Synthesis of the NAG-NAM disaccharide via a versatile intermediate

Article abstract of DOI:10.1016/j.carres.2013.12.007

A simple strategy for the synthesis of a β-GlcNAc-(1→4)-MurNAc (NAG-NAM) moiety, crucial for the preparation of synthetic components of a bacterial peptidoglycan, was achieved. This strategy relies on the use of three O-protecting groups, 4,6-O-benziliden

Full text of DOI:10.1016/j.carres.2013.12.007

Carbohydrate Research 384 (2014) 112–118
Contents lists available at ScienceDirect
Carbohydrate Research
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Note
Synthesis of the NAG–NAM disaccharide via a versatile intermediate
⇑
Ramu Enugala, Marina J. D. Pires, M. Manuel B. Marques
REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple strategy for the synthesis of a b-GlcNAc-(1?4)-MurNAc (NAG–NAM) moiety, crucial for the
preparation of synthetic components of a bacterial peptidoglycan, was achieved. This strategy relies on
the use of three O-protecting groups, 4,6-O-benzilidene acetal, benzyl, and acetyl group, which allows
further regioselective manipulation at O-3, O-4 positions, and on the insertion of the peptide chain at
the lactate moiety in an advanced and versatile intermediate. Overall, a simple route to achieve the bio-
logical relevant NAG–NAM is presented, which may serve as a conceptual framework in the designing of
synthetic strategies of different natural and non-natural polysaccharides.
Received 1 November 2013
Received in revised form 6 December 2013
Accepted 8 December 2013
Available online 12 December 2013
Keywords:
N-Acetyl glucosamine
NAG–NAM
Ó 2013 Elsevier Ltd. All rights reserved.
Protecting group
Glycosylation
Peptidoglycan
The increasing occurrence of drug-resistant infections has stim-
ulated the scientific community to develop new antibiotics against
vancomycin-resistant Enterococci and methicillin-resistant Staphy-
lococcus aureus, as well as to identify novel drug targets, or alterna-
tive strategies, that may help fighting bacterial infections.1,2
Peptidoglycan (PGN), a major constituent of the bacterial cell
wall, has been well-known as a strong immunopotentiator.3 PGN
is recognized by invertebrate and vertebrate innate immune sys-
tem (IIS) and is capable of inducing an innate immune response.
PGN is able to induce many types of mediators, such as prostaglan-
dins, platelet activation factor, NO, and cytokines that stimulate
the immune system. Thus, PGN constitutes an excellent target to
study recognition mechanisms and pathways by the IIS and novel
strategies to modulate an IIS response. PGN is composed of poly-
saccharide chains linked through a peptide network that together
assembles the rigid three-dimensional structure. The polysaccha-
ride chain is constituted of repeating units of N-acetyl glucosamine
(NAG) and N-acetylmuramic acid (NAM) linked by a b(1-4) glyco-
sidic bond, which is crosslinked to a peptide network via the car-
boxy group of the NAM moiety (Fig. 1).4 Unfortunately the study
of PGN recognition has been hampered by the lack of pure and
homogeneous fragments of PGN. Isolation of substantial amounts
of these structures from natural sources is difficult, while the
assembly of this complex structure has been extremely challenging
from the synthetic point of view.
to date involve long multi-step sequences (up to 37 steps) with
many protection–deprotection steps. In fact glycosylation of the
4-hydroxyl group of NAG derivatives is notoriously difficult, due
to the well-known lack of reactivity of this hydroxyl group which
is due to a combination of steric hindrance and to the involvement
of the N-acetyl group in
a
hydrogen-bonded network.6 The
glycosylation of complex aglycones with glycosyl donors bearing
a 2-acetamido-2-deoxy functionality is usually unfeasible, due to
the formation of a rather stable 1,2-O,N-oxazoline intermediate
during glycosylation, which significantly decreases the rate of gly-
cosylation and yields.7
Properly functionalized glucosamine disaccharides constitute
key scaffolds in the synthesis of complex and biologically impor-
tant oligosaccharides, such as the S. aureus bacterial PGN (Fig. 1).
Recently we have explored a one-pot regioselective protection
of glucosamine moieties (both donor and acceptor).8 However,
the glycosylation failed to occur under one-pot conditions. A syn-
thetic strategy was also explored to prepare O-3-hydroxyl free
N-acetyl glucosamine disaccharide.9 Although, the introduction of
the lactate moiety after glycosylation turned to be more difficult
than anticipated and lower yields were obtained when compared
to the introduction on the monomeric residue. The use of a fully
acetylated glucosamine moiety limited a straightforward function-
alization at this unit.
Pursuing our program directed toward the chemical synthesis
of glucosamine building blocks, which is required for the synthesis
of monomeric and dimeric muropeptides8–10 (the smallest compo-
nents of the PGN macromolecule), we report herein an
improved and versatile preparation of b-GlcNAc-(1?4)-MurNAc
(NAG–NAM) building blocks (Scheme 1). A suitable and selective
protection/deprotection strategy involves as few functional group
Excellent synthetic strategies for general PGN fragments have
been reported.5 In order to control the regioselective and enantio-
selective glycosylation, most of the synthetic sequences developed
⇑
Corresponding author. Tel.: +351 21 2948300; fax: +351 21 2948384.
E-mail address: mmbmarques@fct.unl.pt (M. Manuel B. Marques).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.12.007

R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
113
OH
versatile NAG–NAM derivative disaccharide (after glycosylation
with a NAM based acceptor).
OH
OH
OH
O
O
O
O
O
O
O
HO
O
O
O
HO
O
NHAc
NHAc
NHAc
O
NHAc
The synthesis of a NAG–NAM disaccharide started with the
selection of suitable protecting groups for the monomeric building
blocks (donor and acceptor). The strategy for the preparation of
valuable N-acetyl glucosamine disaccharide involved the synthesis
of a glycosyl trichloro-acetamidate donor 4 (Scheme 2) with an
acetate group at the O-3 position and a glucosamine acceptor 10
(Scheme 3). Troc (2,2,2-trichloro-ethoxycarbonyl) group was cho-
sen as N-protecting group for both donor and acceptor due to the
higher reactivity and b-selectivity at glycosylation when compared
to N-acetyl-, N-Phth-glucosamines, and other N-protecting groups
as well can be removed under mild conditions.7 Thus, starting from
O
L-Ala
L-Ala
D-GluNH2
D-GluNH2
L-Lys (Gly)5
D-Ala
L-Lys (Gly)5
D-Ala
D-Ala
D-Ala
OH
OH
O
HO
OH
OH
O
O
O
O
O
O
O
O
O
HO
O
NHAc
NHAc
NHAc
O
NHAc
O
L-Ala
L-Ala
D-GluNH2
D-GluNH2
L-Lys (Gly)5
D-Ala
L-Lys (Gly)5
D-Ala
Figure 1. Structure of the S. aureus peptidoglycan (a Lys-type PGN).
D-glucosamine hydrochloride, protection of amino group was per-
formed using NaHCO3 and TrocCl for the donor moiety 1 and with
(Allyl chloroformate) AllocCl for the acceptor moiety 5.5c
OH
OH
O
O
HO
O
HO
OH
NHAc
O
For the preparation of the donor moiety 4 (Scheme 2), after
N-protection, the benzylidene acetal formation was achieved with
zinc chloride and benzaldehyde followed by a classical acetylation
to give 2 with 78% yield after two steps.11 Selective removal of the
anomeric acetyl group was performed by treatment with morpho-
line,12 and 3 was obtained after column chromatography purifica-
tion in 87% yield. Treatment of 3 with CCl3CN and Cs2CO3 afforded
the desired glycosyl trichloro-acetamidate 4 that was used in the
glycosylation step without further purification.
