06 (1H, m), 4.52 (1H, m), 4.59 (1H, m),
5.84 (1H, d, J 7.3), 5.85 (1H, m), 6.21 (1H, m), 7.53 (1H, d, J 7.6), 7.93 (1H, s) and
8.22 (1H, s); δC(D2O) 37.6 (1C), 38.0 (1C), 63.8 (1C), 64.4 (1C), 70.2 (1C), 75.2
(1C), 82.5 (1C), 84.1 (1C), 84.9 (1C), 85.0 (1C), 95.5 (1C), 117.3 (1C), 138.9 (1C),
140.8 (1C), 147.7 (1C), 151.5 (2C), 154.1 (1C) and 163.4 (1C); HRMS [M+1]+,
Found: 621.1234. C19H26N8O12P2 requires m/z, 621.1224.
2ꢀ-O-tert-butyldimethylsilyluridylyl-{3ꢀ-OP-2-cyanoethyl)]→5ꢀ}-3-N,2ꢀ-
O,3ꢀ-O-tribenzoyl-uridine (20) Following a similar procedure described for 4,
20 (288 mg, 70%, 2 diastereoisomers, 62:38 ratio) was obtained from 18 (0.40
mmol scale) as a white solid. UV (CH2Cl2) λ 254 nm (ε 30 800), λmax 240 nm (ε 32
300). δH(CDCl3) major isomer: 0.09 (3H, s), 0.10 (3H, s), 0.86 (9H, s), 2.79 (2H,
m), 3.87 (1H, m), 3.95 (1H, m), 4.33-4.41 (3H, m), 4.47-4.56 (3H, m), 4.68-4.71
(1H, m), 4.90 (1H, m), 5.63-5.71 (3H, m), 5.81 (1H, m), 5.97 (1H, d, J 8.2), 6.30
(1H, d, J 5.8), 7.29-7.32 (2H, m), 7.35-7.39 (2H, m), 7.39-7.44 (2H, m), 7.50-
7.57 (2H, m), 7.60 (1H, m), 7.67 (1H, m), 7.76 (1H, d, J 8.1), 7.85 (2H, d, J
7.4), 7.92 (4H, m) and 8.90 (1H, s); minor isomer: 0.06 (3H, s), 0.08 (3H, s), 0.84
(9H, s), 2.79 (2H, m), 3.87 (1H, m), 3.95 (1H, m), 4.33-4.41 (3H, m), 4.47- 4.56
(3H, m), 4.68-4.72(1H, m), 4.94(1H, m), 5.63-5.71(3H, m), 5.81(1H, m), 5.82(1H,

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
209
d, J 8.2), 6.25 (1H, d, J 5.4 ), 7.29-7.32 (2H, m), 7.35-7.39 (2H, m), 7.39-7.44 (2H,
m), 7.50-7.57 (2H, m), 7.60 (1H, m), 7.67 (1H, m), 7.73 (1H, d, J 8.2), 7.84 (2H,
d, J 7.1), 7.92 (4H, m) and 8.84 (1H, s); δC(CDCl3) major isomer: -4.9 (2C), 18.0
(1C), 19.8 (1C), 25.6 (3C), 60.9 (1C), 63.0 (1C), 63.1 (1C), 67.2 (1C), 70.6 (1C),
73.3 (1C), 76.3 (1C), 81.1 (1C), 81.2 (1C), 83.2 (1C), 91.7 (1C), 102.5 (1C), 103.7
(1C), 116.5 (1C), 128.1 (arom.C, 1C), 128.3 (arom.C, 1C), 128.5 (arom.C, 2C),
128.6 (arom.C, 2C), 129.2 (arom.C, 2C), 129.8 (arom.C, 2C), 129.9 (arom.C, 2C),
130.5 (arom.C, 2C), 131.1 (arom.C, 1C), 134.0 (arom.C, 2C), 135.2 (arom.C,
1C), 139.9 (1C), 141.7 (1C), 149.4 (1C), 150.3 (1C), 161.6 (1C), 163.0 (1C),
165.4 (1C), 165.5 (1C) and 168.3 (1C); minor isomer: -5.0 (2C), 18.0 (1C), 19.7
(1C), 25.6 (3C), 61.1 (1C), 62.8 (1C), 62.9 (1C), 67.1 (1C), 70.5 (1C), 73.4 (1C),
76.3 (1C), 81.0 (1C), 81.1 (1C), 83.2 (1C), 92.1 (1C), 102.5 (1C), 103.6 (1C),
116.6 (1C), 128.1 (arom.C, 1C), 128.3 (arom.C, 1C), 128.5 (arom.C, 2C), 128.6
(arom.C, 2C), 129.2 (arom.C, 2C), 129.8 (arom.C, 2C), 129.9 (arom.C, 2C), 130.5
(arom.C, 2C), 131.1 (arom.C, 1C), 134.0 (arom.C, 2C), 135.2 (arom.C, 1C), 140.1
(1C), 142.1 (1C), 149.3 (1C), 150.2 (1C), 161.7 (1C), 162.9 (1C), 165.4 (1C), 165.6
(1C) and 168.4 (1C); HRMS [M+Na]+, Found: 1052.2783. C48H52N5NaO17PSi
requires m/z, 1052.2763.
