Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides

Article abstract of DOI:10.1016/j.bmcl.2018.08.024

Glucosamino nucleic acids (GANAs) bearing a β-N-glycoside bond between carbon 1 of the glucosamine and the nucleobase nitrogen were synthesized and incorporated into oligonucleotides (4′,6′-GANA and 3′,6′-GANA). The thermal stability of oligonucleotide duplexes containing the GANA zwitterionic nucleotides was also investigated.

Full text of DOI:10.1016/j.bmcl.2018.08.024

Accepted Manuscript
Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides
Yoshiaki Kitamura, Shuichi Moribe, Yukio Kitade
PII:
S0960-894X(18)30701-7
https://doi.org/10.1016/j.bmcl.2018.08.024
BMCL 26004
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2018
22 August 2018
24 August 2018
Please cite this article as: Kitamura, Y., Moribe, S., Kitade, Y., Synthesis of cationic glucosamino nucleic acids for
stabilizing oligonucleotides, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.08.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides
Yoshiaki Kitamuraa,b,*, Shuichi Moribea, Yukio Kitadea,b,c,*
aDepartment of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan
bDepartment of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
cDepartment of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247
Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan
 To whom correspondence should be addressed:
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
Tel.: +81-58-293-2641; E-mail: kitamura@gifu-u.ac.jp
Department of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247
Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan
Tel.: +81-565-48-7611; E-mail: ykkitade@aitech.ac.jp

Keywords
Glucosamine; Nucleic acids; Oligonucleotides; Pyranosyl nucleosides; Zwitterionic nucleotides

Abstract
Glucosamino nucleic acids (GANAs) bearing a β-N-glycoside bond between carbon 1 of the
glucosamine and the nucleobase nitrogen were synthesized and incorporated into oligonucleotides (4′,6′-
GANA and 3′,6′-GANA). The thermal stability of oligonucleotide duplexes containing the GANA
zwitterionic nucleotides was also investigated.

Chemically modified oligonucleotides (ONs) are currently attracting intense interest because they are
tools in molecular biology, gene probes for diagnosis, and potential drugs. Many chemical modification
strategies have been employed to improve the nuclease stability, RNA-binding affinity, and
pharmacokinetic properties of ONs for therapeutic applications. In particular, the sugar moieties are
often modified to increase nuclease resistance or increase affinity for the complementary target. Several
series of modified nucleosides with a six-membered carbohydrate moiety instead of the five-membered
sugar ring contained in natural nucleosides have been synthesized and incorporated into ONs (Figure
1).1 Examples include homo-DNAs,2 hexitol nucleic acids (HNA),3 mannitol nucleic acids (MNA),4 and
altritol nucleic acids (ANA).4 Herdewijn5,6 and Eschenmoser7,8 developed pioneering synthetic access to
ONs containing hexopyranosyl and dideoxyhexopyranosyl nucleosides. Moreover, ONs that contain
pyranosyl nucleoside analogues may form stable duplexes.9
Glucosamine, an amino derivative of glucose, has a typical pyranose ring structure. The
polysaccharides chitosan and chitin contain glucosamine as a structural element that has high membrane
affinity and low toxicity.10 The positive charge on glucosamine can also neutralize the negatively
charged phosphates on ONs. Therefore, we have focused our research on the thermal stability of ONs
containing glucosamino nucleic acids (GANAs). We have previously synthesized glucosamine
derivatives with thymine at the 6-position for an RNAi study.11 Here, we synthesize the thymine
analogue (GANA-T) bearing a β-N-glycoside bond between carbon 1 of the glucosamine and nitrogen 1
of the nucleobase thymine to prepare ONs with two types of linkage between the glucosamine and
natural nucleotides, 4′,6′-linked ONs and 3′,6′-linked ONs (Figure 1). We investigated the hybridization
of ONs containing GANA-T either in the center or at the end of the sequence.

