Versatile synthetic route to carbocyclic N-Acetylneuraminic acid and its derivatives

Article abstract of DOI:10.1016/j.tet.2018.05.065

Sialic acid (N-acetylneuraminic acid) is a carbohydrate that possess a nine carbon backbone, and it is often found at the termini of glycoconjugates in biological systems. Because of this prominence many syntheses have reported routes to sialic acid and m

Full text of DOI:10.1016/j.tet.2018.05.065

Accepted Manuscript
Versatile synthetic route to carbocyclic N-Acetylneuraminic acid and its derivatives
Sankar Mohan, John R. Thompson, B. Mario Pinto, Andrew J. Bennet
PII:
S0040-4020(18)30613-6
10.1016/j.tet.2018.05.065
TET 29572
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 18 February 2018
Revised Date: 18 May 2018
Accepted Date: 22 May 2018
Please cite this article as: Mohan S, Thompson JR, Pinto BM, Bennet AJ, Versatile synthetic route
to carbocyclic N-Acetylneuraminic acid and its derivatives, Tetrahedron (2018), doi: 10.1016/
j.tet.2018.05.065.
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ACCEPTED MANUSCRIPT

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Versatile Synthetic Route to Carbocyclic N-Acetylneuraminic Acid
and its Derivatives#
Sankar Mohan, John R. Thompson, B. Mario Pinto* and Andrew J. Bennet*
Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC,
Canada V5A 1S6
*To whom correspondence should be addressed
Email: bpinto@sfu.ca, bennet@sfu.ca
#This manuscript is dedicated, with respect and gratitude, to Professor Sir Derek H.R. Barton, a formidable mentor
Keywords: Sialic acid, carbocyclic congener, synthesis, carbasugar
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Abstract:
Sialic acid (N-acetylneuraminic acid) is a carbohydrate that possess a nine carbon backbone, and
it is often found at the termini of glycoconjugates in biological systems. Because of this
prominance many syntheses have reported routes to sialic acid and many of its derivatives. Most
of these compounds retain the endocyclic oxygen atom that becomes part of the ketal glycosidic
lingkage that joins sialic acid to the penultimate residue in the glycoconjugate. With respect to
carba-sialic acid (replacement of the ring oxygen atom with a methylene group) a single
synthesis has been reported (Ogawa, et al. (Carbohydr. Res., 1995, 269, 53-78) in 30 steps and
0.5% yield. The current report details a robust synthesis of 6a-carba-α-D-sialic acid that involves
18 steps and give a 5% yield using
.
D-quinic acid as the starting material.
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Introduction
Sialic acids are a large, structurally diverse, family of nine-carbon α-ketoacid carbohydrates that
are often found as the terminal sugar on numerous glycoconjugates.1-3 In nature, free sialic acids
exist predominantly in the thermodynamically more stable β-anomer.4,5 Whereas, the
configuration of the glycosidic linkage between a sialic acid and the underlying glycoconjugate
is an α-anomer, with the most prevalent sialosides in Humans being based on N-
acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,
Neu5Ac). At biological pH values, this highly functionalized sugar is anionic with the
carboxylic acid attached to the anomeric carbon atom (C2) being ionized. Other structural
features of Neu5Ac are a methylene group at C3, an acetamido group at C5, and a glycerol side
chain as the substituent on the 6-position (1 an α-glycosidic form is shown). Due to its highly
functionalized nature and its prevalence as the terminal sugar on glycoconjugates, sialic acids
play many important roles in numerous biological events, including cell-cell interaction and
communication, cellular aggregation and development, controlling the lifetimes of glycoproteins
in organisms, and mediating pathogenic infection.6-8 Consequently, the enzymes that add these
sugars to or remove them from glycoconjugates are often the target for the development of
therapeutic agents. For example, based on the structure of the natural inhibitor Neu2en5Ac (2a
or DANA)9 two compounds (2b10 and 311) were developed as therapeutic agents for influenza
based on their ability to inhibit the viral sialidase (neuraminidase) that recognizes and
hydrolytically removes host cell sialic acids.12 Thus, the influenza sialidase (neuraminidase) has
been shown to be critical for viral infectivity as newly-budded virus particles aggregate,
requiring the action of the sialidase to effect disaggregation in order to infect further cells and
proliferate.10
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As a result of its critical importance in many biological events several derivatives of sialic acid
have been synthesized as probes of sialic acid processing enzymes, and also as potential ligands
for sialic acid-binding proteins. Indeed, numerous derivatives of sialic acid have been reported
and these include structural or functional group modifications at every position along the nine
carbon backbone.13-19
Figure 1. Sialic acid and various analogues.
Because of its critical importance in the formation of the ketal linkages in glycosides of sialic
acids, substitution of the ring oxygen has been targeted by several groups. For example, sialic
acid congeners containing a cognate nitrogen14 or sulfur20,21 ring atom have been reported. The
effects of such substitutions in glycoconjugates can have profound consequences 22
. Substitution
of the ring oxygen for carbon results in the formation of carbasialic acid derivatives.23
Carbasugars and their analogues are often used to mimic enzymatic substrates as they are
hydrolytically stable and can incorporate functional groups such as carbon-carbon double bonds
that force the six-membered ring into a non-chair conformation.24 Many natural and synthetic
carbasugars with interesting biological activities have been reported in the literature.23,25
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Vasella and co-workers reported an approach to access a close derivative of carbasialic acid,
namely carba-DANA (4),26 the carbocyclic analogue of Neu2en5Ac (2a). Using a chiral pool
approach, they synthesized 4 via an indium-mediated allylation of a suitably protected N-acetyl-
β-D-mannosamine. The key step was performed using a SmI2 mediated radical cyclization to
form the carbocyclic ring. However, this route was developed specifically for the synthesis of
Carba-DANA (and the isomeric cyclohexene iso-Carba-DANA) but not to make carbasialic
acid.26 The synthesis of carbasialic acid 5 (Figure 1) has to the best of our knowledge only been
reported by the group of Ogawa.27,28 Of note, this synthesis of α-carbasialic acid (5) involved
more than 30 steps with an overall yield of 0.48%. Ogawa and co-workers, utilized an optically
resolved Diels-Alder endo-adduct of furan and acrylic acid as their starting material. Key
transformations included: a Horner-Emmons olefination and dihydroxylation sequence to set the
D-glycero side chain; and a vinyl Grignard addition reaction, on an advanced cyclohexanone
intermediate, followed by ozonolysis to establish the pseudo-anomeric α-hydroxy acid.27,28 The
key dihydroxylation, unfortunately, occurred with essentially no stereoselectivity, which
resulted, after separation of the diastereomeric products, in a low yield (44%) at step 12 of the
synthesis. In addition, the protecting group strategy used in this route did not allow any
differentiation of the ring hydroxyls groups from those in the glycerol side chain, thus making
this route less amenable for the synthesis of other carbasialic acid analogues. In this report, we
detail the synthesis of "α"-D-carbasialic acid, in 18 steps and 5% yield, using commercially
available -quinic acid as the starting material.
D
Results and Discussion
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Our retrosynthetic analysis of carbasialic acid is outlined in Scheme 1. We noted that the
cyclohexane scaffold of
D-(-)-quinic acid has the correct α-hydroxy carboxylic acid
stereochemistry and that installation of the desired three carbon side chain via a Stille coupling
entails the use of a readily made vinyl triflate (Scheme 1; 6a R1 = MOM, R2 = TBDMS,29 or 6b
R1 = R2 = TBDMS30,31). That is, we planned to introduce the
D-glycero stereochemistry by an
osmium-catalyzed dihydroxylation, followed by installation of the correct stereochemistry at C-4
and C-5 with a double displacement strategy via an allylic oxirane (Scheme 1, numbering in this
scheme is based on sialic acid).
Scheme 1. Retrosynthetic analysis.
The required (Z)-stannane 7 was synthesized by slight modification of a reported procedure for
preparation of the E-isomer.32 Specifically, reaction of prop-2-yn-1-ol with tributyltin hydride in
hexanes in the presence of a catalytic amount of azobisisobutyronitrile (AIBN) under reflux
conditions yielded (Z)-stannane 7 as the major product. Stille coupling of vinyl triflates 6a and
6b with Z-tributyl stannane 7 gave dienes 8a and 8b (Scheme 2). Selective dihydroxylation of
the exocyclic double bond in 8a and 8b using different reaction conditions (Table 1) yielded
either a complex mixture or the undesired,
L-glycero-configured compound 9, as the major
diastereomer. The absolute stereochemistry of 9a was confirmed by
X-ray crystallography
(Figure S2, supporting information, CCDC number 1534788), and that of the other diene
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mixtures formed by dihydroxylation, of 8a, 8b, and 8d using various reagents, also gave the
undesired L-glycero stereochemistry, as inferred by a comparison of the ratios of the olefinic
protons in the 1H NMR spectra of the crude reaction mixtures (Figure S1, Supporting Data).
Scheme 2. Initial attempts with early stage dihydroxylation strategy.
Given that neither 9a nor 9b possessed the required D-glycero stereochemistry, we also tested a
C5-hydroxy compound (8d) to probe whether the TBDMS group at C5 dictated the outcome of
the dihydroxylation reaction, in favour of the undesired diastereomer. Thus, we converted
lactone 8a into compound 8d by protection of the primary hydroxyl group as a methoxymethyl
(MOM) ether 8c followed by removal of the TBDMS to give alcohol 8d. Disappointingly,
dihydroxylation of diene 8d also gave the undesired
L-glyerco stereoisomer 9d as the major
product (Table 1).
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Table 1. Dihydroxylation conditions tried with olefins 8a, 8b, and 8d.
Olefin
8a
Condition
Product ratio
OsO4/NMO, Acetone/H2O (9:1), 0 °C – RT
3:1 (9a and 10a)
RuCl3/NaIO4/CeCl3, EtOAc/CAN/H2O (3:3:1), 0 °C – RT 2:1(9a and 10a)a
8a
8a
AD-mix-α, t-BuOH/H2O (1:1), 0 °C – RT
AD-mix-β, t-BuOH/H2O (1:1), 0 °C – RT
OsO4/NMO, Acetone/H2O (9:1), 0 °C – RT
Complex mixturea
Complex mixturea
3:1 (9b and 10b)a
2.3:1 (9d and 10d)a
8a
8b
OsO4/NMO, Acetone/H2O (9:1), 0 °C – RT
8d
a
1
Determined by H NMR spectroscopy of the crude reaction mixture (Supporting Data Figure
S1).
We therefore decided to pursue a late stage dihydroxylation strategy using an advanced
intermediate in which we had inverted the configurations at C4 and C5, which necessitated
removal of the conformationally restricting lactone ring. The allylic alcohol 8b was converted to
a tri-silyl protected intermediate 8e using t-butyldimethylsilyl triflate and pyridine (Scheme 3).
Scheme 3. Unsuccessful attempt with a tri-silyl protection.
Initial attempts to open the lactone ring using sodium methoxide led to silyl group migration
from C5 to C4. However, the lactone ring was opened successfully by use of a catalytic amount
of K2CO3 in MeOH to give alcohol 11a, whose structure was confirmed by an observed
correlation between the C4 and hydroxyl protons in the COSY spectrum. The alcohol 11a was
