A robust synthesis of N-glycolyl muramyl dipeptide via azidonitration/reduction

Article abstract of DOI:10.1039/c4ob02147a

A novel synthetic route leading to N-glycolyl muramyl dipeptide (MDP), a bacterial glycopeptide of particular interest in studies of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), is described. The synthetic strategy hinges on the alkylation of benzylidene-protected glucal with 2-bromopropionic acid and thus circumvents a challenging and non-reproducible S_N2 step at the C-3 position of glucosamine derivatives. The subsequent sequence includes an azidonitration and an unusual azide reduction/acylation step via an aza ylide/oxaphospholidine intermediate. This approach generates a protected N-glycolyl MDP that can be either subjected to a one-step global deprotection or differentially deprotected to obtain further derivatives.

Full text of DOI:10.1039/c4ob02147a

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DOI: 10.1039/C4OB02147A
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ARTICLE
A Robust Synthesis of N-Glycolyl Muramyl Dipeptide
Via Azidonitration/Reduction
Cite this: DOI: 10.1039/x0xx00000x
Shuo Xing and James L. Gleasona
a Department of Chemistry, McGill University. 801 Sherbrooke St. West, Montreal, Quebec, Canada, H3A 0B8.
Email: jim.gleason@mcgill.ca.
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
A novel synthetic route leading to N-glycolyl muramyl dipeptide (MDP), a bacterial glycopeptide of
particular interest in studies of nucleotide-binding oligomerization domain-containing protein 2
(NOD2), is described. The synthetic strategy hinges on the alkylation of benzylidene-protected glucal
with 2-bromopropionic acid and thus circumvents a challenging and non-reproducible SN2 step at the C-
3 position of glucosamine derivatives. The subsequent sequence includes an azidonitration and an
unusual azide reduction/acylation step via an aza ylide/oxaphospholidine intermediate. This approach
generates a protected N-glycolyl MDP that can be either subjected to a one-step global deprotection or
differentially deprotected to obtain further derivatives.
www.rsc.org/
The synthesis of N-glycolyl MDP (2) has been reported once
previously starting from 2-acetamido-D-glucosamine (3) and is
closely modelled after the traditional synthesis of 1.5,6
N-Acetyl muramyl dipeptide (N-Ac-MDP, 1, Figure 1), a
lactate linked glycopeptide, is a component of the cell wall of
bacteria. N-Ac-MDP has been shown to trigger an immune
response by activating the mammalian receptor nucleotide-
binding oligomerization domain-containing protein 2 (NOD2).
NOD2 is an intracellular protein that signals via the NF-κB
pathway to proximally activate innate immunity through
macrophage response as well as to more distally affect adaptive
immunity through the production of antigen-specific T-cells.1
Mutations in NOD2 have been associated with chronic
inflammatory diseases such as Crohn’s disease and Blau
syndrome.1d,2 Recent studies have shown that in certain
mycobacteria and related Actinomycetes, oxidation of 1 within
the peptidoglycan by N-acetyl muramic acid hydroxylase
(NamH) results in the formation of N-glycolyl MDP (2).3
Glycopeptide 2 has a higher potency and induces significantly
higher activation of NOD2 than 1.4 In order to study the
binding interactions and kinetics of 1, 2 and their analogs with
NOD2, we required an expeditious and flexible synthesis of
Ph
O
HO
HO
HO
a, b
O
O
O
HO
OBn
NHAc
OH
NHAc
3
4
c, d
e
Ph
O
O
HO
O
OBn
Ph
O
O
NH
O
O
MeOOC
O
OBn
NHAc
Me
BnO
6
5
e
f
Ph
MeOOC
O
Ph
O
O
O
O
O
O
MeOOC
O
OBn
OBn
NH
NH
O
O
Me
BnO
BnO
8
9
these glycopeptides.
Scheme 1. Traditional approach to the synthesis of 8. (a) Benzyl alcohol, pTSA,
Dean-Stark trap, C6H6, 80 °C, 73%. (b) benzaldehyde, (EtO)3CH, pTSA, DMF:p-
dioxane, 60 °C, 65%. (c) KOH, EtOH, 78 °C. (d) O-benzylglycolic acid, HBTU, DIPEA,
DMF, 38% over two steps. (e) i. NaH, DMF, 0 °C; ii. (S)-2-bromopropionic acid,
DMF, 0 °C to room temperature; iii. CH2N2, EtOAc, 0 °C. (f) i. NaH, THF; ii. methyl
2-bromoacetate, THF, 60 °C to room temperature, 71%.
