Synthesis of Glycomimetics by Diastereoselective Passerini Reaction

Article abstract of DOI:10.1021/acs.joc.8b01874

We describe the utilization of bis-isopropylidene-protected d-fructose-derived aldehyde in the Passerini reaction with various acids and isocyanides. A library of densely functionalized glycomimetics bearing up to 3 carbohydrate units was obtained in high

Full text of DOI:10.1021/acs.joc.8b01874

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Article
Synthesis of Glycomimetics by Diastereoselective Passerini reaction
Kristina Vlahovicek-Kahlina, Mario Vazdar, Andreja Jakas, Vilko Smre#ki, and Ivanka Jeric
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01874 • Publication Date (Web): 16 Oct 2018
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Synthesis of Glycomimetics by Diastereoselective Passerini reaction
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Kristina Vlahoviček-Kahlinaa, Mario Vazdar,a Andreja Jakas,a Vilko Smrečkib and Ivanka Jerića*
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a Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry, Bijenička cesta 54,
10000 Zagreb, Croatia
b Ruđer Bošković Institute, NMR Centre, Bijenička cesta 54, 10000 Zagreb, Croatia
Corresponding author: ijeric@irb.hr
R1
NH
O
+
R1
R2
N
C-
O
CHO
O
O
O
O
O
O
O
*
O
O
O
O
O
OH
R2
20 examples
up to 83 % yield
up to 94:6 d.r.
ABSTRACT: We describe the utilization of bis-isopropylidene-protected D-fructose-derived
aldehyde in the Passerini reaction with various acids and isocyanides. A library of densely
functionalised glycomimetics bearing up to 3 carbohydrate units was obtained in high yields and
diastereoselectivities. The configuration of newly formed stereocenter was determined and the
diastereoselectivity rationalized by DFT calculations.
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Introduction
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Carbohydrates are the most abundant class of natural products with a distinctive role in different
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biological processes, like protein folding, cell-cell communication and immune response. In
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addition, synthetic carbohydrate-based compounds found wide application in chemical biology and
medicinal chemistry as diagnostics, therapeutics and vaccines,2 drug delivery systems,3,4 and
molecular receptors.4,5 Carbohydrates are highly valuable molecular scaffolds; their intrinsic
stereochemical diversity and polyfunctionality makes them ideal for the generation of structurally
diverse compound collections. However, there is still a limited number of carbohydrate-based
drugs on the market, owing to the poor pharmacodynamic and pharmacokinetic properties of
natural carbohydrates.6 An attractive way to overcome these drawbacks is through design of
glycomimetics, molecules that emulate bioactive function of carbohydrates but with improved
drug-like properties.7
Multicomponent reactions (MCRs) offer an attractive one-pot strategy for generating a library of
highly functionalized and complex compounds like glycomimetics.8 A large number of MCRs
comprising carbohydrates were developed,9 providing access to structurally diverse
glycoconjugates.10 The formation of new stereocentre is inherent to many MCRs, and the utility of
MCR strategy for drug discovery depends on the ability to deliver structurally complex but
stereochemically defined compounds. Carbohydrate-derived aldehydes were reported to affect the
diastereoselectivity in MCRs, due to the proximity to the newly formed stereocentre. However, the
observed stereoselectivity depends on the position of aldehyde group, OH-protecting groups, and
other components in MCRs.11 To our surprise, sugar-derived aldehydes are neglected in
isocyanide-based MCRs (e.g., Passerini, Ugi reaction), otherwise recognized as highly versatile
tools in drug discovery.12 Acetyl- and benzyl-protected galactose- and fucose-derived aldehydes
were used in the synthesis of focused library of sialyl Lewis x glycomimetics, and observed
diastereoselectivities ranged from 4:1 to 1:1.13 In our search for carbohydrate building blocks
suitable for utilization in MCRs, we opted for bis-isopropylidene-protected D-fructose-derived
aldehyde 1 (Scheme 1), structurally analogous to the anticonvulsant drug topiramate, a rare
example of central nervous system-active carbohydrates.14 We anticipated that such bis-
isopropylidene-protected aldehyde could serve as an efficient chiral inductor in MCRs. Thus, we
report herein the Passerini reaction of easy accessible aldehyde 115 with various acids and
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isocyanides, where complex structures are obtained in very good yields and a highly
stereocontrolled manner (Scheme 1).
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Scheme 1 Passerini reaction with D-fructose-derived aldehyde 1.
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R1
NH
O CHO
O
O
O
O
O
O
O
O
DCM, 24h
+
R1
N
C-
R2 OH
O
O
O
O
O
R2
1
Results and discussion
A first set of Passerini reactions was performed with two commercially available isocyanides,
cyclohexyl- and 1,1,3,3-tetramethylbutyl isocyanide, and four different carboxylic acids. Along
with simple acetic and benzoic acid, we utilized Boc-protected phenylalanine as a carboxylic
component, because Passerini products comprising amino acids can be easily modified in post-
condensation reactions.16 Multivalent carbohydrate derivatives are promising mimics of ligands
that recognize and bind to carbohydrate receptors placed on the cell surface,1 so we opted for per-
O-acetylated glucuronic acid to access carbohydrate-rich Passerini products. Reactions performed
with selected isocyanides and carboxylic acids in dichloromethane at room temperature furnished
Passerini products 2-9 in 68-83 % yield (Figure 1). We were pleased to find that even structurally
complex bivalent derivatives 5 and 9, bearing glucuronic acid part, are obtained in a very good
yield in a single step. The most interesting observation is high stereoselectivity of the performed
reactions. The ratio of two diastereoisomers, determined by NMR spectroscopy is  9:1 d.r., and
the major isomer of all Passerini products with the exception of 5 and 9, was isolated in a pure
form. Compounds 5 and 9 were isolated as a mixture of four diastereoisomers, arising from the
newly formed stereocentre and glucuronic acid component, present as a 15:85 mixture of  and -
pyranose forms.
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NH
O
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
8
9
2 (72 %)
d.r. 91:9
3 (83 %)
d.r. 89:11
ccdc 1590069
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NH
O
NH
O
O
O
O
O
O
O
O
O
NH
O
O
O
O
O
O
O
O
AcO
AcO
O
O
O
O
O
O
O
NHBoc
OAc
AcO
5 (68 %)
d.r. 94:6a
4 (74%)
d.r. 89:11
6 (69 %)
d.r. 91:9
NH
O
O
O
O
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
AcO
O
O
O
O
O
O
O
AcO
AcO
NHBoc
OAc
9 (70 %)
d.r. 94:6a,b
7 (79 %)
d.r. 89:11
8 (79 %)
d.r. 91:9
Figure 1. Passerini products obtained with sugar aldehyde 1 (isolated yields are given in
parentheses). d.r. was determined by 1H NMR analysis of reaction mixture (for compounds
a
2-4 and 6-8, or based on ratio of isolated diastereoisomers. Ratio of  and  isomers
(glucuronic acid) is 15:85. bApproximate d.r. due to poor peak resolving.
Encouraged by these results, we decided to explore the scope of the reaction by introducing sugar-
derived isocyanides. Sugar isocyanides have been sporadically used in MCRs,17,18 owing to
challenging synthetic procedure, but also observed low stability of primarily 1-isocyano
carbohydrate derivatives. Recently, anomeric per-O-acetylated glycosyl and arabinosyl
isocyanides were prepared by an elegant Leuckart–Wallach approach and tested in different MCRs,
however, substrate scope of these reaction has not been explored.18 Since anomeric carbohydrate
isocyanides are generally poor nucleophiles due to the proximal electronegative O-atom, we also
consider 2-isocyano and 6-isocyanoglycosyl derivatives as potentially more reactive sugar building
blocks. Passerini reactions performed with 1-isocyano glucose derivative gave di- and trivalent
products 10-13 (Figure 2) in very good yields (57-80 %). Although purification and NMR
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assignment of compounds comprising multiple carbohydrate units can be difficult and incomplete,
Passerini products 10-12 were isolated as single diastereoisomers, while densely functionalised
product 13 was isolated as a mixture of four diastereisomers. Next set of reactions was carried out
with 2-isocyano glucose derivative and the corresponding Passerini products 14-17 were isolated
again in very good yields (61-80 %, Figure 2) as a mixture of four diastereisomers. Inspection of
the 1H-NMR spectra of product 14 revealed nicely resolved signals of all four isomers that allowed
determination of ratio of two isomers (9:1, d.r.). Finally, reactions performed with 6-isocyano
glucose derivative under the same reaction conditions furnished Passerini products 18-21 in 66-76
% yield. Since 1-OBn-Ac3-D-Glc unit is present in -anomeric configuration, compounds 18-20
are isolated as inseparable mixtures of two isomers with d.r. > 9:1, while Passerini product 21
bearing three carbohydrate units is isolated as a mixture of four isomers.
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OAc OAc
OAc OAc
OAc OAc
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NHBoc
9
12 (62 %)c
10 (80 %)
d.r. 90:10
11 (57 %)
d.r. 91:9
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OAc OAc
AcO
AcO
AcO
AcO
O
AcO
AcO
O
OAc
O
OAc
AcO
NH
O
NH
AcO
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
AcO
O
O
O
O
AcO
AcO
OAc
13 (68 %)c
14 (80 %)
d.r. 90:10d
15 (61 %)
d.r. 95:5d
AcO
AcO
AcO
AcO
AcO
OAc
OAc
O
O
BnO
OAc
OAc
O
OAc
NH
NH
O
AcO
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
AcO
AcO
AcO
O
O
O
O
O
NHBoc
O
OAc
16 (68 %)c
17 (75%)c
18 (76 %)
d.r. 90:10
OAc
OAc
OAc
OAc
BnO
BnO
BnO
OAc
OAc
OAc
OAc
O
O
O
OAc
NH
NH
O
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
AcO
AcO
AcO
O
O
O
O
O
O
O
O
NHBoc
OAc
21 (75 %)c
19 (68 %)
d.r. 94:6
20 (70 %)
d.r. 90:10
Figure 2. Passerini products obtained with sugar aldehyde 1 and sugar isocyanides (isolated
yields are given in parentheses). d.r. was determined based on ratio of isolated
diastereoisomers. aRatio of  and  isomers (glucuronic acid) is 15:85. bApproximate d.r. due
to poor peak resolving. cd.r. cannot be determined due to peak overlapping. dRatio of  and
 isomers for 2-NC-Ac4-D-Glc is 60:40.
