Synthesis of partially N-acetylated chitooligosaccharides and muropeptides

Article abstract of DOI:10.1055/s-0033-1340323

Partially N-acetylated chitooligosaccharides and muropeptides are referred to as microbial associated molecular patterns (MAMPs). To shed light on the molecular basis of their recognition by the innate immunity system of living organisms, their production

Full text of DOI:10.1055/s-0033-1340323

LETTER
▌365
lSetyternthesis of Partially N-Acetylated Chitooligosaccharides and Muropeptides
Synthesis of Chitooligosaccharides and Muropeptides
Emiliano Bedini,* Luigi Cirillo, Roberta Marchetti, Sara Basso, Diego Tufano, Antonio Molinaro,
Michelangelo Parrilli
Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,
80126 Napoli, Italy
Fax +39(81)674393; E-mail: ebedini@unina.it
Received: 07.10.2013; Accepted after revision: 06.11.2013
alternating β-(1→4)-linked GlcNAc and 3-O-(R)-lactyl-
Abstract: Partially N-acetylated chitooligosaccharides and muro-
GlcNAc (muramic acid, MurNAc) units. The lactyl moi-
peptides are referred to as microbial associated molecular patterns
ety of MurNAc is covalently attached to a peptide stem
(MAMPs). To shed light on the molecular basis of their recognition
by the innate immunity system of living organisms, their production
that possesses both D- and L-amino acids and is employed
in pure form is necessary. To this end, we present here the first syn- in polymer cross-linking.4 Peptidoglycan oligomeric frag-
thetic strategy for the obtainment of a series of partially N-acetylat-
ed muropeptides as well as of a chitodisaccharide and a
ments (muropeptides) generated during bacterial division
and growth are considered to also constitute MAMP.
chitotetrasaccharide, all possessing a well-defined N-acetylation
They can be detected by mammals,5 insects6 and plants,7
pattern.
thus stimulating host innate immunity response. In many
Key words: bioorganic chemistry, oligosaccharides, glycopep-
tides, glycosylation, chitosan, peptidoglycan
bacterial species the glycan strand presents some modifi-
cations (N-deacetylation, N-glycolylation, O-acetylation
or a δ-lactam group) on a certain number of GlcN, MurN
or both units along the polymeric chain.8 These structural
modifications can affect bacterial recognition by hosts.9
Among them, N-deacetylation at GlcN sites has been
demonstrated to be critical for the bacterium to evade
mammalian innate immune system.10 Human pathogenic
bacteria (i.e., Listeria monocytogenes, Helicobacter pylo-
ri) possessing a peptidoglycan with a certain amount of N-
deacetylated GlcN units can therefore hang over the gas-
trointestinal environment and disseminate to various or-
gans by surviving in human macrophages.11 Conversely,
N-deacetylation at GlcN units of a phytopathogenic bac-
terium (Xanthomonas campestris) seems to enhance the
defense responses in a plant model (Arabidopsis thali-
ana), by eliciting its innate immune system.12 The molec-
ular basis of MAMPs recognition mechanisms in plants
have not yet been elucidated in full detail.3 A greater in-
sight into this would have remarkable impact on the im-
Chitooligosaccharides are partially depolymerized prod-
ucts of chitosan, a linear polymer consisting of β-(1→4)-
linked 2-amino-2-deoxy-D-glucose (GlcN) and 2-acet-
amido-2-deoxy-D-glucose (GlcNAc) units in varying pro-
portions (Figure 1). Chitooligosaccharides conserve many
of the numerous interesting biological properties of chito-
san, including antitumour, antibacterial, antioxidant and
antiadhesive activities.1 Furthermore, they have been
found in fungi to act as potent elicitors of innate immunity
in several plant systems2 and are referred to as a microbial
associated molecular pattern (MAMP).3 The length of the
chitooligosaccharide chain, the degree and, above all, the
pattern of N-acetylation play a crucial role in determining
their biological activities.
