A new approach to the synthesis of ligands of asialoglycoprotein receptor for targeted delivery of oligonucleotides to hepatocytes

Article abstract of DOI:10.1007/s11172-015-1056-6

A new approach to the synthesis of organic ligands of asialoglycoprotein receptor was suggested. It was shown that the azido-containing tervalent N-acetylglucosamine and N-acetylmannosamine derivatives obtained by this method can serve as convenient starting compounds for the synthesis of therapeutical conjugates with oligonucleotides containing a terminal alkyne.

Full text of DOI:10.1007/s11172-015-1056-6

Russian Chemical Bulletin, International Edition, Vol. 64, No. 7, pp. 1655—1662, July, 2015
1655
A new approach to the synthesis of ligands of asialoglycoprotein receptor
for targeted delivery of oligonucleotides to hepatocytes
S. Yu. Maklakova,a F. A. Kucherov,a R. A. Petrov,a V. V. Gopko,a G. A. Shipulin,b T. S. Zatsepin,a,b,c
E. K. Beloglazkina,a,d N. V. Zyk,a A. G. Majouga,a,d and V. E. Kotelianskya,c
aDepartment of Chemistry, M. V. Lomonosov Moscow State University,
Build. 3, 1 Leninskie Gory, 119991 Moscow, Russian Federation
bCentral Research Institute of Epidemiology,
3a ul. Novogireevskaya, 111123 Moscow, Russian Federation
cSkolkovo Institute of Science and Technology,
100 ul. Novaya, 143025 Skolkovo, Russian Federation
dNational University of Science and Technology MISiS,
4 Leninsky prosp, 119991 Moscow, Russian Federation.
Eꢀmail: majouga@org.chem.msu.ru, bel@org.chem.msu.ru
A new approach to the synthesis of organic ligands of asialoglycoprotein receptor was
suggested. It was shown that the azidoꢀcontaining tervalent Nꢀacetylglucosamine and Nꢀacetylꢀ
mannosamine derivatives obtained by this method can serve as convenient starting compounds
for the synthesis of therapeutical conjugates with oligonucleotides containing a terminal alkyne.
Key words: asialoglycoprotein receptor, ligand, hepatocytes, azideꢀalkyne cycloaddition,
oligonucleotide.
Targeted delivery of therapeutical agents suggests seꢀ
lective and efficient localization of a pharmacologically
active compound in the target tissue, organ, or cell in
efficient concentration, with a simultaneous decrease in
its average content in the organism. The advantages of the
drug targeted delivery are as follows: (1) a decrease in the
toxic effects and an increase in the therapeutical index;
(2) a considerable decrease in the required amount of the
agent and, therefore, in the cost of treatment; (3) the abꢀ
sence of the necessity for drug optimization, since it is
already known. For the therapeutical molecule to be effiꢀ
ciently delivered to a certain organ, tissue, or cell, a search
for the specific receptors capable of binding a ligand
(a vector) is necessary. Great hope lies with the conjugation
of therapeutical molecules with antibodies, the positive
results of clinical trials for which (20 agents, phase I, phase II,
and phase III) indicate that the suggested methodology is
efficient.1—3 The use of lowꢀmolecularꢀweight ligands caꢀ
pable of selective binding with the intercellular receptors
is an alternative platform for the preparation of conjuꢀ
gates. At the present time, the biotin,4 folic acid,5 amino
acid ureide,6 and Nꢀacetylgalactosamine (GalNAc)7 deꢀ
rivatives are used as the ligands for targeted delivery.
Hepatocytes are known to express a large amount of
asialoglycoprotein receptors (ASGPr) (up to 500 thouꢀ
sand units per cell),8,9 which recognize the terminal moiꢀ
eties of Dꢀgalactose and NꢀacetylꢀDꢀgalactosamine. In this
connection, the targeted delivery of therapeutical moleꢀ
cules to the liver is possible if the agents with a targeted
module represented by a galactoseꢀcontaining fragment
or its analog is used. In the present work, we develop a new
synthetic approach to the preparation of tervalent ligands
of asialoglycoprotein receptor, using the synthesis of
ligands with the Nꢀacetylglucosamine and Nꢀacetylꢀ
mannosamine moieties as examples.
Results and Discussion
In contrast to numerous lipid and protein glyco derivꢀ
atives, only limited amount of DNA glyco derivatives are
found in the nature and only in phages and protozoa. As
a rule, glycosylation increases the stability of DNA of
phages and protozoa in the host cell; the glycosylated DNA
are involved in the regulation of protozoa transcription.10
At the same time, there are literature examples of the
preparation of synthetic conjugates of carbohydrates with
oligonucleotides, which are used for targeted delivery of
DNA and RNA derivatives to a certain type of cells (hepaꢀ
tocytes, macrophages), as well as for the studies of the
in vitro functioning of glycosylated DNA.10,11 A selective
inhibition of gene expression through the blocking or
hydrolysis of mRNAs, microRNAs, long nonꢀcoding RNAs,
as well as binding of aptamers with proteins are theraꢀ
peutical methods based on oligonucleotides (Macugen,
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1655—1662, July, 2015.
1066ꢀ5285/15/6407ꢀ1655 © 2015 Springer Science+Business Media, Inc.

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Maklakova et al.