Having the general scope of the diastereoselective Passerini reaction on fructose-derived aldehyde
1 in hands, we applied acid- and base-mediated deprotection schemes on selected mono- and
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bivalent Passerini products. Treatment of the Passerini product 2 (single diastereoisomer) with
NaOH gave -hydroxy amide derivative 22 in 81 % yield (Scheme 2). -Hydroxy amides and their
derivatives are recognized as privileged structures in medicinal chemistry and pharmaceutical
industry, and are widely used as chiral ligands in the asymmetric synthesis.19 1H NMR spectrum
of -hydroxy amide 22 (Supporting Information) revealed that the chiral information derived from
diastereoselective Passerini reaction is preserved, so this chiral synthon is of high value for further
modifications. Acid-based cleavage of isopropylidene groups was performed with TFA in DCM at
room temperature and the Passerini product 23 was obtained in 67 % as a mixture of anomeric
forms. Deprotection of bivalent Passerini products was expected to be more challenging owing to
the presence of different carbohydrate units and protecting groups. Deacetylation of the Passerini
product 10 afforded -hydroxy amide derivative 24 in excellent yield (97 %, Scheme 2), with
preservation of chiral information (Supporting Information). Treatment of compound 10 with
TFA/H2O (9/1) resulted in removal of isopropylidene groups, while NMR spectrum and mass
spectra point toward presence of two compounds; a fructose moiety deprotected derivative 25 and
the corresponding -hydroxy amide 26, but full assignment is hampered by the presence of
multiple anomeric forms. Fully deprotected compound 27 was obtained by acid treatment of
deacetylated compound 24 in 68 % yield, again as a mixture of anomers. It is also worth noting
that O-deacetylation of per-acetylated carbohydrates can be performed enzymatically. Dunne and
Palomo reported recently a mild and efficient strategy for deprotection of per-acetylated
carbohydrates by the using commercially available lipase A from Aspergillus niger.20 A high
selectivity to the O-deacetylation instead of the N-deacetylation was observed, whereas isolated
yields were > 99 % for -glycopyranosides and 75–80 % for -glycopyranosides. Therefore, a
combination of chemical and enzymatic deprotection is available to access partially of fully
deprotected carbohydrate-derived Passerini products.
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Scheme 2. Deprotection schemes for the selected Passerini products.
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6
7
8
9
NH
NH
1M NaOH
MeOH
O
O
O
O
O
O
O
O
O
O
HO
O
O
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reflux, 10 min
O
O
2
22
(81 %)
NH
NH
TFA in DCM
r.t. 6 h
O
O
O
OH
O
HO
O
O
O
OH
O
O
O
O
O
OH
3
23
(67 %)
OAc OAc
OH
OH
AcO
AcO
HO
HO
O
O
0.25M NaOMe
MeOH
NH
NH
O
O
O
O
O
O
r.t. 2 h
O
O
O
O
HO
O
O
O
O
24
(97 %)
10
TFA/H2O (9/1)
4 °C, 5 h
TFA/H2O (9/1)
r.t. 2 h
OH
OH
OAc OAc
OAc OAc
HO
HO
AcO
AcO
AcO
AcO
O
O
O
NH
NH
NH
+
O
OH
HO
O
OH
O
OH
HO
HO
O
OH
HO
O
OH
OH
O
HO
O
O
OH
OH
OH
27
25 + 26
(68 %)
To ascertain the stereochemistry of the newly created stereocentre, NOE contacts observed
in 2D NOESY NMR experiments were compared to calculated interatomic distances in optimized
structures of four different Passerini products (2, 4, 6, and 8). The observed NOE contacts between
hydrogen atom of the new stereocentre and other hydrogen atoms are likely to be observed in
both diastereoisomers (as shown for compound 6 in Supporting Information), since calculated
interatomic distances show similar values for both optimized diastereoisomers. However,
according to calculated total energy the S configuration is more stable compared to
the R configuration, which points towards the S configuration of the product (Supporting
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Information). The S configuration of the new chiral centre was unambiguously determined by
single-crystal x-ray diffraction analysis of product 3.
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In order to elucidate the observed stereoselectivity of Passerini reactions performed with sugar
aldehyde 1, we conducted density functional theory (DFT) calculations at the SMD/M062X/6-
311++G(2d,p)//M062X/6-31G(d) level of theory21 employing dichloromethane as a solvent
starting from two different configurations leading to the final product 2 with S configuration (path-
a) and R configuration (path-b), respectively, following the mechanistic pathway proposed
recently by Ramozzi and Morokuma.22,23 Figure 3a shows calculated free energy profiles for the
reaction. The first step, e. g. complexation of cyclohexane isocyanide with sugar aldehyde 1 and
two acetic acid molecules, results in two pre-complexes of different stabilities – for the path-a, the
pre-complex is by 2.9 kcal mol-1 more stable than the pre-complex which serves as a starting point
for the path-b. The remaining reaction profiles for both reaction paths have similar energetics (with
free energy differences up to 10 kJ mol-1), except for the transition state TS3, which is by ca. 10 kJ
mol-1 more stable in the path-a vs path-b, and the intermediate INT5 which is by ca. 13 kJ mol-1
more stable in the path-b vs path-a (Figure 3a). Based on the presented energy profile, we
identified the rearrangement of imidate-acid cluster (INT3) to dioxolane-acid cluster (INT4) via the
transition state TS3 as the rate-determining step of the reaction with the free energy barrier of 82.3
kJ mol-1, in accordance to previous studies of the mechanism of Passerini reaction.24,25 In addition,
the free energy difference is observed in the final product with the S configuration (path-a), which
is by ca. 6.5 kJ mol-1 more stable than the final product with the R configuration (path-b). We
should also mention here that we tried to elucidate the mechanism with only one carboxylic acid
instead of two as presented here (Figure 3). However, these attempts were not successful since we
were not able to obtain the transition state TS3 with reasonable energetics, similar to Ramozzi and
Morokuma .22
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There is also one important mechanistic step discriminating these two pathways – namely, in the
path-b, the intermediate INT5 is directly converted into the final product via transition state TS4
in contrast to the path-a where additional intermediates INT6 and INT7 are located. In this reaction
step in path-a, the proton transfer from O-atom to N-atom occurs via transition state TS4 resulting
in an additional intermediate INT6 where proton is located on the imine nitrogen atom. A
subsequent conformational change results in the intermediate INT7 which finally converts to the
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final product with the S configuration via the barrier-less transition state TS5 (Figure 3c).23
However, regardless of the mechanistic details of each of the reaction paths, the strong stabilization
of the TS3 in the path-a vs path-b, is the main factor explaining the diastereoselectivity of the
Passerini reaction and the formation of the product with S configuration. Figure 3c and 3d show
the proposed mechanistic pathway for the Passerini reaction leading to S and R products.
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Figure 3. (a) Free energy profiles calculated at the SMD/M062X/6-311++G(2d,p)//M062X/6-
31G(d) level of theory for two different starting configurations leading to final products
with S configuration (path-a) and R configuration (path-b). (b) Optimized geometries of final
products obtained at SMD/M062X/6-31G(d) level of theory. Qualitative indication of
noncovalent van der Waals interactions is shown with red arrows. (c) Proposed mechanistic
pathway for the formation of S product in the Passerini reaction. (d) Proposed mechanistic
pathway for the formation of R product in the Passerini reaction.
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To further explore the utility of here described multicomponent procedure, we performed Passerini
reactions by using D-galactose-, D-ribose and D-xylose derived aldehydes, with acetic acid and
either tert-butyl or cyclohexyl isocyanide. Reactions were conducted under the same reaction
conditions as described previously, in dichloromethane at room temperature, and the corresponding
Passerini products 28-30 were obtained in high yields (Figure 4), thus proving that approach can
be easily expanded to different sugar-derived aldehydes.
O
O
O
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(79 %)
(78 %)
(82 %)
Figure 4. Passerini product obtained with D-galactose, D-ribose and D-xylose-derived
aldehydes.
Recently, Riva et al. reported Passerini reactions on acyclic isopropylidene protected aldehyde,
where 0.4 equiv of Lewis acid (ZnBr2) was required to improve diastereoselectivities up to 92:8.26
Authors presumed that chelation of the metal by the carbonyl oxygen and one of the dioxolane
oxygens accounts for the observed relative configuration of the main product. To reveal the role of
the isopropylidene group in diastereoselectivity of MCRs, we are currently focused on the
utilization of other carbohydrate-derived carbonyl compounds, and these results will be published
in due course.
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In conclusion, we developed a multicomponent approach to densely functionalised glycomimetics
in a high diastereoselective manner. The approach is based on the Passerini reaction of bis
isopropylidene-protected D-fructose-derived aldehyde with various acids and isocyanides and the
observed diastereoselectivity was rationalized by DFT calculations.
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Experimental section
General methods
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All experiments were monitored by analytical thin layer chromatography (TLC) on Silica Gel 60
F254 plates (Merck; Darmstadt, Germany) after spraying with 10 % H2SO4 and heating. Flash
column chromatography was performed on silica gel (Merck, 40−63 μm particle size) by standard
techniques eluting with solvents as indicated. All NMR experiments were carried out by using
Bruker Avance 600 spectrometer (600.13 MHz, 1H; 150.91 MHz, 13C). Samples in CDCl3 solutions
were recorded in 5 mm NMR tubes at 298 K. Chemical shifts in parts per million were referenced
to TMS as internal standard. Spectra were assigned based on 2D homonuclear (COSY, NOESY)
and heteronuclear (HMQC, HMBC) experiments. 13C (APT) spectra were recorded with broadband
proton decoupling (WALTZ) using a broadband-observed probe (BBO; BB, 1H; inner coil tuned
for 13C) with a built-in z-gradient coil. Typically, spectra at a spectral width of 35 kHz and a digital
resolution of 2.1 Hz per point were measured with 10,000 scans using a standard pulse sequence
(jmod) from the Bruker TopSpin library. All 1H and 2D NMR spectra were recorded using a triple
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resonance inverse detection probe (TBI; H, C,BB; inner coil tuned for H) with a built-in z-
gradient coil. 1H spectra at a spectral width of 6,600 Hz and a digital resolution of 0.4 Hz per point
were measured with 128 scans. Standard gradient enhanced pulse programs supplied in the Bruker
TopSpin library were used for HMQC (hmqcgpqf) and HMBC (hmbcgplpndqf) spectra. In the 1H
observation dimension (F2), 2 K data points were acquired at a spectral width of 6,600 Hz. In the
indirect carbon dimension (F1), 256 data points (HMQC) or 512 data points (HMBC) at a spectral
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width of 35 kHz were used. GARP decoupling was employed for C during proton detection in
HMQC. 16 (HMQC) or 32 (HMBC) transients were recorded per evolution time increment. COSY
(cosygpqf) and NOESY (noesyph) were applied using 2 K data points in F2 dimension and 256
(COSY) or 512 (NOESY) increments in F1 dimension at a spectral with of 6,600 Hz. 8 (COSY) or
16 (NOESY) transients were recorded per evolution time increment. NOESY mixing times where
600 ms. All experimental data were zero-filled to double the number of experimental points and
then baseline-corrected using automatic baseline correction. The software TopSpin (Bruker
BioSpin) version 2.1 was used for all acquisition and processing. Since Passerini products are three-
component coupling products, 1H chemical shifts are assigned to the particular starting component,
and the following abbreviations are used: cyclohexyl = CyHex; tetramethylbutyl = tMeBu; fructose
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derived aldehyde 1 = Fru; glucuronic acid = GlaA; 1-, 2-, and 6-isocyano glucose derivatives =
Glc1NC, Glc2NC, and Glc6NC, respectively. High resolution mass spectrometry (HRMS) was
performed on a MALDI-TOF/TOF spectrometer in positive ionization mode. Calibration type was
internal with calibrants produced by matrix ionization dissolved in α-cyano-4-hydroxycinnamic
acid matrix. Accurately measured spectra were internally calibrated and elemental analysis was
performed on Data Explorer v. 4.9 software with mass accuracy better than 5 ppm.