OH
O
Me
O
O
H
N
HO
Me
O
OH
H2N
N
H
NHR
O
COOH
1 N-acetyl muramyl dipeptide R=Ac
2 N-glycolyl muramyl dipeptide R=COCH2OH
Protection of 3 as its 4,6-O-benzylidine-O-benzyl glycoside
Fig. 1 Structure of N-substitutued muramyl dipeptide.
affords acetamide 4 as an inseparable mixture of anomers
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

Organic & Biomolecular Chemistry
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ARTICLE
DOI: 10.1039/C4OB02147A
and thus we prepared 21 following the established 4-step
method of Chambers starting from α-methyl D-glucoside.9 We
were pleased to find that alkylation of 21 with (S)-2-
bromopropionic acid proceeded readily at room temperature in
the presence of NaH in DMF in a much shorter reaction time
compared to 4. After esterification of the resulting acid with
diazomethane, ether 22 was isolated in 96% yield. The ease
and high reproducibility of this alkylation underscore the
influence of the C2 substituent in slowing down alkylations of
both 4 and 6. With the lactate unit installed, we next
incorporated the C2 nitrogen via an azidonitration reaction
(Scheme 4).10 Treatment of 22 with sodium azide and cerium
ammonium nitrate (CAN) provided 23 as a separable mixture
of three diastereomers (23α:23β:24α/β, 4:2.2:1) in 72% overall
yield, with a 62% yield of the desired diastereomers, 23α and
23β. Reduction of the azide in 23α and/or 23β proved to be
problematic under traditional conditions (Ph3P, H2O), as the
(Scheme 1). Deacetylation of 4 occurs easily under alkaline
conditions, presumably due to the presence of the adjacent
hydroxyl group.7 Acylation of the resulting free amine with O-
benzylglycolic acid affords 6 in good yield over two steps.
However, while we were easily able to alkylate 4 with (S)-2-
bromopropionic acid (7) in the presence of NaH to form
protected N-Ac muramic acid 5,6 and in contrast to the reports
of Kobayashi et al.,5a N-glycolyl functionalized 6 resisted all
efforts at O-alkylation with 7, including heating in the presence
of excess NaH and electrophile in a variety of solvents (THF,
DMF, dioxane). Attempts with 2-chloropropionic acid and
methyl 2-halopropionates also afforded no alkylation products,
even under forcing conditions. The difference in reactivity
between 4 and 6 was surprising and although some of the
difference might arise from the additional chelating oxygen, the
facile alkylation of 6, as well as 4, with less hindered methyl
bromoacetate to form 9 suggested that increased sterics from
the benzyl group were the main influence hindering the SN2
displacement.
In order to reduce the steric demands on ether formation, we
prepared the protected 2-azido-2-deoxyglucose derivative 12, as
it was presumed that the smaller azide group would not
interfere in the alkylation step. To this end, D-glucosamine
(10) was azidated by treatment with triflyl azide in the presence
of copper sulfate (Scheme 2). The resulting azide 11 was
subjected to per-acetylation, glycosylation, deacetylation and
4,6-O-benzylidene formation, all under standard conditions, to
afford 12.8 Unfortunately, 12 proved to be wholly unreactive in
alkylation reactions with 7 and NaH, even in the presence of
excess electrophile and prolonged heating.
hydrolysis of the intermediate aza-ylide was quite slow.
OSi(OH)tBu2
HO
O
tBu
Si
OAc
O
MeOOC
O
a, b
c
tBu
Ph
AcO
AcO
O
O
O
HO
Me
16
15
14
Ph
O
O
O
HO
HO
HO
HO
O
O
O
O
HO
d
e
TfO
HO
HO
OMe
OMe
OMe
19
18
17
f
O
Ph
MeOOC
O
O
I
Ph
O
O
HO
h
g
O
O
O
Ph
O
O
HO
21
Me
OMe
22
20
Scheme 3. Synthesis of alkylated glucal 22. (a) NEt3, MeOH:H2O. (b)
tBu2Si(OTf)2, 2,6-lutidine, 98% over two steps. (c) i. NaH, DMF, 0 °C to room
temperature; ii. (S)-2-bromopropionic acid, DMF; iii. CH2N2, EtOAc, 45% (with
55% unalkylated, opened siloxylane). (d) PhCH(OMe)2, pTSA, DMF, 60 °C, 60%.
(e) Tf2O, py, CH2Cl2, -30 °C, 94%. (f)TBAI, toluene, 80 °C, 80%. (g) Zn, AcOH,
HO
HO
HO
AcO
AcO
AcO
O
O
a-c
OH
NH2 HCl
OPMP
11
N3
10
CH2Cl2, 46%. (h) i. NaH, DMF,
bromopropionic acid, DMF; iii. CH2N2, EtOAc, 96%.
