Towards the complete synthetic O-Antigen of: Vibrio cholerae O1, serotype inaba: Improved synthesis of the conjugation-ready upstream terminal hexasaccharide determinant

Article abstract of DOI:10.1039/c9ra08232h

Synthesis of the upstream terminal hexasaccharide part of the lipopolysaccharides (LPS) of Vibrio cholerae O1, serotype Inaba has been improved. The key improvements include but are not limited to optimized conditions for the stereoselectivity of glycosylation reactions involved and fewer number of synthetic steps, compared to previous approaches. Particularly noteworthy is conducting the glycosylation of the very reactive glycosyl acceptor 8-Azido-3,6-dioxaoctanol with the fully assembled hexasaccharide trichloroacetimidate under thermodynamic control. It produced the desired α glycoside with an α:β ratio of 7:1, compared with the ratio of 1.1:1, observed when the coupling was conducted conventionally. Several substances, which were previously obtained in purity acceptable only for synthetic intermediates, were now obtained in the analytically pure state and were fully characterized. The structure of the key trisaccharide glycosyl acceptor was confirmed by single-crystal X-ray structure determination.

Full text of DOI:10.1039/c9ra08232h

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Towards the complete synthetic O-antigen of
Vibrio cholerae O1, serotype inaba: improved
synthesis of the conjugation-ready upstream
terminal hexasaccharide determinant†
Cite this: RSC Adv., 2019, 9, 36440
a
a
Peng Xu,a Edwin D. Stevensb and Pavol Kovac
*
*
Mana Mohan Mukherjee,
´ˇ
Synthesis of the upstream terminal hexasaccharide part of the lipopolysaccharides (LPS) of Vibrio cholerae
O1, serotype Inaba has been improved. The key improvements include but are not limited to optimized
conditions for the stereoselectivity of glycosylation reactions involved and fewer number of synthetic
steps, compared to previous approaches. Particularly noteworthy is conducting the glycosylation of the
very reactive glycosyl acceptor 8-azido-3,6-dioxaoctanol with the fully assembled hexasaccharide
trichloroacetimidate under thermodynamic control. It produced the desired a glycoside with an a : b
ratio of 7 : 1, compared with the ratio of 1.1 : 1, observed when the coupling was conducted
conventionally. Several substances, which were previously obtained in purity acceptable only for
synthetic intermediates, were now obtained in the analytically pure state and were fully characterized.
The structure of the key trisaccharide glycosyl acceptor was confirmed by single-crystal X-ray structure
determination.
Received 9th October 2019
Accepted 5th November 2019
DOI: 10.1039/c9ra08232h
rsc.li/rsc-advances
essential immune responses of conjugates therefrom in mice.5
Chemical structures of O-SPs of the two strains (Inaba and
Introduction
Ogawa) of Vibrio cholerae O1 are the same, except that the
terminal, upstream perosamine residue in the Ogawa strain
carries a methyl group at O-2. To be able to compare immune
responses of the conjugates from the hexasaccharide fragments
of the O-SP of both serotypes of Vibrio cholerae O1 with those of
similar conjugates from synthetic polymers representing the
complete O-SPs, we intend to synthesize glycoconjugates from
the analogous octadecasaccharides. Syntheses of such struc-
tures are much more involved undertakings. Previous syntheses
of the hexasaccharide antigens comprised up to more than 40
linear steps, depending on the individual approach.1,2 The key
intermediates within our strategy towards the octadeca-
saccharides will be synthons derived from the related hex-
asaccharides. With the aim to increase the feasibility of a large-
scale synthesis required by future immunization studies and
decrease the number of synthetic steps involved in making such
substances, the objective of this work was to test the practica-
bility and scalability of the current, new synthetic scheme.
Thus, we synthesized on large scale the trisaccharide glycosyl
donor and acceptor 5 and 4, respectively, and used these to
prepare, also on large scale, the related hexasaccharide 3
(Fig. 1). These substances are versatile intermediates, which we
intend to use to make considerably larger fragments of the O-
SP, up to the complete bacterial O-specic antigen, octadeca-
saccharide. Using hexasaccharide 3, we proceeded to complete
the synthesis of the title hexasaccharide 1. The present pathway
The upstream, terminal part of the lipopolysaccharides (LPS) of
most Gram-negative bacteria consist of several copies of oligo-
saccharide, sometimes monosaccharide repeating units. The
said macromolecules are termed O-specic polysaccharides (O-
SP) and are recognized as protective antigens of these patho-
gens. O-SPs confer specicity to their homologous antibodies
and they, or fragments thereof, are paramount in the develop-
ment of synthetic/conjugate vaccines. We have been involved in
work towards vaccines from synthetic fragments of O-SPs for
a number of years. One of our major targets has been a conju-
gate vaccine for cholera from synthetic fragments of O-SPs of
Vibrio cholerae.1–3
The O-SP of Vibrio cholerae O1, serotype Inaba consists of 12–
18 repeats4 of (1/2)-a-linked perosamine (4-amino-4,6-
dideoxy-D-mannose) whose amino group is acylated with 3-
deoxy-L-glycero-tetronic acid. We have previously synthesized
hexasaccharide fragments of the O-SP and have determined
aLBC, NIDDK, National Institutes of Health, Bethesda, MD 20892-0815, USA. E-mail:
mana.mukherjee@nih.gov; kpn@helix.nih.gov
bDepartment of Chemistry, Western Kentucky University, Bowling Green, KY, 42101,
USA
† Electronic supplementary information (ESI) available: Copy of 1H, 13C NMR of
all compounds, 1H, 13C, COSY, and HSQC NMR spectra of new compounds and
table for crystallographic and structural renement parameters (pdf). CCDC
1939745. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c9ra08232h
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Fig. 1 Retrosynthetic analysis of the hexasaccharide 1.
is the shortest published to date (33 linear steps). An additional was not sought, but in their extensive synthetic study towards
advantage of the approach described here is that the stereo- this class of substances, Saksena et al.11 observed that formation
selectivity of the critical glycosylation reaction, which converted of 2-trimethylsilylethyl mannopyranosides is a complex process
the terminal determinant to a conjugation-ready form, was
substantially increased by controlling it thermodynamically.2,6
Results and discussion
Synthesis of the title hexasaccharide started with the known7
methyl
4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-a-D-man-
nopyranoside (10). It was treated with Ac2O in presence of
H2SO4, and the formed (Scheme 1) 1-O-acetyl-4-azido-2-O-
benzoyl-3-O-benzyl-4,6-dideoxy-a,b-D-mannopyranose
(11)7
where the a-anomer largely predominated was resolved by
chromatography. Unlike previously, the a-acetate 11a was now
obtained crystalline for the rst time. We planned to use the
latter as a glycosyl donor under BF3$Et2O or TMSOTf catalysis8,9
to make the corresponding 2-trimethylsilylethyl (SE) glycoside
13a, and perhaps also higher oligosaccharides in this series,
thereby improving the economy of the overall synthesis.
Unfortunately, this approach was unsuccessful: only the corre-
sponding 1-OH compound was formed. Similarly unsuccessful
was the reaction of the corresponding 2-O-acetyl derivative 12
(/13b), prepared conventionally10 (these reactions are not
described in the Experimental). Explanation for these failures Scheme 1 Synthesis of the monosaccharide building blocks.
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whose outcome is largely unpredictable. In contrast with the chain, then would be the case if extensive hydrogen bonding
unsuccessful Lewis acid mediated reaction of 11 or 12 with 2- were present. Signicantly, the relative conformations of the
trimethylsilylethanol, the BF3$Et2O-catalyzed reaction of 11 trisaccharide chains of both independent molecules are very
with ethanethiol readily produced ethyl 4-azido-2-O-benzoyl-3- similar, while the orientations of the substituent groups
O-benzyl-4,6-dideoxy-1-thio-a,b-D-mannopyranoside (7a and (especially the benzyl and trimethylsilylethyl groups) show
hitherto unknown 7b),11 which was fully characterized. The greater variation (Fig. 2). Further analysis of the conformation
latter mixture of anomers was converted to its corresponding SE of X-ray data for compound 4 will be reported in a subsequent
glycoside 13a as described,11 and the pure a-anomer 7a was communication.
used as glycosyl donor towards oligosaccharide synthesis
Compared to the NMR spectra of parent compound 15,
(Schemes 2 and 3). The initial glycosyl acceptor 87 (Scheme 1) disappearance of the carbonyl carbon signal at 165.3 ppm in the
was prepared by conventional debenzoylation (Zemplen ) from 13C NMR spectrum of 4 and upeld shi of H-2III (from
12
´
the foregoing 2-O-benzoate 13a.
d 5.59 ppm, dd, J2–3 ¼ 2.9 Hz, J2–1 ¼ 2.0 Hz to d 3.98 ppm, ddd,
1
With suitably equipped glycosyl donor and acceptor at hand, J2–3 ¼ 2.8 Hz, J2-OH-2 ¼ 1.7 Hz, J2–1 ¼ 1.3 Hz) in the H NMR
we set out for oligosaccharide synthesis on the multigram scale spectrum conrmed the removal of the benzoyl group and
(see Experimental). Accordingly, glycosyl acceptor 8 and thio- formation of the corresponding 2-hydroxy product 4. Treatment
glycoside donor 7a were coupled using NIS/TMSOTf as of the foregoing 2-trimethylsilyl ethyl glycoside 15 with tri-
promoter, affording disaccharide 14 (94%, Scheme 2). The NMR uoroacetic acid (TFA) produced the corresponding trisaccha-
spectra of the disaccharide showed signals characteristic of the ride hemiacetal 16 (89%). Absence of signals for the 2-
presence of both donor and acceptor moieties (165.3 ppm for (trimethylsilylethyl) group in the 1H NMR spectrum of 16,
the benzoyl carbonyl carbon from the donor and ꢀ1.32 ppm for together with presence of two anomeric C-1I signals in the 13C
SiMe3 carbon from the acceptor), while the a-conguration of NMR spectrum (d 93.5 and 93.2 ppm) conrmed the successful
the interglycosidic linkage was conrmed from the corre- hydrolysis and formation of the desired hemiacetal 16. Subse-
sponding NMR spectra, mainly JC,H at the newly formed glyco- quent base-catalyzed reaction of 16 with trichloroacetonitrile
sidic linkage (173.3 Hz).13 Zemplen debenzoylation of 14 and DBU produced the corresponding trichloroacetimidate
´
furnished disaccharide acceptor 6,7 which was subsequently donor 5 (Scheme 3).
used for chain elongation (Scheme 3). Coupling of disaccharide
Large-scale glycosylation (24.3 g of trichloroacetimidate
acceptor 6 and thioglycoside donor 7a in presence of NIS- donor 5 with 19 g of trisaccharide acceptor 4) produced hex-
TMSOTf at 0 ꢁC produced trisaccharide 15 (93%, Scheme 3). asaccharide 3 (92%, Scheme 4) with excellent stereoselectivity
Formation of the desired trisaccharide 15 was conrmed by (a : b ꢂ 34 : 1, as shown from integration of signals at
HRMS and the stereochemistry of the newly formed inter- d 5.32 ppm and 5.59 ppm for H-1V of 3a and H-2VI of 3b,
glycosidic linkage followed from the 13C–1H coupling constant respectively, in the NMR spectrum of the crude reaction
for the anomeric carbon center at 99.2 ppm (C-1III, JC–H ¼ 173.3 mixture). Strong contours for anomeric carbons at 101.2 ppm
Hz). Zemplen debenzoylation of 15 furnished trisaccharide (C-1II), 101.06 ppm (C-1III), 101.04 ppm (C-1IV), 101.03 ppm (C-
´
acceptor 4, which was obtained crystalline (CCDC no. 1V), 100.3 ppm (C-1VI), 99.16 ppm (C-1I) in the HSQC spectrum
1939745†). Compound 4 crystallizes with 2 independent mole- conrmed the formation of the hexasaccharide. That the
cules in the asymmetric unit of the unit cell. Only one free –OH predominant isomer contained the desired a-conguration
group is available on each molecule for hydrogen bonding, and followed from the 13C–1H coupling constant for the 13C carbon
in each case, a hydrogen bond is formed to an O5 acceptor on involved in the newly formed interglycosidic linkage. For the
a mannopyranoside ring in an adjacent molecule. A modest major isomer, 13C signal of C-1IV at d 101.04 ppm showed
number of weaker C–H/O and C–H/N interactions are also coupling constant JC-1–H-1 ¼ 174.4 Hz whereas the same for the
observed. Given the limited number of free hydroxyl groups minor isomer was at d 96.3 with JC-1–H-1 ¼ 155.5 Hz (Fig. 3).
´
Debenzoylation of hexasaccharide 3a under Zemplen
available for hydrogen bonding, the majority of the intermo-
lecular interactions will be weaker non-polar van der Waals type conditions produced the 2-hydroxyl group-free intermediate
interactions. In this case, the conformations of the poly- 17.14 The six azido groups in the foregoing hexasaccharide were
saccharide chains of the two independent molecules are more transformed, into amines by H2S reduction (/18,14 92%,
likely to reect a conformational energy minimum for the Scheme 5). The next task was to introduce the N-3-deoxy-L-
Scheme 2 Synthesis of the disaccharide acceptor 6.
