Conformational studies of glycosylated cyclic oligomers of furanoid sugar amino acids

Article abstract of DOI:10.1016/j.tet.2016.07.071

Glycosylation of molecules improve their pharmacological and pharmacokinetic properties. In the current manuscript, we have explored the effect of glycosylation on the structure and function of conformationally well-defined small ring homooligomers derived from a structurally diverse library of sugar amino acids (SAA). Conformational analyses carried out by NMR suggested that these cyclic dimers and trimers have well-defined structures in solution. MD simulations performed based on the restraints obtained from NMR revealed that C2H and CO are positioned outside the plane of the ring and NHs are pointed inside the ring. It was encouraging to note that while the cyclic non-glycosylated homooligomers did not show any antimicrobial activity at all, their glycosylated counterparts showed relatively better activity. The modular design developed here is amenable to further improvement and can serve as a tool to investigate many molecular recognition processes.

Full text of DOI:10.1016/j.tet.2016.07.071

Accepted Manuscript
Conformational studies of glycosylated cyclic oligomers of furanoid sugar amino acids
Nishant Singh, Pancham Singh Kandiyal, Praveen Kumar Shukla, Ravi Sankar
Ampapathi, Tushar Kanti Chakraborty
PII:
S0040-4020(16)30729-3
10.1016/j.tet.2016.07.071
TET 27967
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 June 2016
Revised Date: 28 July 2016
Accepted Date: 29 July 2016
Please cite this article as: Singh N, Kandiyal PS, Shukla PK, Ampapathi RS, Chakraborty TK,
Conformational studies of glycosylated cyclic oligomers of furanoid sugar amino acids, Tetrahedron
(2016), doi: 10.1016/j.tet.2016.07.071.
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Graphical Abstract
Conformational studies of glycosylated cyclic
oligomers of furanoid sugar amino acids
Leave this area blank for abstract info.
Nishant Singh,a Pancham Singh Kandiyal,b Praveen Kumar Shukla,c Ravi Sankar Ampapathib,* and Tushar Kanti
Chakrabortya,d,
*
a Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India; b Centre for Nuclear Magnetic Resonance,
SAIF, CSIR-Central Drug Research Institute, Lucknow 226031, India; c Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow
226031, India; d Department of Organic Chemistry, Indian Institute of Science, Bengaluru 560012, India

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1
Tetrahedron
journal homepage: www.elsevier.com
Conformational studies of glycosylated cyclic oligomers of furanoid sugar amino
acids
Nishant Singh,a Pancham Singh Kandiyal,b Praveen Kumar Shukla,c Ravi Sankar Ampapathib,* and Tushar
Kanti Chakrabortya,d,*
a Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India;
b Centre for Nuclear Magnetic Resonance, SAIF, CSIR-Central Drug Research Institute, Lucknow 226031, India
c Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow 226031, India
d Department of Organic Chemistry, Indian Institute of Science, Bengaluru 560012, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Glycosylation of molecules improve their pharmacological and pharmacokinetic properties. In
the current manuscript, we have explored the effect of glycosylation on the structure and
function of conformationally well-defined small ring homooligomers derived from a structurally
diverse library of sugar amino acids (SAA). Conformational analyses carried out by NMR
suggested that these cyclic dimers and trimers have well-defined structures in solution. MD
simulations performed based on the restraints obtained from NMR revealed that C2H and CO
are positioned outside the plane of the ring and NHs are pointed inside the ring. It was
encouraging to note that while the cyclic non-glycosylated homooligomers did not show any
antimicrobial activity at all, their glycosylated counterparts showed relatively better activity. The
modular design developed here is amenable to further improvement and can serve as a tool to
investigate many molecular recognition processes.
Available online
Keywords:
Sugar amino acid
NMR
Conformation
Glycosylation
Cyclic peptide
2016 Elsevier Ltd. All rights reserved.
1. Introduction
MeO
O
MeO
Glycosylated natural products are abundant displaying wide
ranging antimicrobial, antifungal and/or anticancer activities.1
Many bacteria also use glycosylated small molecules as chemical
weapons to gain a selective advantage or as signaling molecules
for intra- and interspecie1s communication.2 Sugar moieties in
these natural products and metabolites dramatically improve their
pharmacological and pharmacokinetic properties, such as
solubility, cellular permeability, distribution and metabolic
stability.3 They also impact the delivery of the natural product to
the target, present high affinity and specificity for a given target,
tissue, cell, as well as modulate both mechanism and in vivo
properties.4 Given that carbohydrates occupy a very large
chemical space, differential glycosylation of natural products
and/or synthetic small molecules offers a viable strategy to
produce new chemical entities with improved pharmacological
properties and biological activities.5 This encouraged us to
explore the effect of glycosylation on the structure and function
of conformationally well-defined small ring homooligomers
derived from a structurally diverse library of sugar amino acids
(SAA). To begin with, the choice of the carbohydrate to be
attached to these cyclic peptides stemmed from many naturally
occurring C2-symmetric cyclic diolides isolated from marine
O
MeO
MeO
MeO
O
O
MeO
N
H
O
O
O
O
OMe
O
O
O
OMe
OMe
O
NH
HN
O
NH
O
HN
O
O
O
O
OMe
OMe
OMe
OMe
OMe
OMe
O
O
3
1
O
O
MeO
MeO
O
OMe
N
H
MeO
OMe
OMe
OMe
O
O
O
O
O
OMe
OMe
O
NH
HN
O
O
O
O
O
O
NH
O
HN
O
MeO
MeO
O
O
MeO
4
O
MeO
MeO
2
MeO
R2
OMe
R1
R3O
O
OMe
O
O
H
Clavosolide A: R1
R2 = H; R3 = Me
Cyanolide A: R1 = Et; R2, R3 = Me
Cocosolide: R1
=
H
=
O
O
O
O
H
O
O
MeO
MeO
O
———
OR3
R1
R2
* Corresponding author. Tel.: +91 80 22932215; E-mail: ravi_sa@cdri.res.in
(RS Ampapathi), tushar@orgchem.iisc.ernet.in (TK Chakraborty)
R2 = Me, R3 = H
H

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2
Tetrahedron
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Figure 1. Structures of glycosylated cyclic peptides of sugar
paper10d that similar kind of substrates with ‘2,5-cis’ ring lead to
amino acids, 1-4 and C2-symmetric diolide natural products.
the bicyclic lactam formation, but linear dimers from such
substrates furnished the requisite products on cyclization. Thus,
the monomer 19 was subjected, separately in equimolar amounts,
to LiOH-mediated saponification in THF-MeOH-H2O solvent
system and TFA-mediated Boc-deprotection in CH2Cl2 to
provide the acid and the TFA-salt, respectively, in quantitative
yields. Next, the acid and TFA-salt were coupled using
conventional solution phase peptide synthesis method using 1-
ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole (HOBt) as coupling reagents
and diisopropylethylamine (DIPEA) as base in CH2Cl2 to provide
the linear precursor 23 in 72% yield (Scheme 2).
cyanobacteria, like clavosolides,6 cyanolides,7 cocosolide,8
etc. (Figure 1), all of which carry methylated xylopyranosides. In
this paper, we describe the synthesis, conformational studies and
biological activities of cyclic homooligomers 1-4 of furanoid
sugar amino acids,9 glycosylated with tri-O-methyl-D-
xylopyranosyl sugar.