0 °C to room temperature; ii. (S)-2-
d, e
Ph
O
Ph
O
O
HO
f
O
O
Heating the reaction or treatment with aqueous base afforded
only a lactam resulting from Staudinger ligation with the lactate
ester.11 Treatment of 25 with aqueous acid was also ineffective,
resulting in only slow cleavage of the benzylidene. The source
of the puzzling stability of the aza ylide was eventually
revealed through 1H-, 31P-NMR and 31P/1H HMBC experiments
(Figure 2). As the nitrate group at C1 position is readily
hydrolyzed, the free hydroxyl group stabilizes the aza ylide
through the formation of an oxaphospholidine (26), which is in
O
MeOOC
O
OPMP
OPMP
N3
N3
12
13
Scheme 2. Attempted synthesis of 2-azido muramic acid 13. (a) TfN3, CuSO4,
NaHCO3, toluene: H2O, 0 °C. (b) acetic anhydride, pyridine, 0 °C, 97% over two
steps. (c) p-methoxyphenol, SnCl4, CH2Cl2, 76%. (d) NaOMe, MeOH. (e)
benzaldehyde, (EtO)3CH, pTSA, DMF:p-dioxane, 60 °C, 74% over two steps. (f) i.
NaH, DMF, 0 °C; ii. (S)-2-bromopropionic acid, DMF, 0 °C to room temperature;
iii. CH2N2, EtOAc, 0 °C.
equilibrium with the open aza ylide form (25). Two distinct 31
P
signals can be observed in the NMR spectrum, one of which
(40.45 ppm) shows HMBC correlation to H-2 (2.74 ppm) alone,
while the second 31P signal (36.22 ppm) correlates to both H-2
Given the significant influence of the identity of the group at
C2, we opted to remove the amine altogether and install it at a
later stage in the synthesis. Preliminary studies using the 4,6-
di-tert-butylsiloxane glucal 15, prepared easily in two steps
from 3,4,6-tri-O-acetyl-D-glucal, revealed that the desired
alkylation with (S)-2-bromopropionic acid was possible,
affording ether 16 where the siloxane had become partially
deprotected, in moderate yield (Scheme 3). The partial
deprotection was difficult to control and resulted in a
significant amount of unalkylated open siloxane side product.
The lability of the protecting group could be addressed through
the use of a more robust 4,6-O-benzylidene protecting group.
Direct installation of the benzylidene on D-glucal was difficult
(3.09
ppm)
and
2.74ppm
an
NH
triplet
(6.34
ppm).
3.09ppm
H
H
Ph
O
O
Ph
MeOOC
O
O
O
O
O
O
MeOOC
OH
N
O
N
36.22ppm
PPh3
Me
Me
Ph3P
H
40.45ppm
6.34ppm
25
26
Fig. 2 31P-1H HMBC correlations in 25 and 26.
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4OB02147A
Ph
O
Ph
O
O
O
O
N
O
Ph
O
O
O
MeOOC
O
O
MeOOC
O
OH
MeOOC
b
O
ONO2
NH
Me
N3
Me
PPh3
Ph3P
Me
O
Ph
O
23α/β 22%/40%
a
26
O
25
MeOOC
O
+
c
Me
N3
O
Ph
O
O
22
O
MeOOC
Ph
O
O
ONO2
O
O
MeOOC
OH
Me
NH
O
Me
24α/β
OBn
27
d, e
Ph
OH
O
H
N
Me
O
O
O
O
Me
O
O
O
H
N
f
O
HO
Me
O
OH
OH
H2N
N
H
H2N
N
H
NH
NH
O
O
O
Me
O
COOBn
COOH
BnO
HO
28
2
While oxaphospholidine formation slowed attack of water, we (400MHz, CDCl3) 2.99 (bs, 1H, OH), 3.48 (ddd, 1H, J1 5.2 Hz,
expected that N-2 would remain nucleophilic, particularly in J2 10.0 Hz, J3 14.8 Hz), 3.61 (ddd, 1.5H, J1 2.0 Hz, J2 9.6 Hz, J3
the cyclic form, and might be acylated directly and this would 11.2 Hz), 3.88 (dt, 1H, J1 10.4 Hz, J2 24.4 Hz), 3.95 (ddd, 1H,
in turn facilitate the desired hydrolysis.11 To our delight, N- J1 4.8 Hz, J2 10.0 Hz, J3 19.6 Hz), 4.11 (m, 2H), 4.17 (s, 1H),
glycolylation with O-benzylglycolic acid in the presence of 4.30 (m, 1H), 4.43, (m, 1H), 4.52-3.77 (m, 6H), 4.94 (m, 1H),
HBTU, although necessitating long reaction times (>48 h), 5,23 (s, 0.8H, anomeric H), 5.59 (s, 1H), 5.60 (s, 0.2H,
proceeded to afford the desired protected glycolamide in 60% anomeric H), 6.88 (d, 0.4H, J 5.2 Hz, NH), 7.05 (d, 0.6H, J 8.8
yield. Conversion of 27 to N-glycolyl-MDP was then achieved Hz, NH), 7.283-7.53 (m, 15H) δC (500MHz, CDCl3) 53.4, 58.7,
through a standard sequence of ester saponification with KOH, 62.7, 66.4, 66.6, 67.2, 68.6, 68.8, 69.3, 70.6, 70.9, 71.3, 73.4,
coupling the resulting acid to the g-benzyl ester of D-isoGlu-L- 73.6, 81.5, 82.0, 96.9, 99.4, 101.98, 102.0, 126.33, 126.35,
Ala with HBTU and then global deprotection of all benzyl and 127.9, 128.0, 128.2, 128.29, 128.31, 128.39, 128.44, 128.47,
benzylidene protecting groups with H2-Pd/C, affording 2 in 128.5, 128.6, 129.21, 129.24, 136.5, 136.6, 136.7, 137.02,
71% yield over the final three steps.
