FIELD OF THE INVENTION
The present invention relates to novel oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit (‘hereinafter Men-Y
oligomer). The present invention also relates to a process for synthesizing
novel N. meningitidis serogroup Y oligomers. 5 More specifically, the present
invention relates to the chemical synthesis of the tetramer of N.
meningitidis serogroup Y (‘hereinafter Men-Y tetramer) capable of being
used as a candidate in the development of semisynthetic and synthetic
conjugate vaccines against meningococcal serogroup Y bacterial infection.
10
BACKGROUND OF THE INVENTION
Bacterial meningitis causes approximately 1, 70, 000 annual deaths, with at
least 5-10% of case fatality in industrialized countries and a 20% case
fatality in the developing world. Streptococcus pneumoniae, Haemophilus
15 influenzae type b (Hib) and N. meningitidis are responsible for most of the
cases of bacterial meningitis worldwide. However, N. meningitidis still
remains a major cause of bacterial meningitis and other invasive bacterial
infections despite the availability of modern antibiotics.
20 Meningococcal disease is a medical emergency requiring immediate
diagnosis and treatment.
In total 13 different serogroups namely A, B, C, D, 29E, H, I, K, L, W135, X,
Y and Z of N. meningitidis have so far been identified, but about 90% of the
25 infections are due to serogroups A, B, C, Y and W135.
The following types of vaccines are available for meningococcal infection:
 Polysaccharide vaccines which are available for over 30 years to
prevent the disease. Meningococcal polysaccharide vaccines are
30 available in various combinations e.g. either bivalent (groups A and
3
C), trivalent (groups A, C and W), or tetravalent (groups A, C, Y
and W) forms to control the disease.
 Meningococcal conjugate vaccines against group C have been
available since 1999. Tetravalent A, C, Y and W conjugate vaccines
are widely used and 5 have been licensed since 2005 for use in
children and adults in different parts of world including Canada,
the United States of America, Europe and India. Other
combinations of the serogroups are also available in licensed
conjugate vaccines. The currently available meningococcal
10 conjugate vaccines except monovalent group A conjugate vaccine
are out of reach for needy people in low resource countries due to
their high cost.
Presently, vaccines available for Men-Y utilize the polysaccharide isolated
15 from bacterial source which are associated with various risks like danger
in handling the live bacterial culture. Also, many a times the isolated
bacterial polysaccharide is so heterogeneous that it fails to pass the desired
homogeneity criteria. The heterogeneous nature of the bacterial
polysaccharide provides the heterogeneity in the conjugates prepared
20 using it leading to large variations and hence, disqualification of the
conjugate batches.
During the last decade there has been an increasing focus on organic
synthesis of bacterial antigens including analogues of capsular
25 oligosaccharides. In view of the existing state of art, the meningococcal
conjugate vaccines containing synthetic Men-Y oligosaccharide are
advantageous over conventional vaccines. The synthetic antigens are
uniform in size and well characterised which reduce the heterogeneity of
the conjugates produced with less batch to batch variation in the final
4
conjugates. One of the main advantages with synthetic antigens is that
these can be engineered with desired linker for simplifying conjugation as
well as resulting into better yields.
Presently there is no prior art disclosing 5 the production and purification
procedures of organic synthesis of Men-Y. The International patent
application no. PCT/US2013/042428 “Multivalent meningococcal
conjugates and methods for preparing conjugates” relates to immunogenic
conjugates including at least one polysaccharide or protein conjugated to a
10 Neisseria surface protein, which can elicit immune responses against
meningococcal polysaccharides (PS) from groups A, C, W-135, and Y. The
said International patent application discloses only general statement on
preparation of Men Y oligomers but there is no enabling disclosure on the
preparation of Men-Y oligomer. Moreover, the existing art available for
15 other serogroups of N. meningitidis are either time consuming or give rise
to a mixture of different sizes of oligomers.
OBJECT OF THE INVENTION
The main object of present invention is to provide novel oligomers of
20 Neisseria meningitidis serogroup Y capsular polysaccharide repeating unit.
Another object of present invention is to provide novel oligomers of
Neisseria meningitidis serogroup Y capsular polysaccharide repeating unit
capable of being used as candidates in the development of synthetic and
25 semisynthetic conjugate vaccines against meningococcal bacterial
infection.
Yet another object of the present invention is to provide novel Men-Y
tetramer capable of being used as a candidate in the development of
5
semisynthetic or fully synthetic vaccine against meningococcal serogroup
Y bacterial infection.
Yet another object of the present invention is to provide a chemical process
of synthesizing Men-Y oligomers using 5 purified saccharides with specific
chain length.
Yet another object of the present invention is to provide a chemical
process of synthesizing Men-Y oligomers with improved antigenicity
10 capable of producing immunogenic conjugate vaccine.
Yet another object of the present invention is to provide a process for the
preparation of synthetic Men-Y capsular oligomers which meet the
physico-chemical quality standards for the purity.
15
Yet another object of the invention is to provide cost effective Men-Y
oligomers with increased efficacy and improved shelf-life when
conjugated to a carrier protein.
20 SUMMARY OF THE INVENTION
Accordingly, the present invention discloses N. meningitidis serogroup Y
oligomers and synthetic process to obtain thereof. Said Men-Y oligomers
are capable of being used as candidates in the development of
semisynthetic and synthetic conjugate vaccine against meningococcal
25 serogroup Y bacterial infection after conjugation to a suitable carrier
protein. Said vaccine can be administered by parental routes.
6
The Men-Y oligomers of the present invention possess improved
antigenicity and improved shelf-life. Said Men-Y oligomers also meet the
physico-chemical quality standards for the purity and are cost effective.
