Synthesis of covalent conjugates of hexaarabinofuranoside with proteins and their testing as antigens for serodiagnosis of tuberculosis

Full text of DOI:10.1007/s11172-010-0397-4

Russian Chemical Bulletin, International Edition, Vol. 59, No. 12, pp. 2333—2337, December, 2010
2333
Letters to the Editor
Synthesis of covalent conjugates of hexaarabinofuranoside with proteins
and their testing as antigens for serodiagnosis of tuberculosis
P. I. Abronina,a N. M. Podvalnyy,a T. M. Mel´nikova,a A. I. Zinin,a K. G. Fedina,a,b V. V. Kachala,a V. I. Torgov,a
L. O. Kononov,a E. A. Panfertsev,c E. V. Baranova,c V. V. Mochalov,c V. I. Dyatlova,c and S. F. Biketovc
aN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 5328. Eꢀmail: kononov@ioc.ac.ru
bHigher Chemical College of the Russian Academy of Sciences,
9 Miusskaya pl., 125047 Moscow, Russian Federation.
cState Scientific Center for Applied Microbiology and Biotechnology,
Obolensk, 142279 Moscow Region, Russian Federation.
Fax: +7 (496) 736 0065. Eꢀmail: biketov@yandex.ru
Specificity and sensitivity are crucial in diagnosis of
tuberculosis. An insufficiently sensitive method will "miss"
too many persons really sick with active tuberculosis. If
the method is insufficiently specific, its use will give many
false positive results among healthy people or among nonꢀ
tubercular patients (the latter fact is not too critical for
mass screening of population, because the primary diagꢀ
nosis is always checked by alternative methods). Enzymeꢀ
linked immunosorbent analysis (ELISA), immunochroꢀ
matography (IC), and dotꢀblotting technique using one or
several recombinant protein antigens of Mycobacterium
tuberculosis were developed in the recent time for serodiꢀ
agnosis of tuberculosis. In particular, an array of recombiꢀ
nant antigens (38ꢀ, 63ꢀ, 64ꢀ, 14ꢀ, and 59ꢀkDa) is successꢀ
fully used in commercial serological assays TBꢀDOT
(Upper Bioꢀtech, China, http://www.poct.cn) and TBꢀ
Checkꢀ1 (VEDA.LAB, France, http://www.vedalab.com);
the declared assay specificity reaches 99.42%, and the senꢀ
sitivity is 98.52% (see Ref. 1). However, it was found when
using these tests that this level of sensitivity is reached
only for certain human populations, which can be associꢀ
ated with national and ethnic peculiarities of the humoral
immune response to tuberculosis.2 For example, 11 reꢀ
combinant proteins of M. tuberculosis have earlier3 been
obtained and used as antigens for the analysis of 320 sera
of sick and healthy peoples from the central region of
Russia. None of them provided the sensitivity higher than
55%, and various combinations of antigens increased this
parameter by at most 7%.
It was assumed that an insufficient sensitivity is related
to the absence of a carbohydrate component in the antiꢀ
gens used, since it was shown that antibodies to the carboꢀ
hydrate part of M. tuberculosis lipoarabinomannan (LAM)
were observed for the majority of tubercular patients.4
Therefore, we attempted to synthesize (Schemes 1 and 2)
covalent conjugates of the terminal hexaarabinofuranoꢀ
side fragment of LAM, viz., the main epitope of LAM,5
with recombinant proteins of M. tuberculosis in order to
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2276—2280, December, 2010.
1066ꢀ5285/10/5912ꢀ2333 © 2010 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Abronina et al.
obtain in vitro glycosylated antigens for serodiagnosis of
tuberculosis.