2ꢀ-O-tert-butyldimethylsilyl-5ꢀ-O-[di(2-cyanoethyloxy)phosphoryl]-
uridylyl-{3ꢀ-OP-2-cyanoethyl)]→ 5ꢀ}-3-N,2ꢀ-O,3ꢀ-O-tribenzoyluridine (21)
Following a similar procedure described for 6, 21 (170 mg, 96%, 2 diastereoiso-
mers, 62:38 ratio) was obtained from 20 (0.146 mmol scale) as a white solid. UV
(CH2Cl2) λ 256 nm (ε 32 000), λmax 240 nm (ε 33 800). δH(CDCl3) major isomer:
0.09 (3H, s), 0.11 (3H, s), 0.86 (9H, s); minor isomer: 0.08 (3H, s), 0.10 (3H, s),
0.85 (9H, s); the remaining protons have the identical chemical shifts: 2.71-2.86
(6H, m), 4.29-4.62 (13H, m), 4.94 (1H, m), 5.64-5.81 (4H, m), 5.97 (1H, m), 6.30
(1H, d, J 4.6), 7.28-7.31 (2H, m), 7.35-7.38 (2H, m), 7.40-7.44 (2H, m), 7.49- 7.56
(3H, m), 7.60 (1H, m), 7.72 (1H, d, J 8.2), 7.84 (2H, m), 7.92 (4H, m) and 9.29
(1H, br s); δC(CDCl3) major isomer: -4.98 (1C), -4.96 (1C), 18.0 (1C), 19.7 (3C),
25.5 (3C), 62.8 (3C), 63.1 (1C), 66.1 (1C), 67.2 (1C), 70.5 (1C), 73.4 (1C), 74.9
(1C), 80.1 (1C), 81.0 (1C), 88.0 (1C), 91.1 (1C), 102.7 (1C), 103.5 (1C), 116.7
(3C), 128.2 (arom.C, 1C), 128.4 (arom.C, 1C), 128.5 (arom.C, 2C), 128.6 (arom.C,
2C), 129.2 (arom.C, 2C), 129.7 (arom.C, 2C), 129.8 (arom.C, 2C), 130.5 (arom.C,
2C), 131.13 (arom.C, 1C), 133.9 (arom.C, 2C), 135.2 (arom.C, 1C), 140.1 (1C),
140.6 (1C), 149.4 (1C), 150.2 (1C), 161.7 (1C), 163.0 (1C), 165.3 (1C), 165.4
(1C) and 168.4 (1C); minor isomer: -4.94 (1C), -4.92 (1C), 18.0 (1C), 19.6 (3C),
25.5 (3C), 62.8 (3C), 63.0 (1C), 66.5 (1C), 67.4 (1C), 70.4 (1C), 73.3 (1C), 74.9
(1C), 80.1 (1C), 81.0 (1C), 88.0 (1C), 92.2 (1C), 102.8 (1C), 103.5 (1C), 116.7
(3C), 128.1 (arom.C, 1C), 128.4 (arom.C, 1C), 128.5 (arom.C, 2C), 128.6 (arom.C,
2C), 129.2 (arom.C, 2C), 129.7 (arom.C, 2C), 129.8 (arom.C, 2C), 130.53 (arom.C,
2C), 131.1 (arom.C, 1C), 133.9 (arom.C, 2C), 135.23 (arom.C, 1C), 140.1 (1C),
141.1 (1C), 149.5 (1C), 150.1 (1C), 161.7 (1C), 162.9 (1C), 165.3 (1C), 165.4
(1C) and 168.6 (1C); HRMS [M+Na]+, Found: 1238.2907. C54H59N7NaO20P2Si
requires m/z, 1238.2957.

ORDER
REPRINTS
210
ZHU AND SCOTT
ACKNOWLEDGMENTS
This work was supported by grants from the Texas Advanced Technology
Research Program and the Robert A. Welch Foundation. We thank Dr. Clotide
Pichon-SantanderforhelpfuldiscussionandDr. HowardJ. Williamsforhisvaluable
suggestion of NMR data assignments.
REFERENCES
1. Noren, C.J.; Anthony-Cahill, S.J.; Griffith, M.C.; Schultz, P.G. Science 1989, 244,
182–188.
