Concurrent Hydrogenation of Three Functional Groups Enables Synthesis of C3′-Homologated Nucleoside Amino Acids

Article abstract of DOI:10.1021/acs.orglett.7b01934

Internucleoside amide linkages are excellent mimics of phosphodiesters in RNA and may be used to optimize the properties of short interfering RNAs. Herein we report a remarkably straightforward, efficient and step economic synthesis of C3′-homologated uri

Full text of DOI:10.1021/acs.orglett.7b01934

Letter
pubs.acs.org/OrgLett
Concurrent Hydrogenation of Three Functional Groups Enables
Synthesis of C3′-Homologated Nucleoside Amino Acids
Venubabu Kotikam* and Eriks Rozners*
Department of Chemistry, Binghamton University, The State University of New York, Binghamton, New York 13902, United States
S
* Supporting Information
ABSTRACT: Internucleoside amide linkages are excellent mimics of phosphodiesters in RNA and may be used to optimize the
properties of short interfering RNAs. Herein we report a remarkably straightforward, efficient and step economic synthesis of
C3′-homologated uridine and adenosine amino acids starting from nucleosides in six steps (31% overall yield) and eight steps
(16% overall yield), respectively. The key enabling step is a one-pot multifunctional group transformation including a
stereoselective hydrogenation, termed “Global Hydrogenation”.
RNA interference (RNAi) has become a broadly used and
powerful tool in functional genomics.1 With more than 20
currently active clinical trials, RNAi also has intriguing potential
to become a new therapeutic approach.2−4 For therapeutic and
other in vivo applications, short interfering RNAs (siRNAs) need
chemical modifications to optimize target specificity, enzymatic
stability, cellular uptake, biodistribution, and pharmacoki-
netics.5−7 Phosphate backbone modifications are a remarkably
successful example of such modifications. Antisense drugs
recently approved by FDA contain phosphorothioate (Fomi-
virsen and Mipomersen8) and neutral phosphorodiamidate
morpholino (Eteplirsen9) linkages that demonstrate the cogency
Figure 1. Structure of amide-linked RNA and its synthetic precursors,
C3′-homologated nucleoside amino acids.
of backbone modifications. Another example of highly successful
backbone modification is peptide nucleic acid (PNA), which
entails the replacement of the entire sugar−phosphate moiety
with a neutral N-(2-aminoethyl)glycine backbone and makes
PNA highly resistant to nucleases and proteases.10,11
of both guide and passenger strands of siRNAs and increased the
silencing activity when placed near the 5′-end of the passenger
strand and in the middle of the guide strand.23 Even three
consecutive amide linkages at the 5′-end of the passenger strand
did not significantly decrease the siRNA activity.24 These results
were encouraging for future studies on amide-linked siRNAs and
also suggested that amide linkages may be useful modifications
for the optimization of other emerging RNA-based gene control
technologies such as CRISPR-Cas.25−27 However, the problem
for future studies on amide-modified siRNAs and CRISPR-Cas
RNAs is the complex, lengthy, and low yielding synthesis of C3′-
homologated nucleoside amino acids 1 (Figure 1c), the
Amide linkages have been explored as nonionic replacements
for phosphodiesters in antisense deoxyoligonucleotides.12
Isolated amide linkages (such as those in Figure 1b) were well
accommodated in DNA−RNA heteroduplexes.13−17 Rozners
and co-workers18,19 showed that amide linkages were excellent
structural mimics of phosphodiesters in RNA and did not
significantly change the structure, thermal stability, and
hydration of short all-RNA duplexes.
Initial tests of amide linkages as backbone modifications in
siRNAs have given promising results. Iwase and co-workers20−22
modified the 3′-overhangs of a siRNA with two amide linkages,
which resulted in increased enzymatic stability and no significant
change in RNAi activity. Recently, our group showed that
isolated amide linkages were well tolerated at internal positions
Received: June 24, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01934
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
monomers required for introduction of consecutive amide
linkages in RNA.