NHAc
O
OH
OR
O
Ph
O
O
AcO
O
O
OR
NHAc
NHAc
O
OEt
peptide chain insertion
O-3 or O-4 glycosylation
In order to prepare the acceptor moiety, N-alloc glucosamine 5
was treated with acetyl chloride in benzyl alcohol at 0 °C, followed
by benzylidene acetal formation to give 7 (Scheme 3). While allyl
ether group has been often used as anomeric protecting group,
the benzyl group was selected to avoid the use of expensive metal
catalysts frequently employed in deallylation procedures,13 in
addition to a full disaccharide deprotection in just one step
(hydrogenation) in a late stage of the synthesis. Lactate moiety
insertion at O-3 required the preparation of (ꢀ)-ethyl (S)-2-trif-
luromethanesulfonyl propionate. Thus, compound 7 was treated
with NaH and then the freshly prepared OTf-ester was added
drop-wise to afford 8 in 68% yield.5c The next step consisted on
the replacement of N-Alloc group by N-Troc, via addiction of
OR
O
R1O
NH
CCl3
O
Ph
R = Bn
R1 = Ac
O
HO
O
+
OR
O
O
NHPG
NHPG
O
OEt
Scheme 1. Proposed approach toward NAG–NAM.
manipulations as possible, though affording regioselective control
over further functionalization. We envisaged that introduction of
an acetyl group at O-3 of the donor moiety would lead to a
OH
O
O
O
i
ii
iii
O
Ph
O
O
O
O
Ph
Ph
O
HO
HO
TrocHN
O
NH
CCl3
AcO
TrocHN
AcO
TrocHN
AcO
TrocHN
OH
78%
87%
74%
O
OH
OAc
2
3
1
4
Donor
i) a) PhCHO, ZnCl2, 3 A MS, RT, overnight; b) Ac2O, Pyridine, RT, overnight; ii) Morpholine, EtOAc,
RT, overnight; iii) CCl3CN, CS2CO3, DCM, RT, 2h.
Scheme 2. Synthesis of the glucosamine donor (NAG).
O
OH
O
OH
HO
HO
AllocHN
O
Ph
O
O
Ph
O
O
O
HO
i
ii
iii
O
HO
HO
AllocHN
AllocHN
OBn
AllocHN
OH
OBn
OBn
61%
72%
68%
CO2Et
6
7
5
8
82% iv
BnO
O
v
O
O
Ph
HO
O
O
O
TrocHN
TrocHN
OBn
72%
OBn
CO2Et
CO2Et
10
Acceptor
9
i) BnOH, AcCl, 80ºC, 3h; ii) PhCHO, ZnCl2, overnight; iii) Ethyl L-(S)-2- trifluoromethanesulphonyloxy-
propionate, NaH, DCM, RT, 3h; iv) Pd(PPh3)4, AcOH, TrocCl, RT; v) BH3Me2N, BF3OEt2, RT, 3h.
Scheme 3. Synthesis of the glucosamine acceptor (NAM).

114
R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
BnO
HO
AcO
AcHN
Pd(PPh3)4 to remove alloc group followed by in situ N-protection
with the TrocCl to afford 9 in 82% yield. The acceptor 10 was
obtained by regioselective benzylidene acetal ring opening under
BnO
O
O
O
O
AcHN
OBn
CO2Et
O-4 glycosylation
reductive conditions, by treatment of
9 with BH3NMe2 and
16
BF3ꢁOEt2.
regioselective ring opening
Glycosylation of donor 4 and acceptor 10 was achieved by treat-
ment with TMSOTf at ꢀ15 °C giving the desired b(1-4)-linked
disaccharide 11 in 60% yield (Scheme 4).
O
BnO
O
Ph
Exchange of both trichloroethyl carbamate groups by the natu-
ral N-acetyl group was performed after the glycosylation. Thus,
Troc groups were removed by addiction of freshly prepared
Zn–Cu couple14 in AcOH/Ac2O/THF, followed by N-acetylation to
obtain 12 in 63% yield. Hydrolysis of 12 was performed by treat-
ment with LiOH, which simultaneously led to the removal of the
O-3 acetyl group and lactic acid formation, giving disaccharide
13 in 69% yield. The last step of the NAG–NAM synthesis consisted
on the removal of the benzyl groups at O-1, O-6 of the muramyl
residue and the benzylidene ring at O-6 and O-4 of the NAG unit
by hydrogenation using Pd(OH)2 on charcoal. The NAG–NAM disac-
charide (14) was isolated in quantitative yield.15
O
O
O
AcO
AcHN
O
NAG-NAM
key building block
AcHN
OBn
CO2Et
12
transesterification
hydrolysis
O
O
BnO
O
Ph
BnO
O
O
Ph
O
O
O
O
HO
AcHN
O
HO
AcHN
O
O
AcHN
AcHN
OBn
OBn
CO2R
CO2H
13
15
O-3 glycosylation
hydrogenation
peptide chain insertion
Disaccharide 12, a NAG–NAM precursor, constitutes a valuable
intermediate for the assembly of peptidoglycan fragments and its
non-natural derivatives. This is a highly useful strategy to achieve
scaffolds possessing different substitution patterns on behalf of
exploring key interactions with various biological targets and for
molecular recognition studies to understand the innate immune
response to bacterial infections, an emergent area of research.16–18
In this sense, 12 is a key intermediate in the synthesis of
NAG–NAM derivatives since it allows selective removal of O-3 pro-
tecting group (acetyl) by a transesterification reaction to give 15,
opening the possibility of a regioselective glycosylation at this po-
sition to obtain trisaccharides (Scheme 5). Additionally, 12 can be
fully hydrolyzed to 13 to which a peptide chain can be coupled
at the lactate group giving a straightforward access to a glycopep-
tide scaffold (unpublished results). Regioselective ring opening of
12 furnishes 16 with a O-4 free position for further glycosylation,
according to a reported procedure.5a
NAG-NAM
14
Scheme 5. Versatility of the NAG–NAM building block 12.
added by traditional SPPS activation protocols, as well as disaccha-
ride 13. The reaction was monitored by high-resolution magic an-
gle spinning NMR (HR-MAS NMR).10 The glycopeptide 17 was
isolated after cleavage from the resin with a TFA solution.
Despite the admirable synthetic approaches reported so far to-
ward PGN fragments,5 the orthogonal protecting group approach is
still a puzzling strategy. In the synthesis of PGN there are some
intricate steps, in particular the lactate insertion at O-3 and the
establishment of a b(1-4) glycosidic bond for which a proper
N-protecting group in both donor and acceptor is demanding.