2. Robertson, S.A.; Noren, C.J.; Anthony-Cahill, S.J.; Griffith, M.C.; Schultz, P.G. Nu-
cleic Acids Res. 1989, 17, 9649–9660.
3. Mendel, D.; Ellman, J.A.; Schultz, P.G. J. Am. Chem. Soc. 1991, 113, 2758–2760.
4. Robertson, S.A.; Ellman, J.A.; Schultz, P.G. J. Am. Chem. Soc. 1991, 113, 2722–2729.
5. Ellman, J.A.; Mendel, D.; Schultz, P.G. Science 1992, 255, 197–200.
6. Mendel, D.; Ellman, J.A.; Chang, Z.; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G.
Science 1992, 256, 1798–1802.
7. Cload, S.T.; Liu, D.R.; Froland, W.A.; Schultz, P.G. Chemistry & Biology 1996, 3,
1033–1038.
8. Bain, J.D.; Glabe, C.G.; Dix, T.A.; Chamberlin, A.R. J. Am. Chem. Soc. 1989, 111,
8013–8014.
9. Bain, J.D.; Wacker, D.A.; Kuo, E.E.; Chamberlin, A.R. Tetrahedron 1991, 47, 2389–
2400.
10. Bain, J.D.; Diala, E.S.; Glabe, C.G.; Wacker, D.A.; Lyttle, M.H.; Dix, T.A.;
Chamberlin, A. R. Biochemistry 1991, 30, 5411–5421.
11. Steward, L.E.; Collins, C.S.; Gilmore, M.A.; Carlson, J.E.; Ross, J.B.A.; Chamberlin,
A.R. J. Am. Chem. Soc. 1997, 119, 6–11.
12. Hecht, S.M. Acc. Chem. Res. 1992, 25, 545–552.
13. Cornish, V.W.; Mendel, D.; Schultz, P.G. Angew. Chem., Int. Ed. Engl. 1995, 34,
621–633.
14. England, P.M.; Lester, H.A.; Davidson, N.; Dougherty, D.A. Proc. Natl. Acad. Sci.
USA 1997, 94, 11025–11030.
15. Hohsaka, T.; Kajihara, D.; Ashizuka, Y.; Murakami, H.; Sisido, M. J. Am. Chem. Soc.
1999, 121, 34–40.
16. Noren, C.J.; Anthony-Cahill, S.J.; Suich, D.J.; Noren, K.A.; Griffith, M.C.; Schultz,
P.G. Nucleic Acids Res. 1990, 18, 83–88.
17. Bain, J.D.; Wacker, D.A.; Kuo, E.E.; Lyttle, M.H.; Chamberlin, A.R. J. Org. Chem.
1991, 56, 4615–4625.
18. Heckler, T.G.; Chang, L.H.; Zama, Y.; Naka, T.; Hecht, S.M. Tetrahedron 1984, 40,
87–94.
19. Heckler, T.G.; Chang, L.H.; Zama, Y.; Naka, T.; Chorghade, M.S.; Hecht, S.M. Bio-
chemistry 1984, 23, 1468–1473.
20. Roesser, J.R.; Xu, C.; Payne, R.C.; Surratt, C.K.; Hecht, S.M. Biochemistry 1989, 28,
5185–5195.
21. Niinomi, T.; Sisido, M. Chem. Lett. 1993, 1305–1308.

ORDER
REPRINTS
SYNTHESIS OF pdCpA AND pdCpdA
211
22. Nielsen, J.; Dahl, O. Nucleic Acids Res. 1987, 15, 3626.
23. Gu, Q.-M.; Prestwich, G.D. J. Org. Chem. 1996, 61, 8642–8647.
24. Imai, J.; Torrence, P.F. J. Org. Chem. 1981, 46, 4015–4021.
25. Scott, A.I. Tetrahedron Lett. 1997, 38, 4961–4964.
26. Sen, S.E.; Roach, S.L.; Boggs, J.K.; Ewing, G.J.; Magrath, J. J. Org. Chem. 1997, 62,
6684–6686.
27. Uhlmann, E.; Engls, J. Tetrahedron Lett. 1986, 27, 1023–1026.
28. Zhu, X.-F.; Williams, H.J.; Scott, A.I. J. Chem. Soc. Perkin Trans. 1 2000, (15), 2305–
2306.
Received May 10, 2000
Accepted October 27, 2000

Request Permission or Order Reprints Instantly!
Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article’s rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly. Simply click on the "Request
Permission/Reprints Here" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers’
(AAP) website for guidelines on Fair Use in the Classroom.
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as
part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our Website
User Agreement for more details.
Order now!
Reprints of this article can also be ordered at
http://www.dekker.com/servlet/product/DOI/101081NCN100002081