transformed into its mesylate with the expectation that removal of the TBDMS group would
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form the desired oxirane 12 directly. However, upon treatment of the mesylate with tetra n-butyl
ammonium fluoride (TBAF), the reaction did not stop at the epoxide stage, instead yielding a
bicyclic compound 13, which results from an intramolecular nucleophilic opening of the epoxide
1
(Scheme 3). The structure of 13 was established with the aid of H-1H COSY, HSQC, HMBC
and APT analyses (Figures S18–S22 Supporting Data). For instance, a distinctive HMBC
correlation between C9 and the H-5 proton confirmed the bicyclic structure to be 13.
Scheme 4. Synthesis of key intermediate 18.
To avoid this side reaction, the primary hydroxyl group in 8b was protected as a para-
methoxybenzyl (PMB) ether using PMB-trichloroacetimidate33 and La(OTf)3, as shown in
Scheme 4. The resulting lactone (8e) was then cleaved to give the corresponding methyl ester
(11b) using K2CO3 in MeOH and the secondary alcohol was converted into mesylate 14.
Gratifyingly, treatment of 14 with TBAF gave the desired oxirane 15, which underwent
regioselective ring opening with sodium azide and ammonium chloride to give the desired
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secondary alcohol 16 in a 94% yield from oxirane 15. OsO4-catalyzed dihydroxylation of 16
gave an inseparable mixture of diastereomers (17), which were converted into the corresponding
acetonides 18 and 19 using 2,2-dimethoxypropane and a catalytic amount of para-
toluenesulfonic acid (PTSA) in ethyl acetate. The diastereomeric acetonides were successfully
separated by column chromatography (18 and 19, 2.5:1, 70%). The major diastereomer was
confirmed to be the desired diastereomer 18, possessing the required D-glycero configuration, by
converting it into the completely protected 6a-carba-α-sialic acid 21 (Scheme 5) whose NMR
data matched those reported by Ogawa and co-workers.27,28
Compound 18 was converted to the corresponding amine by bubbling hydrogen gas into a stirred
heterogeneous mixture of Lindlar catalyst and the azide 18 in ethanol. This material was
transformed into an unsaturated analogue of 6a-carba-sialic acid (20) by acetylation (acetic
anhydride and pyridine), ester hydrolysis (aqueous sodium hydroxide in MeOH), and PMB and
acetonide deprotection (trifluoroacetic acid) in a combined yield of 81% from azide 18 (Scheme
5). Azide 18 was also converted into the 5-azidocyclohexene analogue 22 in an 80% yield over
two steps (Scheme 5). Similarly, the minor diastereomer 19 was converted into the
corresponding N-acetyl and azido derivatives, 23 and 24 (Scheme S1, Supporting Data).
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HO COOH
HO COOMe
1. Lindlar's catalyst, H2, EtOH
2. Ac2O, Py
3. NaOH, MeOH, Water (1:1)
4. TFA, CH2Cl2, H2O
HO
HO
PMBO
O
OH
OH
OH NHAc
O
N3
18
81%
20
Pd-C,H2
50 psi
EtOH/H2O
1. NaOH, MeOH, Water (1:1)
2. TFA, CH2Cl2, H2O
80%
HO COOH
HO COOH
HO
HO
HO
OH
H
OH
N3
HO
OH NHAc
HO
dr ~3:1 (5 major)
22
1. Ac2O, DMAP, Py
2. TMS-CHN2
EtOAc/MeOH
H
HO
HO
H
COOH
OH
AcO COOMe
OH
noe
5
6
AcO
AcO
3
6
1
4
2
1
AcHN
5
2
3
4
OH
3
OAc
H
1
H
H
OAc NHAc
5 major
21, 25% for 3 steps
Scheme 5. Synthesis of 20 and 6a-carba-α-
Finally, we completed the synthesis of 6a-carba-α- -sialic acid (5) by hydrogenation of 20 using
10% Pd-C and 50 psi H2 to give a mixture of compounds with the major component identified as
D-sialic acid (5).
D
1
1
5 using H NMR coupling constant analysis, H-1H COSY and 1D NOE spectroscopy (Scheme
5). Thus, the H4 resonance (numbering based on Ogawa's assignments) was observed as a
doublet of doublets with two large coupling constants, 11.9 Hz and 12.1 Hz, confirming its
trans-diaxial relationship with H3 and H5 (Scheme 5). NOE correlations between H3 and H5
also corroborated the assignment of the R-configuration for the newly generated stereocenter at
C5. This assignment was further confirmed by the isolation of 21 as the major diastereomer by
treating the mixture with acetic anhydride and pyridine, followed by esterification using
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trimethylsilyl diazomethane, as shown in Scheme 5. Detailed 1D and 2D NMR experiments and
HRMS analysis confirmed the structure of compound 21.
1
The H NMR data of 21 are essentially identical to those reported by Ogawa and co-workers;27
all chemical shifts agree to within ± 0.03 ppm (Table 2), which supports our earlier assignment
of absolute configuration of the glycerol side chain stereocenters (vide supra). Complete 1H and
13C NMR spectral assignments are provided (see experimental section).
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1
Table 2. Comparison of H NMR data of per-acetylated carba-sialic acid methyl ester 21 with
the reported data.
1H NMR data (CDCl3)
Position
Ogawa et al {Ogawa, 1995 #1171}
This work (600 MHz)
(270 MHz)
H-1'
NH
H-3
5.24 (dd, J1',5 = 2.4, J1',2' = 9.0 Hz, 1H) 5.24 (dd, J1',5 = 2.6, J1',2' = 8.4 Hz, 1H)
5.22 (d, JNH,4 = 10.5 Hz, 1H)
Not reported
Not reported
5.15 (ddd, J3,2ax = 11.4, J3,2eq = 4.8, J3,4
= 10.5 Hz, 1H)
5.13 (ddd, J2',3'a = 3.0, J2',3'b = 6.6 Hz,
1H)
H-2'
Not reported
4.23 (dd, J3'a,2' = 3.0, J3'a,3'b = 12.0 Hz, 4.23 (dd, J2',3'a = 2.9, J3'a,3'b = 12.5 Hz,
H-3'a
1H)
1H)
H-3'b
3.98 (dd, 1H)
Not reported
3.95 (ddd, J4,5 = J4,NH = J4,3 = 10.5 Hz, 3.98 (ddd, J4,5 = J4,NH = J4,3 = 10.5 Hz,
H-4
1H)
1H)
COOCH3
H-2eq
3.74 (s, 3H)
3.74 (s, 3H)
2.77 (ddd, J2eq,3 = 4.8, J2eq,6eq = 2.4, 2.77 (ddd, J2eq,3 = 2.2, J2eq,6eq = 2.6,
J2ax,2eq = 12.6 Hz, 1H) J2ax,2eq = 12.5 Hz, 1H)
2.54 (ddd, J6eq,5 = 3.0, J6ax,6eq = 13.2 2.54 (ddd, J6eq,5 = 2.6, J6ax,6eq = 13.2
H-6eq
Hz, 1H)
Hz, 1H)
H-5
2.11 (m, 1H)
2.11 (m, 1H)
5
x
OAc 2.11, 2.10, 2.06, 2.03, 2.01 (s, each 2.11, 2.10, 2.06, 2.04, 2.01 (s, each
(CH3)
3H)
3H)
NHAc(CH3) 1.91 (s, 3H)
1.91 (3H)
1.78 (dd, J2ax,3 = 11.4, J2ax,2eq = 12.6
Hz, 1H)
H-2ax
H-6ax
1.79 (t, J2ax,3 = J2ax,2eq = 12.5 Hz, 1H)
1.71 (t, J6ax,5 = 13.2 Hz, 1H)
1.71 (dd, J6ax,5 = 13.2 Hz, 1H)
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Conclusions.
In summary, we have developed a versatile synthetic route to carbasialic acid and its derivatives
using Stille coupling and osmium-catalyzed dihydroxylation as key steps. The described
chemistry paves the way for synthesis of a variety of carbasialic acid derivatives. For example,
one can take advantage of epoxide 15 to access a variety of C-5-modified analogues by simply
changing the reacting nucleophile. In addition, the protecting group strategy used here will
permit further modifications of either the side chain or the cyclohexane ring selectively.
Experimental section
1
13
General methods. H and C NMR spectra were recorded at 600 or 500 MHz and 150 or 125
1
MHz, respectively. All assignments were confirmed with the aid of two-dimensional H–1H
1
1
(COSY) and/or H–13C (HSQC) and H–13C (HMBC) experiments and attached proton test
(APT) using standard pulse programs. Processing of the spectra was performed with
MestReNova software. Analytical thin-layer chromatography (TLC) was performed on
aluminum plates precoated with silica gel 60F-254 as the adsorbent. The developed plates were
air-dried, exposed to UV light and/or sprayed with a solution containing 1% Ce(SO4)2 and 1.5%
molybdic acid in 10% aqueous H2SO4, and heated. Column chromatography was performed with
CombiFlash Rf system using RediSep Rf gold columns from Teledyne ISCO. High resolution
mass spectra were obtained by the electrospray ionization method, using an Agilent 6210 TOF
LC/MS high resolution magnetic sector mass spectrometer.
(Z)-3-(Tributylstannyl)-prop-2-en-1-ol (7).
A mixture of tributyltin hydride (21.6 g, 74.2 mmol), prop-2-yn-1-ol (4.5g, 80.4 mmol) and
AIBN (200 mg, catalytic amount) in 200 mL of hexane was refluxed for 12 h. The solvent was
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removed under reduced pressure and the residue was purified by column chromatography (0–
10% EtOAc/Hex) to give the E-isomer (4.9 g, 19%, colourless oil, Rf: 0.5 in 20% EtOAc/Hex)
and an inseparable mixture of Z-isomer (7) and the regioisomer, 2-(tributylstannyl)-prop-2-en-1-
ol in 3:1 ratio (12.1 g, 47%, colourless oil, Rf: 0.6 in 20% EtOAc/Hex). 1H NMR spectra of the
pure E-isomer and the mixture containing Z-isomer (7) and the regioisomer matched the reported
data.34,35 For the Stille coupling reaction, the mixture containing the Z-isomer and its regioisomer
was used without any further purification.
1
Data for (E)-3-(Tributylstannyl)-prop-2-en-1-ol, H NMR (400 MHz, CDCl3): δ 6.25–6.12
(m, 2H, H-2, H-3), 4.20–4.15 (m, 2H, H-1a, H-1b), 1.57–1.38 (m, 6H, Bu3Sn), 1.36–1.24 (m, 6H,
13
Bu3Sn), 0.99–0.80 (m, 15H, Bu3Sn). C NMR (100 MHz, CDCl3): δ 147.2 (C-2), 128.5 (C-3),
66.6 (C-1), 29.2, 27.4, 13.8, 9.6 (C's of Bu3Sn group).
1
Data for (Z)-3-(Tributylstannyl)-prop-2-en-1-ol, H NMR (500 MHz, CDCl3): δ 6.86–6.52
(m, 1H, H-2), 6.16–5.99 (m, 1H, H-3), 4.15–4.07 (m, 2H, H-1a, H-1b), 1.56–1.41 (m, 6H,
Bu3Sn), 1.36–1.24 (m, 6H, Bu3Sn), 0.94–0.85 (m, 15H, Bu3Sn).
1
Data for 2-(Tributylstannyl) prop-2-en-1-ol, H NMR (500 MHz, CDCl3): δ 6.02–5.74 (m,
1H, H-3a), 5.32–5.17 (m, 1H, H-3b), 4.33–4.23 (m, 2H, H-1a, H-1b), 1.56–1.41 (m, 6H, Bu3Sn),
1.36–1.24 (m, 6H, Bu3Sn), 0.94–0.85 (m, 15H, Bu3Sn).
(1R,4R,5R)-1,4-bis((tert-butyldimethylsilyl)oxy)-3-((Z)-3-hydroxyprop-1-en-1-yl)-6-
oxabicyclo[3.2.1]oct-2-en-7-one (8b).
(Pd)2(dba)3 (531 mg, 0.58 mmol) and AsPh3 (1.2 g, 3.84 mmol) were added to NMP (170 mL)
under N2 atmosphere. After 5 min stirring at room temperature, a solution of triflate 6b31 (10.2 g,
19.2 mmol) in NMP (60 mL) was added dropwise and the stirring was continued for additional
10 min at RT. A solution of stannane 7 (7.3 g, 21.1 mmol) in NMP (20 mL) was then added
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dropwise at RT. After the addition, the reaction mixture was stirred at 40 °C for 3 h. The reaction
mixture was cooled to room temperature and quenched with saturated solution of potassium
fluoride (150 mL) and extracted with ether (2 × 150 mL). The ether layer was washed with water
(2 ×100 mL) and then with brine (100 mL). The organic layer was dried over anhydrous Na2SO4,
filtered and condensed to dryness under reduced pressure. The crude material was purified by
flash column chromatography on silica gel (0-50% EtOAc/Hex) to afford the title compound as
pale yellow syrup (6.0 g, 71%).
1H NMR (600 MHz, CDCl3): δ 5.90–5.83 (m, 2H, H-1ʹ and H-2ʹ), 5.75 (br d-like, 1H, H-2),
4.49 (ddd, 1H, J5,6a = 5.5 Hz, J5,4 = 3.2 Hz, J5,6b = 1.0 Hz, H-5), 4.35–4.21 (m, 2H, H-3ʹ), 4.06 (d,
1H, H-4), 2.42–2.32 (m, 2H, H-6a and H-6b), 0.92 and 0.89 (s, 9H, -C(CH3)3), 0.20, 0.17, 0.13
13
and 0.12 (s, 3H, -CH3). C NMR (150 MHz, CDCl3): δ 175.7 (-C=O), 134.9 (C-3), 134.6 (C-
2), 134.3 (HO-CH2-CH=CH-), 127.8 (HO-CH2-CH=CH-), 76.0 (C-5), 74.9 (C-1), 68.1 (C-4),
59.7 (HO-CH2-CH=CH-), 37.1 (C-6), 25.8 and 25.7 (3C, -C(CH3)3), 18.2 and 18.1 (-C(CH3)3), –
2.90, –2.91, –4.4 and –4.5 (-CH3). HRMS calcʹd for C22H41O5Si2 [M+H]+: 441.2493, found:
441.2487, calcʹd for C22H44NO5Si2 [M+NH4]+: 458.2758, found: 458.2746. TLC (10%
EtOAc/Hex), Rf: 0.2
(1R,4R,5R)-1,4-bis((tert-butyldimethylsilyl)oxy)-3-((Z)-3-((4-methoxybenzyl)oxy)prop-1-en-
1-yl)-6-oxabicyclo[3.2.1]oct-2-en-7-one (8e).
To a stirred solution of lactone 8b (3.3 g, 7.49 mmol) and para-methoxybenzyl
trichloroacetimidate (3.2 g, 11.23 mmol) in toluene (150 mL) was added La(OTf)3 (220 mg, 0.37
mmol) at RT and stirring was continued for 2 h. Toluene was removed under reduced pressure
and the crude material was purified by flash column chromatography on silica gel (0-20%
EtOAc/Hex) to afford the title compound as pale yellow syrup along with some minor PMB-
16