In conclusion, the current work represents a simple strategy to-
ward NAG–NAM unit which relies on the different protection of
hydroxyl groups, using benzyl, benzylidene acetal, and acetyl
group, allowing further regioselective manipulation at O-3, O-4
positions and also at the lactate moiety of a versatile intermediate
In order to explore the insertion of a peptide chain at the lactate
moiety of 13, a pentapeptide containing the sequence of the Lys-
type PGN was prepared via Fmoc (9-fluorenylmethoxycarbonyl)
solid-phase peptide synthesis (SPPS) (Scheme 6). Thus HMPB-AM
resin was used and the Fmoc-protected aminoacids sequentially
O
BnO
O
BnO
O
Ph
O
O
HO
O
Ph
O
i
O
O
AcO
+
NH
CCl3
O
AcO
TrocHN
O
TrocHN
TrocHN
TrocHN
OBn
52%
O
OBn
CO2Et
CO2Et
11
10
4
ii 63%
O
O
BnO
BnO
O
O
Ph
Ph
O
iii
O
O
O
O
O
O
O
HO
AcO
AcHN
AcHN
69%
AcHN
AcHN
OBn
OBn
CO2H
CO2Et
13
12
iv
HO
HO
O
HO
HO
AcHN
O
O
O
AcHN
OH
NAG-NAM
CO2H
14
i) TMSOTf, DCM, 3A MS, -15 ºC; ii) Zn-Cu, THF: AcOH: Ac2O (1:1:1), then Ac2O/pyridine; iii) LiOH,
THF:1,4-dioxane:H2O; iv) Pd(OH)2, H2, AcOH, RT.
Scheme 4. Synthesis of the NAG–NAM disaccharide.

R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
115
O
HO
1. Pentapeptide
coupling
BnO
BnO
O
O
Ph
O
HO
O
O
O
O
HO
AcHN
HO
AcHN
O
O
AcHN
AcHN
OBn
OBn
L-Ala-D-isoGln-L-Lys(Ddiv)-D-Ala-D-Ala
CO2H
2. Resin cleavage
O
13
17
Scheme 6. Insertion of a pentapetide to 13 via Fmoc SPPS.
12. The conjugation of these protecting groups reduces the number
of synthetic steps while allowing a complete protecting group re-
moval in a late stage of the synthesis. Overall, this is a useful route
to achieve the biologically relevant NAG–NAM disaccharide moiety
crucial for the preparation of bacterial peptidoglycan as well as
non-natural derivatives.
3.81-3.75 (m, 2H, H-4, H-6a), 2.21 (s, 3H,COCH3), 2.11
(s, 3H,COCH3).13C NMR (100 MHz, CDCl3, 23 °C): dc 171.3 (COCH3),
168.9 (COCH3), 154.2 (COCH2CCl3), 136.6 (ArC), 129.2 (ArC), 128.2
(ArC), 126.0 (ArC), 101.6 (CHPh), 95.2 (CCl3), 90.92 (C-1), 78.51
(C-4), 74.5 (CH2CCl3), 69.3 (C-3), 68.4 (C-6), 64.8 (C-5), 53.8 (C-2),
20.7 (2 ꢂ COCH3). MALDI-TOF: C20H22Cl3NO9+Na: 548.0257.
Found: 548.044.
1. Experimental
1.1. General
1.3. 2-Deoxy-3-O-acetyl-4,6-O-benzylidene-2-(2,2,2-
trichloroethoxycarbonylamino)-a-D-glucopyranoside (3)
Morpholine (200 L, 2.3 mmol) was added to a solution of com-
l
Melting points were determined with a Reichert–Thermovar
hot stage apparatus and are uncorrected. Optical rotation was
measured for solutions in a 1 cm cell with a Perkin-Elmer 241
MC polarimeter. Matrix-assisted laser desorption ionization-time
of flight spectra were recorded on a Voyager DE PRO Biospectrom-
etry Workstation. High resolution mass spectra were recorded on
an AutoSpeQ spectrometer. 1H NMR spectra (400 MHz and
600 MHz) and 13C NMR spectra (100.63 MHz and 150 MHz) were
recorded on a Bruker ARX 400 spectrometer and on a Bruker Ultra-
shield Plus 600, respectively. NMR spectra were calibrated using
solvent signals (CDCl3: d 1H 7.26 and d 13C 77.00). Chemical shifts
reported are relative to tetramethylsilane (TMS) as the internal ref-
erence (1H 0.00) for 1H NMR spectra and are expressed in parts per
million (ppm), downfield from TMS (d = 0). All reagents and sol-
vents were purified and dried by standard methods19 before use.
After classical work-up organic extracts were dried over anhydrous
sodium sulfate or magnesium sulfate, filtered, and concentrated
under reduced pressure (rotary evaporator and vacuum pump).
Analytical thin-layer chromatography and preparative TLC (PTLC)
were performed on E. Merck Kieselgel 60, F254 silica gel (0.2 mm
thick) and 0.5, 1, or 2 mm thick plates (20 ꢂ 20 cm), respectively.
Column chromatography was performed on E. Merck Kieselgel 60
(240–400 mm) normal silica gel. ‘rt’ denotes room temperature.
(ꢀ)-Ethyl (S)-2-hydroxypropionate and Fmoc-aminoacids were
purchased from Sigma–Aldrich.
pound 2 (500 mg, 0.95 mmol) in dry ethyl acetate (1.5 mL). After
stirring overnight at rt the reaction mixture was quenched with a
3 N HCl solution (0.7 mL) and then stirred for 20 min. Extracted
with EtOAc and washed with water, saturated aq NaHCO3 and
brine, dried, and concentrated. The crude was purified by column
chromatography (Toluene/EtOAc (10:2)) to give 3 as a white solid
23
(401 mg, 87%). Mp 148–151 °C (decompose), ½dꢃD ꢀ11.2 (c 1,
CHCl3). 1H NMR (400 MHz, CDCl3, 23 °C): dH 7.46–7.43 (m, 2H,
ArH), 7.38–7.35 (m, 3H, ArH), 5.53 (s, 1H, CHPh), 5.43 (t, 1H, J
9.8 Hz, H-3), 5.33 (t, 1H, J 3.6 Hz, H-1), 4.80 (d, 1H, J 12 Hz, CH2-
CCl3), 4.67 (d, 1H, J 12 Hz, CH2CCl3), 4.29 (dd, 1H, J 10.4 Hz, J
4.8 Hz, H-6b), 4.17 (td, 1H, J 4.8 Hz, H-5), 4.05 (td, J 2.8 Hz, H-2),
3.80-3.69 (m, 2H, H-6a, H-4), 2.06 (s, 3H, COCH3). 13C NMR
(100 MHz, CDCl3) d171.1 (COCH3), 154.5 (COCH2CCl3), 137.0
(ArC), 129.3 (ArC), 128.4 (ArC), 126.3 (ArC), 101.7 (CHPh), 95.5
(CCl3), 92.7 (C-1), 79.31 (C-4), 74.7 (CH2CCl3), 69.9 (C-3), 69.0
(C-6), 62.9 (C-5), 55.0 (C-2), 21.0 (COCH3). HR-MS (FI): m/z:
C
18H20Cll3NO8: 483.0254. Found: 483.0255.
1.4. 1-(2,2,2-Trichloroacetimine) 2-deoxy-3-O-acetyl-4,6-O-
benzylidene-2-(2,2,2-tri-chloroethoxycarbonylamino)-
a-D-
glucopyranoside (4)
To a solution of 3 (260 mg, 0.54 mmol) in dry DCM (5.2 ml), Cs2-
CO3 (82.3 mg, 0.25 mmol) and CCl3CN (520 L, 5.18 mmol) were
l
added. The reaction mixture was stirred for 2 h at rt, then filtered
over celite and concentrated to give donor 4 as a light yellow solid
(248 mg, 74%). Compound 4 was directly used for next reaction
step (synthesis of compound 11) without further purification.