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Scheme 3 Synthesis of trisaccharide donor 5 and acceptor 4.
glycero-tetronoyl groups into the foregoing hexaamine 18. This reducing sugar 20, thus obtained (Scheme 5), to the corre-
was performed conventionally2,6 with 2,4-di-O-acetyl-3-deoxy-L- sponding trichloroacetimidate donor 21 brought the synthesis
glycero-tetronic acid15 and 1-[3-(dimethylamino)propyl]-3- of the title antigen to a synthetic step that proved difficult in the
ethylcarbodiimide hydrochloride (EDAC). For easier isolation past. In most situations across carbohydrate chemistry,
and next reaction, the crude product was directly acetylated, to synthesis of the 1,2-trans-glycosidic linkage is not problematic
give the fully protected hexasaccharide amide 19 (84% over two because a participating group can be introduced into 2-posi-
steps, Scheme 5).
tions of the glycosyl donors. However, in our case, the 2-O-
To convert compound 19 into a conjugation-ready form, position of the donor is glycosylated and, therefore, no
aglycone in the silyl ethyl glycoside had to be replaced with participating group could be introduced. Furthermore, as we
a suitably equipped linker molecule. Cleavage of 19 with tri- found during our previous syntheses of oligosaccharides within
uoroacetic acid (TFA), and subsequent conversion of the the Vibrio cholerae O1 series, the selectivity of a-mannosylation
Fig. 2 Stereo projections of the two independent molecules of compound 4 at 120 K. Thermal ellipsoids are plotted at the 50% level, and
hydrogen atoms have been omitted for clarity. The projection directions have been chosen to highlight the similarity of the core conformations
of the two trisaccharide chains.
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was the reason why more recent syntheses of similar oligosac-
charides utilized glycosyl donors containing 4-azido groups,1,17
unlike in the earlier works where oligosaccharides related to the
O-SP of Vibrio cholerae O1 were synthesized using glycosyl
donors where the tetronamido side chain was already in
place.18,19 Similarly, in the initial reaction of 21 with 8-azido-3,6-
dioxaoctanol (22) under Ogawa's14 conditions, the a and
b glycosides were formed in a ratio of ꢂ1.1 : 1. When we took
advantage of the glycosylation under thermodynamic control
developed in this laboratory,2,6 the stereoselectivity of the same
glycosylation increased by many folds (a : b ꢂ 7 : 1). Conversion
of oligosaccharides into conjugation-ready forms oen involves
a-mannosylation of very reactive aglycons. Such reactions are
characterized by poor stereo selectivity. Performing such reac-
tions under thermodynamic control remarkably increases the
stereo selectivity of such conversions, and constitutes a consid-
erable improvement of the synthesis of this and similar
Scheme 4 Synthesis of the hexasaccharide derivative 3.
is impaired when the glycosylation is conducted with donors
having the 3-deoxy-L-glycero-tetronic acid side chain already in
place.2,6,16 It is especially noteworthy that when we7,14 previously
glycosylated a fully assembled hexasaccharide Vibrio cholerae
O1 donor with a primary hydroxyl group-containing linker
molecule, the reaction showed almost no stereoselectivity. This
conjugation-ready oligosaccharides over existing protocols.
12
´
Conventional deacetylation (Zemplen ) of 2a, followed by
hydrogenation/hydrogenolysis (Pd/C) yielded the nal glycoside
1 (71%, over two steps). The structure of compound 1 was
conrmed by HRMS and NMR spectra.
Fig. 3 (a) Comparison of 13C-NMR spectra of compounds 3a (150 MHz, C6D6) and 3b (150 MHz, CDCl3); (b) 13C–1H coupled 2D-NMR spectrum
of compound 3a (C6D6); (c) 13C–1H coupled 2D-NMR spectrum of compound 3b (CDCl3).
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Scheme 5 Synthesis of the title hexasaccharide.
O1, serotype Inaba was improved. The number of linear
synthetic steps toward the compound was reduced, and the
stereoselectivity of the critical 1,2-trans-glycosylation of the
very reactive 8-azido-3,6-dioxaoctanol with the fully assem-
bled hexasaccharide trichloroacetimidate was markedly
increased, from a : b ¼ 1.1 : 1 to 7 : 1 thereby increasing
considerably the yield of the conjugation-ready title
compound manifold.
Conclusions
We have modied existing approaches leading to antigenic
determinants of Vibrio cholerae O1 and veried scalability of
reactions involved. Intermediates to oligosaccharides in this
series, up to and including the hexasaccharide have been
successfully prepared on multigram scales. Using such hex-
asaccharide, the synthesis of the terminal hexasaccharide
fragment of the O-specic polysaccharide of Vibrio cholerae
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1.4 Hz, H-1), 5.56 (2d, 1H, J2–3 ¼ 2.8, J2–1 ¼ 1.9 Hz, H-2), 4.80 (d, 1H,
J ¼ 11.2 Hz, CH2Ph), 4.59 (d, 1H, J ¼ 11.2 Hz, CH2Ph), 3.91 (dd, 1H,
Experimental
Materials and methods, crystallography
J3–4 ¼ 9.7 Hz, J3–2 ¼ 3.2 Hz, H-3), 3.67 (dq, 1H, J5–4 ¼ 10.1 Hz, J5–6
¼
6.0 Hz, H-5), 3.57 (2d, 1H, J4–3 ¼ 9.8 Hz, J4–5 ¼ 10.0 Hz, H-4), 2.12 (s,
3H, COCCH3), 1.39 (d, 3H, J6–5 ¼ 6.0 Hz, CH3). 13C{1H} NMR (100
MHz, CDCl3): d 168.4 (C]O), 165.4 (C]O), 136.9, 133.4, 129.9
(2C), 129.3, 128.5 (2C), 128.4 (2C), 128.2 (2C), 127.9, 91.1 (C-1), 75.8
(C-3), 71.6 (CH2Ph), 69.3 (C-2), 66.7 (C-5), 63.8 (C-4), 20.9 (COCH3),
18.7 (C-6). HRMS (ESI-TOF): m/z [M + Na]+ calcd for C22H23O6N3Na
448.1485, found 448.1488. Anal. calcd for C22H23O6N3: C, 62.11; H,
5.45; N, 9.88. Found C, 62.20; H, 5.40; N, 10.06.
Unless specied otherwise, all reagents and solvents were
purchased from Sigma-Aldrich Chemical Company and used as
supplied. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on silica gel 60 glass slides. Spots were visualized by
charring with H2SO4 in EtOH (5% v/v) and/or UV light. Melting
points were determined with a Koer hot stage. Optical rota-
tions were measured at ambient temperature with a Jasco P-
ꢁ
2000 digital polarimeter. NMR spectra were measured at 25 C
Continued elution gave the b-linked derivative 11b as
colorless syrup 11.3 g, 9.2%. Rf ¼ 0.43 (hexane/ethyl acetate, 4/
for solutions in benzene-d6, methanol-d4 or CDCl3, at 400 MHz,
500 MHz or 600 MHz for H, and at 100 MHz, 125 MHz or 150
1
1
MHz for 13C with Bruker Avance Spectrometers. Assignments of
NMR signals were aided by 1D and 2D experiments (1H–1H
homonuclear decoupling, APT, COSY, HSQC, TOCSY and
HMBC) run with the soware supplied with the spectrometer.
Chemical shis were referenced to that of tetramethylsilane (0
ppm) or signals of residual non-deuterated solvents. Crystals of
4 suitable for X-ray data collection were obtained by slow
evaporation of ethanol from ethanolic solution. X-ray intensity
measurements were collected at low temperature from a color-
less needle-shaped crystal using a Bruker Kappa APEX II 4K
CCD diffractometer with MoKa radiation. An Oxford Cry-
osystems 700 low temperature system was used to generate
a stream of cold N2 gas that cooled the sample crystal to 120(2) K
during data collection. Data were collected using both u and f
scans with a scan width of 0.50ꢁ per frame and a rate of 30 s per
frame, with the detector center located 40.0 mm from the crystal
at 2q ¼ 30.00ꢁ or 60.00ꢁ. The data were processed using the
Bruker APEX III soware package. The crystal structure was
solved and rened using the SHELXL20 soware package. The
absolute conguration (Flack) parameter, determined from the
X-ray data during the renement, correctly identied the
absolute conguration of the structure, which was also estab-
lished by the known conguration of the a-D-mannopyranoside
rings. The positions of H atoms attached to C and O atoms were
calculated using idealized sp2 or sp3 geometry and included as
riding atoms in the least-squares renement. For methyl and
–OH hydrogens, the torsion angles about the X-Me or C–OH
bond were also optimized during the renement. Details of the
crystal data and structure renement are given in ESI.† The 7 N
solution of NH3 in MeOH was purchased from Sigma-Aldrich.
Solutions in organic solvents were dried with anhydrous
1). [a]D ꢀ51.1 (c 1.0, CHCl3), lit17 [a]D ꢀ50.2 (c 1.2, CHCl3). H
NMR (400 MHz, CDCl3): d 8.11–8.09 (d, 2H, J ¼ 7.9 Hz, Ar–H),
7.59 (t, 1H, J ¼ 7.3 Hz, Ar–H), 7.49–7.43 (t, 2H, J ¼ 7.7 Hz, Ar–H),
7.36–7.24 (m, 5H, Ar–H), 5.82 (d, 1H, J1–2 ¼ 3.1 Hz, H-2), 5.77 (s,
1H, H-1), 4.80 (d, 1H, J ¼ 11.2 Hz, CH2Ph), 4.56 (d, 1H, J ¼
11.2 Hz, CH2Ph), 3.68 (dd, 1H, J3–4 ¼ 9.6 Hz, J3–2 ¼ 3.0 Hz, H-3),
3.53 (t, 1H, J ¼ 9.7 Hz, H-4), 3.39 (m, 1H, H-5), 2.04 (s, 3H,
COCCH3), 1.45 (d, 3H, J6–5 ¼ 6.1 Hz, CH3). 13C{1H} NMR (100
MHz, CDCl3): d 168.8 (C]O), 165.8 (C]O), 136.6, 133.4, 129.9
(2C), 129.5, 128.5 (2C), 128.4 (2C), 128.3 (2C), 128.1, 91.1 (C-1),
78.0 (C-3), 72.1 (C-2), 71.4 (CH2Ph), 66.8 (C-5), 63.6 (C-4), 20.7
(COCH3), 18.6 (C-6). HRMS (ESI-TOF): m/z [M + Na]+ calcd for
C
C
22H23O6N3Na 448.1485, found 448.1481. Anal. calcd for
22H23O6N3: C, 62.11; H, 5.45; N, 9.88. Found C, 62.21; H,
5.47; N, 10.01.
Ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-1-thio-a-D-
mannopyranoside (7a) and ethyl 4-azido-2-O-benzoyl-3-O-benzyl-
4,6-dideoxy-1-thio-b-D-mannopyranoside (7b)
To a stirred suspension of anomeric mixture of compounds 11a
˚
and 11b (115 g, 270.3 mmol) and powdered 4 A molecular sieves
(10.0 g) in dichloromethane (800 mL), ethanethiol (39 mL, 540.6
mmol) followed by boron triuoride etherate (100 mL, 811
mmol) were added dropwise at 0 ꢁC and the mixture was stirred
at room temperature overnight, when TLC (4 : 1 hexane–EtOAc)
showed that the reaction was complete. The mixture was
neutralized with NEt3 (113 mL, 811 mmol), ltered through
a Celite pad and the ltrate was concentrated with Chlorox in
the receiving ask, to give crude product. A solution of the
residue in DCM (300 mL) was washed with aq NaHCO3, and the
aqueous layer was backwashed with DCM (3ꢃ 100 mL).
Concentration of the organic phase and chromatography (10 : 1
hexane–EtOAc) gave rst the a-linked glycoside (7a, 97.3 g, 84%)
as colorless syrup. Rf ¼ 0.70 (hexane/ethyl acetate, 4/1). [a]D
+63.7 (c 1.3, CHCl3), lit11 [a]D +65 (c 1.45, CHCl3), 1H NMR (400
MHz, CDCl3): d 8.07–8.05 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.59 (app t,
ꢁ
MgSO4 and concentrated at reduced pressure at <40 C.