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related impurities (3.4 g, 81%). A small amount the crude material was purified by column
chromatography to give an analytical sample of 8e for characterization purposes.
1H NMR (600 MHz, CDCl3): δ 7.25 (d, 2H, J = 8.6 Hz, Ar), 6.87 (d, 2H, Ar), 5.92–5.84 (m,
2H, -O-CH2-CH=CH), 5.74 (br d-like, 1H, H-2), 4.48 (ddd, 1H, J5,6a = 4.8 Hz, J5,6b = 1.3 Hz, J5,4
= 3.3 Hz, H-5), 4.44–4.40 (m, 2H, Ar-CH2-), 4.14–4.08 (m, 2H, H-3ʹa and H-3ʹb), 4.07 (d, 1H,
H-4), 3.80 (s, 3H, CH3O-Ar), 2.36 (m, 2H, H-6a and H-6b), 0.91 and 0.89 (s, 9H, -C(CH3)3),
0.17 and 0.14 (s, 3H, -CH3), 0.12 (s, 6H, -CH3). 13C NMR (150 MHz, CDCl3): δ 175.7 (-C=O),
159.4 (ipso Ar-C), 135.0 (C-3), 134.6 (C-2), 132.5 (-O-CH2-CH=CH), 130.2 (ipso Ar-C), 129.6
(2C, Ar), 128.4 (-O-CH2-CH=CH), 114.0 (2C, Ar), 76.1 (C-5), 75.0 (C-1), 72.2 (Ar-CH2-), 68.3
(C-4), 66.6 (-O-CH2-CH=CH), 55.4 (CH3O-Ar), 37.1 (C-6), 25.8 and 25.7 (3C, -C(CH3)3), 18.2
and 18.1 (-C(CH3)3), –2.9 (2C, -CH3), –4.4 and –4.5 (-CH3). HRMS calcʹd for C30H52NO6Si2
[M+NH4]+: 578.3333, found: 578.3326. TLC (10% EtOAc/Hex), Rf: 0.6
Methyl
(1R,4R,5R)-1,4-bis((tert-butyldimethylsilyl)oxy)-5-hydroxy-3-((Z)-3-((4-
methoxybenzyl)oxy)prop-1-en-1-yl)cyclohex-2-ene-1-carboxylate (11b).
To a stirred solution of lactone 8e (3.4 g, 6.06 mmol) in MeOH (90 mL) at 0 °C was added solid
anhydrous K2CO3 (3.5 g) in one portion and the stirring was continued at 0 °C for 10 min. The
reaction mixture was then neutralized with glacial acetic acid (2.0 mL) at 0 °C and precipitated
white solid was removed by filtration. The filtrate was concentrated to dryness under reduced
pressure. The crude syrup was taken in ethyl acetate (150 mL) and washed with water (2 × 100
mL). The organic layer was dried over anhydrous Na2SO4, filtered and condensed to dryness
under reduced pressure. The crude material was purified by flash column chromatography on
silica gel (0-20% EtOAc/Hex) to afford the title compound as pale yellow syrup (2.2 g, 74%
based on recovered unreacted starting material, 0.6 g).
17