137.05, 170.8, 171.0. HRMS (ESI) calcd for C29H31NO7
(M+K+), 544.1732, found, 544.1746.
Conclusions
5-O-ditertbutyl(hydroxy)silyl-3-O-R-(1-methyl
carboxylate)ethyl)-D-glucal (16): 1513 (75.0 mg, 0.262 mmol,
1 equiv) was dissolved in dry DMF (3 mL) at 0 °C. NaH (74
mg, 1.83 mmol, 7 equiv) was added and the mixture stirred for
45 min and allowed to warm up to room temperature. (S)-2-
bromopropionic acid (205 mg, 1.31 mmol, 5 equiv) was added
dropwise to the solution. The reaction was stirred at room
temperature and followed by TLC until the disappearance of
the starting material. The mixture was quenched with sat.
NH4Cl, acidified to pH 3 and quickly extracted with three
portions of EtOAc (5 mL x 3). The combined organic layers
were washed with brine, dried over Na2SO4, filtered and
concentrated at reduced pressure. The crude was treated with
diazomethane solution in ether to make the methyl ester for
purification. The product 16 was purified by flash
chromatography to give a colourless oil (46 mg, 45%). Data for
16: δH (500MHz, CDCl3) 1.04 (s, 18H), 1.46 (d, 3H, J 7 Hz),
3.06 (bs, 1H), 3.77 (s, 3H), 3.82 (dt, 2H, J1 3 Hz, J2 10 Hz),
3.97 (dd, 1H, J1 8 Hz, J2 10 Hz), 4.03 (bs, 1H), 4.13 (dd, 1H, J1
3 Hz, J2 12 Hz), 4.22 (dd, 1H, J1 3 Hz, J2 12 Hz), 4.29 (d, 1H, J
8 Hz), 4.41 (q, J 7 Hz), 4.73 (dd, 1H, J1 2 Hz, J2 6 Hz), 6.41
(dd, 1H, J1 1.5 Hz, J2 6 Hz). δC (500MHz, CDCl3) 19.0, 20.6,
20.8, 27.4, 27.6, 52.3, 61.9, 67.0, 71.2, 76.8, 78.9, 100.9, 145.8,
In conclusion we have developed a convenient 11-step
synthetic route to N-glycolyl MDP that significantly
circumvents the difficult SN2 step in the traditional glucosamine
alkylation route to MDP derivatives. This work will enable the
synthesis of N-acetyl and N-glycolyl MDP derivatives for
ongoing studies of NOD2 binding interactions and kinetics.
Experimental
Benzyl 2-(O-benzyl glycolamido)-4,6-O-benzylidene-D-
glucopyranoside
glucosamine12 (115.2 mg, 0.322 mmol, 1 equiv), O-benzyl
glycolic acid (54 mg, 0.322 mmol, equiv) and
(6):
Benzyl
4,6-O-benzylidene-D-
1
diisopropylethylamine (125 mg, 0.967 mmol, 3 equiv) were
dissolved in DMF (0.5 mL) at room temperature. HBTU (147
mg, 0.387 mmol, 1.2 equiv) was added and the mixture stirred
for 16 hours. The reaction was extracted with EtOAc (3 x 5
mL) against water. The organic layer was dried with Na2SO4,
filtered and loaded on Celite. The crude mixture was purified
using reverse phase flash chromatography (H2O:MeCN) to give
6 as a mixture of anomers (114 mg, 67%). Data for 6: δH
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J. Name., 2012, 00, 1-3 | 3

Organic & Biomolecular Chemistry
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Organic & BiomolecVuielawrArCtihcleeOmnliisnetry
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DOI: 10.1039/C4OB02147A
175.2. HRMS (ESI) calcd for C18H34O7Si (M+Na+), 413.1983, 4.02 (dt, J1 6 Hz, J2 13 Hz, J3 13 Hz), 4.10, t, 1H, J 12 Hz),
found 413.1966.