2. Results and discussion
The synthesis of the cyclic glycopeptides 1-4 was carried out
in a similar fashion as reported by us and others10 using solution
phase peptide synthesis in which the glycosylated sugar amino
acid (SAA) monomers 5-8 (Figure 2) were converted to their
cyclic homooligomer counterparts efficiently. The construction
of the monomers 5-8 (5 for 1, 6 for 2, 7 for 3 and 8 for 4) was
initiated using 2-deoxy-D-ribose (for glycosyl acceptor residues
12 and 13) and D-xylose (for glycosyl donor residue 14) as the
starting raw materials (Scheme 1). The monomers 5-6 differ from
monomers 7-8 in the configuration of the stereocenter at C2 of
the furanoid rings of their constituent δ-SAA moieties (Scheme
1). But the monomers 5 (or 7) and 6 (or 8) departed from each
other with respect to the type of O-glycosidic linkage present in
them, i.e. monomer 5 (or 7) possesses α-O-glycosidic linkage
while monomer 6 (or 8) carry β-O-glycosidic linkage. While
monomers 5 and 6 were used to get solely the C2-symmetric
glycosylated homooligomers 1 and 2, respectively, monomers 7
and 8 furnished C3-symmetric glycosylated homooligomers 3 and
4, respectively as the major products (Schemes 2 and 3).
O
2
O
CO2Me
OH
BCl3, CH2Cl2
0 oC, 10 min
N3
Ref. 11
HO
BnO
10 (2R)
HO
10
12
→
9
11
11 → 13
(2S)
OMe
O
CO2Me
O
OMe
MeO
CO2Me
N3
O
OMe
N3
14
BF3.Et2O, CH3CN
oC, 2 h
15 + 16
HO
12
R1/2
O
(87%)
13 (84%)
0
15 (53%), 16 (36%)
17 (
18
(34%)
12
50%),
→
13 → 17 + 18
O
O
CO2Me
CO2Me
BocHN
BocHN
Pd-C, H2, (Boc)2O
NEt3, MeOH, rt, 5 h
OMe
O
O
OMe
15
16 → 20
17 → 21
19
→
O
OMe
OMe
O
(68%)
OMe
OMe
20
(70%)
19
18
22
→
O
CO2Me
O
CO2Me
BocHN
BocHN
R1O
5
R2O
5
OMe
O
O
OMe
2
OMe
2
O
H2N
OMe
OMe
H2N
H2N
R1
=
COOH
COOH
COOH
COOH
O
O
O
O
OMe
OMe
OMe
OMe
21
5
(69%)
22
(64%)
6
R1O
5
R2O
5
OMe
O
2
2
R2
=
OMe
OMe
H2N
Scheme 1. Synthesis of
precursors 19 22
O-glycosylated and protected
O
O
-
.
7
8
Figure 2. Glycosylated δ-SAA monomers 5-8.
The monomer 20 led to the other linear precursor 24 in 69%
yield using the same reagents under identical reaction conditions.
Both the linear precursors 23 and 24, separately, were subjected
sequentially to LiOH-mediated ester hydrolysis followed by
TFA-mediated Boc-deprotection to furnish TFA-salt which
underwent intramolecular cyclization in the presence of
pentafluorophenyl diphenyphosphinate (FDPP) as coupling
reagent15 and DIPEA as base in dry CH3CN (5 x 10-3 M) to
furnish the required products 1 in 54% yield and 2 in 52% yield,
The synthesis of the protected SAA precursors (19-22), each
representing the monomers 5-8 sequentially, was carried out by
O-glycosylation of 12-13 using the glycosyl donor 14 (Scheme
1). The preparation of 12-13 commenced from commercially
available 2-deoxy-D-ribose (9) which was easily and efficiently
converted in to diastereomeric intermediates 10 and 11 as
reported earlier from our lab (Scheme 1).11 BCl3-mediated benzyl
ether deprotection12 of intermediates 10 (C2-R-isomer) and 11
(C2-S-isomer) furnished 12 and 13, respectively, in excellent
yields. Thereafter, BF3·Et2O mediated O-glycosyaltion reaction13
was implemented between substrates 12-13 and the glycosyl
donor substrate, permethylated-D-xylose14 14, resulting in the
formation of α-O-glycosylated product 15 along with β-O-
glycosylated product 16 (from 12, overall 89% yield) and α-O-
glycosylated product 17 along with β-O-glycosylated product 18
(from 13, overall 84% yield), respectively in a ratio of 1.5:1 (α:β)
after chromatographic separation. The azido moiety in the
intermediates 15-18 were subjected to in situ one-pot reduction
and Boc-protection reactions using Pd-C/H2 and (Boc)2O-NEt3,
furnishing compounds 19-22 in good yields (Scheme 1).
1) LiOH.H2O
O
O
O
THF:MeOH:H2O (3:1:1)
CO2Me
19
or
N
H
BocHN
rt, 1 h, quant. yield
2) TFA, CH2Cl2, 0 oC to rt, 2 h,
quant. yield
R1/2
O
R1/2
O
20
23 (R1, 72%)
3) 1 eq each from steps 1 and 2,
EDCI, HOBt, DIPEA, CH2Cl2, 0 o
24
(R2, 69%)
OR1/2
C
to rt,12 h
19 → 23
20
24
→
O
1) LiOH.H2O, THF:MeOH:H2O (3:1:1)
rt, 1 h, quant. yield
2) TFA, CH2Cl2, 0 oC to rt, 2 h, quant. yield
3) FDPP, DIPEA, CH3CN 0 oC to rt, 72h
O
NH
HN
O
O
After acquiring the monomeric building blocks 19-22, we
proceeded for the synthesis of glycosylated cyclic
homooligomers 1-4. Firstly, syntheses of C2-symmetric cyclic
peptides 1 and 2 were accomplished from the monomers 19 and
20, respectively (Scheme 2). As we had reported in our earlier
R1/2
O
23
24 → 2
1
→
1
(R1, 54%)
2 (R2, 52%)

ACCEPTED MANUSCRIPT
3
respectively, as the only isolable products (Scheme 2).
Figure 3. Newman projection and nOe representation for 1.
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Scheme 2. Synthesis of the C2-symmetric glycosylated
cyclodimers 1 and 2.
Aggregation studies of glycopeptides 1 and 2, carried out with
The
syntheses
of
and
C3-symmetric
4 were commenced from the
glycosylated
homooligomers
3
monomers 21 and 22, respectively (Scheme 3). Both the
monomeric precursors were, separately and sequentially,
subjected to LiOH-mediated ester hydrolysis in THF-
MeOH-H2O solvent system to provide the acids which
further underwent TFA-mediated Boc-deprotection in
CH2Cl2 to give TFA-salts in quantitative yields. The TFA-
salts were then subjected to homooligomerization using the
same reagents under identical reaction conditions as
described above (for the synthesis of compounds
1
and
2) to
furnish the glycosylated tricyclic peptides and
3
4
as the
major products in 32% and 29% yield, respectively (the
corresponding C2-symmetric glycosylated dimers were also
formed in both cases, but in negligible yields).
O
R1/2
O
1) LiOH.H2O
THF:MeOH:H2O (3:1:1)
rt, 1 h, quant. yield
a concentration range of 0.625 mM to 20 mM in CDCl3,
N
H
O
O
21
OR1/2
Figure 4. 2D-ROESY expansion and nOe representations for 1;
characteristic nOes between NH-C3H(pro-R), NH-C6H(pro-S), NH-
C4H, NH-C6H(pro-R), NH-C5H, C5H-C6H(pro-S), C5H-C6H(pro-R)
C6H(pro-R)-C4H, C6H(pro-R)-SugC1H, C4H-SugC1H, C6H(pro-S)
or
2) TFA, CH2Cl2, 0 oC to rt
2 h, quant. yield
O
NH
HN
22
,
-
O
3) FDPP, DIPEA, CH3CN
0 oC to rt, 72h
O
SugC1H are marked as 1-11, respectively.
