Improved protocol for the synthesis of flexibly protected morpholino monomers from unprotected ribonucleosides

Article abstract of DOI:10.1080/15257770.2012.724491

An inexpensive and much improved protocol has been developed for the synthesis of protected morpholino monomers from unprotected ribonucleosides in high overall yield, using oxidative glycol cleavage and reductive amination strategy. Unlike the previous methods, the present strategy allows installing the exocyclic amine protections at a later stage, and thus avoids the use of expensive, or commercially unavailable, exocyclic amine-protected ribonucleosides as starting materials. To demonstrate the flexibility of the present method in choosing protecting groups, the monomers have been protected with several such groups of different deblocking properties at the exocyclic amine position.

Full text of DOI:10.1080/15257770.2012.724491

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Improved Protocol for the Synthesis of
Flexibly Protected Morpholino Monomers
from Unprotected Ribonucleosides
Sankha Pattanayak a , Sibasish Paul a , Bappaditya Nandi a & Surajit
Sinha a
a Department of Organic Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata, India
Published online: 12 Nov 2012.
To cite this article: Sankha Pattanayak , Sibasish Paul , Bappaditya Nandi & Surajit Sinha (2012):
Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers from Unprotected
Ribonucleosides, Nucleosides, Nucleotides and Nucleic Acids, 31:11, 763-782
To link to this article: http://dx.doi.org/10.1080/15257770.2012.724491
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Nucleosides, Nucleotides and Nucleic Acids, 31:763–782, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.724491
IMPROVED PROTOCOL FOR THE SYNTHESIS OF FLEXIBLY
PROTECTED MORPHOLINO MONOMERS FROM UNPROTECTED
RIBONUCLEOSIDES
Sankha Pattanayak, Sibasish Paul, Bappaditya Nandi, and Surajit Sinha
Department of Organic Chemistry, Indian Association for the Cultivation of Science,
Jadavpur, Kolkata, India
An inexpensive and much improved protocol has been developed for the synthesis of protected
2
morpholino monomers from unprotected ribonucleosides in high overall yield, using oxidative glycol
cleavage and reductive amination strategy. Unlike the previous methods, the present strategy allows
installing the exocyclic amine protections at a later stage, and thus avoids the use of expensive,
or commercially unavailable, exocyclic amine-protected ribonucleosides as starting materials. To
demonstrate the flexibility of the present method in choosing protecting groups, the monomers have
been protected with several such groups of different deblocking properties at the exocyclic amine
position.
Keywords Morpholino monomer; antisense agents; exocyclic amine protection; perio-
date cleavage
INTRODUCTION
Morpholino antisense oligonucleotides[1] are neutral DNA analogues
that are extensively used as research tools to evaluate the function of a specific
gene because they can inhibit the target mRNA translation predicatively.[2–4]
Since their first introduction to developmental biology by Heasman,[5] mor-
pholinos have continued to attract the attention of researchers who study
a wide range of organisms[6] including sea urchin,[7] zebrafish,[8–10] chick,
and mouse, etc. More recently, these also have found their growing ap-
plications in nanotechnology,[11] surface hybridization,[12,13] morpholino-
modified DNA and siRNA synthesis etc.[14–17] Unlike DNA, in morpholinos,
Received 14 July 2012; accepted 22 August 2012.
S. S. thanks the Department of Biotechnology, Government of India, for financial support by a grant
(No. BT/PR13426/BRB/10/761/2009). S. P., S. P., and B. N. are grateful to CSIR for their fellowships.
Address correspondence to Surajit Sinha, Department of Organic Chemistry, IACS, Raja S. C.
Mullick Road, Jadavpur, Kolkata, 700032 India. E-mail: ocss5@iacs.res.in
763

764
S. Pattanayak et al.
FIGURE 1 Structural differences between DNA and morpholino oligonucleotides.
the (deoxy)ribose rings are replaced with morpholine rings, which are linked
with diamidophosphate linkages (Figure 1).[18]
Developed by Summerton, the morpholino monomer units are prepared
by a routine periodate cleavage-Schiff base formation-reduction cycle on the
exocyclic amine protected ribonucleosides.[19,20] The choice of suitable pro-
tecting groups plays a pivotal role in morpholino oligomer synthesis.[21]
Attempted synthesis of these monomer units from unprotected ribonucle-
osides in aqueous solution was not considered as a practical process as a
major amount of undesired side products were formed.[20] During our on-
going project on modified morpholinos,[22,23] we were in need to synthe-
size morpholino monomers with exocyclic amine protections, which can
be deprotected in unconventional conditions like hydrogenolysis, photoly-
sis, etc. However, exocyclic amine protected ribonucleosides are available
with only a few (mostly base-labile benzoyl, acetyl or phenoxyacetyl, etc.)
protected forms, leaving little choice over protecting groups manipulation,
and they are expensive. Incorporation of some special protecting groups
on bases of ribonucleosides is not always straightforward, requiring addi-
tional protection–deprotection steps (peracylation-selective ester hydrolysis
or transient protection).[24] The search of the literature demonstrates the
need for better methods for exocyclic amines carbamate protection.[25,26]
Moreover, many mild protections like Pac or Fmoc are often not tolerated
in the reaction conditions of morpholine ring formation, and yet using
the protected ribonucleosides by existing procedures, the monomers are
obtained in poor to moderate yields.
These facts clearly indicate that there is further need to improve the
existing procedures. Therefore, we sought to explore the possibilities of
synthesizing morpholino monomers from inexpensive parent ribonucleo-
sides with unprotected heterocyclic bases, so that the protections can be
installed at a later stage. For wider bio or material science application, the
protecting group manipulation would be beneficial to synthesize engineered
morpholino oligomers whose syntheses are not compatible with the current
protection scheme. Herein, we present our findings on the preparation
of morpholino monomers from native ribonucleosides in high yield, and
with complete control over protecting group manipulation of the exocyclic
amines.

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 765
RESULTS AND DISCUSSION
Optimization of the Morpholino Monomer Synthesis. Our initial at-
tempts to synthesize morpholinothymidine monomer from 5-methyluridine
1 by means of the Summerton protocol[20] resulted in unsatisfactory yields
(entry 1, Table 1) and sluggish purification methods, which are consistent
with published works,[19] even if no exocyclic protection is required in the
case of 1. The complexity of the reaction and lower yields are possibly at-
tributed to many potential side reactions such as polymerization and hemi-
acetal formation of the reactive dialdehyde,[27] bisaldimine formation,[28]
no cyclization, glycosidic bond breakage, and overoxidation.[29] A number
of these side reactions can be avoided by protecting the 5ꢁ-OH functionality
as appropriately attempted by Chakhmakhcheva et al.[30] in the cases of uri-
dine and N -Bz adenosine. Accordingly, the 5ꢁ-OH was protected with a trityl
(Tr) group, and notably, significant improvement in yields was achieved by
installing the protections (Entries 1 and 2, Table 1).
On subsequent standardization, tert-butyldiphenylsilyl (TBDPS) was
found to be the protecting group of choice over Tr or TBS, owing to its
stability and ease of handling (Table 1). DMTr or TBS groups degrade to
some extent in the periodate cleavage step.[31,32] However, incomplete imine
reduction[33,34] (5%–10%) poses a problem during purification as the mono-
hydroxy products have very close Rf values to the desired morpholino, and
this was observed in all the reduction conditions employed (Entries 2, 3, 5, 6;
Table 1). Finally, on the TBDPS protected nucleoside 2,[35] the completion
of the reductive amination was achieved by using NaBH3CN in the presence
TABLE 1 Optimization of the protecting groups and reduction conditions
Overall yield from
Entry
R
Reducing agent
Additive
1 (%)a
1
2
3
4
5
6
7
H
Tr
Tr
Tr
TBS
TBDPS
TBDPS
NaCNBH3
NaCNBH3
NaBH(OAc)3
PtO2 – H2
NaCNBH3
NaCNBH3
NaCNBH3
–
–
–
–
–
–
12b
42c,d
38c
Trace
46c
48c
4◦ MS - AcOH
52
aColumn purified yields.
bYield after N-trityl protection.
cIncomplete reduction was observed.
dMixed with unreduced products.