1.2. 1,3-di-O-acetyl 2-deoxy-4,6-O-benzylidene-2-(2,2,2-
trichloroethoxycarbonylamino)-a-D-glucopyranoside (2)
Zinc chloride (763 mg, 5.6 mmol) was added to a solution of
prepared N-Troc glucosamine5c (2 g, 5.6 mmol) in benzaldehyde
(10 ml) and 3 Å MS (200 mg). After stirring overnight at rt satu-
rated aq NaHCO3 (10 mL) and ether (25 mL) were added and the
reaction mixture was stirred for 10 min. The precipitate was fil-
tered, washed with water, then ether, and dried. The residue was
dissolved in pyridine (4.5 mL) cooled to 0 °C, treated with Ac2O
(2.24 mL, 24 mmol) and stirred overnight at rt. The residue was
concentrated with toluene, cooled to 0 °C and extracted with CHCl3
and saturated aq NaHCO3 (3ꢂ), then brine. The combined organic
layers were dried, concentrated, and the crude was purified by col-
umn chromatography (Hexane/EtOAc (1:1)) to give 2 as a white so-
23
½dꢃD +22.3 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3, 23 °C): dH 8.76
(s, 1H, NHCCl3), 7.47–7.36 (m, 5H, ArH), 6.40 (d, 1H, J 3.6 Hz,
H-1), 5.57 (s, 1H, CHPh), 5.45 (t, 1H, J 10.2 Hz, H-3), 5.31 (d, 1H, J
9.2 Hz, NH), 4.72 (q, 2H, J 12 Hz, CH2CCl3), 4.36 (dd, 1H, J 10.4 Hz,
J 4.8 Hz, H-6b), 4.27 (td, 1H, J 4 Hz, J 3.6 Hz, J 3.6 Hz, H-2), 4.08-
4.03 (m, 1H, H-5), 3.86-3.78 (m, 2H, H-4, H-6a), 2.11 (s, 3H, CH3-
CO).13C NMR (100 MHz, CDCl3, 23 °C): d 171.2 (COCH3), 160.8
(C@NH) 154.4 (COCH2CCl3), 136.7 (ArC), 129.3 (ArC), 128.4 (ArC),
126.2 (ArC), 101.7 (CHPh), 95.3 (CCl3) 95.2 (C-1),78.5 (C-4), 74.7
(CH2CCl3), 69.3 (C-3), 68.6 (C-6), 65.4 (C-5), 54.7 (C-2) 21.0
(COCH3).
23
lid (2.3 g, 78%). Mp 98–101 °C; ½dꢃD +44.5 (c 1.0, CHCl3). 1H NMR
(400 MHz, CDCl3, 23 °C): dH 7.46–7.36 (m, 5H, ArH), 6.21 (d, 1H, J
3.6 Hz, H-1), 5.55 (s, 1H, CHPh), 5.39 (t, 1H, J 10.2 Hz, H-3), 5.25
(d, 1H, J 9.6, NH), 4.80 (d, 1H, J 12 Hz, CH2CCl3), 4.65 (d, 1H, J
12 Hz, CH2CCl3), 4.32 (dd, 1H, J 10.4 Hz, J 4.8 Hz, H-6b), 4.19 (td,
1H, J 3.6 Hz, J 3.6 Hz, J 3.2 Hz, H-2), 3.94 (td, 1H, J 4.8 Hz, H-5),
1.5. Benzyl 2-deoxy-2-(allyloxycarbonylamino)-
a-D-
glucopyranoside (6)
Prepared N-Alloc glucosamine5c (9.5 g, 36.3 mmol) was dis-
solved in BnOH (57 mL) and acetyl chloride (9.8 mL, 137 mmol)

116
R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
was added dropwise at 0 °C. After stirring for 3 h at 80 °C the reac-
tion mixture was quenched with cold saturated aq NaHCO3 and
stirred for 30 min. Cold water and ether were added and the reac-
tion mixture was stirred for 30 min. The ppt was filtered and
washed several times with cold ether (until no traces of BnOH).
Product 6 was obtained as a white solid (7.86 g, 61% calculated
yield) and it was directly used for next reaction step without fur-
ther purification. The acquired spectroscopic data is in accordance
mixture was then treated with neat (ꢀ)-ethyl (S)-2-trif-
luromethanesulfonyl propionate dropwise and stirred for 3 h at
rt. The reaction mixture was quenched by addition of ice and ex-
tracted with CHCl3. The organic solution was washed with satu-
rated aq NaHCO3 and brine, dried and concentrated. The residue
was purified by column chromatography (CHCl3/EtOAc 7:0.5) to
25
give 8 as a white solid (145 mg, 68%). Mp 130–132 °C; ½dꢃD +122
(c 1, CHCl3). 1H NMR (400 MHz, CDCl3, 23 °C): dH 7.46–7.33 (m,
10H, ArH), 6.63 (d, 1H, J 5.4 Hz, NH), 6.0–5.89 (m, 1H, CH@CH2),
5.57 (s, 1H, CHPh), 5.35–5.26 (m, 2H, H-1, CH@CH2), 5.20 (d, 1H,
J 11 Hz, CH@CH2), 4.69 (d, 1H, J 11.6 Hz, CH2Ph), 4.57–4.46 (m,
4H, CH2Ph, CHCH3, CH2CH@CH2), 4.23–4.11 (m, 3H, OCH2CH3,
H-6b), 3.89–3.85 (m, 2H, H-3, H-5), 3.78–3.68 (m, 3H, H-2, H-6a,
H-4), 1.40 (d, 3H, J 7.0 Hz, CHCH3), 1.27 (t, 3H, J 7.1 Hz, CH2CH3).
13C NMR (100 MHz, CDCl3, 23 °C): dc 174.2 (COCH2CH3), 156.3
(COCH2CH3), 137.3 (ArC), 137.2 (ArC), 133.1 (ArC), 128.9 (ArC),
128.3 (ArC), 127.7 (ArC), 125.8 (ArC), 117.0 (CH@CH2), 101.2
(CHPh), 97.4 (C-1), 83.3 (C-4)75.1 (CHCH3), 74.7 (C-3), 70.1 (CH2-
Ph), 68.9 (H-6), 65.8 (CH2CH@CH2), 62.8 (C-5), 61.2 (OCH2CH3),
55.3 (C-2), 18.6 (CHCH3), 14.0 (OCH2CH3). MALDI-TOF: C29H35-
NO9+Na: 564.2209. Found: 564.244.
22
with the literature.20 Mp 120–122°C; ½dꢃD +161.4 (c 0.5, MeOH). 1H
NMR (400 MHz, DMSO-d6, 23 °C): dH 7.35–7.25 (m, 5H, ArH), 7.09
(d, 1H, J 8.4 Hz, NH), 5.91–5.84 (m, 1H, CH@CH2), 5.26 (dd, 1H, J
17.2 Hz, J 1.2 Hz, CH@CH2), 5.18–5.15 (m, 1H, OH, CH@CH2), 4.97
(d, 1H, J 5.6 Hz, OH), 4.78 (t, 1H, J 5.6 Hz, OH), 4.73 (d, 1H, J
3.4 Hz, H-1), 4.64 (d, 1H, J 12.5 Hz, CH2Ph), 4.47–4.39 (m, 3H, CH2-
Ph, ACH2ACH@CH2), 3.70–3.34 (m, 5H+H2O-DMSO-d6, H-6b, H-3,
H-6a, H-5, H-2), 3.21–3.12 (m, 1H, H-4). 13C NMR (100 MHz, CDCl3,
23 °C): dc 156.7 (NHCO), 138.2 (ArC), 134.0 (CH@CH2), 128.7 (ArC),
128.0 (ArC), 117.5 (CH@CH2), 96.4 (C-1), 73.3 (C-5), 71.1 (C-4), 70.9
(C-3), 68.2 (CH2Ph), 64.9 (CH2CH@CH2), 61.3 (C-6), 56.2 (C-2). HR-
MS (FI): C17H23NO7: 353.1474. Found: 353.1455.