R1/2
O
→
→
21
22
3
4
3 (R1, 32%)
4 (R2, 29%)
revealed variations in the chemical shifts of the amide protons in
both 1 and 2 (0.038 and 0.037 ppm, respectively). These
observations suggested that the amide protons may be
participating in intermolecular H-bonding and thus both the
glycopeptides were self-aggregating at concentrations > 5 mM
(Figure 5).
Scheme 3. Synthesis of the C3-symmetric glycosylated
cyclotrimers and
3
4.
3. Conformational Analysis: NMR studies.
Conformational studies of cyclic glycopeptides 1-4 were
carried out in CDCl3 with the sample concentrations of 2−5 mM.
Both 1H, 13C spectra of cyclodimers 1 and 2 showed only one set
of sharp resonances corresponding to the constituent δ-SAA
moiety indicating C2-symmetry among these cyclic peptides. The
vicinal coupling constant values (3JNH-C6H) for 1 and 2 showed
either < 3 Hz or > 10 Hz, which indicated that the dihedral angle
3
φ (CO−N−C6−C5) was about 120°. JC6H−C5H values of 4 Hz and
0 Hz clearly supported the dihedral angle θ (N−C6−C5−O) as
−30° (Figure 3). Observations of nOe cross peaks between
NH↔C6H(pro-R), C5H↔C6H(pro-S) and C5H↔C6H(pro-R) suggested
a (−) synclinal (sc) rotation about C5−C6 bond in both the
glycopeptides 1 and 2. Additionally, nOes between NH↔C5H,
C5H↔C2H, C5H↔C2H and C4H↔C6H(pro-R) confirmed the (−)
sc rotation in both these glycopeptides (Figures 3 and 4). Both,
nOe correlations [C5H−C3H(pro-R)] and vicinal coupling constant
Figure 5. 1H-NMR spectra showing NH peaks as a function of
concentration for glycopeptide 1 in CDCl3.
3
3
values JC2H−C3H(pro-S) of 1.1 Hz and JC2H−C3H(pro-R) of 7.4 Hz
suggested that the five-membered furanoid ring was with C4-
endo ring puckering in both 1 and 2.
Conformational studies were also carried out on
glycopeptides 3 and 4. The 1H, 13C NMR spectra of compounds 3
and 4 in CDCl3 also showed only one set of sharp resonances
indicating C3-symmetry in these cyclotrimeric glycopeptides. The
vicinal coupling constant values (3JNH-C6H) were either < 3 Hz or
N
2
H
(R)
3
H(S)
>
6
Hz, which indicated that the dihedral angle
φ
O
1
H
(CO−N−C6−C5) is about 140°. 3JC6H-C5H value of either < 2 Hz or
> 10 Hz indicated that the dihedral angle θ (N−C6−C5−O) was
about +30° (Figure 6). Observations of nOe cross peaks between
NH↔C6H, C5H↔C6H(pro-S) and C5H↔C6H(pro-R) suggested a
(+) synclinal (sc) rotation about C5−C6 bond in both the
glycopeptides. Further nOes between NH↔C5H, NH↔C4H,
C5H↔C2H, C4H↔C2H and C4H↔C6H, C2H↔C6H(pro-R) and
C2H↔C6H(pro-S) confirmed the (+) sc rotation in both
glycopeptides (Figure 6 and 7). Additionally, nOe correlations
O
4
O
6
N
H
5
2
H(pro-R)
H
4
3
H(R)
RO
H
H5
(pro-S)H
O
MeO
MeO
R =
(
) SC
MeO

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4
Tetrahedron
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between C2H↔C6H(pro-S), C2H−C5H, C2H−C4H and the
RMSD values calculated with all atoms and heavy atoms were
coupling constant values of the protons16 further suggested that
the five-membered furanoid ring was with C2-endo ring
puckering in both the cyclotrimeric glycopeptides 3 and 4.
1.018 Å, 0.784 Å and 1.65 Å, 1.28 Å for compounds 1 and 2,
respectively. Likewise, for the cyclic trimers, the RMSD values
calculated for all atoms and heavy atoms are 1.45 Å, 1.12 Å and
1.52 Å, 1.26 Å for compounds 3 and 4, respectively. Similar
coupling constant values and nOes observed in the cyclic dimers
1,2 and the cyclic trimers 3,4 confirm analogous backbone
conformations among these cyclic glycopeptides irrespective of
the anomeric configurations of the attached sugars (Figure 8).
H(R)
N
H(S)
O
1
H
O
O
3
H5
(pro-S)H
6
N
H
5
2
2
H
4
3
H(R)
H(pro-R)
RO
H
4
O
MeO
+ SC
R = MeO
MeO
Figure 6. Newman projection and nOe representation for 3.
A
B
Figure 8. 15 superimposed structures of (A) cyclic dimers 1
(purple) and 2 (green); (B) cyclic trimers 3 (green) and 4
(purple); after superposition the attached sugars were removed
for clarity.
5. Antimicrobial activity
The synthesized glycopeptides 1-4 and their unglycosylated
congeners 2518 and 2618 (Figure 9) were tested for their
antimicrobial activities against 4 bacterial strains and 6 fungal
strains, of which many were multidrug-resistant strains.16 For the
purpose of comparison, gentamycin and norfloxacin were
employed as positive controls for antibacterial activity and
amphotericin-B and fluconazole were employed as positive
controls for antifungal activity.
Figure 7. 2D-ROESY expansion and nOe representations for 3;
characteristic nOes between NH↔C6H(pro-R), NH↔C6H(pro-S)
,
,
,
NH↔C5H, C5H↔C6H(pro-R), C5H↔C6H(pro-R), C4H↔C6H(pro-R)
C5H↔C6H(pro-S)
,
C2H↔C6H(pro-R)
,
SugC1H↔C6H(pro-R)
SugC1H↔C5H are marked as 1-10, respectively.
O
OH
HN
HO
Aggregation studies carried out as a function of concentration
from 0.6 mM to 20 mM revealed the chemical shift changes of
the amide protons in both the glycopeptides 3 and 4 as 0.019 ppm
and 0.024 ppm, respectively. These observations further
suggested that the molecules were self-aggregating and the amide
protons were involved in intermolecular H-bonding.16
N
H
O
O
O
O
O
OH
NH
HO
O
NH
HN
O
O
O
HO
25
26
4. Molecular Dynamics
Figure 9. Non-glycosylated precursors 25 and 26.
While the cyclic non-glycosylated homooligomers 25 and 26
did not show any antimicrobial or antifungal activity at all (both
showing MICs > 100 µM against all strains), their glycosylated
counterparts 1-4 showed relatively better activity. The cyclic
peptides 1-3 {MIC, 1 (100 µM), 2 (50 µM) and 3 (50 µM)}
showed moderate activity only against bacterium E. coli, but
remained inactive against the rest of the bacterial strains. All the
cyclic peptides {MIC, 1 (100 µM), 2 (50-100 µM), 3 (100 µM)
and 4 (100 µM)} showed activity against fungus strains S.
Energy minimization and simulated molecular dynamics
(MD) calculations were performed on Discovery studio 3.0
version,17a using CHARMm force field17b with default parameters
throughout the simulation with the aid of distance dependent
dielectric constant with ε = 4.81 (dielectric constant for CDCl3).
Distance restraints used in the MD were calculated from the
volume integrals of the cross peaks in the ROESY spectra with
the use of two-spin approximation with a reference distance of
1.80 Å for the geminal protons. Force constants of 10 K cal/Å, 5
K cal/Å were employed for distance and torsional restraints
respectively. Minimization was done with steepest descent
algorithm followed by conjugate gradient methods for maximum
1000 iterations each iterations or RMS deviation of 0.001 Kcal
/mol, whichever was earlier. The molecules were initially
equilibrated for 5 pS and then subjected to 1 nS production run.