We synthesized hexasaccharides 22 and 24 as glycoꢀ
sides with the functionalized spacerꢀaglycones (see
Schemes 1 and 2). The role of the spacerꢀaglycone is not
limited by the spatial separation of the carbohydrate ligand
from the polymer support only, which is required for effiꢀ
cient binding of the carbohydrate ligand to the correspondꢀ
ing receptor. The nature of the spacerꢀaglycone that links
the oligosaccharide haptene to the polymer support can
Scheme 1
Reagents, conditions, and yields: a. 32% HBr/AcOH; b. NaBH4, MeCN (90% over two steps); c. 90% TFA, CH2Cl2;
d. (ClCH2CO)2O, symꢀcollidine, CH2Cl2, 96%; e. PhSH, BF3·Et2O, CH2Cl2, 0 °C, 3 h, 83%; f. NIS, TESOTf, MS 4 Å, CH2Cl2,
–20 °C, chromatography on SiO2, 76%; g. Py; H2O, 70 °C, 2 h, chromatography on SiO2, 89%; h. 6, NIS, AgOTf,
MS 4 Å, CH2Cl2, –40 °C → +4 °C, 4 h, chromatography on SiO2, 84%; i. Py, H2O, 70 °C, 2 h, chromatography on SiO2, 89%; j. NIS,
AgOTf, MS 4 Å, CH2Cl2, –40 °C, 1 h, chromatography on SiO2 (HPLC), 55%; k. (ClCH2CO)2O, symꢀcollidine, CH2Cl2, chromatoꢀ
graphy on SiO2, 87%.

Conjugates for serodiagnosis of tuberculosis
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2335
Scheme 2
Reagents, conditions, and yields: a. NaN3, DMF, 70 °C, 30 h, 97 %; b. nꢀBu4NF, THF, 12 h, chromatography on SiO2 (HPLC), 77%;
c. BzCl, Py, chromatography on SiO2, 80%; d. PPh3, THF—H2O (10 : 1), 0 °C → 20 °C; e. (CF3CO)2O, NEt3, THF, gel chromatoꢀ
graphy on BioꢀBeads S×3 (toluene), chromatography on SiO2, 73% over two steps; f. NaOMe, MeOH, ~20 °C, 30 h, gel chromatoꢀ
graphy on Sephadex LHꢀ20 (MeOH); g. H2, Pd/C, MeOH—H2O, 28 °C, 12 h, gel chromatography on Sephadex LHꢀ20 (MeOH),
41% over two steps; h. NaOH, MeOH; i. HOOCCH2(OCH2CH2)6NHTFA, DMTꢀMM, Nꢀmethylmorpholine, MeOH, gel chromatoꢀ
graphy on Sephadex LHꢀ20 (MeOH), 86%; j. NaOH, MeOH; k. diethyl squarate, H2O, pH 7.5, chromatography on SepꢀPak С18
(gradient Н2О→МеОН); l. protein MPBꢀ64 (27a) or Rv0934 (27b), pH 9, dialysis (water).
also determine both the specificity of the elicited antiꢀ
bodies and the possibility of efficient immunization.6 In
this work, for conjugation with proteins we used hexaꢀ
arabinofuranoside 24 with a long, flexible, and hydrophilic
spacer which contained the oligo(ethylene glycol) fragment.
A preꢀspacer approach was used in the synthesis of
hexasaccharide 24. This is based on the preparation of
protected hexasaccharide 14 as 2ꢀchloroethyl glycoside,
removal of the protecting groups, and modification of the
aglycone to 2ꢀaminoethyl glycoside 22 followed by its couꢀ
pling with HOOCCH2(OCH2CH2)6NHTFA (see Ref. 7)
and liberation of the amino group. This approach is exꢀ
tremely divergent and, unlike the synthetic schemes used
earlier8 for the synthesis of oligosaccharide fragments of
LAM, makes it possible to obtain a set of hexaarabinoꢀ
furanosides with various spacerꢀaglycones from the single
hexasaccharide precursor without repeating the optimizaꢀ
tion of the nonꢀtrivial steps of the glycosidic bond formaꢀ
tion. The rational scheme of assembling protected hexaꢀ
saccharide 14 includes two steps of bisꢀglycosylation: with
the formation of 1,2ꢀtransꢀglycosidic bonds [2+1+1] and
1,2ꢀcisꢀglycosidic bonds [4+1+1]. The hydroxyl groups to
be further glycosylated were protected with chloroacetyl
groups, which can selectively be removed in the presence
of the Oꢀbenzoyl protecting groups.