ACCEPTED MANUSCRIPT
1H NMR (600 MHz, CDCl3): δ 7.25 (d, 2H, J = 8.8 Hz, Ar), 6.86 (d, 2H, Ar), 6.10 (br d, 1H,
J1ʹ,2ʹ = 11.7 Hz, -O-CH2-CH=CH), 5.83 (dt, 1H, J2ʹ,3ʹa = 6.5, J2ʹ,3ʹb = 6.2 Hz, -O-CH2-CH=CH),
5.51 (s, 1H, H-2), 4.41 (s, 2H, Ar-CH2-), 4.19 (ddd, 1H, J = 12.4, 1.5 Hz, -O-CH2-CH=CH), 4.14
(ddd, 1H, J = 12.4, 1.5 Hz, -O-CH2-CH=CH), 3.94 (m, 1H, H-5), 3.91 (d, 1H, J4,5 = 5.4 Hz, H-
4), 3.80 (s, 3H, CH3O-Ar), 3.72 (s, 3H, -COOCH3), 2.82 (br s, 1H, -OH), 2.24-2.17 (m, 2H, H-
13
6), 0.90 and 0.87 (s, 9H, -C(CH3)3), 0.12, 0.10, 0.09 and 0.05 (s, 3H, -CH3). C NMR (150
MHz, CDCl3): δ 175.4 (-COOCH3), 159.3 (ipso Ar-C), 138.5 (C-3), 130.9 (-O-CH2-CH=CH),
130.8 (-O-CH2-CH=CH), 130.5 (ipso Ar-C), 129.5 (2C, Ar), 128.0 (C-2), 113.9 (2C, Ar), 75.0
(C-1), 73.4 (C-4), 72.2 (Ar-CH2-), 70.4 (C-5), 66.7 (-O-CH2-CH=CH), 55.4 (CH3O-Ar), 52.7 (-
COOCH3), 38.0 (C-6), 26.0 and 25.8 (3C, -C(CH3)3), 18.4 and 18.3 (-C(CH3)3), –2.7, –2.8, –4.1
and –4.2 (-CH3). HRMS calcʹd for C31H52NaO7Si2 [M+Na]+: 615.3149, found: 615.3148, calcʹd
for C31H52KO7Si2 [M+K]+: 631.2889, found: 631.2881. TLC (20% EtOAc/Hex), Rf: 0.2.
Methyl
(1R,4R,5R)-1,4-bis((tert-butyldimethylsilyl)oxy)-3-((Z)-3-((4-
methoxybenzyl)oxy)prop-1-en-1-yl)-5-((methylsulfonyl)oxy)cyclohex-2-ene-1-carboxylate
(14).
Methanesulfonyl chloride (0.57 mL, 7.42 mmol) was added dropwise to a stirred solution of
compound 11b (2.2 g, 3.71 mmol) in pyridine (30 mL) at 0 °C under N2 atmosphere. After the
addition, the reaction mixture was stirred at room temperature for 3h. The reaction mixture was
diluted with ether (150 mL) and washed with water (2 × 100 mL). The organic layer was dried
over anhydrous Na2SO4, filtered and condensed to dryness under reduced pressure. The crude
material was purified by flash column chromatography on silica gel (0-20% EtOAc/Hex) to
afford the title compound as colourless syrup (2.4 g, 96%).
18