4.34 (dd, 1H, J1 6 Hz, J2 13 Hz), 4.61 (q, 1H, J 8.5 Hz), 5.59 (s,
1H), 6.28 (d, 1H, J 5.5 Hz), 7.40-7.47 (m, 5H). δC (500MHz,
CDCl3) 19.0, 52.2, 60.8, 62.0, 64.9, 68.2, 75.7, 76.2, 81.7, 97.1,
3-O-R-(1-methylcarboxylate)ethyl)-4,6-O-benzylidene glucal 101.7, 110.0, 125.9, 128.4, 129.3, 136.5, 172.8. IR (cm-1)
(22): The alcohol 2113 (683 mg, 2.92 mmol, 1 equiv) was 2918, 2120, 1750, 1657, 1453, 1374, 1280, 1210, 1134, 1094,
dissolved in dry DMF (15 mL) and cooled to 0°C using an ice 1067, 996, 825, 751, 699. HRMS (ESI) calcd for C17H20N4O9
bath. NaH (60% in mineral oil, 1.17 g, 29.1 mmol, 10 equiv) (M+Na+), 477.1128, found 477.1127. Data for 23β: δH
was added slowly to the solution with vigorous stirring. The (500MHz, CDCl3) 1.48 (d, 3H, J 8.5 Hz), 3.57 (dt, J1 6 Hz, J2
mixture was stirred at low temperature for 15 minutes, at which 13 Hz, J3 13 Hz), 3.61 (t, 1H, J 11 Hz), 4.39 (dd, 1H, J1 6 Hz,
point the ice bath was removed and (S)-2-bromopropionic acid J2 13 Hz), 4.60 (q, 1H, J 8.5 Hz), 5.582 (d, 1H, J 11 Hz), 5.583
(2.23 g, 14.6 mmol, 5 equiv) was added dropwise using a (s, 1H), 7.45-7.48 (m, 5H). δC (500MHz, CDCl3) 19.0, 52.1,
microsyringe. The reaction proceeded at room temperature until 60.4, 62.0, 65.6, 68.0, 75.9, 79.5, 80.6, 97.9, 101.4, 110.0,
the starting material was completely consumed. The excess 125.8, 128.4, 129.3, 136.5, 172.8. IR (cm-1) 2920, 2120, 1743,
NaH was quenched by adding small amount of water and the 1669, 1453, 1374, 1308, 1286, 1216, 1152, 1103, 1019, 827,
reaction mixture acidified to pH 2 with 3M aqueous HCl. The 701, 738, 699, 643. HRMS (ESI) calcd for C17H20N4O9
resulting solution was partitioned between EtOAc and water. (M+Na+), 477.1128, found 477.1126. Data for 24α/β: δH
The aqueous layer was further extracted with EtOAc (2 x 5 (500MHz, CDCl3) 1.50 (d, 3H, J 8.5 Hz), 3.77 (s, 3H), 3.84 (t,
mL). The combined organic layer was washed with brine, dried 1H, J 15 Hz), 3.96 (dt, 1H, J1 6 Hz, J2 12.5 Hz, J3 12.5 Hz),
with Na2SO4, filtered and concentrated in vacuo to give a light 4.00 (dd, 1H, J1 5 Hz, J2 12 Hz), 4.16, (t, 1H, J 12 Hz), 4.31
yellow oil. The concentrate was dissolved in EtOAc and treated (dd, 1H, J1 6 Hz, J2 13 Hz), 4.48 (m, 2H), 5.63 (s, 1H), 6.14 (s,
with a solution of diazomethane in ether to form the methyl 1H), 7.40-7.48 (m, 5H). δC (500MHz, CDCl3) 19.0, 52.1, 60.4,
ester. When the addition of diazomethane was complete, the 66.2, 68.1, 76.0, 76.5, 78.2, 98.6, 101.8, 125.9, 128.3, 129.2,
solution was concentrated in vacuo without heating. The crude 136.9, 173.6. HRMS (ESI) calcd for C17H20N4O9 (M+Na+),
reaction was purified with flash chromatography (10% EtOAc 477.1128, found 477.1124.