766
S. Pattanayak et al.
of acetic acid[36] and 4A molecular sieves (Entry 7, Table 1). Column pu-
rification on silica gel afforded the desired monomer 3 in overall 52% yield
from 5-methyluridine. Considering the number of steps involved, this seems
to be an excellent overall yield. Subsequent N -tritylation and silyl deprotec-
tion afforded the morpholinothymidine monomer 5 (Scheme 1 and Entry 1,
Table 2). The developed protocol worked equally well on a larger scale, and
we got consistent yields with 10 g of 5-methyl-uridine.
˚
OR
OR
OH
OH
OR
O
O
O
B
O
B
O
B
B
B
ii
i
v
iii
N
H
N
N
HO
OH
HO
OH
Tr
4, 9, 15,
21, 27
Tr
2, 7, 13,
19, 25
1, 6, 12,
18, 24
3, 8, 14,
20, 26
5, 28
iv
O
O
NH2
NH2
O
N
OR
OH
N
N
N
N
N
N
NH
N
NH
N
N
NH
NH2
O
BPg
O
BPg
B =
N
O
N
O
N
v
N
N
24 - 28
1 - 5
6 - 9
12 - 15
18 - 21
O
Tr
Tr
NHBz
NHBz
10a, 16a, 22a
11, 17, 23
BPg
=
NH
N
N
N
N
N
N
N
N
NHi-Bu
N
O
R = TBDPS
22a, 23
10a, 11
16a, 17
SCHEME 1 General route for the synthesis of protected morpholino monomers. Reagents and con-
ditions: (i) TBDPSCl, imidazole in DMF for 1 and 24; TBDPSCl in Pyridine for 6 and 12; TBDP-
SCl, Et3N, DMAP in DCM+DMF (1:2) for 18. (ii) NaIO4, (NH4)2B4O7.4H20, MeOH, rt.; and then
◦
˚
NaCNBH3, AcOH, 4A MS, MeOH, rt. (iii) TrCl, Et3N, DMF, 0 C. (iv) BzCl, Et3N, THF for 9; BzCl,
pyridine, 0◦C then 30% aq. NH3, pyridine, 0◦C for 15; (i-BuCO)2O, Et3N, DMF, 140◦C for 21. (v) TBAF,
THF, rt.
To establish whether the above described approach is also amenable to
cytidine 6, adenosine 12 and guanosine 18, the same methodology was then
applied on these. Accordingly, the 5ꢁ-OH protected cytidine and adeno-
sine[35,37] with free exocyclic amine functionality were subjected to mor-
pholino ring formation. The overall yields obtained for 8 and 14 were 58%
and 68% from 6 and 12, respectively (entries 2 and 3, Table 2). Subsequent
trityl protection gave tritylated morpholinos 9 and 15 in excellent yields.
At this juncture, the exocyclic amine can be protected with the protect-
ing groups of choice (vide infra). These were benzoylated (Bz) to obtain
10a and 16a (entries 1 and 8, Table 3) and desilylated to obtain regular
morpholino monomers 11 and 17, respectively in excellent overall yields
(Scheme 1).

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 767
TABLE 2 Overall yields of morpholino monomers from ribonucleosides
Ribo-
nucleoside
Overall
Overall
Entry
1
Product
yielda,b (%)
Product
yielda,b (%)
52
44
2
58
37
3
4
68
34
42
24
5
53
43
aColumn purified yields.
bOverall yields calculated from parent ribonucleoside. R = TBDPS.
TABLE 3 Manipulation of exocyclic amine protections
Entry
B
Conditions
BzCI/Et3N
Ac2O/Et3N
FmocCI/Py
BPg
Yieldsa (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
Cyt (9)
CytBZ (10a)
CytAC (10b)
CytFmoc (10c)
CytPac (10d)
CytCbz (10e)
CytAlloc (10f)
CytpNZ (10g)
AdeBz (16a)
AdePac (16b)
AdeNBz (16c)
AdeCbz (16d)
Guai-Bu (22a)
Guadmf (22b)
84
84
67
83
68
50
72
81
68
71
62
75
76
PacCI/Py
CbzCI/HOBt/Et3N
AllocCI/Et3N
pNZCl/HOBt/Et3N
BzCI/Py
Ade (15)
Gua (21)
PacCI/Py
NBzCI/Et3N
CbzCI/LiHMDS
(i-BuCO)2O/Et3N
DMF-DMA
aIsolated yields.

768
S. Pattanayak et al.
Synthesis of morpholinoguanosine monomer required special attention.
A suspension of the protected guanosine 19[38] under the established condi-
tions yielded the desired compound 20 in an inseparable complex mixture
of products, presumably due to the reactivity and self aggregation of free
guanine moiety.[39,40] Exocyclic amine protected, 2-N -[(dimethylamino)-
methylidene]guanosine also resulted in a complex product mixture. After
watchful standardizations of the reactions, an optimum condition was found
where a clear solution of 19 was subjected to the reactions at a tempera-
ture below 10◦C for 1.5 h for both steps to obtain 20. Prolonged reaction
time leads to the formation of highly polar unidentified side products that
were difficult to purify and characterize. Successive steps afforded the mor-
pholinoguanosine monomer 23 (Scheme 1). To further broaden the scope
of our methodology, inosine 24 was converted to the corresponding mor-
pholino unit 28 following the established protocol (Scheme 1 and entry 5,
Table 2).
Manipulation of the Exocyclic Amine Protection. The choice of the ap-
propriate protecting groups has remained a crucial issue in successful exe-
cution of oligonucleotide synthesis.[41] To demonstrate the flexibility of the
present approach over classical methods, we included a range of exocyclic
amine protecting groups (Table 3) having diverse deblocking properties.
Our strategy bypasses both the classical methods to carry out the peracyla-
tions and thus overcomes the limitations.[24]
Base labile, sturdy benzoyl and acetyl protections are most common for
cytidine and were incorporated to 9 using the acylating agents, BzCl and
Ac2O in the presence of triethylamine (entries 1 and 2, Table 3) to obtain
10a and 10b in good yields. 9-Fluorenylmethoxycarbonyl (Fmoc) protection
has gain importance occasionally[42] because this group can be deprotected
under very mild basic conditions. Reacting 9 with an excess of FmocCl in the
presence of triethylamine, a satisfactory yield of 10c was achieved (entry 3,
Table 3). Phenoxyacetyl (Pac)[43] is another mild base labile protecting group
which has been popularized for “ultra mild” DNA synthesis. Pac-protected
cytidine morpholino 10d was prepared by reacting 9 with a solution of PacCl
in pyridine (entry 4, Table 3).
Benzyloxycarbonyl (Cbz)[44] and allyloxycarbonyl (Alloc)[45] are partic-
ularly important because they represent a set of protections that can be
deblocked under hydrogenolytic conditions, and are orthogonal to many
regular nucleoside protecting groups like Trityl, DMTr or TBDMS. Although
useful, introduction of these protections is not so straightforward, requiring
special reagents like l-CBz-3-ethylimidazolium salt[44,46] for Cbz protection
and allyl-1-benzotriazolyl carbonate[45] or 1-[(allyloxy)carbonyl]tetrazole[47]
for Alloc protection. In our case, a moderate yield for Cbz protected mor-
pholino 10e was obtained (entry 5, Table 3) using an excess of normal CbzCl
in combination with HOBt and triethyamine. Similarly, AllocCl was used to
obtain N -Alloc cytidine morpholino 10f (entry 6, Table 3) in modest yield.

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 769
The para–nitrobenzyloxycarbonyl (pNZ) protecting group has never been
used for exocyclic amine protections of nucleosides although it has been
used in peptide chemistry because of its orthogonality to many protecting
groups like Boc, Fmoc, and Alloc, and can be deprotected in near neutral
reductive conditions.[48] pNZ was introduced to obtain 10g in 72% yield.
For adenosine morpholino 15, acid chlorides like benzoyl (Bz), phe-
noxyacetyl (Pac), and para-nitrobenzoyl (NBz) chloride were employed for
the amine protections (entries 8, 9, 10, Table 3). Pac as a blocking group
for adenosine is known to minimize depurination.[49] NBz is expected to be
more labile than Bz under basic conditions.[50]
When we attempted carbamate protection in 15, CbzCl failed to re-
act with the exocyclic amine in many combinations of solvents and bases.
Uses of other activated reagents like l-CBz-3-ethylimidazolium salt,[44] benzyl-
1-benzotriazolyl carbonate—Et3N, benzyl-1-succinimidyl carbonate—Et3N,
etc., remained unsuccessful. Treatment of 15 with a reagent combination of
1,1ꢁ-carbonyldiimidazole (CDI) and benzyl alcohol did not afford the desired
product.[51] An attempt to reach the carbamate through the isocyanate inter-
mediate by reacting 15 with Boc2O, DMAP and then with benzyl alcohol was
also ineffective as the di-Boc protected nucleoside formed.[52] Gratifyingly,
when lithium bis(trimethylsilyl)amide (LiHMDS) was employed as a base
for the first time for this purpose, CbzCl treatment afforded the protected
morpholino 16d in 62% yield (entry 11, Table 3). Similarly, in guanosine
morpholino 21, base labile protections isobutyryl and dimethoxyformami-
dine were incorporated (entries 12 and 13, Table 3).
In summary, by the modification of Summerton’s procedure, we have
developed an improved protocol to the synthesis of morpholino monomers
from easily available, inexpensive starting materials. Though the chemistry
is the same, the present approach provides absolute control over protect-
ing group manipulation, which is sometimes essential during synthesis of
modified oligomers. Many new protecting groups were introduced which
were not easy to reach by conventional methods. Along with these, fewer
number of purification steps has made the monomer synthesis method
cost effective and easy to achieve, and this certainly makes the present
procedure useful in regular and N-functionalized morpholino monomers
synthesis.
EXPERIMENTAL SECTION
General Methods
All reagents were purchased from commercial sources and used without
˚
further purification, unless otherwise mentioned. Powdered 4 A molecular
sieves were purchased from Alfa Aesar and activated in microwave prior to