ACCEPTED MANUSCRIPT
1H NMR (600 MHz, CDCl3): δ 7.25 (d, 2H, J = 8.6 Hz, Ar), 6.87 (d, 2H, Ar), 6.07 (br d, 1H, J
= 11.7 Hz, -O-CH2-CH=CH), 5.87 (dt, 1H, J = 6.5, 6.2 Hz, -O-CH2-CH=CH), 5.67 (s, 1H, H-2),
4.82 (ddd, 1H, J5,6a = 8.3 Hz, J5,6b = 2.8 Hz, J5,4 = 5.5 Hz, H-5), 4.43 (m, 2H, Ar-CH2-), 4.22
(ddd, 1H, J = 12.5, 1.3 Hz, -O-CH2-CH=CH), 4.17 (ddd, 1H, J = 1.5 Hz, -O-CH2-CH=CH), 4.08
(d, 1H, H-4), 3.80 (s, 3H, CH3O-Ar), 3.71 (s, 3H, -COOCH3), 3.0 (s, 3H, -OMs), 2.52 (dd, 1H,
J6a,6b = 13.3 Hz, H-6a), 2.41 (dd, 1H, H-6b), 0.90 and 0.87 (s, 9H, -C(CH3)3), 0.12, 0.10, 0.09
13
and 0.05 (s, 3H, -CH3). C NMR (150 MHz, CDCl3): δ 172.9 (-COOCH3), 159.4 (ipso Ar-C),
136.5 (C-3), 131.7 (-O-CH2-CH=CH), 130.5 (ipso Ar-C), 130.0 (-O-CH2-CH=CH), 129.5 (2C,
Ar), 129.3 (C-2), 113.9 (2C, Ar), 79.9 (C-5), 74.2 (C-1), 72.3 (Ar-CH2-), 70.2 (C-4), 66.7 (-O-
CH2-CH=CH), 55.4 (CH3O-Ar), 52.6 (-COOCH3), 38.6 (-OMs), 36.2 (C-6), 25.9 and 25.7 (3C, -
C(CH3)3), 18.4 and 18.1 (-C(CH3)3), –2.8, –2.9, –4.1 and –4.2 (-CH3). HRMS calcʹd for
C32H58NO9SSi2 [M+NH4]+: 688.3371, found: 688.3352, calcʹd for C32H54NaO9SSi2 [M+Na]+:
693.2925, found: 693.2912, calcʹd for C32H54KO9SSi2 [M+K]+: 709.2664, found: 709.2648. TLC
(20% EtOAc/Hex), Rf: 0.5
Methyl (1R,4R,5S)-4,5-epoxy-1-hydroxy-3-((Z)-3-((4-methoxybenzyl)oxy)prop-1-en-1-yl)-
cyclohex-2-ene-1-carboxylate (15).
Tetrabutylammonium fluoride (TBAF) (3.6 mL, 3.58 mmol, 1M in THF) was added to a stirred
solution of mesylate 14 (2.4 g, 3.58 mmol) in dry THF (250 mL) under N2 atmosphere and the
resulting solution was stirred at room temperature for 2h. The reaction mixture was then
concentrated to dryness under reduced pressure at room temperature and the crude material was
purified by flash column chromatography on silica gel (0-30% EtOAc/Hex) to afford the title
compound as colourless syrup (1.08 g, 87%).
19