in petroleum ether) to give the product as a white solid (820
mg, 88%). Data for 22: δH (500MHz, CDCl3) 1.43 (d, 3H, J 6.5 2-azaylide-4,6-O-benzylidene-3-(O-R-(1-methyl
Hz, CH3 lactate), 3.71 (s, 3H, COOMe), 3.81 (dd, 1H, J6a,b 7.5 carboxylate)ethyl)-D-glucoside
(in
equilibrium
with
Hz, J5,6b 8.5 Hz, H-6b), 3.88 (ddd, 1H, J5,6a 3.0 Hz, J4,5 8.5 Hz, oxazophospholidine) (25, 26): 23α/β (175 mg, 0.41 mmol, 1
J5,6b 8.5 Hz, H-5), 3.96 (dd, 1H, J3,4 8.5 Hz, J4,5 8.5 Hz, H-4), equiv) was dissolved in dry THF (15 mL). PPh3 (172.5 mg,
4.33 (m, 2H, H-3, H-6a), 4.47 (q, 1H, JCH,Me 6.5 Hz, lactate 0.62 mmol, 1.5 equiv) was added and the mixture was stirred at
CH), 4.90 (dd, 1H, J2,3 2.0 Hz, Jvinylic 6.0 Hz, H-2), 5.62 (s, 1H, room temperature for 16 hours. The crude reaction mixture was
benzylidene CH), 6.34 (d, 1H, Jvinylic 6.0 Hz, H-1), 7.38-7.40 directly loaded on Celite for reverse phase purification (MeOH
(m, 3H, benzylidene), 7.41-7.49 (m, 2H, benzylidene). δC in H2O, 0-95%). The product was obtained in an inseparable
(500MHz, CDCl3) 19.2, 51.9, 68.4, 68.5, 73.6, 74.8, 80.3, mixture of 25 and 26, as an off-white fluffy powder after
101.1, 102.6, 125.9, 128.3, 129.0, 137.2, 144.5, 173.9. IR (cm- lyopholization (151 mg, 60%). Data for 25 and 26 as a mixture:
1) 2920, 2120, 1743, 1669, 1453, 1374, 1308, 1286, 1216, 1152, δH (500MHz, CDCl3) 1.28 (d, 3H, CH3, J 7 Hz), 2.75 (ddd,
1103, 1019, 827, 751, 738, 699. MP 98-100°C. HRMS (ESI) 0.5H, 2H 25, J1 9.5 Hz, J2 9.5 Hz, J3 19.0 Hz), 3.09 (dddd,
calcd for C17H20O6 (M+Na+), 343.1158, found 343.1164.
0.5H, 2-H 26, J1 3.5 Hz, J2 10.0 Hz, J3 10.0 Hz, J4 10.0 Hz),
3.35 (ddd, 0.5H, J1 5 Hz, J2 9.5 Hz, J3 9.5 Hz), 3.42 (ddd, 1H,
J1 0.5 Hz, J2 9.5 Hz, J3 16.5 Hz), 3.53 (s, 1.5H, COOMe), 3.56-
α/β-nitro-2S-azido-4,6-O-benzylidene-3-(O-R-(1-
methylcarboxylate)ethyl)-D-glucopyranoside (23α, β): 22 3.64 (m, 1H), 3.64 (s, 1.5H, COOMe), 3.68-3.74 (m, 1H), 4.07-
(357 mg, 1.11 mmol, 1 equiv) was dissolved in dry acetonitrile 4.27 (m, 3H), 4.62 (q, 1H, OCHMeCOOMe, J 7 Hz), 4.82 (d,
(18 mL) in a flame-dried flask and cooled to -15°C. cerium 0.5H, 1-H 25, 3.5 Hz), 5.40 (s, 0.5H, benzylidene CH), 5.42-
ammonium nitrate (CAN, 1.83 g, 3.34 mmol, 3 equiv) and 5.47 (m, 0.5H, 1-H, 26), 5.47 (s, 0.5H, benzylidene CH), 6.19
sodium azide (109 mg, 1.67 mmol, 1.5 equiv) were added to the (dd, 1H, NH, 26, J1 9.5 Hz, J2 9.5 Hz), 7.28-8.08 (m, 20H, Ar).
cooled solution simultaneously, followed by vigorous stirring. δC (500MHz, CDCl3) 19.0, 19.2, 51.9, 52.1, 52.2, 57.5, 60.7,
The reaction was monitored by TLC closely until starting 60.76, 60.78, 61.8, 65.2, 68.7, 69.1, 71.2, 74.78, 74.85, 75.2,
material was completely consumed approximately 3 hours at - 75.3, 77.3, 77.8, 81.8, 82.2, 82.4, 83.1, 83.2, 92.61, 92.61, 96.3,
15°C. The reaction mixture was partitioned between water and 100.98, 101.0, 101.3, 101.4, 120.6, 121.4, 121.7, 122.5, 125.98,
EtOAc. Enough water was added to remove most of the 126.0, 126.1, 126.2, 128.1, 128.2, 128.8, 129.0, 129.4, 129.5,
residual CAN such that the organic layer became mostly 130.0, 130.1, 133.9, 134.0, 134.2, 134.27, 134.33, 134.4,
colourless and the aqueous layer appeared yellow. The organic 135.22, 135.24, 137.2, 137.3, 173.5, 174.5. δP (500MHz,
layer was then washed with saturated NaHCO3 and saturated CDCl3) 38.2 (25), 40.4 (26). HRMS (APCI) calcd for
aqueous Na2S2O3, and dried with anhydrous Na2SO4. The C35H36NO7P (M+H+), 614.2302, found 614.2303.
