Synthesis of mannoheptose derivatives and their evaluation as inhibitors of the lectin LecB from the opportunistic pathogen Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.carres.2015.04.010

Abstract Biofilm formation and chronic infections with Pseudomonas aeruginosa depend on lectins produced by the bacterium. The bacterial C-type lectin LecB binds to the two monosaccharides l-fucose and d-mannose and conjugates thereof. Previously, d-manno

Full text of DOI:10.1016/j.carres.2015.04.010

Carbohydrate Research 412 (2015) 34e42
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of mannoheptose derivatives and their evaluation as
inhibitors of the lectin LecB from the opportunistic pathogen
Pseudomonas aeruginosa
,
y
,
,
b
,
c
,
, b,
Anna Hofmann a z, Roman Sommer a
z, Dirk Hauck a c, Julia Stifel a,
a
a, b, c, *
€
Inigo Gottker-Schnetmann , Alexander Titz
a Department of Chemistry and Graduate School Chemical Biology, University of Konstanz, D-78457 Konstanz, Germany
b Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany
c Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Biofilm formation and chronic infections with Pseudomonas aeruginosa depend on lectins produced by
Received 23 December 2014
Received in revised form
2 April 2015
Accepted 3 April 2015
Available online 5 May 2015
the bacterium. The bacterial C-type lectin LecB binds to the two monosaccharides
mannose and conjugates thereof. Previously, -mannose derivatives with amide and sulfonamide sub-
stituents at C6 were reported as potent inhibitors of the bacterial lectin LecB and LecB-mediated bacterial
surface adhesion. Because -mannose establishes a hydrogen bond via its 6-OH group with Ser23 of LecB
in the crystal structure and may be beneficial for binding affinity, we extended -mannose and syn-
L-fucose and D-
D
D
D
thesized mannoheptoses bearing the free 6-OH group as well as amido and sulfonamido-substituents at
C7. Two series of diastereomeric mannoheptoses were synthesized and the stereochemistry was deter-
mined by X-ray crystallography. The potency of the mannoheptoses as LecB inhibitors was assessed in a
competitive binding assay. The data reveal a diastereoselectivity of LecB for (6S)-mannoheptose de-
Keywords:
Lectin
Pseudomonas aeruginosa
Inhibitor
rivatives with increased activity over methyl a-D-mannoside.
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
For P. aeruginosa the two bacterial lectins LecA and LecB
(formerly named PA-IL and PA-IIL, respectively) are involved in
biofilm formation and knock-out strains show reduced biofilm
mass.7,8
The Gram-negative bacterium Pseudomonas aeruginosa is an
opportunistic human pathogen that is responsible for an extensive
part of nosocomial infections.1,2 In immuno-compromised patients
or individuals with the genetic disease cystic fibrosis, the bacterium
can establish chronic infections, which can lead to organ failure and
death of the patient.3 In addition to its high level of multidrug
resistance P. aeruginosa can live in biofilms, which provide an
additional protection against hostile environment such as the im-
mune defense or antibiotic treatment.4,5 Bacterial biofilms are
multicellular communities in which bacterial cells are embedded in
a self-produced matrix, consisting of exopolysaccharides, extra-
cellular DNA, lipids and proteins.6
LecB was initially isolated by Gilboa-Garber et al. and a glycan-
binding specificity for L-fucose and D
-mannose was found.9,10 The
structure of LecB was solved by X-ray crystallography and revealed
a non-covalent tetrameric assembly with the carbohydrate binding
sites oriented towards the vertices of a tetrahedron.11,12 The car-
bohydrate ligands L-fucose or D-mannose are bound via all three
secondary hydroxy groups in the respective ligand to the two cal-
cium ions in the carbohydrate recognition domain of LecB. Methyl
a
-
L
-fucoside shows an unusual high affinity for LecB (Kd¼430 nM),
whereas mannoside 1 (Fig. 1) is a 150-fold weaker ligand for the
lectin (Kd¼71
m
M).13 The fucose-containing trisaccharide Lewisa
was identified as its strongest monovalent ligand (Kd¼210 nM).14
Consequently, a number of fucose derivatives have been devel-
oped and evaluated for LecB inhibition, often on multivalent display
as a technique to improve binding affinities.15e18 Reymond and co-
* Corresponding author.
E-mail address: alexander.titz@helmholtz-hzi.de (A. Titz).
Current address: Institute of Pharmacy and Food Chemistry, University of
y
Würzburg, D-97074 Würzburg, Germany.
z
workers developed
a
fucosylated multivalent glycopeptide
Both authors contributed equally.
http://dx.doi.org/10.1016/j.carres.2015.04.010
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
35
Fig. 1. The LecB ligand methyl a-D-mannoside (1) forms a hydrogen bond through its 6-hydroxy group with Ser23. Mannose derivatives 2 and 3 are high affinity inhibitors of LecB,
although unable to establish a hydrogen bond with Ser23. Mannoheptose derivatives 4 and 5 were designed to allow possible hydrogen bond formation of their 6-hydroxy group
with Ser23 and attachment of additional pharmacophores (R).
dendrimer, which inhibits the formation of P. aeruginosa biofilms in
a LecB-dependent manner.19 Recently, Vidal and co-workers re-
ported fucosylated tetravalent glycoclusters on a calixarene scaffold
as high-affinity ligands for LecB, which reduced P. aeruginosa-
induced lung injury as well as bacterial load in lung and spleen
tissue.20 All fucose-based inhibitors of LecB bear unmodified ter-
the nitrogen atom at C7 can then be performed to target the
adjacent protein surface.
Here, we report the chemical synthesis of two sets of diaste-
reomeric mannoheptoses and the optimization of the reaction
sequence Swern oxidation, cyanohydrin formation and subsequent
reduction to the amine. Finally, the compounds obtained in the two
diastereomeric series were evaluated in a competitive binding
assay for their binding affinity to LecB.
minal
a-L-fucosyl residues, an epitope, which is recognized by
numerous lectins of the host immune system, e.g. the selectins, DC-
SIGN, MBL and others. In order to potentially increase target
specificity and reduce interference with host lectins, we recently
reported high affinity mannose-based LecB inhibitors 2 and 3 with
a camouflaged mannosyl residue through capping at C6.21
2. Results and discussion
In contrast to its low affinity for this lectin,
D-mannose forms an
The synthesis of the diastereomeric mannoheptosides 4 and 5
additional hydrogen bond in the crystal structure of its O6 with
LecB at residue Ser23.12 Interestingly, this hydrogen bond does not
contribute significantly to the overall binding affinity at ambient
conditions.22 This observation could result from a high degree of
flexibility of 1 in the unbound state, and thus increased entropy
costs upon binding, a concept well established for the C-type lectin
E-selectin and its carbohydrate ligand sialyl Lewisx.23e27 Extension
of mannose-derived ligand 1 with additional pharmacophores at
C6 allows the targeting of adjacent protein surface and potent in-
hibitors, e.g. 2 and 3 (Fig. 1), were obtained.21 Although these
compounds are LecB inhibitors with more than 20-fold improved
affinity, the hydrogen bond with Ser23 cannot be established due
the lack of O6. Additionally exploiting the interaction of Man-O6 by
increasing its attractive potential could lead to superior inhibitors.
For example, rigidification of Man-O6 in its crystal-observed
bioactive orientation through assistance of closely protein bound
adjacent pharmacophores is a promising approach. To analyze the
effect of a hydrogen bonding interaction of O6 in addition to the
presence of further pharmacophores targeting the adjacent protein
surface on LecB-binding, we designed mannoheptosides 4 and 5
(Fig. 1). The synthesis of both diastereomers should allow the
assessment of the influence of the stereochemistry at C6 on the
binding affinity to LecB. Further attachment of pharmacophores on
was initiated by transforming commercial methyl a-D-mannoside
(1) according to published procedures into the selectively protected
mannoside 628 with a free 6-hydroxy group allowing further
manipulation (Scheme 1). Therefore, 1 was trityl-protected at O-6,
subsequently benzylated at the 2-, 3- and 4-hydroxy groups and
detritylated using iron(III) chloride29 without purification of the
intermediates on a 35 g scale in 48% yield over three steps.
Oxidation of the primary hydroxy group in 6 to the aldehyde 7 was
performed under Swern conditions.