Supporting Information (SI). To increase the efficiency, the
undesired 3′-O-TBS compounds were isomerized to mixtures of
2′- and 3′-O-TBS compounds,35 which could be separated again
to yield more of the desired 2′-O-TBS isomer. Recycling of the
3′-O-TBS isomers increased the yields of 3a and 3b from 47%
and 40% to 66% and 55%, respectively; for experimental details,
see SI. The 3′-OH of 3a and 3b was oxidized to its corresponding
ketones, using Dess−Martin periodinane (DMP) in dry
dichloromethane (DCM). Using fresh DMP reagent with a
minute amount of added water36 provided very clean oxidation,
and the resulting ketones did not require purification. The initial
homologation attempts using a Wittig reagent, generated in situ
from triphenyl phosphine and benzylbromoacetate, gave low
yields (15−40%). The Horner−Wadsworth−Emmons reaction
with benzyldiethylphosphonoacetate led to decomposition of the
ketone. After some optimization, efficient Wittig alkenylation
was accomplished using commercially available benzyl-
(triphenylphosphoranylidene)acetate, furnishing the key ad-
vanced intermediates 4a and 4b, substrates for the GH reaction
(Scheme 1). The stereochemistry of the Wittig products was not
determined because both isomers would give the same product
in the next hydrogenation step.
The first synthesis of C3′-homologated ribonucleoside amino
acids, monomers required for the preparation of amide-linked
RNA, was developed by Peterson et al.28 The route started from
D-xylose, required 11 steps, and produced a 5′-azido precursor of
uridine 1a in ∼10% yield. This route was used by the same
authors to prepare short amide-linked oligouridines29 and by
Iwase and co-workers to prepare 3′-amide-modified siR-
NAs.20−22 We developed two synthetic routes to all four C3′-
homologated nucleoside amino acids 1 having the 2′-O-acetyl
protection.30,31 However, the 2′-O-acetyl group was later found
incompatible with solid-phase synthesis of RNA strands having
consecutive amide linkages.24 While it was possible to modify our
synthesis to produce 2′-O-TBS protected uridine, which was
compatible with the synthesis of consecutive amide linkages, the
route required 18 steps and gave 1a in only ∼4% yield.24,30,31
Hence, the laborious and low yielding syntheses of monomers is
a major limitation for future studies on amide linkages in RNA-
based gene control technologies.
In this Letter, we report a short and efficient synthesis of C3′-
homologated uridine and adenosine amino acids from
commercially available nucleosides. The synthesis requires only
six steps for 1a (31% overall yield) and eight steps for 1b (16%
overall yield). The key step enabling our route is a remarkable
one-pot concurrent hydrogenation of three different functional
groups, termed “Global Hydrogenation” (GH), that transforms
azide to amine, stereoselectively hydrogenates an exocyclic
CC bond, and cleaves a benzyl ester protecting group. The use
of benzyl protecting group for carboxylic acid avoids the
troublesome basic hydrolysis of alkyl (Me or Et) esters and is
critical for preparation of compounds having base labile N-
protecting groups (e.g., N6-benzoyladenosine). In contrast to
previous syntheses, the new route is atom- and step-economic
and does not require a separate deprotection reaction to reach
the final product.
Our first attempts at GH (Scheme 2) of 4a using 10% Pd/C
(40% w/w) and hydrogen gas (delivered from a rubber balloon)
Scheme 2. Global Hydrogenation of Advanced Intermediates
Our synthetic route (Scheme 1) started from uridine and N6-
benzoyladenosine32 and followed the C3′-homologation strategy
Scheme 1. Synthesis of Advanced Intermediates for Global
Hydrogenation
in a mixture of MeOH/EtOAc (1:1) furnished the desired amino
acid 5a in ∼50% yield along with a side product that was assigned
as the over-reduced 5,6-dihydrouracil derivative (7, Scheme 2
and Table 1, entry 1) based on NMR analysis of crude reaction
mixtures. Using the same conditions for the GH of adenosine
derivative 4b (Scheme 2) led to the isolation of the partially
reduced compound 6b having an unreacted exocyclic CC as
the sole product. Attempts to promote reduction of the alkene in
4b by increasing the amount of Pd to 100% w/w, extending the
reaction time, or adding acetic acid were not successful.