ACCEPTED MANUSCRIPT
1H NMR (600 MHz, CDCl3): δ 7.25 (d, 2H, J = 8.6 Hz, Ar), 6.87 (d, 2H, Ar), 6.18 (br dd, 1H, J
= 11.7 Hz, 1.3 Hz, -O-CH2-CH=CH), 5.95 (br s, 1H, H-2), 5.91 (dt, 1H, J = 6.5, 6.2 Hz, -O-CH2-
CH=CH), 4.46 (s, 2H, Ar-CH2-), 4.24-4.17 (m, 2H, -O-CH2-CH=CH), 3.80 (s, 6H, CH3O-Ar, -
COOCH3), 3.77-3.74 (m, 1H, H-5), 3.53 (dd, 1H, J4,5 = 4.1 Hz, J4,2 = 2.0 Hz, H-4), 3.10 (s, 1H, -
OH), 2.62 (dd, 1H, J6a,6b = 15.5 Hz, J6a,5 = 2.3 Hz, H-6a), 2.14 (dd, 1H, J6b,5 = 1.0 Hz, H-6b). 13C
NMR (150 MHz, CDCl3): δ 172.9 (-COOCH3), 159.4 (ipso Ar-C), 135.7 (C-3), 131.9 (-O-CH2-
CH=CH), 131.4 (C-2), 130.2 (ipso Ar-C), 129.7 (-O-CH2-CH=CH*), 129.6 (2C, Ar*), 114.0
(2C, Ar), 72.5 (Ar-CH2-), 71.4 (C-1), 66.4 (-O-CH2-CH=CH), 55.4 (CH3O-Ar), 54.0 (C-5), 53.2
(-COOCH3), 50.9 (C-4), 32.5 (C-6). *denotes interchangeable assignments. HRMS calcʹd for
C19H22NaO6 [M+Na]+: 369.1314, found: 369.1307. TLC (30% EtOAc/Hex), Rf: 0.2.
Methyl
(1R,4S,5S)-4-azido-1,5-dihydroxy-3-((Z)-3-((4-methoxybenzyl)oxy)prop-1-en-1-
yl)cyclohex-2-ene-1-carboxylate (16).
To a stirred solution of epoxide 15 (980 mg, 2.83 mmol) in MeOH (24 mL) was added water (6
mL) followed by sodium azide (1.1 g, 16.98 mmol) and NH4Cl (420 mg, 7.92 mmol). The
resulting mixture was stirred at 75 °C for 1 h. MeOH was carefully removed by rotary
evaporation at room temperature and the aqueous mixture was then extracted with ethyl acetate
(2 × 100 mL). Ethyl acetate layer was washed with water (2 × 100 mL). The organic layer was
dried over anhydrous Na2SO4, filtered and condensed to dryness at room temperature under
reduced pressure. The crude material was purified by flash column chromatography on silica gel
(0-40% EtOAc/Hex) to afford the title compound as colourless syrup (1.04 g, 94%). Note:
Heating of the material (neat or in solution) at above 30 °C was avoided at any time during the
workup and purification process as a safety precaution.
20

ACCEPTED MANUSCRIPT
1H NMR (600 MHz, CDCl3): δ 7.25 (d, 2H, J = 8.6 Hz, Ar), 6.87 (d, 2H, J = 8.6 Hz, Ar), 6.07
(br d, 1H, J = 11.7 Hz, -O-CH2-CH=CH), 5.96 (dt, 1H, J = 6.5, 6.2 Hz, -O-CH2-CH=CH), 5.67
(s, 1H, H-2), 4.46-4.42 (m, 2H, Ar-CH2-), 4.22-4.13 (m, 3H, -O-CH2-CH=CH, H-5), 3.84 (d, 1H,
J4,5 = 3.5 Hz, H-4), 3.80 (s, 3H, CH3O-Ar), 3.78 (s, 3H, -COOCH3), 3.70 (s, 1H, -OH), 3.33 (br
d, 1H, -OH), 2.32 (dd, 1H, J6a,6b = 14.4 Hz, J6a,5 = 2.8 Hz, H-6a), 2.09 (dd, 1H, J6b,5 = 5.5 Hz, H-
13
6b). C NMR (150 MHz, CDCl3): δ 174.8 (-COOCH3), 159.4 (ipso Ar-C), 133.5 (C-3), 133.1
(-O-CH2-CH=CH), 130.2 (ipso Ar-C), 130.0 (-O-CH2-CH=CH), 129.6 (2C, Ar), 128.8 (C-2),
114.0 (2C, Ar), 72.3 (Ar-CH2-), 71.8 (C-1), 68.6 (C-5), 66.3 (-O-CH2-CH=CH), 62.5 (C-4), 55.4
(CH3O-Ar), 53.8 (-COOCH3), 35.0 (C-6). HRMS calcʹd for C19H23N3NaO6 [M+Na]+: 412.1485,
found: 412.1479. TLC (40% EtOAc/Hex), Rf: 0.3.
(1R,4S,5S)-4-Acetamido-1,5-dihydroxy-3-((4S,5R) and (4R,5S)-5-(hydroxymethyl)-2,2-
dimethyl-1,3-dioxolan-4-yl)cyclohex-2-ene-1-carboxylic acid (18 and 19).
To a stirred solution of azide 16 (1.04 g, 2.7 mmol) in a mixture of acetone and water (9:1, 50
mL) at room temperature were added N-methylmorpholine N-oxide (344 mg, 2.9 mmol) and
OsO4 (0.5 mL, 0.05 mmol, 2.5 wt% solution of in t-butanol). The reaction mixture was stirred at
room temperature for 3 days before it was quenched with saturated solution of NaHSO3 (35 mL).
After being stirred for an additional 15 min, most of the acetone was carefully removed by rotary
evaporation at ambient temperature, and the aqueous mixture was extracted with ethyl acetate (2
× 100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and condensed to
dryness at room temperature under reduced pressure to afford the title compound as an
1
inseparable mixture of diastereomers 17 (1.08 g, colourless syrup). H NMR spectrum of the
crude material indicated a ~1:3 mixture of diastereomers and it was taken to the next step
without any purification. Note: Heating of the material (neat or in solution) at above 30
°C was
21

ACCEPTED MANUSCRIPT
avoided at any time during the workup as a safety precaution. To a stirred solution of diols 17
(1.07 g, 2.5 mmol) in ethyl acetate (150 mL) was added 2,2-dimethoxypropane (4 mL, 33 mmol)
and a catalytic amount of p-toluenesulfonic acid (22 mg). The reaction mixture was subjected to
slow distillation at room temperature using a rotary evaporator for 30 min. Another portion of
2,2-dimethoxypropane (4 mL) was added and distillation was continued for an additional 15 min.
Continuous removal of ethyl acetate (~50 mL) along with the by-product methanol led to
complete consumption of starting material as indicated by TLC analysis. The reaction was
quenched by the addition of triethylamine (80 µL) and then concentrated to dryness under
reduced pressure. The crude material was purified by flash column chromatography on silica gel
(0-40% EtOAc/Hex) to afford pure diastereomers as colourless syrups (625 mg of the desired,
and more polar, diastereomer 18 [yield 50% over two steps], and 250 mg of less polar
diastereomer 19). Note: Heating of the material (neat or in solution) at above 30 °C was avoided
at any time during the workup and purification process as a safety precaution.
Data for polar diastereomer 18, 1H NMR (600 MHz, CDCl3): δ 7.24 (d, 2H, J = 8.6 Hz, Ar),
6.87 (d, 2H, J = 8.6 Hz, Ar), 5.86 (br dd-like, 1H, H-2), 4.76 (br d, 1H, J1ʹ,2ʹ = 7.5 Hz, H-1ʹ),
4.52–4.43 (m, 3H, Ar-CH2-, H-2ʹ), 4.16–4.12 (m, 1H, H-5), 4.05 (d, 1H, J4,5 = 3.9 Hz, H-4), 3.80
(s, 3H, CH3O-Ar), 3.77 (s, 3H, -COOCH3), 3.71 (s, 1H, -OH), 3.48 (dd, J3ʹa,3ʹb = 10.9 Hz, J3ʹa,2ʹ
=
4.2 Hz, H-3ʹa), 3.45 (dd, J3ʹb,2ʹ = 6.5 Hz, H-3ʹb), 3.31 (d, 1H, J5,OH = 6.2 Hz, -OH), 2.36 (dd, 1H,
J6a,6b = 14.5 Hz, J6a,5 = 2.6 Hz, H-6a), 2.05 (dd, 1H, J6b,5 = 5.5 Hz, H-6b), 1.57 and 1.40 (2 s, 6H,
13
-O-C(CH3)2-O-). C NMR (150 MHz, CDCl3): δ 174.5 (-COOCH3), 159.5 (ipso Ar-C), 134.9
(C-3), 129.9 (ipso Ar-C), 129.8 (2C, Ar), 128.3 (C-2), 114.0 (2C, Ar), 109.3 (-O-C(CH3)2-O-),
78.3 (C-2ʹ), 78.1 (C-1ʹ), 73.4 (Ar-CH2-), 71.5 (C-1), 69.2 (C-5), 68.8 (C-3ʹ), 60.2 (C-4), 55.4
(CH3O-Ar), 53.4 (-COOCH3), 35.2 (C-6), 27.2 and 24.7 (2C, -O-C(CH3)2-O-). HRMS calcʹd for
22