In a first Swern oxidation, the reaction yielded the desired
product 7 in 34% and a,b-unsaturated elimination product 8 in 33%
after workup without neutralization of the triethylamine (Table 1,
entry 1). Mackie and Perlin30 reported similar base-induced elim-
ination products after Swern oxidation for acylated pyranosides.
The acetates reported by Mackie and Perlin are however much
better leaving groups than benzylic alcoholate as it is present as
leaving group at the 4-position in precursor 6. In a series of re-
actions to avoid elimination, the water bath temperature for sol-
vent removal was reduced, excess of triethylamine was reduced
and either volatiles were removed directly or after aqueous workup
(entry 2e4). Aldehyde 7 was finally obtained in 77% yield after
initial neutralization of the reaction and subsequent purification
(entry 5).

36
A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
Scheme 1. Synthesis of methyl
a
-D-manno-7-amino-7-deoxy-heptapyranosides 11a and 11b: (i) Ph3CCl, pyr, rt, (ii) NaH, PhCH2Br, DMF, 0 ꢀCert, (iii) FeCl3*6H2O, CH2Cl2, rt, (iv)
(COCl)2, DMSO, NEt3, CH2Cl2, ꢁ75 ꢀCert, (v) KCN, MeOH, NH4Cl (aq), 0 ꢀCert, (vi) LiAlH4, THF, 0 ꢀCert, (vii) PdeC, H2 (1 bar), H2O, THF, TFA, rt. 11a and 11b were obtained as crude
products and used without further purification.
Table 1
Optimization of the Swern oxidation (6/7) workup procedure to prevent formation of elimination product 8
Entry
NEt3 [eq.]
Workup procedure
7 [%]a
8 [%]a
1
2
3
4
5
5
5
2.5
2.5
2.5
Quenching (H2O), extraction (CH2Cl2), solvent removal in vacuo (40 ꢀC)
Direct solvent removal in vacuo (30 ꢀC)
34
43
68
70
77
33
d
Quenching (H2O), extraction (CH2Cl2), solvent removal in vacuo (30 ꢀC)
Direct solvent removal in vacuo (30 ꢀC)
d
Traces (TLC)
Traces (TLC)
Quenching (satd. NH4Cl aq), wash (H2O), extraction (CH2Cl2), solvent removal in vacuo (30 ꢀC)
a
Isolated yields.
The mannoheptose motif was then established by cyanohydrin
formation with aldehyde 7 and potassium cyanide. Because of its
basicity and potential to induce elimination in 7, we tested various
aqueous buffered conditions for the addition reaction (Table 2). The
transformation (7/9) was performed using common buffers with
pH values ranging from 4.5 to 8.0 and the product was obtained as a
separable diastereomeric mixture 9a and 9b, with slightly varying
diastereoselectivity from 1.8:1 to 2.3:1. The ammonium chloride
buffered system was used on preparative scale and isolated 9a and
isolated 9b could be obtained in 50% and 22% yield, respectively. To
determine the absolute configuration at C6 of diastereomers 9a and
9b, we performed 1H NMR experiments with J-coupling analysis as
well as J-HMBC NMR experiments. In non-polar solvents such as
deuterated chloroform, compounds 9a and 9b can potentially
establish an intramolecular hydrogen bond between the 6-hydroxy
group and the oxygen atom at position 4 (Fig. 2A). Assuming such a
conformation, a gauche orientation between H5 and H6 for 9a and
a trans orientation for both nuclei in 9b would lead to small and
large 3J-coupling constants respectively, following the Karplus
equation. However, only small coupling constants for both di-
astereomers were observed: 3J5,6¼1.1 Hz for 9a, 3J5,6¼4.0 Hz for 9b.
Varying the temperature during NMR analysis to observe the po-
tential 6-membered ring at reduced temperature only gave line
broadening and signal overlap (see Supplementary data). Similar to
1H,1H coupling, the Karplus equation also describes 3J-coupling
constants between 1H and 13C nuclei. To avoid the problem of signal
overlap in 1D spectra, we therefore analyzed 9a and 9b in a two-
dimensional J-HMBC NMR31 experiment to determine the
coupling constants between H5 and the nitrile carbon C7 of the two
isomers. Again, comparable coupling constants were observed:
Table 2
Optimization of the cyanohydrin formation (7/9) to prevent KCN-induced formation of elimination product 8 and to analyze buffer influence on diastereoselectivitya
Entry
Buffer system
Conc. [M]
pH
8 [%]
d.r. 9a:9b (1H NMR)
Isolated yield [%]
1
2
3
4
TriseHCl
K2HPO4/KH2PO4
NaHCO3
1
1
1
6
7.0
7.4
8.0
4.5
d
1.8:1
2:1
2:1
d
d
d
d
d
NH4Cl
Traces
2.3:1
9a 50%, 9b 22%
a
Diastereomeric ratios were determined on crude products by 1H NMR.

A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
37
Fig. 2. Structure determination of diastereomeric cyanohydrins: A) hypothetical intramolecular hydrogen bonding leads to a six-membered ring. Analysis of 1H,1H-homonuclear
and 1H,13C-heteronuclear 3J NMR coupling constants of protons H-4, -5 and -6 and the nitrile carbon atom could explain the stereochemistry at C6 due to trans- and gauche
orientations in a potential 6-membered ring in CDCl3. B) The crystal structure of 9a shows the (6S)-configuration of the cyanohydrin. C) In the packing of the crystal structure, the 6-
hydroxy group forms an intermolecular hydrogen bond and does not form a possible 6-membered ring structure as hypothesized (carbon atoms are shown black, oxygen red,
nitrogen blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3
3JH5,C7¼9.9 Hz for 9a, JH5,C7¼8.8 Hz for 9b (Table S1). Due to the
cyanohydrins to the corresponding amino alcohol was unsuccessful
for the diastereomeric mixture 9ab (Table 3, entry 1e3) and
product could not be detected. Attempts to reduce 9ab using so-
dium borohydride with or without aluminum chloride only gave
the primary alcohol 6, presumably by rapid elimination of the
nitrile and subsequent reduction of the aldehyde 7 (entry 4, 5). The
use of lithium aluminum hydride as reported by Dziewiszek and
Zamojski33 for related mannoheptoses was finally successful to
transform 9a into 10a in 83% yield and 9b into 10b in 60% yield,
respectively (entry 6, 7). Palladium-catalyzed hydrogenolytic
cleavage of the benzyl groups required the presence of 0.35% tri-
fluoroacetic acid in the solvent system tetrahydrofuranewater34 for
successful transformation of benzylated 10a into amino heptose
11a and diastereomer 10b into 11b. Hydrogenation attempts using
methanol or dioxane as solvents and acetic acid as activator were
unsuccessful. Furthermore, direct hydrogenation of benzylated
cyanohydrin 9a to the fully deprotected amine 11a was successful
on analytical scale using the same solvent system TFA/THF/H2O.
However, it was accompanied by a significant fraction of side
product with m/z 430 found in LC-MS, which could be a product of
the reaction of 11a with the intermediate imine, subsequent elim-
ination of ammonia and hydrogenolysis of the N-alkylated imine
absence of signal overlap in the 2D spectrum, temperature varia-
tion and determination of JH5,C7 coupling constant values over a
3
temperature range from 215e292 K could be performed for 9a and
9b. However, no clear trend towards larger coupling constants in
one of the two diastereomers could be detected to unambiguously
assign its stereochemistry. Finally, 9a could be crystallized by slow
evaporation in a methylene chlorideepentane mixture. The crystal
structure determines the (6S)-configuration in 9a (Fig. 2B, Table S2)
and consequently, 9b is the (6R)-diastereomer. Interestingly, in the
crystalline state 9a does not form the anticipated intramolecular
hydrogen bond for the solution experiments, but its 6-OH forms an
intermolecular hydrogen bond with O1 of the neighboring mole-
cule in the crystal lattice (Fig. 2C). The dihedral angle
O5eC5eC6eO6 of cyanohydrin 9a has a value of ꢁ63.18ꢀ in the
crystal structure (Fig. 2B), whereas in case of the anticipated
intramolecular hydrogen bond this dihedral angle corresponds to
180ꢀ in both diastereomers (Fig. 2A).