Alternative approaches, such as transfer hydrogenation and
conjugate reduction of 4b using NiCl2/NaBH4 or Red-Al/CuBr,
failed to give the desired product. Our attempts at conjugate
reduction of the α,β-unsaturated carboxylic acid 6b were also
unproductive. Recognizing that the only chemical difference
between the two substrates 4a and 4b was their different
nucleobases, uracil and N6-benzoyladenine, respectively, we
speculated that the latter might complex with Pd as an electron-
rich ligand, reducing the activity of the catalyst. We hypothesized
that decreasing the electron density of adenine by introducing a
developed by Peterson et al.33 The synthesis began with
installation of the 5′-azido group using an Appel reaction34
followed by protection of the 2′-OH as a TBS ether. The large
difference in yields of 2a and 2b was somewhat puzzling, but
consistent with the original procedure.34 The desired 2′-O-TBS
products were separated from the undesired 3′-O-TBS isomers
using silica gel column chromatography. The identity of the 2′-
and 3′-O-TBS isomers was confirmed by 1H and 2D NMR, see
B
DOI: 10.1021/acs.orglett.7b01934
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Optimization of Global Hydrogenation Conditions
a
s. no
GH substrate
Pd% (w/w)
solvent
time (h)
product ratio (5a/6a/7 or 5b/6b)
yield % of 1a/1b (over 2 steps)
c
1
4a
4a
4a
4a
4a
4a
4a
4c
4c
4c
40
MeOH/EtOAc (1:1)
MeOH/EtOAc (1:1)
MeOH/H2O (1:1)
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
MeOH/EtOAc (1:1)
tBuOH/H2O (1:1)
MeOH/H2O (5:1)
MeOH/H2O (5:1)
20
4
61:0:39
nd
c
2
40
incomplete reaction
50:0:50
nd
c
3
40
4
nd
c
4
40
4
67:0:33
nd
c
5
30
1.5
3.0
3.5
72
16
24
72
56:44:0
nd
6
30
30
90:10:0
60%
c
7
83:0:17
nd
b
c
8
100
100
100
50
68%
nd
c
9
17:83
nd
10
11
95:5
61%
c
4c
90:10
nd
a
b
c
1
Based on H NMR analysis of crude reaction mixture filtered over Celite. Isolated yield. nd: not determined. The compound was not used to
make 1a/1b.
second benzoyl group, as in 4c, could block the putative
complexation and restore the catalyst activity. Treatment of 4b
with benzoyl chloride in dry pyridine (Scheme 1) gave the
dibenzoyl-protected derivative 4c as a new GH substrate.
Hydrogenation of this new substrate using 10% Pd/C (100%
w/w) in MeOH/EtOAc (1:1) over 72 h yielded the desired GH
product 5b in 68% isolated yield (Table 1, entry 8). Consistent
with the pioneering studies of Peterson et al.,33 we observed
complete stereoselectivity of the hydrogenation of the exocyclic
double bond in 4a and 4c, most likely caused by the bulky α-2′-
O-TBS group. We obtained the desired α-C3′-homologated
products 5a and 5b (Scheme 2) as sole stereoisomers.
Replacing EtOAc with water in the mixed solvent system
accelerated the reaction and enabled clean and quantitative
conversions in shorter time periods (Table 1, cf. entries 1 vs 3,
and 8 vs 10). In the uridine series, replacement of MeOH with t-
BuOH provided milder and more selective hydrogenation
conditions, which was critical for elimination of the over-reduced
byproduct 7. Further optimizations of the reaction conditions by
fine-tuning catalyst loading and reaction time (Table 1, entry 6)
achieved quantitative conversion and gave a mixture of the
desired product 5a and alkene 6a (9:1).