The process of present invention discloses synthesis 5 of Men-Y oligomers
comprising of the two main building blocks; an initiation unit and a
propagation unit. The initiation unit is prepared by the glycosidation of
suitably protected monosaccharide more particularly but not limited to
neuraminic acid with another suitably protected monosaccharide. Said
10 another suitably protected monosaccharide is selected from hexose, more
particularly but not limited to glucose. The propagation unit is prepared
by glycosidation of suitably protected monosaccharide more particularly
but not limited to neuraminic acid with another suitably protected
monosaccharide more particularly hexose, more particularly but not
15 limited to glucose.
The initiation unit and the propagation unit are coupled at predetermined
temperature together using catalyst to provide the tetrasaccharide or
dimeric unit. The catalyst used in the said coupling is glycosilation
20 reagent/ Lewis acid catalyst selected from but not limited to NIodosuccinimide
(NIS), Trifluoromethanesulfonic acid (TfOH),
Trimethylsilyltrifluoromethanesulfonate (TMSOTf), Silver
trifluoromethanesulfonate (CF3SO3Ag). The dimeric unit is reacted with a
basic reagent such as but not limited to sodium methoxide to facilitate ring
25 opening. The dimeric unit so obtained is again reacted with propagation
unit resulting in the formation of trimer. The trimer undergoes iterative
reactions under similar conditions to get protected higher oligomers (Y)
including tetramer, pentamer, hexamer, heptamer etc.
7
The protected higher oligomers so obtained are subjected to sequential
deprotection of protecting groups resulting in Men-Y higher oligomers.
The higher oligomers of N. meningitidis serogroup Y so obtained have
improved yields, high efficacy 5 and are capable of being used as a
candidate for development of conjugate vaccine which confers protection
against disease due to Men-Y infections.
All the illustrative steps devised for the synthesis of Men-Y oligomers
10 using novel approach result in better yield of tetramer, purity of >95%
shown in Figure 8 and enhanced antigenicity as shown in Figure 9.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 depicts 1H-NMR of Men-Y tetramer (compound 26)
15 Figure 2 depicts 13C-NMR of Men-Y tetramer
Figure 3 depicts COSY NMR spectrum of Men-Y tetramer
Figure 4 depicts HMBC NMR spectrum of Men-Y tetramer
Figure 5 depicts HSQC NMR spectrum of Men-Y tetramer
Figure 6 depicts TOCSY NMR spectrum of Men-Y tetramer
20 Figure 7 depicts DEPT NMR spectrum of Men-Y tetramer
Figure 8 depicts HPSEC analysis for purity of Men-Y tetramer
Figure 9 depicts antigenicity analysis of Men-Y tetramer and Men-Y
tetramer-tetanus toxoid (TT) conjugate by inhibition ELISA
against anti-Men-Y polyclonal sera
25
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention relates to novel Men-Y oligomers. The
present invention further relates to a process for synthesizing novel Men-Y
oligomers. More specifically, the present invention relates to the chemical
8
synthesis of Men-Y tetramer capable of being used as a candidate in the
development of semisynthetic and synthetic vaccine against
meningococcal bacterial infection. Said vaccine can be administered with
suitable mode of administration more particularly via parental route. Said
synthesis 5 being accomplished in the following steps:
Step 1: Synthesis of Propagation Unit (Compound 18 as shown in Scheme I)
Step 2: Synthesis of Initiation Unit (Compound 20 as shown in Scheme II)
Step 3: Synthesis of Higher Oligomers (Compound 21, 22, 23, 24, 25, d26 as
shown in Scheme III)
10
Before the preferred embodiment of the present invention is described, it
is understood that this invention is not limited to the particular materials
described, as they may vary. It is also understood that the terminology
used herein is for the purpose of describing the particular embodiment
15 only, and is not intended to limit the scope of the invention in any way.
It must be noted that as used herein, the singular forms “a”, “an” and
“the” include plural reference unless the context clearly dictates otherwise.
20 In one of the non-limiting example of said process, N-acetyl neuraminic
acid which is compound 1 undergoes methylation in presence of catalyst,
such as Dowex 50W X8 (H+) resin to obtain compound 2. The compound 2
so obtained undergoes acylation in presence of acylating agents such as
acetic anhydride in presence of pyridine to give compound 3. This process
25 helps in shielding of hydroxyl groups.
The compound 3 so obtained undergoes selective halogenation more
particularly chlorination in presence of halogenating agents such as
acetylcholride and HCl to obtain compound 4. Compound 4 reacts with
9
thiolating reagent Such as p-methylphenylthiol (TolSH) in presence of
DIPEA also known as Hunig’s base (poor nucleophile) resulting in
compound 5 with –Stol group attached to 2nd position. The compound 5 so
obtained undergoes deacylation in presence of MeOH, methanesulfonic
acid (MsOH) to give compound 6. The 5 compound 6 so obtained reacts
with carbonylation reagent such as Triphosgene, NaHCO3 in presence of
MeCN/H2O and undergoes oxazolidinones ring formation to yield
compound 7. The compound 7 so obtained is subjected to protection of
hydroxyl groups present at terminal position in presence of protecting
10 reagents such as tert-butyldiphenylsilyl chloride TBDPSCl/DMF, and then
for acetonide protection using 2,2-DMP, followed by acylation using AcCl
to yield compound 8. The compound 8 so obtained is reacted with reacting
agents such as dibutyl phosphate (Bu2 PO4H) in presence of NIS, TfOH,
DCM to obtain a phosphate leaving group at terminal position which
15 increases alpha selectivity as compound 9.
Commercially purchased compound 10 β-D-glucose pentaacetate is
reacted with thiocresol and (catalyst) BF3:OEt2 in DCM (solvent) to protect
alpha position yielding compound 11. Compound 11 undergoes
20 deacetylation in presence of MeONa in MeOH resulting in compound 12.
