A modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of PGL-I from Mycobacterium leprae

Article abstract of DOI:10.1016/j.bmcl.2010.04.072

In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnostic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the corresponding disaccharide. Herein, the non-reduc

Full text of DOI:10.1016/j.bmcl.2010.04.072

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3250–3253
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A modified synthesis and serological evaluation of neoglycoproteins
containing the natural disaccharide of PGL-I from Mycobacterium leprae
,
*
*
Jian Zhang , Delphi Chatterjee, Patrick J. Brennan, John S. Spencer, Avraham Liav
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnos-
tic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the
corresponding disaccharide. Herein, the non-reducing disaccharide segment of phenolic glycolipid I from
Received 17 March 2010
Accepted 15 April 2010
Available online 21 April 2010
Mycobacterium leprae, O-(3,6-di-O-methyl-b-D-glucopyranosyl)-(1?4)-O-2,3-di-O-methyl-a-L-rhamno-
pyranose was synthesized by an improved procedure. The disaccharide was efficiently conjugated to
bovine/human serum albumin, via acyl-azide intermediate, to form natural disaccharide-BSA/HSA neo-
glycoproteins that showed a high activity in serodiagnosis of leprosy. The disaccharide incorporated into
the proteins was accurately measured by MALDI-TOF mass spectrometry. The serological activities of the
neoglycoproteins against pooled human lepromatous leprosy sera were measured by ELISA and they
were detectable at picogram amounts.
Keywords:
Leprosy
PGL-I
Natural disaccharide
Neoglycoprotein
Serological activities
Ó 2010 Elsevier Ltd. All rights reserved.
Leprosy, also known as Hansen’s disease, was the first human
disease shown to be caused by bacterium. The prime infectious tar-
gets of Mycobacterium leprae (M. leprae), the causative pathogen of
leprosy, are peripheral nerves and more specifically the Schwann
cells where M. leprae colonizes by attaching to several receptors,
albumin (BSA).5–8 In this work, we have described an efficient and
improved synthesis of the constituents of the natural disaccharide
(ND) 1 from PGL-I. Furthermore, the number of glycosyl residues
incorporated on the proteins was measured accurately using mass
spectrometry.
such as
a-dystroglycan, a laminin-2 receptor in the Schwann cell
plasma membrane.1 The bacterial ligand that binds to these recep-
tors on host cell is a glycolipid termed as phenolic glycolipid I
(PGL-I) that was only found in M. leprae.2 The structure of PGL-I,
O
MeOH2C
O
H3C
O
HO
MeO
O
a
trisaccharide [O-(3,6-di-O-methyl-b-D-glucopyranosyl)-(1?4)-
OH
MeO
OMe
O-(2,3-di-O-methyl- -rhamnopyranosyl)-(1?2)-3-O-methyl-a-
a
-
L
1
L
-rhamnopyranose] glycosidically linked to a phenolic phthiocerol
core has been firmly established.3 PGL-I is present in copious
amounts in M. leprae and it invades human nerves by binding to
the receptors on the Schwann cells,2 and a specific antibody
against PGL-I is produced in sera of leprosy patients. Therefore,
the trisaccharide as well as the corresponding disaccharide [O-
The key intermediate in our synthesis of the disaccharide 1 is
1,4-di-O-acetyl- 2,3-di-O-methyl- -rhamnopyranose (5). -Rham-
nose was converted into methyl 4-O-benzyl-2,3-O-isopropyli-
dene-
-rhamnopyrsnoside (2) by published methods.9,10
Removal of the isopropylidene group followed by methylation
yielded the corresponding 2,3-dimethoxy derivative 4. The main
feature of this improved synthesis is the one-step conversion of 4
into the desired 1,4-diacetate 5 by acetolysis with 2% sulfuric acid
in acetic anhydride and acetic acid (Scheme 1). Purification of the
product by chromatography on silica gel removed a trace amount
of a byproduct which was found to be the corresponding 1-acet-
oxy-4-O-benzyl derivative. Continued elution gave the main prod-
uct 5 in 60% yield.11
L
L
a-L
(3,6-di-O-methyl-b-
D
-glucopyranosyl)-(1?4)-O-2,3-di-O-methyl-
a
-L-rhamnopyranose], located at the non-reducing end of PGL-I,
were found to be serologically active.4 The possibility of using
PGL-I based neoglycoconjugates (di-, or tri-saccharides) in the
serodiagnosis of leprosy infection by an enzyme-linked immuno-
sorbent assay (ELISA) prompted several research groups to synthe-
size the haptens and link them to the protein carrier bovine serum
* Corresponding authors. Tel.: +1 970 491 5537; fax: +1 970 491 1815.
E-mail addresses: jian.zhang@adatech.com (J. Zhang), avraham.liav@colostate.
edu (A. Liav).
Present address: ADA technologies, Inc. 8100 Shaffer Parkway, Littleton, CO
80127, United States. Tel.: +1 303 874 7372.
The other intermediate needed for the synthesis of the disac-
charide is 1,2,4-tri-O-acetyl-3,6-di-O-methyl-
In the previous synthesis, this intermediate had been obtained by
a nine-step process from 3-O-methyl-
-glucose.12 In the present
D-glucopyranose (6).
D
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.072