1-O-Acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranose (11a) and 1-O-acetyl-4-azido-2-O-benzoyl-3-O-
benzyl-4,6-dideoxy-b-D-mannopyranose (11b)
This compound was prepared from 115 g (289.4 mmol) of 10 as 1H, J ¼ 7.5, 7.3 Hz, Ar–H), 7.49–7.43 (t, 2H, J ¼ 7.7 Hz, Ar–H),
described,7 yielding 115.7 g (94%) of anomeric mixture of 11a and 7.36–7.24 (m, 5H, Ar–H), 5.62 (t, 1H, J ¼ 1.5 Hz, H-2), 5.34 (s, 1H,
11b (a/b ꢂ 9 : 1) as white solid. Rf ¼ 0.45 (hexane/ethyl acetate, 4/ H-1), 4.74 (d, 1H, J ¼ 11.3 Hz, CH2Ph), 4.56 (d, 1H, J ¼ 11.2 Hz,
1). Chromatography gave rst 11a (104.4 g, 85%). m.p. 107–108 ꢁC CH2Ph), 3.96 (dq, 1H, J5–4 ¼ 10.0 Hz, J5–6 ¼ 6.2 Hz, H-5), 3.84 (dd,
(EtOAc–hexane). [a]D +1.5 (c 1.0, CHCl3), lit17 [a]D +1.9 (c 1.9, CHCl3) 1H, J3–4 ¼ 10.1 Hz, J3–2 ¼ 3.2 Hz, H-3), 3.55 (t, 1H, J ¼ 9.9 Hz, H-
1
for amorphous material. H NMR (400 MHz, CDCl3): d 8.07–8.05 4), 2.71–2.56 (m, 2H, SCH2CH3), 1.38 (d, 3H, J6–5 ¼ 6.3 Hz, CH3),
(d, 2H, J ¼ 7.9 Hz, Ar–H), 7.59 (t, 1H, J ¼ 7.3 Hz, Ar–H), 7.49–7.43 (t, 1.29 (t, 3H, J ¼ 7.4 Hz, SCH2CH3). 13C{1H} NMR (100 MHz,
2H, J ¼ 7.4 Hz, Ar–H), 7.36–7.24 (m, 5H, Ar–H), 6.15 (d, 1H, J1–2
¼
CDCl3): d 165.6 (C]O), 137.0, 133.3, 129.9 (2C), 129.7, 128.43
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(2C), 128.4 (2C), 128.2 (2C), 127.9, 82.4 (C-1), 76.4 (C-3), 71.5
(CH2Ph), 69.8 (C-2), 67.5 (C-5), 64.6 (C-4), 25.7 (SCH2CH3), 18.6
(C-6), 14.9 (SCH2CH3). HRMS (ESI-TOF): m/z [M + Na]+ calcd for
2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranoside (8)7
This compound was prepared as described7 from 38 g (78.6
mmol) of 13a giving pure compound 8 as colorless syrup (28 g,
94%). Rf ¼ 0.47 (hexane/ethyl acetate, 4/1). [a]D +122.1 (c 1.6,
CHCl3), lit7 [a]D +121 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3):
d 7.41–7.30 (m, 5H, Ar–H), 4.82 (d, 1H, J1–2 ¼ 1.5 Hz, H-1), 4.71,
4.66 (2d, 1H each, 2J ¼ 11.3 Hz, CH2Ph), 3.95 (t, 1H, J ¼ 1.5 Hz,
H-2), 3.76 (dd, 1H, J ¼ 9.9, 6.2 Hz, OCH2CH2Si), 3.73 (dd, 1H, J3–4
¼ 9.7 Hz, J3–2 ¼ 3.5 Hz, H-3), 3.58–3.37 (m, 3H, OCH2CH2Si, H-
4,5), 2.39 (d, 1H, J ¼ 1.6 Hz, OH), 1.32 (d, 3H, J6–5 ¼ 6.2 Hz, CH3),
0.98–0.82 (m, 2H, OCH2CH2Si), 0.01 [s, 9H, (CH3)3Si]. 13C{1H}
NMR (100 MHz, CDCl3): d 137.3, 128.6, 128.2 (2C), 128.1 (2C),
98.5 (C-1), 78.4 (C-3), 72.0 (CH2Ph), 67.4 (C-2), 66.5 (C-5), 65.2
C
22H25O4N3SNa 450.1463, found 450.1459.
Continued elution gave the b-linked glycoside 7b as colorless
syrup (2.1 g, 1.8%, Total 99.4 g, 86% overall, a/b ꢂ 46 : 1). Rf ¼
0.69 (hexane/ethyl acetate, 4/1). Data for 7b, [a]D ꢀ97.2 (c 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3): d 8.13–8.09 (d, 2H, J ¼
7.8 Hz, Ar–H), 7.57 (app t, 1H, J ¼ 7.5, 7.4 Hz, Ar–H), 7.47–7.42 (t,
2H, J ¼ 7.7 Hz, Ar–H), 7.38–7.25 (m, 5H, Ar–H), 5.86 (d, 1H, J2–3
¼ 3.2 Hz, H-2), 4.86 (d, 1H, J ¼ 11.2 Hz, CH2Ph), 4.70 (s, 1H, H-1),
4.56 (d, 1H, J ¼ 11.8 Hz, CH2Ph), 3.61 (dd, 1H, J3–4 ¼ 9.9 Hz, J3–2
¼ 3.3 Hz, H-3), 3.49 (t, 1H, J ¼ 9.5 Hz, H-4), 3.61 (dq, 1H, J5–4
¼
9.7 Hz, J5–6 ¼ 6.2 Hz, H-5), 2.77–2.68 (m, 2H, SCH2CH3), 1.45 (d,
3H, J6–5 ¼ 6.3 Hz, CH3), 1.30–1.24 (t, 3H, J ¼ 7.3 Hz, SCH2CH3).
13C{1H} NMR (125 MHz, CDCl3): d 165.8 (C]O), 136.9, 133.3
(2C), 130.1, 129.4 (2C), 128.42 (2C), 128.39 (2C), 128.37, 127.9,
82.2 (C-1), 79.3 (C-3), 75.4 (C-5), 71.4 (CH2Ph), 69.2 (C-2), 63.9 (C-
4), 25.6 (SCH2CH3), 18.9 (C-6), 14.8 (SCH2CH3). HRMS (ESI-
TOF): m/z [M + Na]+ calcd for C22H25O4N3SNa 450.1463, found
450.1461. Anal. calcd for C22H25O4N3S: C, 61.81; H, 5.89; N, 9.83.
Found C, 61.93; H, 5.89; N, 9.58.
(OCH2Si), 64.1 (C-4), 18.4 (CH3), 17.9 (CH2Si), ꢀ1.34 [3C,
+
(CH3)3Si]. HRMS (ESI-TOF): m/z [M
+
NH4 ] calcd for
C
18H33O4N4Si 397.2271, found 397.2275.
2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1/2)-4-azido-3-O-benzyl-
4,6-dideoxy-a-D-mannopyranoside (14)
To a solution of the glycosyl acceptor 8 (26 g, 68.5 mmol) and
thioglycoside donor 7a (32.2 g, 75.3 mmol) in dry DCM (500 mL)
2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside (13a)11
˚
was added 10 g of 4 A powdered molecular sieves, and the
mixture was stirred for 15 min. N-Iodosuccinimide (22.1 g, 90.4
Method 1. Attempted use of anomeric acetates 11a and 11b mmol) was added, which resulted in slight pink color develop-
ment. The stirring was continued for another 5 min, the reac-
(a/b ꢂ 9 : 1), prepared as described above, as glycosyl donors.
When compounds 11a and 11b (0.25 g, 0.6 mmol) were tion mixture was cooled at 0 ꢁC, and TMSOTf (5.3 mL, 29.4
treated with 2-(trimethylsilyl) ethanol (0.25 mL, 1.8 mmol) in mmol) was added dropwise. Red color developed immediately
presence of either boron triuoride etherate (0.12 mL, 0.9 and, aer 20 min, when TLC (solvent 1, Rf ¼ 0.72 at 4 : 1
mmol) or TMSOTf (0.1 mL, 0.6 mmol) as described above for hexane–EtOAc. Solvent 2, Rf ¼ 0.26 at neat toluene) in both
the synthesis of thioglycosides 7a and 7b, TLC showed that solvents showed complet conversion, the reaction was
a complex mixture was formed where the product of hydro- quenched by addition of triethylamine (4 mL, 29.4 mmol). The
lysis of the anomeric OAc group largely predominated. Opti- precipitate formed was ltered off (a pad of Celite) directly into
mization of reaction conditions for this approach was not a separating funnel containing excess of 2 : 1 (v/v) sodium
attempted.
thiosulfate (10%)-sodium bicarbonate (saturated) solution. The
Method 2. From anomeric mixture of compounds 7a and 7b. organic layer was extracted with DCM (3ꢃ 50 mL), dried and
This compound was prepared as described11 from 36 g (84.2 concentrated. The residue was chromatographed (10 : 1
mmol) of anomeric mixture of 7a and 7b resulting in 38.3 g hexane–EtOAc) and title compound 14 was obtained as colorless
(94%) of pure compound 13a as white solid. Rf ¼ 0.45 (hexane/ syrup (47.9 g, 94%), [a]D +23.8 (c 1.0, CHCl3). 1H NMR (600 MHz,
ethyl acetate, 9/1). Mp. 75–76 ꢁC (hexane), lit11 mp. 73–75 ꢁC. CDCl3): d 8.05–8.03 (d, 2H, J ¼ 7.5 Hz, Ar–H), 7.58 (t, 1H, J ¼
[a]D ꢀ0.6 (c 1.0, CHCl3), lit11 [a]D ꢀ1 (c 1, CHCl3). 1H NMR (400 7.5 Hz, Ar–H), 7.48–7.45 (t, 2H, J ¼ 7.5 Hz, Ar–H), 7.38–7.33 (m,
MHz, CDCl3): d 8.06–8.05 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.57 (app t, 4H, ArH), 7.31–7.24 (m, 5H, ArH), 7.17 (t, 1H, J ¼ 7.5 Hz, Ar–H),
1H, J ¼ 7.5, 7.2 Hz, Ar–H), 7.47–7.43 (m, 2H, Ar–H), 7.34–7.24 5.62 (t, 1H, J ¼ 2.5 Hz, H-2II), 4.94 (d, 1H, J1–2 ¼ 1.6 Hz, H-1II),
(m, 5H, Ar–H), 5.52 (s, 1H, H-2), 4.89 (s, 1H, H-1), 4.76 (d, 1H, J ¼ 4.78 (d, 1H, J ¼ 11.2 Hz, CH2Ph), 4.71 (d, 1H, J1–2 ¼ 1.4 Hz, H-1I),
11.7 Hz, CH2Ph), 4.56 (d, 1H, J ¼ 11.3 Hz, CH2Ph), 3.93 (dd, 1H, 4.69 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.61 (d, 1H, J ¼ 11.5 Hz,
J3–4 ¼ 9.8 Hz, J3–2 ¼ 3.0 Hz, H-3), 3.78 (ddd, 1H, J ¼ 6.8, 6.6, CH2Ph), 4.57 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 3.90 (dd, 1H, J3–4
¼
6.4 Hz, OCH2CH2Si), 3.63 (ddd, 1H, J ¼ 6.0, 6.2, 4.1 Hz, OCH2- 9.8 Hz, J3–2 ¼ 3.1 Hz, H-3II), 3.83 (t, 1H, J ¼ 2.2 Hz, H-2I), 3.75
CH2Si), 3.56–3.47 (m, 2H, H-4,5), 1.37 (d, 3H, J6–5 ¼ 6.5 Hz, CH3), (dd, 1H, J3–4 ¼ 9.8 Hz, J3–2 ¼ 2.9 Hz, H-3I), 3.71 (m, 1H, H-5I),
1.01–0.87 (m, 2H, OCH2CH2Si), 0.28 (s, 9H, (CH3)3Si). 13C{1H} 3.66 (m, 1H, OCHaCH2Si), 3.52–3.47 (m, 2H, H-4II, H-5II), 3.43
NMR (100 MHz, CDCl3): d 165.9 (C]O), 137.5, 133.3, 129.9 (2C), (m, 1H, OCHbCH2Si), 3.33 (t, 1H, J ¼ 9.9 Hz, H-4I), 1.34 (d, 3H,
129.7, 128.4 (2C), 128.3 (2C), 128.2 (2C), 127.8, 97.3 (C-1), 76.2 J6–5 ¼ 6.2 Hz, CH3, H-6I), 1.29 (d, 3H, J6–5 ¼ 6.3 Hz, CH3, H-6II),
(C-3), 71.4 (CH2Ph), 68.1 (C-2), 66.9 (C-5), 65.5 (OCH2Si), 64.4 (C- 0.94–0.82 (m, 2H, CH2CH2Si), 0.01 [s, 9H, (CH3)3Si]. 13C{1H}
4), 18.7 (CH3), 17.9 (CH2Si), ꢀ1.33 [3C, (CH3)3Si]. HRMS (ESI- NMR (150 MHz, CDCl3): d 165.3 (C]O), 137.6, 137.1, 133.2,
TOF): m/z [M + Na]+ calcd for C25H33O5N3SiNa 506.2087, 129.9 (2C), 129.7, 128.5 (2C), 128.4 (2C), 128.3 (2C), 127.94 (2C),
found 506.2089.
127.90 (2C), 127.8 (2C), 99.5 (C-1II, JC-1,H-1 ¼ 173.3 Hz), 98.2 (C-1I,
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JC-1,H-1 ¼ 167.7 Hz), 77.8 (C-3I), 75.3 (C-3II), 74.4 (C-2I), 72.1 7.20–7.15 (m, 2H, Ar–H), 5.59 (dd, 1H, J2–3 ¼ 2.9 Hz, J2–1
¼
¼
(CH2Ph), 71.4 (CH2Ph), 67.7 (C-2II), 67.6 (C-5I), 66.9 (C-5II), 65.2 2.0 Hz, H-2III), 4.97 (d, 1H, J1–2 ¼ 1.6 Hz, H-1II), 4.89 (d, 1H, J1–2
(OCH2CH2Si), 64.2 (C-4I), 64.1 (C-4II), 18.7 (CH3, C-6I), 18.6 (CH3, 1.6 Hz, H-1III), 4.76 (d, 1H, J ¼ 11.0 Hz, CH2Ph), 4.71 (d, 1H, J ¼
C-6II), 17.5 (CH2Si), ꢀ1.32 [3C, (CH3)3Si]. HRMS (ESI-TOF): m/z 11.7 Hz, CH2Ph), 4.65 (d, 1H, J1–2 ¼ 1.6 Hz, H-1I), 4.63 (d, 1H, J ¼
+
[M + NH4 ] calcd for C38H52O8N7Si 762.3647, found 762.3637. 11.6 Hz, CH2Ph), 4.60–4.55 (3d, 3H, J ¼ 11.5 Hz, 11.4 Hz,
Anal. calcd for C38H48O8N6Si: C, 61.27; H, 6.5; N, 11.28. Found 11.3 Hz, 3CH2Ph), 3.88–3.85 (m, 2H, H-2II, H-3III), 3.82 (t, 1H, J ¼
C, 61.54; H, 6.36; N, 11.09.