1.6. Benzyl 2-deoxy-4,6-O-benzylidene-2-
(allyloxycarbonylamino)-
a-
D
-glucopyranoside (7)
1.8. Benzyl 2-deoxy-3-O-((R)-10-ethoxycarbonylethyl)-4,6-O-
benzylidene-2-(2,2,2-tri-chloroethoxycarbonylamino)-
a-D-
Zinc chloride (313 mg, 2.3 mmol) was added to a solution of 6
(802 mg, 2.3 mmol) in benzaldehyde (4 mL) and 3 Å MS
(325 mg). After stirring for 12 h at rt the reaction mixture was trea-
ted with saturated aq NaHCO3 (5 mL), petroleum ether (30 mL),
and stirred for 10 min. The ppt was filtered, washed with petro-
leum ether and dissolved in CHCl3. The organic solution was ex-
tracted with saturated aq NaHCO3, water, and brine, dried and
concentrated. The crude was purified by column chromatography
glucopyranoside (9)
Tetrakis(triphenylphosphine) palladium (476 mg, 0.41 mmol)
and AcOH (124 L, 2.17 mmol) were added to a solution of 8
(764 mg, 1.4 mmol) in dry DCM (14 mL). The reaction mixture
was stirred at rt for 15 min and then TrocCl (395 L, 2.87 mmol)
l
l
was added dropwise and stirred for 1 h at rt. The reaction mixture
was quenched with saturated aq NaHCO3, extracted with CHCl3,
washed with H2O and brine. After being concentrated the residue
was dissolved in ether and insoluble materials were filtered off (re-
peated until no insoluble materials were observed). The organic
phase was dried, concentrated, and purified by column chromatog-
raphy (Toluene:Acetone 10:1) to give 9 as a white solid (730 mg,
(10:1?10:2?10:3 CHCl3/EtOAc) to give
7 as a white solid
25
(733 mg, 72%). Mp 182–184 °C; ½dꢃD +92 (c 1, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.51–7.32 (m, 5H, ArH), 5.95–5.87 (m, 1H,
CH@CH2), 5.56 (s, 1H, CHPh), 5.31 (dd, 1H, J 17.2 Hz, J 1.4 Hz,
CH@CH2), 5.23 (d, 1H, J 10.4 Hz, CH@CH2), 5.10 (d, 1H, J 7.0 Hz,
NH), 4.96 (d, 1H, J 2.4 Hz, H-1), 4.74 (d, 1H, J 11.7 Hz, CH2Ph),
4.58 (d, 1H, J 5.6 Hz, CH2ACH@CH2), 4.51 (d, 1H, J 11.7 Hz, CH2Ph),
4.24 (dd, 1H, J 10.1 Hz, J 4.7 Hz, H-6a), 3.95–3.84 (m, 3H, H-2, H-4,
H-6b), 3.76 (t, 1H, J 10.2 Hz, H-3), 3.59 (t, 1H, J 8.9 Hz, H-5). 13C
NMR (100 MHz, CDCl3, 23 °C): dc 156.9 (NHCO), 134.6 (ArC),
132.6 (CH@CH2), 129.0 (ArC), 128.4 (ArC), 128.1 (ArC), 127.9
(ArC), 126.4 (ArC), 117.6 (CH@CH2), 101.9 (CHPh), 97.4 (C-1), 82.0
(C-4), 69.8 (CH2Ph), 69.2 (C-3), 68.3 (C-6), 62.6 (C-5), 65.7 (OCH2-
CH@CH3), 55.9 (C-2). MALDI-TOF: C24H27NO7+Na: 464.1685.
Found: 464.266.
25
82%). Mp 121–123 °C, ½dꢃD +125.2 (c 0.5, CHCl3). 1H NMR
(400 MHz, CDCl3, 23 °C): dH 7.46–7.29 (m, 10H, ArH), 6.85 (d, 1H,
J 5.1 Hz, NH), 5.58 (s, 1H, CHPh), 5.31 (d, 1H, J 3.6 Hz, H-1), 4.83
(d, 1H, J 12 Hz, CH2CCl3), 4.67 (dd, 2H, J 17.1 Hz, 12.0 Hz, CH2Ph,
CH2CCl3), 4.55–4.50 (m, 2H, CH2Ph, CHCH3), 4.26–4.11 (m, 3H, H-
6b, OCH2CH3,) 3.94–3.85 (m, 2H, H-3, H-5), 3.81–3.68 (m, 3H, H-
2, H-6a, H-4), 1.41 (d, 3H, J 7.0 Hz, CHCH3), 1.28 (t, 3H, J 7.2 Hz,
OCH2CH3). 13C NMR (100 MHz, CDCl3, 23 °C): dc 174.3 (COCH2CH3),
154.9 (COCH2CCl3), 137.3 (ArC), 129.1 (ArC), 128.5 (ArC), 128.4
(ArC), 128.0 (ArC), 126.0 (ArC), 101.5 (CHPh), 97.3 (C-1), 95.9
(CCl3), 83.4 (C-4), 75.5 (CHCH3), 74.8 (C-3), 74.6 (CH2CCl3), 70.4
(CH2Ph), 69.1 (C-6), 63.1 (C-5), 61.5 (OCH2CH3), 55.7 (C-2), 18.8
(CHCH3), 14.3 (COCH2CH3). MALDI-TOF: C28H32Cl3NO9+Na:
654.1040. Found: 654.106.
1.7. Benzyl 2-deoxy-3-O-((R)-10-ethoxycarbonylethyl)-4,6-O-
benzylidene-2-(allyloxy-carbonylamino)-
(8)
a-D-glucopyranoside
Preparation of (ꢀ)-ethyl (S)-2-trifluromethanesulfonyl propio-
nate: To
solution of (ꢀ)-ethyl (S)-2-hydroxypropionate
(0.23 mL, 2 mmol) in dry DCM (0.56 mL) was added 2,6-lutidine
(236
L, 2 mmol). The reaction mixture was kept at ꢀ78 °C and
Tf2O (344 L, 2 mmol) was added dropwise, stirred for 40 min,
warmed up to rt and stirred for 1 h. The mixture was diluted with
dry DCM: Hexane ((1:1) 0.7 mL) and filtered through a short silica
gel column (approx. pack 1 cm of silica gel pad). Then the reaction
mixture was washed with 0.7 mL of dry DCM: Hexane (1:1) and
concentrated to give (ꢀ)-ethyl (S)-2-trifluromethanesulfonyl pro-
pionate quantitatively.