J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3250–3253
3251
OMe
OMe
OMe
MeI, NaH
H3C
H3C
H3C
O
O
O
30 % HOAc
95 %
BnO
BnO
BnO
THF
O
MeO
HO
L-Rhamnose
82 %
O
OMe
OH
4
3
2
OAc
Ac2O, H2SO4, HOAc
60 %
H3C
O
AcO
MeO
OMe
5
Scheme 1.
O(CH2)8CO2Me
O(CH2)8CO2Me
OAc
H3C
HO
HO(CH
2
)8CO2Me
H3C
MeONa, MeOH
91 %
O
H3C
O
O
AcO
AcO
MeO
Hg(CN)2
78 %
MeO
MeO
OMe
OMe
OMe
9
8
5
MeOH2C
AcO
O
O(CH2)8CO2Me
MeOH2C
HO
O(CH2)8CO2Me
MeONa, MeOH
94 %
MeOH2C
AcO
MeO
O
H3C
OAc
O
O
H3C
O
O
MeO
7
Cl
O
MeO
OH
MeO
OAc
MeO
OMe
Hg(CN)2
74%
OMe
11
10
Scheme 2.
O(CH2)8CO2Me
O(CH2)8CONHNH2
MeOH2C
HO
MeOH2C
NH2NH2
81 %
O
O
H3C
H3C
O
O
HO
O
O
MeO
MeO
OH
OH
MeO
MeO
OMe
OMe
11
12
O(CH2)8CON3
MeOH2C
tert-Butyl nitrite
HCl-1,4-dioxane
O
H3C
HO
O
O
MeO
DMF, -30 o
C
OH
MeO
OMe
BSA/HSA
0.08 M Na2B4O7-0.3 M KHCO3, pH 9.2
O(CH2)8CONH
BSA/HSA
MeOH2C
HO
O
H3C
O
O
MeO
OH
MeO
n
OMe
ND-O-BSA, n=32
ND-O-HSA, n=40
Scheme 3.
work, compound 6 was synthesized by a five-steps procedure
starting from the commercially available 6,3- -glucurono lactone
as described.13 Compound 6 was then converted to the chloroace-
tate derivative of 3,6-di-O-methyl- -glucose (7, not shown).
D
D