2.3 Hz, H-2I), 3.73 (dd, 1H, J3–4 ¼ 9.9 Hz, J3–2 ¼ 2.9 Hz, H-3II),
3.73–3.68 (m, 2H, H-3I, OCHaCH2Si), 3.60–3.51 (m, 2H, H-5III, H-
5II), 3.49–3.40 (m, 3H, H-4III, H-5I, OCHbCH2Si), 3.35 (t, 1H, J ¼
10.0 Hz, H-4II), 3.22 (t, 1H, J ¼ 9.9 Hz, H-4I), 1.28, 1.27 (over-
lapped 2d, 3H each, J6–5 ¼ 6.1 Hz, CH3, H-6II, H-6III), 1.23 (d, 3H,
J6–5 ¼ 6.1 Hz, CH3, H-6I), 0.93–0.82 (m, 2H, CH2CH2Si), 0.01 [s,
9H, (CH3)3Si]. 13C{1H} NMR (150 MHz, CDCl3): d 165.3 (C]O),
137.4, 137.3, 137.1, 133.3, 129.9, 129.8 (2C), 128.6, 128.5 (2C),
128.46 (2C), 128.45 (2C), 128.4 (2C), 128.3 (2C), 128.1 (2C), 128.0
2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranosyl-(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranoside (6)7
This compound was prepared as described7 from 47.5 g (63.8
mmol) of 14, to give 38.4 g (94%) of pure compound 6 as
colorless syrup. Rf ¼ 0.41 at hexane/ethyl acetate, 4/1. [a]D
+101.7 (c 1.1, CHCl3), lit7 [a]D +102 (c 1.0, CHCl3). 1H NMR (500
(2C), 127.9 (2C), 100.4 (C-1II, JC–H ¼ 173.7 Hz), 99.2 (C-1III, JC–H
¼
MHz, CDCl3): d 7.41–7.29 (m, 10H, Ar–H), 4.94 (d, 1H, J1–2
¼
173.3 Hz), 98.3 (C-1I, JC–H ¼ 168.6 Hz), 77.7 (C-3I), 76.7 (C-3II),
75.3 (C-3III), 74.1 (C-2II), 74.0 (C-2I), 72.1 (2C, 2 CH2Ph), 71.4
(CH2Ph), 67.7 (C-5II), 67.64 (C-5III), 67.6 (C-2III), 67.0 (C-5I), 65.2
(OCH2CH2Si), 64.5 (C-4I), 64.1 (C-4III), 64.0 (C-4II), 18.7 (CH3, C-
1.4 Hz, H-1II), 4.72, 4.68, 4.64, 4.61 (partially overlapped 4d, 4H,
2J ꢂ 11.5 Hz, 2 CH2Ph), 4.66 (partially overlapped d, 1H, J1–2
¼
4.4 Hz, H-1I), 3.99 (m, 1H, H-2II), 3.88 (t, 1H, J ¼ 2.5 Hz, H-2I),
3.75–3.68 (m, 3H, H-3I,II, OCHaCH2Si), 3.61 (m, 1H, H-5I), 3.51–
3.40 (m, 3H, H-4I, H-5II, OCHbCH2Si), 3.29 (t, 1H, J ¼ 10.0 Hz, H-
4II), 2.29 (d, 1H, J ¼ 1.7 Hz, OH), 1.30, 1.29 (overlapped 2d, 6H,
J6–5 ¼ 6.2 Hz, CH3, H-6I,II), 0.94–0.82 (m, 2H, CH2CH2Si), 0.01 [s,
9H, (CH3)3Si]. 13C{1H} NMR (125 MHz, CDCl3): d 137.4, 137.1,
128.6 (2C), 128.5 (2C), 128.3 (2C), 128.2 (2C), 127.9 (2C), 100.7
(C-1II), 98.3 (C-1I), 77.9 (C-3I), 77.6 (C-3II), 73.8 (C-2I), 72.1 (2C,
CH2Ph), 67.2 (C-2II), 67.1 (C-5I), 66.9 (C-5II), 65.2 (OCH2Si), 64.3
(C-4II), 63.8 (C-4I), 18.6 (CH3), 18.4 (CH3), 17.7 (CH2Si), ꢀ1.32
6I), 18.6 (CH3, C-6II), 18.58 (CH3, C-6III), 17.8 (CH2Si), ꢀ1.32 [3C,
+
(CH3)3Si]. HRMS (ESI-TOF): m/z [M
+
NH4 ] calcd for
C
C
51H67O11N10Si 1023.4760, found 1023.4742. Anal. calcd for
51H63O11N9Si: C, 60.88; H, 6.31; N, 12.53. Found C, 61.17; H,
6.05; N, 12.32.
2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranosyl-(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranosyl-(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-
a-D-mannopyranoside (4)
+
[3C, (CH3)3Si]. HRMS (ESI-TOF): m/z [M + NH4 ] calcd for
C
31H48O7N7Si 658.3384, found 658.3389.
To the solution of trisaccharide 15 (23 g, 22.8 mmol) in
a mixture of dry DCM (50 mL) and dry MeOH (50 mL), meth-
anolic NaOMe (1 M, 10 mL) was added under Ar, and the
mixture was stirred at room temperature overnight, when TLC
(Rf ¼ 0.54 at 4 : 1 hexane : EtOAc) showed that the reaction was
2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1/2)-4-azido-3-O-benzyl-
4,6-dideoxy-a-D-mannopyranosyl-(1/2)-4-azido-3-O-benzyl-
4,6-dideoxy-a-D-mannopyranoside (15)
To a solution of disaccharide acceptor 6 (38 g, 59.3 mmol) and complete and that a much slower moving product was formed.
thioglycoside donor 7a (28 g, 65.2 mmol) in dry DCM (500 mL) The mixture was neutralized with Dowex 50W resin, ltered,
˚
was added 10 g of 4 A powdered molecular sieves, and the and the solvent was removed. The crude product was passed
mixture was stirred for 15 min. N-Iodosuccinimide (19.2 g, 78.2 through a short pad of silica and elution with 5 : 1 hexane–EA
mmol) was added, which resulted in slight pink color develop- afforded pure 4 as white solid. Crystallization from hot hexane
ꢁ
ment. The stirring was continued for another 5 min, and the gave needles (19.4 g, 94%), mp. 72–73 C, [a]D +102.86 (c 1.0,
reaction mixture was cooled at 0 ꢁC. TMSOTf (4.7 mL, 26.1 CHCl3). 1H NMR (600 MHz, CDCl3): d 7.40–7.29 (m, 15H, Ar–H),
mmol) was added, whereupon red color developed immediately. 4.96 (d, 1H, J1–2 ¼ 1.3 Hz, H-1III), 4.95 (d, 1H, J1–2 ¼ 1.6 Hz, H-1II),
Aer 20 min at 0 ꢁC, when TLC in 2 solvents (1. Rf ¼ 0.72 at 4 : 1 4.72 (d, 1H, J ¼ 11.4 Hz, CH2Ph), 4.69–4.64 (m, 3H, 2 CH2Ph, H-
hexane–EtOAc; 2. Rf ¼ 0.32 at neat toluene) showed that the 1I), 4.62 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.61 (d, 1H, J ¼ 11.6 Hz,
reaction was complete, the reaction was terminated by addition CH2Ph), 4.57 (d, 1H, J ¼ 11.6 Hz, CH2Ph), 3.98 (ddd, 1H, J2–3
¼
of triethylamine (3.7 mL). The precipitate formed was ltered 2.8 Hz, J2-OH-2 ¼ 1.7 Hz, J2–1 ¼ 1.3 Hz, H-2III), 3.93 (t, 1H, J ¼
through a pad of Celite directly into a separating funnel con- 2.2 Hz, H-2II), 3.81 (t, 1H, J ¼ 2.3 Hz, H-2I), 3.74–3.71 (m, 1H, H-
taining excess of 2 : 1 (v/v) sodium thiosulfate (10%)–sodium 3II), 3.71–3.67 (m, 3H, H-3III, H-3I, OCHaCH2Si), 3.56–3.51 (m,
bicarbonate (saturated) solution. The mixture was extracted 1H, H-5II), 3.51–3.47 (m, 1H, H-5III), 3.47–3.38 (m, 3H, H-5I,
with DCM (3ꢃ 50 mL), the combined organic layers were dried, OCHbCH2Si, H-4III), 3.32 (t, 1H, J ¼ 10.0 Hz, H-4II), 3.22 (t, 1H, J
concentrated, and chromatography (12 : 1 hexane : EtOAc) gave ¼ 9.9 Hz, H-4I), 2.27 (d, 1H, J2-OH-2 ¼ 1.8 Hz, OH), 1.28 (2d, 3H
product 15 as colorless syrup (55.8 g, 93%). [a]D +34.0 (c 1.0, each, J6–5 ¼ 6.1 Hz, CH3, H-6II, H-6I), 1.18 (d, 3H, J6–5 ¼ 6.1 Hz,
CHCl3). 1H NMR (600 MHz, CDCl3): d 8.07–8.05 (d, 2H, J ¼ CH3, H-6III), 0.92–0.83 (m, 2H, CH2CH2Si), 0.01 [s, 9H, (CH3)3Si].
7.3 Hz, Ar–H), 7.61 (t, 1H, J ¼ 7.4 Hz, Ar–H), 7.50–7.47 (t, 2H, J ¼ 13C{1H} NMR (150 MHz, CDCl3): d 137.31, 137.26, 137.1, 128.6
7.7 Hz, Ar–H), 7.38–7.37 (m, 2H, ArH), 7.34–7.25 (m, 11H, ArH), (2C), 128.57, 128.3 (2C), 128.26 (2C), 128.21 (2C), 128.17 (2C),
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128.1 (2C), 128.0 (2C), 100.42 (C-1II), 100.4 (C-1III), 98.3 (C-1I), material and formation of a faster moving product. The mixture
77.6 (C-3II), 77.5 (C-3I), 76.9 (C-3III), 73.9 (C-2II), 73.2 (C-2I), 72.2 was concentrated to a small volume and applied onto a short
(2C, 2 CH2Ph), 72.1 (CH2Ph), 67.7 (C-5II), 67.3 (C-5III), 67.1 (C- column of silica gel. Elution with 10 : 1 hexane–EtOAc gave
2III), 67.0 (C-5I), 65.2 (OCH2CH2Si), 64.4 (C-4I), 64.2 (C-4II), 63.8 trichloroacetimidate donor 5 as colorless syrup (24.8 g, 91%).
(C-4III), 18.64 (CH3, C-6I), 18.56 (CH3, C-6III), 18.3 (CH3, C-6II), 1H NMR for the major isomer (600 MHz, CDCl3): d 8.59 (s, 1H,
17.7 (CH2Si), ꢀ1.32 [3C, (CH3)3Si]. HRMS (ESI-TOF): m/z [M + NH), 8.07–8.06 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.61 (t, 1H, J ¼ 7.5 Hz,
+
NH4 ] calcd for C44H63O10N10Si 919.4498; found 919.4483. Anal. Ar–H), 7.50–7.47 (t, 2H, J ¼ 7.6 Hz, Ar–H), 7.41–7.39 (m, 2H, Ar–
calcd for C44H59O10N9Si: C, 58.58; H, 6.59; N, 13.97. Found C, H), 7.36–7.21 (m, 13H, Ar–H), 6.07 (s, 1H, H-1I), 5.61 (s, 1H, H-
58.77; H, 6.44; N, 13.77.
2III), 4.99 (s, 1H, H-1II), 4.98 (s, 1H, H-1III), 4.78 (d, 1H, J ¼
11.5 Hz, CH2Ph), 4.75 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.67–4.56 (m,
4H, 4CH2Ph), 3.93–3.86 (m, 3H, H-2I, H-2II, H-3III), 3.75–3.71 (m,
2H, H-3I, H-3II), 3.66–3.55 (m, 3H, H-5I, H-5II, H-5III), 3.49 (t, 1H, J
¼ 9.9 Hz, H-4III), 3.38 (t, 1H, J ¼ 10.0 Hz, H-4II), 3.33 (t, 1H, J ¼
10.0 Hz, H-4I), 1.32 (d, 3H, J6–5 ¼ 6.3 Hz, CH3, H-6II), 1.30 (d, 3H,
J6–5 ¼ 6.1 Hz, CH3, H-6I), 1.28 (d, 3H, J6–5 ¼ 6.2 Hz, CH3, H-6III).
13C{1H} NMR (150 MHz, CDCl3): d 165.3 (C]O), 159.9 (C]O),
137.3, 137.1, 136.8, 133.3, 129.9 (2C), 129.7, 128.6 (2C), 128.5
(2C), 128.44 (2C), 128.42 (2C), 128.4 (2C), 128.3 (2C), 128.2 (2C),
128.1 (2C), 127.9, 100.4 (C-1II), 99.2 (C-1III), 96.2 (C-1I), 76.7 (C-
3I), 76.5 (C-3II), 75.3 (C-3III), 73.8 (C-2I), 72.5 (CH2Ph), 72.2
(CH2Ph), 71.9 (C-2II), 71.4 (CH2Ph), 70.1 (C-5II), 68.1 (C-5III), 67.7
(C-2III), 67.6 (C-5I), 64.0 (C-4I), 63.9 (C-4III), 63.7 (C-4II), 18.64
(CH3, C-6I), 18.62 (CH3, C-6II), 18.5 (CH3, C-6III). HRMS (ESI-
TOF): m/z [M + 18]+ calcd for C48H51O11N10Cl3 1048.2804;
found 1048.2810.
4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-D-mannopyranose (16)
A solution of glycoside 16 (30 g, 30 mmol) in triuoroacetic acid
(TFA, 120 mL) was stirred at room temperature for 2 hours,
when TLC (4 : 1 hexane–EtOAc) showed that the reaction was
complete and that a much slower moving product was formed.
