Preparation of carbohydrate-oligonucleotide conjugates using the squarate spacer

Article abstract of DOI:10.1016/j.bmcl.2007.09.078

Attachment of carbohydrates to oligonucleotides has proven to induce receptor-mediated endocytosis. A facile method for the formation of covalent linkages between glycans and oligonucleotides is herein described. Thus, use of 3,4-diethoxy-3-cyclobutene-1,2-dione as a linking reagent provides easy conjugation between carbohydrates bearing an amino group at the reducing end and oligonucleotides bearing an aminoalkyl modification.

Full text of DOI:10.1016/j.bmcl.2007.09.078

Bioorganic & Medicinal Chemistry Letters 17 (2007) 6535–6538
Preparation of carbohydrate–oligonucleotide conjugates
using the squarate spacer
*
´
´
Hongbin Yan, Aime Lopez Aguilar and Yuyan Zhao
Department of Chemistry, Brock University, St. Catharines, Ont., Canada L2S 3A1
Received 8 September 2007; accepted 24 September 2007
Abstract—Attachment of carbohydrates to oligonucleotides has proven to induce receptor-mediated endocytosis. A facile method for
the formation of covalent linkages between glycans and oligonucleotides is herein described. Thus, use of 3,4-diethoxy-3-cyclobutene-
1,2-dione as a linking reagent provides easy conjugation between carbohydrates bearing an amino group at the reducing end and
oligonucleotides bearing an aminoalkyl modification.
ꢀ 2007 Elsevier Ltd. All rights reserved.
In recent years, nucleic acids have been investigated as
potential therapeutic agents in various forms, such as
gene therapy, antisense oligonucleotides, antigene oligo-
nucleotides, aptamers, and most recently RNA interfer-
ence.1 However, the effectiveness of these nucleic acids
as therapeutic agents has been hampered by a number
of limitations. Because nucleic acids are relatively large
molecules bearing multiple negative charges, their cellu-
lar uptake is not efficient, which results in poor bioavail-
ability. In addition, their cell targeting is not specific. A
variety of structural modifications on nucleic acids have
been developed which confer novel properties, such as
enhanced resistance to nucleases, increased ability to
hybridize with complementary sequences, and higher
internalization efficiency through cell membranes. Addi-
tionally, use of delivery vehicles, such as lipids, lipo-
some, peptide, virus and viral vectors, and antibodies,1
have also shown promise in addressing the bioavailabil-
ity of therapeutic nucleic acids.
drug molecule, the drug–carbohydrate conjugate can
be transported into the cell with increased efficiency.
This approach, which is called glycotargeting,3 has been
shown to improve both the internalization efficiency and
specificity of cell targeting. A number of conjugation
chemistry have been reported in the literature,4,5 how-
ever, in our efforts to address the drug delivery issue
of small interference RNAs (siRNA), we requested a
method that allows for easy incorporation of carbohy-
drate moieties into the RNA molecules under mild
conditions. 3,4-Diethoxy-3-cyclobutene-1,2-dione 1 (or
diethyl squarate) has been shown to be an efficient link-
ing reagent in the formation of glycopeptides and
glycoproteins.6 By using 3,4-diethoxy-3-cyclobutene-
1,2-dione 1 as a linking reagent, a glycan moiety 2 can
be readily attached first to the squarate to form the acti-
vated glycan 3, which can further react with a protein or
peptide to form a glycoconjugate 4 (Scheme 1). Because
it is relatively straightforward to introduce an amino
group into oligonucleotide using either the phospho-
One useful method that has received a lot of attention is
to utilize the carbohydrate-binding proteins—lectins—
that reside on cell membranes of certain cell types.2
These lectins show specific binding affinity toward car-
bohydrate of defined structures. Upon binding, carbo-
hydrates are internalized through receptor-mediated
endocytosis. When the carbohydrate is attached to a
Keywords: Drug delivery; Glycoconjugate; Lectin; Oligonucleotide;
Squarate.
*
Corresponding author. Tel.: +1 905 688 5550x3545; fax: +1
9056829020; e-mail: tyan@brocku.ca
Scheme 1. Squarate linker in glycoprotein synthesis.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.078