The compound 12 so obtained reacts with silyl protecting group such as
tert-butyldiphenylsilyl chloride (TBDPS-Cl) thereby protecting –OH group
at 6th position of compound 12 gives compound 13 with 80% yield. The
compound 13 so obtained undergoes alkylation and protection of specific
25 position with benzylation to yield compound 14 with 76% yield of reaction
at 00C to room temperature.
The above mentioned process is a known process.
10
Compound 15 is then obtained with yield in the range of 85% to 95% by
recovering one of the protected hydroxyl group of Compound 14 by
reacting compound 14 with deprotecting reagents such as Tetra-nbutylammoniumfluoride
(TBAF) in Tetrahydrofuran (THF). The
compound 14 acts as an intermediate in the 5 synthesis of Propagation Unit
18.
Remaining amount of compound 14 so obtained undergoes glycosidation
with 5-azido-hexanol to yield compound 16. The yield of compound 16 so
10 obtained is in the range of 65% to 75%, more particularly 70%. The
compound 16 is subjected to silyl group deprotection reagent such as
TBAF in THF solvent resulting in the removal of sterically hindering –
OTBDPS group to yield compound 17. The above reaction yield
compound 17 with an overall increase in Alpha:Beta ratio (95:5) along
15 with yield of reaction in the range of 55% to 65% more particularly 60%
The compound 17 so obtained acts as an intermediate in the synthesis of
Initiation Unit 20.
One part of Compound 9 is then reacted with compound 15 at low
20 temperatures, more particularly in the range of -800C to -600C in presence
of catalyst such as TMSOTf to yield Propagation unit (compound 18).
Other remaining part of Compound 9 is then reacted with compound 17 at
low temperatures, more particularly in the range of -800C to -600C in
25 presence of catalyst such as TMSOTf to yield compound 19.
Compound 19 so obtained is reacted with a basic reagent such as NaOMe
in presence of Methanol (MeOH)/ Dichloromethane (DCM) to yield
Initiation Unit (compound 20).
30
11
The Initiation Unit (20) and the Propagation Unit (18) are coupled together
using catalyst at low temperatures, more particularly in the range of -500C
to --300C to provide the tetrasaccharide or dimeric unit (21). The catalyst
used in said coupling is but not limited to NIS/TfOH
/Trimethylsilyltrifluoromethanesulfonate 5 (TMSOTf)/ Silver
trifluoromethanesulfonate (CF3SO3Ag) in presence of solvent. The catalyst
activates thiotolyl (Stol) group of compound 18. After activation it is prone
to removal and then the Stol group is replaced by free hydroxy group of
Initiation Unit (20).
10
The dimeric unit (21) is reacted with a basic reagent, such as but not
limited to Sodium methoxide, which facilitate ring opening resulting into
protected dimeric unit (22). Thereafter the dimer so obtained is subjected
to iterative reactions under similar conditions in presence of said catalyst
15 and said basic reagent to yield a higher synthetic oligomers including
trimer, tetramer, pentamer, hexamer, heptamer, 24, 26, 28….n) etc.
The dimeric unit (22) so obtained is again reacted with propagation unit
under similar conditions of temperature and atmospheric pressure
20 resulting in the formation of trimer (23, 24). The trimer so obtained is
again reacted with propagation unit under similar conditions to obtain
tetramer unit (25). The tetramer undergoes iterative reactions under
similar conditions to get higher oligomers (Y) including tetramer,
pentamer, hexamer, heptamer, etc.
25
The higher oligomers so obtained are subjected to final deprotection of
protecting groups in presence of deprotecting reagents such as BF3:OEt2
ACN, NaOH, MeOH, H2/Pd(OH)2. The sequential deprotection of
protecting groups in presence of a deprotecting reagent resulting in
30 deprotected Men-Y higher oligomers (d22, d24, d26, d28.... dn)
12
The total time taken for said process is in the range of 330 hours to 400
hours and more particularly is 370 hours, resulting in significantly low
cost of production.
The higher oligomers so obtained are 5 linked to carrier proteins by
conjugation through in-built linker to obtain a semisynthetic conjugate
vaccine against meningococcal serogroup Y bacterial infection. This
chemical process of synthesis can also be employed for the preparation of
synthetic protein/peptide to prepare fully synthetic conjugate vaccine as
10 well. It is noted that such conjugation with carrier proteins result in
vaccines with good yield and enhanced antigenicity.
In one of the embodiment Men-Y tetramer is tested for antigenicity by
conducting inhibition enzyme-linked immunosorbent assay (Inhibition
15 ELISA) and the tetramer and its conjugate with tetanus toxoid is found to
give neutralization of specific antibodies against Men-Y capsular
polysaccharide.
Complete scheme for MenY tetramer synthesis:
20 Scheme I: Synthesis of propagation unit 18
13
5
10
Synthesis of the sialic acid phosphate donor:
OH Step-1 OH H Step-,2 OAc Step-3
H0--(_~~-_11 H0--(_~~-1.: AczO/ Py Ac0-.....1.._'E.__ _ T CH3COCIIHCI
AcirN~COOH AcirN~-COOMe - AcJl-N~COOMe
HO DowexW50X8{H+) HO AcO
' - 2 3 Stap4
ToiSH,
DIPEA
~~-__rx'Me
AoliN~-s--("1
......