Scheme 3. Completion of Synthesis of U and A Amino Acid
Building Blocks
BuOH/H2O (4:1) failed to reduce the exocyclic double bond.
Replacing t-BuOH by MeOH gave a mixture of the desired C3′-
homologated cytidine amino acid along with over-reduced
(C5C6) product. The synthesis of a guanosine substrate for
the GH reaction is currently in progress.
In summary, we have developed a short and efficient synthesis
of C3′-homologated uridine and adenosine amino acids 1a and
1b, monomers for solid-phase synthesis of amide-linked RNA.
The key innovation and enabling step is a global hydrogenation
that achieves three concurrent reactions: transformation of azide
to amine, stereoselective hydrogenation of an exocyclic CC
bond, and cleavage of a benzyl ester protecting group, all in a one-
pot single-step reaction. Compared to previous syntheses, our
new route is remarkably efficient and atom- and step-economic,
achieving 1a in only six steps with 31% overall yield and 1b in
eight steps with 16% overall yield from commercially available
nucleosides. The new route will open the door to more extensive
studies on amide-modified siRNAs that have shown promising
results in initial tests.20−24
In the adenosine series, more forceful hydrogenation in
MeOH/H2O using 100% w/w of Pd catalyst over 24 h was
required to achieve quantitative conversion (entry 10, Table 1).
The GH in aqueous methanol led to partial loss of the more labile
second benzoyl group,37 resulting in a ∼3:1 mixture of 5b and 5c.
After a workup with ammonia to completely convert 5c to 5b,
NMR analysis of crude GH reaction mixture showed the desired
product 5b along with ∼5% of 6b. Reducing the amount of Pd to
50% w/w while increasing reaction time directly furnished the
desired monobenzoylated adenine compound 5b and eliminated
the need for ammonia workup (Table 1, entry 11), albeit in a less
favorable ratio of 5b to 6b. During optimization of the GH
reaction, we found that isolation of the polar amino acids was
cumbersome. Therefore, we optimized the GH reactions based
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b01934.
1
Experimental procedures and supporting spectral data
(PDF)
on H NMR analysis of crude products. The crude reaction
1
mixtures of successful hydrogenations (based on H NMR
analysis) were used directly in the next reaction. The final step of
synthesis of the C3′-homologated uridine and adenosine amino
acid monomers 1a and 1b involved protection of the 5′-amino
group of crude GH products using methoxytrityl chloride
(MMTr-Cl) in pyridine (Scheme 3). Compounds 1a and 1b are
ready for use in our recently developed solid-phase synthesis of
RNA containing consecutive amide linkages.24 Our initial
attempts at the global hydrogenation of cytidine substrate in t-
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: erozners@binghamton.edu.
*E-mail: vkotikam@binghamton.edu.
ORCID
Eriks Rozners: 0000-0001-7649-0040
C
DOI: 10.1021/acs.orglett.7b01934
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(28) Robins, M. J.; Doboszewski, B.; Timoshchuk, V. A.; Peterson, M.
A. J. Org. Chem. 2000, 65, 2939−2945.
Notes
The authors declare no competing financial interest.
(29) Robins, M. J.; Doboszewski, B.; Nilsson, B. L.; Peterson, M. A.
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 69−86.
(30) Rozners, E.; Liu, Y. J. Org. Chem. 2005, 70, 9841−9848.
(31) Tanui, P.; Kullberg, M.; Song, N.; Chivate, Y.; Rozners, E.
Tetrahedron 2010, 66, 4961−4964.
ACKNOWLEDGMENTS
■
We thank NIH (R01 GM071461) for financial support of this
research. The Regional NMR Facility (600 MHz instrument) at
Binghamton University is supported by NSF (CHE-0922815).
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DOI: 10.1021/acs.orglett.7b01934
Org. Lett. XXXX, XXX, XXX−XXX