Novel 1,2,3-triazole-tethered Pam3CAG conjugates as potential TLR-2 agonistic vaccine adjuvants

Article abstract of DOI:10.1016/j.bioorg.2021.104838

A focused library of water soluble 1,2,3-triazole tethered glycopeptide conjugates derived from variety of azido-monosaccharides and aliphatic azido-alcohols were synthesized through manipulation at the C-terminus of Pam₃CAG and screened for their potential as TLR2 agonistic adjuvants against HBsAg antigen. In vitro ligand induced TLR2 signal activation was observed with all the analogues upon treatment with HEK blue TLR2 cell lines. Conjugate derived from ribose (6e), which exhibited pronounced HBsAg specific antibody (IgG) titer also shown enhanced CD8+ population indicating superior cell mediated immunity compared to standard adjuvant Pam₃CSK₄. Further, docking studies revealed ligand induced heterodimerization between TLR1 and 2. Overall, the result indicates the usefulness of novel conjugates as potential vaccine adjuvant.

Full text of DOI:10.1016/j.bioorg.2021.104838

Bioorganic Chemistry 111 (2021) 104838
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 1,2,3-triazole-tethered Pam3CAG conjugates as potential TLR-2
agonistic vaccine adjuvants
,
b
,
,
b
,
,
Tukaram B. Mhamanea 1, Shainy Sambyala 1, Sravanthi Vemireddya b, Imran A. Khanc,
Syed Shafic, Sampath Kumar Halmuthur M. a
,b,*
a Vaccine Immunology Laboratory, OSPC Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
b Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201 002, India
c Department of Chemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi
A R T I C L E I N F O
A B S T R A C T
Keywords:
A focused library of water soluble 1,2,3-triazole tethered glycopeptide conjugates derived from variety of azido-
monosaccharides and aliphatic azido-alcohols were synthesized through manipulation at the C-terminus of
Pam3CAG and screened for their potential as TLR2 agonistic adjuvants against HBsAg antigen. In vitro ligand
induced TLR2 signal activation was observed with all the analogues upon treatment with HEK blue TLR2 cell
lines. Conjugate derived from ribose (6e), which exhibited pronounced HBsAg specific antibody (IgG) titer also
shown enhanced CD8+ population indicating superior cell mediated immunity compared to standard adjuvant
Pam3CSK4. Further, docking studies revealed ligand induced heterodimerization between TLR1 and 2. Overall,
the result indicates the usefulness of novel conjugates as potential vaccine adjuvant.
TLR-2
Pam3CAG-triazole conjugate
Click chemistry
Adjuvant
HBsAg
IgG
CD4
CD8
1. Introduction
not fully understood so far, amhiphilicity of these cysteinyl glycolipids
has been recognized as key feature that influence their TLR activation
Importance of new immune adjuvants is steadily increasing with the
growing demand for safe and highly efficacious vaccines against old as
well as emerging viral diseases. With the availability of repository of
subunit antigens for various diseases, there has been a need for devel-
oping novel PRR agonists with varying degrees of Th1/Th2 activation,
required for eliciting qualitatively specific immune response with
particular antigens [1–3]. Activation of innate and adaptive immune
response through heterodimerization of TLRs induced by these PRR
agonists is well established. Inspired by the structure of bacterial lipo-
protein derived N-terminal amino acid S-(2,3 dihydroxypropyl)-L-
cysteine lipid head group, libraries of novel triacylated and diacylated
cysteinyl lipopeptides have been developed, which act through TLR1/2
and TLR2/6 heterodimerization respectively [4,5]. Evolutionarily, such
TLR heterodimerization enable recognition of continuous ligand field
expansion of pathogen associated molecular patterns encompassing
structural changes in lipid head group. Such glycolipid adjuvants can be
administered either in combination with an antigen or can be covalently
conjugated to an antigenic peptide to elicit desired immune response.
Even though the molecular mechanism for ligand recognition by TLRs is
propensity. The important role played by TLR2 in regulating the host
immune response to pathogens, led researchers to develop several new
structural analogues of natural N-apalmitoyl-S-[2,3-bis(palmitoyloxy)-
(2R)-propyl]-L-cysteinyl peptides tuning it’s amphiphilicity to suit their
application in different vaccine developments. In this context, the N-
terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium
or Braun lipoprotein, have all been described as TLR2 agonists, their