organic layer was then filtered and concentrated in vacuo
without heating. The products were separated by flash 2-N-(2-benzyloxyacetyl)-4,6-O-benzylidene-3-(O-R-(1-
chromatography (15% EtOAc in petroleum ether) to provide methyl carboxylate)ethyl)-D-glucopyranoside (27):
A
four azidonitration products: 2R-azido-4,6-O-benzylidene-α/β- mixture of azaylide 25 and 26 (72 mg, 0.12 mmol, 1 equiv) was
methoxy-D-glucoside (24α/β, inseparable mixture, 49 mg dissolved in dry DMF (1 mL). 2-Benzyloxyacetic acid (23 mg,
~10.5%), 2S-azido-4,6-O-benzylidene-α-methoxy-D-glucoside 0.12 mmol, 1 equiv) and diisopropylethyl amine (78 µL, 0.47
(23α, 104 mg, 22.1%), 2S-azido-4,6-O-benzylidene-β-methoxy- mmol, 4 equiv) were added to the solution. HBTU (53 mg, 0.14
D-glucoside (23β, 189 mg, 40.0%). Data for 23α: δH (500MHz, mmol, 1.2 equiv) was added at the end. The whole mixture was
CDCl3) 1.45 (d, 3H, J 8.5 Hz), 3.79 (s, 3H), 3.72-3.83 (m, 3H), stirred at room temperature for 48 hours, after which the
4 | J. Name., 2012, 00, 1-3
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View ArticAleROTnlIinCeLE
DOI: 10.1039/C4OB02147A
reaction mixture was directly loaded on Celite. The solvent was 18.2, 26.5, 30.1, 49.3, 49.4, 52.4, 52.6, 53.4, 61.1, 61.27, 61.34,
removed as much as possible on high vacuum with gentle 61.43, 69.9, 70.2, 71.9, 76.4, 76.6, 77.0, 77.6, 77.8, 78.9, 81.8,
heating. The crude reaction was purified on reverse phase 91.1, 95.2, 173.9, 174.0, 174.9, 175.0. HRMS (ESI) calcd for
chromatography (MeOH in H2O, 0-95%). The product (1:4 C19H32N4O12 (M+Na+), 531.1914, found, 531.1909.
mixture of α/β anomers) was obtained as a white powder after
lyopholization (34 mg, 60%). Data for 27: δH (500MHz,
CDCl3) 1.44 (d, 2.4 H, J 6.5 Hz), 1.54 (d, 0.6H, J 6.5 Hz), 3.52
We thank CIHR for funding of this research. S.X. thanks CIHR
(dt, J1 5 Hz, J2 9.5 Hz), 3.76 (s, 2.4H), 3.72 (s, 0.6H), 3.85
Acknowledgements
and NSERC for graduate fellowships.
(ddd, J1 10.5 Hz, J2 10.5 Hz, J3 20.5 Hz), 4.12 (q, 1H, J 6.5
Hz), 4.18 (d, 1H, J 15.5 Hz), 4.24 (d, 1H, J 15.5 Hz), 4.29 (m,
Notes and references
2H), 4.57 (q, 1H, J 7 Hz), 4.67 (s, 0.4H), 4.78 (2, 1.6H), 5.60
(s, 1H), 5.74 (d, 0.8H, J 8.5 Hz), 5.83 (d, 0.2H, J 8.5 Hz), 7.33-
7.65 (m, 10H), 8.04 (d, 1H, J 8.5 Hz) δC (500MHz, CDCl3)
18.8, 29.7, 52.0, 54.0, 66.5, 68.3, 69.7, 73.6, 75.4, 76.0, 82.0,
101.4, 106.8, 109.7, 120.0, 124.8, 125.9, 127.97, 128.0, 128.29,
128.35, 128.36. 128.50, 128.53, 128.8, 129.0, 129.2, 129.8,
136.7, 137.2, 171.5, 173.6. HRMS (ESI) calcd for C26H31NO9
(M+Na+), 524.1897, found 524.1891.