Figure 1.
Partial structures of DNA, RNA, and chemically modified ONs bearing various pyranosyl nucleoside
analogs.
The synthetic route of the GANA-T derivatives, thymine analogues of 4′,6′-GANA (X) and 3′,6′-
GANA (Y) (Base = T in Figure 1), is shown in Scheme 1. First, thymine was introduced into the
anomeric position of 212,13, which was easily prepared from glucosamine hydrochloride (1), using
trimethylsilyl trifluoromethanesulfonate (TMSOTf) to obtain 3 with complete stereoselectivity. After all
acetyl groups were removed by treatment with NaOMe, the 4'- and 6'-positions of the sugar moiety were
protected by a benzylidene group, and subsequent benzoyl protection of the N3 position of the thymine
moiety produced key intermediate 6. We chose cyanoethyl as the protecting group for the free 3′- or 4′-
hydroxyl group in the phosphoramidite derivatives. Compound 6 was treated with acrylonitrile to afford
7. The benzylidene acetal was removed by treatment with (±)-10-camphorsulfonic acid (CSA) to give 8
in 81% yield. Next, the 6′-hydroxyl group of 8 was protected by a 4,4′-dimethoxytrityl (DMTr) group to
give corresponding 6′-DMTr derivative 9, which was phosphitylated with 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite to produce 10. In addition, 6 was treated with benzyl bromide and
sodium hydride in DMF to afford corresponding benzyl compound 11 in 80% yield. Subsequent
regioselective reductive ring opening of the benzylidene acetal14 gave 3′,6′-dibenzyl-protected derivative
12. Cyanoethyl protection of 4′-hydroxyl group failed; therefore, we protected the 4′-OH with a tert-
butyldimethylsilyl (TBDMS) group, obtaining corresponding 4′-silyl-protected derivative 13, although
partial debenzoylation occurred. Next, the benzyl groups and N-benzoyl group of thymine were removed
by hydrogenation with Pd(OH)2/C in EtOH. Finally, DMTr protection of the primary 6′-hydroxyl group

of 15 followed by phosphitylation reactions provided phosphoramidite derivative 17.
To investigate the hybridization properties of GANA-T-modified ONs with a complementary strand, a
set of dT15-mers modified with GANA-T at the 5′-end, at the center, or at the 3′-end (ONs 19–23) were
synthesized by an automated nucleic acid synthesizer using phosphoramidite derivative 10 or 17 (Table
1). The structures of these nucleic acid oligomers were confirmed by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry analysis. In the hybridization experiments, the natural dT15
(ON 18) and each synthetic GANA-T-modified ON (ONs 19–23) were incubated with complementary
15-mer DNA (dA15) and 15-mer RNA (rA15) (Table 1).
Scheme 1. Reagents and conditions: (a) i) CF3COOEt, Et3N, MeOH, rt; ii) Ac2O, pyridine, rt, 99%; (b)
(TMS)2-thymine, TMSOTf, MeCN, reflux, 74%; (c) NaOMe, MeOH, rt, 99%; (d) PhCH(OMe)2, CSA,
MeCN, reflux, 79%; (e) i) TMSCl, pyridine, rt; ii) BzCl, rt, iii) H2O, rt, 75%; (f) acrylonitrile, Cs2CO3, t-
BuOH, DMF, rt, 49%; (g) CSA, MeOH, rt, 81%; (h) DMTrCl, pyridine, rt, 44%; (i) (i-
Pr2N)P(Cl)O(CH2)2CN, i-Pr2NEt, CH2Cl2, rt, 77%; (j) BnBr, NaH, DMF, −20 °C, 80%; (k) CF3CO2H,

(CF3CO)2O, Et3SiH, CH2Cl2, rt, 52%; (l) TBDMSCl, imidazole, DMF, 80 °C, 36% for 13 and 41% for
14; (m) H2, Pd(OH)2/C, EtOH, rt, 54%; (n) DMTrCl, pyridine, rt, 48%; (o) (i-Pr2N)P(Cl)O(CH2)2CN, i-
Pr2NEt, CH2Cl2, rt, 56%.