The known methyl 2,3,5ꢀtriꢀОꢀbenzoylꢀαꢀDꢀarabinoꢀ
furanoside (1)9 was transformed into 3,5ꢀdiꢀОꢀbenzoylꢀ
1,2ꢀОꢀbenzylideneꢀβꢀDꢀarabinofuranose (3), which served
as a key monosaccharide building block for the synthesis
of phenyl 3,5ꢀdiꢀOꢀbenzoylꢀ2ꢀOꢀchloroacetylꢀ1ꢀthioꢀαꢀ
Dꢀarabinofuranoside (6) (see Scheme 1) containing the
selectively removable chloroacetyl protecting group for
subsequent chain elongation at O(2). (1ꢀ5)ꢀLinked diꢀ
saccharide 10 was obtained in 76% yield by glycosylation
of 2ꢀchloroethyl 2,3ꢀdiꢀОꢀbenzoylꢀαꢀDꢀarabinofuranoside
(8)10 with thioglycoside 7b (synthesized by chloroacetylaꢀ
tion of alcohol 7a)10 followed by removal of the chloroꢀ
acetyl groups. The glycosylation of the synthesized diꢀ
saccharide 10 with thioglycoside 6 afforded tetrasacchaꢀ
ride 11 in 84% yield. The 13C NMR spectrum of tetraꢀ

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Abronina et al.
saccharide 11 contains signals for the anomeric carbon
atoms of the αꢀlinked arabinofuranose residues (δC 105.30,
105.47, 105.51, and 105.57), and the H NMR spectrum
(26a) and Ara6ꢀRv0934 (26b) were isolated by dialysis
against water at +4 °C and stored in the frozen state. The
inclusion of the squaric acid fragment and, hence, hexaꢀ
saccharide into the obtained conjugates was indicated by
a considerable increase in the absorption band intensity
1
exhibits signals for four anomeric protons (δH 5.27 (2 H),
5.37, and 5.44). The monosaccharide residues linked by
1,2ꢀcisꢀglycosidic bonds were introduced into tetrasacꢀ
charide 12 with two hydroxyl groups by glycosylation with
thioglycoside 13 (see Ref. 8a) to form an anomeric mixture
of hexasaccharides in 95% yield. The required β,βꢀlinked
isomer of hexasaccharide 14 was isolated by HPLC in
a yield of 55%. Subsequent steps included modification
of the aglycone in the hexasaccharide (14→15), deꢀ
silylation (15→16), benzoylation of the free hydroxyl
groups (16→17), reduction of the azido group in the
aglycone to the amino group (17→18), trifluoroacetylaꢀ
tion of the amino group (18→19), Oꢀdeacylation (19→20),
hydrogenolysis (20→21), and liberation of the amino
group to form unprotected hexasaccharide 22 with
the aminoethyl aglycone, which was coupled with
HOOCCH2(OCH2CH2)6NHTFA7 to form amide 23 in
86% yield (15% based on protected hexasaccharide 14)
([α]D28 +36.0 (c 0.44, MeOH); MS (ESI): m/z 1288.5024
[M + NH4]. Calculated for C48H81F3N2O33 [M + NH4]:
1288.5012). The 13C NMR spectrum of hexasaccharide
23 contains signals for two anomeric carbon atoms of the
βꢀlinked arabinofuranose residues (δC 102.62 and 102.72)
and four anomeric carbon atoms of the αꢀlinked arabinoꢀ
furanose residues (δC 107.27, 107.57, and 109.73 (2 С));
(λ
= 277 nm) in the UV spectrum of conjugate 26a
max
compared to the starting protein 27a (at the same concenꢀ
tration), as well as the appearance in the UV spectrum of
conjugate 26b of an intense absorption band (λmax = 274 nm),
which was absent from the spectrum of the starting proꢀ
tein 27b.
The synthesized conjugates were used as antigens for
ELISA of the sera of tubercular patients. We tested 200 sera
collected in the central region of Russia: 100 sera from
turbercular patients and 100 sera from healthy donors vacꢀ
cinated with the antiꢀtuberculosis BCG vaccine. Artificial
M. tuberculosis antigens 26a and 26b provided sensitivity
of 68 and 55%, respectively, which exceeds the sensitivity
for nonꢀconjugated proteins 27a and 27b by more than
10%. Some decrease in the specificity of the obtained conꢀ
Table 1. Results of determination of the sensitivity and specificiꢀ
ty of recombinant proteins 27a,b and their conjugates 26a,b in
ELISA with the sera from tubercular patients and from healthy
donorsa
Antigen
Sensitivityb
Specificityc
(%)
1
the H NMR spectrum exhibits signals for two anomeric
protons of the βꢀlinked arabinofuranose residues (δH 5.03
(2 H, br.s) and four anomeric protons of the αꢀlinked
arabinofuranose residues (δH 4.90 (s), 4.96 (s), 5.09 (s),
and 5.17 (s)), whose presence confirms the structure of
the synthesized compound. Removal of the Nꢀtrifluoroꢀ
acetyl protecting group in amide 23 by alkaline hydrolysis
gave spacered hexasaccharide 24, which was used without
isolation for the conjugation with proteins.