ACCEPTED MANUSCRIPT
calcʹd for C22H29N3NaO8 [M+Na]+: 486.1852, found: 486.1847. TLC (5% MeOH/CH2Cl2), Rf:
0.4.
1
Data for less polar diastereomer 19, H NMR (600 MHz, CDCl3): δ 7.23 (d, 2H, J = 8.6 Hz,
Ar), 6.87 (d, 2H, J = 8.6 Hz, Ar), 6.14 (br dd-like, 1H, H-2), 4.85 (dd, 1H, J1ʹ,2ʹ = 6.2 Hz, J1ʹ,2
=
1.6 Hz, H-1ʹ), 4.45 and 4.40 (2 d, 2H, JAB = 11.6 Hz, Ar-CH2-), 4.42–4.38 (m, 1H, H-2ʹ), 4.24–
4.18 (m, 1H, H-5), 3.84 (d, 1H, J4,5 = 3.9 Hz, H-4), 3.81 (s, 3H, -COOCH3), 3.80 (s, 3H, CH3O-
Ar), 3.74 (s, 1H, -OH), 3.47 (dd, J3ʹa,3ʹb = 10.2 Hz, J3ʹa,2ʹ = 7.9 Hz, H-3ʹa), 3.32 (dd, J3ʹb,2ʹ = 4.5 Hz,
H-3ʹb), 3.22 (br d-like, 1H, -OH), 2.34 (dd, 1H, J6a,6b = 14.5 Hz, J6a,5 = 2.8 Hz, H-6a), 2.06 (dd,
1H, J6b,5 = 5.8 Hz, H-6b), 1.45 and 1.39 (2 s, 6H, -O-C(CH3)2-O-). 13C NMR (150 MHz,
CDCl3): δ 174.6 (-COOCH3), 159.6 (ipso Ar-C), 132.5 (C-3), 129.8 (2C, Ar), 129.5 (ipso Ar-C),
126.4 (C-2), 114.1 (2C, Ar), 108.8 (-O-C(CH3)2-O-), 76.2 (C-2ʹ), 76.04 (C-1ʹ), 73.2 (Ar-CH2-),
71.4 (C-1), 68.9 (C-5), 68.8 (C-3ʹ), 60.7 (C-4), 55.4 (CH3O-Ar), 53.6 (-COOCH3), 35.5 (C-6),
27.8 and 25.4 (2C, -O-C(CH3)2-O-). HRMS calcʹd for C22H33N4O8 [M+NH4]+: 481.2298, found:
481.2291, calcʹd for C22H29N3NaO8 [M+Na]+: 486.1852, found: 486.1844. TLC (5%
MeOH/CH2Cl2), Rf: 0.5.
(1R,4S,5S)-4-acetamido-1,5-dihydroxy-3-((1S,2R)-1,2,3-trihydroxypropyl)cyclohex-2-ene-1-
carboxylic acid (20).
Hydrogen gas was bubbled through a mixture of compound 18 (107 mg, 0.23 mmol) and Lindlar
catalyst (290 mg) in ethanol (10 mL) at room temperature for 4 h. The reaction mixture was
filtered through Celite and washed with MeOH (15 mL). The filtrates were combined and
concentrated to dryness. The crude amino compound (100 mg) was dissolved in pyridine (2.5
mL) and acetic anhydride (0.4 mL) was added at 0 °C. The stirring was continued at room
temperature for 2h. Pyridine was then removed under reduced pressure and the residue was
23

ACCEPTED MANUSCRIPT
subjected to a filter column (silica gel, 10% MeOH/CH2Cl2) to give a mixture of diacetyl
derivative resulting from the acetylation of both C-4 amino and C-5 hydroxyl groups along with
a small amount of 4-hydroxy 5-N-acetyl derivative. The mixture (98 mg, 0.18 mmol) was then
dissolved in methanol (4 mL) and a solution of NaOH (22 mg, 0.54 mmol) in water (4 mL) was
added at 0 °C. After being stirred at 0 °C for 4 h, the reaction mixture was neutralized with IR-
120 (H+ form) resin at 0 °C. The resin was filtered off and washed with methanol (3 × 5 mL).
The filtrates were combined and concentrated under reduced pressure. The residue (90 mg) was
then dissolved in a mixture of dichloromethane (10 mL) and water (1 mL) and trifluoroacetic
acid (3 mL) was added at 0 °C. The reaction mixture was stirred at room temperature for 3 h
before it was concentrated under reduced pressure. The residue was taken in water (5 mL) and
washed with ethyl acetate (2 × 5 mL). The aqueous layer was concentrated to give an off-white
foam (57 mg, 81% yield over 4 steps) and 1H NMR indicated the purity of the crude material is
greater than 95%. An analytical sample was purified by reverse phase HPLC (a gradient of 2–
100% ACN/H2O) to afford the title compound 20 as a colourless gum.
1H NMR (600 MHz, D2O): δ 5.98 (br s, 1H, H-2), 4.56 (br d, 1H, J4,5 = 5.4 Hz, H-4), 4.11 (m,
2H, H-5, H-1ʹ), 3.84 (br d, 1H, J3ʹa,3ʹb = 11.9 Hz, H-3ʹa), 3.80 (br-ddd-like, 1H, J2ʹ,3ʹb = 6.3 Hz,
J2ʹ,1ʹ = 7.6 Hz, H-2ʹ), 3.67 (br dd, 1H, H-3ʹb), 2.44 (br d, 1H, J6a,6b = 13.5 Hz, H-6a), 2.05 (s, 3H,
NHCOCH3), 1.99 (dd, 1H, J6b,5 = 8.4 Hz, H-6b). 13C NMR (150 MHz, D2O): δ 178.5 (-COOH),
174.0 (-NHCOCH3), 137.4 (C-3), 129.9 (C-2), 72.9 (C-1), 72.7 (C-5), 72.0 (C-2ʹ), 68.3 (C-1ʹ),
62.6 (C-3ʹ), 50.9 (C-4), 37.5 (C-6), 22.1 (-NHCOCH3). HRMS calcʹd for C12H20NO8 [M+H]+:
306.1189, found: 306.1185, calcʹd for C12H19NNaO8 [M+Na]+: 328.1008, found: 328.1004.
Methyl
(1S,3S,4S,5R)-4-acetamido-1,3-diacetoxy-5-((1S,2R)-1,2,3-
triacetoxypropyl)cyclohexane-1-carboxylate (21).
24