Peptidoglycan is an essential component of the bacterial
cell wall. It possesses a polymeric glycan structure with
HO
HO
HO
HO
O
O
HO
O
HO
HO
O
O
O
O
GlcN
GlcN
HO
HO
GlcNAc
MurNAc
NH2
NH2
H3C
AcHN
AcHN
On-Pr
On-Pr
O
1
X
3: X = OH
4: X = L-Ala
5: X = L-Ala-D-Glu
HO
HO
O
HO
O
HO
O
GlcN
HO
O
HO
O
HO
GlcNAc
HO
O
NH2
GlcN
O
HO
GlcNAc
AcHN
NH2
2
AcHN
On-Pr
Figure 1 Partially N-acetylated chitooligosaccharides and muropeptides as the target of the synthesis
SYNLETT 2014, 25, 0365–0370
Advanced online publication: 16.12.2013
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0033-1340323; Art ID: ST-2013-B0944-L
© Georg Thieme Verlag Stuttgart · New York

366
E. Bedini et al.
LETTER
provement of plant disease resistance. To this end, the
obtainment of pure MAMP-related molecules with well-
defined structure is mandatory.
Ph
ref.18
O
O
O
N-acetyl-
D-glucosamine
HO
H2N
OAll
The production of single chitooligosaccharides with a
well-defined chain length and N-acetylation pattern has
been reported. The strategies for their production usually
rely upon the combination of well-tailored chemical or en-
zymatic degradation and/or N-acetylation/N-deacety-
lation reactions on chitin or chitosan, together with
extensive use of purification techniques.13 As a valuable
alternative, in the last years the chemical assembly of
GlcN and GlcNAc building blocks through multistep syn-
theses have allowed access to partially N-acetylated
9
ref.17j
O
ref.17j
BnO
PMBO
BnO
BnO
HO
CbzO
O
N3
TrocHN
OAll
O
CCl3
8
7
NH
chitodisaccharides14 and, in one case, to
a
BF3⋅OEt2 (0.2 equiv)
CH2Cl2–n-hexane (3:2), –78 °C
71%
chitotetrasaccharide15 with a well-defined N-acetylation
pattern. Similarly, several chemical syntheses of muro-
peptides have been reported.16 Among them, the synthesis
of a partially O-acetylated fragment16o as well as a muro-
peptide containing a δ-lactam moiety16a as examples of
fragments with a typical peptidoglycan structural modifi-
cation have been reported. Nonetheless, to the best of our
knowledge, no synthesis of N-deacetylated muropeptides
has been published, in spite of the interest in this molecu-
lar feature of several pathogenic bacteria. In light of our
decade-long interest in the chemical synthesis and phyto-
pathological study of MAMP-related compounds,17 we
embarked on the synthesis of a series of partially N-acet-
ylated chitooligosaccharides and muropeptides. In partic-
ular, since the N-deacetylation modification in the studied
phytopathogenic bacteria occurred exclusively at GlcN
sites, we designed the target muropeptide structures 3–5
with a common GlcN-MurNAc disaccharide unit. Analo-
gously, the target chitooligosaccharides 1 and 2 possessed
a GlcN-GlcNAc motif (Figure 1).
BnO
PMBO
BnO
O
BnO
O
O
N3
CbzO
TrocHN
OAll
6
Scheme 1 Synthesis of intermediate disaccharide 6
groups were cleaved under alkaline hydrolysis conditions
(aqueous KOH in THF), followed by chemoselective
acetylation of the free amine to give 10 (68% over two
steps) and oxazolidinone 11 as byproduct (12%), the latter
being presumably formed by cyclization of a 3-OH-2-
NHTroc intermediate (Scheme 2).21 Interestingly, the cy-
clic carbamate 11 was stable under the employed hydroly-
sis conditions and could not be converted into 10. Global
deprotection of 10 by hydrogenolysis then furnished tar-
get chitodisaccharide 122 in 66% yield.