MeOH, MsOH
8tep..7-8
1\TBDPSCUim/DM(
11)2,2-DMP
iii)AcCI
5 AcO ~
TBDPSO~OC~O OM~ 8tep..10 TBDPso-t_"1.___~0MeO
.· o N c"~"~,PO~·~":,-. A:;N~o~p·
AcN}r- S NIS, TfOH, AcNh--O Bud 'OBu
8Q DCM B/j
Svpthes!s gf Glycose acceDI:p[J J.i All
OAc 8tep..11 OAc step..12 OH step..13 OTBDPS step-14
XxOAo
thiocresol,
XxSTol
MoONa
BFa:OEt2 XxSTol
TBDP8-CI yoySTol BnBr, NaH TBDPSO"'(OySTol
AoO 'OAo 70% AoO OAc Ho···Y·''OH DMF, rt, o•c Bno···y··'Oen
OAc OAc
1 0 commercial "
Synthesis of the building propagaUon unit 18
X' OH
TBDPS_O9 ~OCO OMeO XxSTol
· o o~ '' +
AcN
0
euJ'oBu Bno··· ···oen
9 r OBn
"
MoOH HO OH 80%
qual
Step-18
TMSOTl, CH~~
-70"C
OH
t2
OH 76% OBn
13 14 ~Step-15
BAF, THF
90%
Step-11
NIS, TfOH
70%
OTBDPS step-17
Bno-< -0 TBAF, THF
eno~
BnO 0 60%
.. ~., Alpha:beta ratio (65:35) "
HO"'(OySTol
Bno···y··'Oen
OBo
"
Alpha:beta ratio (95:5)
o'>: TBDPSO.~ 0f'CO OM#e AoN
18 r~ 0 STol
OBo
14
Scheme II: Synthesis of Initiation unit 20
Scheme III: S 5 ynthesis of dimer trimer and tetramer
15
The above detailed description of process is illustrated by non-limiting
5 examples as follows:
Examples:
Example 1: Step 1-Preparation of compound 2
10
Procedure: To a stirred solution of N-acetyl Neuraminic acid (2.0 kg, 6.472
mol) in methanol (32 lit); Dowex 50W X8 resin is added. Reaction is stirred
at RT for 24h. Reaction is monitored by TLC (20% methanol; DCM). After
completion, reaction mixture was filtered, resin was washed with
15 methanol (2 lit). The filtrate was concentrated to give required product as
white solid (2.0 kg; 95%).
Example 2: Step 2-Preparation of compound 3
20
O COOMe
OH
HO
AcHN
HO OH
OH
2
O COOMe
OAc
AcO
AcHN
AcO OAc
OAc
3
16
Procedure: To a mechanically stirred solution of compound 2 (1 kg, 3.095
mol) in pyridine (10 lit), DMAP (38 g, 0.0309 mol) was added at 0°C
followed by acetic anhydride (11.7 lit). Reaction 5 was stirred at RT for 24 h.
After completion reaction mixture was concentrated under vacuum (10
mbar). Crude compound was purified by flash column chromatography
over silica-gel (230-400 mesh) eluting with 30% ethyl acetate-hexanes to
100% ethyl acetate. Compound-3 was obtained as white foamy solid (1.4
10 kg; 87%).
Example 3: Step 3-Preparation of compound 4
15 Procedure: A stirred solution of compound-3 (1.4 kg, 2.626 mol) in acetyl
chloride (7.5 lit) was cooled to 0°C; and a solution of freshly prepared HCl
in acetyl chloride (4.5 lit) was added. The resulting reaction mixture was
warmed to RT and stirred for 16h. After completion, reaction mixture was
concentrated under reduced pressure. Obtained compound was co20
distilled with toluene (2*2.5 lit), and the crude product was forwarded to
the next step (1.4 kg, 99%).
Example 4: Step 4: Preparation of compound 5
17
Procedure: To a mechanically stirred solution of compound-4 (1.35 kg,
2.652 mol) in DCM (13.5 lit) p-thiocresol (324 g, 2.652 mol) was added. The
solution was cooled to 0°C and diisopropylethyl amine (548 ml, 3.135 mol)
was added slowly with dropping funnel 5 for 30 mins under nitrogen
atmosphere. The resulting reaction mixture was warmed to RT and stirred
for 18 h. Reaction was monitored by TLC, after completion; reaction
mixture was cooled to 0°C and diluted with DCM (5.0 lit), washed with
water (2*5 lit). Organic layer was separated, washed with brine solution,
10 dried over sodium sulphate and concentrated. Obtained compound was
treated with 25% ethyl acetate/petroleum ethers. Resulting white solid
was filtered and dried (900 g, 60%).
Example 5: Step 5-Preparation of compound 6
15
Procedure: To a stirred solution of compound-5 (500 g, 0.8375 mol) in
methanol (5.0 lit), methane sulphonic acid (322 g, 3.35 mol) was added
slowly at 0°C. The resulting reaction mixture was heated to reflux for 36 h.
The reaction mixture was cooled to 0 °C and quenched with triethyl amine
20 (790.0ml). The mixture was concentrated and the residue was taken to next
step without any further purification. LC MS purity 96 %. (Crude weight:
650 g)
18
Example 6: Step 6-Preparation of compound 7
Procedure: Compound-6 was dissolved in a mechanically stirred mixture
of acetonitrile (4.0 lit), water (5.0 lit) and NaHCO3 (2.352 kg, 46.51 moles),
(1.8 kg, 4.651 moles) at room temperature. 5 Reaction mixture was cooled
with ice-bath to 0°C and a solution of triphosgene (0.895 kg, 4.651 moles)
in acetonitrile (1.0 lit) was added drop wise over a period of 60 mins.
Reaction was stirred at 0°C for additional 3 h. After completion, the
reaction mixture was neutralized with 10% HCl (3 lit). Product was
10 extracted with ethyl acetate (2*4.0 lit). Organic layer was washed with
water, brine, dried over Na2SO4 and concentrated. Crude compound was
purified by flash column chromatography, eluting with 2-5 % methanol;
DCM. Compound-6 was obtained as off-white solid (700 g, 56% for 2
steps).
