An efficient method for the synthesis of some chlorinated and heteroatom rich triazole-linked β-lactam glycoconjugates

Article abstract of DOI:10.1016/j.tet.2016.10.036

The synthetic utility of chlorinated bicyclic C-fused tetrahydrofuro[3,2-c] azetidin-2-ones synthesized in our laboratory by Cu(I)-catalyzed halogen atom transfer radical cyclization (ATRC) has been illustrated through the synthesis of some novel β-lactam glycoconjugates. The chlorine atom of the chloromethyl side chain of the bicyclic C-fused tetrahydrofuro[3,2-c] azetidin-2-ones was subjected to nucleophilic substitution with azide group followed by Cu(I)-catalyzed azide-alkyne click reaction (CuAAC) with propargyl glycosides to generate the desirous β-lactam glycoconjugates. A sequential optimization of the reaction procedure for CuAAC was carried out to obtain an efficient catalyst system to achieve β-lactam glycoconjugates in good yields. The β-lactam glycoconjugates are the compounds of interesting architecture and structurally suitable for bioactivity evaluation. Therefore, these β-lactam glycoconjugates were screened for in vitro antibacterial activity. Additionally, a representative β-lactam glycoconjugate was also tested for biocompatibility and cytotoxic activity against L929 cancer cell lines.

Full text of DOI:10.1016/j.tet.2016.10.036

Accepted Manuscript
An efficient method for the synthesis of some chlorinated and heteroatom rich
triazole-linked β-lactam glycoconjugates
Nisha Dawra, Ram N. Ram
PII:
S0040-4020(16)31070-5
10.1016/j.tet.2016.10.036
TET 28177
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 5 June 2016
Revised Date: 12 October 2016
Accepted Date: 14 October 2016
Please cite this article as: Dawra N, Ram RN, An efficient method for the synthesis of some chlorinated
and heteroatom rich triazole-linked β-lactam glycoconjugates, Tetrahedron (2016), doi: 10.1016/
j.tet.2016.10.036.
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ACCEPTED MANUSCRIPT
OAc
N
N3
Cl
R5
R4
N
O
N
OAc
O
O
N
R1
O
Cl
R5
R4
CuCl/PMDETA
O
R3
OR6
+
N
(1:1, 50 mol%)
R2
O
R2
R3
R1
O
R1 = Ph; R2 = Ph, p-MeOC6H4
R3 = OAc, H (ax/eq); R4 = OAc (ax/eq); R3- R4 = Π-bond
R5 = OAc (ax/eq); R6 = H2C
(ax/eq)
ax : axial; eq : equatorial

ACCEPTED MANUSCRIPT
An efficient method for the synthesis of some chlorinated and heteroatom rich
triazole-linked β-lactam glycoconjugates
Nisha Dawraa,b* and Ram N. Rama
aDepartment of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016,
India
bSchool of Engineering and Technology, BML Munjal University, Gurgaon, Haryana, 122413
Abstract: The synthetic utility of chlorinated bicyclic C-fused tetrahydrofuro[3,2-c] azetidin-2-
ones synthesized in our laboratory by Cu(I)-catalyzed halogen atom transfer radical cyclization
(ATRC) has been illustrated through the synthesis of some novel β-lactam glycoconjugates. The
chlorine atom of the chloromethyl side chain of the bicyclic C-fused tetrahydrofuro[3,2-c]
azetidin-2-ones was subjected to nucleophilic substitution with azide group followed by Cu(I)-
catalyzed azide-alkyne click reaction (CuAAC) with propargyl glycosides to generate the
desirous β-lactam glycoconjugates. A sequential optimization of the reaction procedure for
CuAAC was carried out to obtain an efficient catalyst system to achieve β-lactam
glycoconjugates in good yields. The β-lactam glycoconjugates are the compounds of interesting
architecture and structurally suitable for bioactivity evaluation. Therefore, these β-lactam
glycoconjugates were screened for in vitro antibacterial activity. Additionally, a representative β-
lactam glycoconjugate was also tested for biocompatibility and cytotoxic activity against L929
cancer cell lines.
1. Introduction
Heteroatom rich organic compounds particularly containing N, O and Cl atoms are of
immense importance both in chemistry and biology. The majority of the natural products, drugs
and biologically active compounds are rich in heteroatoms. β-Lactams (azetidin-2-ones) occupy
1

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an important place in chemistry and medicine.1 The appeal of working with β-lactams stems
from their ability to exhibit a diverse range of biological activities1a,2-4 and interesting
pharmacological activities.1a,4,5 Furthermore, β-lactams are the useful kits in the tool-box of
synthetic organic chemists for the synthesis of a variety of heterocyclic compounds of biological
interest.6 A chlorine substituent at the α-position of the β-lactam ring is well known to enhance
the chemical reactivity and it also offers a great diversity regarding its biological activity.7
Conjugation of β-lactams has been emerging as a new strategy in drug discovery for
increasing the effectiveness of β-lactam antibiotics and modification of their pharmacological
potential.8 Additionally, it provides an opportunity to design and synthesize diverse molecular
architectures through modification of one or both the components.8 Cu(I)-catalyzed azide-alkyne
click reaction (CuAAC) has been widely used as the most powerful technique nowadays for the
conjugation of β-lactams with some other bioactive molecules in pursuit of more potent
bioactivity.9 In addition to conjugation, mere the covalent attachment of a triazole ring to the β-
lactam scaffold has been reported to make a significant enhancement in the biological properties
of β-lactams.10 Our laboratory has reported the synthesis of a triazolyl-β-lactam through CuAAC
of an azido C-fused bicylic β-lactam with phenylacetylene.11
Sugars are the most important and most diverse class of biomolecules. Glycoconjugates
are the promising scaffolds in drug discovery and development as they are involved in crucial
life governing processes such as molecular recognition, inflammation, immunological response,
pathogen defense, tumorigenesis and metastasis.12 Furthermore, triazolyl glycoconjugates
synthesized by CuAAC exhibit a broad spectrum of potential synthetic, pharmaceutical and
biological applications.13
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To the best of our knowledge, not many reports have appeared in the literature that
reveals the presence of a sugar unit and a β-lactam scaffold in a single molecule.14 However,
some of the reported β-lactam sugar derivatives exhibit interesting biological and medicinal
properties.14 Furthermore, the β-lactam glycoconjugates are rarely reported in the literature.
Curiously, the glycoconjugation of β-lactams through CuAAC which would bring together all
the three medicinally significant components, the β-lactam, sugar and 1,2,3-triazole, together is
scarcely reported in the literature. In fact we could locate only one such report in which the
triazolyl-β-lactam glycoconjugate synthesized by CuAAC, acted as lectin inhibitor.14d
Enlightened by such innovative and promising work and in continuation of our interest to
use the bicyclic azido β-lactam synthesized previously in our laboratory as a substrate, we
envisioned to synthesize a series of chlorinated and heteroatom rich β-lactam glycoconjugates
via click chemistry and explore their biological competences.
2. Results and Discussion
To begin with, the synthesis of azidotetrahydrofurano fused β-lactams 2 (Scheme 1) was
carried out by the bimolecular substitution of chlorine atom of the chloromethyl side chain of
chlorinated C-fused bicyclic β-lactams 1 with azido nucleophile as demonstrated earlier by this
laboratory.11
Cl
Cl
N3
N3
Cl
Cl
Cl
Cl
O
O
NaN3 (5 equiv)
O
O
+
+
DMF, 65 °C, 12 h
N
N
N
N
O
R2
R2
R2
O
O
R2
O
R1
R1
R1
R1
1a,b
2a,b
(69, 65%)
a: R1 = Ph, R2 = Ph; b: R1 = Ph, R2 = p-MeOC6H4
Scheme 1: Synthesis of azidotetrahydrofurano fused β-lactams 2
3