ACCEPTED MANUSCRIPT
A solution of compound 20 (9 mg, 0.03 mmol) in EtOH (3 mL) and water (0.1 mL) was
subjected to hydrogenation using 10% Pd/C (12 mg) and 50 psi hydrogen atmosphere for 2h. The
reaction mixture was filtered and washed with methanol (10 mL). The filtrates were combined
1
and concentrated under reduced pressure. H NMR analysis of the crude material indicated a
mixture of diastereomers in 3:1 ratio. 1D NOE experiments confirmed that the major
diastereomer is the target compound 6a-carba-α-D-sialic acid (5) that possesses the desired
configuration at the newly generated stereogenic centre, C-6. This crude material (7 mg) was
then dissolved in pyridine (1 mL). N,N-Dimethyl-4-aminopyridine (0.5 mg, catalytic amount)
and acetic anhydride (0.5 mL) were added to the reaction mixture and the stirring was continued
at rt for 12 h. Pyridine was removed under reduced pressure and the crude material was subjected
to a filter column (EtOAc:MeOH:H2O, 6:3:1) to obtain a colourless syrup (4.7 mg). It was then
dissolved in EtOAc/MeOH (2 mL, 1:1) and trimethylsilyl diazomethane reagent was added at
0 °C until the yellow colour was just apparent. The solution was then stirred at rt for 12 h before
it was concentrated to dryness. The crude material was purified by flash column chromatography
on silica gel (0–5% MeOH/DCM) to afford the fully protected 6a-carba-α-D-sialic acid 20 as a
colourless foam (3.9 mg, 25% yield).
1H NMR (600 MHz, CDCl3): δ 5.24 (dd, 1H, J1',5 = 2.4, J1',2' = 9.0 Hz, H-1'), 5.22 (d, 1H, JNH,4
10.5 Hz, NH), 5.15 (ddd, 1H, J3,2ax = 11.4, J3,2eq = 4.8, J3,4 = 10.5 Hz, H-3), 5.13 (ddd, 1H, J2',3'a
=
=
3.0, J2',3'b = 6.6 Hz, H-2'), 4.23 (dd, 1H, J3'a,2' = 3.0, J3'a,3'b = 12.0 Hz, H-3'a), 3.98 (dd, 1H, H-3'b),
3.95 (ddd, 1H, J4,5 = J4,NH = J4,3 = 10.5 Hz, H-4), 3.74 (s, 3H, -COOCH3), 2.77 (ddd, 1H, J2eq,3
=
4.8, J2eq,6eq = 2.4, J2ax,2eq = 12.6 Hz, H-2eq), 2.54 (ddd, 1H, J6eq,5 = 3.0, J6ax,6eq = 13.2 Hz, H-6eq),
2.11 (m, 1H, H-5), 2.11, 2.10, 2.06, 2.03, 2.01 (s, each 3H, 5 × OAc), 1.91 (s, 3H, -NHCOCH3),
13
1.78 (dd, 1H, J2ax,3 = 11.4, J2ax,2eq = 12.6 Hz, H-2ax), 1.71 (dd, 1H, J6ax,5 = 13.2 Hz, H-6ax). C
25

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NMR (150 MHz, CDCl3): δ 171.5 (-COOMe), 171.0, 170.7, 170.2, 169.9, 169.7 (5C, 5 × -
OCOMe), 170.6 (-NHCOMe), 78.5 (C-1), 70.7 (C-3), 69.3 (C-2ʹ), 67.7 (C-1ʹ), 62.9 (C-3ʹ), 52.8
(-COOCH3), 52.2 (C-4), 37.6 (C-2), 36.5 (C-5), 32.2 (C-6), 23.3 (-NHCOCH3), 21.0, 21.0,
20.93, 20.88, 20.84 (5C, 5 × -OCOCH3). HRMS calcʹd for C23H34NO13 [M+H]+: 532.2025,
found: 532.2027, calcʹd for C23H37N2O13 [M+NH4]+: 549.2290, found: 549.2291, calcʹd for
C23H33NNaO13 [M+Na]+: 554.1844, found: 554.1847. TLC (5% MeOH/CH2Cl2), Rf: 0.6.
(1R,4S,5S)-4-azido-1,5-dihydroxy-3-((1S,2R)-1,2,3-trihydroxypropyl)cyclohex-2-ene-1-
carboxylic acid (22).
Compound 18 (30 mg, 0.06 mmol) was dissolved in methanol (1.5 mL) and a solution of NaOH
(5.2 mg, 0.13 mmol) in water (1.5 mL) was added at 0 °C. After being stirred at 0 °C for 2 h, the
reaction mixture was neutralized with IR-120 (H+ form) resin at 0 °C. The resin was filtered off
and washed with methanol (3 × 5 mL). The filtrates were combined and concentrated under
reduced pressure. The residue (25 mg) was then dissolved in a mixture of dichloromethane (5
mL) and water (0.2 mL) and trifluoroacetic acid (1 mL) was added at 0 °C. The reaction mixture
was stirred at room temperature for 3 h before it was concentrated under reduced pressure. The
residue was taken in water (5 mL) and washed with ethyl acetate (2 × 5 mL). The aqueous layer
was concentrated and the crude was purified by reverse phase HPLC (a gradient of 2–100%
ACN/H2O) to afford the title compound 22 as a colourless gum (14.9 mg, 80%).
1H NMR (600 MHz, D2O): δ 5.92 (br s, 1H, H-2), 4.26–4.20 (m, 2H, H-5, H-1ʹ), 4.09 (br-dd-
like, 1H, J4,5 = 6.9 Hz, H-4), 3.90 (ddd, 1H, J2ʹ,1ʹ = 7.2 Hz, J2ʹ,3ʹa = 3.0 Hz, J2ʹ,3ʹb = 6.6 Hz, H-2ʹ),
3.82 (dd, 1H, J3ʹa,3ʹb = 11.9 Hz, H-3ʹa), 3.65 (dd, 1H, H-3ʹb), 2.47 (dd, 1H, J6a,5 = 3.5 Hz, J6a,6b
=
13
13.6 Hz, H-6a), 1.94 (dd, 1H, J6b,5 = 9.9 Hz, H-6b). C NMR (150 MHz, D2O): δ 176.3 (-
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COOH), 137.5 (C-3), 129.8 (C-2), 72.9 (C-5), 72.8 (C-1), 72.0 (C-2ʹ), 68.9 (C-1ʹ), 62.7 (C-4),
62.6 (C-3ʹ), 38.3 (C-6). HRMS calcʹd for C10H15N3NaO7 [M+Na]+: 312.0808, found: 312.0804.
Acknowledgment. AJB and BMP gratefully acknowledge the Canadian Institutes of Health
Research (CIHR) for funding this research work (MOP-259122).
Appendix A Supporting Information Data. Full synthetic and characterization details for
compounds 8a, 8c, 9a, 9b, 9d, 10a, 10b, 10d, 13, 23, and 24. Single crystal diffraction
experiments, ORTEP diagram, and Table of data for the structure of 9a. 1H and 13C NMR spectra
for all new compounds, 1D NOE spectra for 5 and COSY, HSQC, HMBC for selected
compounds.
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