Mipomersen).12 Unfortunately, the use of unmodified
oligonucleotides is inefficient because of their low stability
in vivo and rapid excretion from the organism, as well as
impossibility of their targeted delivery to a certain tissue
and/or cell. This is the latter disadvantage which limited
the application of oligonucleotides in therapeutical pracꢀ
tice until recently despite significant achievements in the
use of antisense oligonucleotides, short interfering RNA,
and aptamers.13,14 Several approaches were suggested to
increase the efficiency of penetration through the cell
membrane: preparation of liposomes with cationic pepꢀ
tides, synthesis of complexes with different polyamines,
cationic lipids, conjugation with hydrophobic molecules
and peptides.15—17 Despite significant success achieved
during testing in vitro, in the case of the in vivo testing
most synthesized derivatives have proved to be too toxic
for the application in therapeutical practice. Apart from
that, in the case when complexes or polymer derivatives
are used it is difficult to accurately establish their structure
(stoichiometry of complexes and polymerization degree).
Nonetheless, oligonucleotide complexes, as before, are
widely represented in the early stages of clinical trials,
despite considerable disadvantages of their use because of
substantial complications in the synthesis of conjugates.
Recently, the conjugates of oligonucleotides with a terꢀ
valent Nꢀacetylgalactosamine derivative18—20 of the folꢀ
lowing structure were synthesized (see below).
Such oligonucleotideꢀcarbohydrate conjugates conꢀ
siderably faster penetrated the cells and distributed in the
organism in vivo, as well as had low toxicity and immunoꢀ
genicity even at high concentrations. It is necessary to
note that in this case, the authors used stable in vivo chemꢀ
ically modified oligonucleotides containing thiophosphate
groups, as well as substituents (fluoro, alkoxy) at 2´ꢀposiꢀ
tion of the carbohydrate fragment. The principal advanꢀ
tages of such derivatives are extremely high selectivity and
rapid penetration into hepatocytes. Already 1.5—2 min
after the intravenous administration into the mouse body,
98% of the conjugate were in the hepatocytes, that makes
it possible to considerably decrease (by 10—50 times) the
single dose of the agent.18 In future, this approach can
"reanimate" a large number of oligonucleotides removed
earlier from the II—III stages of clinical trials because of
their high toxicity. The subcutaneous administration of
the agent turned out to be even more efficient. Since the
inhibition of expression occurs already at about 0.1 g kg–1
dose of the oligonucleotide, such an application of chemꢀ
ically modified conjugates as a single administered dose
makes it possible to observe therapeutical effect for
30—40 days due to their slow liberation.
The oligonucleotideꢀcarbohydrate conjugates can be
synthesized applying two strategies: 1) a standard oligoꢀ
nucleotide synthesis which uses carbohydrate amidophosꢀ
phite derivatives or preparation of solidꢀphase supports
with immobilized carbohydrates; 2) a postsynthetic addiꢀ
tion of active carbohydrate derivatives to modified oligoꢀ
nucleotides.10,11 The use of the first approach is optimal
for the preparation of large amounts of oligonucleotide
derivatives, whereas the second approach is more suitable
for the screening of ligands. Since in the future we plan to
obtain derivatives of a number of carbohydrates with difꢀ
ferent linkers and cluster geometry, we used the second
strategy. The Cu(I)ꢀcatalyzed 1,3ꢀdipolar cycloaddition
of azides and alkynes21,22 lately widely used for bioconjuꢀ
gation23 was chosen as a key reaction.
Earlier, the tervalent ligands of asialoglycoprotein reꢀ
ceptor containing Nꢀacetylgalactosamine fragments were
obtained using an approach based on the sequential addiꢀ
tion of structural units of the target molecule24 to the
2ꢀaminoꢀ2ꢀ(hydroxymethyl)propaneꢀ1,3ꢀdiol derivative
(TRIS). The principal disadvantage of this method conꢀ
sisted in the formation in each step of a poorly separable
mixture of monoꢀ, bisꢀ, and trisꢀderivatives, which reꢀ
quired a chromatographic separation of compounds with
similar properties. Despite the yields in each step are
satisfactory, the overall yield of the product is no more
than 30%.
To prepare the target tervalent ligands, we suggested
a convergent strategy of the synthesis, which allowed us
for the first time to obtain structural analogs of the known
X is an oligonucleotide, Y is a linker.

Synthesis of the ligands for the ASGPr
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
1657
ligand containing the Nꢀacetylglucosamine or Nꢀacetylꢀ
mannosamine moieties, rather than the Nꢀacetylgalacꢀ
tosamine fragment. Such structures are of interest as the
models for the studies of binding with ASGPr. Apart from
that, the ligand based on Nꢀacetylmannosamine is a poꢀ
tential antiviral agent.25
with glycosyl acetates 2a,b in the presence of TMSOTf
(Me3SiOSO2CF3) through the intermediate formation of
oxazoline.26 The amino group protection was removed
upon treatment with trifluoroacetic acid, and the resulting
amines 4a,b (as trifluoroacetates) were involved in subseꢀ
quent reactions without additional purification.
The first step of the synthesis of ligands included the
ꢀvalerolactone ring opening with monoꢀBocꢀ1,3ꢀdiaminoꢀ
propane in the presence of a catalytic amount of pꢀtolueꢀ
nesulfonic acid (Scheme 1). Alcohol 1 was glycosylated
In the case of transformation of Nꢀacetylglucosamine
derivative 2a, the ꢀanomer of glycoside 3a was exclusiveꢀ
ly obtained. When a mixture of ,ꢀanomers of peracetyꢀ
lated NꢀacetylꢀDꢀmannosamine 2b was used, an anomer
Scheme 1
Reagents and conditions: i. TsOH, toluene, 50 C; ii. 1) TMSOTf, Cl(CH2)2Cl, 50 C, 12 h; 2) Et3N, 1.5 h; iii. 1, MS 4 Å, TMSOTf,
Cl(CH2)2Cl, 24 h; iv. TFA, CH2Cl2, 1.5 h.