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The acetylenic components needed for the glycoconjugation of azido β-lactams 2 via
click reaction were easily prepared diastereoselectively from the appropriate sugar derivatives
through glycosylation or Ferrier reaction with propargyl alcohol according to the reported
literature procedures.15 Thus, five different propargyl glycosides 3 (Figure 1) including both the
saturated and unsaturated and the α as well as β-anomeric propargylic sugars were employed to
prepare β-lactam glycoconjugates.
OAc
O
AcO
AcO
OAc
OAc
O
O
OAc
O
O
AcO
AcO
AcO
AcO
AcO
AcO
O
3a
β-propargyl glucoside
3b
3c
β-propargyl galactoside
α−propargyl mannoside
OAc
OAc
O
OAc
O
AcO
AcO
O
O
3e
α-2-deoxygalactoside
3d
α-∆3,4-glycoside
Figure 1. Different propargyl glycosides employed for glycoconjugation of azido β-lactam 2
2.1. Optimization of the reaction procedure for preparation of β-lactam glycoconjugate by
CuAAC:
Next, the crucial Cu(I)-catalyzed azide-alkyne click reaction of the azidotetrahydrofurano
fused bicyclic β-lactams with the sugar based alkynes was investigated. The reaction conditions
for the glycoconjugation of the β-lactam through CuAAC were optimized by exploring the
reaction of azido-β-lactam 2a (Scheme 2) with the propargyl glucoside 3a (Table 1).
4