1.9. 1,6-Di-O-benzyl 2-deoxy-3-O-((R)-10-ethoxycarbonylethyl)-
a
2-(2,2,2-trichloroetho-xycarbonylamino)-
(10)
a-D-glucopyranoside
l
l
To a solution of 9 (218 mg, 0.34 mmol) in dry CH3CN (3.4 mL) at
0 °C was added a solution of Me3NBH3 (30 mg, 0.41 mmol) in CH3-
CN (0.2 mL) and then a solution of BF3ꢁOEt2 (260
lL, 2.1 mmol) in
CH3CN (0.73 mL) was added dropwise. After stirring for 3 h at
0 °C, the mixture was quenched with cold saturated aq NaHCO3
(1.8 mL), diluted with ethyl acetate, and washed with saturated
aq NaHCO3, 5% citric acid (4 ꢂ 4.4 mL), saturated aq NaHCO3
(3.6 mL), and brine (2.2 mL). The organic layer was dried, concen-
trated, and the crude was purified by column chromatography
(Toluene:Acetone, 10:1) to give 10 as a colorless foam (158 mg,
To a solution of 7 (175 mg, 0.4 mmol) in dry DCM (2.6 mL) was
added NaH (35 mg, 1.5 mmol, 60% oil dispersion), stirred for 1.5 h,
added more NaH (10 mg, 0.4 mmol) and stirred for 1 h at rt. The

R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
117
25
72%). Mp 105–108 °C, ½dꢃD +91.6 (c 1, CHCl3). 1H NMR (400 MHz,
OCH2CH3), 4.14–4.11 (m, 1H, OCH2CH3), 3.97 (dd, 1H, J 18.6 Hz, J
10 Hz, H-20), 3.90 (t, 1H, J 9.5 Hz, H-4), 3.79–3.74 (m, 2H, H-2, H-
60a), 3.64–3.58 (m, 2H, H-40, H-3), 3.55–3.49 (m, 2H, H-5, H-6b),
3.35 (td, 1H, J 9.8 Hz, J 5.0 Hz, H-50), 3.33 (dd, 1H, J 10.6 Hz, J
2.2 Hz, H-6a), 2.03 (s, 3H, NHCOCH3), 1.98 (s, 3H, COCH3), 1.68 (s,
3H, NH0COCH3), 1.35 (d, 3H, J 7.0 Hz, OCHCH3), 1.29 (t, 3H, J
7.2 Hz, OCH2CH3). 13C NMR (150 MHz, CDCl3, 23 °C): dc 175.9
(COCH2CH3), 170.7 (COCH3, NHCOCH3), 169.8 (NH0COCH3), 137.8
(ArC), 137.3 (ArC), 136.8 (ArC), 129.6 (ArC), 129.5 (ArC),129.2
(ArC), 128.3 (ArC), 128.2 (ArC), 127.6 (ArC), 126.1 (ArC), 101.4
(CHPh), 101.0 (C-10), 96.6 (C-1), 78.5 (C-40), 78.1 (C-4),74.9 (C-3),
74.5 (CHCH3), 74.1 (CH2Ph),72.2 (C-30), 70.4 (CH2Ph), 70.1 (C-5),
68.7 (C-60), 66.9 (C-6), 66.2 (C-50), 61.4 (OCH2CH3), 54.4 (C-2),
54.2 (C-20), 23.1 (COCH3, NHCOCH3), 20.8 (NH0COCH3), 18.9
(CHCH3), 14.1 (OCH2CH3),. MALDI-TOF: C44H54N2O14+Na:
857.3472. Found: 857.375.
CDCl3, 23 °C): dH 7.39–7.27 (m, 10H, ArH), 6.77 (d, 1H, J 4.5 Hz,
NH), 5.23 (d, 1H, J 2.8 Hz, H-1), 4.79 (d, 1H, J 12.0 Hz, CH2CCl3),
4.68–4.59 (m, 4H, ½ CH2CCl3, CHCH3, CH2Ph), 4.52 (dd, 2H, J
12.2, 4.7 Hz, CH2Ph), 4.27–4.17 (m, 2H, OCH2CH3), 3.74–3.60 (m,
6H, H-6b,H-3, H-4,H-2, H-5, H-6a), 1.41 (d, 3H, J 6.9 Hz, CHCH3),
1.28 (t, 3H, J 7.2 Hz, CH2CH3).13C NMR (100 MHz, CDCl3, 23 °C): dc
175.0 (COCH2CH3), 154.8 (COCH2CCl3), 137.5 (ArC), 137.4, (ArC)
128.6 (ArC), 128.5 (ArC), 128.1(ArC), 128.0 (ArC), 127.9 (ArC),
96.6 (C-1), 95.9 (CCl3),77.3 (C-3) 74.8 (CHCH3), 74.5 (CH2CCl3),
74.6 (C-4), 73.9 (CH2Ph), 71.1 (C-6), 70.1 (CH2Ph), 69.4 (C-5), 61.5
(OCH2CH3), 54.4 (C-2), 19.1 (CHCH3), 14.2 (OCH2CH3). MALDI-
TOF: C28H34Cl3NO9+Na: 656.1196. Found: 656.029.
1.10. Benzyl 6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-O-
benzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-b-
D-
glucopyranosyl]-2-deoxy-3-O-[(R)-1’-ethoxycarbonylethyl]-2-
(2,2,2-trichloroethoxycarbonylamino)-
(11)
a-
D
-glucopyranoside
1.12. Benzyl 6-O-benzyl-4-O-[2-deoxy -4,6-O-benzylidene-2-
acetylamino-b-
ethoxycarbonylethyl]-2-acetylamino-
D
-glucopyranosyl]-2-deoxy-3-O-[(R)-10-
a-D-glucopyranoside (13)
The donor 4 (235 mg, 0.37 mmol) and acceptor 10 (158 mg,
0.25 mmol), with 3 Å molecular sieves (27 mg) in dry DCM
To a solution of 12 (91 mg, 0.109 mmol) in THF/1,4-dioxane/
H2O 4:2:1 (2.8 mL) was added LiOHꢁH2O (56 mg, 1.34 mmol). After
stirring for 2 h at rt, the reaction mixture was filtered over Dowex
H+ (freshly activated with 1 N HCl). The residue was purified by
diaion HP-20 column chromatography (2 ꢂ 7 cm) previously
washed with water and MeOH and then water. Column was eluted
with H2O (50 mL) and then eluted with MeOH (30 mL). The meth-
anol fractions were concentrated to give 13 as a colorless solid
(7 mL) were treated with TMSOTf (27
l
L, 0.15 mmol) at ꢀ15 °C.
After stirring for 20 min, the mixture was quenched with a cold
saturated aq of NaHCO3 (1 mL) and extracted with CHCl3
(6.7 mL). The organic layer was washed with saturated aq NaHCO3
and brine, dried and concentrated. The residue was purified by col-
umn chromatography (Toluene/Acetone (10:2)) to give 11 as a
25
white foam (164 mg, 60%). Mp 96–98 °C, ½dꢃD +12 (c 0.5, CHCl3).