Table 1. Tm values of double-stranded ONs containing GANA-T.
No.of ON Sequence
dA15
rA15
Tm (°C)
Tm (°C)
ON 18
ON 19
ON 20
ON 21
ON 22
ON 23
5'-d(TTTTTTTTTTTTTTT)-3'
37.4
35.9 (−1.5)
26.2 (−11.2)
36.6
5'-d(XTTTTTTTTTTTTTT)-3'
5'-d(TTTTTTTXTTTTTTT)-3'
33.8 (−2.8)
24.7 (−11.9)
35.8 (−0.8)
33.2 (−3.4)
24.9 (−11.7)
5'-d(TTTTTTTTTTTTTTX)-3' a 37.5 (+0.1)
5'-d(YTTTTTTTTTTTTTT)-3'
35.7 (−1.7)
23.9 (−13.5)
5'-d(TTTTTTTYTTTTTTT)-3'
aThe structure is the same as that of 5'-d(TTTTTTTTTTTTTTY)-3'.
bThe values in parentheses indicate Tm relative to the unmodified duplex.
cMeasurements were carried out in 10 mM Na2HPO4/NaH2PO4 (pH 7.0), and 100 mM NaCl, with 3.0
μM of each ON.
The Tm value of the modified ON bearing GANA-T at the 3′-end (ON 21) with dA15 and rA15 was
similar to that of unmodified dT15 (ΔTm +0.1 and −0.8 °C, respectively). The binding affinities of
modified ONs with GANA-T at the 5′-end (ONs 19 and 22) toward the complementary DNA and RNA
were lower than that of the corresponding 3′-GANA-T-modified ON (21). The ΔTm values were similar
for the DNA and RNA complementary duplexes of 4′,6′-linked (X) ON 19 and 3′,6′-linked (Y) ON 22
containing GANA-T at the 5′-end. The thermal stabilities of the duplexes of ONs containing GANA-T in
the center (ON 20 and ON 23) with complementary DNA or RNA were lower than those of the
unmodified natural duplexes in all strands. The stability of ON 20/rA15 (Tm = 24.7 °C) was similar to
that of ON 23/rA15 (Tm = 24.9 °C). In contrast, the binding affinities for the DNA complementary
duplexes of 4′,6′-linked (X) ON 20 and 3′,6′-linked (Y) ON 23 containing GANA-T in the center were
different. The stability of ON 20/dA15 (Tm = 26.2 °C) was greater than that of ON 23/dA15 (Tm =
23.9 °C). The stability of duplexes with DNA complements were slightly greater than those of duplexes
with RNA complements in ONs with GANA-T at the 3′- or 5′-end. However, the DNA/DNA duplex
with Y in the center (ON 23/dA15) was slightly less stable than the DNA/RNA duplex with Y in the
center (ON 23/rA15). DNA/DNA duplexes usually possess B-type duplex geometries under
physiological conditions, whereas RNA/DNA duplexes have A-type duplex geometries. Thus, the

GANA-T conformation in ONs should differ between A- and B-type duplexes, and the thymine moiety
in GANA-T, especially X, can form more favorable base pairs with the complementary adenine base of
the opposite strand in B-type DNA/DNA duplexes than in A-type DNA/RNA duplexes.

Table 2. Tm values of duplexes formed between ONs and single-stranded DNA or RNA.
d(A7-Z-A7)
r(A7-Z-A7)
No.of ON
Z = A
37.4
26.2
23.9
Z = T
Z = C
27.9
16.7
21.2
Z = G
28.4
18.5
22.3
Z = A
36.6
24.7
24.9
Z = U
Z = C
22.2
14.6
18.2
Z = G
31.5
24.2
23.9
ON 18
ON 20
ON 23
27.8
18.1
21.0
24.5
18.9
19.7
aMeasurements were carried out in 10 mM Na2HPO4/NaH2PO4 (pH 7.0), and 100 mM NaCl, with 3.0
μM of each ON.
To measure the base recognition activity of X or Y in ONs, we investigated the binding affinity of ON
20 with X and ON 23 with Y for single-stranded DNA [d(A7-Z-A7)] bearing a deoxyribonucleoside (Z =
A, G, C, or T) or RNA [r(A7-Z-A7)] bearing a ribonucleoside (Z = A, G, C, or U) at the center of each
strand. The Tm values are summarized in Table 2. In general, ON 20 with X and ON 23 with Y formed
the most stable duplexes with ONs bearing guanine in the opposite position [d(A7-G-A7) and r(A7-G-
A7)], with the exception of complementary sequences (Z = A). Remarkably, the duplex formed between
ON 20 and r(A7-G-A7) furnished a Tm value of 24.2 °C, which is only 0.5 °C lower than that obtained
for the corresponding ON 20/rA15 duplex (Tm = 24.7 °C). In addition, the ΔTm value for the duplexes ON
23/r(A7-G-A7) and ON 23/rA15 is 1.0 °C. This result may be explained by a G-T wobble base pair
forming in DNR/RNA duplexes. In contrast, the Tm values of ON 23 with single base mismatches in the
DNA counter strands [d(A7-Z-A7)] (Z = T, C, and G) were close to those of unmodified dT15 (ON 18)
(ΔTm −2.9, −2.7, and −1.6 °C, respectively). Thus, ON 23 was relatively insensitive to single-base
mismatches in the DNA counter strand.
In conclusion, we have described a robust synthetic route for new GANAs bearing a β-N-glycoside
bond between carbon 1 of the glucosamine and the nitrogen of the nucleobase. Several thymidine
oligomers bearing two types of linkage between the glucosamine nucleoside (X and Y) and natural
nucleoside were synthesized. Substituting these glucosamine nucleotides into the center of a 15-mer had
a greater effect on duplex stability than end-substitution. The Tm experiments showed that the base
recognition activity of X or Y in ONs differs between DNA/DNA duplexes and DNA/RNA duplexes.