770
S. Pattanayak et al.
use. Solvents were purified and dried according to recommended proce-
dures. All reactions were carried out in oven-dried glassware under argon
atmosphere using anhydrous solvents unless otherwise mentioned. TLC was
monitored on aluminum backed Silica Gel 60 (F254) 0.25 mm plates (Merck,
Darmstadt, Germany) using methanol and dichloromethane mixtures. Com-
pounds were visualized using UV light (254 nm) and staining with a solution
of cerium ammonium molybdate or ninhydrin. Column chromatography
was performed on silica gel (100–200 mesh). The column was treated with
0.5% triethyamine in dichloromethane or pet ether prior to purify tritylated
compounds. Melting points are uncorrected. 1H and 13C NMR spectra were
recorded at 300 or 500 MHz and 75 or 125 MHz, respectively, using CDCl3
or DMSO-d6 as solvent. Chemical shifts (δ) are given in ppm relative to the
solvent residual peak or TMS or PPh3 as internal standard. The following ab-
breviations are used for multiplicity of NMR signals: s = singlet, d = doublet,
t = triplet, sept = septet, m = multiplet, br = broad.
Procedure for Morpholine-Ring Formation
General Procedure for the Synthesis of 7ꢁ-O-(tert-butyldiphenylsilyl) Mor-
pholino Monomers (3, 8, 14, 20, 26). 2.0 Mmol of 5-methyl uridine 1,
adenosine 6, cytidine 12, guanosine 18 and inosine 24 were protected with
tert-butyldiphenylsilyl group at 5ꢁ-OH position following literature proce-
dure. The crude 5ꢁ-O-(tert-butyldiphenylsilyl)-protected nucleoside [5ꢁ-O-
(tert-butyldiphenylsilyl)-5-methyl uridine 2,[53] 5ꢁ-O-(tert-butyldiphenylsilyl)-
cytidine 7,[54] 5ꢁ-O-(tert-butyldiphenylsilyl)-adenosine 13,[55] 5ꢁ-O-(tert-
Butyldiphenylsilyl)-guanosine 19,[56] 5ꢁ-O-(tert-butyldiphenylsilyl)-inosine
25[57]] was dissolved in methanol by slight heating in water bath. The so-
lution was cooled to room temperature (10◦C for 19) and were added
sodium meta-periodate (2.2 mmol) and ammonium biborate tetrahydrate
(2.2 mmol). The reaction mixture was stirred at room temperature (10◦C
for 19) for 1.5 h when the starting material was consumed and an envelope
of very closely spaced ninhydrin active spots (TLC) was generated. The re-
action mixture was filtered through a pad of celite and were added sodium
˚
cyanoborohydride (4.4 mmol) followed by activated powdered 4A molec-
ular sieves (300 mg/mmol) and glacial acetic acid (4.4 mmol) dropwise.
This was allowed to stir for another 2 h when the intermediate product was
completely reduced. This was again filtered through celite and evaporated
to dryness. The residue was dissolved in EtOAc or CHCl3 and washed with
water and brine. The solvent was dried over sodium sulfate and removed
under reduced pressure. Chromatographic purification (SiO2, elution with
1%–5% methanol in CH2Cl2) afforded the desired products (3, 8, 14, 20, 26)
as white solid in indicated yield (Table 2). Alternatively, the crude product
can be used in the next step for tritylation without further purification.

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 771
7ꢁ-O-(tert-Butyldiphenylsilyl) morpholinothymidine (3). White solid, yield
52% from 5-methyl uridine 1: mp 156–159◦C; IR (KBr) νmax 3312, 1701 cm−1;
1H NMR (500 MHz, CDCl3) δ 1.06 (s, 9H), 1.85 (br s, 1H), 1.88 (s, 3H), 2.59
(dd, 1H, J = 12.3, 10.3 Hz), 2.72 (dd, 1H, J = 13.0, 10.5 Hz), 3.05 (dd,
1H, J = 12.5, 2.0 Hz), 3.12 (dd, 1H, J = 12.5, 2.5 Hz), 3.70 (dd, 1H, J =
10.8, 5.7 Hz), 3.74 (dd, 1H, J = 10.8, 4.3 Hz), 3.86–3.89 (m, 1H), 5.68 (dd,
1H, J = 10.0, 2.5 Hz), 7.22 (s, 1H), 7.35–7.39 (m, 4H), 7.41–7.44 (m, 2H),
7.63 (t, 4H, J = 9.7 Hz); 13C NMR (125 MHz, CDCl3) δ 12.6, 19.4, 26.9,
46.7, 49.3, 64.8, 79.0, 80.7, 110.8, 127.8, 130.0, 133.3, 135.64, 135.67, 150.1,
163.8; HRMS (ESI) m/z [M + H]+, calcd for C26H34N3O4Si 480.2319, found
480.2313. Anal. Calcd for C26H33N3O4Si: C, 65.11; H, 6.93; N 8.76. Found:
C, 64.77; H, 6.98; N, 8.92.
7ꢁ-O-(tert-Butyldiphenylsilyl)-morpholinocytidine (8). White solid, yield
52% from cytidine 6: mp 212◦C (decomp.); IR (KBr) νmax 3458, 3383,
1
1660 cm−1; H NMR (500 MHz, CDCl3) δ 1.06 (s, 9H), 2.42 (dd, 1H, J =
12.3, 9.7 Hz), 2.68 (dd, 1H, J = 12.3, 11.3 Hz), 3.05 (d, 1H, J = 11.0 Hz),
3.24 (dd, 1H, J = 12.0, 1.5 Hz), 3.68 (dd, 1H, J = 10.7, 5.3 Hz), 3.75 (dd,
1H, J = 10.8, 4.3 Hz), 3.87–3.89 (m, 1H), 5.72 (dd, 1H, J = 9.5, 2.5 Hz),
5.78 (d, 1H, J = 7.5 Hz), 7.36–7.40 (m, 5H), 7.42–7.45 (m, 2H), 7.65 (t, 4H,
J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.8, 46.8, 49.8, 64.8, 78.8,
81.7, 94.8, 127.8, 129.8, 129.9, 133.2, 133.4, 135.6, 140.7, 155.5, 165.8; HRMS
(ESI) m/z [M + Na]+, calcd for C25H32N4O3SiNa 487.2141, found 487.2145.
7ꢁ-O-(tert-Butyldiphenylsilyl)-morpholinoadenosine (14). White solid,
yield 68% from cytidine 12: mp 65–67◦C; IR (KBr) νmax 3325, 1649,
1
1600 cm−1; H NMR (500 MHz, CDCl3) δ 1.05 (s, 9H), 2.79 (dd, 1H, J =
12.5, 11.0 Hz), 2.99 (dd, 1H, J = 12.0, 10.5 Hz), 3.13 (dd, 1H, J = 12.5,
2.0 Hz), 3.32 (dd, 1H, J = 12.4, 2.3 Hz), 3.70 (dd, 1H, J = 10.5, 5.5 Hz),
3.78 (dd, 1H, J = 10.8, 4.7 Hz), 3.95–3.98 (m, 1H), 5.83 (dd, 1H, J = 10.0,
2.5 Hz), 5.91 (br s, 2H), 7.33–7.37 (m, 4H), 7.39–7.43 (m, 2H), 7.64 (t, 4H,
J = 7.5 Hz), 7.91 (s, 1H), 8.35 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.4,
26.9, 47.1, 50.7, 64.8, 78.8, 80.8, 119.5, 127.8, 129.9, 133.33, 133.39, 135.68,
135.72, 138.3, 149.4, 153.3, 155.6; HRMS (ESI) m/z [M + H]+, calcd for
C26H33N6O2Si 489.2422, found 489.2427.
7ꢁ-O-(tert-Butyldiphenylsilyl)-morpholinoguanosine (20). White solid: mp
1
183–186◦C; IR (KBr) νmax 3331, 1692 cm−1; H NMR (500 MHz, CDCl3) δ
0.88 (s, 9H), 1.47 (t, 1H, J = 10.7 Hz), 1.57 (t, 1H, J = 10.5 Hz), 3.23
(d, 1H, J = 11.0 Hz), 3.32 (d, 1H, J = 11.0 Hz), 3.50 (dd, 1H, J = 10.5,
6.0 Hz), 3.68 (dd, 1H, J = 10.5, 4.0 Hz), 4.21–4.22 (m, 1H), 5.94 (d, 1H,
J = 9.5 Hz), 6.17 (br s, 2H), 7.05–7.57 (m, 26H), 11.80 (br s, 1H); 13C NMR
(125 MHz, CDCl3) δ 18.2, 25.9, 49.0, 52.1, 63.6, 75.9, 79.2, 117.0, 125.5,
126.6, 126.7, 126.8, 127.0, 128.3, 128.66, 128.72, 128.9, 132.2, 132.3, 134.0,
134.3, 134.4, 134.5, 150.0, 152.6, 158.1; HRMS (ESI) m/z [M + Na]+, calcd
for C45H46N6O3SiNa 769.3298, found 769.3297.