Scheme 2
Reagents and conditions: i. N3(CH2)10COOH, Me2N(CH2)3N=C=NEt•HCl, Et3N, CH2Cl2, 24 h; ii. HCOOH, 12 h.

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Maklakova et al.
of glycoside 3b was isolated as the only product, which
-anomer ( 69.1—80.1 for the structurally similar derivaꢀ
tives27); in the 1H NMR spectrum of product 3b, the spinꢀ
spin coupling constant of the equatorial protons at carbon
atoms C(1) and C(2) is equal to 1.4 Hz (the values for the
structurally similar Nꢀacetylꢀ1ꢀpropylꢀꢀDꢀmannosamine
1
was identified as having ꢀconfiguration based on the H
and 13C NMR spectra. In the 13C NMR spectrum of comꢀ
pound 3b, the signals for atoms C(3) and C(5) are shifted
to the highꢀfield region ( 68.0—69.5) as compared to
Scheme 3
Reagents and conditions: i. HBTU, DIPEA, THF, 12 h; ii. TBTA—CuSO4—ascorbic acid, 18 h; iii. aqueous NH3, 6 h.

Synthesis of the ligands for the ASGPr
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
1659
3200 V, respectively). IR spectra were recorded on a Termoꢀ
Nicolet IR200 Fourierꢀtransform IR spectrometer (USA) with
a 4 cm–1 resolution. Laser ionization mass spectra (MALDIꢀTOF)
were recorded on a Bruker Autoflex II instrument, using
ꢀcyanoꢀ4ꢀhydroxycinnamic acid as a matrix.
and Nꢀacetylꢀ1ꢀpropylꢀꢀDꢀmannosamine were found to
be J1,2 = 0.9 Hz and J1,2 = 4.5 Hz, respectively27).
For further conjugation of the target organic ligands
with oligonucleotides, compound 7 containing a linker
with the terminal azido group was obtained (Scheme 2).
To achieve this, amine 5 (see Ref. 28) was involved in the
reaction with 11ꢀazidoundecanoic acid. Such an approach
makes it possible in future vary the linkers introduced in
the structure. In the present work, we used a linker conꢀ
taining ten methylene groups, since the ligands with such
fragments earlier have demonstrated good results in bioꢀ
logical trials.19 tertꢀButyl ester 6 was hydrolyzed to acid 7,
which was involved in the reaction with amines 4a,b
(Scheme 3).
Analysis and purification of oligonucleotide conjugates were
carried out by HPLC on a Jupiter C18 column (4.6×250 mm;
5 m, Phenomenex); buffer A: 0.05 M solution of ammonium
acetate (pH 7), 5% MeCN; buffer B: 0.03 M solution of ammoꢀ
nium acetate (pH 7), 80% MeCN; gradient B: 015% (1 volꢀ
ume of the column), 1550% (10 volumes of the column); the
flow rate 1 mL min–1 at 45 C. Mass spectrometric analysis of
oligonucleotides was carried out using a Bruker Maxis Impact
qꢀTOF GLCꢀMS spectrometer connected to an Agilent 1260
chromatograph similarly to that described in the work.29
Commercially available reactants (SigmaꢀAldrich) were used
in the work without additional purification. Compounds 2a,b
were obtained according to the known procedure,30 compound 5
was synthesized according to the described procedure.28
tertꢀButyl {3ꢀ[(5ꢀhydroxypentanoyl)amino]propyl}carbamate
(1). ꢀValerolactone (0.6 mL, 7 mmol) and monoꢀBocꢀ1,3ꢀdiꢀ
aminopropane31 (1.2 mL, 7 mmol) were dissolved in toluene
(50 mL). The reaction mixture was stirred for 24 h at 70 C in the
presence of TsOH•H2O (10 mg), then the solvent was evaporatꢀ
ed. A yellow oil obtained was purified by flashꢀchromatography
(eluent ethyl acetate—methanol, 10 : 1) to isolate compound 1
(1.7 g, 90%) as a white crystalline compound, m.p. 58—60 C.
IR (CDCl3), /cm–1: 3338, 3361 (N—H); 1689, 1637 (C=O).
1H NMR (CDCl3), : 1.42 (s, 9 H, OC(CH3)3); 1.54—1.65
(m, 4 H, CH2CH2NH, CH2CH2C(O)); 1.68—1.78 (m, 2 H,
CH2CH2OH); 2.25 (t, 2 H, CH2C(O), J = 7.2 Hz); 3.10—3.18
(m, 2 H, CH2NHBoc); 3.19 (br.s, 1 H, CH2CH2OH); 3.27
(q, 2 H, CH2NHC(O), J = 6.1 Hz); 3.63 (t, 2 H, CH2CH2OH,
J = 6.2 Hz); 5.10 (br.s, 1 H, NHBoc); 6.72 (br.s, 1 H, NHC(O)).