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N3
Cl
N3
OAc
O
O
Cl
AcO
AcO
O
O
+
+
AcO
N
N
O
O
Ph
Ph
Ph
Ph
3a
2a
(racemic mixture)
Cu(I)
OAc
N
OAc
N
N
O
AcO
AcO
N
AcO
AcO
N
O
O
O
N
Cl
O
N
O
Cl
+
AcO
AcO
N
Ph
Ph
O
Ph
O
Ph
4aa
(1:1 diastereomeric mixture)
Scheme 2: Optimization of reaction procedure for preparation of β-lactam glycoconjugate
4aa
Unfortunately, the reaction failed under the reaction conditions previously used in our laboratory
for the synthesis of a triazolyl-β-lactam11 (entry 1, Table 1). This might be ascribed to the poor
solubility of the reactants in solvent system used and the insufficient catalyst loading. We then
tended to optimize the reaction condition by changing the solvent and increasing the catalyst
loading. To our delight, the partial formation of the β-lactam glycoconjugate 4aa was observed
in CH2Cl2/H2O (1:1 v/v) system using CuSO4.5H2O (2 mol equiv) and Na-Asc (4 mol equiv) at
1
room temperature (entry 2, Table 1), as evidenced by H NMR analysis of the crude reaction
mixture. Presumably, the incomplete conversion of 2a to 4aa despite of using excess of catalyst
and longer reaction times might be due to the deactivation of Cu(I) ions to Cu(0) particles and
Cu(II) ions. This was evidenced by the change in the color of the reaction mixture from the
original light yellow color to dark brown and the presence of some suspended brownish particles
in the reaction mixture.
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Table 1. Optimization of reaction procedure for glycoconjugation of β-lactam 2aa
Yield (%)b
Entry Azide Alkyne
Reaction Conditions
Time
(h)
β
-Lactam
Recovered
substrates
2a/3a
glycoconjugate
4aa
1.
2.
3.
CuSO4.5H2O (10 mol%)/
Sodium ascorbate
24
12
1
-
76/75
42/42
-
2a
2a
2a
3a
3a
3a
(Na-Asc) (20 mol%),
t-BuOH/H2O (5:1), 65 ˚C
CuSO4.5H2O (2 mol
equiv)/Na-Asc (4 mol
equiv), CH2Cl2/H2O (1:1),
rt (25-30 ˚C)
CuSO4.5H2O (1 mol
equiv)/ N,N'N'N"N"-
pentamethyldiethylene
triamine (PMDETA)
(1 mol equiv)/Na-Asc (2
mol equiv), CH2Cl2/H2O
(1:1), rt (25-30 ˚C)
CuI (0.5 mol equiv)/
N,N-diisopropyl
35
64
4.
5
-
74/72
2a
3a
ethylamine (DIPEA) (0.75
mol equiv), CH3CN,
N2 atm, rt (25-30 ˚C)
CuCl (60 mol%)/ bipyridyl
(bpy) (60 mol%), 1,2-
dichloroethane (DCE),
N2 atm, reflux
5.
6.
12
12
-
-
79/72
78/73
2a
2a
3a
3a
CuCl (20 mol%)/
N,N,N',N'-tetramethyl
ethylenediamine
(TMEDA) (40 mol%),
DCE, N2 atm, reflux
CuCl (50 mol%)/
PMDETA (50 mol%),
DCE, N2 atm, rt
7.
8.
1
78
-
2a
2a
3a
3a
(25-30 ˚C)
CuCl (30 mol%)/
24
37
43/45
PMDETA (30 mol%),
DCE, N2 atm, rt
(25-30 ˚C)
b
aAll the reactions were performed by taking 2a (1 mmol) and 3a (1 mmol). Isolated yield after purification by
column chromatography.
The literature survey has shown that the nitrogen containing ligands have been the first
and the most popular ligands for CuAAC and their affinity for copper is well-known.16
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Alternatively, such additives increase the solubility of copper(I) species in the reaction media
and also stabilize the copper(I) active species even in reactions carried out in water. Arguably, it
was envisaged to use some nitrogen based ligand in the reaction medium of CuAAC to ascertain
completion of the reaction. A triamine ligand, N,N'N'N"N"-pentamethyldiethylenetriamine
(PMDETA) is probably the most commonly employed ligand in CuAAC, due to the well-
established efficiency of CuCl/PMDETA in ATRC as previously demonstrated in our
laboratory.11
Therefore, employing the use of CuSO4.5H2O/PMDETA/Na-Asc (1:1:2 mol equiv), in
CH2Cl2/H2O (1:1 v/v) as the solvent system at room temperature (25-30 ˚C), delightfully the
reaction got completed in 1 h and resulted in isolation of the pure β-lactam glycoconjugate 4aa in
64% yield (entry 3, Table 1). However, the requirement of high catalyst loading in the above
biphasic reaction media seemed unsatisfactory, and therefore this method was not preceded
subsequently.
Consequently, it was thought to perform the click reaction in monophasic non-aqueous
conditions using the pre-formed Cu(I) salt in the presence of a nitrogen based ligand. The
screening of Cu(I)-salt and ligand showed that only CuCl/PMDETA catalyst system was found
to be effective for the aforesaid click reaction. Gratifyingly, by using CuCl/PMDETA (1:1 mol
ratio, 50 mol%) in DCE at room temperature (25-30 ˚C) (entry 7, Table 1), the remarkable
increase in the product yield was observed as the desired β-lactam glycoconjugate 4aa was
obtained in 78% isolated yield. Thus, CuCl/PMDETA (1:1 mol ratio, 50 mol%), was found to be
the most efficient catalyst system for the synthesis of β-lactam glycoconjugates in terms of
reasonable improvement in product yield and an appreciable decrease in the amount of catalyst
required for the completion of glycoconjugation reaction.
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The click reaction proceeded smoothly with complete retention of the anomeric
configuration and was highly regioselective as only 1,4-disubstituted-1,2,3-triazole was isolated.
No 1,5-disubstituted-1,2,3-triazole could be detected. However, the reaction furnished a mixture
of two diastereomers in 1:1 ratio because the azido β-lactam 2a was racemic.
In the 1H NMR spectrum of the compound 4aa, the two most downfield singlets at δ 7.80
and 7.78 ppm, integrating together for one proton were assigned to the methine proton of the
triazole ring of the two diastereomers with equal contribution to the total signal area from each of
the diastereomer indicating that the two diastereomers were formed in 1:1 ratio. Other signals
due to the diastereomeric protons were not well separated. Therefore, COSY NMR spectrum was
used to help the assignments of the remaining protons. In the proton-decoupled 13C NMR
spectrum of 4aa, few signals appeared as twins, further confirming the presence of two
diastereomers of the β-lactam glycoconjugate 4aa. The presence of a requisite number of
13
carbons in the C NMR spectrum further corroborates the assigned structure. IR spectrum and
HRMS data also supports the assigned structure.
Having established the exact chemical procedure for synthesis of a β-lactam
glycoconjugate 4aa, we set out to synthesize a variety of β-lactam glycoconjugates with diversity
in both β-lactam and sugar moieties. Nevertheless, the procedure was found to be more general,
being equally applicable to the saturated and unsaturated and the α as well as β anomers of the
propargylic sugars employed. Various β-lactam glycoconjugates synthesized under the optimized
reaction conditions are shown in Table 2.
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Table 2. Generalization of the synthesis of β-lactam glycoconjugates 4a
Entry
Azide 2
Alkyne 3
Product 4
Time Yield
(h)
(%)b
OAc
OAc
O
O
N
N
N
AcO
AcO
O
N3
AcO
AcO
O
Cl
O
Cl
AcO
O
AcO
1.
2
78
N
O
Ph
N
O
Ph
Ph
3a
3b
Ph
4aa
2a
OAc
AcO
OAc
OAc
N
N
N
O
O
AcO
AcO
O
AcO
O
Cl
O
AcO
2.
3.
6
80
82
2a
2a
N
O
Ph
Ph
4ab
OAc
OAc
AcO
AcO
OAc
O
AcO
O
AcO
N
N
N
O
N
Cl
AcO
O
O
2.5
Ph
3c
O
Ph
4ac
OAc
O
OAc
O
N
N
O
N
Cl
AcO
AcO
O
N
O
4.
5.
1.5
0.5
65
75
2a
2a
O
Ph
3d
4ad
OAc
OAc
OAc
OAc
O
O
AcO
AcO
N
N
N
O
Cl
O
O
3e
N
Ph
O
Ph
4ae
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N3
O
OAc
O
O
OAc
N
N
N
AcO
AcO
Cl
O
AcO
O
Cl
O
AcO
O
AcO
N
AcO
N
6.
3.5
86
O
OMe
OMe
Ph
3a
3b
4ba
2b
2b
OAc
AcO
OAc
O
O
OAc
N
N
O
AcO
N
Cl
AcO
O
O
AcO
AcO
7.
8.
N
7
4
79
92
O
OMe
Ph
4bb
OAc
AcO
OAc
AcO
OAc
O
AcO
N
O
AcO
N
N
O
N
Cl
AcO
O
2b
O
O
OMe
Ph
3c
4bc
OAc
O
OAc
O
N
O
N
N
Cl
O
N
AcO
AcO
AcO
O
O
9.
1
89
76
2b
2b
Ph
OMe
3d
4bd
OAc
OAc
OAc
O
OAc
O
AcO
N
N
N
O
N
Cl
O
10.
O
3.5
O
OMe
Ph
3e
4be
aAll the reactions were performed with 2 mmol each of the azido-β-lactam 2 and the propargyl glycoside 3 using 50
mol% each of CuCl and PMDETA in DCE at 25-30 ˚C under a nitrogen atmosphere. Isolated yield of 4 after
b
purification by column chromatography.
The structures of all the newly synthesized β-lactam glycoconjugates 4ab-4be were supported by
IR, 1H, and 13C NMR spectroscopy and HRMS data. COSY NMR spectra were used for detailed
assignment of all the protons in the β-lactam glycoconjugates. The scope of the synthesis of β-
lactam glycoconjugates can be further expanded by installation of alkyne functionality on to
10

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other positions of carbohydrate ring or on to different carbohydrate partners and or using other
substituted azido β-lactams.
2.2. Biological studies
2.2.1. Evaluation of antibacterial activity
In order to study the preliminary biological activity of β-lactam glycoconjugates
synthesized in the present work, a representative β-lactam glycoconjugate 4aa was screened for
antibacterial activity. Surprisingly, the β-lactam glycoconjugate 4aa did not display any
antibacterial activity against both Gram-positive (Staphylococcus aureus and Bacillus subtilis)
and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains even at
much higher concentrations. The similar studies for antibacterial activity when extended for
other derivatives of β-lactam glycoconjugates showed negative results. This observation revealed
that the β-lactam glycoconjugates synthesized in the present work do not exhibit any
antibacterial activity.
2.2.2. Evaluation of biocompatibility
Thereafter, a representative β-lactam glycoconjugate 4aa was examined for in vitro
hemolytic activity on human erythrocytes via hemolytic assay test. Human erythrocytes were
treated with various concentrations of 4aa and the cell viability was then determined by
hemolytic assay and was expressed as mean of three separate experiments. This study showed
that the β-lactam glycoconjugate 4aa is biocompatible.
2.2.3. Evaluation of cytotoxicity
Further, the tumor cell growth inhibition activity of a representative β-lactam
glycoconjugate 4aa against L929 cancer cell line was studied in vitro via XTT assay. L929
cancer cells were exposed to different concentrations of 4aa and the % cell viability was
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observed by XTT assay and expressed as mean of three experiments. The study revealed the
cytotoxic activity of β-lactam glycoconjugate 4aa against L929 cancer cell lines.
However, the study in the present publication is mainly focused on the development of a
facile chemical procedure and its utility that would enable the development of a relatively new
small family of heteroatom rich β-lactam glycoconjugates for the potential bioanalytical
applications. The biological competence of the synthesized β-lactam glycoconjugates was
illustrated through antibacterial studies on all the compounds and hemolytic assay and XTT
assay studies on a representative β-lactam glycoconjugate. The detailed biological studies of
these β-lactam glycoconjugates are clearly outside the scope of the present publication.
3. Conclusion
In conclusion, we have developed a convenient route to synthesize a small ensemble of
chlorinated and heteroatom rich β-lactam glycoconjugates using the well proven chemistry
involved (Staudinger reaction, Cu(I)-catalyzed ATRC, glycosylation, Ferrier reaction and
CuAAC). The appropriate optimization of the reaction procedure for CuAAC revealed that
CuCl/PMDETA was the best catalyst to obtain maximum yield of the β-lactam glycoconjugates.
The present method is mild, economical, wide in scope, and regioselective. The starting
materials used are inexpensive and easily accessible. β-Lactam glycoconjugates synthesized in
the present work represent a rare and interesting assembly of four different biologically active
structural units, the tetrahydrofuran ring, the β-lactam ring, the triazole ring and a sugar moiety
in a single molecular framework. The preliminary biological studies showed that the β-lactam
glycoconjugates are non-antibacterial. The hemolytic assay and XTT assay studies on a
representative β-lactam glycoconjugate revealed its good biocompatibility and cytotoxicity
against L929 cancer cell lines. These results offer good opportunity to medicinal chemists for
12