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Synthesis of sugar isocyanides
Per-O-acetylated 1-isocyano-glucose derivative (1-NC-Ac4-D-Glc) was prepared by the Leuckart–
Wallach approach.18
Per-O-acetylated 2-isocyano-glucose derivative (2-NC-Ac4-D-Glc) was prepared by the
modified approach described in the literature (Scheme S1 in the Supporting Information):27
Glucosamine hydrochloride (432 mg, 2 mmol) was dissolved in 8 mL of saturated NaHCO3
solution, and methylformate (1.23 mL) was added and mixed at room temperature. Methylformate
was added twice more, after 16 h and after 24 h (total volume was 4.23 mL). After 48 h, the reaction
mixture was evaporated and dried for several hours in a desiccator. To the residue 5 mL of
pyridine/Ac2O 1:1, v/v mixture was added and stirred overnight at room temperature. The solvent
was evaporated with addition of toluene and the residue was dried over H2SO4. To the residue,
DCM (4.6 mL), Et3N (820 μL) and POCl3 (224 μL) were added. The reaction was stirred at room
temperature for 1 h, and then Na2CO3 (400 mg) dissolved in water (1.6 mL) was added. The
reaction mixture was stirred for 1 h at room temperature. DCM and water were added and extracted.
The organic layers were collected and dried over K2CO3 filtered and evaporated. The residue was
purified on silica gel column in a solvent system toluene/EtOAc 1:1, v/v. The product was obtained
in 40 % yield. Spectral data are consistent with those published in Ref 27.
1-OBz-6-NC-Ac3-D-Glc was prepared by the Leuckart–Wallach approach18 starting from 1-O-Bn-
6-NH2-Glc (obtained by standard carbohydrate chemistry, as depicted at Scheme S2 in the
Supporting Information).
1-O-Bn-6-NH2-Glc (400 mg, 1.488 mmol) was dissolved in a saturated solution of NaHCO3 (12
mL) and methylformate (12 mL, 194.4 mmol) was added and stirred overnight at room
temperature. During this time methylformate was added twice (2×1 mL). After 48 h, the reaction
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mixture was evaporated and purified by flash chromatography in a solvent system:
EtOAc/EtOH/AcOH/H2O 70:15:15:15. To the residue a mixture of pyridine/acetic anhydride 1:1,
v/v (12 mL) was added and stirred at room temperature for 5 h. The solvent was evaporated with
addition of toluene and the residue was purified by flash chromatography on silica gel column in a
solvent system: EtOAc/AcOH/H2O 70:1:1, v/v. A product of 1-O-Bn-6-NCO-Ac3-Glc (70 %) was
obtained and dried for 24 h in a desiccator.
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1-O-Bn-6-NCO-Ac3-Glc (370 mg, 0.87 mmol) was dissolved in DCM (2.4 mL), and Et3N (472
μL) and POCl3 (128 μL) were added. The reaction was stirred at room temperature for 1h, followed
by the addition of Na2CO3 (232 mg) dissolved in water (0.920 mL). The reaction mixture was
stirred for further 1 h at room temperature. DCM and water were added and extracted. The organic
layers were collected and dried over K2CO3, filtered and evaporated. The residue was purified on
silica gel column in a solvent system toluene/EtOAc 1:1, v/v. The product was obtained in 67 %.
1H NMR (CDCl3): δ 7.38–7.26 (m, 5H, CH-benzyl), 5.16 (t, J = 9.5 Hz, 1H, H-3), 5.04 (dd, J =
9.5, 8.0 Hz, 1H, H-2), 4.92 (d, J = 7.8 Hz, 1H, H-6a), 4.90 (d, J = 10.0 Hz, 1H, H-4), 4.66 (d, J =
12.3 Hz, 1H, H-5), 4.56 (d, J = 7.9 Hz, 1H, H-1β), 3.70–3.65 (m, 1H, H-6b), 3.55 (d, J = 5.4 Hz,
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2H, CH2-benzyl), 2.10 – 1.86 (m, 9H, CH3-acetyl). C NMR (CDCl3): δ 170.33, 169.8, 169.4,
159.7, 136.5, 128.8, 128.4, 128.2, 99.2, 72.5, 71.7, 71.4, 71.0, 70.3, 43.3, 20.8, 20.8. (ESI+): m/z =
406,4. HRMS (MALDI-TOF/TOF) m/z: [M+H]+ calcd. for C20H24NO8 406.1509; found 406.1507.
General procedure for Passerini reactions
To a glass vial containing 1 M solution of aldehyde 1 (0.1 mmol) in DCM under nitrogen were
added acid (0.1 mmol) and the isocyanide component (0.1 mmol) dissolved in 100 µL DCM. With
all reactants added, the solution was allowed to stir for 8-24 h. The reactions were concentrated
under reduced pressure and reaction mixtures were purified by flash column chromatography.
(S)-2-(cyclohexylamino)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)ethyl acetate (2)
Yield 72 % (31 mg); colorless oil; Rf = 0.39 (toluene/acetone 5:1, v/v).1H NMR (CDCl3): δ 6.25
(d, J = 7.5 Hz, 1H, NH), 5.05 (s, 1H, H1 Fru), 4.58 (dd, J = 7.9, 2.6 Hz, 1H, H4 Fru), 4.38 (d, J =
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2.6 Hz, 1H, H3 Fru), 4.22 (d, J = 7.8 Hz, 1H, H 5 Fru), 3.92 (dd, J = 12.9 Hz, 1.4 Hz, 1H, H6 Fru),
3.78 (d, J = 12.9 Hz, 1H, H6' Fru), 3.75 (m, 1H, CyHex), 2.17 (s, 3H, CH3 Ac), 1.87 (m, 2H,
CyHex), 1.63 (m, 3H, CyHex), 1.53 (m, 1H, CyHex), 1.50 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.44
(s, 3H, CH3), 1.32 (m, 4H, CH3, CyHex), 1.16 (m, 3H, CyHex). 13C NMR (CDCl3): δ 169.4, 164.5,
109.7, 109.5, 102.3, 73.9, 70.8, 70.3, 61.8, 48.5, 32.9, 32.9, 26.7, 26.3, 25.8, 25.5, 24.8, 24.8, 24.2,
21.0. MS (ESI+): m/z = 428.3. HRMS (MALDI-TOF/TOF) m/z: [M+H]+ calcd. for C21H33NO8
428.2284; found 428.2278. HPLC analysis: n-hexane/i-PrOH = 80/20, υ = 0.5 mLmin-1, λ = 215
nm, t =17.4 min (95 %). [α]D20 = -14 (CHCl3, c=0.5).
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(S)-2-(cyclohexylamino)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)ethyl benzoate (3)
Yield 83 % (41 mg); white solid; Rf = 0.58 (toluene/acetone 5:1, v/v); m.p. 123-124 °C. 1H NMR
(CDCl3): δ 8.08 (dd, J = 8.2, 1.1 Hz, 2H, Ph), 7.54 (dd, J = 10.6, 4.2 Hz, 1H, Ph), 7.42 (t, J = 7.8
Hz, 2H, Ph), 6.39 (d, J = 7.8 Hz, 1H, NH), 5.43 (s, 1H, H1 Fru), 4.59 (dd, J = 7.9 Hz, 2.7 Hz, 1H,
H4 Fru), 4.47 (d, J = 2.8 Hz, 1H, H3 Fru), 4.24 (d, J = 8.0 Hz, 1H, H5 Fru), 3.99 (dd, J = 13.0, Hz,
1.7 Hz, 1H, H6 Fru), 3.84 (d, J = 13.0 Hz, 1H, H6' Fru), 3.82–3.76 (m, 1H, CyHex), 1.89 (s, 2H,
CyHex), 1.63 (m, 2H, CyHex), 1.60 (s, 1H, CyHex), 1.55 (m, 1H, CyHex), 1.54 (s, 3H, CH3), 1.53
(s, 6H, CH3), 1.32 (s, 4H, CH3, CyHex), 1.19 (m, 3H, CyHex). 13C NMR (CDCl3): δ 165.6, 164.6,
133.4, 130.2, 129.9, 128.6, 109.9, 102.4, 74.2, 70.8, 70.2, 61.9, 48.6, 33.00, 26.8, 26.3, 26.0, 25.8,
24.8, 24.2. MS (ESI+): m/z = 490.3. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for
C26H35NO8Na 512.2260; found 512.2242.HPLC analysis: n-hexane/i-PrOH = 80/20, υ = 0.5
mLmin-1, λ = 215 nm, t = 24.4 min (99 %). [α]D20 = -12 (CHCl3, c=0.5). Crystals suitable for x-ray
analysis were obtained by slow evaporation from n-hexane/i-PrOH (9/1. v/v) solution.
(S)-((S)-2-(cyclohexylamino)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-
3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)ethyl)
phenylpropanoate (4)
2-(tert-butoxycarbonylamino)-3-
Yield 79 % (50 mg); colorless oil; Rf = 0.52 (toluene/acetone 5:1, v/v). 1H NMR (CDCl3): δ 7.27-
7.25 (m, 2H, Ph), 7.21-7.18 (m, 3H, Ph), 6.39 (d, J = 7.8 Hz, 1H, NH), 5.14 (s, 1H, H1 Fru), 4.90
(d, J = 8.4 Hz, 1H, NH Phe), 4.61 (dd, J = 8.3 Hz, 3.5 Hz, 1H, α Phe ), 4.58 (dd, J = 7.8 Hz, 2.5
Hz, 1H, H4 Fru), 4.36 (d, J = 2.4 Hz, 1H, H3 Fru), 4.22 (d, J = 7.8 Hz, 1H, H5 Fru ), 3.93 (d, J =
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12.9 Hz, 1H, H6 Fru), 3.79 (m, 2H, H6' Fru, CyHex), 3.40 (dd, J = 14.3 Hz, 4.5 Hz, 1H, β Phe),
3.02 (dd, J = 14.2 Hz, 8.5 Hz, 1H, β' Phe), 1.89 (d, J = 11.6 Hz, 2H, CyHex), 1.67-1.54 (m, 2H,
CyHex), 1.50 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.43 (s, 2H, CyHex), 1.34 (s, 9H, CH3 Boc), 1.32 (s,
3H, CH3), 1.28 (br s, 1H, CyHex), 1.18 (m, 3H, CyHex). 13C NMR (CDCl3): δ 170.9, 164.2, 155.4,
136.6, 129.7, 128.7, 127.0, 109.9, 109.5, 102.1, 80.1, 74.2, 70.8, 70.7, 70.2, 62.0, 54.6, 48.6, 38.5,
33.0, 28.4, 26.7, 26.3, 25.8, 25.6, 24.9, 24.8, 24.3. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+
calcd. for C33H48N2O10Na 655.3207; found 655.3203. HPLC analysis: n-hexane/i-PrOH = 80/20,
υ = 0.5 mLmin-1, λ = 215 nm, t = 29.8 min (95 %). [α]D20 = -10 (CHCl3, c= 0.5).