The mixture was concentrated and a solution of the residue in
DCM was washed with saturated Na2CO3 solution. The aqueous
layer was extracted with DCM (3ꢃ 50 mL), and the combined
organic phase was dried and concentrated. The residue was
chromatographed (Rf ¼ 0.27 at 4 : 1 hexane–EtOAc) to give pure
1
product 16 as white foam (23.9 g, 89%). H NMR for the major
a anomer (600 MHz, CDCl3): d 8.07–8.06 (d, 2H, J ¼ 7.3 Hz, Ar–
H), 7.61 (t, 1H, J ¼ 7.5 Hz, Ar–H), 7.50–7.47 (t, 2H, J ¼ 7.7 Hz, Ar–
H), 7.39–7.27 (m, 13H, Ar–H), 7.20–7.16 (m, 2H, Ar–H), 5.59 (dd,
2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranosyl-(1/2)-[4-azido-3-O-benzyl-
4,6-dideoxy-a-D-mannopyranosyl-(1/2)]4-4-azido-3-O-benzyl-4,6-
dideoxy-a-D-mannopyranoside (3a) and 2-(Trimethylsilyl)ethyl 4-
azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-b-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranoside
(3b)
1H, J2–3 ¼ 2.9 Hz, J2–1 ¼ 2.0 Hz, H-2III), 5.08 (dd, 1H, J1-1-OH
¼
3.2 Hz, J1–2 ¼ 1.8 Hz, H-1I), 4.98 (d, 1H, J1–2 ¼ 1.6 Hz, H-1II), 4.89
(d, 1H, J1–2 ¼ 1.6 Hz, H-1III), 4.77 (d, 1H, J ¼ 11.6 Hz, CH2Ph),
4.71 (d, 1H, J ¼ 11.7 Hz, CH2Ph), 4.65–4.55 (m, 4H, 4CH2Ph),
3.89–3.84 (m, 3H, H-2I, H-2II, H-3III), 3.75 (dd, 1H, J3–4 ¼ 10.0 Hz,
J3–2 ¼ 2.8 Hz, H-3I), 3.73 (dd, 1H, J3–4 ¼ 10.0 Hz, J3–2 ¼ 2.9 Hz, H-
3II), 3.68 (m, 1H, H-5I), 3.58 (m, 1H, H-5III), 3.52 (m, 1H, H-5II),
3.47 (t, 1H, J ¼ 10.0 Hz, H-4III), 3.35 (t, 1H, J ¼ 10.0 Hz, H-4II),
3.22 (t, 1H, J ¼ 10.0 Hz, H-4I), 2.54 (d, 1H, J1-OH-1 ¼ 3.5 Hz, OH),
1.28, 1.27 (overlapped 2d, 3H each, J6–5 ¼ 6.1 Hz each, CH3, H-
6II, H-6I), 1.23 (d, 3H, J6–5 ¼ 6.1 Hz, CH3, H-6III). 13C{1H} NMR
(150 MHz, CDCl3): d 165.3 (C]O), 137.4, 137.3, 137.1, 133.3,
129.9, 129.7, 128.52, 128.5 (2C), 128.44 (2C), 128.43, 128.4 (2C),
128.3 (2C), 128.2 (2C), 128.1 (2C), 128.04 (2C), 128.0, 127.9, 100.3
(C-1II), 99.2 (C-1III), 93.5 (C-1I), 77.0 (C-3I), 76.6 (C-3II), 75.3 (C-
3III), 74.0 (C-2II), 73.9 (C-2I), 72.2 (CH2Ph), 72.0 (CH2Ph), 71.3
(CH2Ph), 67.7 (C-5II), 67.63 (C-5III), 67.6 (C-2III), 67.2 (C-5I), 64.4
(C-4I), 64.1 (C-4III), 64.0 (C-4II), 18.7 (CH3, C-6I), 18.6 (CH3, C-6II),
18.5 (CH3, C-6III). HRMS (ESI-TOF): m/z [M + NH4+] calcd for
To a solution of the trisaccharide acceptor 4 (19 g, 21 mmol) and
trisaccharide trichloroacetimidate donor 5 (24.3 g, 23.2 mmol)
˚
in dry DCM (300 mL) was added 5 g of 4 A powdered molecular
sieves, and the mixture was stirred for 15 min. The mixture was
cooled to ꢀ40 ꢁC, TMSOTf (1.7 mL, 9.3 mmol) was added
dropwise at the rate of 0.1 mL minꢀ1 over a period of 17 min
using a syringe pump. Aer additional 30 min at ꢀ40 ꢁC TLC in
two solvents (1. Rf ¼ 0.74 at 4 : 1 hexane–EtOAc and 2. Rf ¼ 0.40
at neat toluene) showed that the glycosyl acceptor was
completely consumed. Two faster moving spots were formed,
one of which strongly predominated, indicating formation of
anomeric hexasaccharides. The mixture was neutralized with
NEt3 (1.3 mL, 9.3 mmol), ltered through Celite pad and ltrate
combined with the washings were concentrated. The residue
was chromatographed (8 : 1 hexane–EtOAc) to give rst the a-
C
46H55O11N10 923.4052; found 923.4054.
4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-4-azido-3-O-benzyl-4,6-dideoxy-D-mannopyranosyl
trichloroacetimidate (5)
1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU, 1.95 mL, 13 mmol) linked hexasaccharide (3a, 33.72 g, 90%) as colorless syrup, [a]D
1
was added at 0 ꢁC to a solution of hemiacetal 16 (23.5 g, 26 +73.0 (c 2.0, CHCl3). H NMR (600 MHz, C6D6): d 8.20–8.19 (d,
mmol) and trichloroacetonitrile (3.9 mL, 39 mmol) in dry DCM 2H, J ¼ 7.3 Hz, Ar–H), 7.43–7.38 (d, 2H, J ¼ 7.4 Hz, Ar–H), 7.38–
(100 mL). The mixture was stirred for 2 h, when TLC (Rf ¼ 0.57 at 7.31 (m, 10H, Ar–H), 7.31–7.19 (m, 11H, Ar–H), 7.19–7.08 (m,
4 : 1 hexane–EtOAc) showed complete consumption of starting 5H, Ar–H), 7.06–7.01 (m, 5H, Ar–H), 5.93 (dd, 1H, J2–3 ¼ 2.9 Hz,
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J2–1 ¼ 1.9 Hz, H-2VI), 5.32 (d, 1H, J ¼ 1.3 Hz, H-1V), 5.25 (d, 1H, J 1H, J3–4 ¼ 9.5 Hz, J3–2 ¼ 3.0 Hz, H-3VI), 3.81 (dd, 1H, J3–4
¼
¼ 1.1 Hz, H-1IV), 2.23 (d, 1H, J ¼ 1.2 Hz, H-1III), 5.21 (d, 1H, J ¼ 11.3 Hz, J3–2 ¼ 3.0 Hz, H-3V), 3.77–3.69 (m, 4H, OCHaCH2Si,
1.2 Hz, H-1II), 5.16 (d, 1H, J ¼ 1.3 Hz, H-1VI), 4.89 (d, 1H, J ¼ H-1IV, H-3II, H-3I), 3.68 (dd, 1H, J3–4 ¼ 10.1 Hz, J3–2 ¼ 3.4 Hz,
1.3 Hz, H-1I), 4.59 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.49–4.43 (dd, 2H, H-3III), 3.63 (t, 1H, J ¼ 10.0 Hz, H-4III), 3.54 (m, 1H, H-5II),
I ¼ 16.6, 11.5 Hz, CH2Ph), 4.42–4.33 (m, 7H, CH2Ph), 4.31 (d, 1H, 3.51–3.36 (m, 6H, OCHbCH2Si, H-4VI, H-4V, H-5VI, H-5III, H-
J ¼ 11.8 Hz, CH2Ph), 4.30 (d, 1H, J ¼ 11.3 Hz, CH2Ph), 4.15 (t, 1H, 5I), 3.34 (t, 1H, J ¼ 10.0 Hz, H-4II), 3.33 (t, 1H, J ¼ 10.0 Hz, H-
J ¼ 2.4 Hz, H-2IV), 4.12 (dd, 1H, J3–4 ¼ 10.0 Hz, J3–2 ¼ 3.2 Hz, H- 4I), 3.29 (t, 1H, J ¼ 9.9 Hz, H-4IV), 3.00 (dd, 1H, J3–4 ¼ 9.8 Hz,
3VI), 4.10 (t, 1H, J ¼ 2.2 Hz, H-2III), 4.09 (t, 1H, J ¼ 2.4 Hz, H-2II), J3–2 ¼ 2.3 Hz, H-3IV), 2.46 (dq, 1H, J5–4 ¼ 3.8 Hz, J5–6 ¼ 6.1 Hz,
4.05 (t, 1H, J ¼ 2.3 Hz, H-2V), 3.98 (t, 1H, J ¼ 2.4 Hz, H-2I), 3.96– H-5IV), 1.34 (d, 3H, J6–5 ¼ 6.1 Hz, CH3, H-6V), 1.31 (d, 3H, J6–5
3.88 (m, 6H, H-5VI, H-3V, H-3IV, H-3III H-3II, H-3I), 3.80 (m, 1H, H- ¼ 6.5 Hz, CH3, H-6I), 1.29 (d, 3H, J6–5 ¼ 6.5 Hz, CH3, H-6II),
5V), 3.77–3.68 (m, 5H, OCHbCH2Si, H-4VI, H-5IV, H-5III, H-5I), 1.22 (d, 3H, J6–5 ¼ 6.1 Hz, CH3, H-6IV), 1.17 (d, 3H, J6–5
¼
3.67–3.61 (m, 2H, H-5II, H-4I), 3.60–3.51 (m, 4H, H4V, H-4IV, H- 6.2 Hz, CH3, H-6III), 1.12 (d, 3H, J6–5 ¼ 6.0 Hz, CH3, H-6VI),
4III, H-4II), 3.34 (m, 1H, OCHbCH2Si), 1.33 (d, 3H, J6–5 ¼ 6.4 Hz, 0.95–0.83 (m, 2H, CH2CH2Si), 0.01 (s, 9H, (CH3)3Si). 13C{1H}
CH3, H-6II), 1.32 (d, 3H, J6–5 ¼ 6.5 Hz, CH3, H-6V), 1.30 (2d, 6H, J NMR (150 MHz, CDCl3): d 165.4 (C]O), 138.2, 137.9, 137.4,
¼ 6.1 Hz, H-6VI, H-6I), 1.28 (d, 3H, J6–5 ¼ 6.5 Hz, CH3, H-6IV), 1.26 137.2, 137.1, 136.9, 133.1, 129.9 (2C), 129.8, 128.7 (2C), 128.6
(d, 3H, J6–5 ¼ 6.0 Hz, CH3, H-6III), 0.85–0.76 (m, 2H, CH2CH2Si), (2C), 128.5 (2C), 128.45 (2C), 124.43 (3C), 128.1 (3C), 128.06
0.06 (s, 9H, (CH3)3Si). 13C{1H} NMR (150 MHz, C6D6): d 165.9 (3C), 128.03 (3C), 128.0 (3C), 127.8 (3C), 127.7 (3C), 127.6
(C]O), 138.3, 138.1, 137.9, 137.89, 137.88 (2C), 133.7, 130.7, (2C), 127.3, 100.1 (C-1II, JC-1,H-1 ¼ 170.5 Hz), 99.4 (C-1VI, JC-1,H-1
130.5 (2C), 129.3 (2C), 129.28 (2C), 129.24 (2C), 129.22 (2C), ¼ 173.7 Hz), 98.33 (C-1I, JC-1,H-1 ¼ 169.3 Hz), 98.26 (C-1III, JC-1,H-1
129.2 (2C), 129.17 (2C), 129.15 (2C), 129.1 (2C), 129.07 (2C), ¼ 170.8 Hz), 97.4 (C-1V, JC-1,H-1 ¼ 177.3 Hz), 96.3 (C-1IV, JC-1,H-1
¼
129.05 (3C), 129.0 (2C), 128.9 (3C), 128.8 (3C), 128.7 (2C), 128.6, 155.5 Hz), 80.8 (C-3IV), 78.3 (C-3I), 77.8 (C-3II), 77.7 (C-3V), 75.5
101.2 (C-1II, JC-1,H-1 ¼ 173.2 Hz), 101.06 (C-1III, JC-1,H-1 ¼ 173.8 (C-3VI), 75.3 (C-3III), 74.1 (C-2V), 72.9 (C-2I), 72.7 (CH2Ph), 72.5
Hz), 101.04 (C-1IV, JC-1,H-1 ¼ 174.4 Hz), 101.03 (C-1V, JC-1,H-1
174.2 Hz), 100.3 (C-1VI, JC-1,H-1 ¼ 172.8 Hz), 99.16 (C-1I, JC-1,H-1
¼
¼
(C-2II), 72.2 (CH2Ph), 71.9 (CH2Ph), 71.5 (CH2Ph), 71.3 (CH2Ph),
70.6 (C-5IV), 69.9 (CH2Ph), 69.2 (C-2III), 67.7 (C-5II), 67.6 (C-5VI),
170.9 Hz), 78.7 (C-3I), 77.9 (C-3IV), 77.8 (2C, C-3II, C-3III), 77.6 (C- 67.43 (C-5III), 67.40 (C-2VI), 67.10 (C-5I), 66.9 (C-5V), 66.6 (C-2IV),
3V), 76.1 (C-3VI), 75.0 (C-2V), 74.6 (C-2I), 73.99 (C-2II), 73.97 (C- 65.2 (OCH2CH2Si), 64.5 (C-4I), 64.4 (C-4II), 64.3 (C-4V), 64.05 (C-
2IV), 73.7 (C-2III), 72.8 (CH2Ph), 72.7 (3C, 3ꢃ CH2Ph), 72.3 4VI), 64.01 (C-4IV), 62.9 (C-4III), 18.9 (CH3, C-6V), 18.6 (CH3, C-6I),
(CH2Ph), 71.6 (CH2Ph), 68.8 (2C, C-5I, C-5IV), 68.7 (C-5V), 68.6 (C- 18.5 (CH3, C-6II), 18.3 (CH3, C-6IV), 18.23 (CH3, C-6III), 18.21
5III), 68.5 (2C, C-5VI, C-2VI), 67.9 (C-5II), 65.7 (OCH2CH2Si), 65.5 (CH3, C-6VI), 17.7 (CH2Si), ꢀ1.33 [3C, (CH3)3Si]. HRMS (ESI-
(C-4V), 65.2 (C-4IV), 65.1 (C-4III), 65.05 (C-4VI), 65.02 (C-4II), 64.9 TOF): m/z [M + NH4+] calcd for C90H112O20N19Si 1806.8100;
(C-4I), 19.14 (3ꢃ CH3, C-6VI, C-6III, C-6II), 19.11 (CH3, C-6V), 19.09 found 1806.8089. Anal. calcd for C90H108O20N18Si: C, 60.39; H,
(CH3, C-6IV), 19.08 (CH3, C-6I), 18.2 (CH2Si), ꢀ0.98 [3C, 6.08; N, 14.08. Found C, 60.46; H, 6.15; N, 14.06.