After establishing the stereochemical configuration of the cya-
nohydrins, 9a and 9b were reduced to the primary amines 10a and
10b, respectively (Scheme 1). Nickel-catalyzed borane reduction in
iso-propanol as reported by Lu et al.32 for structurally more simple
Table 3
Reaction conditions tested to reduce cyanohydrins 9a and 9b to amines 10a and 10b
Entry
Starting material
Reaction conditions
Product
1
2
3
4
5
6
7
9ab
9ab
9ab
9ab
9ab
9a
BH3*THF (12 equiv), iso-PrOH, NiCl2 (satd), 18 h, 0 ꢀCert.
BH3*THF (12 equiv), iso-PrOH, NiCl2 (3 equiv), 6 h, 0 ꢀCert.
BH3*THF (16 equiv), iso-PrOH, NiCl2 (3 equiv), 6 h, 0 ꢀCert.
NaBH4 (4 equiv), THF, 6 h, rt.
d
d
d
6a
NaBH4 (4 equiv), THF, AlCl3 (0.5 equiv), 6 h, rt.
6a
LiAlH4 (4.5 equiv), THF, 15 min, 0 ꢀCert.
10a (83%)b
10b (60%)b
9b
LiAlH4 (4.5 equiv), THF, 15 min, 0 ꢀCert.
Identity was determined by 1H NMR analysis of the crude product.
b Isolated yields.
a

38
A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
Table 4
leading to a secondary amine (MW¼429 g/mol) as described by
Rylander35 for hydrogenolysis of nitriles and their common side
products. Therefore, the two-step procedure using first a lithium
aluminum hydride reduction of the nitriles and subsequent
hydrogenolytic cleavage of the benzyl groups was performed.
Subsequently, crude amines 11a and 11b were transformed into
amides 4aec and 5aeb respectively (Scheme 2). The resulting
amides of the (6S)-4- and the (6R)-5-series were then tested in the
competitive binding assay for LecB inhibition (Table 4). We recently
developed this binding assay, which relies on the competitive
displacement of a fluorescein-fucose conjugate from the carbohy-
drate binding site of LecB and detection by fluorescence polariza-
tion.21 Interestingly, all tested compounds bound to LecB, although
in the high micromolar range. Furthermore, the influence of the
stereocenter at C6 on binding affinity towards LecB favors (6S)-
mannoheptoses, with a selectivity of a factor of three. The data
suggest either a reduced importance of the hydrogen bond estab-
lished between 6-OH and Ser23 on binding affinity or a sterically
unfavored interaction of the amide substituents introduced at C7. A
combination of both effects could also lead to the observed nega-
tive influence on binding affinity, although large amide sub-
stituents introduced at the shorter C6 as in 2 generally led to
increased binding affinities compared to 1.21 Due to the better af-
finities in the (6S)-4 series of amides, sulfonamides 4d and 4e were
synthesized and evaluated in the binding assay for LecB. The
introduction of sulfonamides at C7 of (6S)-mannoheptoses (4d, 4e)
lead to an additional two- to threefold improvement as compared
to the (6S)-amides 4aec.
Biochemical evaluation of heptosides 4aee and 5aeb for LecB binding. IC50 values
are averages of at least three independent experiments, standard deviations are
given. Data for 1, 2 and 3 have been previously reported21
Entry
Compound
Configuration
IC50 [mM]
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
5a
5b
1
6S
6S
6S
6S
6S
6R
6R
d
d
d
180 25
217 57
198 111
101 29
82 23
553 29
546 60
158 53
37 5.0
3.4 0.4
2
3
10
bond with Ser23 and increase binding affinity due to additional
pharmacophores attached at C7. The synthesis followed the route
Swern oxidation at C6, cyanohydrin formation and subsequent
reduction of the nitrile to the amine. All three reactions were
optimized and the stereochemistry of one diastereomer (9a) was
determined by X-ray crystallography. All final compounds were
inhibitors of LecB in the micromolar range. Sulfonamides 4d and 4e
displayed increased affinities compared to the lead structure 1. The
effect of the orientation of the hydroxy group at C6 on binding af-
finity was established with
a selectivity of LecB for 6S-di-
astereomers, the magnitude of the effect indicated a reduced
importance to the overall binding affinity in solution. The results
obtained in this study will help to design more potent inhibitors of
LecB as future therapeutics against chronic P. aeruginosa infections.
In summary, we have designed and synthesized a series of
diastereomeric mannoheptoses with amide and sulfonamide sub-
stituents at C7 for the inhibition of the bacterial lectin LecB. Pre-
viously,
substituents at C6 (e.g., 2 and 3) were reported as potent inhibitors
of LecB. Because -mannose establishes a hydrogen bond via its 6-
OH group with Ser23 of LecB in the crystal structure,12 we extended
-mannose and synthesized mannoheptoses bearing the free 6-OH
D-mannose derivatives with amide and sulfonamide
3. Experimental section
D
3.1. General experimental details
D
Commercially available chemicals were used without further
purification. Dry solvents were acquired from VWR and Acros Or-
ganics. Thin layer chromatography (TLC) was performed using silica
group as well as amido and sulfonamido-substituents at C7 (4aee,
5aeb). Such compounds could allow the formation of a hydrogen
Scheme 2. Synthesis of amide and sulfonamide derivatives 4 and 5: (i) for 4a and 5a: benzoyl chloride, NEt3, DMF; for 4b and 5b: cinnamic acid, EDC*HCl, NEt3, DMF, 0 ꢀCert.; for
4c: acetic anhydride, NEt3, DMF, 0 ꢀCert; for 4d and 4e: sulfonyl chloride, NEt3, DMF, 0 ꢀCert; Isolated yields over two steps from 10a and 10b, respectively.

A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
39
gel 60 aluminum plates containing fluorescent indicator from
Merck and detected either with UV light at 254 nm or by charring in
molybdate solution (0.02 M solution of (NH4)4Ce(SO4)4$2H2O and
(NH4)6Mo7O24$4H2O in aqueous 10% H2SO4), in Ekkert's reagent
(1.5% p-anisaldehyde, 1.5% concentrated H2SO4 and 15% glacial
acetic acid in EtOH), or in ninhydrin (0.05 M in EtOH) with heating.
Solvents for flash chromatography were distilled before use. Flash
chromatography was carried out at Combiflash Rf200 from Tele-
Structure Visualisation 3.1.0.0 or ORTEP-3 V2.02 for Windows
XP.38,39 The drawn ellipsoids represent 50% probability.
3.3. Synthesis
3.3.1. Methyl 2,3,4-tri-O-benzyl-
a-D-mannopyranoside (6)
Methyl -manno-pyranoside (30.0 g, 155 mmol) and trityl
a-D
chloride (90.6 g, 323 mmol) were stirred in pyridine (180 mL) at rt
for 41 h under nitrogen. The brown suspension was diluted with
CH2Cl2 (500 mL) and washed with 150 mL brine, the organic layer
was separated and the aqueous layer was extracted with CH2Cl2
(300 mL). The organic layers were combined, dried over Na2SO4,
filtered, concentrated in vacuo and coevaporated with toluene to
yield a brown solid residue. The crude product was diluted in dry
DMF (100 mL) and this solution was slowly added to a suspension
of NaH (60% in oil, 37.9 g, 926 mmol) in dry DMF (250 mL) at 0 ꢀC.