Starting from 50 K, they were heated to 300 K in five steps,
increasing the temperature 50 K at each step. 20 structures were
stored from the production run and were again energy minimized
with the above-mentioned protocol. Superposition was done on
to the average structure of these minimized structures. The
schenckii, T. mentagrophytes, A.
fumigatus, except C.
parapsilosis. The data suggested that the cyclic glycopeptides 1-3
displayed moderate antimicrobial activities only against E. coli,
but all of them showed moderate antifungal activities against
three out of four strains.
6. Conclusion
The glycosylated furanoid sugar amino acid based cyclic
homooligomers constitute a new class of novel molecular
scaffolds that displayed interesting 3D structures in CDCl3.
Conformational studies of cyclodimeric compounds 1 and 2 with

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5
2R and 5R stereocenters revealed that the C2H and CO atoms
has influenced the activity and up to what extent the impact of
ACCEPTED MANUSCRIPT
were placed on same side of the ring while the NHs were
pointing to other side. The dihedral angle ψ noticed between NH-
CO-C2H-O was ≈ 50˚. Both the CO and NH were arranged
perpendicular to the ring plane. Conformational studies of cyclic
trimers 3 and 4 with 2S and 5R stereocenters in CDCl3 showed
that the C2H and CO were positioned outside the plane of the
ring similar to the cyclic dimer and NHs pointed inside the ring.
The dihedral angle ψ noticed between NH-CO-C2H-O was ≈ 15˚.
such influence can be generated via implementing variable sugar
moieties on different SAA backbones is yet to be ascertained.
Besides, the conformational studies carried out here will also be
useful in developing new SAA based scaffolds amenable to
glycosylation for further enhancement of their biological,
pharmacological and pharmacokinetic properties. NMR analysis
suggested that these cyclic glycopeptides have well defined
structures in solution that are independent of the anomeric
configurations of the attached sugar and can serve as simple, and
yet versatile, tool to investigate many molecular recognition
processes of biological significance, especially taking advantage
of the unlimited structural diversities accessible for the appended
carbohydrates. Further work is under progress.
That the cyclic glycopeptides showed better activity than their
non-glycosylated precursors lends support to the plausible role of
the attached sugars on modulating their antimicrobial and
antifungal activities. But the manner in which O-glycosylation
7. Experimental section
7.1. General
(neat liquid) 3409, 3019, 2399, 1644, 1403, 1215, 1108, 928,
758, 669 cm–1; δH (300 MHz, CDCl3) 4.68 (1H, dd, J 8.9, 2.9
Hz), 4.32 - 4.22 (2H, m), 3.78 (3H, s), 3.48 (1H, dd, J 12.9, 4.1
Hz), 3.31 (1H, dd, J 12.9, 4.1 Hz), 2.93 (1H, br), 2.56 - 2.43 (1H,
m), 2.23 - 2.12 (1H, m); δC (100 MHz, CDCl3) 174.5, 86.5, 77.0,
73.7, 52.7, 52.6, 38.8; HRMS: [M+Na]+, found 224.0647.
C7H11N3O4Na requires 224.0642.
All the reactions were carried out under an inert atmosphere in
oven-dried glassware using dry solvents, unless otherwise stated.
All chemicals purchased from commercial suppliers were used as
received unless otherwise stated. Reactions and chromatography
fractions were monitored by silica gel 60 F-254 glass TLC plates
and visualized using UV light, 7% ethanolic phosphomolybdic
acid-heat or 2.5% ethanolic anisaldehyde (with 1% AcOH and
3.3% conc.H2SO4)-heat as developing agents. Flash column
chromatography was performed with 100-200 mesh silica gel and
yields refer to chromatographically and spectroscopically pure
compounds. All NMR spectra were recorded in CDCl3 on 300,
400 and 500 MHz instruments at 300 K and are calibrated to
chloroform solvent residual peak (7.26 ppm) as a reference for
the proton NMR spectra and deuterated chloroform peak (77.16
ppm) as a reference for carbon NMR spectra. Multiplicities are
abbreviated as: s = singlet; d = doublet; t = triplet; q = quartet; m
= multiplate. All IR data were recorded as neat liquid or KBr
pellets. HRMS spectra were taken under ESI-Q-TOF conditions.
Optical rotations were measured using sodium (589 nm, D line)
lamp and are reported as follows: [α]D25 [c (g/100 mL), CHCl3].
7.4. Synthesis of compounds 15 and 16
Compound 12 (48 mg, 0.24 mmol) was dissolved in dry
CH3CN (3 mL) and cooled to 0 °C under Argon atmosphere.
Then the solution of the glycosyl donor 14 (75 mg, 0.36 mmol) in
CH3CN (2 mL) was added by cannulation followed by drop wise
addition of BF3·Et2O (45 µL, 0.36 mmol) at the same
temperature. The reaction mixture was stirred at room
temperature for 2 h and then quenched by saturated aqueous
NaHCO3 solution (5 mL) and extraction was done with EtOAc (2
x 20 mL). The combined organic extracts were washed with
water (5 mL), brine (5 mL), dried (Na2SO4), filtered and
concentrated in vacuo to give the crude product, which was
purified by flash chromatography (20% EtOAc/Hexane) to give
the title compounds 15 (48 mg, 53%) and 16 (32 mg, 36%) as
yellow oils in a ratio of 1.5:1 (15:16) with overall 89% yield;
28
7.2. Synthesis of compound 12
Data for 15: Rf (40% EtOAc/hexane) 0.45; [α]D +42.6 (c 0.32,
CHCl3); νmax (neat liquid) 3410, 3019, 2399, 2105, 1644, 1403,
1215, 1107, 929, 758, 669 cm–1; δH (300 MHz, CDCl3) 4.88 (1H,
d, J 3.3 Hz), 4.68 (1H, t, J 7.6 Hz), 4.30 - 4.15 (2H, m), 3.76 (3H,
s), 3.71 (1H, dd, J 10.7, 5.0 Hz), 3.60 (3H, s), 3.49 (3H, s), 3.47
(3H, s), 3.46 – 3.42 (2H, m), 3.42 – 3.33 (2H, m), 3.30 – 3.20
(1H, m), 3.14 (1H, dd, J 9.3, 3.3 Hz), 2.41 – 2.19 (2H, m); δC
(100 MHz, CDCl3) 172.2, 95.9, 83.9, 83.3, 81.3, 79.8, 77.8, 61.0,
60.2, 59.2, 59.1, 52.7, 52.5. 35.8; HRMS: [M+Na]+, found
398.1525. C15H25N3O8Na requires 398.1539.
To a stirred solution of 10 (2.33 g, 8.02 mmol) in dry CH2Cl2
(24 mL) was slowly added BCl3 solution (24 mL, 24 mmol, 1.0
M in CH2Cl2) at 0 °C and stirring was continued for 10 min at the
same temperature. The reaction mixture was quenched at 0 °C by
slow addition of saturated aqueous NaHCO3 solution (50 mL)
and extraction was done with EtOAc (2 x 200 mL). The
combined organic extracts were washed with water (60 mL),
brine (50 mL), dried (Na2SO4), filtered and concentrated in vacuo
to give the crude product, which was purified by flash
chromatography (30% EtOAc/Hexane) to give the title
compound 12 (1.40 g, 87%) as a yellow oil; Rf (50% EtOAc
Data for 16: Rf (40% EtOAc/hexane) 0.5; [α]D29 +7.17 (c 0.36,
CHCl3); νmax (neat liquid) 3409, 3019, 2399, 1644, 1403, 1215,
1107, 928, 758, 669 cm–1; δH (300 MHz, CDCl3) 4.65 (1H, t, J
7.9 Hz), 4.29 - 4.13 (3H, m), 3.96 (1H, dd, J 11.4, 4.8 Hz), 3.76
(3H, s), 3.60 (3H, s), 3.53 (3H, s), 3.46 (3H, s), 3.45 - 3.38 (2H,
m), 3.30 - 3.18 (1H, m), 3.16 - 3.03 (2H, m), 2.94 (1H, dd, J 8.7,
7.6 Hz), 2.52 - 2.39 (1H, m), 2.33 - 2.19 (1H, m); δC (100 MHz,
CDCl3) 172.3, 103.7, 85.3, 84.1, 83.4, 80.8, 79.3, 63.6, 60.9,
60.8, 58.9, 52.8, 52.4, 37.3; HRMS: [M+Na]+, found 398.1518.