13C NMR (CDCl3), : 21.80 (CH2CH2C(O)); 28.36 (OC(CH3)3);
30.09 (CH2CH2NH); 31.93 (CH2CH2OH); 35.91 (CH2NHBoc);
35.97 (CH2C(O)); 37.13 (CH2NHC(O)); 61.96 (CH2CH2OH);
79.43 (OC(CH3)3); 156.68 (CH2NHC(O)); 173.68 (NHC(O)O).
MS (ESI) found: m/z 297.1785 [M + Na]+; C13H26N2O4Na;
calculated: 297.1785.
Synthesis of tertꢀbutyl carbamates 3a,b (general procedure).
Trimethylsilyl triflate (1.2 equiv.) was added to a solution of the
corresponding 2ꢀacetamidoꢀ1,3,4,6ꢀtetraꢀOꢀacetylꢀ2ꢀdeoxyꢀDꢀ
hexopyranose 2a or 2b in 1,2ꢀdichloroethane. The mixture was
stirred for 12 h at 50 C and cooled to 20 C, followed by the
addition of triethylamine (2.5 equiv.) and stirring for 1.5 h. The
resulting mixture was applied to a short layer of silica gel, thorꢀ
oughly washed with dichloromethane, and eluted with ethyl
acetate to isolate the intermediate oxazoline as a light yellow oil.
The oil was dissolved in 1,2ꢀdichloroethane, followed by the
addition of alcohol 1 (1.2 equiv.) and stirring for 30 min with
molecular sieves 4Å (beads, 50 mg). After addition of TMSOTf
(0.5 equiv.), the mixture was allowed to stand for 24 h at 20 C.
Molecular sieves were filtered off, the reaction mixture was conꢀ
centrated at reduced pressure. The product was isolated by flashꢀ
chromatography (eluent ethyl acetate  dichloromethane—
methanol (20 : 1)).
The structure of all the compounds obtained was conꢀ
firmed by a combination of physicochemical methods,
including 1H and 13C NMR spectroscopy and mass specꢀ
trometry.
In the final stage, ligand 8a was used as an example to
accomplish a copperꢀcatalyzed [3 + 2] azidoꢀacetylene
cycloaddition reaction with a model oligonucleotide dT20
containing a terminal alkyne on the 5´ꢀend of 9. The reacꢀ
tion was carried out for 18 h at room temperature in the
system TBTA (trisꢀ[(1ꢀbenzylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)ꢀ
methyl]amine)—CuSO4—ascorbic acid in the presence of
two equivalents of azide. The formation of conjugate 10a
was confirmed using HPLC and mass spectrometry
(calculated weight [(Ac)3GlcNAc]3—dT20 = 8294.14,
found = 8293.50). The conversion was more than 95%.
The conjugation was carried out with derivative 8a, which
contains the acetyl protections to simplify purification
process of the product by reversedꢀphase HPLC because
of the increased hydrophobicity. Then, the acetyl protectꢀ
ing groups were removed with an aqueous solution of amꢀ
monia at room temperature with the formation of conjuꢀ
gate 11a (calculated weight [GlcNAc]3—dT20 = 7917.05,
found = 7915.56).
In conclusion, we suggested an alternative approach to
the synthesis of tervalent ligands of ASGPr, its universal
character was demonstrated by the preparation of strucꢀ
tures containing Nꢀacetylglucosamine and Nꢀacetylmanꢀ
nosamine moieties. We also showed a possibility of the
application of [3 + 2] azidoꢀacetylene cycloaddition for
the preparation of conjugates of obtained tervalent carboꢀ
hydrate azido derivatives with oligonucleotide containing
a terminal alkyne.
Experimental
1
H and 13C NMR spectra were recorded on Bruker DPXꢀ300
(400 (1H) and 100 (13C) MHz) and Agilent 400 MR (400 (1H)
and 100 (13C) MHz) spectrometers. Electrospray ionization (ESI)
high resolution mass spectra were recorded on a Bruker
micrOTOF II instrument. Measurements were carried out in
positive and negative ion modes (capillary potential 4500 and
tertꢀButyl Nꢀ[9ꢀ(2ꢀacetamidoꢀ3,4,6ꢀtetraꢀOꢀacetylꢀ2ꢀdeꢀ
oxyꢀꢀDꢀglucopyranosyloxy)ꢀ5ꢀoxoꢀ4ꢀazanonyl]carbamate (3a).
The reaction and the workꢀup were carried out according to the
general procedure. Glycoside 3a (1.4 g, 60%) was obtained as

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Maklakova et al.
a colorless oil from compound 2a (1.5 g, 4.0 mmol) and comꢀ
pound 1 (1.3 g, 4.9 mmol); []D –100 (c 0.2, CHCl3).