+
(CH3)3Si]. HRMS (ESI-TOF): m/z [M
+
NH4 ] calcd for
2-(Trimethylsilyl)ethyl [4-azido-3-O-benzyl-4,6-dideoxy-a-d-
mannopyranosyl-(1/2)]5-4-azido-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranoside (17)14
C
C
90H112O20N19Si 1806.8100; found 1806.8083. Anal. calcd for
90H108O20N18Si: C, 60.39; H, 6.08; N, 14.08. Found C, 60.22; H,
5.92; N, 13.89.
Continued elution gave the b-linked hexasaccharide 3b as This compound was prepared as described14 from 2 g (1.1
colorless syrup (0.98 g, 2.5%, Total yield of the glycosylation, mmol) of 3a giving 1.8 g (94%) of pure compound 17 as color-
34.7 g, 92%, a/b ꢂ 34 : 1). Data for 3b, [a]D +17.6 (c 2.0, less syrup. Rf ¼ 0.55 at 4 : 1 hexane–EtOAc. [a]D +115.3 (c 1.0,
CHCl3). 1H NMR (600 MHz, CDCl3): d 8.02–7.98 (d, 2H, J ¼ CHCl3), lit14 [a]D +112. 1H NMR (600 MHz, C6D6): d 7.38–7.34 (m,
7.1 Hz, Ar–H), 7.56 (t, 1H, J ¼ 7.5 Hz, Ar–H), 7.50–7.47 (d, 2H, 4H, Ar–H), 7.34–7.29 (m, 6H, Ar–H), 7.29–7.24 (m, 7H, Ar–H),
J ¼ 7.5 Hz, Ar–H), 7.46–7.43 (t, 2H, J ¼ 7.8 Hz, Ar–H), 7.43– 7.24–7.18 (m, 7H, Ar–H), 7.18–7.12 (m, 4H, Ar–H), 7.12–7.08 (m,
7.36 (m, 8H, Ar–H), 7.36–7.31 (m, 6H, Ar–H), 7.31–7.22 (m, 2H, Ar–H), 5.29 (d, 1H, J ¼ 1.3 Hz, H-1VI), 5.25 (brs, 1H, H-1V),
5H, Ar–H), 7.22–7.17 (m, 3H, Ar–H), 7.17–7.09 (m, 5H, Ar–H), 5.21 (d, 1H, J ¼ 1.3 Hz, H-1II), 5.19 (brs, 2H, H-1III, H-1IV), 4.88 (d,
7.04 (m, 1H, Ar–H), 5.59 (dd, 1H, J2–3 ¼ 2.8 Hz, J2–1 ¼ 2.0 Hz, 1H, J ¼ 1.4 Hz, H-1I), 4.47–4.31 (m, 8H, CH2Ph), 4.31–4.27 (m,
H-2VI), 5.31 (d, 1H, J ¼ 0.9 Hz, H-1V), 4.99 (d, 1H, J ¼ 1.0 Hz, 2H, CH2Ph), 4.19–4.13 (m, 3H, 2 CH2Ph, H-2V), 4.01 (t, 1H, J ¼
H-1II), 4.87 (s, 1H, H-1VI), 4.87 (s, 1H, H-1III), 4.79 (d, 1H, J ¼ 2.2 Hz, H-2II), 4.09–4.06 (m, 2H, H-2III, H-2IV), 4.01 (brs, 1H, H-
10.8 Hz, CH2Ph), 4.76 (d, 1H, J ¼ 11.0 Hz, CH2Ph), 4.71 (d, 2VI), 3.97 (t, 1H, J ¼ 2.3 Hz, H-2I), 3.96–3.86 (m, 5H, H-3I–V), 3.80–
1H, J ¼ 11.8 Hz, CH2Ph), 4.70 (d, 1H, J ¼ 10.8 Hz, CH2Ph), 3.60 (m, 9H, H-5I–VI, H-4II, H-3VI, OCHaCH2Si), 3.59–3.49 (m, 4H,
4.68 (d, 1H, J ¼ 1.5 Hz, H-1I), 4.66 (d, 1H, J ¼ 10.8 Hz, CH2Ph), H-4I–V), 3.46 (t, 1H, J ¼ 10.0 Hz, H-4VI), 3.33 (m, 1H, OCHbCH2Si),
4.60 (d, 1H, J ¼ 11.6 Hz, CH2Ph), 4.57 (d, 1H, J ¼ 11.0 Hz, 2.08 (d, 1H, J ¼ 1.8 Hz, H-2VI-OH), 1.32 (d, 3H, J6–5 ¼ 6.3 Hz, CH3,
CH2Ph), 4.54 (d, 1H, J ¼ 11.6 Hz, CH2Ph), 4.52 (d, 1H, J ¼ H-6), 1.31–1.23 (m, 15H, 5 CH3, H-6), 0.83–0.76 (m, 2H, CH2-
11.60 Hz, CH2Ph), 4.46 (d, 1H, J ¼ 10.8 Hz, CH2Ph), 4.24 (d, CH2Si), 0.06 (s, 9H, (CH3)3Si). 13C{1H} NMR (150 MHz, C6D6):
1H, J ¼ 11.3 Hz, CH2Ph), 4.15 (dd, 1H, J ¼ 3.3, 1.5 Hz, H-2III), d 138.1 (2C), 138.0 (2C), 137.9 (2C), 129.33 (3C), 129.3, 129.28
4.10 (t, 1H, J ¼ 2.2 Hz, H-2II), 4.03 (d, 1H, J ¼ 1.9 Hz, H-2IV), (3C), 129.23 (3C), 129.2 (3C), 129.13 (3C), 129.1 (2C), 128.98 (2C),
3.99 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 3.92 (m, 1H, H-5V), 3.89 (t, 128.96 (2C), 128.8 (3C), 128.75 (2C), 128.7 (2C), 128.6 (2C), 101.5
1H, J ¼ 2.2 Hz, H-2I), 3.86 (t, 1H, J ¼ 2.0 Hz, H-2V), 3.83 (dd, (C-1VI), 101.2 (C-1IV), 101.1 (C-1V), 100.06 (C-1II), 100.04 (C-1III),
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99.2 (C-1I), 78.7 (C-3I), 78.5 (C-3VI), 77.82 (C-3IV), 77.8 (2C, C- acid15 (0.5 g, 2.5 mmol) in dry dichloromethane (20 mL).
3II,II), 77.7 (C-3V), 74.6 (C-2I), 74.0 (C-2IV), 73.8 (C-2III), 73.7 (C- Stirring was continued overnight, when TLC showed
2II), 73.3 (C-2V), 72.8 (CH2Ph), 72.7 (CH2Ph), 72.69 (CH2Ph), 72.6 complete consumption of starting material and formation
(CH2Ph), 72.3 (CH2Ph), 71.8 (CH2Ph), 68.83 (C-5V), 68.78 (C-5VI), of a much faster moving product (15 : 1 DCM–MeOH). The
68.72 (2C, C-5I,III), 68.3 (C-5II), 67.9 (C-5IV), 67.6 (C-2VI), 65.7 mixture was diluted with DCM (25 mL), washed with aq
(OCH2CH2Si), 65.5 (C-4II), 65.14, 65.12, 65.1 and 65.07, (4C, C-4I, NaHCO3 solution (3ꢃ 10 mL), brine (3ꢃ 10 mL), dried and
C-4III, C-4IV and C-4V), 64.5 (C-4VI), 19.14 (2ꢃ CH3, 2ꢃ C-6), 19.11 concentrated. HRMS analysis of the crude product
(CH3, C-6), 19.1 (CH3, C-6), 19.08 (CH3, C-6), 18.9 (CH3, C-6), 18.2 conrmed formation of the desired coupling product only
+
(CH2Si), ꢀ0.98 [3C, (CH3)3Si]. HRMS (ESI-TOF): m/z [M + NH4
]
(HRMS (ESI-TOF): m/z [M + H+] calcd for C131H177O49N6Si
2646.1312; found 2646.1313). The crude product was dis-
solved in dry pyridine (2 mL) and treated with acetic
anhydride (0.1 mL) overnight at room temperature. TLC
showed complete conversion of the starting material and
formation of a faster moving product (Rf ¼ 0.54 at 15 : 1
calcd for C83H108O19N19Si 1702.7838; found 1702.7826.
2-(Trimethylsilyl)ethyl [4-amino-3-O-benzyl-4,6-dideoxy-a-
D-mannopyranosyl-(1/2)]5-4-amino-3-O-benzyl-4,6-dideoxy-a-D-
mannopyranoside (18)14
Compound 17 (0.75 g, 0.45 mmol) was dissolved in pyridine/
triethylamine 7 : 3 (20 mL). Hydrogen sulde (H2S) was passed
through the above solution for 2 h and the resulting dark solution
was stirred at room temperature, in the same ask equipped with
an empty balloon, to ensure exclusion of atmospheric oxygen,
overnight. TLC (Rf ¼ 0.51 at DCM–MeOH 10 : 1) showed that the
reaction was complete and that a slower moving product was
formed. The reaction mixture was concentrated to dryness and
coevaporated with toluene. The crude product was chromato-
graphed and eluted with 1–3% ammonia (7 N solution in MeOH)
in DCM (v/v). Compound 18 was obtained as a white foam (0.62 g,
92%), [a]D ꢀ5.0 (c 2.1, CHCl3), 1H NMR (600 MHz, CDCl3): d 7.37–
7.27 (m, 30H, Ar–H), 5.09 (s, 1H, H-1), 5.08–5.06 (s, 3H, 3ꢃ H-1),
5.01 (s, 1H, H-1VI), 4.78 (s, 1H, H-1I), 4.70–4.65 (m, 6H, CH2Ph),
4.51 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.47 (d, 1H, J ¼ 12.0 Hz, CH2Ph),
4.44–4.38 (m, 4H, CH2Ph), 4.08–4.06 (m, 2H, 2ꢃ H-2), 4.03 (brs, 3H,
3ꢃ H-2), 3.92 (t, 1H, J ¼ 2.3 Hz, H-2I), 3.77–3.72 (m, 1H, OCHa-
CH2Si), 3.65–3.46 (m, 12H, 6ꢃ H-3, 6ꢃ H-5), 3.46–3.42 (m, 1H,
OCHbCH2Si), 2.88–2.79 (m, 6H, 6ꢃ H-4), 1.38–1.21 (m, 18H, 12ꢃ
NH2, 2ꢃ CH3, 2ꢃ H-6), 1.18–1.14 (m, 12H, 4ꢃ CH3, 4ꢃ H-6), 0.96–
0.90 (m, 1H, CH2CHaSi), 0.89–0.84 (m, 1H, CH2CHbSi), 0.02 (s, 9H,
(CH3)3Si). 13C{1H} NMR (150 MHz, CDCl3): d 137.9 (2C), 137.8 (2C),
137.7, 137.6, 128.6 (2C), 128.56 (3C), 128.53 (2C), 128.5 (3C), 128.4
(2C), 128.36 (3C), 128.32 (3C), 128.2 (2C), 128.1 (2C), 128.0 (3C),
127.98 (3C), 127.9 (2C), 101.1 (C-1), 100.98 (C-1), 100.96 (C-1),
100.94 (2C, 2ꢃ C-1), 98.8 (C-1), 79.7 (C-3), 79.2 (3C, 3ꢃ C-3), 78.8 (C-
3), 78.7 (C-3), 73.2 (C-2), 73.1 (C-2), 73.0 (C-2), 72.9 (C-2), 72.8 (C-2),
71.4 (CH2Ph), 71.2 (CH2Ph), 71.15 (CH2Ph), 71.1 (CH2Ph), 71.0 (2C,
2ꢃ CH2Ph), 70.3 (4C, 4 C-5), 69.6 (C-5), 69.5 (C-5), 66.5 (C-2), 64.7
(OCH2CH2Si), 53.7 (2C, 2ꢃ C-4), 53.6 (3C, 3ꢃ C-4), 53.3 (C-4), 18.3
(CH3, C-6), 18.2 (3C, CH3, 3ꢃ C-6), 18.1 (CH3, C-6), 17.9 (CH3, C-6),
17.8 (CH2Si), ꢀ1.3 [3C, (CH3)3Si]. HRMS (ESI-TOF): m/z [M + H+]
calcd for C83H117O19N6Si 1529.8143; found 1529.8136.