15
Example 7: Step 7-Preparation of compound 7A
Procedure: To a stirred solution of compound-7 (700 g, 1.694 mol) in DMF
(6.0 lit), imidazole (288 g, 4.237 mol) was added. t-ButylDiphenylsilyl
20 chloride (557 g, 2.033 mol) was added slowly at 0°C for 10 mins. Reaction
temperature was raised to RT and stirred for 16h. The progress of the
reaction was monitored by TLC. After completion, RM was diluted with
19
water (10 lit). Product was extracted with ethyl acetate (2*4 lit). Organic
layer was separated, washed with brine, dried over Na2SO4 and
concentrated. Product was purified by flash column chromatography over
silica-gel using 60% ethyl acetate-hexanes as eluent to yield off-white solid
5 (1000 g, 90%).
Example 8: Step 8- Preparation of compound 7B
O
COOMe
S
O
HN
TBDPSO O
O
O 7B
Procedure: To a stirred solution of Compound-7A (1050 g, 1.697 mol) in 2,
10 2 –dimethoxy propane (7 lit) camphorsulphonic acid (470 g, 2.036 mol)
was added. The mixture was stirred at RT for 3h. The progress of the
reaction was monitored by TLC. After completion by TLC; reaction
mixture was quenched with triethyl amine (350 mL; 2.515 mol). Reaction
mixture was concentrated. Crude compound was purified by flash column
15 chromatography over silica gel. Compound-7B was obtained as off-white
foamy solid (1000 g, 92%).
Example 9: Step 9-Preparation of compound 8
20
Procedure: To a stirred solution of compound-7B (590g, 0.8538 mol) in
DCM (5.0 lit) diisopropylethyl amine (1500 ml, 8.538 mol) was added. The
solution was cooled to 0°C, acetyl chloride (490 ml, 6.83 mol) was added
slowly with dropping funnel for 10 mins under nitrogen atmosphere. The
resulting reaction mixture was 5 warmed to RT and stirred for 18 h.
Reaction was monitored by TLC, after completion; reaction mixture was
cooled to 0°C and diluted with DCM (3 lit), washed with water (3 lit).
Organic layer was separated, washed with brine solution, dried over
sodium sulphate and concentrated. Product was purified on silica column
10 chromatography; 10-20% ethyl acetate; hexanes as eluent (Yield: 500 g,
80%).
Example 10: Step 10-Preparation of compound 9
15 Procedure: To a stirred mixture of Compound-8 (100 g, 0.1364 mol) and di
butyl phosphate (54 mL; 0. 2728 mol) in DCM (1 Lit.) under nitrogen
atmosphere, 4A° MS (Activated Under high vacuum at 140°C for 2h; 100 g,
wt/wt) was added. The mixture was stirred at RT for 2h then cooled to
0°C and NIS (61.11 g, 0.2728 mol) followed by triflic acid (3.62 mL, 0.0409
20 mol) was added. Reaction temperature was maintained at 0°C for 5h. The
progress of the reaction was monitored by TLC. After completion, reaction
mixture was quenched with saturated hypo solution (250 ml). It was
filtered through celite, and extracted. Organic layer was separated and
then washed with water, dried using Na2SO4 and concentrated. Crude
21
compound was purified by flash column chromatography over silicagel.
Compound-9 was obtained as sticky material (80.0 g, 70%).
Example 11: Step 11-Preparation of compound 11
5
Procedure: β-D-Glucose pentaacetate 10 (500 g, 1.2816 mol), p-thiocresol
(255 g, 2.050mol) and 4A° Molecular sieves powder (500 g) were added in
dichloromethane (5 lit) under argon atmosphere in a flame dried flask.
The solution was cooled to 0 °C and BF3·Et2O (49%; 1000 ml, 3.5886 mol)
10 was added. The mixture was stirred for 16 h. The reaction mixture was
diluted with dichloromethane (2 lit), washed with sodium hydrogen
carbonate (2×2.5 lit of a saturated aqueous solution) and then with water
(3 lit). The organic layer dried over Na2SO4 filtered and concentrated in
vacuum. The residue was purified by flash column chromatography to
15 afford compound-11(417 g, 72%) as a white solid.
Example 12: Step 12-Preparation of compound 12
22
Procedure: Compound 11 (417 g, 0.9182mol) was dissolved in methanol
(4.0 lit) under argon atmosphere in a flame dried RB flask. The solution of
sodium methoxide in methanol (39.6 ml of a 25% solution) was added and
the resulting mixture stirred for 2 h. After completion, amberlite IR-120+
ion exchange resin was added portion wise 5 until the solution becomes
neutral. The reaction mixture filtered to remove resin and the filtrate was
concentrated in vacuum to afford compound 12 (254 g, 95%) as a white
crystalline solid.
10 Example 13: Step 13-Preparation of compound 13
Procedure: To a stirred solution of compound-12 (200 g, 0.402 moles) in
DMF (2.0 lit), imidazole (114 g, 1.678 moles) was added. To above solution
t-butyl diphenylsilyl chloride (269 g, 0.979 moles) was added slowly at 0°C
15 for 10 mins. Reaction temperature was raised to RT and stirred for 16 h.
The progress of the reaction was monitored by TLC. After completion,
reaction mixture was diluted with water (4 lit). Product was extracted with
ethyl acetate (2*1 lit). Organic layer was separated, washed with brine,
dried over Na2SO4 and concentrated. Product was purified by flash
20 column chromatography over silica-gel using 60% ethyl acetate-hexanes as
eluent to yield off-white solid (260 g, 72%).