772
S. Pattanayak et al.
7ꢁ-O-(tert-Butyldiphenylsilyl)-morpholinoinosine (26). White solid, yield
53% from inosine 24: mp 177◦C (decomp.); IR (KBr) νmax 3283, 1692 cm−1;
1H NMR (500 MHz, DMSO-d6) δ 0.96 (s, 9H), 2.55 (dd, 1H, J = 12.5,
10.5 Hz), 2.92 (dd, 1H, J = 12.8, 2.0 Hz), 3.04 (dd, 1H, J = 12.0, 2.5 Hz),
3.16 (dd, 1H, J = 12.0, 10.5 Hz), 3.64–3.69 (m, 2H), 3.89–3.92 (m, 1H), 5.69
(dd, 1H, J = 10.0, 2.5 Hz), 7.33–7.37 (m, 4H), 7.41–7.45 (m, 2H), 7.58 (t,
4H, J = 7.4 Hz), 8.07 (s, 1H), 8.20 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ
19.4, 27.1, 46.6, 49.0, 65.2, 78.5, 80.3, 124.5, 128.3, 130.4, 133.4, 133.5, 135.6,
146.5, 148.4, 157.1; HRMS (ESI) m/z [M + H]+, calcd for C26H32N5O3Si
490.2274, found 490.2268.
Procedure for N-Tritylation
General Procedure for the Synthesis of 7ꢁ-O-(tert-Butyldiphenylsilyl)-N-
trityl Morpholino Monomers (4, 9, 15, 21, 27). To a solution of the 7ꢁ-O-(tert-
Butyldiphenylsilyl) morpholino monomers (3, 8, 14, 20, 26; 1.0 mmol) in
dry DMF (2.5 mL) were added triethylamine (1.50 mmol) and trityl chloride
(1.2 mmol) portion-wise at 0◦C. After stirring for 1 h, the reaction mixture
was quenched with methanol (0.5 mL) and concentrated under reduced
pressure. This was diluted with EtOAc (50 mL) and washed with water (3 ×
20 mL) and brine (20 mL). The solvent was dried over Na2SO4 and removed
in vacuo, and the residue was purified by SiO2 column chromatography
(0%–2% MeOH/CH2Cl2) to give the products (4, 9, 15, 21, 27) as foamy
white solid.
7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinothymidine (4). White
foamy solid, yield 89% from 3: IR (KBr) νmax 3188, 1692 cm−1; 1H NMR
(500 MHz, CDCl3) δ 0.88 (s, 9H), 1.28 (t, 1H, J = 10.3 Hz), 1.44 (t, 1H, J =
11.0 Hz), 1.67 (s, 3H), 3.18 (d, 1H, J = 12.0 Hz), 3.24 (dd, 1H, J = 10.5,
2.0 Hz), 3.52 (dd, 1H, J = 10.8, 5.8 Hz), 3.65 (dd, 1H, J = 10.8, 4.3 Hz),
4.15–4.18 (m, 1H), 6.01 (dd, 1H, J = 9.3, 2.3 Hz), 6.85 (s, 1H), 7.11–7.50 (m,
25H), 7.95 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 12.5, 19.4, 27.1, 50.0,
52.3, 64.7, 77.0, 77.3, 80.7, 110.5, 126.6, 127.8, 127.99, 128.05, 128.08, 128.18,
128.21, 129.4, 129.8, 129.9, 133.3, 133.4, 135.2, 135.59, 135.64, 135.7, 149.9,
163.6; HRMS (ESI) m/z [M + Na]+, calcd for C45H47N3O4SiNa 744.3234,
found 744.3233.
7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinocytidine (9). White
foamy solid, yield 85% from 8: IR (KBr) νmax 3333, 1645 cm−1; 1H NMR
(500 MHz, CDCl3) δ 0.87 (s, 9H), 1.11 (t, 1H, J = 10.3 Hz), 1.38 (t, 1H,
J = 11.0 Hz), 3.17 (d, 1H, J = 11.5 Hz), 3.31 (d, 1H, J = 11.0 Hz), 3.48
(dd, 1H, J = 10.5, 6.0 Hz), 3.65 (dd, 1H, J = 10.5, 4.5 Hz), 4.13–4.15 (m,
1H), 5.42 (d, 1H, J = 7.0 Hz), 6.09 (dd, 1H, J = 7.0, 1.5 Hz), 7.01 (d, 1H,
J = 7.0 Hz), 7.07–7.44 (m, 24H), 7.49 (d, 2H, J = 7.0 Hz), 7.93 (s, 1H);
13C NMR (125 MHz, CDCl3) δ 19.3, 26.7, 50.0, 52.8, 64.7, 76.88, 76.91, 81.4,
93.8, 126.3, 126.9, 127.2, 127.6, 127.7, 127.8, 127.9, 128.0, 128.8, 129.3, 129.6,

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 773
129.8, 133.1, 133.5, 135.50, 135.55, 141.2, 155.1, 165.5; HRMS (ESI) m/z [M
+ Na]+, calcd for C44H46N4O3SiNa 729.3237, found 729.3239.
7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinoadenosine (15). White
foamy solid, yield 87% from 14: IR (KBr) νmax 3327 cm−1; 1H NMR (500 MHz,
CDCl3) δ 0.94 (s, 9H), 1.59 (t, 1H, J = 11.0 Hz), 1.75 (t, 1H, J = 10.5 Hz),
3.34 (d, 1H, J = 12.0 Hz), 3.41 (d, 1H, J = 11.5 Hz), 3.58 (dd, 1H, J = 11.0,
6.0 Hz), 3.75 (dd, 1H, J = 9.8, 4.3 Hz), 4.32–4.34 (m, 1H), 5.91–5.96 (m,
2H), 6.30 (d, 1H, J = 9.5 Hz), 7.18 (t, 3H, J = 6.8 Hz), 7.26–7.56 (m, 22H),
7.62 (s, 1H), 8.35 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.8, 50.0,
53.3, 64.6, 77.0, 77.1, 79.9, 119.2, 126.5, 127.7, 127.9, 129.3, 129.7, 129.8,
133.2, 133.4, 135.6, 138.1, 149.4, 153.3, 155.5; HRMS (ESI) m/z [M + Na]+,
calcd for C52H50N6O3SiNa 857.3611, found 857.3611.
7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinoguanosine (21). White
solid, yield 34% from guanosine 18: mp 183–186◦C; IR (KBr) νmax 3331,
1
1692 cm−1; H NMR (500 MHz, CDCl3) δ 0.88 (s, 9H), 1.47 (t, 1H, J =
10.7 Hz), 1.57 (t, 1H, J = 10.5 Hz), 3.23 (d, 1H, J = 11.0 Hz), 3.32 (d, 1H,
J = 11.0 Hz), 3.50 (dd, 1H, J = 10.5, 6.0 Hz), 3.68 (dd, 1H, J = 10.5, 4.0 Hz),
4.21–4.22 (m, 1H), 5.94 (d, 1H, J = 9.5 Hz), 6.17 (br s, 2H), 7.05–7.57 (m,
26H), 11.80 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 18.2, 25.9, 49.0,
52.1, 63.6, 75.9, 79.2, 117.0, 125.5, 126.6, 126.7, 126.8, 127.0, 128.3, 128.66,
128.72, 128.9, 132.2, 132.3, 134.0, 134.3, 134.4, 134.5, 150.0, 152.6, 158.1;
HRMS (ESI) m/z [M + Na]+, calcd for C45H46N6O3SiNa 769.3298, found
769.3297.
7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinoinosine (27). White
1
solid, yield 94% from 26: mp 145–148◦C; IR (KBr) νmax 1699 cm−1; H
NMR (500 MHz, CDCl3) δ 0.97 (s, 9H), 1.61 (t, 1H, J = 11.3 Hz), 1.74 (t,
1H, J = 10.5 Hz), 3.34 (d, 1H, J = 12.0 Hz), 3.42 (d, 1H, J = 11.0 Hz), 3.61
(dd, 1H, J = 10.8, 5.7 Hz), 3.77 (dd, 1H, J = 10.8, 4.7 Hz), 4.32–4.34 (m,
1H), 6.25 (dd, 1H, J = 10.0, 2.0 Hz), 7.19–7.58 (m, 25H), 7.66 (s, 1H), 8.23
(s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.2, 26.8, 49.9, 53.4, 64.6, 76.9, 77.2,
80.3, 126.6, 127.7, 127.9, 129.2, 129.7, 129.8, 133.3, 135.5, 137.8, 146.0, 148.3,
159.3; HRMS (ESI) m/z [M + Na]+, calcd for C45H45N5O3SiNa 754.3189,
found 754.3188.
Procedures for Exocyclic Amine Protection
N4-Benzoyl-7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl
Morpholinocytidine
(10a). To a solution of 9 (75 mg, 0.106 mmol) in dry THF (1 mL) were
added Et3N (30 μL, 0.212 mmol) and benzoyl chloride (19 μL, 0.160 mmol)
dropwise at 0◦C. The reaction mixture was stirred at room temperature for
1.5 h and quenched with few drops of water. The residual mass obtained
after solvent removal was diluted with CHCl3 (5 mL) and washed with
saturated NaHCO3 and brine. SiO2 column purification (0%–2.5% MeOH
in DCM) yielded the product 10a as a foamy white solid (72 mg, 84%): IR