For the synthesis of partially N-acetylated chitotetrasac-
charide 2, the selective cleavage of the orthogonal PMB
and allyl protecting groups of 6 was first accomplished to
give disaccharide acceptor 12 (81%) and hemiacetal 13
(80%), respectively, the latter then being converted into
disaccharide trihaloacetimidate donors 14 (80%) and 15
(73%). Several reactions conditions were tested for the
[2+2] glycosylation (Table 1). By employing N-phenyl
trifluoroacetimidate 14 as disaccharide donor, no glyco-
sylation conditions (activator amount, temperature, time)
could be found to afford fully protected tetrasaccharide 16
in good yields without concurrent cleavage of the PMB
ether giving tetrasaccharide alcohol 17 (entries 1–4).
However, trichloroacetimidate 15 could be activated un-
der milder conditions (entry 5) with respect to 14, thus en-
abling the obtainment of 1623 in good yield (72%).
Compound 16 was subjected to Cbz and Troc protecting
group cleavage followed by N-acetylation to furnish 18 in
moderate yield (44%), due to the formation of 2,3-oxa-
zolidinone byproducts. Compound 18 was then deprotect-
ed by hydrogenolysis to afford tetrasaccharide target 224
in 55% yield.
All the known syntheses of peptidoglycan fragments ne-
cessitate the introduction of the acetamido moiety on
GlcN and MurN units before their coupling with the pep-
tide chain.16 Therefore, a different, new strategy should be
developed for the synthesis of partially N-acetylated mu-
ropeptides. The reported syntheses of partially N-acetylat-
ed chitooligosaccharides14,15 could not be exploited
because they do not allow the selective insertion of an (R)-
lactyl group at position O-3 of one or more GlcN units to
convert them into MurN derivatives.
To have access to the designed targets, nitrogen atoms had
to be protected with orthogonal protecting groups. Com-
pound 6 (Scheme 1) was designed as a suitable disaccha-
ride building block, possessing orthogonally cleavable
protecting groups not only on nitrogen atoms but also at
position O-1I and O-4II to allow oligomerization, as well
as at O-3I to ensure the regioselective insertion of the (R)-
lactyl moiety. Disaccharide 6 could be obtained by stereo-
selective coupling between suitably protected GlcN ac-
ceptor 717j and donor 8 under SN2-type conditions.18,19
For the synthesis of partially N-acetylated muropeptides
3, 4 and 5, an (R)-lactyl moiety was first introduced on al-
cohol 10 by reaction with triflyl (S)-2-propionic acid ethyl
ester16k and NaH to afford 19 (61%), which was, in turn,
The synthesis of the first target, partially N-acetylated chi-
todisaccharide 1, was accomplished in three steps from
6.20 Firstly, Troc carbamate and Cbz carbonate protecting
Synlett 2014, 25, 365–370
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Chitooligosaccharides and Muropeptides
367
i. 2 M aq KOH, THF, 60 °C
ii. CH2Cl2–MeOH–Ac2O (10:10:1)
6
PdCl2, CH2Cl2–MeOH (1:1)
77%, α/β = 20:1
BnO
PMBO
BnO
O
BnO
O
O
BnO
O
R2O
BnO
O
N3
PMBO
O
R1HN
BnO
OAll
10: R1 = Ac, R2 = H, 68% (2 steps)
11: R1 = R2 = CO, 12% (2 steps)
H2, Pd(OH)2/C
CH2Cl2–MeOH–AcOH–H2O
(1:2:1:1)
CbzO
N3
66%
OR1
TrocHN
13: R1 = H
14: R1 = C(NPh)CF3
15: R1 = α-C(NH)CCl3
CF3C(NPh)Cl
Cs2CO3, acetone
80%, α/β = 1:1
TFA, CH2Cl2
81%
1
Cl3CCN, DBU
CH2Cl2
73%
BnO
HO
BnO
O
BnO
O
O
CbzO
N3
TrocHN
see Table 1
OAll
12
BnO
R3O
BnO
O
BnO
O
O
BnO
O
BnO
O
BnO
O
R2O
N3
O
R1HN
R2O
N3
i. 2 M aq KOH, THF, 60 °C
ii. CH2Cl2–MeOH–Ac2O (10:10:1)
44% (2 steps)
16: R1 = Troc, R2 = Cbz, R3 = PMB
17: R1 = Troc, R2 = Cbz, R3 = H
18: R1 = Ac, R2 = H, R3 = PMB
H2, Pd(OH)2/C
R1HN
OAll
CH2Cl2–MeOH–AcOH–H2O (1:5:2:1)
55%
2
Scheme 2 Synthesis of chitooligosaccharides 1 and 2
treated with LiOH to liberate the carboxylic acid moiety muropeptide 2728 was obtained in good yield (79%). Ester
and give 20 (82%; Scheme 3). Hydrogenolysis of 20 fur- hydrolysis with LiOH (28; 95%) and global deprotection
nished target compound 3 (60%). Elongation of the lactyl by hydrogenolysis furnished target 5 (60%).