After stirring for 10 min at 0 ꢀC benzyl bromide (100 mL, 924 mmol)
was added slowly and the suspension was stirred 5 min at 0 ꢀC and
21 h at rt. The suspension was quenched with water (5 L), split in
two fractions and each extracted with ethyl acetate (3ꢂ800 mL),
large solvent volumes were required to separate the phases con-
taining DMF. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo to obtain a brown oil. The residue was dis-
solved in CH2Cl2 (500 mL) and FeCl3$6H2O (194 g, 719 mmol) was
added and the mixture was stirred at rt for 20 h. The reaction was
quenched with saturated NaHCO3 solution (2 L) and extracted with
CH2Cl2 (3ꢂ3 L), large solvent volumes were required to separate the
phases due to floating iron salts. The combined organic layers were
dried over Na2SO4, filtered and concentrated in vacuo to a brown
oil, which was purified by silica gel chromatography (PE to PE/
EtOAc 1/1). The product was obtained as orange oil (34.7 g, 48%
over three steps): Rf¼0.3 (PE/EtOAc 2/1); 1H NMR (CDCl3,
dyne Isco using silica gel 60 M (particle size 40e63 mm). High res-
olution mass spectra were obtained on ESI Bruker MicrOTOF II. NMR
spectra were recorded on Bruker Avance III 400, 500 or 600 at 400/
500/600 MHz (1H) and 101/125/151 MHz (13C). Chemical shifts are
given in parts per million and were calibrated on internal standards
of deuterium labeled solvents. Multiplicities were specified as s
(singlet), d (doublet), dd (doublet of a doublet), ddd (doublet of a
doublet of a doublet), t (triplet), m (multiplet). NMR assignments of
new compounds were confirmed by 1H,1H COSY, 1H,13C HSQC and
1H,13C HMBC experiments. IR spectra of solid compounds were
recorded on a Spectrum 100 FT-IR spectrometer from Perkin Elmer
with ATR sampling accessory. Optical rotation was measured on
JASCO P-2000 polarimeter. Purification of compounds 4be4e and
5a/5b by reversed phase HPLC was performed on a Shimadzu HPLC
with an Agilent C18 column (30* 100 mm, 10 mm) using H2O/MeCN
as mobile phase (0e5 min 5% MeCN; 5e25 min 5e60% MeCN;
25e35 min 60% MeCN; 35e40 min 60e95% MeCN; 40e45 min 95%
MeCN; 45e50 min 95e5% MeCN; flow: 4 mL/min).
3.2. Crystal structure analysis of 9a
Complete crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic Data Centre,
CCDC no.1026815. Copies of thisinformation may be obtainedfree of
charge from the Director, Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge, CB2 1EZ, UK. (fax: þ44 1223 336033, e-
mail: deposit@ccdc.cam.ac.uk or via: www.ccdc.cam.ac.uk).
400 MHz):
d
7.39e7.28 (m, 15H, Ph), 4.96 (d, J¼10.9 Hz, 1H,
OCH2Ph), 4.80 (d, J¼12.3 Hz, 1H, OCH2Ph), 4.72e4.65 (m, 5H, H1,
OCH2Ph), 4.71 (m, 1H, OCH2Ph), 4.67 (m, 1H, OCH2Ph), 4.65 (s, 2H,
OCH2Ph), 3.99 (t, J¼9.4 Hz, 1H, H4), 3.92 (dd, J¼2.9, 9.4 Hz, 1H, H3),
3.86 (dd, J¼2.9, 11.6 Hz, 1H, H6), 3.82e3.77 (m, 2H, H2, H6), 3.64
(ddd, J¼3.0, 4.5, 9.2 Hz, 1H, H5), 3.32 (s, 3H, OCH3); 13C NMR (CDCl3,
X-ray diffraction analysis was performed at 100 K on a STOE
IPDS II diffractometer equipped with a graphite-monochromated
radiation source (
l¼0.71073 Å) and an image plate detection sys-
tem. Crystals of 9a suitable for X-ray diffraction analysis were
grown from methylene chloride/pentane (1/7) solutions by slow
evaporation of the solvent. A 0.45x0.25x0.15 mm crystal fragment
of a 1.2x0.25x0.15 mm needle was mounted on the top of a glass-
fiber by means of silicon-grease and placed on the goniometer
head. A total of 240 frames with a width of 1ꢀ each (180 frames at
phi¼0ꢀ, and 60 frames at phi¼90ꢀ) with an exposure time of
2.5 min was then collected. The selection, integration and averaging
procedure of the measured reflex intensities, the determination of
101 MHz): d 138.6, 138.5, 138.4, 128.5, 128.5 (3C), 128.1, 127.9, 127.8,
127.7 (2C), 99.4 (C1), 80.3 (C3), 75.3 (OCH2Ph), 75.0 (C4), 74.8 (C2),
73.0 (OCH2Ph), 72.3 (OCH2Ph), 72.1 (C5), 62.5 (C6), 54.9 (OCH3);
HRMS [MþNa]þ: calcd 487.2091, found 487.2072.
3.3.2. Methyl 2,3,4-tri-O-benzyl-a-D-manno-hexodialdo-
pyranoside (7)
DMSO (5.2 mL, 73.9 mmol) was added to a solution of oxalyl
chloride (3.0 mL, 35.2 mmol) in dry CH2Cl2 (60 mL) at ꢁ75 ꢀC and
stirred for 15 min. A solution of 6 (13.5 g, 29.1 mmol) in 75 mL
CH2Cl2 was added dropwise and stirring was continued for 1 h
at ꢁ75 ꢀC. Afterwards NEt3 (10 mL, 73.9 mmol) was added and
stirred for 40 min. After warming to rt the reaction mixture was
quenched with saturated NH4Cl solution (50 mL). The separated
organic layer was then washed with H2O (150 mL), the aqueous
phases were extracted with CH2Cl2 (200 mL) and the combined
organic layers were dried over Na2SO4, filtered and concentrated in
vacuo at 30 ꢀC. The light brown oil was purified by silica gel chro-
matography (PE/EtOAc 9/1 to PE/EtOAc 3/2) to give a yellow oil
(10.4 g, 77%): Rf¼0.2 (PE/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz):
the unit cell dimensions and a least-square fit of the 2q values as
well as data reduction, LP-correction and space group determina-
tion were performed using the X-Area software package delivered
with the diffractometer.36 A semiempirical absorption correction
was performed. The structure was solved by direct methods
(SHELXS-9737), completed with difference fourier syntheses, and
refined with full-matrix least-square cycles using SHELXL-97 by
minimizing u
(F20-F2c)2 including an extinction correction. Weighted
R factor (wR2) and the goodness of fit GooF are based on F2. The
hydrogen atom H30 attached to O6 was located in the electron
density map and refined isotropically without constraints. All other
hydrogen atoms were refined in a riding model. The absolute ste-
reochemistry of the model was not reliably determined by the X-
ray diffraction experiment due to the absence of heavy atoms (>Si).
However, the synthetic procedure for compound 9a starts from
d
9.74 (d, J¼0.7 Hz, 1H, CHO), 7.35e7.30 (m, 15H, Ph), 4.86e4.83 (m,
2H, H1, OCH2Ph), 4.71e4.61 (m, 5H, OCH2Ph), 4.10e4.03 (m, 2H, H4,
H5), 3.95 (dd, J¼2.9, 7.9 Hz, 1H, H3), 3.77 (t, J¼2.8 Hz, 1H, H2), 3.38
methyl
a
-D-mannoside (1) and therefore the absolute stereo-
(s, 3H, OCH3); 13C NMR (CDCl3, 101 MHz):
d 198.0 (CHO), 138.2 (2C),
chemistry is assumed as given in the model. Graphical output
(Ortep plots) were created using Diamond Crystal or Molecular
137.9, 128.5, 128.5, 128.5, 128.4, 128.0, 128.0, 127.9, 127.8, 127.8, 99.6
(C1), 79.3 (C3), 76.2 (C4), 74.8 (OCH2Ph), 74.5 (C5), 74.3 (C2), 73.0

40
A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
(OCH2Ph), 72.4 (OCH2Ph), 55.6 (OCH3): HRMS [MþNa]þ: calcd
in dry THF (2 mL/mmol nitrile) was added dropwise and stirred for
5 min at 0 ꢀC and further 10 min at rt. The reaction was first
quenched with 0.1 mL of 0.1 M NaOH solution per 1 mmol LiAlH4
and extracted with EtOAc. The separated organic phase was dried
over Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel chromatography (CH2Cl2/EtOAc/MeOH 1/
1/0 to CH2Cl2/EtOAc/MeOH 1/1/1).
485.1935, found 485.1916.