C15H25N3O8Na requires 398.1539.
27
/hexane) 0.5; [α]D +73.1 (c 0.60, CHCl3); νmax (neat liquid)
3409, 3019, 2105, 1740, 1641, 1403, 1215, 1109, 928, 758, 669
cm–1; δH (300 MHz, CDCl3) 4.69 (1H, t, J 7.7 Hz), 4.37 - 4.30
(1H, m), 4.08 - 3.99 (1H, m), 3.76 (3H, s), 3.51 - 3.36 (2H, m),
2.27 (2H, dd, J 7.7, 4.8 Hz), 2.18 (1H, br); δC (100 MHz, CDCl3)
172.6, 85.9, 76.7, 73.4, 52.7, 52.4, 38.7; HRMS: [M+Na]+, found
224.0642. C7H11N3O4Na requires 224.0642.
7.3. Synthesis of compound 13
7.5. Synthesis of compounds 17 and 18
Compound 13 was synthesized from 11 (2.50 g, 8.58 mmol)
following the same procedure described above for the synthesis
of 12 with the same reagents under identical reaction conditions
to give the title compound 13 (1.45 g, 84%) as a yellow oil; Rf
Compounds 17 and 18 were synthesized from 13 (74 mg, 0.37
mmol) and 14 (115 mg, 0.56 mmol), respectively following the
same procedure described above for the synthesis of 15 and 16
with the same reagents under identical reaction conditions to give
the title compounds 17 (69 mg, 50%) and 18 (45 mg, 34%) as
28
(50% EtOAc/hexane) 0.45; [α]D +88.5 (c 0.44, CHCl3); νmax

ACCEPTED MANUSCRIPT
6
Tetrahedron
yellow oils in a ratio of 1.5:1 (17:18) with overall 84% yield;
of 20 with the same reagents under identical reaction conditions
ACCEPTED MANUSCRIPT
28
Data for 17: Rf (40% EtOAc/hexane) 0.3; [α]D +121.5 (c 0.30,
CHCl3); νmax (neat liquid) 3409, 3019, 2106, 1641, 1402, 1385,
1215, 1160, 1097, 758, 668 cm–1; δH (300 MHz, CDCl3) 4.85
(1H, d, J 3.3 Hz), 4.68 (1H, dd, J 8.4, 4.2 Hz), 4.40-4.32 (1H, m),
4.26 - 4.19 (1H, m), 3.77 (3H, s), 3.71 (1H, dd, J 10.7, 5.3 Hz),
3.57 (3H, s), 3.55 – 3.51 (1H, m), 3.46 (3H, s), 3.43 (3H, s), 3.37
– 3.26 (3H, m), 3.25 – 3.16 (1H, m), 3.09 (1H, dd, J 9.4, 3.5 Hz),
2.55 – 2.41 (1H, m), 2.30 (1H, dt, J 13.3, 3.9 Hz); δC (100 MHz,
CDCl3) 172.8, 95.8, 83.4, 82.1, 81.3, 79.6, 77.4, 76.6, 60.9, 60.2,
59.1, 58.8, 52.4, 52.2, 36.3; HRMS: [M+Na]+, found 398.1532.
C15H25N3O8Na requires 398.1539.
to give the title compound 21 (236 mg, 69%) as a colourless oil;
30
Rf (60% EtOAc/hexane) 0.45; [α]D +64.5 (c 0.34, CHCl3); νmax
(neat liquid) 3410, 3019, 2399, 1640, 1385, 1215, 1159, 1097,
758, 669 cm–1; δH (300 MHz, CDCl3) 4.86 (1H, d, J 3.3 Hz),
4.82 (1H, br), 4.62 (1H, dd, J 8.4, 4.2 Hz), 4.30 - 4.22 (1H, m),
4.19 - 4.11 (1H, m), 3.76 (3H, s), 3.71 (1H, dd, J 10.7, 5.4 Hz),
3.57 (3H, s), 3.45 (3H, s), 3.43 (3H, s), 3.38 – 3.16 (5H, m), 3.08
(1H, dd, J 9.3, 3.4 Hz), 2.46 – 2.26 (2H, m), 1.43 (9H, s); δC
(100 MHz, CDCl3) 172.9, 156.0, 95.5, 83.9, 81.9, 81.2, 79.6,
76.2, 60.7, 60.0, 58.7, 58.5, 52.1, 42.3, 36.1, 28.3; HRMS:
[M+H]+, found 450.2344. C20H36NO10 requires 450.2339.
Data for 18: Rf (40% EtOAc/hexane) 0.4; [α]D25 +34.0 (c 0.34,
CHCl3); νmax (neat liquid) 3408, 3019, 2105, 1639, 1402, 1385,
1215, 1158, 1071, 758, 669 cm–1; δH (300 MHz, CDCl3) 4.65
(1H, dd, J 8.6, 4.6 Hz), 4.34 - 4.27 (1H, m), 4.25 - 4.17 (2H, m),
3.95 (1H, dd, J 11.4, 4.8 Hz), 3.74 (3H, s), 3.58 (3H, s), 3.57 –
3.53 (1H, m), 3.51 (3H, s), 3.45 (3H, s), 3.32 (1H, dd, J 13.2, 3.8
Hz), 3.26 - 3.17 (1H, m), 3.15 - 3.03 (2H, m), 2.88 (1H, dd, J 8.7,
7.6 Hz), 2.63 - 2.50 (1H, m), 2.36 (1H, dt, J 13.6, 4.2 Hz); δC
(100 MHz, CDCl3) 172.8, 103.9, 85.2, 83.5, 83.4, 80.0, 79.4,
76.6, 63.4, 60.8, 60.7, 58.8, 52.3, 37.7; HRMS: [M+Na]+, found
398.1536. C15H25N3O8Na requires 398.1539.
7.9. Synthesis of compound 22
Compound 22 was synthesized from 18 (229 mg, 0.61 mmol)
following the same procedure described above for the synthesis
of 21 with the same reagents under identical reaction conditions
to give the title compound 22 (175 mg, 64%) as a yellow oil; Rf
29
(60% EtOAc/hexane) 0.5; [α]D -7.9 (c 0.70, CHCl3); νmax (neat
liquid) 3409, 3019, 1643, 1402, 1385, 1215, 1157, 1069, 769,
669 cm–1; δH (300 MHz, CDCl3) 4.83 (1H, br), 4.58 (1H, dd, J
8.6, 4.6 Hz), 4.27 (1H, d, J 7.4 Hz), 4.22 - 4.09 (2H, m), 3.93
(1H, dd, J 11.4, 4.8 Hz), 3.73 (3H, s), 3.59 (3H, s), 3.52 (3H, s),
3.45 (3H, s), 3.39 - 3.27 (2H, m), 3.26 - 3.17 (1H, m), 3.15 -
3.05 (2H, m), 2.88 (1H, dd, J 8.5, 7.4 Hz), 2.56 - 2.43 (1H, m),
2.35 (1H, dt, J 13.6, 4.1 Hz), 1.44 (9H, s); δC (75 MHz, CDCl3)