appendage with an L-alanine unit was accomplished by
In conclusion, five partially N-acetylated chitooligosac-
charides and muropeptides have been obtained by exploit-
ing suitably orthogonally protected disaccharide 6 as a
key building block for the synthesis of all the target mol-
ecules. Even more complex structures could be obtained
in the future by further sugar and/or peptide chain elonga-
coupling of 20 and L-alanyl methyl ester25 under activa-
tion with HOBt, pyBOP and DIPEA to give 21 (92%),
which was, in turn, treated with LiOH (22; 85%) and fully
deprotected by hydrogenolysis to complete the synthesis
of target 426 (49%).
The elongation of the lactyl appendage with an L-Ala-D- tion of the protected intermediate herein described. Work
Glu dipeptide was performed by HOBt/pyBOP/DIPEA is in progress towards this end, as well as to shed light on
activation of 20 in the presence of dipeptide 23, which the molecular basis of chitooligosaccharide and muropep-
was obtained, in turn, by coupling of commercially avail- tide recognition in plants by testing the already obtained
able L-alanine derivative 24 with D-glutamic acid diester molecules on a plant model (Arabidopsis thaliana). The
2527 followed by Boc carbamate removal. Fully protected results will be reported in due course.
Table 1 [2+2] Glycosylation Reaction
Entrya
Donor
Catalyst (equiv)b
T (°C)
Time (h)
16 (%)c
17 (%)c
1
2
3
4
5
14
14
14
14
15
TMSOTf (0.2)
TMSOTf (0.025)
TMSOTf (0.075)
Bi(OTf)3e (0.1)
TMSOTf (0.04)
–20
4
trace
80
–
–50 to 5
–50
overnight
–
4
37 (85)d
6
–10 to r.t.
–30
overnight
1
–
–
72
–
a Reactions conducted with acceptor 12 in CH2Cl2 in the presence of freshly activated AW-300 4 Å MS.
b Molar equivalents calculated with respect to the glycosyl donor.
c Isolated yield.
d Yield calculated on reacted acceptor.
e See ref. 29 for the activation of glycosyl trihaloacetimidates with catalytic Bi(OTf)3.
© Georg Thieme Verlag Stuttgart · New York
Synlett 2014, 25, 365–370