1.62—1.69 (both m, 6 H, OCH2CH2CH2, CH2CH2NH3+); 1.76,
1.90, 1.96, 2.01 (all s, 3 H each, 4 C(O)CH3); 2.05—2.10 (m, 2 H,
CH2C(O)NH); 2.72—2.80 (m, 2 H, CH2NH3+); 3.09 (q, 2 H,
C(O)NHCH2, J = 6.4 Hz); 3.42 (dt, 1 H, OCHaHb(CH2)2,
J = 10.0 Hz, J = 5.9 Hz); 3.65—3.76 (m, 2 H, OCHaHb(CH2)2,
Hꢀ2); 3.81 (ddd, 1 H, Hꢀ5, J = 9.8 Hz, J = 4.8 Hz, J = 1.9 Hz);
4.00 (dd, 1 H, Hꢀ6, J = 12.2 Hz, J = 1.9 Hz); 4.18 (dd, 1 H,
Hꢀ6´, J = 12.2 Hz, J = 4.8 Hz); 4.58 (d, 1 H, Hꢀ1, J = 8.6 Hz); 4.82
(t, 1 H, Hꢀ3, J = 9.8 Hz); 5.07 (t, 1 H, Hꢀ4, J = 9.8 Hz); 7.71
(br.s, 3 H, NH3+); 7.92 (t, 1 H, C(O)NH, J = 5.7 Hz), 7.96 (d, 1 H,
NHAc, J = 9.3 Hz). 13C NMR (DMSOꢀd6), : 20.33, 20.41,
20.51, 21.83 (CH3C(O)); 22.62 (CH2CH2C(O)NH); 27.55
(OCH2CH2); 28.54 (CH2CH2NH); 34.94 (CH2C(O)NH); 35.43
(CH2NHBoc); 36.81 (CH2NHC(O)); 53.15 (Cꢀ2); 61.88 (Cꢀ6);
68.65 (OCH2CH2); 68.71 (Cꢀ4); 70.67 (Cꢀ3); 72.67 (Cꢀ5);
100.30 (Cꢀ1); 169.30, 169.31, 169.63, 170.06 (4 C(O)CH3);
172.60 (C(O)NH). MS (ESI) found: m/z 504.2549 [M + H]+;
C22H38N3O10; calculated: 504.2552.
Nꢀ(3ꢀAmmoniopropyl)ꢀ5ꢀ[3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ
2ꢀdeoxyꢀꢀDꢀmannopyranosyloxy]pentanamide trifluoroacetate
(4b). A yellow oil, quantitative yield. 1H NMR (DMSOꢀd6),
: 1.48—1.60, 1.62—1.71 (both m, 6 H, OCH2CH2CH2,
CH2CH2NH3+); 1.90, 2.02 (both s, 6 H each, 4 C(O)CH3);
2.09—2.15 (m, 2 H, CH2C(O)NH); 2.71—2.79 (m, 2 H,
CH2NH3+); 3.11 (q, 2 H, C(O)NHCH2, J = 6.5 Hz); 3.37—3.46
(m, 1 H, OCHaHb(CH2)2); 3.52—3.61 (m, 1 H, OCHaHbꢀ
(CH2)2); 3.79—3.86 (m, 1 H, Hꢀ5); 4.01 (dd, 1 H, Hꢀ6, J = 11.7
Hz, J = 2.5 Hz); 4.27 (dd, 1 H, Hꢀ6´, J = 11.7 Hz, J = 7.2 Hz);
4.35—4.39 (m, 1 H, Hꢀ2); 4.64 (s, 1 H, Hꢀ1); 5.05 (dd, 1 H,
Hꢀ3, J = 10.1 Hz, J = 4.7 Hz); 5.21 (t, 1 H, Hꢀ4, J = 10.1 Hz); 7.70
(br.s, 3 H, NH3+); 7.99 (t, 1 H, C(O)NH J = 5.6 Hz); 8.14 (d, 1 H,
NHAc, J = 9.4 Hz). Found: m/z 526 [M + Na]+ (MALDIꢀTOF);
C22H37N3O10Na; calculated: 526.
25
IR (CDCl3), /cm–1: 3313 (N—H); 1751, 1687, 1660 (C=O).
1H NMR (CDCl3), : 1.41 (s, 9 H, (CH3)3C); 1.55—1.64,
1.70—1.79 (both m, 6 H, OCH2(CH2)2, CH2CH2NHBoc); 1.93,
1.99, 1.99, 2.05 (all s, 3 H each, 4 C(O)CH3); 2.14—2.34 (m, 2 H,
CH2NHBoc); 3.11—3.16 (m, 2 H, CH2C(O)NH); 3.22—3.31
(m, 2 H, C(O)NHCH2); 3.46—3.52 (m, 1 H, OCHaHb(CH2)2);
3.68 (ddd, 1 H, Hꢀ5, J = 9.7 Hz, J = 4.8 Hz, J = 2.1 Hz);
3.83—3.94 (m, 2 H, OCHaHb(CH2)2, Hꢀ2); 4.09 (dd, 1 H, Hꢀ6,
J = 12.2 Hz, J = 2.1 Hz); 4.24 (dd, 2 H, Hꢀ6´, J = 12.2 Hz,
J = 4.8 Hz); 4.63 (d, 1 H, Hꢀ1, J = 8.3 Hz); 5.03 (t, 1 H, Hꢀ3,
J = 9.7 Hz); 5.21 (br.s, 1 H, NHBoc); 5.24 (t, 1 H, Hꢀ4, J = 9.7 Hz);
6.74 (br.d, 1 H, NHAc, J = 8.7 Hz); 6.85 (br.s, C(O)NH).
13C NMR (CDCl3), : 20.55, 20.62, 20.69, 22.40 (4 CH3C(O));
23.11 (CH2CH2C(O)NH); 27.97 (CH2CH2NH)); 28.34
((CH3)3C); 29.96 (OCH2CH2); 35.66 (CH2C(O)NH); 35.97
(CH2NHBoc); 37.08 (CH2NHC(O)); 54.44 (Cꢀ2); 62.08 (Cꢀ6);
68.70 (OCH2CH2); 69.26 (Cꢀ4); 71.64 (Cꢀ3); 72.57 (Cꢀ5); 79.17
((CH3)3C); 100.41 (Cꢀ1); 156.52 (C(O)But); 169.35, 170.66, 170.69,
170.82 (4 C(O)CH3); 173.62 (C(O)NH). MS (ESI) found:
m/z 626.2892 [M + Na]+; C27H45N3O12Na; calculated: 626.2895.
tertꢀButyl Nꢀ[9ꢀ(2ꢀacetamidoꢀ3,4,6ꢀtetraꢀOꢀacetylꢀ2ꢀdeꢀ
oxyꢀꢀDꢀmannopyranosyloxy)ꢀ5ꢀoxoꢀ4ꢀazanonyl]carbamate (3b).