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(3R,4S,5S,6S)-6-((2-(cyclohexylamino)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-
tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-
yl)ethoxy)carbonyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (5)
Yield 68 % (49 mg); colorless oil; Rf = 0.38 (toluene/acetone 5:1, v/v); : = 15:85.Chemical shifts
are given for major isomer. 1H NMR (CDCl3): δ 6.37 (d, J = 3.6 Hz, H1α GlcA), 6.31 (d, J = 7.8
Hz, β NH), 6.22 (d, J = 8.0 Hz, α NH), 5.79 (d, J = 7.5 Hz, H1β GlcA), 5.45 (t, J = 9.5 Hz, H4α
GlcA), 5.40 (t, J = 9.5 Hz, H4β GlcA), 5.25 (t, J = 9.1 Hz, H3 GlcA), 5.11 (d, J = 7.8 Hz, H2
GlcA), 5.09 (s, H1 Fru), 4.58 (dd, J = 7.9 Hz, 2.7 Hz, H4 Fru), 4.39 (d, J = 9.8 Hz, H5 GlcA), 4.37
(d, J = 2.8 Hz, H3 Fru), 4.21 (d, J = 7.8 Hz, H5 Fru), 3.92 (d, J = 12.9 Hz, H6 Fru), 3.77 (d, J =
13.0 Hz, H6' Fru), 3.72 (m, H1 CyHex), 2.07-1.99 (s, CH3-Ac), 1.83 (m, CyHex), 1.61 (m, CyHex),
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1.48 (s, 3H, CH3), 1.45 (s, CH3), 1.41 (s, CH3), 1.31 (s, CH3), 1.29-1.16 (m, CyHex). C NMR
(CDCl3): δ 170.3, 169.6, 169.4, 168.7, 165.2, 163.3, 110.1, 109.5, 102.0, 91.8, 88.9, 74.8, 73.1,
72.5, 70.8, 70.7, 70.5, 70.2, 70.2, 68.5, 62.0, 48.5, 32.8, 26.7, 26.3, 25.8, 25.4, 24.8, 24.7, 24.3,
20.9, 20.9, 20.8, 20.8. MS (ESI+): m/z = 730.3. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd.
for C33H47NO17 752.2742; found 752.2743.HPLC analysis: (n-hexane/i-PrOH = 50/50, υ = 0.5
mLmin-1, λ = 215 nm, t = 20.7 min (95 %).
(S)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-
b:4',5'-d]pyran-3a-yl)-2-(2,4,4-trimethylpentan-2-ylamino)ethyl acetate (6)
Yield 69 % (32 mg); colorless oil; Rf = 0.69 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
6.36 (s, 1H, NH), 5.01 (s, 1H, H1 Fru), 4.58 (dd, J = 7.9 Hz, 2.7 Hz, 1H, H4 Fru), 4.32 (d, J = 2.7
Hz, 1H, H3 Fru), 4.22 (d, J = 7.0 Hz, 1H, H5 Fru), 3.92 (dd, J = 12.9 Hz, 1.6 Hz, 1H, H6 Fru), 3.77
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(d, J = 12.9 Hz, 1H, H6' Fru), 2.17 (s, 3H, CH3), 1.68 (d, J = 15.0 Hz, 1H, H2 tMeBu), 1.60 (d, J
= 15.0 Hz, 1H, H2' tMeBu), 1.50 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.40 (s, 3H,
CH3 tMeBu), 1.38 (s, 3H, CH3 tMeBu), 1.32 (s, 3H, CH3), 0.98 (s, 9H, H4, H3',H3' CH3 tMeBu).
13C NMR (CDCl3): δ 169.2, 164.0, 109.7, 109.6, 102.4, 74.0, 70.9, 70.9, 70.3, 61.8, 55.5, 53.1,
31.8, 29.2, 29.0, 26.7, 26.2, 25.6, 24.4, 21.0. HRMS (MALDI-TOF/TOF) m/z: [M+H]+ calcd. for
C23H39NO8 458.2754; found 458.2761. HPLC analysis: n-hexane/i-PrOH = 80/20, υ = 0.5 mLmin-
1, λ = 215nm, t = 16.4 min (96 %). [α]D20 = -10 (CHCl3, c= 0.5).
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(S)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-
b:4',5'-d]pyran-3a-yl)-2-(2,4,4-trimethylpentan-2-ylamino)ethyl benzoate (7)
Yield 79 % (41 mg); colorless oil; Rf = 0.77 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
8.11-8.07 (m, 2H, Ph), 7.54 (t, J = 7.4 Hz, 1H, Ph), 7.41 (t, J = 7.8 Hz, 2H, Ph), 6.49 (s, 1H, NH),
5.39 (s, 1H, H1 Fru), 4.58 (dd, J = 7.9 Hz, 2.8 Hz, 1H, H4 Fru), 4.41 (d, J = 2.8 Hz, 1H, H3 Fru),
4.24 (d, J = 7.9 Hz, 1H, H5 Fru), 3.99 (dd, J = 12.9 Hz, 1.6 Hz, 1H, H6 Fru), 3.82 (d, J = 12.9 Hz,
1H, H6' Fru), 1.70 (d, J = 14.9 Hz, 1H, H2 tMeBu), 1.60 (d, J = 15.0 Hz, 1H, H2' tMeBu), 1.54 (s,
3H, CH3), 1.53 (s, 6H, CH3), 1.41 (s, 3H, CH3 tMeBu), 1.39 (s, 3H, CH3 tMeBu), 1.32 (s, 3H, CH3),
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0.99 (s, 9H, H4, H3',H3' CH3 tMeBu). C NMR (CDCl3): δ 165.4, 164.1, 133.4, 130.3, 130.0,
128.6, 109.9, 109.7, 102.4, 74.3, 70.9, 70.9, 70.2, 61.9, 55.5, 53.0, 31. 8, 31.7, 29.2, 29.1, 26.8,
26.2, 26.1, 24.3. MS (ESI+): m/z = 520.3. HRMS (MALDI-TOF/TOF) m/z: [M+H]+ calcd. for
C28H41NO8 520.2910; found 520.2936. HPLC analysis: n-hexane/i-PrOH = 80/20, υ = 0.5 mLmin-
1, λ = 215nm, t = 20.9 min 96 %. []D20 = -12 (CHCl3, c=0.5).
(S)-((S)-2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-
b:4',5'-d]pyran-3a-yl)-2-(2,4,4-trimethylpentan-2-ylamino)ethyl)
butoxycarbonylamino)-3-phenylpropanoate (8)
2-(tert-
Yield 79 % (52 mg); colorless oil; Rf = 0.83 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
7.31-7.17 (m, 5H, Ph), 6.35 (s, 1H, NH), 5.06 (s, 1H, H1 Fru), 4.90 (d, J = 8.4 Hz, 1H, NH Phe),
4.61 (d, J = 5.3 Hz, 1H, H Phe), 4.57 (dd, J = 7.8 Hz, 2.7 Hz, 1H, H4 Fru), 4.29 (d, J = 2.8 Hz,
1H, H3 Fru), 4.21 (d, J = 7.7 Hz, 1H, H5 Fru), 3.93 (d, J = 12.9 Hz, 1H, H6 Fru), 3.76 (d, J = 12.9
Hz, 1H, H6' Fru), 3.37 (dd, J = 14.1 Hz, 4.9 Hz, 1H,  Phe), 3.07 (dd, J = 14.2 Hz, 7.6 Hz, 1H, '
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Phe), 1.74 (d, J = 14.9 Hz, 1H, H2 tMeBu), 1.59 (d, J = 15.0 Hz, 1H, H2' tMeBu), 1.50 (s, 3H,
CH3), 1.47 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.42 (s, 3H, CH3 tMeBu), 1.39 (s, 3H, CH3 tMeBu),
1.35 (s, 9H, CH3 Boc), 1.32 (s, 3H, CH3), 0.99 (s, 9H, H4, H3', H3' CH3 tMeBu). 13C NMR (CDCl3):
δ 170.7, 163.6, 155.3, 136.5, 130.0, 129.5, 128.6, 127.0, 109.9, 109.5, 102.1, 80.0, 74.3, 70.9, 70.7,
70.2, 62.0, 55.6, 54.5, 52.9, 52.4, 38.3, 31.8, 31.8, 29.1, 29.1, 28.5, 26.7, 26.3, 25.6, 24.3. MS
(ESI+): m/z = 663.4. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C35H54N2O10Na
685.3676; found 685.3656. [α]D20 = -5 (CHCl3, c=0.5).
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(3R,4S,5S,6S)-6-((2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)-2-(2,4,4-trimethylpentan-2-
ylamino)ethoxy)carbonyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (9)
Yield 70 % (53 mg); colorless oil; Rf = 0.60 (ethyl acetate/toluene 1:1, v/v); : = 15:85. Chemical
shifts are given for major isomer. 1H NMR (CDCl3): δ 6.35 (br s, NH), 5.78-5.74 (m, H1 GlcA),
5.42 (t, J = 9.4 Hz, H4 GlcA), 5.28-5.22 (m, H3 GlcA), 5.12–5.10 (m, H2 GlcA), 5.00 (br s, 1H,
H1 Fru), 4.57 (dd, J = 7.8, 2.8 Hz, H4 Fru), 4.36 (d, J = 9.7 Hz, H5 GlcA), 4.31 (d, J = 2.7 Hz, H3
Fru), 4.20 (d, J = 7.8 Hz, H5 Fru), 3.91 (dd, J = 12.8, 1.4 Hz, H6 Fru), 3.76-3.72 (m, H6 Fru, H1
CyHex), 2.06–1.99 (m, CH3-Ac), 1.67 (d, J = 14.9 Hz, H2 tMeBu), 1.58 (d, J = 14.2 Hz, H2'
tMeBu), 1.48 (s, CH3), 1.44 (s, CH3), 1.41 (s, CH3), 1.35 (m, CH3), 1.34 (s, CH3), 1.31 (s, CH3),
0.96 (s, H4, H3',H3' CH3 tMeBu). 13C NMR (CDCl3): δ 170.3, 169.5, 169.4, 168.7, 165.0, 162.7,
110.1, 109.6, 102.0, 91.8, 89.1, 75.0, 73.2, 72.6, 72.1, 70.8, 70.8, 70.4, 70.2, 69.1, 68.5, 61.9, 55.5,
53.2, 52.8, 31.7, 29.0, 29.0, 26.7, 26.2, 25.5, 24.3, 20.9, 20.8, 20.7. MS (ESI+): m/z = 782.4. HRMS
(MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C35H53NO17Na 782.3211; found 782.3192.