774
S. Pattanayak et al.
(KBr) νmax 3333, 1645 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.97 (s, 9H), 1.23
(t, 1H, J = 10.7 Hz), 1.54 (t, 1H, J = 11.3 Hz), 3.26 (d, 1H, J = 12.0 Hz),
3.53 (d, 1H, J = 11.0 Hz), 3.63 (dd, 1H, J = 11.0, 5.5 Hz), 3.76 (dd, 1H, J =
10.8, 4.3 Hz), 4.26–4.28 (m, 1H), 6.21 (dd, 1H, J = 9.3, 1.7 Hz), 7.17–7.59
(m, 30H), 7.88 (d, 2H, J = 7.5 Hz), 8.81 (br s, 1H); 13C NMR (125 MHz,
CDCl3) δ 19.3, 26.9, 49.9, 53.0, 64.8, 77.1, 82.2, 96.7, 126.4, 126.5, 127.82,
127.84, 127.96, 128.1, 128.2, 128.3, 128.5, 129.0, 129.4, 129.9, 130.2, 130.3,
133.2, 133.5, 135.5, 135.6, 135.7, 135.8, 135.9, 136.0, 144.7, 154.0, 162.5,
171.0; HRMS (ESI) m/z [M + Na]+, calcd for C51H50N4O4SiNa 833.3499,
found 833.3490.
N4-Acetyl-7ꢁ-O-(tert-Butyldiphenylsilyl)-N-trityl Morpholinocytidine (10b).
To a stirred solution of 9 (200 mg, 0.28 mmol) in dry THF (5 mL) were
added Et3N (117 μL, 0.84 mmol) and acetic anhydride (60 μL, 0.65 mmol)
dropwise at 0◦C. After stirring at room temperature for overnight, usual work
up and chromatographic purification (0%–2.5% MeOH in DCM) afforded
the product 10b as a foamy white solid (176 mg, 84%): mp 118–120◦C; IR
(KBr) νmax 1722, 1672 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.89 (s, 9H), 1.10
(dd, 1H, J = 10.8, 9.7 Hz), 1.45 (t, 1H, J = 11.3 Hz), 2.13 (s, 3H), 3.18 (d,
1H, J = 11.5 Hz), 3.42 (d, 1H, J = 11.5 Hz), 3.55 (dd, 1H, J = 10.5, 5.5 Hz),
3.66 (dd, 1H, J = 10.8, 4.3 Hz), 4.17–4.19 (m, 1H), 6.11 (dd, 1H, J = 9.3,
1.7 Hz), 7.11–7.49 (m, 25H), 7.50 (d, 2H, J = 7.0 Hz), 8.59 (br s, 1H); 13
C
NMR (125 MHz, CDCl3) δ 19.3, 25.0, 26.9, 49.9, 53.0, 64.7, 77.0, 77.4, 82.2,
96.5, 126.5, 127.8, 127.9, 128.0, 128.2, 129.4, 129.87, 129.94, 133.2, 133.5,
135.6, 135.7, 144.5, 154.4, 162.7, 171.0; HRMS (ESI) m/z [M + Na]+, calcd
for C46H48N4O4SiNa 771.3343, found 771.3344.
N4-(9-Fluorenylmethoxycarbonyl)-7ꢁ-O-(tert-butyldiphenylsilyl)-N -trityl
Morpholinocytidine (10c). To a solution of 9 (75 mg, 0.106 mmol) in dry
pyridine (1 mL) was added FmocCl (44 mg, 0. 170 mmol) portion-wise
at 0◦C. The reaction mixture was stirred at room temperature for 3 h
and quenched with few drops of water. The residual mass obtained after
solvent removal was diluted with CHCl3 (15 mL) and washed with saturated
NaHCO3, brine and dried over Na2SO4. The solvent was removed and
SiO2 column purification (5–20% EtOAc in petroleum ether) of the crude
product yielded 10c as a white foamy solid (66 mg, 67%): IR (KBr): νmax
1
1746, 1678, 1626 cm−1. H NMR (500 MHz, CDCl3) δ 0.96 (s, 9H), 1.17
(t, 1H, J = 10.0 Hz), 1.52 (t, 1H, J = 11.3 Hz), 3.24 (d, 1H, J = 12.0 Hz),
3.49 (d, 1H, J = 9.0 Hz), 3.61 (dd, 1H, J = 10.5, 5.5 Hz), 3.73 (dd, 1H,
J = 11.0, 4.5 Hz), 4.22–4.25 (m, 2H), 4.42–4.50 (m, 2H), 6.18 (d, 1H, J =
8.5 Hz), 6.98 (d, 1H, J = 7.0 Hz), 7.15–7.18 (m, 3H), 7.27–7.35 (m, 12H),
7.39–7.49 (m, 10H), 7.50–7.58 (m, 7H), 7.76 (d, 2H, J = 7.5 Hz); 13C NMR
(125 MHz, CDCl3) δ 19.3, 26.9, 46.8, 49.9, 53.5, 64.8, 68.2, 77.0, 82.2, 94.6,
120.2, 120.3, 125.1, 126.5, 127.3, 127.8, 127.9, 128.1, 129.4, 129.9, 133.2,
133.5, 135.6, 135.7, 141.5, 143.3, 144.5; HRMS (ESI) m/z [M + Na]+, calcd
for C59H57N4O5SiNa 952.3996, found 952.3948.