6536
H. Yan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6535–6538
ramidite or H-phosphonate chemistry, we decided to
explore the use of squarate as linker in the preparation
of glycosylated oligonucleotides. Once the glycan is suc-
cessfully linked to oligonucleotides through the squarate
spacer, further elaboration of the conjugate can be
effected by glycosyltransferases.7 This approach will
provide easy access to oligonucleotides with complex
glycan structures that are required in glycotargeting.
were fully characterized by reverse phase HPLC, 1H
and 13C NMR, and mass spectroscopy.
As can be seen in the COSY–NMR spectrum of lacto-
side 9b in Figure 1, single b-anomer was obtained, which
is indicated by a doublet at 4.35 ppm, representing the b-
anomeric proton of lactoside.
We then carried out the conjugation reaction between
squarate-activated glycan 9 and 50-deoxy-50-aminot-
hymidine 1014 and dinucleotide 12 bearing an amino
modification at the 50-terminus (Scheme 4).15 The ami-
no-modified dinucleotide 12 was prepared by the modi-
fied H-phosphonate chemistry in solution.16,17 The
conjugation was carried out in a mixture of sodium
hydrogen carbonate (20 mM) and sodium tetraborate
buffer (20 mM) (Scheme 4). The reactions were followed
by reverse phase HPLC on a 4.6 · 150 mm Acclaim PA
C18 3l column, eluted with a mixture of triethylammo-
nium acetate (TEAA) buffer (0.1 M, pH 7.0)—acetoni-
trile under a linear gradient of TEAA–acetonitrile
(100:0 to 85:15 v/v over 15 min and then isocratic
For method development purpose, we chose to use b-(2-
aminoethyl)glucoside 8a and b-(2-aminoethyl)lactoside
8b as glycans because these two glycosides can be readily
obtained by catalytic hydrogenation of corresponding
b-(2-azidoethyl)glycosides 7 (Scheme 2),8–10 which in
turn were conveniently prepared in good overall yields
from glucose 5a (or lactose 5b) using the trichloroace-
timidate chemistry.11
The reactions between the b-(2-aminoethyl)glycosides 8
and 3,4-diethoxy-3-cyclobutene-1,2-dione 1 were carried
out in aqueous methanol solution in the presence of
trace amount of triethylamine (Scheme 3).12,13 The reac-
tions were found to be complete within 5–10 min as indi-
cated by TLC (CHCl3–MeOH–H2O, 65:35:5 v/v). The
products were readily purified by size exclusion chroma-
tography on Bio-Gel P2 fine gel (Bio-Rad), eluted with
ammonium bicarbonate buffer. Lyophilization of the
fractions that contain the products typically gives the
products in good yield. The squarate-activated glyco-
sides 9 appear to be colorless hygroscopic froth, and
Figure 1. COSY–NMR spectrum of squarate-activated lactoside 9b.
Scheme 2. Preparation of 20-aminoethylglucoside 8a and lactoside 8b.
Reagents and conditions: (i) (Ac)2O, C5H5N, rt; (ii) NH2NH2ÆH2O,
AcOH, DMF, rt, 45 min; (iii) CCl3CN, DBU, CH2Cl2; (iv) BF3ÆEt2O,
HOCH2CH2Cl, CH2Cl2; (v) NaN3, DMF, 55 ꢁC, 3d; (vi) NaOMe,
MeOH; (vii) Pd/C, H2, H2O.
Scheme 3. Activation of glycan 8 by diethyl squarate 1. Reagents and
conditions: (i) H2O, MeOH, NEt3, rt, 5 min.
Scheme 4. Conjugation reaction (a,b). Reagents and conditions: (i)
NaHCO3, Na2B4O7, pH 8.5.