368
E. Bedini et al.
LETTER
O
O
10
O
O
HOBt, pyBOP
DIPEA, DMF
OH
MeO
OBn
+
MeO
OBn
NH2
BocHN
triflyl (S)-2-propionic acid ethyl ester
61%
NH
O
70%
NaH, CH2Cl2
24
25
X
O
BnO
O
H2, Pd(OH)2/C
BnO
PMBO
O
TFA, CH2Cl2
99%
26: X = NHBoc
O
CH2Cl2–MeOH–
AcOH–H2O
(1:3:2:2)
BnO
23: X = NH3+CF3COO–
N3
AcHN
O
OAll
3
O
60%
RO
HOBt, pyBOP
DIPEA, DMF
LiOH
19: R = Et
20: R = H
THF–1,4-dioxane–H2O (4:2:1)
82%
79%
L-alanyl methyl ester
HOBt, pyBOP, DIPEA, DMF
BnO
PMBO
BnO
92%
O
BnO
O
O
O
N3
AcHN
H2, Pd(OH)2/C
CH2Cl2–MeOH–
AcOH–H2O
OAll
O
HN
BnO
PMBO
BnO
O
BnO
O
H2, Pd(OH)2/C
CH2Cl2–MeOH–
AcOH–H2O
O
(1:3:2:2)
O
4
N3
NH
AcHN
O
OAll
49%
OR'
RO
(1:3:2:2)
5
O
O
O
60%
HN
O
LiOH
27: R = Bn, R' = Me
28: R = R' = H
OR
THF–1,4-dioxane–H2O (4:2:1)
95%
LiOH
21: R = Me
22: R = H
THF–1,4-dioxane–H2O (4:2:1)
85%
Scheme 3 Synthesis of muropeptides 3, 4 and 5
(7) Gust, A. A.; Biswas, R.; Lenz, H. D.; Rauhut, T.; Ranf, S.;
Kemmerling, B.; Götz, F.; Glawischnig, E.; Lee, J.; Felix,
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Hugot, J.-P.; Giovannini, M.; Coyle, A.; Bertin, J.; Namane,
A.; Rousselle, J.-C.; Cayet, N.; Prévost, M.-C.; Balloy, V.;
Chignard, M.; Philpott, D. J.; Cossart, P.; Girardin, S. E.
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Acknowledgment
NMR and MS facilities of CIMCF (Centro Interdipartimentale di
Metodologie Chimiche Fisiche) of Università degli Studi di Napoli
‘Federico II’ are acknowledged.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnoIufproig
m
iotSrat
ungIifoop
r
t
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Synlett 2014, 25, 365–370
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Chitooligosaccharides and Muropeptides
369
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(17) (a) Bedini, E.; Parrilli, M.; Unverzagt, C. Tetrahedron Lett.
2002, 43, 8879. (b) Bedini, E.; Barone, G.; Unverzagt, C.;
Parrilli, M. Carbohydr. Res. 2004, 339, 393. (c) Bedini, E.;
Carabellese, A.; Corsaro, M. M.; De Castro, C.; Parrilli, M.
Carbohydr. Res. 2004, 339, 1907. (d) Bedini, E.; De Castro,
C.; Erbs, G.; Mangoni, L.; Dow, J. M.; Newman, M.-A.;
Parrilli, M.; Unverzagt, C. J. Am. Chem. Soc. 2005, 127,
2414. (e) Bedini, E.; Carabellese, A.; Barone, G.; Parrilli, M.
J. Org. Chem. 2005, 70, 8064. (f) Bedini, E.; Carabellese,
A.; Schiattarella, M.; Parrilli, M. Tetrahedron 2005, 61,
5439. (g) Bedini, E.; Esposito, D.; Parrilli, M. Synlett 2006,
825. (h) Bedini, E.; Carabellese, A.; Comegna, D.; De
Castro, C.; Parrilli, M. Tetrahedron 2006, 62, 8474.
(i) Comegna, D.; Bedini, E.; Parrilli, M. Tetrahedron 2008,
64, 3381. (j) Cirillo, L.; Bedini, E.; Molinaro, A.; Parrilli, M.