The genes that encode MPBꢀ64 and Rv0934 proteins
of М. tuberculosis were cloned in E. coli by the polymerase
chain reaction (PCR) using DNA of strain H37Rv and
were expressed from plasmid pET under control of a strong
promoter. Recombinant MPBꢀ64 and Rv0934 proteins
were purified by affinity chromatography. The conjugaꢀ
tion of hexaarabinofuranoside 24 with the hydrophilic
spacerꢀaglycone with МРВꢀ64 (27a) and Rv 0934 (27b)
proteins was carried out using the squaric acid fragment as
a linker.11 In the first step, the reaction of hexasaccharide
haptene 24 with diethyl squarate in a 0.1 М phosphate
buffer (pH 7.0) at ~20 °C (10 h) afforded monoethyl hapꢀ
26a
26b
27a
27b
68
55
55
44
80
80
84
85
a We tested 200 sera collected in the central region of Russia:
100 sera from tuberculosis patients and 100 sera from healthy
donors vaccinated with the antiꢀtuberculosis vaccine BCG. Testꢀ
ing was carried out by indirect ELISA. Antigens were immobiꢀ
lized in wells of 96ꢀwell plates (100 μL per well) from a concenꢀ
tration of 3 μg mL–1 in a carbonate buffer (pH 9.6) at 4 °С for
16 h. After washing off, samples of the sera diluted in a ratio of
1 : 200 were introduced into the wells and incubated for 1 h at
37 °С. The immune complexes formed were revealed by the
peroxidase conjugate of antibodies against human IgG in the
dilution 1 : 10 000. The immune complexes formed were detectꢀ
ed with the substrate containing tetramethylbenzidine recording
the absorbance at 450 nm when the reaction ceased. The cutꢀoff
point was determined by the results of ELISA of the studied
proteins with the sera of the reference group as an average value
of the detected absorbance of the sera of the reference group plus
three standard deviations. The sensitivity and specificity of the
diagnosis were determined from the value of cutꢀoff.
tene squarate 25 (λ
= 259 nm). After purification by
max
b The diagnosis sensitivity of the antigens is the percent of posiꢀ
tive samples in the serological assay of the sera from patients
with the confirmed "tuberculosis" diagnosis.
c The diagnosis specificity of the antigens is the percent of negaꢀ
tive (truly negative) responses in the serological assay of the sera
of healthy donors or nonꢀtubercular patients.
reversedꢀphase chromatography on a SepꢀPak C18 carꢀ
tridge (gradient water→MeOH), compound 25 was conꢀ
jugated (separately) with proteins 27a (λ
= 274 nm)
max
and 27b (λ
≈ 265 nm, shoulder) in a 0.05 М borate
max
buffer (pH 9.0) at +4 °C (60 h). Conjugates Ara6ꢀMPBꢀ64

Conjugates for serodiagnosis of tuberculosis
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2337
7. M. SibrianꢀVazquez, T. J. Jensen, R. P. Hammer, M. G. H.
Vicente, J. Med. Chem., 2006, 49, 1364.
jugates (by ~5%) compared to that of nonꢀconjugated proꢀ
teins 27a and 27b should be mentioned: 80% in the case of
conjugates 26a and 26b (see Table 1).
Thus, the introduction of LAM carbohydrate epitopes
of M. tuberculosis (hexaarabinofuranoside) into tubercuꢀ
losis protein antigens can increase the diagnostic sensitivꢀ
ity of the serological tuberculosis assays developed for the
use in the central region of Russia, and the work in this
direction seems promising.
8. (a) X.ꢀY. Liang, L.ꢀM. Deng; X. Liu, J.ꢀS. Yang, Tetraꢀ
hedron, 2010, 66, 87; (b) M. Joe, Y. Bai, R. C. Nacario, T. L.