DCM–MeOH). Aer concentration,
a solution of the
residue in DCM (10 mL) was washed with cold 4 N HCl (3ꢃ
10 mL), aq NaHCO3 solution (3ꢃ 10 mL), the phases were
separated, and the aqueous phase was backwashed with
DCM. The organic phase was dried, concentrated, and
chromatography (DCM–MeOH–Py 10 : 1 : 0.1) gave pure 19
as colorless syrup (738 mg, 84% over 2 steps), [a]D ꢀ11.6 (c
1.0, CHCl3), 1H NMR (600 MHz, CD3OD): d 7.45–7.41 (m,
4H, Ar–H), 7.39–7.37 (m, 6H, Ar–H), 7.36–7.32 (m, 6H, Ar–
H), 7.31–7.25 (m, 10H, Ar–H), 7.25–7.18 (m, 4H, Ar–H), 5.40
(t, 1H, J ¼ 2.0 Hz, H-2VI), 5.10–5.00 (m, 10H, 6ꢃ H-20, H-1II,
H-1III, H-1IV, H-1V), 4.77 (s, 1H, H-1I), 4.65–4.55 (m, 12H,
11ꢃ CH2Ph and H-1VI at 4.56), 4.41 (d, 1H, J ¼ 11.3 Hz,
CH2Ph), 4.21–4.03 (m, 22H, 12ꢃ H-40, 6ꢃ H-4I–VI, 4ꢃ H-2II–V),
3.98–3.91 (m, 6H, 6ꢃ H-3I–VI), 3.88–3.76 (m, 7H, H-2I, 5ꢃ H-5,
OCHaCH2Si), 3.71 (m, 1H, H-5), 4.48 (m, 1H, OCHbCH2Si), 2.15–
1.98 (m, 51H, 6ꢃ Ha,b-30 incl. 12 s at 2.14, 2.11, 2.08, 2.07, 2.06,
2.05, 2.04, 2.03, 2.02, 2.00, 1.998, 1.99 for 13ꢃ COCH3), 1.14–
1.11 (m, 6H, 2ꢃ H-6), 1.06–1.02 (m, 12H, 4ꢃ H-6), 0.96–0.92 (m,
1H, CH2CHaSi), 0.90–0.86 (m, 1H, CH2CHbSi), 0.02 (s, 9H,
(CH3)3Si). 13C{1H} NMR (150 MHz, CD3OD): d 172.9 (CH3C]O),
172.87 (CH3C]O), 172.8 (CH3C]O), 172.79 (CH3C]O), 172.77
(CH3C]O), 172.7 (CH3C]O), 172.61 (3C, 3ꢃ CH3C]O), 172.60
(CH3C]O), 172.5 (2C, 2ꢃ CH3C]O), 171.84 (NHC]O), 171.8
(NHC]O), 171.78 (NHC]O), 171.76 (NHC]O), 171.73 (NHC]
O), 171.69 (NHC]O), 171.5 (CH3C]O), 139.8, 139.7, 139.68,
139.63 (2C), 139.6, 129.7, 129.67 (2C), 129.6 (2C), 129.5 (2C),
129.4 (3C), 129.23 (3C), 129.2 (3C), 129.1 (2C), 129.0 (2C), 128.9
(2C), 128.8 (2C), 128.7 (2C), 128.6 (2C), 128.5, 102.5 and 102.3
(4C, 4ꢃ C-1II–V), 100.9 (C-1VI), 99.8 (C-1I), 77.0, 76.9, 76.6, 76.5,
76.4 (10C, 5ꢃ C-3, 5ꢃ C-2), 75.6 (C-3VI), 73.0 (CH2Ph), 72.8 (3C,
3ꢃ CH2Ph), 72.6 (CH2Ph), 72.56 (CH2Ph), 72.5 (C-20), 72.43 (2C,
2ꢃ C-20), 72.4 (3C, 3ꢃ C-20), 69.8 (C-5), 69.7 (C-5), 69.6 (C-5), 69.5
(C-5), 68.9 (2C, C-5, C-2VI), 68.8 (C-5), 66.1 (OCH2CH2Si), 61.3
(4C, 4ꢃ C-40), 61.25 (C-40), 61.2 (C-40), 53.6 (C-4), 53.4 (C-4), 53.3
(C-4), 53.2 (3C, 3ꢃ C-4), 32.4 (C-30), 32.3 (5C, 5ꢃ C-30), 20.9
(COCH3), 20.83 (5C, 5ꢃ COCH3), 20.81 (COCH3), 20.78 (COCH3),
20.73 (COCH3), 20.71 (3C, 3ꢃ COCH3), 20.70 (COCH3), 18.7
(CH3, C-6), 18.57 (CH3, C-6), 18.54 (CH2Si), 18.48 (2C, CH3, 2ꢃ C-
6), 18.4 (CH3, C-6), 18.3 (CH3, C-6), ꢀ1.2 [3C, (CH3)3Si]. HRMS
2-(Trimethylsilyl)ethyl 2-O-acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-
3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-mannopyranosyl-
(1/2)-[3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranosyl-(1/2)]4-3-O-
benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-
dideoxy-a-D-mannopyranoside (19)
EDAC (0.45 g, 2.4 mmol) was added portion wise at room (ESI-TOF): m/z [M + H+] calcd for C133H179O50N6Si 2688.1418;
temperature to a stirred solution of amino sugar 18 (0.5 g, found 2688.1423. Anal. calcd for C133H178O50N6Si: C, 59.41; H,
0.33 mmol) and 2,4-di-O-acetyl-3-deoxy-L-glycero-tetronic 6.67; N, 3.13. Found C, 59.51; H, 6.66; N, 3.06.
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(DCM : MeOH ¼ 19 : 1) containing a few drops of triethylamine,
to obtain pure compound 21 as light brown syrup (448 mg,
85%). HRMS: m/z [M + NH4 ] calcd for C130H170O50N8Cl3
2748.0071; found 2748.0081. The material was sufficiently pure
for the next step.
2-O-Acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranosyl-(1/2)-[3-
O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-
4,6-dideoxy-a-D-mannopyranosyl-(1/2)]4-3-O-benzyl-4-
(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-
mannopyranose (20)
+
Method 1. To
a solution of the hexasaccharide tri-
chloroacetimidate donor 21 (100 mg, 0.04 mmol) and glycosyl
acceptor 22 (35 mg, 0.2 mmol) in dry DCM (7 mL) was added
A solution of 19 (700 mg, 0.26 mmol) in TFA (12 mL) was kept at
room temperature for 2 hours, when TLC (Rf ¼ 0.31 at 15 : 1
DCM–MeOH) showed that the reaction was complete and that
a much slower moving product was formed. The mixture was
processed as described above for a similar reaction, and chro-
matography (19 : 1 DCM–MeOH) gave 20 as foam (565 mg,
84%). 1H NMR (600 MHz, CD3OD) for the major anomer: d 8.14–
7.92 (m, 6H, NH), 7.43–7.17 (m, 30H, Ar–H), 5.39 (brs, 1H, H-2VI),
5.09–5.00 (11H, 6ꢃ H-20, 5ꢃ H-1I–V at 5.083, 5.076, 5.056, 5.056,
5.012), 4.69–4.55 (m, 12H, 11ꢃ CH2Ph and H-1VI at 4.56), 4.41 (d,
1H, J ¼ 11.4 Hz, CH2Ph), 4.20–4.03 (m, 21H, 12ꢃ H-40, 4ꢃ H-2,
4ꢃ H-4 and H-5VI), 3.97–3.78 (m, 14H, H-2I, 6ꢃ H-3, 2ꢃ H-4, 5ꢃ
H-5), 2.17–1.95 (m, 51H, 6ꢃ Ha,b-30 incl. 12 s at 2.136, 2.13, 2.10,
2.09, 2.08, 2.07, 2.06, 2.05, 2.03, 2.02, 2.01, 2.00, 1.99 for 13ꢃ
COCH3), 1.16–0.99 (m, 18H, 6ꢃ H-6). 13C{1H} NMR (150 MHz,
CD3OD): d 172.98 (CH3C]O), 172.95 (CH3C]O), 172.9 (CH3C]
O), 172.87 (CH3C]O), 172.84 (CH3C]O), 172.80 (CH3C]O),
172.78 (CH3C]O), 172.76 (CH3C]O), 172.7 (CH3C]O), 172.6
(2C, 2ꢃ CH3C]O), 172.5 (CH3C]O), 171.85 (NHC]O), 171.8
(NHC]O), 171.78 (NHC]O), 171.76 (NHC]O), 171.73 (NHC]
O), 171.7 (NHC]O), 171.6 (CH3C]O), 139.8, 139.76, 139.72,
139.64, 139.64, 139.6, 129.7, 129.6 (2C), 129.55 (2C), 129.5 (2C),
129.4 (2C), 129.2 (2C), 129.19 (2C), 129.1 (2C), 129.0 (3C), 128.86
(3C), 128.84 (2C), 128.80 (2C), 128.7 (2C), 128.6 (2C), 128.5, 102.4
and 102.2 (4C, 4ꢃ C-1II–V), 100.9 (C-1VI), 94.5 (C-1I), 76.9, 76.8,
76.6, 76.56, 76.5, 75.6, 75.5 (11C, 6ꢃ C-3, 5 C-2), 72.9 (2C, 2ꢃ
CH2Ph), 72.8 (3C, 3ꢃ CH2Ph), 72.6 (CH2Ph), 72.5 (C-20), 72.47 (C-
20), 72.45 (2C, 2ꢃ C-20), 72.4 (2C, 2ꢃ C-20), 69.76 (C-5), 69.7 (C-5),
69.5 (2 C, C-5), 68.9 (2C, C-5, C-2VI), 68.3 (C-5), 61.3 (4C, 4ꢃ C-40),
61.25 (C-40), 61.2 (C-40), 53.9 (C-4), 53.8 (C-4), 53.5 (C-4), 53.4 (C-
4), 53.3 (C-4), 53.2 (C-4), 32.4 (6C, 6ꢃ C-30), 20.9 (COCH3), 20.8
(6C, 6ꢃ COCH3), 20.78 (COCH3), 20.74 (COCH3), 20.72 (2C, 2ꢃ
COCH3), 20.70 (2C, 2ꢃ COCH3), 18.7 (CH3, C-6), 18.6 (CH3, C-6),
18.5 (3C, CH3, 3ꢃ C-6), 18.3 (CH3, C-6). HRMS (ESI-TOF): m/z [M
+ H+] calcd for C128H167O50N6 2588.0710; found 2588.0725.
˚
0.1 g of 4 A powdered molecular sieves, and the mixture was
stirred for 15 min. The mixture was cooled to 0 ꢁC, and TMSOTf
(3 mL, 0.016 mmol) was added. Aer 30 min at 0 ꢁC, when TLC
(3 : 2 toluene–acetone) showed complete consumption of the
glycosyl donor, the reaction was terminated by addition of
triethylamine (1 drop). The mixture was ltered through a pad
of Celite directly into a separating funnel containing excess of
sodium bicarbonate (saturated) solution. The mixture was
extracted with DCM (3ꢃ 5 mL), the combined organic layers
were dried and concentrated, to give crude product (65.3 mg,
65%). NMR showed that the reaction produced the two anomers
with poor selectivity (a : b ¼ 1.1 : 1).