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their further study and biological evaluation. Thus, the versatility and potential of the synthesis
has been realized in the present publication, providing an expedient access to an interesting and
relatively new family of biologically relevant β-lactam glycoconjugates.
4. Experimental Section
4.1. General Remarks. The entire data analysis of all the compounds synthesized in the present
work was done in Chemistry Instrumentation Lab, IIT Delhi, New Delhi. Biological studies were
done in Biomedical engineering Lab, IIT Delhi, New Delhi. IR spectra were recorded on an FT-
IR spectrometer by taking solid samples as KBr pellets and liquids as thin films on KBr disks.
NMR spectra were recorded on a 300 MHz FT NMR spectrometer in CDCl3 with TMS as
internal standard. Multiplicities are indicated by the following abbreviations: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), dd (doublet doublet). DEPT spectra were
routinely recorded to identify different types of carbons. COSY NMR spectrum was also
recorded to help the assignments of different protons. Mass spectra were recorded on a high-
resolution mass spectrometer (ESI-TOF) in positive-ion mode. Melting points were determined
on an electrically heated apparatus by taking the samples in a glass capillary sealed at one end
and are uncorrected. The progress of the reaction was monitored by TLC using a glass plate
coated with a TLC grade silica gel. Iodine was used for visualizing the spots. For column
chromatography, silica gel (60-120 mesh) or neutral alumina (60-325 mesh) was used as the
stationary phase, and n-hexane-ethyl acetate mixtures were used as the mobile phase. Solvents
were evaporated on a rotary evaporator under reduced pressure using an aspirator.
Dichloroacetyl chloride was commercially available and used as received. Et3N was dried over
KOH pellets overnight and distilled over CaH2. CH2Cl2 and DCE were dried by distilling over
anhydrous P2O5. Tetrahydrofuran (THF) and benzene were dried by distillation after a persistent
13

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blue color was observed on treating them with enough amount of sodium in the presence of
benzophenone.
4.2. Preparation of azidotetrahydrofurano fused bicyclic β-lactams 2a and 2b
These were prepared according to a procedure previously used in our laboratory.11 To a
solution of the β-lactam 1a or 1b (0.004 mol) in DMF (25 mL) was added sodium azide (1.30 g,
0.020 mol) and the solution was stirred at 65 ˚C for 12 h. The solution was diluted with water (30
mL) and extracted with ethyl acetate (2×50 mL). The combined organic extract was washed with
brine (2×30 mL), dried (Na2SO4) and evaporated under reduced pressure. The crude yellowish
solid product was purified by flash column chromatography using a short band of silica gel as the
stationary phase and a mixture of n-hexane-ethyl acetate (9:1 v/v) as the solvent for elution. The
azido β-lactams thus obtained were further purified by recrystallization from n-hexane-diethyl
ether to provide the crystalline products 2a and 2b in 69 and 65% yields, respectively.
4.2.1. 3-Chloro-4-azidomethyl-1-(4-methoxyphenyl)-6-phenyltetrahydrofuro[3,2-c]-azetidin-2-
one (2a). Colorless needles, 0.979 g, 69%; mp 107-108 ˚C (n-
hexane−diethyl ether); Rf (5% n-hexane/EtOAc, 5:1 v/v) 0.54; IR (KBr) νmax
3066(m), 2925(w), 2103(s), 1768(s), 1595(w), 1501(m), 1451(w), 1383(m),
1290(m), 1241(w), 1186(w), 1133(m), 1057(m), 972(w), 897(w), 748(m),
H5 N3
H4
H3
H2
H1
Cl
O
N
O
Ph
Ph
2a
689(m), 622(w) cm-1; δH (300 MHz, CDCl3) 7.52-7.44 (m, 7H, aromatic), 7.31-7.26 (m, 2H,
aromatic), 7.14 (t, J = 7.2 Hz, 1H, aromatic), 4.63 (t, J = 9.6 Hz, 1H, H1), 3.98-3.84 (m, 2H, H4,
H2), 3.60 (t, J = 10.2 Hz, 1H, H5), 2.86-2.75 (m, 1H, H3) ppm; δC (75.5 MHz, CDCl3) 159.7,
134.8, 131.7, 129.9, 129.2, 128.7, 127.2, 125.4, 118.5, 101.2, 81.1, 69.6, 48.9, 48.8 ppm; HRMS
(ESI-TOF): m/z [M + Na]+, found 377.0769. C18H15ClN4O2Na requires 377.0776.
14

ACCEPTED MANUSCRIPT
4.2.2. 3-Chloro-4-azidomethyl-1-(4-methoxyphenyl)-6-phenyltetrahydrofuro[3,2-c]-azetidin-2-
one (2b).11 Colorless cubes, 1.0 g, 65%; mp 90-92 ˚C (n-hexane−diethyl ether) (Lit.11 mp. 90-92
˚C); IR (KBr) νmax 3067(w), 3064(w), 2923(w), 2102(s), 1764(s), 1593(w), 1495(m), 1442(w),
H5 N3
H4
H3
H2
H1
1381(m), 1292(m), 1243(w), 1184(w), 1123(m), 1056(m), 974(w),
898(w), 746(m), 690(m), 647(w) cm-1; δH (300 MHz, CDCl3) 7.51-
7.49 (m, 3H, aromatic), 7.42-7.36 (m, 4H, aromatic), 6.79 (dd, J =
7.2, 2.1 Hz, 2H, aromatic), 4.61 (dd, J = 9.9, 7.5 Hz, 1H, H1), 3.93
Cl
O
N
O
Ph
OMe
2b
(dd, J = 12.9, 4.8 Hz, 1H, H4), 3.84 (t, J = 10.2 Hz, 1H, H2), 3.73 (s, 3H, OMe), 3.60 (dd, J =
12.6, 9.9 Hz, 1H, H5), 2.84-2.74 (m, 1H, H3) ppm; δC (75.5 MHz, CDCl3) 159.1, 157.1, 131.7,
129.8, 128.6, 128.0, 127.2, 119.9, 114.4, 101.0, 81.1, 69.5, 55.3, 48.9, 48.7 ppm.
4.3. Preparation of propargyl glycosides 3
All the propargyl glycosides used are known in the literature and were prepared
according to the reported procedures.15a-d
4.4. Typical procedure for preparation of β-lactam glycoconjugates 4
A two-neck round bottom flask fitted with a rubber septum was connected to a Schlenk
tube through a condenser and was evacuated and filled with dry nitrogen. The flask was charged
with the azido-β-lactam 2 (0.002 mol), propargyl glycoside 3 (0.002 mol), CuCl (0.01 g, 0.001
mol, 50 mol%) and degassed dichloroethane (20 mL) under a slow stream of the nitrogen gas.
PMDETA (0.173 g, 0.208 mL, 0.001 mol, 50 mol%) was then injected into the mixture and the
reaction mixture was stirred at room temperature (25-30 ˚C). The progress of the reaction was
periodically monitored by TLC. After completion of the reaction in 0.5-7 h, as shown in Table 2,
the solvent was evaporated. The residual material was taken up in ethyl acetate (30 mL), washed
with brine (2×20 mL), dried (Na2SO4), filtered and evaporated. Purification of the crude
15