25
1H NMR (400 MHz, CDCl3) dH 7.60–7.27 (m, 16 H, NH, 15 ArH),
5.49 (s, 1H, CHPh), 5.29 (s, 1H, H-1), 4.95 (d, 1H, J 12.1 Hz, ½ CH2-
Ph), 4.78–4.73 (m, 3H, NH0, CH2CCl3), 4.67 (d, 1H, J 12.0 Hz, ½ CH2-
CCl3), 4.61–4.56 (m, 3H, ½ CH2Ph, ½ CH2CCl3, CHCH3), 4.50 (d, 1H, J
12.0 Hz, ½ CH2Ph), 4.43 (dd, 1H, J 10.3, 5.0 Hz, H-6b), 4.37–4.23 (m,
2H, ½ OCH2CH3, ½ CH2Ph), 4.19–4.10 (m, 2H, ½ OCH2CH3, H-10),
3.95–3.91 (m, 1H, H-6b0), 3.81–3.66 (m, 6H, H-2, H-40, H-30, H-3,
H-50, H-6a), 3.60–3.53 (m, 3H, H-20, H-4, H-6a0), 3.42–3.29 (m,
1H, H-5), 2.03 (s, 3H, COCH3), 1.35 (d, 3H, J 6.9 Hz, CHCH3), 1.29
(t, 1H, J 7.1 Hz, CH2CH3). 13C NMR (100 MHz, CDCl3, 23 °C): dc
174.8 (COCH2CH3), 169.8 (COCH3), 154.8 (COCH2CCl3), 154.0
(COCH2CCl3), 101.3 (CHPh), 100.9 (C-10), 95.9 (2 ꢂ CCl3), 96.0
(C-1), 78.8 (C-40), 78.1 (C-4), 75.2 (C-50), 74.9 (CHCH3), 74.6 (CH2-
CCl3), 74.4 (CH2Ph), 74.1 (CH2CCl3), 71.6 (C-30), 70.3 (CH2Ph), 70.1
(C-3), 68.8 (C-60), 66.7 (C-6), 66.1 (C-5), 61.6 (OCH2CH3), 56.7
(C-2), 55.5 (C-20), 20.6 (COCH3), 18.9 (CHCH3), 14.0 (OCH2CH3).
MALDI-TOF: C46H52Cl6N2O16+Na: 1121.1345. Found: 1121.099.
(57.4 mg, 69%). Mp 102–105 °C, ½dꢃD +40.1 (c 0.75, CH3OH). 1H
NMR (600 MHz, CD3OD) dH 7.51–7.27 (m, 15H, ArH), 5.59 (s, 1H,
CHPh), 5.36 (d, J 3.1 Hz, H-1), 4.85 (m, (overlaped by solvent impu-
rity) H2O, H-10, ½ CH2Ph), 4.66–4.57 (m, 3H, J 12.1 Hz, CHCH3, CH2-
Ph), 4.46 (d, 1H, J 12.2 Hz, ½ CH2Ph), 4.29 (dd, 1H, J 10.3 Hz, J
5.0 Hz, H-60b), 4.07 (t, 1H, J 9.6 Hz, H-30), 3.95 (t, 1H, J 9.1 Hz,
H-4), 3.82–3.77 (m, 2H, H-6b, H-3), 3.72–3.62 (m, 3H, H-60a, H-5,
H-6a), 3.55–3.40 (m, 3H, H-20, H-40,H-2), 3.30–3.27 (m, 1H, H-50),
1.98 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.37 (d, J 6.9 Hz, 3H,
CHCH3). 13C NMR (150 MHz, CDCl3, 23 °C): dc 182.6 (COOH),
173.7 (2 ꢂ NHCOCH3), 139.8 (ArC), 139.2 (ArC), 139.1 (ArC), 129.9
(ArC), 129.4 (ArC), 129.2 (ArC), 129.0 (ArC), 128.9 (ArC), 128.7
´
(ArC), 128.6 (ArC), 127.5 (ArC), 103.0 (CHPh), 101.5 (C-1), 97.0
(C-1), 83.1 (C-40), 79.3 (CHCH3), 78.3 (C-4), 75.8 (C-3), 74.1 (CH2Ph),
72.6 (C-5), 71.0 (C-30), 69.9 (C-60), 69.3 (C-6), 67.6 (C-50), 60.0 (C-20),
56.3 (C-2), 23.1 (NHCOCH3), 22.8 (NHCOCH3), 20.2 (CHCH3).
MALDI-TOF: C40H48N2O13+Na: 787.3054. Found: 787.8830.
1.11. Benzyl 6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-O-
1.13. 4-O-[2-deoxy-2-acetylamino-b-D-glucopyranosyl]-2-
benzylidene-2-acetylamino-b-
D
-glucopyranosyl]-2-deoxy-3-O-
deoxy-3-O-[(R)-10-carboxyethyl]-2-acetylamino-
a-D-
[(R)-10-ethoxycarbonylethyl]-2-acetylamino-
a-
D-
glucopyranoside (14)
glucopyranoside (12)
Compound 13 (20 mg, 0.026 mmol) was dissolved in acetic acid
(8 mL), and then Pd(OH)2/C (58 mg) was added. The mixture was
stirred at rt for 6 h under H2 atmosphere (3 balloons were used).
The mixture was filtered over celite and concentrated. Compound
14 was isolated in quantitative yield. The data found was identical
to that reported in the literature.21
A solution of 11 (150 mg, 0.136 mmol) and freshly activated
zinc–copper couple (390 mg) in AcOH/Ac2O/THF 1:1:1 (1.5 mL)
was stirred for 4 h at rt. The reaction mixture was filtered over cel-
ite, washed with ethyl acetate, and concentrated. Then the crude
was dissolved in Pyridine: Ac2O (2:1, 374 lL) and stirred overnight
at rt. After completion of the reaction, the crude was concentrated
and purified by column chromatography (CHCl3/Acetone (7:1)) to
1.14. Protected disaccharide pentapeptide (17)
25
give 12 as a white solid (71 mg, 63%). ½dꢃD +31.4 (c 0.5, CHCl3).
1H NMR (600 MHz, CDCl3) dH 8.03 (d, 1H, J 4.4 Hz, NH), 7.56–
7.27 (m, 15H, ArH), 5.50 (s, 1H, CHPh), 5.41 (d, 1H, J 3.4 Hz, H-1),
4.94 (d, 1H, J 12.1 Hz, CH2Ph), 4.84 (t, 1H, J 9.9 Hz, H-30), 4.61–
4.57 (m, 2H, CHCH3, CH2Ph), 4.52 (d, 1H, J 12.3 Hz, CH2Ph), 4.44
(dd, 1H, J 10.4 Hz, J 5.0 Hz, H-60b), 4.40 (d, 1H, J 9.9 Hz, NH0), 4.29
(dd, 2H, J 10.3 Hz, J 7.7 Hz, H-10, CH2Ph), 4.24–4.21 (m, 1H,
The peptide chain
[Ddiv
(L-Ala-D-isoGln-L-Lys(Ddiv)-D-Ala-D-Ala)
(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbu-
tyl)] was assembled using Fmoc SPPS protocol22 using HMPB-AM
(NovaBiochem) resin. The peptide assembly was monitored by
high-resolution magic angle spinning NMR (HR-MAS NMR).10 The
disaccharide was coupled under the conditions used for aminoacid

118
R. Enugala et al. / Carbohydrate Research 384 (2014) 112–118
Tetrahedron 2011, 67, 977–9778; (j) Hadi, T.; Pfeffer, J. M.; Clarke, A. J.; Tanner,
M. E. J. Org. Chem. 2011, 76, 1118–1125.
6. Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819–6825.
7. For a review, see: (a) Enugala, R.; Carvalho, L. C. R.; Pires, M. J. D.; Marques, M.
M. B. Chem. Asian J. 2012, 7, 2482–2501; (b) Bongat, A. F. G.; Demchenko, A.
Carbohydr. Res. 2007, 342, 374–406.