(2R,3R,4S,5R,6R)-2-((S)-2-acetoxy-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-
3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)-6-(acetoxymethyl)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (10)
Yield 80 % (54 mg); colorless oil; Rf = 0.50 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
6.94 (d, J = 9.4 Hz, 1H, NH), 5.27-5.20 (m, 2H, H1 Glc1NC, H3 Glc1NC), 5.07 (t, 1H, H4
Glc1NC), 5.02 (s, 1H, H1 Fru), 4.97 (t, 1H, H2 Glc1NC), 4.58 (dd, J = 7.9, 2.8 Hz, 1H, H4 Fru),
4.33 (dd, J = 12.4, 4.4 Hz, 1H, H6 Glc1NC), 4.30 (d, J = 2.8 Hz, 1H, H3 Fru)), 4.24 – 4.21 (m, 1H,
H5 Fru), 4.04 (dd, J = 12.4, 2.2 Hz, 1H, H6 Glc1NC), 3.88 (dd, J = 12.9, 1.7 Hz, 1H, H6 Fru), 3.83
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(d, J = 12.8 Hz, 1H, H6 Fru), 3.75 (ddd, J = 10.1, 4.3, 2.2 Hz, 1H, H5 Glc1NC), 2.16, 2.07, 2.02,
2.00, 1.97 (m, 15H, CH3), 1.53, 1.50, 1.44, 1.32 (m, 12H, CH3). 13C NMR (CDCl3): δ 170.8, 170.1,
169.8, 169.1, 166.3, 110.2, 109.7, 101.8, 78.6, 74.1, 73.9, 73.1, 70.8, 70.7, 70.4, 70.2, 68.7, 61.9,
61.9, 26.7, 26.1, 25.4, 24.4, 20.9, 20.9, 20.8, 20.7. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+
calcd. for C29H41NO17Na 698.2272; found 698.2289.
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(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((S)-2-(benzoyloxy)-2-((3aR,5aR,8aR,8bS)-2,2,7,7-
tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)tetrahydro-
2H-pyran-3,4,5-triyl triacetate (11)
Yield 57 % (42 mg); colorless oil; Rf = 0.62 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
8.06 (m, 2H, Ph), 7.57 (t, J = 7.5 Hz, 1H, Ph), 7.44 (t, J = 7.8 Hz, 2H, Ph), 7.20 (d, J = 9.2 Hz, 1H,
NH), 5.42 (s, 1H, H1 Fru), 5.26 (m, 2H, H1 Glc1NC, H3 Glc1NC), 5.08 (t, J = 9.7 Hz, 1H, H4
Glc1NC), 5.01 (t, J = 9.6 Hz, 1H, H2 Glc1NC), 4.59 (dd, J = 7.8 Hz, 2.7 Hz, 1H, H4 Fru), 4.41 (d,
J = 2.8 Hz, 1H, H3 Fru), 4.31 (dd, J = 12.4 Hz, 4.2 Hz, 1H, H6 Glc1NC), 4.25 (d, J = 7.8 Hz, 1H,
H5 Fru), 4.03 (dd, J = 12.4 Hz, 1.7 Hz, 1H, H6’ Glc1NC), 3.94 (d, J = 11.6 Hz, 1H, H6 Fru), 3.88
(d, J = 13 Hz, 1H, H6’ Fru), 3.74 (dd, J = 10.0 Hz, 1.9 Hz, 1H, H5 Glc1NC), 2.08, 2.05, 2.00, 1.99,
1.60, 1.54, 1.51, 1.32 (s, 24H, CH3). 13C NMR (CDCl3): δ 170.9, 170.9, 170.2, 169.8, 166.4, 165.4,
133.8, 130.2, 129.3, 128.7, 110.3, 109.7, 101.8, 78.7, 77.4, 77.2, 77.0, 74.0, 73.9, 73.0, 70.8, 70.7,
70.3, 70.1, 68.5, 62.0, 61.9, 26.8, 26.1, 25.9, 24.3, 21.0, 20.8. HRMS (MALDI-TOF/TOF) m/z:
[M+K]+ Calcd. for C34H43NO17K 776.2168; found 776.2135.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((S)-2-((S)-2-(tert-butoxycarbonylamino)-3-
phenylpropanoyloxy)-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)tetrahydro-2H-pyran-3,4,5-triyl
triacetate (12)
Yield 62 % (55 mg); colorless oil; Rf = 0.52 (ethyl acetate/toluene 1:1, v/v). 1H NMR (CDCl3): δ
7.28 (t, J = 7.3 Hz, 2H, Ph), 7.22 (m, 1H, Ph), 7.18 (d, J = 7.2 Hz, 2H, Ph), 7.02 (d, J = 9.4 Hz, 1H,
NH), 5.28-5.22 (m, 2H, H1 Glc1NC, H3 Glc1NC), 5.09 (s, 1H, H1 Fru), 5.08 (m, 1H, H4 Glc1NC),
4.99-4.96 (m, 2H, NH Phe, H2 Glc1NC), 4.58 (m, 2H,  Phe, H4 Fru), 4.34 (dd, J = 12.5 Hz, 4.3
Hz, 1H, H6 Glc1NC),4.29 (d, J = 2.5 Hz, 1H, H3 Fru), 4.22 (d, J = 7.8 Hz, 1H, H5 Fru), 4.01 (d, J
= 12.3 Hz, 1H, H6 Glc1NC), 3.86 (dd, J = 12.6 Hz, 2H, H6’ Fru), 3.76 (m, 1H, H5 Glc1NC), 3.32
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(dd, J = 14.1 Hz, 4.9 Hz, 1H,  Phe), 2.98 (dd, J = 14.1 Hz, 8.2 Hz, 1H, ’ Phe), 2.02, 2.01, 2.00,
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1.98 (s, 12H, CH3), 1.54, 1.51, 1.44, 1.36, 1.33 (s, 21H, CH3, Boc). C NMR (CDCl3): δ 171.0,
170.7, 170.2, 169.8, 165.985, 155.3, 136.3, 129.7, 128.8, 127.1, 110.4, 109.7, 101.5, 80.2, 78.5,
77.4, 77.2, 77.0, 74.2, 74.0, 73.0, 70.8, 70.5, 70.4, 70.1, 68.6, 62.1, 61.9, 54.5, 38.4, 28.4, 26.7,
26.1, 25.5, 24.4, 20.9, 20.8, 20.7. HRMS (MALDI-TOF/TOF) m/z: [M+K]+ calcd. for
C41H56N2O19K 919.3114; found 919.3116.
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(3R,4S,5S,6S)-6-((2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)-2-((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-ylamino)ethoxy)carbonyl)tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate (13)
Yield 68 % (67 mg); colorless oil; Rf = 0.37 (ethyl acetate/toluene 1:1, v/v); : 15:85; d.r. cannot
be determined due to peak overlap. Chemical shifts are given for major isomer. 1H NMR (CDCl3):
δ 7.34 (d, J = 8.9 Hz, α NH), 7.06 (d, J = 9.2 Hz, β NH), 6.37 (d, J = 3.5 Hz, H1α GlcA), 5.77 (d,
J = 7.5 Hz, H1β GlcA), 5.35 (t, J = 9.5 Hz, 1H, H4 GlcA), 5.15 (m, 1H, H1 Glc1NC), 5.11 (m, 1H,
H3 Glc1NC), 5.06 (m, 1H, H4 Glc1NC), 5.06-5.00 (m, 2H, H1 Fru, H3 GlcA), 4.94 (t, 1H, J = 9.5
Hz, H2 Glc1NC), 4.58 (dd, J = 7.7, 2.6 Hz, 1H, H4 Fru), 4.36 (d, J = 9.7 Hz, 1H, H5 GlcA), 4.32
(d, J = 4.2 Hz, 1H, H6 Glc1NC), 4.30 (d, J = 2.7 Hz, 1H, H3 Fru), 4.22 (d, J = 7.7 Hz, 1H, H5 Fru),
4.04 (dd, J = 12.4, 1.8 Hz, 1H, H6 Glc1NC), 3.87 (m, 2H, H6 Fru), 3.74 (dd, J = 8.1, 1.9 Hz, 1H,
H5 Glc1NC), 2.08-1.97 (m, 21H, CH3), 1.61-1.31 (m, 12H, CH3). 13C NMR (CDCl3): δ 170.9,
170.7, 170.2, 170.1, 169.8, 169.5, 169.4, 168.8, 165.2, 165.0, 110.5, 109.7, 101.4, 91.7, 78.6, 74.6,
73.9, 72.9, 72.5, 70.7, 70.6, 70.4, 70.2, 70.0, 68.6, 68.5, 62.0, 61.9, 26.7, 26.1, 25.4, 24.4, 20.9,
20.9, 20.9, 20.8, 20.8, 20.7. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C41H55NO26Na
1000.2910; found 1000.2930.
(3R,4R,5S,6R)-3-(2-acetoxy-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)-6-(acetoxymethyl)tetrahydro-2H-
pyran-2,4,5-triyl triacetate (14)
Yield 60 % (41mg); colorless oil; Rf = 0.33 (ethyl acetate/ petrol ether 1:1, v/v); : 60:40; approx
(peak overlap) d.r. 90:10. Chemical shifts are given for  and  isomer. 1H NMR (CDCl3): δ 6.32
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(d, J = 9.2 Hz, 0.6H,  NH), 6.14 (d, J = 3.6 Hz, 0.6H, H1 Glc2NC), 6.06 (d, J = 9.2 Hz, 0.4H, 
NH), 5.65 (d, J = 8.8 Hz, 0.4H, H1 Glc2NC), 5.28-5.23 (m, 0.6H, H3 Glc2NC), 5.14-5.08 (m,
1.4H, H3 Glc2NC, H4, Glc2NC), 4.99 (s, 0.6H, H1 Fru), 4.90 (s, 0.4H, H1 Fru), 4.58-4.56
(m, 1H, H4, Fru), 4.49-4.45 (m, 0.6H, H2 Glc2NC), 4.37-4.35 (m, 0.4H, H2 Glc2NC), 4.30
(d, 0.4H, J = 2.7 Hz, H3 Fru), 4.26 (d, 0.6H, J = 2.6 Hz, H3 Fru), 4.23-4.20 (m, 2.4H, H5 Fru,
H6 Glc2NC), 4.11 (dd, J = 12.5 Hz, 1.9 Hz, 0.4H, H6 Fru), 4.04 (dd, 0.6H, J = 12.5 Hz, 2.1 Hz,
H6 Fru), 3.98 (m, 0.6H, H5 Fru), 3.84-3.80 (dd, J = 13.0 Hz, 1.4 Hz, 0.6H, H6’ Fru), 3.80-
7.76 (m, 1.4H, H6’ Fru, H5, Glc2NC), 2.15-1.99 (m, 15H, CH3), 1.58-1.31 (m, 12H, CH3). 13C
NMR (CDCl3): δ 171.2, 170.9, 169.4, 169.1, 169.1, 168.4, 165.7, 165.6, 110.0, 109.9, 109.7, 109.7,
101.9, 101.9, 92.7, 90.8, 77.4, 77.2, 77.0, 74.0, 73.7, 73.4, 72.0, 70.8, 70.8, 70.6, 70.5, 70.2, 70.2,
69. 9, 68.2, 68.1, 61.9, 61.9, 61.7, 52.4, 50.6, 26.8, 26.7, 26.3, 25.5, 24.4, 24.3, 21.0, 20.9, 20.9,
20.8, 20.8. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C29H41NO17Na 698.2272; found
698.2278.