ACCEPTED MANUSCRIPT
brownish solid products, thus obtained, by column chromatography using silica gel as the
stationary phase and a mixture of n-hexane-ethyl acetate (1:1 v/v) as the eluting solvent afforded
the pure β-lactam glycoconjugates 4 in 65-92% yields as a mixture of two diastereomers in 1:1
ratio.
4.4.1.
β
-Lactam glycoconjugate (4aa). White solid, 1.156 g, 78%; mp 88-90 ˚C; Rf (50%
EtOAc/n-hexane) 0.44; IR (KBr) νmax 2953(w),
H6
H7
H4
OAc
2361(w), 1757(s), 1634(w), 1498(w), 1377(m),
1229(w), 1045(m), 759(w), 694(w) cm-1; δH (300
MHz, CDCl3) [7.80 (s) (one diastereomer) + 7.78 (s)
(other diastereomer), 1H, H10], 7.48-7.43 (m, 7H,
H2
H11
N
N
N
AcO
AcO
H12
O
O
Cl
H13
H10
H14
H15
O
H5
H1
H8 H9
H3
N
O
Ph
Ph
4aa
aromatic), 7.32-7.27 (m, 2H, aromatic), 7.15 (t, J = 7.5 Hz, 1H, aromatic), 5.20 (t, J = 9.3 Hz,
1H, H3), 5.10 (t, J = 9.3 Hz, 1H, H4), 5.02 (t, J = 9.0 Hz, 1H, H2), 4.94 (d, J = 12.6 Hz, 1H, H8),
4.84 (d, J = 13.2 Hz, 1H, H9), 4.79-4.68 (m, 3H, H1, H11, H12), 4.64-4.58 (m, 1H, H14), 4.31-4.26
(m, 1H, H6), 4.19-4.10 (m, 1H, H7), 4.05-3.97 (m, 1H, H15), 3.75 (d, J = 7.8 Hz, 1H, H5), 3.29-
3.18 (m, 1H, H13), [2.09 (s) (one diastereomer) + 2.08 (s) (other diastereomer), 3H, COCH3],
2.02 (s, 3H, COCH3), 2.00 (s, 6H, COCH3) ppm; δC (75.5 MHz, CDCl3) 170.6, 170.1, 169.3
159.6, 144.0, 134.5, 131.2, 130.0, 129.2, 128.6, 127.1, 125.6, 124.2, 118.5, [101.31 (one
diastereomer) + 101.26 (other diastereomer)], 99.6, 80.7, 72.7, 71.8, 71.1, 69.2, 68.2, 62.6, 61.6,
49.1, 47.6, 20.7, 20.6, 20.5 ppm; HRMS (ESI-TOF): m/z [M + Na]+, found 763.1955.
C35H37ClN4O12Na requires 763.1989.
4.4.2.
β-Lactam glycoconjugate (4ab). White solid, 1.185 g, 80%; mp 89-91 ˚C; Rf (50%
EtOAc/n-hexane) 0.44; IR (KBr) νmax 3145(w), 3069(w), 2924(w), 2852(w), 2360(w), 1754(s),
1637(w), 1513(m), 1451(w), 1372(m), 1297(w), 1248(s), 1228(s), 1126(w), 1177(w), 1135(w),
16

ACCEPTED MANUSCRIPT
1048(m), 833(w), 739(w), 697(w), 671(w), 600(w) cm-1; δH (300 MHz, CDCl3) [7.78 (s) (one
diastereomer) + 7.74 (s) (other diastereomer),
H6
H7
1H, H10], 7.51-7.42 (m, 7H, aromatic), 7.33-7.30
(m, 2H, aromatic), 7.16 (t, J = 7.2 Hz, 1H,
aromatic), [5.41 (s) (one diastereomer) + 5.40 (s)
(other diastereomer), 1H, H4], 5.25 (dd, J = 9.9,
8.1 Hz, 1H, H2), 5.03-4.96 (m, 2H, H3, H1), 4.85-
OAc
H2
OAc
H11
N
N
H4
AcO
N
H12
O
O
Cl
H13
H10
H14
H15
O
H5
H1
H8 H9
H3
N
O
Ph
Ph
4ab
4.59 (m, 5H, H8, H9, H11, H12, H14), 4.20-4.15 (m, 2H, H6, H7), 4.04-3.93 (m, 2H, H15, H5), 3.29-
3.17 (m, 1H, H13), 2.15 (s, 3H, COCH3), 2.07 (s, 3H, COCH3), [2.02 (s) (one diastereomer) +
2.01 (s) (other diastereomer), 3H, COCH3], 1.98 (s, 3H, COCH3) ppm; δC (75.5 MHz, CDCl3)
170.4, 170.2, 170.0, [169.50 (one diastereomer) + 169.46 (other diastereomer)], 159.7, [144.2
(one diastereomer) + 144.1 (other diastereomer)], 134.5, 131.3, 130.1, 129.3, 128.7, 127.2,
125.7, [124.2 (one diastereomer) + 124.1 (other diastereomer)], 118.5, [101.4 (one diastereomer)
+ 101.3 (other diastereomer)], [100.13 (one diastereomer) + 100.06 (other diastereomer)], 80.8,
70.8, 69.3, 68.7, 67.0, [62.52 (one diastereomer) + 62.46 (other diastereomer)], [61.27 (one
diastereomer) + 61.25 (other diastereomer)], 49.2, 47.7, 20.74, 20.66, 20.62, 20.5 ppm; HRMS
(ESI-TOF): m/z [M + Na]+, found 763.1982. C35H37ClN4O12Na requires 763.1989.
4.4.3.
β
-Lactam glycoconjugate (4ac). White solid, 1.215 g, 82%; mp 86-88 ˚C; Rf (50%
H6
H7
EtOAc/n-hexane) 0.45; IR (KBr) νmax 3144(w),
H4
OAc
OAc
AcO
H3
AcO
O
2931(w), 2365(w), 1749(s), 1647(w), 1598(w),
1497(m), 1447(w), 1375(s), 1225(s), 1135(m), 1052(s),
752(m), 694(w), 605(w) cm-1; δH (300 MHz, CDCl3)
H1
H11
N
H12
O
H2
N
5
O
H
Cl
N
H13
N
H8 H9
H10
H14
Ph
O
Ph
H15
4ac
17