Further investigation of the properties of GANA-T is currently in progress and the results will be
reported in due course.
Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists (B) (no. 16K18911 [Y.K.]) and a
Grant-in-Aid for Scientific Research (B) (no. 24390025 [Y.K.]) from the Japan Society for the
Promotion of Science (JSPS). We are grateful to Professor M. Ikeda (Gifu University) for helpful
discussions and Dr. A. Shibata (Gifu University) for providing universal solid supports used in
synthesizing ONs. We also acknowledge the Division of Instrumental Analysis and the Division of
Genomics Research, Life Science Research Center, Gifu University, for maintaining the instruments
used in this study.

References and notes
1 For a review on pyranose nucleic acids, see: Herdewijn, P. Chem. Biodivers. 2010, 7, 1−59.
2 Eschenmoser, A.; Dobler, M. Helv. Chim. Acta 1992, 75, 218−259.
3 De Bouvere, B.; Kerremans, L.; Hendrix, C.; De Winter, H.; Schepers, G.; Van Aerschot, A.,
Herdewijn, P. Nucleosides Nucleotides 1997, 16, 973−976.
4 Froeyen, M.; Wroblowski, B.; Esnouf, R.; De Winter, H.; Allart, B.; Lescrinier, E.; Herdewijn, P.
Helv. Chim. Acta 2000, 83, 2153−2182.
5 Augustyns, K.; Van Aerschot, A.; Herdewijn, P. Nucleosides Nucleotides 1991, 10, 587−588.
6 Augustyns, K.; Vandendriessche, F.; Van Aerschot, A.; Busson, R.; Urbanke, C.; Herdewijn, P.
Nucleic Acids Res. 1992, 20, 4711−4716.
7 Eschenmoser, A. Pure Appl. Chem. 1993, 65, 1179−1188.
8 Beier, M.; Reck, F.; Wagner, T.; Krishnamurthy, R.; Eschenmoser A. Science 1999, 284,
2118−2124.
9 Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van Aerschot, A.; Herdewijn, P. Chem. Eur.
J. 1997, 3, 110−120.
10 Anderson, J. W.; Nicolosi, R. J.; Borzelleca, J. F. Food Chem. Toxicol. 2005, 43, 187−201.
11 Luo, X.; Sugiura, T.; Nakashima, R.; Kitamura, Y.; Kitade, Y. Bioorg. Med. Chem. Lett. 2013, 23,
4157−4161.
12 Masuko, S.; Bera, S.; Green, D. E.; Weiwer, M.; Liu, J.; DeAngelis, P. L.; Linhardt, R. J. J. Org.
Chem. 2012, 77, 1449−1456.
13 Goodnow, R. A. Jr.; Tam, S. Y.-K. U.S. Patent 5 780 607, 1998.
14 Oberli, M. A.; Bindschaedler, P.; Werz, D. B.; Seeberger, P. H. Org. Lett. 2008, 10, 905−908.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Leave this area blank for
Synthesis of
cationic
glucosamino
nucleic acids for
stabilizing
oligonucleotides
Yoshiaki Kitamura, Shuichi Moribe, Yukio Kitade

Highlights
∙ A robust synthetic route for new glucosamino nucleic acids (GANAs)
∙ ONs with two types of linkage between the glucosamine and natural nucleotides
∙ Hybridization of ONs containing GANA-T either in the center or at the end of the sequence
∙ Thermal stability of oligonucleotide duplexes containing GANA zwitterionic nucleotides
∙ Measurement of base recognition activity of GANA-T in ONs