3252
J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3250–3253
Figure 1. MALDI-TOF mass spectrum of HSA (A) and ND-O-HSA (B).
steps of conversion. Finally, deacetylation of 10 quantitatively gave
the methoxycarbonyloctyl derivative of the natural disaccharide
(11).
MeOH2C
O
AcO
MeO
OAc
OAc
Conjugation of the methoxycarboxyl-octyl intermediate (11,
Scheme 3) to the carrier proteins (either BSA or HSA) proceeded
by converting it into the corresponding hydrazide derivative 12
(which could be stored for several months at À20 °C) in 81% yield.
The hydrazide was then converted into the acyl-azide, which was
immediately conjugated to BSA or HSA, according to the estab-
lished procedure.12,14 The carbohydrate content in these two neo-
glycoproteins, ND-O-BSA and ND-O-HSA was measured by the
carbohydrate assay (phenol–H2SO4 assay) as described5 and MAL-
DI-TOF mass spectrometry. As shown in Figure 1, ND-O-HSA (B)
exhibited higher heterogeneity compared with HSA (A) in the
MALDI-TOF mass spectra. The single charged (m/z, 66,372 and
87,132) and double charged (m/z, 33,186 and 43,566) monomer
of HSA and ND-O-HSA were the major components. The number
of disaccharide molecules incorporated in the neoglycoproteins
was calculated from the difference in the molecular weights of
the neoglycoproteins and BSA or HSA. ND-O-HSA showed higher
incorporation of the disaccharide (n = 40) than ND-O-BSA
(n = 32). Thus, MALDI-TOF mass spectrometry provides an accurate
6
Previously,8 the 1,4-disaccharide was synthesized by coupling
of a protected rhamnoside intermediate (a debenzylation product
of compound 2) and a derivative of 3-O-methyl-D-glucosyl bro-
mide. The methylation process was completed only after the
deprotection of the disaccharide and selective methylation of the
2,3-OH groups of rhamnose and the 6-OH of glucose. However, in
this improved approach, the methyl groups were introduced before
the coupling of the two hexose moieties. The building blocks 5 and
7 were obtained in high-yield from compound 2 and 6,3-D-glucur-
ono lactone, respectively.
The synthesis of disaccharide intermediate (11) to be conju-
gated to the BSA was initiated by the conversion of the building
block 5 into the corresponding 8-methoxycarbonyloctyl glycoside
8 in 78% yield (Scheme 2). Subsequent deacetylation of compound
8 produced compound 9, which was coupled to the gluco-interme-
diate 7 to give the disaccharide derivative 10 in 67% yield for two
LL sera ND-O-BSA
LL sera ND-O-HSA
control sera ND-O-BSA
control sera ND-O-HSA
ND-O-BSA
New
ND-O-HSA
ND-O-HSA AL
2
Old
2
0
ND-O-HSA AL
0
100
1000
10000
100
1000
10000
Antigen (pg/well)
Antigen (pg/well)
Figure 2. Reactivity of pooled lepromatous leprosy (LL) patient sera with a known
high antibody titer to native PGL-I and normal control sera with ND-O-BSA (32
sugar residues per molecule) and ND-O-HSA (40 sugar residues per molecule) by
ELISA. The wells were coated with twofold serial dilutions of the antigen ranging
from 50 ng to 100 pg. The antigens were each tested with the pooled serum samples
at a 1:200 dilution. The O.D. readings were determined using a plate reader at
405 nm.
Figure 3. Reactivity of monoclonal antibody CS-48 specific for ND-O-BSA and ND-
O-HSA by ELISA. The glycoconjugates tested were prepared by using an improved
synthesis described in this work (new batches) or by an earlier coupling procedure
(old batches). The wells were coated with twofold serial dilutions of the antigen
ranging from 50 ng to 100 pg. The O.D. readings were determined using a plate
reader at 405 nm.