Glycoside 3b (320 mg, 25%) was obtained as a colorless oil from
compound 2b (820 mg, 2.1 mmol) and compound 1 (690 mg,
2.5 mmol). The product was additionally purified by column
chromatography (gradient elution with dichloromethane 
dichloromethane—methanol, 20 : 1). IR (CDCl3), /cm–1: 3319
(N–H); 1747, 1691, 1655 (C=O). 1H NMR (CDCl3), : 1.43
(s, 9 H, (CH3)3C); 1.58—1.68, 1.70—1.78 (both m, 6 H,
OCH2CH2CH2, CH2CH2NHBoc); 1.99, 2.04, 2.04, 2.10 (all s,
3 H each, 4 C(O)CH3); 2.26 (t, 2 H, CH2C(O)NH, J = 7.2 Hz);
3.15 (q, 2 H, CH2NHBoc, J = 5.8 Hz); 3.29 (q, 2 H,
C(O)NHCH2, J = 6.2 Hz); 3.45 (dt, 1 H, OCHaHb(CH2)2,
J = 9.7 Hz, J = 6.0 Hz); 3.70 (dt, 1 H, OCHaHb(CH2)2, J = 9.7 Hz,
J = 5.9 Hz); 3.97 (ddd, 1 H, Hꢀ5, J = 10.1 Hz, J = 5.3 Hz,
J = 2.4 Hz); 4.05 (dd, 1 H, Hꢀ6, J = 12.3 Hz, J = 2.4 Hz); 4.27
(dd, 1 H, Hꢀ6´, J = 12.3 Hz, J = 5.3 Hz); 4.59 (ddd, 1 H, Hꢀ2,
J = 9.0 Hz, J = 4.5 Hz, J = 1.4 Hz); 4.73 (d, 1 H, Hꢀ1, J = 1.4 Hz);
5.09 (t, 1 H, Hꢀ4, J = 10.1 Hz); 5.10 (br.s, 1 H, NHBoc); 5.29
(dd, 1 H, Hꢀ3, J = 10.1 Hz, J = 4.5 Hz); 5.81 (d, 1 H, NHAc,
J = 9.0 Hz); 6.39 (br.s, 1 H, C(O)NH). 13C NMR (CDCl3), : 20.58,
20.65, 20.67, 22.57 (4 CH3C(O)); 23.16 (CH2CH2C(O)NH);
28.30 ((CH3)3C); 28.38 (CH2CH2NH); 29.97 (OCH2CH2);
35.86 (CH2C(O)NH); 36.08 (CH2NHBoc); 37.03 (CH2NHC(O));
50.26 (Cꢀ2); 62.53 (Cꢀ6); 66.03 (OCH2CH2); 68.02 (Cꢀ4); 68.36
(Cꢀ3); 69.30 (Cꢀ5); 79.02 ((CH3)3C); 98.93 (Cꢀ1); 156.41
(C(O)But); 169.79, 170.07, 170.15, 170.47 (4 C(O)CH3);
172.97 (C(O)NH). MS (ESI) found: m/z 604.3078 [M + H]+;
C27H46N3O12; calculated: 604.3076.
11ꢀAzidoundecanoic acid Nꢀtrisꢀ{[2ꢀ(tertꢀbutoxycarbonyl)ꢀ
ethoxy]methyl}methylamide (6). Amine 5 (500 mg, 1 mmol) was
added to a mixture of 11ꢀazidoundecanoic acid (235 mg,
1 mmol), Nꢀ(3ꢀdimethylaminopropyl)ꢀN´ꢀethylcarbodiimide
hydrochloride
((CH3)2N(CH2)3N=C=NCH2CH3•HCl)
(190 mg, 1 mmol), and triethylamine (0.2 mL, 1.3 mmol) in
CH2Cl2 (2 mL). The resulting mixture was stirred for 36 h at
room temperature (20 C). The solvent was evaporated at reꢀ
duced pressure. The residue was dissolved in diethyl ether and
washed with 0.5 M aqueous solution of HCl and a saturated
aqueous NaCl. Amide 6 (350 mg, 60%) was isolated by column
chromatography of the residue (gradient elution with chloroꢀ
form  chloroform—methanol, 50 : 1) as a clear oil. IR (CDCl3),
/cm–1: 3421, 3361, (NꢀH); 2096 (N3); 1677, 1730 (C=O).