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(3R,4R,5S,6R)-6-(acetoxymethyl)-3-(2-(benzoyloxy)-2-((3aR,5aR,8aR,8bS)-2,2,7,7-
tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)tetrahydro-
2H-pyran-2,4,5-triyl triacetate (15)
Yield 61 % (45 mg); colorless oil; Rf = 0.43 (ethyl acetate/ petrol ether 1:1, v/v); : 60:40; approx
(peak overlap) dr 95:5. Chemical shifts are given for  and  isomer. 1H NMR (CDCl3): δ 8.07-
8.03 (m, 2H, Ph), 7.58-7.55 (m, 1H, Ph), 7.42-7.45 (m, 2H, Ph), 6.56 (d, J = 9.1 Hz, 0.6H,  NH),
6.30 (d, J = 9.7 Hz, 0.4H,  NH), 6.19 (d, J = 3.6 Hz, 0.6H, H1 Glc2NC), 5.68 (d, J = 8.8 Hz,
0.4H, H1 Glc2NC), 5.43 (s, 0.6H, H1 Fru), 5.28 (s, 0.4H, H1 Fru), 5.30-5.27 (m, 0.6H, H3
Glc2NC), 5.16-5.07 (m, 1.4H, H3 Glc2NC, H4, Glc2NC), 4.58 (dd, J = 7.8 Hz, 3.0 Hz, 1H,
H4, Fru), 4.49-4.44 (m, 0.6H, H2 Glc2NC), 4.40 (d, J = 2.7 Hz, 0.4H, H3β Fru), 4.37 (m,
0.4H, H2β Glc2NC), 4.33 (d, J = 2.7 Hz, 0.6H, H3 Fru), 4.23 (m, 2.6H, H5 Fru, H6 Glc2NC),
4.11 (dd, J = 12.5 Hz, 2.0 Hz, 0.4H, H6 Fru), 4.04 (dd, J = 12.4 Hz, 2.0 Hz, 0.6H, H6 Fru), 3.98
(m, 1H, H5 Fru), 3.89 (m, 1H, H6, Fru), 3.86-3.81 (m, 1H, H5, Glc2NC), 2.16-1.88 (m,
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12H, CH3), 1.58-1.31 (m, 12H, CH3). C NMR (CDCl3): δ 171.1, 170.9, 170.7, 169.8, 169.5,
169.4, 168.4, 165.9, 165.7, 165.3, 165.2, 133.7, 133.6, 130.2, 130.1, 128.7, 110.2, 110.0, 109.8,
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109.7, 101.9, 101.9, 92.8, 90.7, 74.2, 73.8, 73.3, 71.9, 70.8, 70.8, 70.7, 70.4, 70.1, 70.1, 69.9, 68.3,
68.2, 62.0, 61.9, 61.7, 52.5, 50.8, 26.9, 26.8, 26.2, 26.0, 24.4, 24.2, 20.9, 20.9, 20.9, 20.9, 20.7.
HRMS (MALDI-TOF/TOF) m/z: [M+K]+ calcd. for C34H43NO17K 776.2168; found 776.2145.
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(3R,4R,5S,6R)-6-(acetoxymethyl)-3-((R)-2-((S)-2-(tert-butoxycarbonylamino)-3-
phenylpropanoyloxy)-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamido)tetrahydro-2H-pyran-2,4,5-triyl
triacetate (16)
Yield 68 % (60 mg); colorless oil; Rf = 0.46 (ethyl acetate/toluene 1:1, v/v); : = 80:20. 1H NMR
(CDCl3): δ 7.38-7.08 (m, 5H, Ph), 6.55 (d, J = 9.1 Hz, 0.8H, α NH), 6.45 (d, J = 9.3 Hz, 0.2H, β
NH), 6.17 (d, J = 3.6 Hz, 0.8H, H1α Glc2NC), 5.67 (d, J = 8.8 Hz, 0.2H, H1-β Glc2NC), 5.28 (m,
1H, H3α,β Glc2NC), 5.14 (m, 0.8H, H4α Glc2NC), 5.11 (s, 0.8H, H1α Fru), 5.08 (m, 0.2H, H4β
Glc2NC), 4.99 (s, 0.2H, H1β Fru), 4.89 (d, J = 8.6 Hz, 0.8H, α NH Phe), 4.83 (d, J = 7.8 Hz 0.2H,
β NH Phe), 4.63-4.85 (m, 1H, α H Phe), 4.56 (dd, J = 7.8, 2.8 Hz, 1H, H4α,β Fru), 4.53-4.49 (m,
1.2H, H3β Glc2NC, H4α,β Glc2NC), 4.39 (dd, J = 19.9, 9.5 Hz, 0.2H, H2β Glc2NC), 4.30 (d, J =
2.4 Hz, 0.8H, H3β Fru), 4.23 (m, 2.8H, H5β Fru, H6 Glc2NC), 4.11 (dd, J = 12.5 Hz, 0.2H, H6β
Fru), 4.04 (dd, J = 12.4, 2.1 Hz, 0.8H, H6α Fru), 4.01-3.98 (m, 0.2H, H5β Fru), 3.87-3.85 (dd,J =
13.0, 1.5Hz, 0.8H, H6α Fru), 3.82-3.85 (m, 1.2H, H6β Fru, H5α,β Glc2NC), 3.38 (dd, J = 14.2,
4.4Hz, 0.8H, Hβ Phe), 3.33 (dd, J = 14.1 Hz, 0.2H, Hβ Phe). 3.06 (dd. J = 13.7, 7.8 Hz, 0.2H, Hβ’
Phe), 2.99 (dd, J = 14.2, 8.5 Hz, 0.8H, Hβ’ Phe), 2.14-2,00 (m, 12H, Ac), 1.63-1.30 (m, 24H, CH3-
isop, CH3 Boc). 13C NMR (CDCl3): δ 171.0, 170.9, 170.8, 169.4, 168.6, 165.4, 155.3, 136.4, 129.8,
129.7, 128.8, 128.7, 127.1, 110.3, 110.0, 109.5, 101.8, 92.7, 90.8, 80.2, 77.4, 77.2, 77.0, 74.1, 73.4,
70.8, 70.7, 70.5, 70.4, 70.1, 70.0, 68.1, 62.1, 61.8, 54.4, 50.6, 38.2, 28.5, 28.4, 26.7, 26.7, 26.3,
25.5, 25.5, 24.3, 21.1, 21.0, 20.9, 20.8, 20.7. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for
C41H56N2O19Na 903.3375; found 903.3346.
(3R,4S,5S,6S)-6-((2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)-2-((3R,4R,5S,6R)-2,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-3-ylamino)ethoxy)carbonyl)tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate (17)
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Yield 75 % (73 mg); colorless oil; Rf = 0.37 (ethyl acetate/toluene 1:1, v/v); : 80:20. Chemical
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shifts are given for the major isomer. H NMR (CDCl3): δ 6.50 (d, J = 9.1Hz, α NH), 6.11 (d, J =
3.5 Hz, H1α Glc2NC), 5.78 (d, J = 7.5 Hz, H1β GlcA), 5.38 (t, J = 9.4 Hz, H4β GlcA), 5.27-5.23
(m, H3 GlcA, H3 Glc2NC), 5.12-5.09 (m, H2 GlcA, H4 Glc2NC), 5.02 (s, H1 Fru), 4.92 (s, H1
Fru), 4.57 (dd, J = 7.8 Hz, 2.6 Hz, H4 Fru), 4.46-4.42 (m, H2 Glc2NC), 4.38 (dd, J = 9.7, 4.2 Hz,
H5 GlcA), 4.31-4.28 (d, J = 2.6 Hz, H3 Fru), 4.20 (m, H5 Fru), 4.09 (d, J = 12.2 Hz, H6 Glc2NC,),
4.03 (dd, J = 12.5, 2.1 Hz, H6 Glc2NC), 3.96 (m, H6 Fru), 3.87-3.74 (m, H5 Glc2NC, H6 Fru),
2.12-1.97 (m, CH3), 1.61-1.30 (m, CH3). 13C NMR (CDCl3): δ 170.9, 170.3, 169.7, 169.4, 169.4,
168.8, 168.5, 165.1, 164.4, 110.4, 109.7, 101.6, 91.8, 90.8, 74.0, 72.9, 72.4, 70.7, 70.7, 70.5, 70.3,
70.0, 69.9, 68.3, 68.0, 62.0, 61.8, 52.4, 50.5, 26.8, 26.2, 25.4, 24.3, 21.0, 20.9, 20.8, 20.8, 20.8.
HRMS (MALDI-TOF/TOF) m/z: [M+K]+ calcd. for C41H55NO26K 1016.2649; found 1016.2605.