3.3.3. Methyl 2,3-di-O-benzyl-4-deoxy-6-aldehydo-b-L-erythro-
hex-4-enodialdo-1,5-pyranoside (8)
The reaction was performed as described for 7 with the
following amounts: oxalyl chloride (0.7 mL, 8.2 mmol), DMSO
(1.1 mL, 16 mmol), NEt3 (4.4 mL, 32 mmol) and 6 (1.84 mg,
4.0 mmol) in CH2Cl2 (20 mL). The workup was performed as fol-
lows: The reaction mixture was washed with H2O (40 mL) and the
aqueous layer was extracted with CH2Cl2 (40 mL). The combined
organic phases were dried over Na2SO4, filtered and concentrated
in vacuo at 40 ꢀC. After the purification by silica gel chromatog-
raphy (PE/EtOAc 9/1 to 1/1), 7 (0.63 g, 34%) and a yellowish oil of 8
was obtained (0.47 g, 33%): Rf¼0.3 (PE/EtOAc 3/1); IR (ATR): 2933
(w), 2863 (w),1698 (s), 1640 (m),1496 (m),1454 (m),1347 (m),1313
(m), 1200 (m), 1180 (m), 1143 (s), 1121 (s), 1074 (s), 1026 (s), 959 (s),
3.3.5.1. Methyl 2,3,4-tri-O-benzyl-(6S)-a-D-manno-7-amino-7-
deoxy-heptapyranoside (10a). LiAlH4 (2.00 g, 52.7 mmol) in
50 mL THF and 9a (5.80 g, 11.8 mmol) in 100 mL THF yielded 4.87 g
(84%) of a yellowish oil: [
a]
24 þ21.2 (c 0.87, CHCl3); Rf¼0.4 (CH2Cl2/
D
MeOH/EtOAc 2/1/1); 1H NMR (CDCl3, 400 MHz):
d 7.38e7.24 (m,
15H, Ph), 4.97 (d, J¼10.8 Hz, 1H, OCH2Ph), 4.80e4.68 (m, 4H, H1,
OCH2Ph), 4.64 (s, 2H, OCH2Ph), 4.15 (t, J¼9.5 Hz, 1H, H4), 3.88 (dd,
J¼3.0, 9.3 Hz, 1H, H3), 3.84 (ddd, J¼1.2, 4.1, 7.8 Hz, 1H, H6), 3.79 (dd,
J¼1.9, 2.9 Hz, 1H, H2), 3.50 (d, J¼9.6 Hz, 1H, H5), 3.28 (s, 3H, OCH3),
2.93 (dd, J¼8.0, 12.9 Hz, 1H, H7), 2.76 (dd, J¼4.2, 12.9 Hz, 1H, H7);
894 (s), 817 (m), 736 (s); 1H NMR (CDCl3, 400 MHz):
d 9.18 (s, 1H,
CHO), 7.38e7.30 (m, 10H, Ph), 5.93 (dd, J¼1.6, 2.5 Hz, 1H, H4), 5.10
(d, J¼2.9 Hz, 1H, H1), 4.74 (s, 2H, OCH2Ph), 4.63 (d, J¼1.9 Hz, 2H,
OCH2Ph), 4.42 (dd, J¼2.5, 4.1 Hz, 1H, H3), 3.83 (ddd, J¼1.6, 3.0,
13C NMR (CDCl3, 101 MHz):
d 138.7, 138.6, 138.4, 128.5 (3C), 128.0,
127.9, 127.8, 127.7, 127.7, 127.6, 99.6 (C1), 80.3 (C3), 75.3 (OCH2Ph),
74.7 (2C, C2, C4), 73.0 (OCH2Ph), 72.7 (C5), 72.3 (OCH2Ph), 70.3
(C6), 54.8 (OCH3), 45.3 (C7); HRMS [MþH]þ: calcd 494.2537, found
494.2516.
4.4 Hz,1H, H2), 3.46 (s, 3H, CH3); 13C NMR (CDCl3,101 MHz):
d 186.3
(CHO), 148.8 (C5), 137.8, 128.6, 128.6, 128.5, 128.1, 128.1, 128.0, 127.7,
121.1 (C4), 99.9 (C1), 73.0 (OCH2Ph), 71.6 (OCH2Ph), 70.4 (C2), 70.2
(C3), 56.7 (CH3); HRMS [MþNa]þ: calcd 377.1359, found 377.1337.
3.3.5.2. Methyl 2,3,4-tri-O-benzyl-(6R)-a-D-manno-7-amino-7-
deoxy-heptapyranoside (10b). LiAlH4 (432 mg, 11.4 mmol) in
10 mL THF and 9b (1.30 g, 2.65 mmol) in 20 mL THF yielded 777 mg
3.3.4. Methyl 2,3,4-tri-O-benzyl-(6S)-cyano-
a-D
-mannopyranoside
(9a), methyl 2,3,4-tri-O-benzyl-(6R)-cyano-
a-
D-mannopyranoside
(60%) of a yellowish oil: [
a
]
24 þ41.7 (c 0.38, CHCl3); Rf¼0.4 (CH2Cl2/
D
(9b)
MeOH/EtOAc 2/1/1); 1H NMR (CDCl3, 400 MHz):
d 7.36e7.27 (15H,
KCN (113 mg, 1.74 mmol) was dissolved in 0.15 mL saturated
aqueous NH4Cl solution and cooled to 0 ꢀC. 7 (96.0 mg, 0.20 mmol)
in MeOH (0.4 mL) was added dropwise and stirred for 5 min at 0 ꢀC
and 1 h at rt. The reaction mixture was adjusted to pH¼8 and
extracted with 30 mL EtOAc. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo at 30 ꢀC. The crude
product war purified by silica gel chromatography (PE/EtOAc 4/1) to
give a colorless crystalline solid 9a (49.0 mg, 50%) and a colorless oil
Ph), 4.99 (d, J¼11.0 Hz, 1H, OCH2Ph), 4.77e4.56 (m, 5H, H1,
OCH2Ph), 3.98e3.90 (m, 2H, H3, H4), 3.82 (dd, J¼5.2, 10.2 Hz, 1H,
H6), 3.78 (m, 1H, H2), 3.66 (dd, J¼4.7, 8.7 Hz, 1H, H5), 3.32 (s, 3H,
OCH3), 2.83 (d, J¼5.3 Hz, 2H, H7); 13C NMR (CDCl3, 101 MHz):
d
138.3, 138.3, 138.1, 128.6, 128.5, 128.5, 128.3, 128.2, 128.0, 127.8,
127.8 (2C), 99.1 (C1), 80.6 (C3), 76.5 (C4), 74.9 (OCH2Ph), 74.7 (C2),
72.9 (OCH2Ph), 72.8 (C6), 72.3 (C5), 72.1 (OCH2Ph), 55.0 (OCH3),
43.2 (C7); HRMS [MþH]þ: calcd 494.2537, found 494.2518.
9b (22.0 mg, 22%). 9a: [
a
]
D
24 þ23.4 (c 0.61, CHCl3); Rf¼0.5 (PE/EtOAc
3/1); 1H NMR (CDCl3, 400 MHz):
d
7.42e7.26 (m, 15H), 5.00 (d,
3.3.6. General procedure of debenzylation
J¼11.0 Hz, 1H, OCH2Ph), 4.80 (d, J¼1.7 Hz, 1H, H-1), 4.78 (d,
J¼12.4 Hz, 1H, OCH2Ph), 4.68 (d, J¼12.5 Hz, 1H, OCH2Ph), 4.68 (d,
J¼1.1 Hz, 1H, H-6), 4.65 (d, J¼11.9 Hz, 1H, OCH2Ph), 4.63 (s, 2H,
OCH2Ph), 4.11 (t, J¼9.6 Hz,1H, H-4), 3.93 (dd, J¼9.5, 3.0 Hz,1H, H-3),
To a mixture of methyl 2,3,4-tri-O-benzyl-a-D-manno-7-amino-
7-deoxy-heptapyranoside in H2O and THF, TFA and Pd/C 10% were
added. The reaction mixture was stirred under H2 atmosphere at rt
and atmospheric pressure. After TLC indicated completion of the
reaction, it was filtered through a Celite pad and concentrated in
vacuo to yield a colorless solid, which was used as starting material
for the next steps without further purification.