Lowary, J. Am. Chem. Soc., 2007, 129, 9885; (c) D. Crich,
C. M. Pedersen, A. A. Bowers, D. J. Wink, J. Org. Chem.,
2007, 72, 1553; (d) X. Zhu, S. Kawatkar, Y. Rao, G.ꢀJ. Boons,
J. Am. Chem. Soc., 2006, 128, 11948; (e) A. Ishiwata,
H. Akao, Y. Ito, Org. Lett., 2006, 8, 5525; (f) B. FraserꢀReid,
J. Lu, K. N. Jayaprakash, J. C. Lopez, Tetrahedron: Asymꢀ
metry, 2006, 17, 2449; (g) Y. J. Lee, K. Lee, E. H. Jung, H. B.
Jeon, K. S. Kim, Org. Lett., 2005, 7, 3263; (h) K. Marotte,
S. Sanchez, T. Bamhaoud, J. Prandi, Eur. J. Org. Chem.,
2003, 3587; (i) R. R. Gadikota, C. S. Callam, B. J. Apꢀ
pelmelk, T. L. Lowary, J. Carbohydr. Chem., 2003, 22, 459;
(j) H. Yin, F. W. D´Souza, T. L. Lowary, J. Org. Chem.,
2002, 67, 892; (k) S. Sanchez, T. Bamhaoud, J. Prandi, Eur.
J. Org. Chem., 2002, 3864; and references cited therein.
9. (a) R. K. Ness, H. G. Fletcher, J. Am. Chem. Soc., 1958, 80,
2007; (b) C. S. Callam, T. L. Lowary, J. Chem. Educ., 2001,
78, 73.
10. N. M. Podvalnyy, S. L. Sedinkin, P. I. Abronina, A. I. Zinin,
K. G. Fedina, V. I. Torgov, L. O. Kononov, Carbohydr. Res.,
2011, 346, 7.
11. (a) L. F. Tietze, M. Arlt, M. Beller, K.ꢀH. Glüsenkamp,
E. Jähde, M. F. Rajewsky, Chem. Ber., 1991, 124, 1215;
(b) L. F. Tietze, C. Schröter, S. Gabius, U. Brinck, A. Goꢀ
erlachꢀGraw, H.ꢀJ. Gabius, Bioconjugate Chem., 1991, 2,
148; (c) V. P. Kamath, P. Diedrich, O. Hindsgaul, Glycoconꢀ
References
1. F. Abebe, C. HolmꢀHansen, H.G. Wiker, G. Bjune, Scand.
J. Immunol., 2007, 66, 176.
2. (a) E. A. Talbot, D. C. Hay Burgess, N. M. Hone, M. F.
Iademarco, M. J. Mwasekaga, H. J. Moffat, T. L. Moeti,
R. A. Mwansa, P. Letsatsi, N. T. Gokhale, T. A. Kenyon, C. D.
Wells, Clin. Infect. Dis., 2004, 39, e1; (b) M. D. Perkins, J.
Cunningham, J. Infect. Dis., 2007, 196, Suppl. 1, S 15;
(c) S. Pottumarthy, V. C. Wells, A. J. Morris, J. Clin. Microꢀ
biol., 2000, 38, 2227.
3. A. V. Zherdev, A. A. Mart´yanov, V. G. Avdienko, S. S.
Babayan, V. Ya. Gergert, A. S. Kalyukina, D. K. Laurinaviꢀ
chute, N. V. Pozdnyakova, A. N. Fedorov, E. S. Severin,
E. A. Panfertsev, S. F. Biketov, N. A. Byzova, B. B. Dzanꢀ
tiev, Fiziologiya i Patologiya Immunnoi Sistemy [Physiology
and Pathology of Immune System], 2006, 9, 3 (in Russian).
4. E. D. Chan, R. Reves, J. T. Belisle, P. J. Brennan, W. E.
Hahn, Am. J. Respir. Crit. Care Med., 2000, 161, 1713.
5. D. Kaur, T. L. Lowary, V. D. Vissa, D. C. Crick, P. J. Brenꢀ
nan, Microbiology, 2002, 148, 3049.
jugate J., 1996, 13, 315; (d) S. Hou, R. Saksena, P. Kov
Carbohydr. Res., 2008, 343, 196.
,
6. S. Svedhem, L. Öhberg, S. Borrelli, R. Valiokas, M. Andersꢀ
son, S. Oscarson, S. C. T. Svensson, B. Liedberg, P. Konꢀ
radsson, Langmuir, 2002, 18, 2848.
Received October 25, 2010;
in revised form November 25, 2010