H. Yan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6535–6538
6537
Table 1. Glycosylated oligonucleotides
squarate-activated lactoside 9b (middle profile), and
the isolated conjugate 13b (top profile).
Entry
Mass (ESIꢀ)
Yield (%)
Observed
Calculated
The exact mass of the conjugates was determined by
electrospray mass spectroscopy to detect negative ions.
All the observed masses are in close agreement with their
theoretical values (Table 1).
11a
11b
13a
13b
541.191
703.249
968.233
1130.266
541.178
703.231
968.232
1130.286
86
84
91
95
In conclusion, we have demonstrated a facile method for
the preparation of glycan–oligonucleotide conjugates
that utilizes diethyl squarate as the linking reagent. Fur-
ther elaboration of these conjugates by glycosyltransfe-
rases will open an avenue for the incorporation of
complex glycans into oligonucleotides. It is conceivable
that modification of therapeutic oligonucleotides by
these complex glycans may serve to improve the bio-
availability of nucleic acids as therapeutic agents.
elution). After incubation at room temperature for 24 h,
the reactions were found to be complete.
The conjugation products 11 and 13 were readily iso-
lated in good yields (Table 1) by size exclusion chroma-
tography on Bio-Gel P2 fine gel, eluted with ammonium
bicarbonate buffer (5 mM). These products were charac-
terized, where appropriate, by 1H, and 31P NMR,
COSY, reverse phase HPLC, and mass spectrometry.
As expected, the lactoside conjugate 13b showed two
peaks at 0.28 and ꢀ1.05 ppm, respectively, in 31P
NMR, and each peak integrated one phosphorus
(Fig. 2).
Acknowledgments
This work was supported by Research Corporation,
Canada Foundation for Innovation and Natural
Sciences, and Engineering Research Council of Canada.
The authors thank Tim Jones and Razvan Simionescu
for mass spectrometric and NMR analysis, respectively.
In C18 reverse phase HPLC, the squarate-activated gly-
can 9, 50-deoxy-50-aminothymidine 10 (or dinucleotide
12), and the conjugation products 11 and 13 were very
well resolved. Figure 3 shows the HPLC profiles of the
50-amino-modified dinucleotide 12 (bottom profile),
References and notes
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7, E61.
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9, 237.
3. Monsigny, M.; Midoux, P.; Mayer, R.; Roche, A. C.
Biosci. Rep. 1999, 19, 125.
4. Yan, H.; Tram, K. Glycoconjugate J. 2007, 24, 107.
5. Zatsepin, T. S.; Oretskaya, T. S. Chem. & Biodivers. 2004,
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1996, 13, 315.
7. Kathryn, M. K.; Wong, C.-H. Chem. Rev. 2000, 100, 4465.
8. Ladmiral, V.; Mantovani, G.; Clarkson, G. J.; Cauet, S.;
Irwin, J. L.; Haddleton, D. V. J. Am. Chem. Soc. 2006,
128, 4823.
Figure 2. 1H (left) and 31P (right) NMR spectra of conjugate 13b.
9. Chong, P. Y.; Petillo, P. A. Org. Lett. 2000, 8, 1093.
10. Blixt, O.; Vasiliu, D.; Allin, K.; Jacobsen, N.; Warnock,
D.; Razi, N.; Paulson, J. C.; Bernatchez, S.; Gilbert, M.;
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Chemistry; Hanessian, S., Ed.; Marcel Dekker, Inc: New
York, 1997; pp 283–312.
1
2
3
C3-d(CpT)
Lac-Squa
Lac-Squa-C3-d(CpT)
WVL:260 nm
12. Andersen, S. M.; Ling, C. C.; Zhang, P.; Townson, K.;
Willison, H. J.; Bundle, D. R. Org. Biomol. Chem. 2004, 2,
1199.
13. Preparation of squarate-activated lactoside 9b. b-(2-Ami-
noethyl)-lactoside 8b (20 mg, 0.0519 mmol) was dissolved
in distilled water (50 ll), followed by addition of methanol
(0.8 ml), 3,4-diethoxy-3-cyclobutene-1,2-dione 1 (50 ll,
0.338 mmol), and triethylamine (3 ll). After 5 min, the
solvents were quickly removed by a flow of air. The
residue was diluted with distilled water (1.0 ml) and
purified by size exclusion column on Bio-Gel P2 (fine,
1.6 · 75 cm), eluted with aqueous ammonium bicarbonate
buffer (5 mM). The appropriate fractions were combined
and freeze-dried to give the title compound as a hygro-
scopic solid (23 mg, 87%).
3
2
1
min
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
Retention Time [min]
Figure 3. Stack of HPLC profiles of amino-modified d(CpT) dimer 12
(bottom), squarate-activated lactoside 9b (middle), and conjugation
product 13b (top).