3.83 (dd, J¼2.9, 1.8 Hz, 1H, H-2), 3.74 (dd, J¼9.7, 1.1 Hz, 1H, H-5); 13
C
NMR (CDCl3, 101 MHz):
d 138.1, 138.0, 137.9, 128.6, 128.6, 128.5,
128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 118.5 (CN), 99.8 (C1), 80.0 (C3),
75.5 (OCH2Ph), 74.6 (C2), 73.4 (C4), 73.2 (OCH2Ph), 72.3 (C5), 72.3
(OCH2Ph), 61.1 (C6), 55.3 (OCH3); HRMS [MþNa]þ: calcd 512.2044,
3.3.6.1. Methyl (6S)-a-D-manno-7-amino-7-deoxy-heptapyranoside
found 512.2047. 9b: [
a
]
24 þ42.2 (c 0.54, CHCl3); Rf¼0.3 (PE/EtOAc 3/
(11a). 10a (4.49 g, 9.09 mmol) in a mixture of H2O (200 mL) and
THF (800 mL), TFA (3.5 mL) and 10% (w/w) Pd/C (451 mg) yielded
after 3 d stirring under H2 atmosphere a colorless solid: Rf¼0.05
(CH2Cl2/MeOH/NEt3/H2O 8/3/0.5/0.5); 1H NMR (MeOH-d4,
D
1); 1H NMR (CDCl3, 400 MHz):
d 7.39e7.27 (m, 15H, Ph), 5.01 (d,
J¼11.0 Hz, 1H, OCH2Ph), 4.80e4.75 (m, 2H, H-1, OCH2Ph), 4.72 (d,
J¼4.0 Hz, 1H, H-6), 4.68 (d, J¼12.4 Hz, 1H, OCH2Ph), 4.67 (d,
J¼11.0 Hz, 1H, OCH2Ph), 4.63 (d, J¼11.7 Hz, 1H, OCH2Ph), 4.60 (d,
J¼11.7 Hz, 1H, OCH2Ph), 4.09 (t, J¼9.5 Hz, 1H, H-4), 3.93 (dd, J¼9.2,
3.0 Hz, 1H, H-3), 3.82e3.81 (m, 1H, H-2), 3.80 (dd, J¼10.0, 4.0 Hz,
400 MHz):
d
4.69 (d, J¼1.7 Hz, 1H, H1), 4.14 (ddd, J¼2.1, 3.4, 9.4 Hz,
1H, H6), 3.83 (t, J¼9.6 Hz, 1H, H4), 3.80 (dd, J¼1.9, 3.5 Hz, 1H, H2),
3.67 (dd, J¼3.3, 9.5 Hz, 1H, H3), 3.43 (dd, J¼2.2, 9.7 Hz, 1H, H5), 3.36
(s, 3H, OCH3), 3.16 (dd, J¼9.4, 12.7 Hz, 1H, H7), 3.09 (dd, J¼3.5,
1H, H-5), 3.33 (s, 3H, OCH3); 13C NMR (CDCl3, 101 MHz):
d 138.1,
138.1, 137.7, 128.7, 128.6, 128.5, 128.2, 128.2, 127.9, 127.8, 127.8 (2C),
117.3 (CN), 99.5 (C1), 80.0 (C3), 75.5 (OCH2Ph), 75.5 (C4), 74.4 (C2),
72.9 (OCH2Ph), 72.1 (OCH2Ph), 71.8 (C5), 63.0 (C6), 55.4 (OCH3);
HRMS [MþNa]þ: calcd 512.2044, found 512.2011.
12.8 Hz, 1H, H7); 13C NMR (MeOH-d4, 101 MHz):
d 103.1 (C1), 74.2
(C5), 72.4 (C3), 71.8 (C2), 67.7 (C4), 67.2 (C6), 55.5 (OCH3), 44.1 (C7),
HRMS [MþH]þ: calcd 224.1129, found 224.1125.
3.3.6.2. Methyl (6R)-
(11b). 10b (777 mg, 1.57 mmol) in a mixture of H2O (45 mL) and
THF (120 mL), TFA (500 L) and 10% (w/w) Pd/C (80.0 mg)
a-D-manno-7-amino-7-deoxy-heptapyranoside
3.3.5. General procedure for synthesis of methyl 2,3,4-tri-O-benzyl-
a-D
-manno-7-amino-7-deoxy-heptapyranoside (10a, 10b)
m
LiAlH4 (4.5 equiv) was suspended in dry THF (1 mL/mmol hy-
yielded after 2 d stirring under H2 atmosphere a colorless solid:
dride LiAlH4) and cooled to 0 ꢀC. A solution of cyanohydrin (1 equiv)
Rf¼0.1 (CH2Cl2/MeOH/NEt3/H2O 8/3/0.5/0.5); 1H NMR (MeOH-d4,

A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
41
400 MHz):
d
4.66 (d, J¼1.6 Hz, 1H, H1), 4.17 (ddd, J¼1.7, 4.0,
1H, H-7), 1.97 (s, 3H, Ac). 13C NMR (MeOH-d4, 101 MHz):
103.0 (C1), 73.7 (C5), 72.6 (C3), 72.0 (C2), 68.7 (C6), 67.8 (C4), 55.5
(OCH3), 44.3 (C7), 22.6 (CH3); HRMS [MþNa]þ: calcd 288.1054,
found 288.1048.
d
173.6,
6.2 Hz, 1H, H6), 3.80 (dd, J¼1.7, 2.9 Hz, 1H, H2), 3.67e3.61 (m, 3H,
H3, H4, H5), 3.39 (s, 3H, OCH3), 3.20 (dd, J¼6.7, 13.1 Hz, 1H, H7),
3.14 (dd, J¼4.1, 13.1 Hz, 1H, H7); 13C NMR (MeOH-d4, 101 MHz):
d
102.8 (C1), 75.7 (C5), 72.5 (C3), 71.8 (C2), 69.0 (C4), 68.1 (C6),
55.4 (OCH3), 41.9 (C7); HRMS [MþH]þ: calcd 224.1129, found
3.3.10. Methyl (6S)-a-D-manno-7-(2-mesitylenesulfonamido)-7-
deoxy-heptapyranoside (4d)
224.1126.
To crude 11a (70.0 mg) in DMF (1.5 mL), NEt3 (44
mL,
3.3.7. Methyl (6S)-
heptapyranoside (4a)
Crude 11a (50 mg) was dissolved in 2 mL DMF, NEt3 (78
0.56 mmol) was added and the mixture was cooled to 0 ꢀC. After
dropwise addition of benzoyl chloride (31 L, 0.27 mmol) the re-
a-D-manno-7-(benzamido)-7-deoxy-
0.32 mmol) was added and the mixture was cooled to 0 ꢀC. Af-
terwards the 2-mesitylenesulfonyl chloride (45.3 mg,
0.21 mmol) in DMF (0.5 mL), was added dropwise and the re-
action mixture stirred 30 min under cooling and 26 h at rt. After
removal of the solvent the crude product was directly purified by
HPLC to obtain 20.1 mg (0.05 mmol, 32% over two steps): Rf¼0.6
(EtOAc/EtOH/NH4OH(25%) 2/2/1); 1H NMR (MeOH-d4, 500 MHz):
mL,
m
action mixture was stirred for 1 h at 0 ꢀC and was then allowed to
warm to rt. The mixture was diluted with EtOAc (10 mL) and
washed with saturated NH4Cl solution (30 mL). The organic phase
was dried over Na2SO4, filtered and concentrated in vacuo. Incu-
bation of the crude product with acidic ion exchange resin
Amberlite IR-120 in 2 mL MeOH to remove excess NEt3 and puri-
fication by silica gel chromatography (EtOAc to EtOAc/EtOH/NH4OH
d
7.02 (s, 2H, Ph), 4.61 (d, J¼1.5 Hz, 1H, H1), 3.95 (ddd, J¼1.8, 5.3,
7.4 Hz, 1H, H6), 3.78e3.73 (m, 2H, H2, H4), 3.63 (dd, J¼3.4,
9.5 Hz, 1H, H3), 3.39 (dd, J¼1.7, 9.8 Hz, 1H, H5), 3.30 (s, 3H,
OCH3), 3.07 (dd, J¼5.2, 12.7 Hz, 1H, H7), 2.96 (dd, J¼7.8, 12.7 Hz,
1H, H7), 2.62 (s, 6H, PhCH3), 2.30 (s, 3H, PhCH3); 13C NMR
aq (25%) ratio 5/5/1) yielded 25.6 mg of
a colorless solid
(MeOH-d4, 101 MHz):
d 143.5, 140.3 (2C), 135.1, 132.9 (2C), 102.9
(0.08 mmol, 71% over two steps): Rf¼0.2 (CH2Cl2/EtOH 4/1); 1H
(C1), 73.4 (C5), 72.6 (C3), 72.01 (C2), 68.8 (C6), 67.8 (C4), 55.5
(OCH3), 46.7 (C7), 23.1 (2C, CH3), 20.9 (CH3); LCMS [MþH]þ:
calcd 406.2, found 405.9.