Example 14: Step 14-Preparation of compound 14
23
Procedure: A stirred mixture of compound 13 (260 g, 0.4962 mol) and
benzyl bromide (273 ml, 2.2328 mol) in DMF (2.5 lit) was cooled to 0°C,
sodium hydride (99 g, 2.480 mol) was added portion wise at 0°C. RM was
stirred at RT for 16h. TLC was showing 5 SM consumed, then RM was
quenched with ice cold water (4 lit), and extracted with ethyl acetate
(2X1lit). Organic layer was separated and concentrated under reduced
pressure. Crude compound was purified by flash column
chromatography, product was eluted with 5% ethyl acetate-hexanes gave
10 Compound 14 as a colorless liquid (300 g, 76% yield).
Example 15: Step 15-Preparation of compound 15
Procedure: Compound 14 (145 g, 0.1826 mol) was dissolved in THF (1.4
15 lit), was cooled to 0°C and 1M TBAF in THF (219 mL, 0.2191 mol) was
added. Cooling bath was removed and the reaction mixture was stirred
for 16h at RT. Reaction was monitored by TLC, after completion reaction
was diluted with water, extracted with ethyl acetate (2*500 ml). Organic
24
layer was dried over Na2SO4, filtered and concentrated. Crude product
was purified by FCC, eluted with 20% EtOAc/hexane. Compound was
obtained as viscous liquid (90 g, 89%).
5 Example 16: Step 16-Preparation of compound 16
Procedure: To a stirred mixture of Compound-14 (50 g, 0.0629 mol) and 6-
azidohexanol (18 g; 0.1259 mol) in diethyl ether (500 ml) under nitrogen
atmosphere, 4A° MS (Activated under high vacuum at 140°C for 2h; 50 g,
10 wt/wt) was added. The mixture was stirred at RT for 2h and then was
cooled to -40°C. NIS (28.2 g, 0.1259 mol) followed by triflic acid (2.8 ml,
0.0314 mol) was added and stirring continued for 2h. After completion by
TLC, reaction mixture was quenched with addition of saturated hypo
solution (100 ml). The reaction mixture filtered through celite and the
15 filtrate transferred to a separating funnel. Organic layer was separated,
washed with water, dried over Na2SO4 and concentrated. Crude
compound was purified by flash column chromatography, eluted with
10% ethyl acetate and hexane. Compound-16 the anomeric mixture was
obtained as sticky material (35 g, 70%).
20
Example 17: Step 17-Preparation of compound 17
25
Procedure: Compound 16 (100 g, 0.123 mol) was dissolved in THF (1.0 Iit),
cooled to 0°C and 1M TBAF in THF (148 mL, 0.1476 mol) was added.
Cooling bath was removed and stirred for 16h at RT. Reaction was
monitored by TLC. After completion, 5 reaction was diluted with water,
extracted with ethyl acetate (2*500ml). Organic layer was dried over
Na2SO4, filtered and concentrated. Crude product was purified by flash
column chromatography, eluted with 20% EtOAc/hexane. Compound
was obtained as viscous liquid (55g, 78%).
10
Example 18: Step 18-Preparation of compound 18
Procedure: A mixture of compound-15 (43.3 g, 0.0778 mol) and
compound-9 (84 g; 0.1307 mol) was dissolved in DCM (430 ml) and was
15 added MS 4A° (45 g). The reaction mixture was stirred at RT for 2h. It was
then cooled to -78°C and TMSOTf (15.58 mL, 0.0855 mol) was added.
Reaction was maintained at -78°C and stirred for 1h. The progress of the
reaction was monitored by TLC, after completion; reaction mixture was
quenched with triethyl amine (1.2 eq.) filtered through celite. Organic
20 layer was washed with saturated sodium bicarbonate solution (300 mL),
26
organic layer was separated and washed with water and brine solution,
dried over Na2SO4, filtered and concentrated. Crude compound was
purified by flash column chromatography; eluted with 30% ethyl acetate
and hexane gave compound-18 as a white solid (85 g, 93%).
5
Example 19: Step 19- Preparation of compound 19
Procedure: To the mixture of compound-17 (45 g, 0.0778 mol) and
compound-9 (85 g; 0.1037 mol) in DCM (450 mL) was added MS 4A° (45 g)
10 and the resulting reaction mixture stirred at RT for 2h. It was then cooled
to -78°C, and TMSOTf (15.66 mL, 0.0855 mol) was added. Stirring
continued for 2h at same temperature. The progress of the reaction was
monitored by TLC. After completion, reaction mixture was quenched with
triethyl amine (1.2 eq.) filtered through celite. Organic layer was washed
15 with saturated sodium bicarbonate solution (500 mL), followed by water
and brine solution, dried over Na2SO4, filtered and concentrated. Crude
compound was purified by flash column chromatography, eluted with
30% ethyl acetate and hexane to provide compound-19 as a white foamy
solid (80 g, 86%).
20
Example 20: Step 20- Preparation of compound 20
27
Procedure: Compound-19 (80 g, 0.0676 mol) was dissolved in methanol
(533 mL) and DCM (267 mL) under argon atmosphere. Solution of sodium
methoxide in methanol (14.6 mL, 25% NaOMe in methanol) was added
and the mixture stirred at RT for 1 h. After 5 completion of reaction, Ion
exchange resin (IR 120+) was added portion wise until the solution was
neutralized. The reaction mixture was filtered, the filtrate concentrated
and the crude product purified by flash column chromatography (ethyl
acetate: petroleum ether 2:1 as eluent) to provide compound-20 (55 g, 70%)
10 as a white foamy solid.
Example 21: Step 21- Preparation of compound 21
15 Procedure: To a stirred solution of compound-18 (53 g, 0.0453 mol) and
Compound-20 (35 g; 0.0302 mol) in diethyl ether (350 mL) 4A° molecular
28
sieves (35 g) was added and stirred at room temperature for 2h. The
mixture was cooled to 0°C. To this mixture NIS (13.54 g, 9.030 mmol) was
added in one lot followed by triflic acid (1.34 mL, 0.0151 mol). Reaction
temperature was raised to RT and maintained for 16h. The progress of the
reaction was monitored by TLC. After 5 completion, the reaction mixture
was quenched with saturated sodium thiosulfate solution (250 ml), diluted
with ethyl acetate (300 ml). It was filtered through celite and the organic
layer was separated. The organic layer was washed with water, dried over
anhydrous Na2SO4, filtered and concentrated. Crude compound was
10 purified by flash column chromatography eluted with 30% ethyl acetate
and hexane. Compound-21 was obtained as white solid (32 g, 48%).