172.9, 156.1, 103.6, 85.1, 83.9, 83.4, 80.0, 79.4, 76.3, 63.4, 60.8,
60.7, 58.8, 52.2, 42.4, 37.5, 28.5; HRMS: [M+H]+, found
450.2342. C20H36NO10 requires 450.2339.
7.6. Synthesis of compound 19
To a stirred solution of compound 15 (250 mg, 0.67 mmol),
(Boc)2O (0.31 mL, 1.34 mmol) and NEt3 (0.23 mL, 1.68 mmol)
in MeOH (10 mL) was added 10% Pd-C (100 mg) and subjected
to hydrogenation condition using a hydrogen filled balloon for 5
h. The reaction mixture was filtered through a short pad of Celite
and further washed with EtOAc (3 x 30 mL). The combined
organic extracts were concentrated in vacuo to give the crude
product, which was purified by flash chromatography (35%
EtOAc/Hexane) to give the title compound 19 (205 mg, 68%) as
7.10. Synthesis of compound 23
To a solution of 19 (290 mg, 0.65 mmol) in THF:MeOH:H2O
(3:1:1, 5 mL) at 0 °C was added LiOH·H2O (164 mg, 3.9 mmol)
and the mixture was stirred at room temperature for 1 h. The
reaction mixture was then acidified to pH 2 with aqueous 1 N
HCl. The reaction mixture was extracted with EtOAc (2 x 100
mL) and the combined organic extracts were washed with water
(50 mL), brine (30 mL), dried (Na2SO4), filtered and
concentrated in vacuo to obtain the acid.
To a solution of 19 (306 mg, 0.68 mmol) in dry CH2Cl2 (10
mL) at 0 °C was added TFA (3.3 mL) and the mixture was stirred
for 2 h at room temperature. The reaction mixture was then
concentrated and co-evaporated using dry CH2Cl2 in vacuo to
obtain the TFA-salt.
28
a colourless oil; Rf (55% EtOAc/hexane) 0.45; [α]D +18.9 (c
0.24, CHCl3); νmax (neat liquid) 3425, 2925, 1637, 1398, 1219,
1098, 769 cm–1; δH (300 MHz, CDCl3) 5.27 (1H, br), 4.88 (1H,
d, J 3.3 Hz), 4.65 (1H, t, J 7.8 Hz), 4.26 - 4.12 (2H, m), 3.76 (3H,
s), 3.71 (1H, dd, J 11.0, 5.5 Hz), 3.60 (3H, s), 3.48 (3H, s), 3.46
(3H, s), 3.43 – 3.30 (4H, m), 3.28 – 3.18 (1H, m), 3.13 (1H, dd, J
9.5, 3.5 Hz), 2.44 – 2.30 (1H, m), 2.24 - 2.14 (1H, m), 1.43 (9H,
s); δC (100 MHz, CDCl3) 173.0, 156.1, 95.7, 84.0, 82.1, 81.4,
79.7, 76.4, 60.9, 60.2, 58.7, 52.4, 42.4, 36.2, 28.5; HRMS:
[M+Na]+, found 472.2185. C20H35NO10Na requires 472.2159.
To a stirred solution of the acid in dry CH2Cl2 (5 mL) at 0 °C
was added HOBt (132 mg, 0.98 mmol) and EDCI (188 mg, 0.98
mmol). The mixture was stirred for another 30 min at room
temperature followed by addition of a solution of TFA-salt in dry
CH2Cl2 (5 mL) by cannulation and slow addition of DIPEA (0.57
mL, 3.25 mmol) at 0 °C. After stirring for 12 h at room
temperature, the reaction mixture was quenched with aqueous 1
N HCl (5 mL) and the solvent was evaporated and extraction was
done with CHCl3 (3 x 50 mL). The combined organic extracts
were washed with aqueous 1 N HCl (2 x 10 mL), saturated
aqueous NaHCO3 solution (2 x 10 mL), water (40 mL), brine (30
mL), dried (Na2SO4), filtered and concentrated in vacuo to give
the crude product, which was purified by flash chromatography
(2% MeOH/CHCl3) to give the title compound 23 (358 mg, 72%)
7.7. Synthesis of compound 20
Compound 20 was synthesized from 16 (200 mg, 0.53 mmol)
following the same procedure described above for the synthesis
of 19 with the same reagents under identical reaction conditions
to give the title compound 20 (167 mg, 70%) as a colourless oil;
27
Rf (55% EtOAc/hexane) 0.5; [α]D +2.14 (c 0.28, CHCl3); νmax
(neat liquid) 3425, 3021, 2925, 1637, 1399, 1216, 1091, 768, 670
cm–1; δH (300 MHz, CDCl3) 5.30 (1H, br), 4.63 (1H, t, J 7.7 Hz),
4.31 - 4.19 (2H, m), 4.17 - 4.09 (1H, m), 3.94 (1H, dd, J 11.4, 4.7
Hz), 3.76 (3H, s), 3.59 (3H, s), 3.53 (3H, s), 3.46 (3H, s), 3.41 -
3.34 (1H, m), 3.32 – 3.18 (2H, m), 3.16 - 3.03 (2H, m), 2.93
(1H, dd, J 8.6, 7.6 Hz), 2.53 - 2.41 (1H, m), 2.26 - 2.12 (1H, m),
1.44 (9H, s); δC (100 MHz, CDCl3) 173.4, 156.3, 103.5, 85.3,
84.5, 83.5, 80.3, 79.4, 76.5, 63.5, 60.9, 60.7, 58.9, 52.4, 42.5,
37.7, 28.5; HRMS: [M+Na]+, found 472.2141. C20H35NO10Na
requires 472.2159.
26
as a yellow oil; Rf (4% MeOH/EtOAc) 0.3; [α]D +97.2 (c 0.20,
CHCl3); νmax (neat liquid) 3409, 1643, 1402, 1385, 1216, 1157,
1069, 770, 668 cm–1; δH (400 MHz, CDCl3) 7.52 (1H, br), 5.46
(1H, br), 4.90 (1H, d, J 3.6 Hz), 4.87 (1H, d, J 3.6 Hz), 4.67 (1H,
t, J 7.6 Hz), 4.57 (1H, dd, J 9.1, 7.1 Hz), 4.28 - 4.17 (2H, m),
4.16 – 4.09 (2H, m), 3.77 (3H, s), 3.71 (1H, dd, J 10.8, 5.7 Hz),
3.60 (3H, s), 3.59 (3H, s), 3.51 – 3.48 (2H, m), 3.48 (3H, s), 3.47
7.8. Synthesis of compound 21
Compound 21 was synthesized from 17 (285 mg, 0.76 mmol)
following the same procedure described above for the synthesis

ACCEPTED MANUSCRIPT
7
(3H, s), 3.46 (3H, s), 3.45 (3H, s), 3.43 - 3.34 (6H, m), 3.28 -
Compound 2 was synthesized from 24 (135 mg, 0.18 mmol)
ACCEPTED MANUSCRIPT
3.19 (3H, m), 3.17 - 3.09 (2H, m), 2.47 - 2.35 (2H, m), 2.25 -
2.12 (1H, m), 2.09 - 1.98 (1H, m), 1.43 (9H, s); δC (100 MHz,
CDCl3) 174.2, 172.3, 170.5, 156.4, 96.2, 95.3, 84.6, 82.3, 81.3,
79.8, 78.0, 75.9, 75.8, 61.0, 60.2, 60.1, 59.1, 59.0, 58.9, 52.7,
52.6, 43.1, 40.2, 36.2, 28.5; HRMS: [M+H]+, found 767.3798.