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 775
N4-Phenoxyacetyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Morpholinocyti-
dine (10d). A suspension of phenoxyacetic acid (1 g, 6.57 mmol) in toluene
was treated with oxalyl chloride (0.68 mL, 7.9 mmol) in the presence of one
drop of DMF at 0◦C and left to stir at rt for overnight. The volatile substances
were evaporated in low vacuum to obtain a 1.3 M solution of phenoxyacetyl
chloride in toluene. The solution of this reagent (75 μL, 0.098 mmol)
was added dropwise to a stirred solution of 9 (25 mg, 0.035 mmol) in dry
pyridine at 0◦C and kept for 2 h. Usual work up and silica gel purification
(0%–2% MeOH in DCM) yielded the product 10d (24 mg, 83%) as a white
foamy solid. IR (KBr): νmax 3389, 1666 cm−1. 1H NMR (500 MHz, CDCl3) δ
0.99 (s, 9H), 1.21 (t, 1H, J = 10.3 Hz), 1.56 (t, 1H, J = 11.3 Hz), 3.27 (d,
1H, J = 11.5 Hz), 3.54 (d, 1H, J = 11.0 Hz), 3.64 (dd, 1H, J = 10.8, 5.8 Hz),
3.77 (dd, 1H, J = 10.8, 4.3 Hz), 4.26–4.28 (m, 1H), 4.61 (s, 2H), 6.23 (d,
1H, J = 8.5 Hz), 6.94 (d, 2H, J = 8.0 Hz), 7.07 (t, 1H, J = 7.3 Hz), 7.21 (t,
3H, J = 6.8 Hz), 7.26–7.38 (m, 14H), 7.42–7.55 (m, 10H), 7.60 (d, 2H, J =
8.0 Hz), 9.12 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.9, 49.9, 53.1,
64.8, 67.4, 77.4, 82.3, 96.1, 114.8, 122.8, 126.6, 127.81, 127.84, 128.0, 129.4,
129.8, 129.9, 130.0, 133.2, 133.5, 135.6, 135.7, 145.1, 154.5, 156.7, 161.2,
168.5; HRMS (ESI) m/z [M + Na]+, calcd for C52H52N4O5SiNa 863.3605,
found 863.3604.
N4-Benzyloxycarbonyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Morpholino-
cytidine (10e). A solution of 9 (25 mg, 0.035 mmol), HOBt (9.5 mg,
0.070 mmol) and Et3N (24 μL, 0.72 mmol) in DCM was treated with CbzCl
(15 μL, 0.106 mmol), stirred for 8 h and quenched with water (0.5 mL).
Usual work up and elution with 0%–3% MeOH in DCM over silica gel yielded
the desired product 10e (20 mg, 68%) as a white foamy solid. IR (KBr): νmax
3419, 1747, 1662 cm−1. 1H NMR (500 MHz, CDCl3) δ 0.96 (s, 9H), 1.17 (t,
1H, J = 10.0 Hz), 1.52 (t, 1H, J = 11.3 Hz), 3.25 (d, 1H, J = 11.5 Hz), 3.51
(d, 1H, J = 11.0 Hz), 3.61 (dd, 1H, J = 10.8, 5.8 Hz), 3.74 (dd, 1H, J = 10.8,
4.8 Hz), 4.24–4.26 (m, 1H), 5.19 (s, 2H), 6.18 (d, 1H, J = 8.5 Hz), 7.02 (d,
1H, J = 7.5 Hz), 7.17–7.19 (m, 3H), 7.28–7.47 (m, 25H), 7.51 (d, 2H, J =
7.0 Hz), 7.58 (d, 2H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.9,
49.9, 53.0, 64.8, 68.1, 77.4, 82.2, 94.6, 126.6, 127.8, 127.9, 128.0, 128.5, 128.9,
129.4, 129.9, 133.2, 133.5, 135.1, 135.6, 135.7, 144.4, 152.3, 154.4, 162.1;
HRMS (ESI) m/z [M + Na]+, calcd for C52H52N4O5SiNa 863.3605, found
863.3604.
N4-Allyloxycarbonyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Morpholinocy-
tidine (10f). To a stirred solution of 9 (50 mg, 0.071 mmol) in dry CH2Cl2
were added Et3N (39 μL, 0.28 mmol) and AllocCl (9 μL, 0.085 mmol) drop-
wise at 0◦C. After 6 h at room temperature another batch of AllocCl (18 μL,
0.17 mmol) was added and left to stir for another 24 h. The mixture was
diluted with CHCl3 and washed with saturated NaHCO3 solution and brine.
The organic layer was dried over Na2SO4 and column purification over sil-
ica gel (0%–3% MeOH in DCM) afforded the title compound 10f (28 mg,

776
S. Pattanayak et al.
50%) as a white foamy solid. IR (KBr): νmax 3321, 1745, 1670 cm−1. 1H NMR
(500 MHz, CDCl3) δ 0.94 (s, 9H), 1.16 (t, 1H, J = 10.0 Hz), 1.50 (t, 1H, J =
11.3 Hz), 3.23 (d, 1H, J = 12.0 Hz), 3.49 (d, 1H, J = 11.0 Hz), 3.59 (dd, 1H,
J = 10.8, 5.8 Hz), 3.72 (dd, 1H, J = 11.0, 4.5 Hz), 4.22–4.24 (m, 1H), 4.64 (d,
1H, J = 5.5 Hz), 5.27 (d, 1H, J = 10.5 Hz), 5.33 (dd, 1H, J = 17.5, 1.0 Hz),
5.87–5.95 (m, 1H), 6.18 (d, 1H, J = 8.5 Hz), 6.99 (d, 1H, J = 7.5 Hz), 7.16
(t, 3H, J = 7.0 Hz), 7.26–7.34 (m, 11H), 7.38–7.51 (m, 12H), 7.60 (d, 2H,
J = 7.3 Hz); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.9, 49.9, 53.0, 64.8, 66.9,
77.4, 82.2, 94.5, 119.3, 126.5, 127.81, 127.83, 128.0, 129.4, 129.86, 129.94,
131.5, 133.2, 133.5, 135.6, 135.7, 144.4, 152.1, 154.4, 162.1; HRMS (ESI) m/z
[M + Na]+, calcd for C48H50N4O5SiNa 813.3448, found 813.3448.
N4-(4-Nitrobenzyloxycarbonyl)-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Mor-
pholinocytidine (10g). To a solution of the nucleoside 9 (50 mg,
0.071 mmol), HOBt (11 mg, 0.078 mmol) and Et3N (40 μL, 0.28 mmol) in
DCM (2 mL) was added a solution of 4-nitrobenzyl chloroformate (17 mg,
0.078 mmol) in DCM (0.25 mL) dropwise and stirred at rt for 6 h. Another
batch of HOBt (11 mg, 0.078 mmol) and chloroformate reagent (17 mg,
0.078 mmol) was added and left to stir for 5 h. The reaction was quenched
with few drops of water, diluted with CHCl3 and usual work up was per-
formed. Silica gel chromatography yielded the desired compound (10g) as
a foamy white solid (45 mg, 72%). IR (KBr): νmax 3439, 1751, 1670, 1522,
1
1347 cm−1. H NMR (500 MHz, CDCl3) δ 0.95 (s, 9H), 1.17 (m, 1H), 1.52
(t, 1H, J = 11.3 Hz), 3.25 (d, 1H, J = 11.5 Hz), 3.49 (m, 1H), 3.61 (dd, 1H,
J = 10.8, 5.3 Hz), 3.73 (dd, 1H, J = 10.8, 4.3 Hz), 4.23–4.25 (m, 1H), 5.25
(dd, 2H, J = 20, 13 Hz), 6.16 (d, 1H, J = 7.5 Hz), 6.98 (m, 2H), 7.17 (t,
3H, J = 7.0 Hz), 7.26–7.35 (m, 10H), 7.39–7.54 (m, 12H), 7.56 (d, 2H, J =
7.5 Hz), 8.15 (br s, 1H), 8.22 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 18.6,
26.1, 49.2, 52.3, 64.0, 65.6, 76.7, 81.5, 93.8, 123.1, 123.3, 125.8, 126.4, 127.1,
127.2, 127.9, 128.6, 129.1, 129.2, 132.4, 132.8, 134.9, 135.0, 141.6, 144.0,
147.4, 151.3, 161.2; HRMS (ESI) m/z [M + Na]+, calcd for C52H51N5O7SiNa
908.3455, found 908.3455.
N6-Benzoyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Morpholinoadenosine
(16a). The compound 15 (150 mg, 0.21 mmol) was co-evaporated with
pyridine twice. To a stirred solution of 15 in dry pyridine (3 mL) at 0◦C was
added benzoyl chloride (73 μL, 0.63 mmol) dropwise under Ar. After 18 h of
stirring at room temperature, the diprotection was found to be completed.
The mixture was again cooled to 0◦C and 0.5 mL of 30% aqueous ammonia
was added and stirred at this temperature for 1 h 45 min. The solvent
was then evaporated to near dryness and usual work up was performed.
The crude residue was purified by silica gel column chromatography using
0%–2% MeOH in CH2Cl2 as eluent to yield 16a as a white foamy solid
1
(142 mg, 81% over two steps from 15): IR (KBr) νmax 1722 cm−1; H NMR
(500 MHz, CDCl3) δ 0.87 (s, 9H), 1.54 (t, 1H, J = 11.3 Hz), 1.67 (t, 1H,