Method 2. To
a solution of the hexasaccharide tri-
chloroacetimidate donor 21 (200 mg, 0.08 mmol) and acceptor
22 (70 mg, 0.4 mmol) in dry toluene (10 mL with drops of dry
˚
DCM to aid solubility of synthons) was added 0.1 g of 4 A
powdered molecular sieves, and the mixture was stirred for
15 min. The reaction mixture was warmed to 100 ꢁC (bath), and
TMSOTf (6 mL, 0.03 mmol) was added. Aer 1.5 hours at that
temperature, when TLC (Rf ¼ 0.43 at 3 : 2 toluene–acetone)
showed complete consumption of the glycosyl donor, the
mixture was processed as described above. 1H NMR of the crude
mixture showed the ratio of the a and b anomers formed was
7 : 1. Chromatography (3 : 2 toluene–acetone) gave rst the a-
linked hexasaccharide (2a, 132 mg, 66%) as colorless syrup. [a]D
1
ꢀ10.8 (c 2.0, CHCl3). H NMR (600 MHz, CD3OD): d 7.74–7.71
(m, 2H, Ar–H), 7.40–7.38 (m, 5H, Ar–H), 7.36–7.33 (m, 5H, Ar–H),
7.30–7.26 (m, 6H, Ar–H), 7.23–7.19 (m, 6H, Ar–H), 7.16–7.14 (m,
4H, Ar–H), 7.13–7.10 (m, 2H, Ar–H), 5.40 (brs, 1H, H-2VI), 5.09–
5.00 (m, 10H, 6ꢃ H-20, 4ꢃ H-1II–V at 5.09, 5.065, 5.06 and 5.02),
4.83 (d, 1H, J1–2 ¼ 1.5 Hz, H-1I), 4.66–4.55 (m, 12H, 11ꢃ CH2Ph
and H-1VI at 4.57), 4.41 (d, 1H, J ¼ 11.3 Hz, CH2Ph), 4.20–4.05
(m, 22H, 12ꢃ H-40, 4ꢃ H-2II–V, 6ꢃ H-4I–VI), 3.99–3.92 (m, 6H, 5ꢃ
H-3II–V, H-2I), 3.91–3.88 (m, 2H, H-3, H-5), 3.86–3.79 (m, 4H, 4ꢃ
H-5), 3.78–3.73 (m, 2H, OCHa, H-5), 3.69–3.63 (m, 8H, 8ꢃ OCH2),
3.59 (m, 1H, OCHb), 3.37 (t, 2H, J ¼ 5.0 Hz, CH2N3), 2.15–1.98
(m, 51H, 6ꢃ Ha,b-30 incl. 12 s at 2.145, 2.143, 2.12, 2.08, 2.07,
2.06, 2.04, 2.03, 2.02, 2.00, 1.999 and 1.99 for 13ꢃ COCH3), 1.15–
1.11 (m, 6H, 2ꢃ CH3, 2ꢃ H-6), 1.09–1.02 (m, 12H, 4ꢃ CH3, 4ꢃ H-
6). 13C{1H} NMR (150 MHz, CD3OD): d 172.88 (CH3C]O),
172.84 (CH3C]O), 172.82 (CH3C]O), 172.77 (CH3C]O),
172.75 (CH3C]O), 172.68 (CH3C]O), 172.59 (2C, 2ꢃ CH3C]
8-Azido-3,6-dioxaoctyl 2-O-acetyl-3-O-benzyl-[4-(2,4-di-O-acetyl-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-
mannopyranosyl]-(1/2)-[3-O-benzyl-[4-(2,4-di-O-acetyl-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-
mannopyranosyl-(1/2)]4-3-O-benzyl-4-(2,4-di-O-acetyl-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-a-D-
mannopyranoside (2)
1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU, 15 mL, 0.1 mmol) was O), 172.58 (2C, 2ꢃ CH3C]O), 172.54 (2C, 2ꢃ CH3C]O), 171.81
added at 0 ꢁC to a stirred solution of hemiacetal 21 (500 mg, 0.19 (2C, 2ꢃ NHC]O), 171.76 (NHC]O), 171.74 (NHC]O), 171.71
mmol) and trichloroacetonitrile (30 mL, 0.29 mmol) in dry DCM (NHC]O), 171.67 (NHC]O), 171.5 (CH3C]O), 139.74, 139.72,
(10 mL). The stirring was continued for 2 hours when TLC 139.70 (2C), 139.6 (2C), 129.9, 129.7 (2C), 129.6 (2C), 129.56 (2C),
(15 : 1 DCM–MeOH) showed complete formation of a faster 129.5 (2C), 129.4 (2C), 129.22 (2C), 129.21 (2C), 129.18 (2C),
moving product. The mixture was concentrated, and passed 129.1 (2C), 129.0 (2C), 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6,
through a small pad of silica gel which was eluted with 128.5, 126.3, 102.4 and 102.3 (4C, 4ꢃ C-1II–V), 100.9 (C-1VI), 100.5
36452 | RSC Adv., 2019, 9, 36440–36454
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(C-1I), 76.98, 76.9, 76.6, 76.5, 76.4, 75.6 (11C, 6ꢃ C-3, 5 C-2),
72.86 (2C, 2ꢃ CH2Ph), 72.8 (2C, 2ꢃ CH2Ph), 72.6 (CH2Ph), 72.54
(CH2Ph), 72.5 (C-20), 72.4 (3C, 3ꢃ C-20), 72.3 (2C, 2ꢃ C-20), 71.7
(OCH2), 71.5 (OCH2), 71.4 (OCH2), 71.2 (OCH2), 69.8 (C-5), 69.7
(2C, 2ꢃ C-5), 69.6 (C-5), 69.5 (C-5), 68.9 (C-2VI), 68.8 (C-5), 68.1
(OCH2), 61.3 (4C, 4ꢃ C-40), 61.24 (C-40), 61.2 (C-40), 53.5 (C-4),
53.4 (C-4), 53.3 (2C, 2ꢃ C-4), 53.2 (2C, 2ꢃ C-4), 51.7 (CH2N3),
32.42 (C-30), 32.4 (2C, 2ꢃ C-30), 32.36 (3C, 3ꢃ C-30), 21.5
(COCH3), 20.9 (COCH3), 20.84 (5C, 5ꢃ COCH3), 20.82 (COCH3),
20.8 (COCH3), 20.73 (3C, 3ꢃ COCH3), 20.7 (COCH3), 18.7 (CH3,
C-6), 18.6 (CH3, C-6), 18.5 (CH3, C-6), 18.47 (CH3, C-6), 18.4 (CH3,
C-6), 18.3 (CH3, C-6). HRMS (ESI-TOF): m/z [M + NH4+] calcd for
8-Amino-3,6-dioxaoctyl [4-(3-deoxy-L-glycero-tetronamido)-4,6-
dideoxy-a-D-mannopyranosyl-(1/2)]5-4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-a-D-mannopyranopyranoside (1)
To a solution of the linker-equipped hexasaccharide 2a
(100 mg, 0.04 mmol) in dry MeOH (10 mL), methanolic
NaOMe (1 M, 1 mL) was added with exclusion of moisture and
atmospheric CO2, and the mixture was kept at room
temperature for 6 hours, when TLC (3 : 2 toluene–acetone)
showed that the starting material was consumed and that
a much slower moving product was formed (Rf ¼ 0.34 at 4 : 1
DCM–methanol with 1 drop of acetic acid). Aer neutraliza-
tion (Dowex 50W H+ resin) and ltration, the solvent was
removed, and the residue, showing correct HRMS (ESI-TOF:
m/z [M + H+] calcd for C108H152O39N9 2199.0187; found
2199.0188), was used for the next step without further
purication.
To a solution of the above product in MeOH (2 mL), 10 mg
of Pd–C was added, and the mixture was stirred at room
temperature under 100 Psi pressure of hydrogen gas for 4
hours. TLC showed complete consumption of starting mate-
rial and presence of a much more polar product. HRMS
analysis conrmed the completion of global reduction. The
mixture was ltered over a Celite pad, the solids were washed
several times with methanol and the solvent was removed. A
solution of the product in MeOH : H2O (2 : 1) was ltered
through a 0.2 mm porosity syringe lter and lyophilized to
collect pure product as white foam (42 mg, 71% over 2 steps),
[a]D +1.8 (c 1.0, CH3OH). 1H NMR (600 MHz, CD3OD): d 5.16–
5.11 (m, 4H, 4ꢃ H-1II–V), 4.98 (s, 1H, H-1VI), 4.87 (s, 1H, H-1I),
4.22–4.14 (m, 6H, 6ꢃ H-20), 4.13–4.08 (m, 4H, 4ꢃ H-2II–V),
4.05–3.98 (m, 5H, 4ꢃ H-3II–V, H-2VI at 4.02), 3.98–3.83 (m, 13H,
2ꢃ H-3I,VI, 6ꢃ H-4I–VI, 5ꢃ H-5II–VI), 3.83–3.78 (m, 3H, H-2I, H-
5I, OCHa), 3.77–3.71 (m, 12H, 12ꢃ H-40), 3.71–3.52 (m, 10H,
10ꢃ OCH2), 3.13 (t, 1H, J ¼ 4.8 Hz, OCH2CHaNH2), 2.06–1.97
(m, 6H, 6ꢃ H-30a), 1.87–1.78 (m, 6H, 6ꢃ H-30b), 1.22–1.10 (m,
18H, 6ꢃ H-6). 13C{1H} NMR (150 MHz, CD3OD): d 178.0 (5C,
5ꢃ NHC]O), 177.9 (NHC]O), 103.8 (C-1VI), 102.7, 102.5,
105.46, 102.4 (4C, 4ꢃ C-1II–V), 100.4 (C-1I), 79.6, 79.23, 79.2
(5C, 5ꢃ C-2), 71.6, 71.4, 71.30 (3C, 3ꢃ OCH2), 70.9 (C-2), 70.7
(6C, 6ꢃ C-20), 70.0, 69.5, 69.4, 69.3 (11C, 6ꢃ C-3, 5ꢃ C-5), 68.7
(C-5), 68.1 (OCH2), 67.8 (OCH2), 59.4 (6C, 6ꢃ C-40), 54.8–54.2
(6C, C-4I–VI), 40.7 (CH2NH2), 38.3 (6C, 6ꢃ C-30), 18.4 (2C, CH3,
2ꢃ C-6), 18.3 (3C, CH3, 3ꢃ C-6), 18.2 (CH3, C-6). HRMS (ESI-
TOF): m/z [M + H+] calcd for C66H118O39N7 1632.7465; found
1632.7467.
C
C
134H181O52N10 2762.1826; found 2762.1836. Anal. calcd for
134H177O52N9: C, 59.61; H, 6.50; N, 4.59. Found C, 59.48; H,
6.60; N, 4.47.
Continued elution gave the b-linked hexasaccharide 2b as
colorless syrup (19 mg, 9%, total yield of glycosylation, 151 mg,
1
75%, a/b ꢂ 6.9 : 1). Data for 2b: [a]D ꢀ18.3 (c 0.9, CHCl3). H
NMR (600 MHz, CD3OD): d 7.52–7.17 (m, 30H, Ar–H), 5.41 (t,
1H, J ¼ 2.1 Hz, H-2VI), 5.21 (d, 1H, J1–2 ¼ 1.9 Hz, H-1I), 5.15–4.98
(m, 9H, 6ꢃ H-20, 3ꢃ H-1II–V at 5.10, 5.08 and 5.04), 4.73–4.55
(m, 12H, 11ꢃ CH2Ph and H-1VI at 4.58), 4.49 (s, 1H, H-1I), 4.43
(d, 1H, J ¼ 11.6 Hz, CH2Ph), 4.24–4.03 (m, 22H, 12ꢃ H-40, 4ꢃ H-
2II–V, 6ꢃ H-4I–VI), 3.99–3.75 (m, 13H, H-2I, 6ꢃ H-3II–V, 6ꢃ H-5),
3.69–3.63 (m, 8H, 8ꢃ OCH2), 3.59 (m, 2H, OCH2), 3.38 (2d, 2H,
J ¼ 4.5 Hz, CH2N3), 2.15–1.98 (m, 51H, 6ꢃ Ha,b-30 incl. 11 s at
2.11, 2.09, 2.08, 2.06, 2.057, 2.05, 2.04, 2.03, 2.02, 2.01 and 2.00
for 13ꢃ COCH3), 1.15–1.11 (m, 6H, 2ꢃ CH3, 2ꢃ H-6), 1.09–1.02
(m, 12H, 4ꢃ CH3, 4ꢃ H-6). 13C{1H} NMR (150 MHz, CD3OD):
d 172.9 (CH3C]O), 172.88 (CH3C]O), 172.84 (CH3C]O),
172.78 (4C, 4ꢃ CH3C]O), 172.6 (2C, 2ꢃ CH3C]O), 172.59 (3C,
3ꢃ CH3C]O), 171.82 (NHC]O), 171.81 (NHC]O), 171.77
(NHC]O), 171.75 (NHC]O), 171.72 (NHC]O), 171.7 (NHC]
O), 171.6 (CH3C]O), 139.8, 139.73, 139.71, 139.65, 139.6,
139.5, 129.7 (2C), 129.6 (2C), 129.5 (2C), 129.4 (2C), 129.3 (2C),
129.24 (2C), 129.2 (2C), 129.1 (2C), 129.0 (2C), 128.96 (2C),
128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6 (2C), 128.5 (2C), 101.5
(3C, 3ꢃ C-1II–V), 100.8 (3C, 3ꢃ C-1II–V), 76.9, 76.6, 76.2, 75.6
(11C, 6ꢃ C-3, 5 C-2), 73.2 (2C, 2ꢃ CH2Ph), 72.8 (3C, 3ꢃ CH2Ph),
72.6 (CH2Ph), 72.54, 72.5, 72.4, 72.40, 72.39 (6C, 6ꢃ C-20), 71.6
(OCH2), 71.5 (OCH2), 71.4 (OCH2), 71.1 (OCH2), 69.8 (OCH2),
69.8 (C-5), 69.7 (3C, 3ꢃ C-5), 69.6 (C-5), 69.5 (C-5), 68.9 (C-2VI),
61.3 (C-40), 61.28 (2C, 2ꢃ C-40), 61.26 (2C, 2ꢃ C-40), 61.23 (C-40),
53.7 (C-4), 53.4 (2C, 2ꢃ C-4), 53.3 (3C, 3ꢃ C-4), 51.7 (CH2N3),
32.5 (C-30), 32.4 (2C, 2ꢃ C-30), 32.38 (3C, 3ꢃ C-30), 20.98
(COCH3), 20.85 (2C, 2ꢃ COCH3), 20.83 (3C, 3ꢃ COCH3), 20.8
(COCH3), 20.78 (COCH3), 20.75 (COCH3), 20.7 (4C, 4ꢃ COCH3),
18.7 (CH3, C-6), 18.6 (CH3, C-6), 18.5 (3CH3, 3ꢃ C-6), 18.4 (CH3,
Conflicts of interest
+
C-6). HRMS (ESI-TOF): m/z [M
+
NH4
]
calcd for
There are no conicts to declare.
C134H181O52N10 2762.1826; found 2762.1838. Anal. calcd for
134H177O52N9: C, 59.61; H, 6.50; N, 4.59. Found C, 58.99; H,
C
6.50; N, 4.49.
Acknowledgements
This research was supported by the Intramural Research
Program of the National Institutes of Health, NIDDK.
This journal is © The Royal Society of Chemistry 2019
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