NMR (MeOH-d4, 400 MHz):
d 7.85e7.83 (m, 2H, Ph), 7.55e7.51 (m,
1H, Ph), 7.48e7.44 (m, 2H, Ph), 4.70 (s, 1H, H1), 4.20 (dd, J¼4.9,
7.2 Hz, 1H, H6), 3.87 (t, J¼9.6 Hz, 1H, H4), 3.79 (dd, J¼1.5, 3.0 Hz, 1H,
H2), 3.73e3.67 (m, 2H, H3, H7), 3.56e3.49 (m, 2H, H5, H7), 3.38 (s,
3H, OCH3); 13C NMR (MeOH-d4, 101 MHz):
d 170.5, 135.7, 132.6,
3.3.11. Methyl (6S)-
heptapyranoside (4e)
To crude 11a (70.0 mg) in DMF (1.5 mL), NEt3 (44
a-D-manno-7-(p-toluylsulfonamido)-7-deoxy-
129.5 (2C), 128.2 (2C), 103.1 (C1), 73.9 (C5), 72.6 (C3), 72.1 (C2), 68.8
(C6), 67.9 (C4), 55.5 (OCH3), 44.9 (C7); HRMS [MþNa]þ: calcd
350.1210, found 350.1203.
mL, 0.32 mmol)
was added and the mixture was cooled to 0 ꢀC. Afterwards tosyl
chloride (39.8 mg, 0.21 mmol) in DMF (0.5 mL) was added dropwise
and the reaction mixture stirred 30 min under cooling and 26 h at
rt. After removal of the solvent the crude product was directly
purified by HPLC to obtain 31.6 mg (0.08 mmol, 54% over two
steps): Rf¼0.6 (EtOAc/EtOH/NH4OH(25%) 2/2/1); 1H NMR (MeOH-
3.3.8. Methyl (6S)-
heptapyranoside (4b)
A mixture of 11a (70.0 mg) and NEt3 (44
a-D-manno-7-(cinnamido)-7-deoxy-
m
L, 0.32 mmol) in
DMF (1.5 mL) was cooled to 0 ꢀC. After the slow addition of
cinnamoyl chloride (33.3 mg, 0.20 mmol) in 0.5 mL DMF the
reaction mixture was stirred further 30 min at 0 ꢀC and 24 h at rt.
After removal of the solvent in vacuo the crude product was
directly purified by HPLC to obtain 18.3 mg (0.05 mmol, 34% over
two steps) of a colorless solid: Rf (CH2Cl2/EtOH 8/1) 0.1; 1H NMR
d4, 400 MHz):
d
7.74 (d, J¼8.4 Hz, 2H, Ph), 7.38 (d, J¼8.2 Hz, 2H, Ph),
4.63 (d, J¼1.8 Hz, 1H, H1), 3.98 (ddd, J¼7.5, 6.2, 1.7 Hz, 1H, H6), 3.79
(dd, J¼9.7, 9.7 Hz, 1H, H4), 3.76e3.74 (m, 1H, H2), 3.65 (dd, J¼9.6,
3.2 Hz, 1H, H3), 3.47 (dd, J¼9.8, 1.7 Hz, 1H, H5), 3.33 (s, 3H, OCH3),
3.10 (dd, J¼12.5, 6.2 Hz, 1H, H7), 2.90 (dd, J¼12.5, 7.4 Hz, 1H, H7),
2.42 (s, 3H, PhCH3); 13C NMR (MeOH-d4, 101 MHz):
d 144.6, 138.5,
(MeOH-d4, 600 MHz):
d 7.57e7.53 (m, 3H), 7.41e7.36 (m, 3H),
130.7 (2C), 128.1 (2C), 102.9 (C1), 73.0 (C5), 72.6 (C3), 72.0 (C2), 68.9
(C6), 67.7 (C4), 55.6 (OCH3), 47.1 (C7), 21.4 (PhCH3), LCMS [MþNa]þ:
calcd 400.1, found 399.8.
6.67 (d, J¼15.8 Hz, 1H), 4.69 (s, 1H, H1), 4.12e4.09 (m, 1H, H6).
3.84 (t, J¼3.8 Hz, 1H, H4), 3.78 (dd, J¼1.7, 3.4 Hz, 1H, H2), 3.67
(dd, J¼3.4, 9.6 Hz, 1H, H3), 3.63 (dd, J¼4.8, 13.5 Hz, 1H, H7),
3.48e3.42 (m, 2H, H5, H7), 3.38 (s, 3H, OCH3); 13C NMR (MeOH-
d4, 151 MHz):
d
168.9, 141.7, 136.3, 130.8, 129.9 (2C), 128.8 (2C),
3.3.12. Methyl (6R)-
heptapyranoside (5a)
To a solution of crude 11b (49.0 mg) in 7 mL DMF, NEt3 (89.0
0.64 mmol) was added and the mixture was cooled to 0 ꢀC. Benzoyl
chloride (31.1 L, 0.27 mmol) was added dropwise and stirred for
a-D-manno-7-(benzamido)-7-deoxy-
121.9, 103.0 (C1), 73.8 (C5), 72.7 (C3), 72.1 (C2), 68.9 (C6), 67.9
(C4), 55.5 (CH3), 44.4 (C7); HRMS [MþNa]þ: calcd 376.1367,
found 376.1340.
mL,
m
3.3.9. Methyl (6S)-
heptapyranoside (4c)
To a mixture of crude 11a (70 mg) in DMF (1.5 mL) NEt3 (44
0.32 mmol) was added dropwise and the reaction was cooled to
0 ꢀC. After slow addition of acetic anhydride (20
L, 0.21 mmol) in
a
-
D
-manno-7-(acetamido)-7-deoxy-
30 min at 0 ꢀC and further 30 min at rt. After removing the solvent
in vacuo the product was directly purified by silica gel chroma-
tography (CH2Cl2/EtOH 95/5 to 8/1). Incubation of the product with
acidic ion exchange resin Amberlite IR-120 in 5 mL MeOH to
remove excess NEt3 for 1 h at rt, filtration, concentration in vacuo
and subsequent purification by HPLC gave the title compound as a
colorless solid (16.0 mg, 0.05 mmol, 17% over two steps): Rf¼0.5
mL,
m
DMF (0.5 mL), the reaction mixture continued stirring for further
45 min at 0 ꢀC and 24 h at rt. Afterwards the mixture was quenched
with saturated NaHCO3 solution and was allowed to stir further
30 min. After concentration in vacuo the crude product was directly
purified by preparative HPLC to obtain colorless solid (27.4 mg, 67%
over two steps): Rf¼0.4 (CH2Cl2/EtOH 2/1); 1H NMR (MeOH-d4,
(CH2Cl2/EtOH 4/1); 1H NMR (MeOH-d4, 400 MHz):
d 7.86e7.83 (m,
2H, Ph), 7.53e7.51 (m, 1H, Ph), 7.48e7.44 (m, 2H, Ph), 4.66 (d,
J¼1.5 Hz, 1H, H1), 4.13 (dt, J¼3.8, 7.5 Hz, 1H, H6), 3.82e3.76 (m, 3H,
H2, H4, H7), 3.67 (dd, J¼3.3, 9.3 Hz, 1H, H3), 3.62e3.55 (m, 2H, H5,
500 MHz):
d
4.67 (d, J¼1.7 Hz, 1H, H1), 4.02 (ddd, J¼8.6, 4.7, 1.6 Hz,
H7), 3.38 (s, 3H, OCH3); 13C NMR (MeOH-d4, 101 MHz):
d 170.6,
1H, H-6), 3.82 (t, J¼9.7 Hz, 1H, H-4), 3.77 (dd, J¼3.4, 1.7 Hz, 1H, H-2),
3.66 (dd, J¼9.6, 3.4 Hz, 1H, H-3), 3.47 (dd, J¼13.6, 4.7 Hz, 1H, H-7),
3.41 (dd, J¼9.8, 1.6 Hz, 1H, H-5), 3.36 (s, 3H, OCH3), 3.34e3.25 (m,
135.7, 132.6, 129.5, 128.3, 102.8 (C1), 74.9 (C5), 72.7 (C3), 71.8 (C2),
71.7 (C6), 69.7 (C4), 55.3 (OCH3), 43.3 (C7); HRMS [MþNa]þ: calcd
350.1210, found 350.1196.