ACCEPTED MANUSCRIPT
[7.82 (s) (one diastereomer) + 7.81 (s) (other diastereomer), 1H, H10], 7.55-7.37 (m, 7H,
aromatic), 7.32-7.28 (m, 2H, aromatic), 7.16 (t, J = 7.2 Hz, 1H, aromatic), 5.32-5.24 (m, 3H, H2,
H3, H4), 4.99 (s, 1H, H1), 4.96-4.81 (m, 2H, H8, H9), 4.79-4.60 (m, 3H, H11, H12, H14), 4.34-4.27
(m, 1H, H6), 4.15-4.04 (m, 3H, H7, H5, H15), 3.31-3.18 (m, 1H, H13), 2.14 (s, 3H, COCH3), 2.12
(s, 3H, COCH3), [2.03 (s) (one diastereomer) + 2.02 (s) (other diastereomer), 3H, COCH3], 1.98
(s, 3H, COCH3) ppm; δC (75.5 MHz, CDCl3) 170.6, [169.94 (one diastereomer) + 169.91 (other
diastereomer)], 169.8, 169.6, 159.7, 143.5, 134.5, 131.3, 130.0, 129.2, 128.6, 127.2, 125.6,
124.2, 118.5, 101.3, [96.8 (one diastereomer) + 96.7 (other diastereomer)], 80.8, 69.4, 68.9, 68.7,
66.0, [62.4 (one diastereomer) + 62.3 (other diastereomer)], [60.8 (one diastereomer) + 60.7
(other diastereomer)], [49.21 (one diastereomer) + 49.18 (other diastereomer)], 47.7, 20.8, 20.7,
20.6 ppm; HRMS (ESI-TOF): m/z [M + Na]+, found 763.1997. C35H37ClN4O12Na requires
763.1989.
4.4.4.
β-Lactam glycoconjugate (4ad). White solid, 0.810 g, 65%; mp 65-67 ˚C; Rf (50%
6 H7
EtOAc/n-hexane) 0.45; IR (KBr) νmax 3144(w),
3065(w), 2924(w), 2360(w), 1768(s), 1743(s),
1636(w), 1598(w), 1498(m), 1451(w), 1376(s),
1236(s), 1103(w), 1038(s), 758(w), 694(w),
H
OAc
O
H11
H4 H5
AcO
H3
H12
Cl
N
H1
O
N
N
N
H13
H10
O
H2
Ph
H9
H8
H14
O
Ph
H15
4ad
608(w) cm-1; δH (300 MHz, CDCl3) 7.80 (s, 1H, H10), 7.52-7.40 (m, 7H, aromatic), 7.33-7.27 (m,
2H, aromatic), 7.16 (t, J = 7.2 Hz, 1H, aromatic), 5.91 (d, J = 10.2 Hz, 1H, H2), 5.83 (d, J = 10.2
Hz, 1H, H3), 5.34 (d, J = 9.6 Hz, 1H, H4), 5.18 (s, 1H, H1), 4.93 (d, J = 12.3 Hz, 1H, H8), 4.83-
4.60 (m, 4H, H9, H11, H12, H14), 4.30-4.15 (m, 3H, H6, H7, H5), 4.01 (t, J = 10.8 Hz, 1H, H15),
3.28-3.17 (m, 1H, H13), 2.11 (s, 3H, COCH3), 2.08 (s, 3H, COCH3) ppm; δC (75.5 MHz, CDCl3)
170.7, 170.1, 159.6, [144.5 (one diastereomer) + 144.4 (other diastereomer)], 134.5, 131.2,
18

ACCEPTED MANUSCRIPT
130.0, 129.4, 129.2, 128.6, 127.3, 127.1, 125.6, 124.0, 118.4, 101.3, [93.61 (one diastereomer) +
93.56 (other diastereomer)], 80.7, 69.3, 67.0, 65.2, 62.8, [61.24 (one diastereomer) + 61.20
(other diastereomer)], 49.1, 47.5, 20.8, 20.7 ppm; HRMS (ESI-TOF): m/z [M + Na]+, found
645.1696. C31H31ClN4O8Na requires 645.1723.
4.4.5.
β
-Lactam glycoconjugate (4ae). White solid, 1.024 g, 75%; mp 73-75 ˚C; Rf (50%
EtOAc/n-hexane) 0.46; IR (KBr): νmax 3144(w),
H7
H8
AcO
OAc
H3
2928(w), 1749(s), 1634(w), 1598(w), 1497(w),
1449(w), 1375(m), 1236(s), 1157(w), 1123(w),
1049(m), 752(w), 690(w), 613(w) cm-1; δH (300 MHz,
CDCl3) [7.79 (s) (one diastereomer) + 7.77 (s) (other
H5
AcO
O
H1
N
H12
H13
H14
N
2 H6
N
H
O
N
Cl
O
H4
H9 H10
H11
Ph
H15
O
H16
Ph
4ae
diastereomer), 1H, H11], 7.52-7.40 (m, 7H, aromatic), 7.33-7.27 (m, 2H, aromatic), 7.17 (t, J =
7.2 Hz, 1H, aromatic), 5.34-5.26 (m, 2H, H4, H5), 5.17 (s, 1H, H1), 4.85-4.61 (m, 5H, H9, H10,
H12, H13, H15), 4.22 (t, J = 6.0 Hz, 1H, H6), 4.12 (d, J = 6.3 Hz, 2H, H7, H8), 4.00 (t, J = 12.0 Hz,
1H, H16), 3.31-3.21 (m, 1H, H14), 2.15 (s, 3H, COCH3), 2.15-2.07 (m, 1H, H2, overlapped
between two singlets at 2.15 and 2.07), 2.07 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.91 (dd, J =
10.2, 5.1 Hz, 1H, H3) ppm; δC NMR (75.5 MHz, CDCl3) 170.6, 170.3, 170.0, 159.8, 144.3,
134.6, 131.3, 130.1, 129.3, 128.7, 127.2, 125.6, [124.0 (one diastereomer) + 123.9 (other
diastereomer)], 118.5, 101.4, [96.9 (one diastereomer) + 96.8 (other diastereomer)], 80.9, 69.4,
66.9, 66.6, 66.0, 62.3, 60.5, 49.3, 47.7, 29.7, 20.84, 20.77, 20.73 ppm; HRMS (ESI-TOF): m/z
[M + Na]+, found 705.1944. C33H35ClN4O10Na requires 705.1934.
4.4.6.
β-Lactam glycoconjugate (4ba). White solid, 1.326 g, 86%; mp 76-78 ˚C; Rf (50%
EtOAc/n-hexane) 0.44; IR (KBr) νmax 2934(w), 2340(w), 1757(s), 1622(w), 1513(w), 1450(w),
1377(w), 1235(s), 1129(w), 1047(m), 831(w), 692(w), 605(w) cm-1; δH (300 MHz, CDCl3) δ
19