†
Electronic Supplementary Information (ESI) available. See
DOI: 10.1039/b000000x/
1. (a) Grimes, C. L.; Ariyananda, L. D. Z.; Melnyk, J. E.; O'Shea, E. K.,
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2-N-(2-benzyloxyacetyl)-4,6-O-benzylidene-3-(O-R-(2-
propionyl-L-alanyl-D-isoglutamine
benzyl
ester)-D-
glucopyranoside (28): To a solution of 27 (13 mg, 0.0259
mmol, 1 equiv) in dry THF (1.5 mL) 1M aqueous KOH
solution (0.5 mL) and MeOH (0.5 mL) was added. The mixture
was stirred at room temperature until no more starting material
was observable on TLC. The solution was then acidified to pH
3 and quick extracted with EtOAc (3 mL x 3) and the combined
organic layer dried with Na2SO4 and concentrated in vacuo.
The crude carboxylic acid was then dissolved in dry DMF, to
which L-alanyl-D-isoglutamine benzyl ester TFA salt was
added as a solution in DMF (12 mg, 0.0285 mmol, 1.1 equiv).
DIPEA (18.2 µL, 0.104 mmol, 4 equiv) and HBTU (12 mg,
0.0311 mmol, 1.2 equiv) were added in that sequence. The
reaction was stirred at room temperature for 20 hrs. The
mixture was extracted with EtOAc (5 mL x 3) and water. The
combined organic layer was washed with brine, dried with
Na2SO4 and concentrated in vacuo. The crude mixture was
purified by reverse phase chromatography (H2O:MeCN) to give
28 (15 mg, 74%). Data for 28: δH (500MHz, CDCl3) 1.34 (d,
3H, J 7.5 Hz), 1.41 (d, 3H, J 6.5 Hz), 2.02 (m, 1H), 2.21 (m,
1H), 2.49 (dt, 1H, J1 6.5 Hz, J2 17.5 Hz), 2.62 (m, 1H), 3.51 (td,
1H, J1 4.5 Hz, J2 9.5 Hz), 3.77 (d, 1H, J 11.5 Hz), 3.83 (d, 1H,
J 11.5 Hz), 4.11-4.31 (m, 5H), 4.48 (dt, 1H, J1 4 Hz, J2 8 Hz),
4.76 (dd, 1H, J 12 Hz), 5.13 (d, 1H, J 12.5 Hz), 5.18, (d, 1H, J
12.5 Hz), 5.40 (bs, 1H), 5.59 (s, 1H), 5.71 (d, 1H, J 9 Hz), 6.70
(bs, 1H), 6.92 (d, 1H, J 7 Hz), 7.17 (d, 1H, J 8 Hz), 7.35-7.45
(m, 15H), 8.05 (d, 1H, J 8.5 Hz), 9.12 (s, 1H). HRMS (ESI)
calcd for C40H48N4O12 (M+Na+), 799.3161, found, 799.3164.
2. Miceli-Richard, C.; Lesage, S.; Rybojad, M.; Prieur, A. M.;
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Yamamura, Y., Bull. Chem. Soc. Jap. 1980, 53, 2570-2577. For a
related route to N-glycolylated Lipid II, see (b) Meng, F.-C.; Chen,
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N-glycolyl muramyl dipeptide (2): To a solution of 28 (10
mg) in MeOH (5 mL) and EtOAc (5 mL), was added small
amount of acetic acid and catalytic amount of Pd/C. The
mixture was stirred at room temperature under H2 atmosphere
for 24 hrs. The reaction mixture was filtered through Celite and
purified using reverse phase HPLC (H2O:MeOH) to give 2 as a
mixture of anomers (5.8 mg, 96%). Data for 2: δH (500MHz,
CD3OD) certain alcohol and carboxylic acid peaks are not
detected. 1.38-1.42 (2 overlapping sets of 2 doublets, 6H) 1.40
(d, J 6.5 Hz), 1.41 (d, J 6.5 Hz), 1.42 (d, J 7 Hz), 1.43(d, J 7
Hz), 1.97 (m, 1H), 2.25 (m, 1H), 2.43 (t, 1H, J 7.5 Hz), 2.42 (t,
1H, J 7.5 Hz), 3.37 (s, 1H), 3.45-3.61 (m, 2H), 3.68-3.73 (m,
2H), 3.80-3.91 (m, 2H), 3.96-4.03 (m, 2H), 4.27-4.42 (m, 3H),
4.71 (d, 0.5H, J 8.5 Hz), 5.18 (d, 0.5H, J 3.5 Hz), 7.42 (m, 1H),
8.08 (d, 1H, J 8.5 Hz). δC (500MHz, CD3OD) 16.2, 16.4, 18.1,
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This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

Organic & Biomolecular Chemistry
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ARTICLE
Organic & BiomolecVuielawrArCtihcleeOmnliisnetry
DOI: 10.1039/C4OB02147A
13. Wehlan, H.; Dauber, M.; Fernaud, M. T. M.; Schuppan, J.; Keiper,
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6 | J. Name., 2012, 00, 1-3
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