42
A. Hofmann et al. / Carbohydrate Research 412 (2015) 34e42
3.3.13. Methyl (6R)-
a-
D-manno-7-(cinnamido)-7-deoxy-
References
heptapyranoside (5b)
To a mixture of 11b (49.0 mg) in DMF (7 mL) NEt3 (87
1. Yahr T, Parsek M. The prokaryotes volume 6: Proteobacteria: gamma subclass. 3
ed., vol. 6. Springer-Verlag; 2006.
mL,
0.63 mmol) and EDC*HCl (66.0 mg, 0.34 mmol) were added. After
cooling to 0 ꢀC, cinnamic acid (44.0 mg, 0.29 mmol) was added
portion wise and the reaction mixture was allowed to warm to rt
and stirring was continued for 20 h. After removal of the volatiles
and purification by silica gel chromatography (CH2Cl2/EtOH 19/1)
the product was dissolved in MeOH (4 mL) and incubated with
acidic ion exchange resin Amberlite IR-120 to remove excess NEt3
for 1 h at rt. Filtration, concentration in vacuo and subsequent
purification by HPLC gave the title compound as a colorless solid
(10.0 mg, 0.03 mmol, 10% over two steps) was isolated: Rf (CH2Cl2/
2. Peleg AY, Hooper DC. New Engl J Med 2010; 362: 1804e13.
3. Tümmler B, Kiewitz C. Mol Med Today 1999; 5: 351e8.
4. Davies D. Nat Rev Drug Discov 2003; 2: 114e22.
5. Poole K. Front Microbiol 2011; 2: 65.
6. Flemming H-C, Wingender J. Nat Rev Microbiol 2010; 8: 623e33.
7. Tielker D, Hacker S, Loris R, Strathmann M, Wingender J, Wilhelm S, et al.
Microbiology 2005; 151: 1313e23.
ꢀ
8. Diggle SP, Stacey RE, Dodd C, Camara M, Williams P, Winzer K. Environ
Microbiol 2006; 8: 1095e104.
9. Gilboa-Garber N, Mizrahi L, Garber N. Can J Biochem 1977; 55: 975e81.
10. Gilboa-Garber N. Methods Enzymol 1982; 83: 378e85.
ꢀ
11. Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Perez S, et al. Nat
Struct Biol 2002; 9: 918e21.
EtOH 8/1) 0.2; 1H NMR (MeOH-d4, 400 MHz):
d 7.58e7.53 (m, 3H),
12. Loris R, Tielker D, Jaeger K-E, Wyns L. J Mol Biol 2003; 331: 861e70.
ꢀ
ꢀ
13. Sabin C, Mitchell EP, Pokorna M, Gautier C, Utille J-P, Wimmerova M, et al. FEBS
Lett 2006; 580: 982e7.
7.41e7.36 (m, 3H), 6.69 (d, J¼15.8 Hz, 1H), 4.65 (d, J¼1.2 Hz, 1H, H1),
4.05 (dt, J¼3.7, 7.6 Hz, 1H, H6), 3.79e3.65 (m, 4H, H2, H3, H4, H7),
3.56 (dd, J¼3.9, 9.7 Hz,1H, H5), 3.50 (dd, J¼7.9,13.9 Hz,1H, H7), 3.38
ꢀ
14. Perret S, Sabin C, Dumon C, Pokorna M, Gautier C, Galanina O, et al. Biochem J
2005; 389: 325e32.
(s, 3H, OCH3); 13C NMR (MeOH-d4, 151 MHz):
d 169.0, 141.7, 136.3,
15. Imberty A, Chabre YM, Roy R. Chem Eur J 2008; 14: 7490e9.
ꢀ
16. Bernardi A, Jimenez-Barbero J, Casnati A, De Castro C, Darbre T, Fieschi F, et al.
130.8, 129.9, 128.8, 121.9, 102.8 (C1), 74.5 (C5), 72.6 (C3), 72.0 (C6),
71.8 (C2), 70.0 (C4), 55.3 (CH3), 42.8 (C7); HRMS [MþNa]þ: calcd
376.1367, found 376.1343.
Chem Soc Rev 2012; 42: 4709e27.
17. Sommer R, Joachim I, Wagner S, Titz A. CHIMIA 2013; 67: 286e90.
18. Titz A. In: Seeberger PH, Rademacher C, editors. Carbohydrates as drugs topics in
medicinal chemistry12. Springer Berlin Heidelberg; 2014. p. 169e86.
19. Johansson EMV, Crusz SA, Kolomiets E, Buts L, Kadam RU, Cacciarini M, et al.
Chem Biol 2008; 15: 1249e57.
3.4. Evaluation of compounds for LecB inhibition
ꢀ
20. Boukerb AM, Rousset A, Galanos N, Mear J-B, Thepaut M, Grandjean T, et al.
J Med Chem 2014; 57: 10275e89.
21. Hauck D, Joachim I, Frommeyer B, Varrot A, Philipp B, Moller HM, et al. ACS
All final compounds described in this study were evaluated in a
competitive binding experiment for inhibition of LecB. The assay is
run in black 384-well plates (Greiner BioOne, Germany) and is
based on the displacement of a LecB-bound FITC-labelled tracer
(fluorescein-fucose conjugate, details see Hauck et al.21) and fluo-
rescence polarization readout in a microplate reader (Pherastar FS,
BMGlabtech, Germany; ex. 485 nm, em. 525 nm) as previously
described.21
€
Chem Biol 2013; 8: 1775e84.
22. Sommer R, Exner TE, Titz A. PLoS One 2014; 9: e112822.
ꢁ
23. Titz A, Marra A, Cutting B, Smiesko M, Papandreou G, Dondoni A, et al. Eur J Org
Chem 2012: 5534e9.
24. Titz A, Patton J, Alker AM, Porro M, Schwardt O, Hennig M, et al. Bioorg Med
Chem 2008; 16: 1046e56.
25. Titz A, Ernst B. CHIMIA 2007; 61: 194e7.
26. Schwizer D, Patton JT, Cutting B, Smiesko M, Wagner B, Kato A, et al. Chem Eur J
ꢁ
2012; 18: 1342e51.
27. Binder FPC, Lemme K, Preston RC, Ernst B. Angew Chem Int Ed Engl 2012; 51:
7327e31.
28. Boren HB, Eklind K, Garegg PJ, Lindberg B, Pilotti A. Acta Chem. Scand 1972; 26:
4143e6.
Acknowledgments
The authors are grateful to Philipp Voss for initial experiments,
Holger Bußkamp for HR-MS measurements and Dr. Josef Zapp and
Anke Friemel for excellent support in NMR analyses. Dr. Anne
Imberty is acknowledged for sharing the expression plasmid of
LecB. We thank the Helmholtz Association [VH-NG-934], the Kon-
stanz Research School Chemical Biology, the Zukunftskolleg, and
the Deutsche Forschungsgemeinschaft [Ti756/2-1] for financial
support.
29. Ding X, Wang W, Kong F. Carbohydr Res 1997; 303: 445e8.
30. Mackie DM, Perlin AS. Carbohydr Res 1972; 24: 67e85.
31. Meissner A, Sørensen OW. Magn Res Chem 2001; 39: 49e52.
32. Lu Y, Miet C, Kunesch N, Poisson JE. Tetrahedron: Asymmetry 1993; 4:
893e902.
33. Dziewiszek K, Zamojski A. Carbohydr Res 1986; 150: 163e71.
ꢀ
34. Deunf E, Zaborova E, Guieu S, Bleriot Y, Verpeaux J-N, Buriez O, et al. Eur J Inorg
Chem 2010: 4720e7.
35. Rylander. Hydrogenation methods. Academic Press; 1985.
36. X-RED version 1.31. Stoe data reduction program. Darmstadt, Germany.
2005.
37. Sheldrick GM. SHELXS-97 program for crystal structure analysis. Germany:
Supplementary data
€
University of Gottingen; 1997.
38. Faruggia LJ. ORTEP-3 V2.02 for windows XP. Scotland: University of Glasgow;
2008.
39. Diamondecrystal and molecular structure visualization, crystal impact. Dr. H.
Putz & Dr. K. Brandenburg. GbR: Bonn, Germany.
Supplementary data (1H NMR and 13C NMR traces of all syn-
thesized compounds, temperature dependent 1H NMR spectra and
data of J-HMBC measurements for 9a and 9b) related to this article
can be found at http://dx.doi.org/10.1016/j.carres.2015.04.010.