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 777
J = 10.5 Hz), 3.27 (d, 1H, J = 12.0 Hz), 3.39 (d, 1H, J = 11.0 Hz), 3.53 (dd,
1H, J = 10.5, 6.0 Hz), 3.69 (dd, 1H, J = 10.8, 4.3 Hz), 4.26–4.28 (m, 1H),
6.30 (dd, 1H, J = 9.8, 1.7 Hz), 7.11–7.53 (m, 28H), 7.75 (s, 1H), 7.92 (d,
2H, J = 7.0 Hz), 8.73 (s, 1H), 9.04 (br s, 1H); 13C NMR (125 MHz, CDCl3)
δ 19.3, 26.9, 50.0, 53.5, 64.6, 77.4, 80.4, 122.7, 126.7, 127.7, 127.8, 128.0,
128.03, 128.5, 128.6, 128.9, 129.3, 129.6, 129.8, 129.9, 130.0, 130.1, 132.8,
133.3, 133.4, 133.8, 135.6, 135.9, 140.7, 149.5, 151.3, 152.9, 164.7; HRMS
(ESI) m/z [M + Na]+, calcd for C52H50N6O3SiNa 857.3611, found 857.3611.
N6-Phenoxyacetyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Morpholinoade-
nosine (16b). To a solution of 15 (25 mg, 0.034 mmol) in dry pyridine
was added previously prepared phenoxyacetyl chloride (100 μL of 1.3 M
soln. in toluene) at 0◦C and stirred at rt for 6 h. The reaction was quenched
with water, diluted with EtOAc and washed with NaHCO3 and brine. The
organic layer was dried and concentrated. Silica gel purification afforded
the title compound 16b as a white solid (20 mg, 68%). IR (KBr): νmax 3379,
1732, 1615 cm−1. 1H NMR (500 MHz, CDCl3) δ 0.95 (s, 9H), 1.62 (t, 1H, J =
11.3 Hz), 1.75 (t, 1H, J = 10.8 Hz), 3.34 (d, 1H, J = 12.0 Hz), 3.45 (d, 1H, J
= 11.5 Hz), 3.61 (dd, 1H, J = 10.8, 5.8 Hz), 3.76 (dd, 1H, J = 10.5, 4.5 Hz),
4.33–4.35 (m, 1H), 4.84 (s, 2H), 6.36 (dd, 1H, J = 9.8, 1.3 Hz), 7.04 (m, 2H),
7.20 (t, 3H, J = 6.8 Hz), 7.27–7.41 (m, 23H), 7.56 (d, 2H, J = 7.0 Hz), 7.85
(s, 1H), 8.80 (s, 1H), 9.38 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.3,
26.9, 50.0, 53.5, 64.6, 68.3, 77.4, 80.4, 115.1, 121.6, 122.5, 126.7, 127.8, 128.0,
129.4, 129.84, 129.89, 130.0, 133.3, 133.4, 135.6, 141.1, 148.3, 150.1, 151.4,
152.8, 157.2, 166.7; HRMS (ESI) m/z [M + Na]+, calcd for C53H52N6O4SiNa
887.3717, found 887.3715.
N6-(4-Nitrobenzoyl)-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl
Morpholino-
adenosine (16c). A solution of 15 (25 mg, 0.034 mmol) in dry DCM was
treated with Et3N (14.2 μL, 0.102 mmol) and 4-nitrobenzoyl chloride
(14 mg, 0.075 mmol) and left to stir for 8 h. Usual work-up and silica gel
purification (0%–3% MeOH in DCM) afforded the protected nucleoside
16c (21 mg, 71%) as a white solid. IR (KBr): νmax 3429, 1707, 1611,
1
1528 cm−1. H NMR (500 MHz, CDCl3) δ 0.88 (s, 9H), 1.55 (t, 1H, J =
11.3 Hz), 1.67 (t, 1H, J = 10.5 Hz), 3.28 (d, 1H, J = 12.0 Hz), 3.41 (d,
1H, J = 9.5 Hz), 3.54 (dd, 1H, J = 10.8, 5.8 Hz), 3.69 (dd, 1H, J = 10.8,
4.8 Hz), 4.27–4.29 (m, 1H), 6.31 (d, 1H, J = 9.0 Hz), 7.13–7.50 (m, 25H),
7.79 (s, 1H), 8.06–8.10 (m, 2H), 8.21–8.28 (m, 2H) 8.71 (br s, 1H), 9.18
(br s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.4, 26.9, 50.0, 53.5, 64.6, 80.5,
124.1, 126.7, 127.8, 128.0, 129.3, 129.9, 133.2, 133.4, 135.6, 135.7, 135.9,
141.2, 148.8, 150.4, 161.6, 152.9, 163.2; HRMS (ESI) m/z [M + Na]+, calcd
for C52H49N7O5SiNa 902.3462, found 902.3459.
N6-Benzyloxycarbonyl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl
Morpholi-
noadenosine (16d). To a solution of 15 (50 mg, 0.068 mmol) in dry THF
(1.5 mL) was added LiHMDS (150 μL of a 1 M solution in THF, 0.15 mmol)

778
S. Pattanayak et al.
at −40◦C under Ar atomosphere. The temperature was raised to −20◦C
over 15 min and again lowered to −30◦C when a solution of CbzCl (13 μL,
0.090 mmol) in 0.3 mL THF was added dropwise. The mixture was left to
stir for 1 h at −15◦C and quenched with citric acid (7 mg in 0.5 mL water)
and extracted with EtOAc (2 × 15 mL). The combined organic layers were
washed with water, brine and dried over Na2SO4. Purification over silica gel
(15%–35% EtOAc in pet ether) afforded the protected morpholino 16d
1
(36 mg, 62%) as a white solid. IR (KBr): νmax 3418, 1763, 1612 cm−1. H
NMR (500 MHz, CDCl3) δ 0.94 (s, 9H), 1.60 (t, 1H, J = 11.0 Hz), 1.73 (t,
1H, J = 10.8 Hz), 3.34 (d, 1H, J = 11.5 Hz), 3.44 (d, 1H, J = 11.5 Hz), 3.58
(dd, 1H, J = 10.8, 5.8 Hz), 3.74 (dd, 1H, J = 10.5, 4.5 Hz), 4.32–4.33 (m,
1H), 5.26 (dd, 2H, J = 21.2, 12.3 Hz), 6.34 (d, 1H, J = 9.5 Hz), 7.19–7.21
(m, 3H), 7.25–7.51 (m, 25H), 7.54 (d, 2H, J = 7.5 Hz), 7.75 (s, 1H), 8.52
(br s, 1H), 8.76 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.3, 26.9, 50.0, 53.4,
64.6, 67.9, 77.1, 77.3, 80.3, 121.6, 126.7, 127.8, 128.0, 128.6, 128.7, 129.3,
129.8, 129.9, 133.2, 133.4, 135.6, 140.4, 149.4, 150.7, 151.0, 153.1; HRMS
(ESI) m/z [M + Na]+, calcd for C53H52N6O4SiNa 887.3717, found 887.3716.
N2-Isobutyryl-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl
Morpholinoguano-
sine (22a). To a stirred solution of 21 (100 mg, 0.134 mmol) in dry
DMF (1 mL) were added isobutyryl anhydride (44 μL, 0.268 mmol) and
triethylamine (56 μL, 0.402 mmol) under Ar. After heating at 140◦C for
3 h, another batch of isobutyryl anhydride (44 μL, 0.268 mmol) and
triethylamine (75 μL, 0.536 mmol) was added and again heated for 2 h. The
reaction mixture was concentrated under reduced pressure, diluted with
chloroform (25 mL), washed with water (3 × 10 mL), saturated NaHCO3
(10 mL) and brine (15 mL). The organic layer was dried over Na2SO4, and
evaporated to dryness. Chromatographic purification (SiO2, 0%–2% MeOH
in DCM) furnished 22a as a yellowish white solid product (82 mg, 75%): mp
145–148◦C; IR (KBr) νmax 1681 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.91 (s,
9H), 1.26 (dd, 6H, J = 6.5, 3.0 Hz) 1.52 (t, 1H, J = 11.3 Hz), 1.68 (t, 1H, J
= 11.0 Hz), 2.58 (sept, 1H, J = 7.0 Hz), 3.26 (d, 1H, J = 12.0 Hz), 3.36 (d,
1H, J = 11.0 Hz), 3.54 (dd, 1H, J = 11.0, 6.0 Hz), 3.71 (dd, 1H, J = 11.0,
4.5 Hz), 4.22–4.24 (m, 1H), 5.93 (dd, 1H, J = 10.0, 2.0 Hz), 7.16 (t, 3H, J =
7.0 Hz), 7.23–7.48 (m, 21H), 7.51 (dd, 2H, J = 7.5, 1.5 Hz), 7.91 (br s, 1H),
11.82 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ 19.1, 19.3, 19.4, 26.9, 36.8,
50.0, 52.9, 64.7, 77.0, 80.8, 121.4, 126.7, 127.8, 128.0, 129.4, 129.9, 133.26,
133.29, 135.6, 136.6, 147.3, 155.5, 177.8; HRMS (ESI) m/z [M + Na]+, calcd
for C49H52N6O4SiNa 839.3717, found 839.3718.
N2-Dimethylaminomethylene-7ꢁ-O-(tert-butyldiphenylsilyl)-N-trityl Mor-
pholinoguanosine (22b). A solution of 21 (100 mg, 0.134 mmol) in dry
methanol was treated with N,N-dimethylformamide dimethyl acetal (89 μL,
0.67 mmol) for 2 days. The solvent was evaporated and work up was per-
formed as usual way to obtain the title product 22b (82 mg, 76%). IR (KBr)