8. Enugala, R.; Carvalho, L. C. R.; Marques, M. M. B. Synlett 2010, 2711–2716.
9. Enugala, R.; Marques, M. M. B. Arkivoc 2012, vi, 90–100.
10. Carvalho, L. R.; Corvo, M. C.; Enugala, R.; Cabrita, E. J.; Marques, M. M. B. Magn.
Reson. Chem. 2010, 48, 323–330.
coupling and the resulting glycopeptide 17 was detached from the
solid support by treatment with a trifluoroacetic acid solution
(1% in dichloromethane). 1H NMR (600 MHz, D2O) dH 7.52–7.34
(m, 10H, ArH), 5.02 (s, 1H), 4.96–4.60 (overlaped by water peak,
5H), 4.37–4.10 (m, 9 H), 3.96–3.51 (m, 9H), 3.42–3.35 (m, 2H),
3.25–3.19 (m, 1H), 2.94 (d, J 6.8 Hz, 2H), 2.38 (s, 4H), 2.32–2.24
(m, 2H), 1.99 (s, 3H), 1.92 (s, 3H), 1.88–1.63 (m, 8H), 1.45–1.30
(m, 12H) , 0.99 (s, 6H), 0.91 (d, J 6.6 Hz, 6H). MALDI-TOF:
ˇ
11. Carbohydrate Chemistry: Proven Synthetic Methods; Kovác, P., Ed.; CPR Press,
C53H79N9O19+Na: 1168.5389. Found: 1168.4369.
Taylor & Francis Group: Boca Raton, 2012. Vol. 1, pp 199–204.
12. Johnson, D. A.; Johnson, C. L.; Helene, B.-L. G.; Sowell, C.G. World Intellectual
Property Organisation Patent W.O 005308, 2004.
Acknowledgments
13. (a) Nelson, G. S.; Bungard, C. J.; Wang, K. J. Am. Chem. Soc. 2003, 125, 13000–
13001; (b) Higashino, T.; Sakaguchi, S.; Ishii, Y. Org. Lett. 2000, 2, 4193–4195;
(c) Gigg, R. J. Chem. Soc., Perkin Trans. 1 1980, 738–740; (d) Boullanger, P.;
Chateland, P.; Descotes, G.; Kloosterman, M.; Boom, V. H. J. J. Carbohydr. Chem.
1986, 5, 541–559; (e) Nishiguchi, T.; Tachi, K.; Fukuzumi, K. J. Org. Chem. 1975,
40, 237–240; (f) Mereyala, H. B.; Guntha, S. Tetrahedron Lett. 1993, 34, 6929–
6930; (g) Carless, H. A.; Haywood, D. J. J. Chem. Soc., Chem. Commun. 1980, 980–
981; (h) Lee, J.; Cha, J. K. Tetrahedron Lett. 1996, 37, 3663–3666; (i) Honda, M.;
Morita, H.; Nagagura, I. J. Org. Chem. 1997, 62, 8932–8936; (j) Ito, H.; Taguchi,
T.; Hanzawa, Y. J. Org. Chem. 1993, 58, 774–775; (k) Rao, G. V.; Reddy, D. S.;
Mohan, G. H.; Iyengar, D. S. Synth. Commun. 2000, 30, 3565–3568; (l) Bailey, W.
F.; England, M. D.; Mealy, M. J.; Thongsornkleeb, C.; Teng, L. Org. Lett. 2000, 4,
489–491; (m) Diaz, R. R.; Melagatejo, C. R.; Espinosa, M. T. P. L.; Cubro, I. J. Org.
Chem. 1994, 59, 7928–7929.
The authors thank the Fundação para a Ciência e Tecnologia for
the fellowships (SFRH/BPD/42134/2007 and SFRH/BD/89518/2012)
and for the projects PTDC/SAU-IMU/111806/2009 and PTDC/QEQ-
QOR/2132/2012. The authors would also like to thank Dr. Sérgio
Filipe for the helpful discussions.
References
1. Allen, H. K.; Donato, J.; Wang, H. H.; Cloud-Hansen, K. A.; Davies, J.;
Handelsman, J. Nat. Rev. Microbiol. 2010, 8, 251–259.
14. Le Goff, E. J. Org. Chem. 1964, 29, 2048–2050.
2. Crunkhorn, S. Nat. Rev. Drug Discovery 2013, 12, 99.
15. Inamura, S.; Fujimoto, Y.; Kawasaki, A.; Shiokawa, Z.; Woelk, E.; Heine, H.;
Lindner, B.; Inohara, N.; Kusumoto, S.; Fukase, K. Org. Biomol. Chem. 2006, 4,
232–242.
3. Rietschel, E. T.; Schletter, J.; Weidmann, B.; El-Samalouti, V.; Mattern, T.;
Zähringer, U.; Seydel, U.; Brade, H.; Flad, H. D.; Kusumoto, S.; Gupta, D.;
Dziarski, R.; Ulmer, A. J. Microb. Drug Resist. 1998, 4, 37–44.
16. Boudreau, M. A.; Fisher, J. F.; Mobashery, S. Biochemistry 2012, 51, 2974–2990.
17. Kusumoto, S.; Fukase, K.; Shiba, T. Proc. Jpn. Acad., Ser. B 2010, 86, 322–337.
18. Siegrist, M. S.; Whiteside, S.; Jewett, J. C.; Aditham, A.; Cava, F.; Bertozzi, C. R.
ACS Chem. Biol. 2013, 8, 500–505.
19. Purification of Laboratory Chemicals; Perrin, D. D., Armarego, W. L. F., Perrin, D.
R., Eds., 2nd ed.; Pergamon: Oxford, 1980.
20. Lafont, D.; Boullanger, P.; Fenet, B. J. Carbohydr. Chem. 1994, 3, 565–584.
21. Kantoci, D.; Keglevic´, D. Carbohydr. Res. 1987, 162, 227–235.
22. Swaminathan, C. P.; Brown, P. H.; Roychowdhury, A.; Wang, Q.; Guan, R.;
Silverman, N.; Goldman, W. E.; Boons, G.-J.; Mariuzza, R. A. PNAS 2006, 103,
684–689.
4. Höltje, J.-V. Microbiol. Mol. Biol. Rev. 1998, 62, 181–203.
5. (a) Hesek, D.; Lee, M.; Morio, K.-I.; Mobashery, S. J. Org. Chem. 2004, 69, 2137–
2146; (b) Zhang, Y.; Fechter, E. J.; Wang, T.-S. A.; Barrett, D.; Walker, S.; Kahne,
D. E. J. Am. Chem. Soc. 2007, 129, 3080–3081; (c) Inamura, S.; Fukase, K.;
Kusumoto, S. Tetrahedron Lett. 2001, 42, 7613–7616; (d) Fujimoto, Y.; Konishi,
Y.; Kubo, O.; Hasegawa, M.; Inohara, N.; Fukase, K. Tetrahedron Lett. 2009, 50,
3631–3634; (e) Shih, H.-W.; Chen, K.-T.; Cheng, T.-J. R.; Wong, C.-H.; Cheng,
W.-C. Org. Lett. 2011, 13, 4600–4603; (f) Chowdhury, A. R.; Siriwardena, A.;
Boons, G.-J. Tetrahedron Lett. 2002, 43, 7805–7807; (g) Hesek, D.; Lee, M.;
Zhang, W.; Noll, B. C.; Mobashery, S. J. Am. Soc. Chem. 2009, 131, 5187–5193; (h)
Cirillo, L.; Bedini, E.; Molinaro, A.; Parrilli, M. Tetrahedron Lett. 2010, 51, 1117–
1120; (i) Gampe, C. M.; Tsukamoto, H.; Wang, T.-S. A.; Walker, S.; Kahne, D.