1H NMR (CDCl3), : 1.27—1.39 (m, 12 H, N3CH2CH2(CH2)6);
1.44 (s, 27 H, 3 (CH3)3C); 1.54—1.62 (m, 4 H, N3CH2CH2,
CH2CH2C(O)NH); 2.14 (t, 2 H, CH2CH2C(O)NH, J = 7.5 Hz);
2.44 (t, 6 H, 3 CH2C(O)OBut, J = 6.3 Hz); 3.25 (t, 2 H, N3CH2,
J = 6.9 Hz); 3.64 (t, 6 H, 3 OCH2CH2C(O), J = 6.3 Hz); 3.70
(s, 6 H, 3 CCH2O); 6.03 (br.s, 1 H, NH). 13C NMR (CDCl3), :
25.65, 26.69, 28.82, 29.11, 29.19, 29.33, 29.38, 29.44 ((CH2)8);
28.09 (OC(CH3)3); 36.15 (OCH2CH2C(O)); 37.26
((CH2)8CH2C(O)); 51.46 (CH2N3); 59.57 (CCH2O); 67.00
(OCH2CH2C(O)); 69.17 (CCH2O); 80.41 (C(CH3)3); 170.92
(C(O)OBut); 173.46 (C(O)NH). MS (ESI) found: m/z 737.4662
[M + Na]+; C36H66N4O10Na; calculated: 737.4671.
Synthesis of compounds 4a,b (general procedure). The correꢀ
sponding tertꢀbutyl carbamate 3a or 3b (600 mg, 1 mmol) was
stirred with trifluoroacetic acid (12 mL) in 1,2ꢀdichloromethane
(48 mL) for 1.5 h. The solvent was evaporated at reduced presꢀ
sure. The product was used in subsequent reactions without adꢀ
ditional purification.
Nꢀ(3ꢀAmmoniopropyl)ꢀ5ꢀ[3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ
2ꢀdeoxyꢀꢀDꢀglucopyranosyloxy]pentanamide trifluoroacetate
(4a). A yellow oil, quantitative yield. IR (CDCl3), /cm–1: 3354,
3282 (N—H); 1747, 1660. 1H NMR (DMSOꢀd6), : 1.44—1.53,
11ꢀAzidoundecanoic acid Nꢀtrisꢀ{[2ꢀcarboxyethoxy]methyl}ꢀ
methylamide (7). A mixture of tertꢀbutyl ester 6 (400 mg,

Synthesis of the ligands for the ASGPr
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
1661
0.5 mmol) and 99% formic acid (7 mL) was stirred for 12 h.
Removal of the acid at reduced pressure at 50 C gave a white
crystalline compound (270 mg, quantitative yield), m.p. 89—90 C.
(t, 3 H, 3 NH, J = 5.7 Hz); 7.30—7.36 (m, 3 H, 3 NH). Found:
m/z 2026 [M + Na]+ (MALDIꢀTOF); C90H147N13O37Na; calꢀ
culated: 2026.
1
IR (CDCl3), /cm–1: 2096 (N3); 1716, 1631 (C=O). H NMR
Synthesis of oligonucleotide and its derivatives 10a and 11a.
Oligonucleotide dT20 containing a terminal alkyne on the 5´ꢀend
(9) was synthesized using a known procedure32 on an ASMꢀ1000
automated oligonucleotide synthesizer according to the amidoꢀ
phosphite scheme using 2´ꢀdeoxythymidine 3´ꢀamidophosphite,
Sꢀethylthioꢀ1Hꢀtetrazole, and a controlledꢀporeꢀglass of 500 Å
with a UnylinkerꢀCPG immobilized universal linker (GlenReꢀ
search, USA). The removal of oligonucleotide from the polyꢀ
meric support and the removal of protecting groups were carried
out upon treatment with saturated aqueous ammonia for 18 h at
55 C. Oligonucleotide was purified using a GlenꢀPak reversedꢀ
phase cartridge (GlenResearch, USA) according to the
producer´s guidance. The conjugation of oligonucleotide with
azido derivative 8a by a Cu(I)ꢀcatalyzed 1,3ꢀdipolar cycloaddiꢀ
tion was carried out according to a modified procedure.33 To
achieve this, oligonucleotide (2 nmol) was dissolved in DI water
(type I) (50 L), followed by the addition of 2 M aqueous triethylꢀ
ammonium acetate (20 L) (рH 7.0), a solution of the catalyst
(20 L of 5 mM solution of TBTA (trisꢀ[(1ꢀbenzylꢀ1Hꢀ1,2,3ꢀ
triazolꢀ4ꢀyl)methyl]amine), 13 mM solution of copper(II) sulꢀ
fate in 60% DMSO), 10 mM solution of azido derivative in
DMSO (4 L), and 50% DMSO in water (100 L). After the
solution was degassed in a centrifugal evaporator and the system
was filled with argon (repeated 3 times), 0.25 M solution of ascorꢀ
bic acid in water (6 L) was added and the reaction mixture was
allowed to stand for 18 h at 20 C. Then, a 4% solution of
lithium perchlorate in acetone (1 mL) was added to the mixture,
which was stirred for 1 h with maintenance at –18 C, the oligoꢀ
nucleotide was precipitated by centrifugation at 12000 g for
5 min. The product was purified by HPLC; the fraction containing
conjugate 10a was concentrated to dryness using a centrifugal
evaporator, the precipitate was dissolved in saturated aqueous
ammonia and allowed to stand at room temperature for 6 h to
remove protecting groups of the carbohydrate fragment. Then,
the solution was concentrated to 100 L, followed by the addiꢀ
tion of 96% ethanol (1 mL) and stirring with maintenance at –18 C
for 1 h. Oligonucleotide 11a was precipitated by centrifugation
at 12000 g for 5 min. The formation of conjugates was confirmed
by mass spectrometry (for conjugate 10a: calculated 8294.14,
found 8293.50; for 11a: calculated 7917.05, found 7915.56).