C34H59N2O17 requires 767.3814.
following the same procedure described above for the synthesis
of 1 with the same reagents under identical reaction conditions to
give the title compound 2 (29 mg, 52%) as a yellow oil; Rf (10%
30
MeOH/EtOAc) 0.5; [α]D -71.9 (c 0.14, CHCl3); νmax (neat
liquid) 3412, 3019, 2399, 1637, 1523, 1475, 1421, 1384, 1215,
1083, 928, 757, 669, 627 cm–1; δH (500 MHz, CDCl3) 7.04 (1H,
dd, J 10.2, 2.6 Hz), 4.55 - 4.48 (2H, m), 4.13 (1H, dd, J 14.9,
10.2 Hz), 3.99 - 3.89 (2H, m), 3.74 - 3.63 (1H, m), 3.60, (3H, s),
3.56 (3H, s), 3.45 (3H, s), 3.26 – 3.14 (4H, m), 2.96 (1H, dd, J
8.7, 7.5 Hz), 2.75 - 2.66 (1H, m), 2.37 - 2.26 (1H, m); δC (125
MHz, CDCl3) 172.8, 104.0, 84.8, 83.4, 83.1, 79.5, 77.7, 76.8,
63.2, 60.8, 60.4, 58.7, 37.4, 36.4; HRMS: [M+Na]+, found
657.2822. C28H46N2O14Na requires 657.2847.
7.11. Synthesis of compound 1
To a stirred solution of 23 (200 mg, 0.26 mmol) in
THF:MeOH:H2O (3:1:1, 5 mL) at 0 °C was added LiOH·H2O (66
mg, 1.56 mmol) and the mixture was stirred at room temperature
for 1 h. The reaction mixture was then acidified to pH 2 with
aqueous 1 N HCl. The reaction mixture was extracted with
EtOAc (2 x 50 mL) and the combined organic extracts were
washed with water (20 mL), brine (20 mL), dried (Na2SO4),
filtered and concentrated in vacuo to obtain the acid. The acid
was again dissolved in dry CH2Cl2 (5 mL) followed by slow
addition of TFA (1.7 mL) at 0 °C and the mixture was stirred for
2 h at room temperature. The reaction mixture was then
concentrated and co-evaporated using dry CH2Cl2 in vacuo to
obtain the TFA-salt.
To a stirred solution of the TFA-salt in dry CH3CN (25 mL, 5
x 10-3 M dilution) at 0 °C was added FDPP (300 mg, 0.78 mmol)
and stirred for 30 min at room temperature followed by the slow
addition of DIPEA (0.32 mL, 1.82 mmol) at 0 °C. After stirring
for 72 h at room temperature, the solvent was evaporated and
extraction was done with CHCl3 (3 x 50 mL). The combined
organic extracts were washed with aqueous 10% NaOH solution
(2 x 10 mL), water (20 mL), brine (20 mL), dried (Na2SO4),
filtered and concentrated in vacuo to give the crude product,
which was purified by flash chromatography (5% MeOH/CHCl3)
to give the title compound 1 (44 mg, 54%) as a yellow oil; Rf
(12% MeOH/EtOAc) 0.5; [α]D30 +2.73 (c 0.22, CHCl3); νmax (neat
liquid) 3415, 3019, 2400, 1637, 1384, 1215, 1084, 758, 669 cm–1;
δH (500 MHz, CDCl3) 7.05 (1H, dd, J 10.0, 2.8 Hz), 5.00 (1H, d,
J 3.7 Hz), 4.57 - 4.52 (1H, m), 4.20 (1H, dd, J 14.6, 10.2 Hz),
4.08 (1H, dd, J 8.2, 4.0 Hz), 3.86 - 3.81 (1H, m), 3.73 (1H, dd, J
11.5, 5.8 Hz), 3.63 – 3.59 (1H, m), 3.60 (3H, s), 3.47 (3H, s),
3.46 (3H, s), 3.40 (1H, t, J 9.2 Hz), 3.26 - 3.17 (2H, m), 3.15
(1H, dd, J 9.7, 3.7 Hz), 2.79 – 2.74 (1H, m), 2.24 (1H, dt, J 12.3,
9.8 Hz); δC (125 MHz, CDCl3) 172.5, 95.6, 83.4, 82.2, 81.4,
79.8, 77.5, 73.7, 61.0, 60.3, 59.0, 58.9, 37.5, 34.6; HRMS:
[M+Na]+, found 657.2824. C28H46N2O14Na requires 657.2847.
7.14. Synthesis of compound 3
Compound 3 was synthesized from 21 (327 mg, 0.72 mmol)
following the same procedure described above for the synthesis
of 1 and 2 with the same reagents under identical reaction
conditions to give the title compound 3 (73 mg, 32%) as a yellow
30
oil; Rf (20% MeOH/EtOAc) 0.5; [α]D +57.9 (c 0.18, CHCl3);
νmax (neat liquid) 3424, 3019, 2399, 1637, 1403, 1215, 1084, 928,
758, 669 cm–1; δH (500 MHz, CDCl3) 6.81 (1H, dd, J 6.4, 2.7
Hz), 4.89 (1H, d, J 3.2 Hz), 4.48 (1H, t, J 8.3 Hz), 4.14 - 4.06
(2H, m), 3.84 - 3.71 (2H, m), 3.61 (3H, s), 3.48 (3H, s), 3.46 (3H,
s), 3.42 - 3.32 (2H, m), 3.28 - 3.11 (3H, m), 2.85 – 2.78 (1H, m),
2.24 - 2.13 (1H, m); δC (125 MHz, CDCl3) 171.5, 95.8, 82.2,
81.3, 81.2, 79.7, 61.1, 60.4, 59.0, 58.9, 42.2, 34.9; HRMS:
[M+Na]+, found 974.4316. C42H69N3O21Na requires 974.4321.
7.15. Synthesis of compound 4
Compound 4 was synthesized from 22 (290 mg, 0.65 mmol)
following the same procedure described above for the synthesis
of 3 with the same reagents under identical reaction conditions to
give the title compound 4 (59 mg, 29%) as a yellow oil; Rf (20%
MeOH/EtOAc) 0.3; [α]D30 -7.9 (c 0.38, CHCl3); νmax (neat liquid)
3430, 3019, 2400, 1637, 1403, 1215, 1084, 928, 758, 669 cm–1;
δH (500 MHz, CDCl3) 6.79 (1H, dd, J 6.8, 2.1 Hz), 4.45 (1H, t, J
8.4 Hz), 4.25 (1H, d, J 7.3 Hz), 4.01 (1H, q, J 7.6 Hz), 3.97 –
3.92 (2H, m), 3.83 (1H, ddd, J 13.6, 7.2, 2.1 Hz), 3.58 (3H, s),
3.52 (3H, s), 3.45 (3H, s), 3.25 - 3.20 (1H, m), 3.17 - 3.05 (3H,
m), 2.94 (1H, dd, J 8.8, 7.3 Hz), 2.75 (1H, dt, J 13.3, 7.5 Hz),
2.28 (1H, dt, J 13.3, 8.6 Hz); δC (100 MHz, CDCl3) 171.6, 104.1,
85.0, 83.2, 81.4, 81.1, 79.3, 77.0, 63.3, 60.8, 60.7, 58.8, 42.3,
36.6; HRMS: [M+Na]+, found 974.4287. C42H69N3O21Na requires
974.4321.