Tetrahedron Lett. 2010, 51, 1117.
concentrated. The residue was subjected to column
chromatography (14:1 to 8:1 v/v toluene–EtOAc) to afford
16 (91.7 mg, 72%) as a white foam: [α]D22 –20.6 (c = 1.0,
CH2Cl2); 1H NMR (600 MHz, CDCl3): δ = 7.37–7.25 (m,
40 H, H-Ar), 7.07 (d, J = 8.6 Hz, 2 H), 6.80 (d, J = 8.6 Hz,
2 H), 5.87 (m, 1 H), 5.31–5.27 (m, 2 H), 5.22 (dd, J = 10.4,
1.2 Hz, 1 H), 5.16 (d, J = 12.5 Hz, 1 H), 5.11–4.98 (m, 5 H),
4.93 (d, J = 3.6 Hz, 1 H), 4.82 (d, J = 10.9 Hz, 1 H), 4.78 (d,
J = 10.9 Hz, 1 H), 4.70–4.57 (m, 8 H), 4.51–4.43 (m, 5 H),
4.37 (d, J = 9.4 Hz, 1 H), 4.34 (d, J = 12.2 Hz, 1 H), 4.30–
4.23 (m, 3 H), 4.19 (dd, J = 14.1, 6.6 Hz, 1 H), 4.17 (d, J =
9.4 Hz, 1 H), 4.06 (dt, J = 10.3, 3.7 Hz, 1 H), 4.01–3.93 (m,
5 H), 3.87–3.80 (m, 2 H), 3.79 (s, 3 H), 3.72–3.60 (m, 5 H),
3.54 (d, J = 8.5 Hz, 1 H), 3.44–3.39 (m, 3 H), 3.29 (d, J =
7.9 Hz, 1 H), 3.25–3.08 (m, 5 H), 2.96 (d, J = 9.7 Hz, 1 H);
13C NMR (50 MHz, CDCl3): δ = 159.3, 155.2, 154.9, 154.1,
153.9, 138.6, 138.4, 138.0, 137.9, 137.7, 137.3, 135.4,
133.1, 130.1, 129.4-127.5, 118.4, 113.8, 100.9, 100.8, 100.3,
96.3, 95.6, 95.4, 83.1, 81.2, 77.6, 77.3, 75.6, 75.3, 75.0, 74.8,
74.4, 73.5, 73.1, 70.3, 69.6, 68.7, 68.6, 67.9, 67.7, 67.6, 66.4,
66.0, 56.5, 55.2, 54.2; MS (MALDI TOF): m/z = 2053.40 [M
+ Na]+ .
(18) Cai, J.; Davison, B. E.; Robin Ganellin, C.; Thaisrivongs, S.;
Wibley, K. S. Carbohydr. Res. 1997, 300, 109.
(19) (a) Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem. 1985,
1537. (b) Wang, L. X.; Li, C.; Wang, Q.-C.; Hui, Y.-Z.
Tetrahedron Lett. 1993, 34, 7763. (c) Yang, Y.; Li, Y.; Yu,
B. J. Am. Chem. Soc. 2009, 131, 12076.
(24) A solution of 18 (16.3 mg, 10.9 μmol) in CH2Cl2 (400 μL)
was diluted with MeOH (2.0 mL), AcOH (800 μL) and H2O
(400 μL), and the resulting monophasic, clear solution was
treated with Pearlman’s catalyst (16.0 mg, 22.8 μmol). The
mixture was stirred under a H2 atmosphere at r.t. overnight,
then filtered on a Celite pad. Volatiles were removed by
rotoevaporation. The residue was freeze-dried to give a
white powder that was purified by HPLC (0.05% v/v TFA–
H2O to 0.05% v/v TFA–MeOH). Pure 2 (4.7 mg, 55%) was
obtained as a white powder: [α]D22 +2.3 (c = 0.4, H2O); 1H
NMR (600 MHz, D2O): δ = 4.88 (d, J = 4.6 Hz, 1 H), 4.82
(d, J = 8.3 Hz, 2 H), 4.58 (d, J = 7.8 Hz, 1 H), 3.94–3.84 (m,
10 H), 3.79–3.75 (m, 5 H), 3.70–3.64 (m, 5 H), 3.57 (m,
2 H), 3.48 (m, 2 H), 3.16 (t, J = 8.3 Hz, 1 H), 3.14 (t, J =
8.3 Hz, 1 H), 2.06 (s, 3 H), 2.04 (s, 3 H), 1.60 (sext., J =
7.0 Hz, 2 H), 0.91 (t, J = 7.0 Hz, 3 H); 13C NMR (150 MHz,
D2O): δ = 175.4, 175.1, 102.2, 99.0, 97.4, 79.3, 77.7, 77.1,
75.8, 75.1, 73.1, 72.1, 70.9, 70.4, 69.8, 61.1, 61.0, 60.4, 56.5,
(20) See the Supporting Information for experimental procedures
and characterization data of all the new products.