ACCEPTED MANUSCRIPT
[7.77 (s) (one diastereomer) + 7.74 (s)
H6
H7
OAc
H4
H2
(other diastereomer), 1H, H10], 7.49-7.37
(m, 7H, aromatic), 6.81 (d, J = 9.0 Hz, 2H,
aromatic), 5.19 (t, J = 9.0 Hz, 1H, H3),
5.09 (t, J = 9.3 Hz, 1H, H4), 5.01 (t, J =
H11
N
N
N
AcO
AcO
H12
O
O
Cl
H13
H10
H14
H15
O
H5
H1
H8 H9
H3
N
O
Ph
OMe
4ba
8.1 Hz, 1H, H2), 4.94 (d, J = 13.2 Hz, 1H, H8), 4.83 (d, J = 12.9 Hz, 1H, H9), 4.81-4.65 (m, 3H,
H11, H1, H12), 4.62-4.56 (m, 1H, H14), 4.30-4.24 (m, 1H, H6), 4.18-4.13 (m, 1H, H7), 3.98 (t, J =
10.2 Hz, 1H, H15), 3.76 (s, 4H, multiplet for 1H (H5), overlapped by singlet for 3H, OMe), 3.26-
3.14 (m, 1H, H13), 2.08 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.99 (s,
3H, COCH3) ppm; δC (75.5 MHz, CDCl3) 170.6, 170.1, 169.33, 169.29, 159.2, 157.2, 144.1,
131.3, 130.0, 128.6, 127.8, 127.2, 124.2, 120.0, 114.5, 101.3, 99.7, 80.8, 72.7, 71.9, 71.1, 69.2,
68.3, 62.6, 61.7, 55.4, 49.2, 47.7, 20.7, 20.6, 20.5 ppm; HRMS (ESI-TOF): m/z [M + Na]+,
found 793.2101. C36H39ClN4O13Na requires 793.2094.
4.4.7.
β-Lactam glycoconjugate (4bb). White solid, 1.218 g, 79%; mp 89-91 ˚C; Rf (50%
EtOAc/n-hexane) 0.44; IR (KBr) νmax 3145(w), 2940(m), 1768(s), 1743(s), 1619(w), 1513(m),
1450(m), 1376(s), 1221(s), 1133(m), 1047(s), 910(w), 830(m), 742(w), 701(w), 598(w) cm-1; δH
(300 MHz, CDCl3) [7.79 (s) (one
H6
H7
OAc
H2
OAc
diastereomer)
+
7.76 (s) (other
H11
N
N
H4
AcO
N
H12
O
O
diastereomer), 1H, H10], 7.51-7.38 (m,
7H, aromatic), 6.82 (d, J = 9.0 Hz, 2H,
aromatic), 5.40 (s, 1H, H4), 5.27-5.21 (m,
Cl
O
H13
H10
H14
H15
O
H5
H1
3 A
H8 H9
c
H
N
O
Ph
OMe
4bb
1H, H2), 5.01 (dd, J = 9.6, 3.3 Hz, 1H, H3), 4.96 (s, 1H, H1), 4.85-4.58 (m, 5H, H8, H9, H11, H12,
H14), 4.19-4.09 (m, 2H, H6, H7), 4.03-3.95 (m, 2H, H15, H5), 3.76 (s, 3H, OMe), 3.24-3.18 (m,
20

ACCEPTED MANUSCRIPT
1H, H13), 2.15 (s, 3H, COCH3), [2.02 (s) (one diastereomer) + 2.01 (s) (other diastereomer), 3H,
COCH3], [2.06 (s) (one diastereomer) + 2.05 (s) (other diastereomer), 3H, COCH3], 1.98 (s, 3H,
COCH3) ppm; δC (75.5 MHz, CDCl3) 170.3, 170.2, 170.0, [169.48 (one diastereomer) + 169.45
(other diastereomer)], 159.1, 157.2, [144.09 (one diastereomer) + 144.03 (other diastereomer)],
131.3, 130.0, 128.6, 127.7, 127.2, [124.2 (one diastereomer) + 124.1 (other diastereomer)],
120.0, 114.5, [101.3 (one diastereomer) + 101.2 (other diastereomer)], [100.1 (one
diastereomer) + 100.0 (other diastereomer)], 80.8, 70.8, 69.2, 68.7, 67.0, [62.5 (one
diastereomer) + 62.4 (other diastereomer)], 61.2, 55.3, 49.2, 47.7, 20.7, 20.62, 20.57, 20.5 ppm;
HRMS (ESI-TOF): m/z [M + Na]+, found 793.2110. C36H39ClN4O13Na requires 793.2094.
4.4.8.
β
-Lactam glycoconjugate (4bc). White solid, 1.419 g, 92%; mp 90-92 ˚C; Rf (50%
EtOAc/n-hexane) 0.45; IR (KBr) νmax
H6
H7
H4
OAc
3146(w), 2930(w), 1752(s), 1634(w),
1498(m), 1451(w), 1373(m), 1301(w),
1227(s), 1126(w), 1057(m), 823(w),
759(w), 694(w), 606(w) cm-1; δH (300 MHz,
OAc
AcO
H3
O
H1
H11
N
H12
O
H2
N
5
H
Cl
N
AcO
O
H13
N
H8 H9
H10
H14
H15
OMe
O
Ph
4bc
CDCl3) [7.814 (s) (one diastereomer) + 7.805 (s) (other diastereomer), 1H, H10], 7.50-7.38 (m,
7H, aromatic), 6.82 (d, J = 9.0 Hz, 2H, aromatic), 5.31-5.24 (m, 3H, H2, H3, H4), 4.95 (s, 1H,
H1), 4.87-4.59 (m, 5H, H8, H9, H11, H12, H14), 4.33-4.27 (m, 1H, H6), 4.14-3.98 (m, 3H, H7, H5,
H15), 3.76 (s, 3H, OMe), 3.28-3.16 (m, 1H, H13), 2.14 (s, 3H, COCH3), 2.12 (s, 3H, COCH3),
[2.02 (s) (one diastereomer) + 2.01 (s) (other diastereomer), 3H, COCH3], 1.98 (s, 3H, COCH3)
ppm; δC (75.5 MHz, CDCl3) 170.6, [169.90 (one diastereomer) + 169.87 (other diastereomer)],
169.8, 169.6, 159.2, 157.2, 143.5, 131.4, 129.9, 128.6, 127.8, 127.3, 124.2, 120.0, 114.5, 101.3,
[96.84 (one diastereomer) + 96.77 (other diastereomer)], 80.8, 69.4, 69.0, 68.7, 66.1, 62.4,
21