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(3R,4S,5R,6R)-2-((2-acetoxy-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-3a-yl)acetamido)methyl)-6-(benzyloxy)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (18)
Yield 66 % (48 mg); colorless oil; Rf = 0.34 (ethyl acetate/toluene 1:1, v/v); d.r. 90:10. Chemical
shifts are given for major isomer. 1H NMR (CDCl3): δ 7.34-7.26 (m, 5H, Bn), 6.98 (br t, 1H, NH),
5.16 (s, 1H, H1 Fru), 5.10 (t, J = 9.5 Hz, 1H, H3 Glc6NC), 4.98-4.95 (m, 2H, H2 Glc6NC, H6
Glc6NC), 4.86 (d, J = 12.2 Hz, 1H, H4 Glc6NC), 4.60-4.58 (m, 2H, H5 Glc6NC, H4 Fru), 4.52 (d,
J = 8.1 Hz, 1H, H1 Glc6NC), 4.42 (d, J = 2.7 Hz, 1H, H3 Fru), 4.23 (d, J = 7.9 Hz, 1H, H5 Fru),
3.97 (dd, J = 13.0 Hz, 1.7 Hz, 1H, H6 Fru), 3.81 (d, J = 13.0 Hz, 1H, H6’ Fru), 3.60-3.57 (m, 2H,
H6 Glc6NC, CH2 Bn), 3.45-3.41 (m, 1H, CH2 Bn), 2.19 (s, 3H, CH3), 1.99-1-94 (m, 9H, CH3),
1.57-1.31 (m, 12H, CH3). 13C NMR (CDCl3): δ 170.4, 169.6, 169.4, 165.7, 136.9, 128.7, 128.3,
128.0, 110.1, 109.4, 102.4, 99.3, 77.4, 77.2, 77.0, 73.9, 73.2, 72.6, 71.4, 71.0, 70.8, 70.6, 70.3,
69.7, 61.8, 39.5, 26.6, 26.3, 25.4, 24.3, 21.0, 20.9, 20.8. HRMS (MALDI-TOF/TOF) m/z: [M+Na]+
calcd. for C34H45NO16Na 746.2636; found 746.2655.
(3R,4S,5R,6R)-2-((2-(benzoyloxy)-2-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-
3aH-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-3a-yl)acetamido)methyl)-6-
(benzyloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19)
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Yield 68 % (54 mg); colorless oil; Rf = 0.51 (ethyl acetate/toluene 1:1, v/v); d.r. 94:6. Chemical
shifts are given for the major isomer. 1H NMR (CDCl3): δ 8.09 (dd, J = 7.4 Hz, 2H, Ph), 7.56 (m,
1H, Ph), 7.43 (t, J = 7.7 Hz, 2H, Ph), 7.35-7.28 (m, 5H, Bn), 7.12 (br t, 1H, NH), 5.53 (s, 1H, H1
Fru), 5.10 (t, J = 9.5 Hz, 1H, H3 Glc6NC), 4.99 (m, 2H, H2 Glc6NC, H6 Glc6NC), 4.88 (d, J =
12.2 Hz, 1H, H4 Glc6NC), 4.62-4.58 (m, 2H, H5 Glc, H4 Fru), 4.54-4.51 (m, 2H, H1 Glc6NC,
H3 Fru), 4.25 (d, J = 7.7 Hz, 1H, H5 Fru), 4.03 (dd, J = 13.0 Hz, 1.7 Hz, 1H, H6 Fru), 3.85 (d, J =
13.0 Hz, 1H, H6’ Fru), 3.62-3.58 (m, 2H, H6 Glc6NC, CH2 Bn), 3.51-3.46 (m, 1H, CH2 Bn), 1.97-
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1-94 (m, 9H, CH3), 1.62-1.30 (m, 12H, CH3). C NMR (CDCl3): δ 170.5, 169.6, 169.6, 165.8,
165.6, 136.9, 133.5, 130.2, 129.8, 128.7, 128.7, 128.3, 128.0, 110.2, 109.5, 102.5, 99.3, 77.4, 77.2,
77.0, 74.2, 73.2, 72.6, 71.4, 70.9, 70.8, 70.6, 70.2, 69.7, 61.9, 39.5, 26.7, 26.3, 25.8, 24.2, 20.9.
HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C39H47NO16Na 808.2793; found 808.2789.
(3R,4S,5R,6R)-2-((7R)-7-benzyl-11,11-dimethyl-3,6,9-trioxo-4-((3aR,5aR,8aR,8bS)-2,2,7,7-
tetramethyltetrahydro-3aH-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-3a-yl)-5,10-dioxa-2,8-
diazadodecyl)-6-(benzyloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (20)
Yield 70 % (65 mg); colorless oil; Rf = 0.58 (ethyl acetate/toluene 1:1, v/v); d.r. 90:10. Chemical
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shifts are given for the major isomer. H NMR (CDCl3): δ 7.23-7.21 (m, 10H, Bn, Ph), 7.11 (m,
1H, NH), 5.23 (s, 1H, H1 Fru), 5.10 (t, J = 9.5 Hz, 1H, H3 Glc6NC), 5.01-4.96 (m, 2H, H2 Glc6NC,
H6 Glc6NC), 4.93 (d, J = 8.4 Hz, 1H, NH Phe), 4.85 (d, J = 12.2 Hz, 1H, H4 Glc6NC), 4.65-4.57
(m, 3H,  Phe, H5 Glc, H4 Fru), 4.50 (d, J = 8.0 Hz, 1H, H1 Glc6NC), 4.40 (d, J = 2.5 Hz, 1H,
H3 Fru), 4.22 (d, J = 7.5 Hz, 1H, H5 Fru), 3.89 (d, J = 11.9 Hz, 1H, H6 Fru), 3.81 (d, J = 13.0 Hz,
1H, H6’ Fru), 3.60-3.51 (m, 3H, H6 Glc6NC, CH2 Bn), 3.41 (dd, J = 14.2 Hz, 4.7 Hz, 1H,  Phe),
3.03 (dd, J = 14.2 Hz, 8.2 Hz, 1H, ’ Phe), 1.97-1-94 (m, 9H, CH3), 1.58-1.30 (m, 21H, CH3 Boc).
13C NMR (CDCl3): δ 170.9, 170.5, 169.6, 169.5, 165.4, 155.3, 136.9, 136.7, 129.8, 128.7, 128.7,
128.3, 128.0, 127.0, 110.3, 109.4, 102.3, 99.3, 80.1, 77.4, 77.2, 77.0, 74.1, 73.2, 72.7, 71.3, 70.9,
70.8, 70.5, 70.1, 69.6, 62.0, 54.7, 39.6, 38.5, 28.4, 26.6, 26.3, 25.4, 24.3, 20.9, 20.9. HRMS
(MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C46H60N2O18Na 951.3739; found 951.3741.
(3R,4S,5S,6S)-6-((2-oxo-1-((3aR,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)-2-(((3R,4S,5R,6S)-3,4,5-triacetoxy-6-
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(benzoyloxy)tetrahydro-2H-pyran-2-yl)methylamino)ethoxy)carbonyl)tetrahydro-2H-
pyran-2,3,4,5-tetrayl tetraacetate (21)
Yield 75 % (76 mg); colorless oil; Rf = 0.37 (ethyl acetate/toluene 1:1, v/v). 4 isomers. Chemical
shifts are given for the major isomer. 1H NMR (CDCl3): δ 7.35-7.25 (m, Bn), 7.00 (t, J = 5.9 Hz,
NH), 5.80 (d, J = 7.7 Hz, H1β GlcA), 5.40 (t, J = 9.5 Hz, H4 GlcA), 5.26 (t, J = 9.1 Hz, H3 GlcA),
5.18-5.08 (m, H1 Fru, H2 GlcA, H3 Glc6NC), 4.98-4.92 (m, H2, H6 Glc6NC), 4.85 (d, J = 12.2
Hz, H4 Glc6NC), 4.60-4.56 (m, H4 Fru, H6 Glc6NC), 4.52 (d, J = 8.0 Hz, H1 Glc6NC), 4.41 (dd,
J = 6.2, 3.4 Hz, H3 Fru, H5 GlcA), 4.21 (d, J = 7.8 Hz, H5 Fru), 3.97 (dd, J = 13.0, 1.6 Hz, H6
Fru), 3.80 (d, J = 13.0 Hz, H6 Fru), 3.56 (m, H6 Glc6NC, CH2 Bn), 3.41 (m, CH2 Bn), 2.08-1.94
(m, CH3), 1.56-1.30 (m, CH3). 13C NMR (CDCl3): δ 170.5, 170.2, 169.6, 169.5, 169.4, 168.9, 165.5,
164.5, 136.9, 128.7, 128.3, 127.9, 110.4, 109.4, 102.1, 99.4, 91.8, 74.8, 73.2, 73.1, 72.6, 72.5, 71.3,
71., 70.8, 70.5, 70.4, 70.1, 69.8, 68.6, 62.0, 39.6, 26.6, 26.3, 25.3, 24.3, 20.9, 20.9, 20.8, 20.8.
HRMS (MALDI-TOF/TOF) m/z: [M+Na]+ calcd. for C46H59NO25Na 1048.3274; found 1048.3307.
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Deprotection of selected Passerini products
Synthesis
of
(S)-N-cyclohexyl-2-hydroxy-2-((3aS,5aR,8aR,8bS)-2,2,7,7-
tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)acetamide (22)
Passerini product 2 (15 mg; 0.035 mmol) was dissolved in MeOH and 1M NaOH (70 μL) was
added. Reaction was stirred under reflux and followed by TLC (petrol ether/ethyl acetate 1:1, v/v).
After 10 min, solvent was evaporated and the product purified by flash column chromatography
(petrol/ethyl acetate 1:1, v/v). Yield 81 % (11 mg); colorless oil; Rf = 0.7 (petrol/ethyl acetate 1:1,
v/v). 1H NMR (600 MHz, CDCl3) δ 6.56 (d, J = 7.5 Hz, 1H, NH), 4.68 (d, J = 2.7 Hz, 1H, H3 Fru),
4.61 (dd, J = 7.9, 2.7 Hz, 1H, H4 Fru), 4.31 (s, 1H, OH), 4.22 (dd, J = 7.9, 1.2 Hz, 1H, H5 Fru),
3.97 (s, 1H, H1 Fru), 3.93 (dd, J = 12.9, 1.8 Hz, 1H, H6 Fru), 3.83 – 3.78 (m, 1H, CyHex), 3.76 (d,
J = 13.0 Hz, 1H, H6 Fru), 1.92 – 1.85 (m, 2H, CyHex), 1.69 – 1.53 (m, 4H, CyHex), 1.46 (d, J =
4.7 Hz, 6H, CH3), 1.38 (s, 3H, CH3), 1.33 (s, 4H, CyHex, CH3), 1.24 – 1.15 (m, 3H, CyHex). 13C
NMR (CDCl3) δ 169.4, 109.4, 109.2, 103.7, 71.1, 71.0, 70.5, 61.9, 49.1, 33.0, 32.8, 26.6, 26.3,
25.8, 25.7, 24.8, 24.7, 24.2. HRMS (MALDI-TOF/TOF) m/z: [M+H]+calcd. for C19H31NO7 386.
2179; found 386.2174.
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Synthesis of (S)-2-(cyclohexylamino)-2-oxo-1-((3S,4R,5R)-2,3,4,5-tetrahydroxytetrahydro-
2H-pyran-2-yl)ethyl benzoate (23)
Compound 3 (20 mg, 0.04 mmol) was dissolved in mixture of DCM and TFA (200 µL, 1:1, v/v),
and stirred at room temperature for 6 h. Product was precipitated with cold di-isopropyl ether. Yield
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67 % (11 mg); colorless oil; C NMR (151 MHz, DMSO) δ 209.1, 169.8, 166.9, 133.7, 129.6,
129.0, 128.7, 127.1, 96.3, 82.6, 77.8, 75.4, 75.3, 72.7, 72.5, 72.3, 70.8, 69.7, 68.7, 66.3, 62.4, 62.2,
60.2, 48.0, 47.5, 40.0, 39.9, 35.3, 34.2, 32.0, 31.9, 31.7, 25.0, 24.7, 24.5, 22.7. HRMS (MALDI-
TOF/TOF) m/z: [M+Na]+calcd. for C20H27NO8Na 432.1634; found 432.1637.