J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3250–3253
3253
2. Ng, V.; Zanazzi, G.; Timpl, R.; Talts, J. F.; Salzer, J. L.; Brennan, P. J.; Rambukkana,
A. Cell 2000, 103, 511.
3. Hunter, S. W.; Fujiwara, T.; Brennan, P. J. J. Biol. Chem. 1982, 257, 15072.
4. Chatterjee, D.; Cho, S. N.; Stewart, C.; Douglas, J. T.; Fujiwara, T.; Brennan, P. J.
Carbohydr. Res. 1988, 183, 241.
5. Chatterjee, D.; Cho, S. N.; Brennan, P. J.; Aspinall, G. O. Carbohydr. Res. 1986, 156,
39.
6. Fujiwara, T.; Aspinall, G. O.; Hunter, S. W.; Brennan, P. J. Carbohydr. Res. 1987,
163, 41.
7. Fujiwara, T.; Hunter, S. W.; Brennan, P. J. Carbohydr. Res. 1986, 148, 287.
8. Fujiwara, T.; Hunter, S. W.; Cho, S. N.; Aspinall, G. O.; Brennan, P. J. Infect.
Immun. 1984, 43, 245.
9. Haines, A. H. Carbohydr. Res. 1969, 10, 466.
10. Liptak, A. N. P.; Neszmelyi, A.; Wagner, H. Tetrahedron 1980, 36, 1261.
11. Synthesis of the diacetate 5: To a cold (ice-bath) solution of methyl 4-O-benzyl-
and simple method to determine the degree of the conjugation of
the disaccharides and the proteins.
The serological activities of these two neoglycoproteins against
pooled human lepromatous leprosy (LL) and normal sera were
tested by ELISA. As shown in Figure 2, ND-O-BSA and ND-O-HSA
showed the detection limit at 100 picogram (pg)/well, where the
assays using LL sera had obvious difference of O.D.405 nm compared
to the assays using normal control sera. The higher sensitivity of
ND-O-HSA compared to ND-O-BSA at the region of 100–
10,000 pg/well may be caused by its higher incorporation of ND
onto HSA. In addition, the activity of the newly synthesized anti-
gens were compared to the old batches provided by Leprosy con-
tract (NIH N01 AI-25469). As shown in Figure 3, compared with
the old batches, both new ND-O-BSA and ND-O-HSA showed high-
er activity along with the increase of antigen concentration from
100 pg to 50 nanogram (ng)/well. The new batches of ND-O-BSA
and ND-O-HSA achieved comparable O.D. values using 5 to 7-fold
less antigen than the old batches, indicating that they were supe-
rior in the ELISA assays.
Due to the need for large amount of leprosy diagnostic reagents,
such as ND-O-BSA and ND-O-HSA we started searching for syn-
thetic and sensitive analogues of PGL antigens. The newly made
neoglycoproteins, ND-O-BSA and ND-O-HSA, carrying high natural
disaccharide content (30–40 ND incorporated) showed higher sen-
sitivity compared to the ones used before to detect anti-PGL-I anti-
bodies. Especially, the ND-O-HSA containing 40 natural
disaccharides possessed the detection limit in the pg range and
had the potential to be developed as a high-throughput diagnostic
kit that can examine 2 Â 105 serum samples for every 10 mg of the
reagent. In addition, the synthetic antigens will serve as an ideal
probe to fabricate carbohydrate microarray that will be used to
diagnose infectious diseases.
2,3-di-O-methyl-a-L-rhamnopyranpside (380 mg) in acetic acid (2 mL) and
acetic anhydride (2 mL) was added a 10% solution of sulfuric acid in acetic acid
(1 mL) and the mixture was then stirred at room temperature overnight. Ice,
solid sodium bicarbonate and ethyl acetate were added and the two layers
were separated. The organic layer was washed with water, dried and
evaporated. The residue was dried under vacuum and chromatographed on
silica gel (70–230 mesh). Elution with ethyl acetate/petroleum ether 1:1
removed trace amount of 1-O-acetyl-4-O-benzyl-2,3-di-O-methyl-
a-L-
rhamnopyranoside. 1H NMR (CDCl3, 300 MHz):
d
7.36–7.34 (m, 5H,
aromatic), 6.18 (s, 1H, H-1), 4.91, 4.62 (2 d, Jgem = 10.8 Hz, OCH2Ph), 3.56,
3.55, (2s, 3H each, OCH3), 2.09 (s, 3H, C(O)CH3), 1.31 (d, 3H, J6,5 = 6.0 Hz, CH3–
C5). Continued elution with the same solvent system gave product 5 (218 mg;
60%). 1H NMR (CDCl3, 300 MHz):
d 6.17 (d, J = 2.1 Hz, 1H, H-1), 5.08 (t,
J = 9.9 Hz, 1H, H-4), 3.80 (dq, J = 6.3, 9.9 Hz, 1H, H-5), 3.48. 3.38 (2s, 6H, OMe),
2.08, 2.04 (2s, OAc), 1.13 (d, J = 6.3, C5-Me).
12. Chatterjee, D.; Douglas, J. T.; Cho, S.-N.; Rea, T. H.; Gelber, R. H.; Aspinall, G. O.;
Brennan, P. J. Glycoconjugate J. 1985, 2, 187.
13. Liav, A.; Goren, M. B. Carbohydr. Res. 1986, 149, C13.
14. Synthesis of neoglycoproteins: A stirred solution of the hydrazide (11
6 mg) in dry DMF (125
L) was cooled to À30 °C, and 3.6 M HCl-1,4-dioxane
(25 L) was added. Then tert-butyl nitrite in DMF (1:10, 40 L) was added, and
lmol,
l
l
l
the solution was stirred for 30 min at À30 °C. TLC in solvent (EtOAc/MeOH/
H2O, 6:2:1) showed the disappearance of the hydrazide and formation of a new
component. The excess of nitrous acid was neutralized with 0.5 M sulfamic
acid (40
solution of BSA/HSA (8 mg, 0.1
l
L). The cold solution (À50 °C) of acyl-azide was added dropwise to a
l
mol) in 0.08 M Na2B4O7 (0.8 mL) and 0.3 M
KHCO3, pH 9.2 at 0 °C, stirred overnight at 0 °C, and dialyzed against four
changes of de-ionized water in an ultrafiltration cell equipped with a PM-10
membrane. Gel filtration of dialysis retentate was conducted in a column
(80 Â 1.6 cm) of sephadex G-75 in Milli-Q water. The neoglycoproteins were
lyophilized and stored at À10 °C.
Acknowledgments
The work was supported by NIH/NIAID contract N01 AI-25469.
References and notes
1. Rambukkana, A.; Yamada, H.; Zanazzi, G.; Mathus, T.; Salzer, J. L.; Yurchenco, P.
D.; Campbell, K. P.; Fischetti, V. A. Science 1998, 282, 2076.