Example 22: Step 22-- Preparation of compound 22
15
Procedure: Compound-21 (10 g, 4.5454 mmol) was dissolved in methanol
(65 mL) and DCM (35 mL) under argon atmosphere in a flame dried flask.
Solution of sodium methoxide in methanol (0.982 mL of a 25% in
methanol) was added and the mixture stirred at RT for 1 h. After
20 completion of reaction, the reaction mixture quenched using amberlite IR
120 was added portion wise until the solution was neutralized. The
reaction mixture filtered, the filtrate was concentrated and purified by
29
flash column chromatography (ethyl acetate: petroleum ether 2:1-3:1 as
eluent) to afford compound-22 (6.1 g, 62%) as a white foamy solid.
Example 23: Step 23- Preparation of compound 23
5
Procedure: To a stirred solution of compound-22 (15 g, 6.899 mmol) and
Compound-18 (12 g; 10.349 mmol) in dry diethyl ether (150 mL) 4A°
molecular sieves (15 g) was added and the mixture stirred for 2h at RT.
The mixture was cooled to 0°C. To this mixture NIS (3.1 g, 13.790 mmol)
10 followed by triflic acid (0.306 ml, 3.450 mmol) was added. The resulting
reaction mixture was stirred at RT for 16h. After completion by TLC, the
reaction mixture was quenched with addition of saturated sodium
thiosulfate solution (150 ml). The reaction mixture filtered through celite
and the filtrate organic layer was washed with water and brine, dried over
15 anhydrous Na2SO4, filtered and concentrated. Crude compound was
purified by flash column chromatography eluting with 50% ethyl acetate
and hexane. Compound-23 was obtained as white foamy solid (12 g, 41%).
Example 24: Step 24- Preparation of compound 24
30
Procedure: Compound 23 (12 g, 3.732 mmol) was dissolved in methanol
(80 mL) and DCM (40 mL) under argon atmosphere in a flame dried flask.
Solution of sodium methoxide in methanol (0.806 mL of a 25%) was added
and the mixture stirred at 0°C for 5 1 h. After completion by TLC, the
reaction mixture quenched by portion wise addition of ion exchange resin
(IR 120+) until the solution was neutralized. The reaction mixture filtered,
concentrated and the crude product purified by flash column
chromatography (ethyl acetate: petroleum ether 2:1 as eluent) to afford
10 compound 24 (7 g, 60%) as a white foamy solid.
Example 25: Step 25- Preparation of compound 25
Procedure: To a stirred solution of Compound-24 (4.1 g, 1.4733 mmol) and
15 Compound-18 (3.1 g; 2.6505 mmol) in diethyl ether (40 mL) 4A° molecular
31
sieves (4.1 g) was added and stirred at room temperature for 2h. NIS (0.34
g, 1.5175 mmol) was added at once followed by addition of triflic acid
(0.13 mL, 1.4733 mmol) at RT. The stirring continued at RT for 60h. The
progress of the reaction was monitored by TLC (35% ethyl acetate;
hexanes). After completion, the reaction 5 mixture was quenched with
saturated sodium thiosulfate solution (40 mL). It was filtered through
celite and the organic layer was separated. The organic layer was washed
with water, dried over anhydrous Na2SO4, filtered and concentrated.
Crude compound was purified by flash column chromatography eluting
10 with 20-30% ethyl acetate and hexane. Compound-25 was obtained as
white solid (2.06g, 38%).
Example 26: Step 26- Preparation of compound 25A
15 Procedure: Compound-25 (3 g, 0.7088 mmol) was dissolved in methanol
(624 mL) and DCM (12 mL) under argon atmosphere. Solution of sodium
methoxide in methanol (0.184 mL, 25%) was added and the mixture
stirred at RT for 1 h. After completion by TLC reaction mixture was
quenched using Ion exchange resin amberlite IR 120 until the solution was
20 neutralized. The mixture was filtered and concentrated. The crude
32
product purified by flash column chromatography (ethyl acetate:
petroleum ether 2:1) to afford compound-25A (1.7 g, 58%) as a white solid.
Example 27: Step 27- Preparation of compound 25B
5
Procedure: Compound 25A (1.7 g, 0.4040 mmol) was dissolved in
acetonitrile (45 mL) and cooled to 0°C. 45% BF3; OEt2 (7.65 mL, 24.2453
mmol) was added and the stirring continued for 3h. The reaction mixture
was diluted with ethyl acetate (100 mL), washed with saturated aqueous
10 NaHCO3 (80 mL) and then with brine solution. The organic layer was then
dried over Na2SO4, filtered and concentrated.
The residue washed with hexane and the resulting solid product obtained
was taken in ethanol and water. Lithium hydroxide was added (0.17g,
4.0408 mmol) and the reaction was stirred at 80°C for overnight. The
15 solvent was removed under reduced pressure; crude material was
dissolved in water (20 mL), washed with diethyl ether (20 mL) and the
aqueous layer separated. pH of the aqueous layer was adjusted to 5 using
1N HCl (0.4 mL) solution. Product was extracted with 10% methanol:DCM
(2*60 mL). Organic layer dried over Na2SO4, filtered and concentrated to
20 yield the compound 25B (0.8 g; 80% for two steps).