Synthesis
of
(S)-2-hydroxy-2-((3aS,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-
bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-3a-yl)-N-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)acetamide (24)
To the stirred solution of compound 10 (20.8 mg, 0.03 mmol) in MeOH (600 mL) sodium metoxide
(25.5 mL, 0.25 M) was added. After stirring for 2 h at room temperature the reaction was quenched
by addition of IR-120 resin (H+). The insoluble materials were filtrated off and the filtrate was
concentrated. The residue was passed through a short column of silica gel to afford compound 24.
Yield 97 % (13 mg); white solid; m.p. = 145-146° C; Rf = 0.28 (EtOAc/EtOH/AcOH/H2O 70:5:2:2,
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v/v). H NMR (600 MHz, DMSO) δ 7.64 (d, J = 8.9 Hz, 1H, NH), 4.70 (t, J = 8.9 Hz, 1H, H-1
Glc), 4.60 (dd, J = 7.9, 2.5 Hz, 1H, H-3 Fru), 4.50 (d, J = 2.5 Hz, 1H, H-4 Fru), 4.23 (t, J = 8.7 Hz,
1H, H-5 Fru), 3.93 (s, 1H, H-1 Fru), 3.74 (m, 1H, H-6 Fru), 3.63 (m, 2H, H-6 Fru, Glc), 3.46 (m,
1H, H-6 Glc), 3.19 (m, 1H, H-3 Glc), 3.10 (m, 3H, H-5, H-4, H-2 Glc), 1.44 (s, 3H, CH3), 1.41 (s,
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3H, CH3), 1.35 (s, 3H, CH3), 1.29 (s, 3H, CH3). C NMR (151 MHz, DMSO) δ 170.2, 108.4,
108.2, 103.1, 80.0, 78.8, 77.2, 72.6, 72.0, 70.0, 69.8, 69.7, 69.5, 60.8, 60.7, 26.3, 25.8, 25.4, 24.0.
HRMS (MALDI-TOF/TOF) m/z: [M+Na]+calcd. for C19H31NO12Na 488.1744; found 488.1748.
Synthesis
of
(2R,3R,4S,5R,6R)-2-((S)-2-acetoxy-2-((3S,4R,5R)-2,3,4,5-
tetrahydroxytetrahydro-2H-pyran-2-yl)acetamido)-6-(acetoxymethyl)tetrahydro-2H-pyran-
3,4,5-triyl triacetate (25) and (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((S)-2-hydroxy-2-
((3S,4R,5R)-2,3,4,5-tetrahydroxytetrahydro-2H-pyran-2-yl)acetamido)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (26)
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Compound 10 (18.3 mg, 0.027 mmol) was dissolved in mixture TFA and H2O (9:1, v/v) and stirred
for 5h at room temperature. The reaction was monitored by TLC in EtOAc/EtOH/AcOHH2O
70:5:2:2. The products were precipitated with cold diisopropylether and centrifuged. The
precipitate was dried, dissolved in water and passed through C-18 silica gel Bond Elute patron to
give inseparable mixture of compounds 25 and 26 (5 mg) as a mixture of anomers. 13C NMR (151
MHz, DMSO) δ 196.2, 169.9, 169.4, 169.2, 169.0, 168.97, 168.2, 104.7, 102.1, 101.4, 96.3, 93.7,
91.2, 89.0, 87.9, 84.1, 82.1, 79.8, 77.0, 76.6, 76.1, 75.5, 72.9, 72.7, 72.6, 72.4, 72.2, 71.1, 70.8,
70.6, 70.4, 69.3, 68.8, 68.5, 67.8, 66.2, 62.7, 62.3, 62.1, 61.8, 40.0, 39.9, 39.9, 39.8, 39.8, 39.7,
39.5, 39.4, 39.2, 39.1, 35.6, 34.6, 20.5, 20.3, 20.2, 15.6.
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Synthesis of (S)-2-hydroxy-2-((3S,4R,5R)-2,3,4,5-tetrahydroxytetrahydro-2H-pyran-2-yl)-N-
((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)acetamide
(27)
Compound 24 (32 mg, 0.688 mmol) was dissolved in mixture TFA and H2O (9:1, v/v) and stirred
for 2h at room temperature. The reaction was monitored by TLC in EtOAc/EtOH/AcOH/H2O
7:2:2:2. The products were precipitated with cold diisopropylether, centrifuged, and dried, to give
compound 27 as TFA salt. Yield 68 % (23 mg); white solid; m.p. = 152-155° C; Rf = 0.35
(EtOAc/EtOH/AcOH/H2O 7:2:2:2, v/v). 13C NMR (151 MHz, DMSO) δ 175.09, 173.73, 171.73,
170.65, 152.96, 118.01, 102.43, 101.52, 100.06, 96.87, 92.19, 81.83, 81.78, 81.65, 81.34, 79.72,
79.36, 79.25, 79.21, 78.85, 78.76, 78.72, 77.18, 77.15, 77.07, 77.02, 76.75, 76.70, 76.17, 75.11,
74.83, 74.64, 74.47, 73.25, 73.07, 72.53, 72.50, 72.36, 72.18, 71.91, 70.90, 70.61, 70.32, 69.91,
69.79, 69.71, 69.65, 69.45, 69.12, 68.01, 67.12, 63.47, 63.41, 62.81, 62.70, 61.22, 60.93, 60.81,
60.48.
Synthesis
of
2-(tert-butylamino)-2-oxo-1-((3aR,5S,5aS,8aS,8bR)-2,2,7,7-
tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'-d]pyran-5-yl)ethyl acetate (28)
To a glass vial containing 1 M solution of 1,2:3,4-di-O-isopropylidene-α-D-galacto-hexadialdo-
1,5-pyranose (0.1 mmol) in DCM under nitrogen were added acetic acid (0.1 mmol) and tert-butyl
isocyanide (0.1 mmol) dissolved in 100 µL DCM. With all reactants added, the solution was
allowed to stir for 8 h. The reactions were concentrated under reduced pressure and reaction
mixtures were purified by flash column chromatography.
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Yield 78 % (31 mg); colorless oil; Rf = 0.60 (EtOAc/PE 1:1, v/v); d.r. 80:20. Chemical shifts are
given for both isomers. 1H NMR (CDCl3) δ 6.19 (s, 0.8H, NH), 6.09 (s, 0.2H, NH), 5.49 (d, J = 4.8
Hz, 1H, H-1 Gal), 5.14 (d, J = 6.5 Hz, 0.2H, H-6 Gal), 4.87 (d, J = 9.8 Hz, 0.8H, H-6 Gal), 4.61 –
4.55 (m, 1H, H-3 Gal), 4.32 – 4.23 (m, 2H, H-2,4 Gal), 4.18 (d, J = 6.4 Hz, 0.2H, H-5 Gal), 4.11
(dd, J = 9.8, 1.4 Hz, 0.8H, H-5 Gal), 2.13 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.36
– 1.25 (m, 15H, CH3). 13C NMR (CDCl3) δ 170.5, 169.7, 167.2, 166.3, 109.8, 109.7, 109.7, 109.3,
96.6, 96.4, 73.9, 71.6, 71.1, 71.0, 70.9, 70.6, 70.6, 67.4, 67.4, 51.7, 28.9, 28.7, 26.3, 26.2, 26.2,
26.1, 25.2, 24.6, 24.3, 21.2, 20.9. HRMS (MALDI-TOF/TOF) m/z: calcd. for C19H31NO8 [M+H]+
402.2128; found 402.2146.
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Synthesis of 2-(cyclohexylamino)-1-((3aR,4S,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-2-oxoethyl acetate (29)
To a glass vial containing 1 M solution of methyl 2,3-O-isopropylidene-β-D-ribo-
pentodialdofuranoside (0.1 mmol) in DCM under nitrogen were added acetic acid (0.1 mmol) and
cyclohexyl isocyanide (0.1 mmol) dissolved in 100 µL DCM. With all reactants added, the solution
was allowed to stir for 8 h. The reactions were concentrated under reduced pressure and reaction
mixtures were purified by flash column chromatography.
Yield 82 % (30 mg); colorless oil; Rf = 0.63 (EtOAc/PE 1:1, v/v); d.r. 77:23. Chemical shifts are given for
both isomers. 1H NMR (CDCl3): δ 5.97 (d, J = 7.5 Hz, 0.77H, NH), 5.90 (d, J = 7.7 Hz, 0.23H, NH), 5.11
(d, J = 7.6 Hz, 0.23H, H-5 Rib), 5.07 (d, J = 6.9 Hz, 0.77H, H-5 Rib), 4.95 (s, 0.23H, H-1 Rib), 4.94 (s,
0.77H, H-1 Rib), 4.87 (dd, J = 6.0, 1.4 Hz, 0.23H, H-2 Rib), 4.71 (dd, J = 6.2, 0.5 Hz, 0.77H, H-2 Rib), 4.60
(d, J = 6.9 Hz, 0.77H, H-4 Rib), 4.55 (d, J = 6.0 Hz, 0.77H, H-3 Rib), 4.53 (d, J = 6.0 Hz, 0.23H, H-3 Rib),
4.50 (dd, J = 7.6, 1.4 Hz, 0.23H, H-4 Rib), 3.79 – 3.72 (m, 1H, CH-1 CyHex), 3.32 (s, 2.3H, OCH3), 3.28
(s, 0.7H, OCH3), 2.15 (s, 0.7H, CH3), 2.13 (s, 2.3H, CH3), 1.89 (m, 2H, CyHex), 1.66 (m, 2H, CyHex), 1.58
(m, 1H, CyHex), 1.45 (s, 3H, CH3), 1.34 (m, 2H, CyHex), 1.29 (s, 2.3H, CH3), 1.28 (s, 0.7H, CH3), 1.16 –
1.14 (m, 3H, CyHex). 13C NMR (CDCl3) δ 170.0, 166.1, 112.9, 111.1, 110.3, 86.2, 86.0, 85.4, 85.3, 81.7,
81.4, 74.8, 73.6, 56.2, 55.5, 48.5, 48.35, 33.1, 33.1, 26.8, 26.7, 25.7, 25.6, 25.3, 24.9, 24.9, 21.4, 21.1.
HRMS (MALDI-TOF/TOF) m/z: calcd. for C18H29NO7Na [M+Na]+ 394.1842; found 394.1843.
Synthesis of 1-((3aR,5S,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-
(cyclohexylamino)-2-oxoethyl acetate (30)
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