(CDCl3), : 1.23—1.36 (m, 12 H, (CH2)6); 1.54—1.61 (m, 4 H,
CH2CH2N3, CH2CH2C(O)); 2.15 (t, 2 H, (CH2)8CH2C(O),
J = 7.6 Hz); 2.57 (t, 6 H, 3 CH2COOH, J = 6.0 Hz); 3.24 (t, 2 H,
CH2N3, J = 6.9 Hz); 3.69 (t, 6 H, 3 OCH2CH2COOH, J = 6.0 Hz);
3.70 (s, 6 H, 3 CCH2O); 6.09 (br.s, 1 H, NH); 10.90 (br.s, 3 H,
3 COOH). 13C NMR (CDCl3), : 25.63, 26.60, 28.73, 28.95,
29.03, 29.20, 29.29, 29.35 ((CH2)8); 34.68 (OCH2CH2C(O));
36.99 ((CH2)8CH2C(O)); 51.36 (CH2N3); 59.74 (CCH2O);
66.44 (OCH2CH2C(O)); 69.13 (CCH2O); 174.69 (C(O)NH);
176.68 (COOH). MS (ESI) found: m/z 569.2798 [M + Na]+;
C24H42N4O10Na; calculated: 569.2793.
Synthesis of compounds 8a,b (general procedure). A mixture
of acid 7 (55 mg, 0.1 mmol), the corresponding amine 4a or 4b
(255 mg, 0.5 mmol), DIPEA (Pri2NEt) (0.2 mL) in THF (8 mL)
and HBTU (Oꢀ(1Hꢀbenzatriazolꢀ1ꢀyl)ꢀN,N,N´,N´ꢀtetramethylꢀ
uronium hexafluorophosphate) (140 mg, 0.37 mmol) was stirred
for 36 h. The solvent was evaporated at reduced pressure, the
residue was dissolved in dichloromethane, washed with 1 M aqueous
H3PO4, 1 M aqueous NaHCO3, and saturated aqueous NaCl.
Compound 8 was isolated by column chromatography (eluent
CH2Cl2—MeOH, the gradient from 50 : 0 to 5 : 1).
Nꢀ(11ꢀAzidoundecylamido)ꢀtrisꢀ{2ꢀoxaꢀ6,10ꢀdiazaꢀ5,11ꢀdiꢀ
oxoꢀ15ꢀ[3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀꢀDꢀglucoꢀ
pyranosyloxy]pentadecyl}methane (8a). The yield was 37 mg
(18%), a white amorphous compound. 1H NMR (CDCl3), :
1.25—1.35 (m, 12 H, (CH2)6); 1.54—1.76 (m, 22 H, CH2CH2N3,
(CH2)7CH2CH2C(O), 3 OCH2CH2CH2, 3 CH2CH2NHC(O));
1.93, 2.01, 2.07 (all s, a total of 36 H, 12 C(O)CH3); 2.12—2.18,
2.25—2.30 (both m, 8 H, (CH2)9CH2C(O), 3 CH2C(O)NH);
2.41—2.46 (m, 6 H, 3 OCH2CH2C(O)NH); 3.23—3.27 (m, 14 H,
CH2N3,
3 NHCH2CH2CH2NH); 3.46—3.51 (m, 3 H,
3 OCHaHb(CH2)2); 3.65—3.71 (m, 15 H, 3 OCHaHb(CH2)2,
3 OCH2CH2C(O), 3 CCH2O); 3.87—3.94 (m, 6 H, 3 Hꢀ2, 3 Hꢀ5);
4.11 (dd, 3 H, 3 Hꢀ6, J = 12.3 Hz, J = 1.2 Hz); 4.26 (dd, 3 H,
3 Hꢀ6´, J = 12.3 Hz, J = 4.5 Hz); 4.64 (d, 3 H, 3 Hꢀ1, J = 8.2 Hz);
5.04 (t, 3 H, 3 Hꢀ3, J = 9.6 Hz); 5.07 (t, 3 H, 3 Hꢀ4, J = 9.6 Hz);
6.48 (br.s, 1 H, (CH2)9C(O)NH); 6.91 (br.d, 3 H, 3 NHAc,
J = 8.8 Hz); 7.03 (t, 3 H, 3 NH, J = 5.6 Hz); 7.28—7.29 (m, 3 H,
3 NH). Found: m/z 2026 [M + Na]+ (MALDIꢀTOF);
C90H147N13O37Na; calculated: 2026.
Nꢀ(11ꢀAzidoundecylamido)ꢀtrisꢀ{2ꢀoxaꢀ6,10ꢀdiazaꢀ5,11ꢀdiꢀ
oxoꢀ15ꢀ[3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀꢀDꢀmannopyrꢀ
anosyloxy]pentadecyl}methane (8b). The yield was 60 mg (30%),
a colorless oil. 1H NMR (CDCl3), : 1.26—1.36 (m, 12 H, (CH2)6);
1.54—1.74 (m, 22 H, CH2CH2N3, (CH2)7CH2CH2C(O),
3 OCH2CH2CH2, 3 CH2CH2NHC(O)); 1.98, 2.04, 2.05, 2.09
(all s, a total of 36 H, 12 C(O)CH3); 2.13—2.28 (m, 8 H,
(CH2)8CH2C(O), 6 CH2C(O)NH); 2.43—2.47 (m, 6 H,
This work was financially supported by the Russian
Science Foundation (Project No. 14ꢀ34ꢀ00017).
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Received March 11, 2015;
in revised form April 10, 2015