33
Example 28: Step 28- Preparation of compound d26
Procedure: Compound-25B (0.8 g, 0.2638 mmol) was dissolved in a
mixture of methanol (50 mL) and 5 water (12 m l) in a 250 mL
hydrogenation flask. 20% Pd (OH) 2/C (0.8 g) was added under nitrogen
atmosphere. The flask was purged with hydrogen for three times and then
the content was stirred under hydrogen atmosphere (100 psi) for 24 h.
After the completion the reaction mixture was filtered through celite and
10 concentrated under reduced pressure at 25°C. Product was purified by gel
filtration through Sephadex-G-10 column, eluted with water. Compound
fractions collected and lyophilized to obtain Men-Y tetramer (d26) as
white solid (350 mg; 70%).
15 Example 29: Antigenicity analysis of Men-Y tetramer and Men-Y
tetramer-tetanus toxoid (TT) conjugate
Procedure: The ELISA plate was coated with bacterial polysaccharide
which was reacted with polyclonal serum raised against anti-Men-Y
34
bacterial capsular polysaccharide with or without inhibition with different
concentrations (0.39-100 microgram/ml) of Men-Y tetramer or Men-Y
tetramer-TT conjugate prepared using thio-ether chemistry. When the
reaction was developed using HRP labelled secondary antibody, both the
Men-Y tetramer as well as its conjugate 5 neutralized the Men-Y specific
IgGs evident with reduction in the optical density (% inhibition) after
adsorption of serum dilution with different antigen concentrations as
compared to no antigen control as shown in Figure

We Claim:
1. A novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit, said process
5 comprising the steps of
(a) synthesizing Propagation Unit (18)
(b) synthesizing Initiation Unit (20)
(c) coupling said Initiation Unit (20) with said Propagation Unit
(18) in presence of at least one catalyst and at least one basic
10 reagent to synthesize higher oligomers
(d) iterating reaction of step (c) in presence of said catalyst and
said basic reagent to yield higher synthetic oligomers (22,
24, 26, 28….n)
(e) subjecting said higher synthetic oligomers of step (d) to
15 sequential deprotection of protecting groups in presence of
at least one deprotecting reagent resulting in deprotected
Men-Y higher oligomers (d22, d24, d26, d28.... dn)
such that said process results in said novel higher synthetic
oligomers with better yield and high purity.
20 2. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1 wherein said higher synthetic oligomer (Y) is a novel
tetramer (d26).
3. The novel process of synthesizing oligomers of Neisseria meningitidis
25 serogroup Y capsular polysaccharide repeating unit as claimed in
claim 2 wherein the time taken to synthesize said tetramer is in the
range of 330 hours to 400 hours, more preferably 370 hours.
36
4. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1 wherein said Initiation Unit (20) is synthesized by:
(a) subjecting monosaccharide compound 1 to methylation in
presence of catalyst to 5 obtain compound 2, subjecting
compound 2 to acylation in presence of acylating reagent to
obtain compound 3, subjecting compound 3 to halogenation
in presence of halogenating reagent to obtain compound 4,
reacting compound 4 with thiolating reagent to obtain
10 compound 5, reacting compound 5 with deacylating reagent
to obtain compound 6, reacting compound 6 with
carbonylation reagent to obtain compound 7, subjecting
compound 7 to protecting reagent to obtain compound 8,
reacting compound 8 to phosphorylation to obtain
15 compound 9 by known process
(b) reacting commercially purchased compound 10 with
acceptor and catalyst to obtain compound 11, subjecting
compound 11 to deacetylating reagents to obtain compound
12, subjecting compound 12 to protecting group to obtain
20 compound 13, subjecting compound 13 to alkylation to
obtain compound 14 by known process
wherein said compound 14 is subjected to :
(c) glycosidation of compound 14 with an alcohol in presence at
least one catalyst to obtain compound 16
25 (d) subjecting compound 16 to at least one deprotecting reagent
to obtain compound 17
(e) reacting compound 17 and compound 9 in presence of said
at least one catalyst to obtain compound 19
37
(f) subjecting compound 19 to at least one basic reagent to
obtain said Initiation unit compound 20
5. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1 wherein s 5 aid Propagation Unit (18) is synthesized by:
(a) subjecting compound 14 to at least one deprotecting reagent
to obtain compound 15
(b) reacting said compound 15 to said compound 9 in presence
of said at least one catalyst to obtain Propagation Unit 18
10 6. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1, claim 4 and claim 5 wherein said catalyst is selected from
NIS, TfOH, TMSOTf.
7. The novel process of synthesizing oligomers of Neisseria meningitidis
15 serogroup Y capsular polysaccharide repeating unit as claimed in
claim 4 wherein said alcohol is 6-azido hexanol.
8. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1 wherein said deprotecting reagent is selected from BF3:OEt2,
20 ACN, NaOH, MeOH, H2/Pd(OH)2.
9. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 4 wherein said monosaccharide is neuraminic acid more
particularly N-acetyl neuraminic acid.
25 10. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 4 and claim 5 wherein said deprotecting reagent is selected
from TABF, THF, CSA, HCl, PTSA.
38
11. The novel process of synthesizing oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit as claimed in
claim 1 and claim 4 wherein said basic reagent is selected from
NaOMe, NaOEt, KOtBu.
12. A novel higher synthetic 5 oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit prepared by
process as claimed in claim 1 wherein said novel higher synthetic
oligomers (Y) are synthetic oligomers of high purity and improved
antigenicity.
10 13. The novel higher synthetic oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit prepared by
process as claimed in claim 1 wherein said novel higher synthetic
oligomer (Y) is tetramer (d26) of more than 95% purity and
improved efficacy having structural formula:
14. The novel higher synthetic oligomers of Neisseria meningitidis
serogroup Y capsular polysaccharide repeating unit prepared by
process as claimed in claim 1 is capable of being used as a candidate
in the development of semisynthetic 5 or fully synthetic conjugate
vaccines against meningococcal serogroup Y bacterial infection.