6538
H. Yan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6535–6538
ESI ꢀ MS foundꢀMꢀ ¼ C20H30NO1ꢀ4508:1789.
tetraborate buffer (0.10 ml, 20 mM). After the reaction
mixture has been shaken for 24 h at room temperature, the
products were purified by size exclusion column on Bio-
Gel P2 (fine, 1.6 · 75 cm), eluted with aqueous ammonium
bicarbonate (5 mM). The appropriate fractions were
combined and freeze-dried to give the conjugate as a
colorless solid (5.7 mg, 95%).
1H3014N16O14requires : 508:1672.
12
C
20
Selected dH[D2O, 500 MHz]: 1.35 (t, J = 7.2), 1.36 (t,
J = 7.2) (these two triplets integrate three protons—
CH3CH2O–Squarate), 3.22 (1H, m, H-2_Gal), 3.45 (1H,
dd, J = 7.8 and 9.9, H-2_Glc), 4.35 (1H, d, J = 7.8, H-
1_Glc), 4.41 (1H, d, J = 8.4, H-1_Gal), 4.65 (2H, hidden
under HOD, CH3CH2O–Squarate).
dC[D2O, 150 MHz]: 14.98 and 15.07 (CH3CH2O–Squa-
rate), 44.13, 44.21, 60.10, 61.03, 68.56, 68.86, 69.14, 70.62,
70.66, 70.96, 72.54, 72.84, 74.35, 74.40, 74.79, 75.37, 78.35,
78.41, 102.14, 102.95, 173.75, 173.79, 177.21, 177.49,
183.44, 183.51, 188.98.
ESI ꢀ MS found Mꢀ ¼ C40H58N7O27Pꢀ2 1130:266.
C O
1H5814N7 31Pꢀ2 requires : 1130:286.
40 27
12
16
dP[D2O, 121 MHz]: 0.28 (1 P), ꢀ1.05 (1 P).
dH[D2O, 300 MHz] includes the following peaks:1.82 (3H,
s, CH3_Thy), 5.97 (1 H, d, J = 7.5, H-5_Cyt), 6.22 (2 H, m,
Hꢀ10_Thy + Hꢀ10_Cyt), 7.63 (1H, s, Hꢀ6_Thy), 7.83
(1H, d, J = 7.5, H-6_Cyt).
Rt: 10.54 min (linear gradient of triethylammonium ace-
tate buffer–acetonitrile (100:0 to 85:15 v/v) over 15 min
and then isocratic elution).
Rt: 9.08 min (linear gradient of triethylammonium acetate
buffer–acetonitrile (100:0 to 85:15 v/v) over 15 min and
then isocratic elution).
14. Challa, H.; Bruice, T. C. Bioorg. Med. Chem. 2004, 12, 1475.
15. Preparation of conjugate 13b. Squarate-activated lactoside
9b (5.4 mg, 10.6 lmol) and amino-modified dinucleotide
12 (3.5 mg, 5.3 lmol) were dissolved in a mixture of
sodium hydrogen carbonate (0.10 ml, 20 mM) and sodium
16. Reese, C. B.; Song, Q. L. Bioorg. Med. Chem. Lett. 1997,
7, 2787.
17. Reese, C. B.; Yan, H. J. Chem. Soc., Perkin Trans. 1 2002,
23, 2619.