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers 779
1
νmax 1699, 1522 cm−1; H NMR (500 MHz, CDCl3) δ 0.94 (s, 9H), 1.55 (t,
3H, J = 11.3 Hz), 1.73 (t, 1H, J = 10.8 Hz), 3.14 (s, 3H), 3.34 (s, 3H),
3.33 (t, 2H, J = 9.5 Hz), 3.57 (dd, 1H, J = 11.0, 5.7 Hz), 3.75 (dd, 1H, J
= 10.5, 4.5 Hz), 4.29–4.30 (m, 1H), 6.07 (d, 1H, J = 9.0 Hz), 7.18 (t, 3H,
J = 7.0 Hz), 7.26–7.56 (m, 21H), 8.59 (s, 1H), 8.94 (br s, 1H); 13C NMR
(125 MHz, CDCl3) δ 19.3, 26.9, 27.0, 35.4, 41.5, 50.1, 53.3, 64.8, 77.4, 80.0,
120.4, 126.6, 127.8, 127.9, 128.2, 129.4, 129.8, 129.9, 133.30, 133.32, 135.5,
135.6, 135.7, 136.0, 149.6, 156.7, 157.8, 162.7.
Procedure for Silyl Deprotection
General Procedure for the Synthesis of 7ꢁ-hydroxy-N-trityl Morpholino
Monomers (5, 11, 17, 23, 28). To a stirred solution of the silylated mor-
pholino units (4, 10a, 16a, 22a, 27; 1.0 mmol) in dry THF was added drop-
wise a solution of 1 M TBAF in THF (1.5 mmol) and left to stir for 1.5 h
at room temperature. The reaction mixture was concentrated and diluted
with EtOAc (30 mL), washed with water (2 × 20 mL) and brine (20 mL).
The organic layer was dried (Na2SO4), concentrated and purified over silica
gel column chromatography (20% EtOAc in petroleum ether and then 5%
MeOH in DCM) to afford the desired products.
7ꢁ-Hydroxy-N-trityl Morpholinothymidine (5). White solid, yield 95%
from 4: mp 171–173◦C; IR (KBr) νmax 3410, 1705 cm−1; 1H NMR (500 MHz,
CDCl3) δ 1.45–1.50 (m, 2H), 1.82 (s, 3H), 2.58 (br s, 1H), 3.11 (d, 1H,
J = 12.0 Hz), 3.36 (d, 1H, J = 11.0 Hz), 3.60–3.63 (m, 2H), 4.30–4.32 (m,
1H), 6.20 (dd, 1H, J = 9.7, 1.7 Hz), 7.03 (s, 1H), 7.21 (t, 3H, J = 6.8 Hz),
7.32 (t, 6H, J = 7.8 Hz), 7.50 (m, 6H), 9.25 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 12.5, 49.0, 52.1, 63.8, 77.0, 77.8, 80.6, 110.7, 126.7, 128.1, 129.4,
135.7, 150.0, 163.9; HRMS (ESI) m/z [M + Na]+, calcd for C29H29N3O4Na
506.2056, found 506.2059.
N4-Benzoyl-7ꢁ-hydroxy-N-trityl Morpholinocytidine (11). White solid,
yield 89% from 10a: mp >225◦C; IR (KBr): νmax 3381, 1651 cm−1. 1H NMR
(500 MHz, DMSO-d6) δ 1.24 (t, 1H, J = 10.3 Hz), 1.39 (t, 1H, J = 11.0 Hz),
3.10 (d, 1H, J = 11.5 Hz), 3.33–3.46 (m, 3H), 4.20–4.22 (m, 1H), 4.77 (t,
1H, J = 5.8 Hz), 6.11 (d, 1H, J = 8.5 Hz), 7.20–7.51 (m, 18H), 7.61 (t, 1H,
J = 7.3 Hz), 7.97 (d, 3H, J = 7.5 Hz), 11.21 (br s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 50.0, 52.9, 62.6, 77.0, 77.5, 81.8, 97.0, 126.9, 128.4, 128.9, 129.0,
129.1, 129.6, 133.3, 133.7, 145.8, 154.3, 163.7, 167.9; HRMS (ESI) m/z [M +
Na]+, calcd for C35H32N4O4Na 595.2321, found 595.2321.
N6-Benzoyl-7ꢁ-hydroxy-N-trityl Morpholinoadenosine (17). White solid,
yield 87% from 16a: mp 172–174◦C; IR (KBr) νmax 3404, 1691 cm−1; 1H NMR
(500 MHz, CDCl3) δ 1.53 (t, 1H, J = 11.3 Hz), 1.74 (t, 1H, J = 10.8 Hz), 3.05
(d, 1H, J = 12.0 Hz), 3.29 (br s, 1H), 3.42 (d, 1H, J = 11.5 Hz), 3.46–3.55 (m,
2H), 4.24–4.26 (m, 1H), 6.29 (d, 1H, J = 9.5 Hz), 7.11 (t, 3H, J = 7 Hz), 7.22
(t, 7H J = 7.5 Hz), 7.40–7.44 (m, 8H), 7.51 (t, 1H, J = 7.3 Hz), 7.81 (s, 1H),

780
S. Pattanayak et al.
7.96 (d, 2H, J = 7.0 Hz), 8.71 (s, 1H), 9.38 (br s, 1H); 13C NMR (125 MHz,
CDCl3) δ 48.9, 53.1, 63.5, 76.9, 77.6, 80.4, 111.1, 122.4, 126.7, 128.01, 128.03,
128.8, 129.2, 132.7, 133.8, 140.3, 149.6, 151.1, 152.9, 164.9; HRMS (ESI) m/z
[M + Na]+, calcd for C36H32N6O3Na 619.2434, found 619.2432.
N2-Isobutyryl-7ꢁ-hydroxy-N-trityl Morpholinoguanosine (23). White
solid, yield 94% from 22a: mp 198◦C (decomp.); IR (KBr) νmax 3408,
1
1682 cm−1; H NMR (500 MHz, CDCl3) δ 1.26 (d, 6H, J = 7.0 Hz) 1.52
(t, 1H, J = 11.0 Hz), 1.78 (t, 1H, J = 10.5 Hz), 2.73 (sept, 1H, J = 6.8 Hz),
3.10 (d, 1H, J = 12.0 Hz), 3.35 (d, 1H, J = 12.0 Hz), 3.53–3.60 (m, 2H),
4.27–4.30 (m, 1H), 5.97 (dd, 1H, J = 10.0, 2.0 Hz), 7.17 (m, 3H), 7.26–7.27
(m, 6H), 7.44 (m, 6H), 7.51 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 18.9,
19.2, 36.5, 48.9, 52.5, 63.6, 77.0, 77.8, 80.6, 120.8, 126.7, 128.0, 129.4, 136.5,
147.8, 155.8, 178.8; HRMS (ESI) m/z [M + Na]+, calcd for C33H34N6O4Na
601.2539, found 601.2535.
7ꢁ-Hydroxy-N-trityl Morpholinoinosine (28). White solid, yield 86% from
1
27a: mp 178◦C (decomp.); IR (KBr) νmax 3385, 1694 cm−1; H NMR
(500 MHz, CDCl3) δ 1.61 (t, 1H, J = 11.3 Hz), 1.85 (t, 1H, J = 10.5 Hz),
3.16 (d, 1H, J = 11.5 Hz), 3.43 (d, 1H, J = 11.0 Hz), 3.59 (dd, 1H, J = 12.0,
6.0 Hz), 3.65 (dd, 1H, J = 10.3, 3.8 Hz), 4.33–4.37 (m, 1H), 6.27 (dd, 1H, J =
10.0, 2.0 Hz), 7.19 (t, 3H, J = 7.3 Hz), 7.30 (t, 6H, J = 7.5 Hz), 7.48 (m, 6H),
7.78 (s, 1H), 8.07 (s, 1H); 13C NMR (125 MHz, DMSO-d6) 50.0, 52.5, 62.4,
76.9, 77.2, 80.4, 124.5, 126.9, 128.4, 129.4, 138.4, 146.5, 148.0, 157.0. HRMS
(ESI) m/z [M + Na]+, calcd for C29H27N5O3Na 516.2012, found 516.2012.
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