7.12. Synthesis of compound 24
Compound 24 was synthesized from 20 (513 mg, 1.14 mmol)
following the same procedure described above for the synthesis
of 23 with the same reagents under identical reaction conditions
to give the title compound 24 (602 mg, 69%) as a yellow oil; Rf
8. Antimicrobial activity: Material and methods
In vitro antimicrobial activities of compounds were tested against
6 medically important fungi; Candida albicans (Ca, patient
isolate), Cryptococcus neoformans (Cn, patient isolate),
Sporothrix schenckii (Ss, patient isolate), Trichophyton
mentagrophytes (Tm, patient isolate), Aspergillus fumigatus (Af,
patient isolate) and C. parapsilosis (Cp, ATCC-22019) and 4
bacteria viz, Escherichia coli (Ec, ATCC 9637), Pseudomonas
aeruginosa (Psu, ATCC BAA-427), Staphyllococcus aureus (Sa,
ATCC 25923), Klebsiella pneumoniae (Kpn, ATCC 27736). The
susceptibility testing was performed by Standard Broth
Microdilution method as per National Committee for Clinical
Laboratory Standards (now CLSI)19 guidelines using RPMI 1640
29
(4% MeOH/EtOAc) 0.5; [α]D -29.5 (c 0.32, CHCl3); νmax (neat
liquid) 3411, 3019, 2399, 1643, 1402, 1385, 1215, 1158, 1069,
928, 757, 669 cm–1; δH (300 MHz, CDCl3) 7.54 (1H, br), 5.49
(1H, br), 4.64 (1H, t, J 7.5 Hz), 4.54 (1H, t, J 7.5 Hz), 4.32 - 4.05
(6H, m), 4.00 - 3.88 (2H, m), 3.76 (3H, s), 3.58 (6H, s), 3.53 (3H,
s), 3.51 (3H, s), 3.45 (6H, s), 3.40 - 3.17 (6H, m), 3.14 - 3.03
(4H, m), 2.97 - 2.88 (2H, m), 2.58 - 2.44 (2H, m), 2.22 - 2.00
(2H, m), 1.42 (9H, s); δC (100 MHz, CDCl3) 172.8, 156.0, 103.6,
103.3, 85.0, 84.9, 83.8, 83.4, 83.3, 83.1, 80.0, 79.8, 79.4, 79.3,
77.7, 76.2, 63.2, 60.7, 60.6, 58.7, 58.6, 52.1, 40.3, 37.6, 37.0,
28.4; HRMS: [(M-Boc)+H]+, found 667.3280. C29H51N2O15
requires 667.3289.
medium
buffered
with
MOPS
[3-(N-morpholino)
propanesulphonic acid] for fungi and Mueller Hinton Broth
(Difco) for bacteria in 96 well microtitre plates. The maximum
concentration of the test peptides tested was 100µg/ml and the
inoculums load in each test well was in the range of 1-5×103
7.13. Synthesis of compound 2

ACCEPTED MANUSCRIPT
8
Tetrahedron
ACCEPTED MANUSCRIPT
R.; Vairamani, M.; Kumar, S. K.; Kunwar, A. C. J. Org.
Chem. 2003, 68, 6257-6263.
cells. The plates were incubated for 24-48 h for yeasts, 72-96 h
for mycelial fungi at 35 ºC and 24 h for bacteria at 37 ºC and read
visually as well as spectrophotometerically (Spectra max) at 492
nm for determination of minimal inhibitory concentrations
(MIC).16
11. Siriwardena, A.; Pulukuri, K. K.; Kandiyal, P. S.; Roy, S.; Bande,
O.; Ghosh, S.; Fernandez, J. M. G.; Martin, F. A.; Ghigo, J. -M.;
Beloin, C.; Ito, K.; Woods, R. J.; Ampapathi, R. S.; Chakraborty,
T. K. Angew. Chem. Int. Ed. 2013, 52, 10221-10226.
12. Desvergnes, S.; Vallée Y.; Py S. Org. Lett. 2008, 10, 2967-2970.
13. Owens, N. W.; Stetefeld, J.; Lattova´, E.; Schweizer, F. J. Am.
Chem. Soc. 2010, 132, 5036-5042.
Acknowledgements
14. (a) Wang, H.; Sun, L.; Glazebnik, S.; Zhao, K. Tetrahedron Lett.
1995, 36, 2953-2956; (b) Chakraborty, T. K.; Reddy, R. K.;
Gajula, P. K. Tetrahedron 2008, 64, 5162-5167.
15. (a) Pal, S.; Mitra, K.; Azmi, S.; Ghosh, J. K.; Chakraborty, T. K.
Org. Biomol. Chem. 2011, 9, 4806-4810; (b) Krishna, Y.; Sharma,
S.; Ampapathi, R. S.; Koley, D. Org. Lett. 2014, 16, 2084-2087.
16. Please see supporting information.
17. (a) Brooks B. R.; Mackerell A. D.; Nilsson L.; Petrella R. J. J.
Comp. Chem. 2009, 30, 1545-1615; (b) Brooks, B. R.; Bruccoleri,
R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M.
J. Comput. Chem. 1983, 4, 187-217.
18. Compounds 25 was synthesized by intramolecular cyclization
(following the same method used for making 1 from 23) of the
linear dimer of O-benzylated SAA monomer derived from 10 by
selective hydrogenation of azide using 10% Pd-C as catalyst and
EtOAc as solvent followed by Boc-protection. The cyclized
product (66% yield in three steps from the N-Boc protected
dimeric methyl ester) was hydrogenated using 10% Pd-C as
catalyst and MeOH as solvent to furnish 25 in 74% yield after
chromatographic purification. Data for 25: Rf (15%
MeOH/EtOAc) 0.5; [α]D27 -21.6 (c 0.40, MeOH); νmax (KBr) 3429,
3018, 2399, 2128, 1637, 1404, 1215, 1052, 1027, 1009, 928, 819,
757, 668, 624 cm–1; δH (300 MHz, DMSO-d6) 8.23 (1H, br), 5.23
(1H, br), 4.36 (1H, d, J 8.6 Hz,), 3.85 (1H, dd, J 13.3, 10.2 Hz),
3.68 - 3.60 (1H, m), 3.58 - 3.39 (1H, m), 3.06 - 2.91 (1H, m), 2.42
- 2.28 (1H, m), 2.04 - 1.07 (1H, m); δC (100 MHz, DMSO-d6)
172.5, 84.5, 76.6, 68.2, 37.3, 36.5; HRMS: [M+Na]+, found
309.1043. C12H18N2O6Na requires 309.1063.
Authors acknowledge SAIF, CDRI for the analytical
facilities. Authors are thankful to SERB, New Delhi, India
for financial support under JC Bose fellowship (No.
SR/S2/JCB-30/2007; T.K.C.), to CSIR, New Delhi for
research fellowships (NS and PSK) and for funding NWP-
BSC-0113. This is CDRI manuscript No. 143/2016/TKC.
Supplementary data
Experimental details, NMR spectral data, chemical shift
tables, restraints used in MD calculations and antibacterial-
antifungal data are provided.
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Synthesis of 26 commenced from 11 which on selective
hydrogenation of azide using 10% Pd-C as catalyst and EtOAc as
solvent followed by Boc-protection gave the requisite monomer
Boc-SAA(OBn)-OMe. Cyclotrimerisation of the monomer,
following the same protocol used for getting 3 from 21, furnished
the O-Bn protected cyclotrimer cyclo-[SAA(OBn)]3 in 54% yield
in three steps, which on hydrogenation using 10% Pd-C as catalyst
and MeOH as solvent furnished 26 in 71% yield after
chromatographic purification. Data for 26: Rf (30%
27
MeOH/EtOAc) 0.45; [α]D +58.9 (c 0.35, MeOH); νmax (KBr)
3426, 3018, 2257, 2129, 1644, 1384, 1215, 1050, 1026, 1007, 821,
757, 668 cm–1; δH (300 MHz, DMSO-d6) 8.27 (1H, br), 5.31 (1H,
d, J 5.4 Hz), 4.41 - 4.32 (1H, m), 3.87 - 3.68 (2H, m), 3.63 - 3.54
(1H, m), 2.92 - 2.79 (1H, m), 1.86 - 1.74 (1H, m); δC (100 MHz,
DMSO-d6) 172.4, 82.3, 75.7, 72.4, 41.7, 37.3; HRMS: [M+Na]+,
found 452.1623. C18H27N3O9Na requires 452.1645.
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