(21) (a) Williams, A. L.; Abad Grillo, T.; Comins, D. L. J. Org.
Chem. 2002, 67, 1972. (b) Cipolla, L.; Redaelli, C.; Nicotra,
F. Lett. Drug Design Discovery 2005, 2, 291. (c) Cipolla, L.;
Reis Fernandes, M.; Gregori, M.; Airoldi, C.; Nicotra, F.
Carbohydr. Res. 2007, 342, 1813. (d) Chen, G.-H.; Pan, P.;
Chen, Y.; Meng, X.-B.; Li, Z.-J. Tetrahedron 2009, 65,
5922. (e) Cirillo, L.; Silipo, A.; Bedini, E.; Parrilli, M. Eur.
J. Org. Chem. 2010, 4062. (f) Pragani, R.; Seeberger, P. H.
J. Am. Chem. Soc. 2011, 133, 102. (g) Bedini, E.; Cirillo, L.;
Parrilli, M. Tetrahedron 2013, 69, 1285.
© Georg Thieme Verlag Stuttgart · New York
Synlett 2014, 25, 365–370

370
E. Bedini et al.
LETTER
56.2, 54.5, 22.9, 22.7, 22.6, 10.6; MS (MALDI TOF): m/z =
HOBt in DMF (250 μL) and finally with DIPEA (65 μL, 280
μmol). After 3 h stirring at r.t., the reaction mixture was
diluted with CH2Cl2 (50 mL) and washed with 0.1 M HCl
(50 mL). The organic phase was collected, dried over
anhydrous Na2SO4, filtered, and concentrated. After column
chromatography (3:1 to 1:3 v/v n-hexane–EtOAc), pure 27
(32.4 mg, 79%) was obtained as a colorless oil: [α]D22 +13.7
(c = 2.0, CH2Cl2); 1H NMR (600 MHz, CDCl3): δ = 7.33–
7.26 (m, 20 H), 7.02 (d, J = 8.6 Hz, 2 H), 6.79 (d, J = 8.6 Hz,
2 H), 5.82 (m, 1 H), 5.23 (dd, J = 17.2, 1.5 Hz, 1 H), 5.16
(dd, J = 10.4, 1.5 Hz, 1 H), 5.11 (d, J = 12.3 Hz, 1 H), 5.08
(d, J = 12.3 Hz, 1 H), 5.07 (d, J = 3.9 Hz, 1 H), 4.80 (s, 2 H),
4.69 (d, J = 12.0 Hz, 1 H), 4.65 (d, J = 10.5 Hz, 1 H), 4.57
(dq, J = 7.9, 5.0 Hz, 1 H), 4.51 (d, J = 11.8 Hz, 1 H), 4.46–
4.41 (m, 4 H), 4.36 (app. quint., J = 7.0 Hz, 1 H), 4.21 (d,
J = 8.2 Hz, 1 H), 4.08 (dd, J = 13.0, 5.2 Hz, 1 H), 4.03–3.93
(m, 4 H), 3.76 (s, 3 H), 3.74 (d, J = 9.9 Hz, 1 H), 3.70–3.56
(m, 8 H), 3.29 (dd, J = 9.8, 8.3 Hz, 1 H), 3.22 (t, J = 9.0 Hz,
1 H), 3.19 (m, 1 H), 2.38–2.29 (m, 2 H), 2.23–2.15 (m, 1 H),
2.05–1.98 (m, 1 H), 1.98 (s, 3 H), 1.35 (d, J = 7.4 Hz, 3 H),
1.33 (d, J = 7.5 Hz, 3 H); 13C NMR (100 MHz, CDCl3): δ =
174.7, 173.3, 172.1, 171.5, 171.4, 159.2, 137.9, 137.7,
137.5, 137.4, 133.7, 130.0, 128.7-127.6, 117.6, 113.9, 100.8,
96.1, 83.2, 76.7, 76.4, 75.3, 74.4, 74.3, 73.4, 73.1, 71.8, 70.6,
68.5, 68.3, 67.8, 67.3, 66.4, 55.2, 53.9, 53.8, 51.9, 51.7, 49.2,
29.8, 26.8, 23.1, 19.3, 17.8; MS (MALDI TOF): m/z =
1238.19 [M + Na]+ .