ACCEPTED MANUSCRIPT
[60.81 (one diastereomer) + 60.78 (other diastereomer)], 55.4, 49.2, 47.8, 20.8, 20.7, 20.6 ppm;
HRMS (ESI-TOF): m/z [M + Na]+, found 793.2112. C36H39ClN4O13Na requires 793.2094.
4.4.9.
β
-Lactam glycoconjugate (4bd). White solid, 1.162 g, 89%; mp 76-78 ˚C; Rf (50%
6 H7
EtOAc/n-hexane) 0.45; IR (KBr) νmax 3143(w),
3068(w), 2933(m), 1765(s), 1736(s), 1616(w),
1513(s), 1451(m), 1377(m), 1298(m), 1246(s),
1184(w), 1135(m), 1042(s), 873(w), 830(m),
H
OAc
O
H11
H4
H5
H12
Cl
N
H1
O
N
N
N
H13
H10
AcO
H3
O
H2
Ph
H9
H8
4bd
H14
H15
O
Ph
740(m), 699(w), 592(w), 548(w) cm-1; δH (300 MHz, CDCl3) 7.74 (s, 1H, H10), 7.42-7.30 (m,
7H, aromatic), 6.74 (d, J = 9.0 Hz, 2H, aromatic), 5.83 (d, J = 10.2 Hz, 1H, H2), 5.75 (d, J =
10.2 Hz, 1H, H3), 5.26 (d, J = 9.3 Hz, 1H, H4), 5.10 (s, 1H, H1), 4.85 (d, J = 12.0 Hz, 1H, H8),
4.75-4.63 (m, 3H, H9, H11, H12), 4.58-4.51 (m, 1H, H14), 4.22-4.07 (m, 3H, H6, H7, H5), 3.92 (t,
J = 10.5 Hz, 1H, H15), 3.69 (s, 3H, OMe), 3.19-3.07 (m, 1H, H13), [2.04 (s) (one diastereomer) +
2.03 (s) (other diastereomer), 3H, COCH3], 2.00 (s, 3H, COCH3) ppm; δC (75.5 MHz, CDCl3)
170.7, 170.2, 159.2, 157.2, [144.6 (one diastereomer) + 144.5 (other diastereomer)], 131.3,
130.0, 129.5, 128.6, 127.7, 127.4, 127.2, 124.0, 120.0, 114.5, 101.3, [93.68 (one diastereomer)
+ 93.63 (other diastereomer)], 80.8, 69.3, 67.0, 65.2, 62.8, [61.32 (one diastereomer) + 61.28
(other diastereomer)], 55.3, 49.2, 47.6, 20.9, 20.8 ppm; HRMS (ESI-TOF): m/z [M + Na]+,
found 675.1812. C32H33ClN4O9Na requires 675.1828.
4.4.10.
β-Lactam glycoconjugate (4be). White solid, 1.083 g, 76%; mp 83-85 ˚C; Rf (50%
EtOAc/n-hexane) 0.46; IR (KBr) νmax 3144(w), 3069(w), 2926(w), 2361(w), 1748(s), 1637(w),
1512(s), 1450(w), 1374(m), 1298(w), 1248(s), 1122(w), 1035(m), 833(w), 739(m), 697(w),
671(w) cm-1; δH (300 MHz, CDCl3) [7.71 (s) (one diastereomer) + 7.70 (s) (other diastereomer),
1H, H10], 7.43-7.29 (m, 7H, aromatic), 6.76-6.71 (m, 2H, aromatic), 5.25-5.17 (m, 2H, H4, H5),
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[5.08 (s) (one diastereomer) + 5.07 (s)
H7
H8
OAc
AcO
H3
(other diastereomer), 1H, H1], 4.76-4.50
(m, 5H, H9, H10, H12, H13, H15), 4.15-4.11
(m, 1H, H6), 4.03 (d, J = 6.0 Hz, 2H, H7,
H8), 3.97-3.90 (m, 1H, H16), 3.68 (s, 3H,
H5
AcO
O
H1
H12
H13
H14
N
N
2 H6
N
H
O
N
Cl
O
H4
H9 H10
H11
H15
H16
4be
O
OMe
Ph
OMe), 3.17-3.11 (m, 1H, H14), 2.06 (s, 3H, COCH3), 2.06-2.00 (m, 1H, H2, overlapped by singlet
at 2.06), 1.98 (s, 3H, COCH3), 1.89 (s, 3H, COCH3), 1.84-1.78 (m, 1H, H2) ppm; δC (75.5 MHz,
CDCl3) 170.5, 170.2, 170.0, 159.2, 157.2, 144.2, 131.4, 130.0, 128.6, 127.7, 127.2, [124.0 (one
diastereomer) + 123.9 (other diastereomer)], 120.0, 114.5, [101.24 (one diastereomer) + 101.21
(other diastereomer)], [96.84 (one diastereomer) + 96.76 (other diastereomer)], 80.8, [69.4 (one
diastereomer) + 69.3 (other diastereomer)], 66.8, 66.5, 66.0, 62.2, 60.4, 55.3, 49.2, 47.7, 29.9,
20.8, 20.70, 20.65 ppm; HRMS (ESI-TOF): m/z [M + Na]+, found 735.2035. C34H37ClN4O11Na
requires 735.2040.
4.5. Antibacterial activity assay
The in vitro antibacterial activities of test compounds were determined by the well diffusion
method. Muller Hinton Agar (MHA) medium was used for preparation of plates. The medium
was poured to the sterile petriplates of 90 mm diameter. Fresh human pathogenic bacterial
cultures of two Gram-positive bacteria, (B. subtilis and S. aureus) and two Gram-negative
bacteria, (E. coli and P. aeruginosa) were spread on surface of the MHA plate. After incubation,
the MHA plates were allowed for the pre-incubation for 10 min using a sterile cork borer, the
well was cut from the agar in the plate. Freshly prepared solutions of β-lactam glycoconjugates 4
with different concentrations were poured into wells. The inoculated plates were initially
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incubated for 15 min at room temperature and then at 37 ˚C for 24 h. Thereafter, the plates were
examined for any zone of growth inhibition.
4.6. Hemolytic assay
Freshly prepared solutions of β-lactam glycoconjugate 4aa with different concentrations were
added to freshly prepared human RBC suspension in a 96-well culture plate. The resulting
mixture was kept at 37 ̊C for 30 min under rotary agitation. Afterwards, the plate was centrifuged
and the supernatant in each well was transferred to a new plate. Hemolysis was monitored by
measuring the absorbance of the released hemoglobin at 414 nm.17 All experiments were done in
triplicate.
4.7. XTT assay
L929 cancer cells cultured into 96-well microculture plates were exposed to β-lactam
glycoconjugate 4aa at different concentrations. Absorbance was measured after 4 h of incubation
at 37 ̊
C. XTT assay was quantitatively used to evaluate % cell proliferation.18 Each experiment
was performed three times.
Acknowledgements
We are thankful to IIT Delhi for providing research facilities and the Council of Scientific and
Industrial Research, New Delhi for a research fellowship for carrying out this research work. The
authors also thank BML Munjal University for providing support to publish this work.
Supplementary data
1
Supplementary data related to this article comprised of copies of H and 13C NMR spectra of
compounds 2a, 2b and 4aa-4be and COSY NMR spectrum of 4aa and data of hemolytic assay
and XTT assay of 4aa.
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