789.13 [M + H]+ .
(25) Li, J.; Sha, Y. Molecules 2008, 13, 1111.
(26) A solution of 22 (26.6 mg, 27.1 μmol) in CH2Cl2 (300 μL)
was diluted with MeOH (900 μL), AcOH (600 μL) and H2O
(600 μL), and the resulting monophasic, clear solution was
treated with Pearlman’s catalyst (26.6 mg, 37.9 μmol). The
mixture was stirred under a H2 atmosphere at r.t. overnight,
then filtered on a Celite pad. Volatiles were removed by
rotoevaporation and the residue was freeze-dried to give a
yellowish powder that was purified by HPLC (0.05% v/v
TFA–H2O to 0.05% v/v TFA–MeOH). Pure 4 (7.5 mg, 53%)
was obtained as a white powder: [α]D22 +44 (c = 0.2, H2O);
1H NMR (600 MHz, D2O): δ = 4.93 (d, J = 3.6 Hz, 1 H), 4.82
(d, J = 8.2 Hz, 1 H), 4.40 (q, J = 7.1 Hz, 1 H), 4.37 (q, J =
8.2 Hz, 1 H), 4.06 (t, J = 9.3 Hz, 1 H), 3.96 (m, 2 H), 3.87
(m, 2 H), 3.82 (t, J = 10.2 Hz, 1 H), 3.76 (m, 2 H), 3.69 (t,
J = 9.6 Hz, 1 H), 3.64 (m, 1 H), 3.51 (m, 1 H), 3.44 (m, 2 H),
3.14 (dd, J = 9.6, 8.2 Hz, 1 H), 1.99 (s, 3 H), 1.59 (sext, J =
7.4 Hz, 2 H), 1.45 (d, J = 8.2 Hz, 3 H), 1.38 (d, J = 7.1 Hz,
3 H), 0.90 (t, J = 7.4 Hz, 3 H); 13C NMR (150 MHz, D2O):
δ = 177.0, 175.7, 174.5, 98.1, 97.4, 78.5, 77.4, 77.3, 75.1,
72.6, 71.3, 70.4, 61.7, 60.9, 57.2, 53.9, 49.2, 22.8, 22.5, 19.1,
17.3, 10.4; MS (MALDI TOF): m/z = 568.16 [M + H]+ .
(27) Kelly, R. C.; Gebhard, I.; Wicnienski, N. J. Org. Chem.
1986, 51, 4590.
(28) A solution of 20 (30.9 mg, 33.9 μmol) and 23 (133 mg, 305
μmol) in DMF (1.25 mL) was treated with a 0.27 M solution
of PyBOP in DMF (250 μL), then with a 0.27 M solution of
(29) Adinolfi, M.; Iadonisi, A.; Ravidà, A.; Valerio, S.
Tetrahedron Lett. 2006, 47, 2595.
Synlett 2014, 25, 365–370
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