simpler synthetic analogues S-[2,3-bispalmitoyloxy-(2R)-propyl]-R-
cysteinyl lipopeptides, like ‘Pam2CAG’ and Pam3CysSK4, have also
shown TLR2 agonist activity and can therefore be considered as more
useful and easily attainable immunoadjuvants. However, non-specific
adhering of highly amphiphilic polycationic lysine peptide entities
make this more unsuitable for vaccine application as they complicate the
preparation of the vaccine formulation cocktail. Amphiphilic lip-
opeptides, such as Pam3CAG (N-palmitoyl-S-[2,3-bis(palmitoyloxy)-
(2R)-propyl]-L-cysteinyl-L-alanylglycine) synthesized by Benoit et al.
effectively induced the proliferation of BALB/c mouse splenocyte in vitro
when incorporated into liposomes [6]. The lipid head group of this
synthetic lipopeptide interacted with lipid bilayer of liposome whereas
* Corresponding author at: Vaccine Immunology Laboratory, OSPC Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India.
E-mail address: sampath.iict@gov.in (S.K. Halmuthur M.).
1
Equal authorship.
https://doi.org/10.1016/j.bioorg.2021.104838
Received 8 December 2020; Received in revised form 4 March 2021; Accepted 16 March 2021
Available online 22 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
their hydrophilic tail was covalently modified to anchor one or two
peptide containing T epitopes against which an immune response was
elicited [7,8]. On the contrary, whenever liposomal formulation is not
desirable, the amphiphilicity of these lipopeptide becomes a serious
drawback, hampering ease of its usage as immune adjuvants both in vitro
and in vivo [9]. Several studies revealed the loss of activity owing to
large heterogeneous aggregates of such amphiphilic lipopeptides in so-
lutions, rendering its activity critically dependent on the presence of
proteins like bovine serum albumin or solubiliser detergents such as
octyl-β-D-glucopyranoside, and even on the protocol of dilution (e.g.,
with tert-butyl alcohol or dimethyl sulfoxide) [10]. As a possible solution
to these difficulties, Benoit et al. further developed hydrophilic PEG
conjugates of Pam3CAG and demonstrated their ability to induce mouse
dendritic cell maturation and B cell proliferation in vitro [6,11]. In spite
of these efforts, their actual usefulness as vaccine adjuvants in vivo -a
true testimony for their adjuvanticity, has not been demonstrated.
In continued efforts of Halmuthur MSK et al. to develop novel vac-
cine adjuvants and delivery systems, first successful attempt of the
amalgamation of carbohydrate-Pam3Cys motifs tethered to a 1,2,3-tria-
zole linker as a peptide free TLR2 adjuvants [12]. The 1,2,3-triazole
based derivatives have been employed in various medicinal chemistry
applications, such as anti HIV [13], antiviral, anticancer [14], anticon-
carbohydrate/aliphatic alcohol conjugates against HBsAg antigen.
When tested in murine model, these ligands show TLR2 agonistic pro-
pensity and DC activation in vitro. Furthermore, ligand induced heter-
odimerization between TLR1 and TLR2 has been demonstrated by in
silico docking studies.
2. Materials and methods
2.1. Chemistry
All reagents which are commercially available were used without
further purification and purchased from Sigma Aldrich, if not indicated
otherwise. All organic solvents were dried and freshly distilled before
use wherever necessary by using standard procedures. Tetrahydrofuran
(THF) was dried and distilled from freshly extruded Na wires and
Dichloromethane (DCM) purified by overnight stirring with CaH2 fol-
lowed by distillation. All the moisture sensitive reactions were per-
formed in the oven dried glass wares under inert atmosphere of nitrogen
or argon. All the chemical transformations were monitored by using
Thin Layer Chromatography (TLC) on a 2–5 cm percolated E. Merck
Silica Gel 60 F254 plates (0.25 mm) and spots were visualized using UV
lamp. Staining was developed with Iodine or by dipping TLC in p-Ani-
saldehyde or 10% Phosphomolybdic acid solution in EtOH and then
charred on a hot plate. Removal of solvent or concentration under
reduced pressure was performed using a rotary evaporator at 25–40 ◦C.
Column chromatography was carried out by using silica gel (Merck 60-
120mesh) and (Merck 100–200 mesh) with technical grade ethyl ace-
tate/hexane and chloroform/methanol as eluents. All melting points
were determined on a Buchi capillary melting point apparatus and are
uncorrected. NMR spectra were recorded with a Bruker AC- 300 MHz, a
Bruker AM-400 MHz and a Bruker AMX-500 MHz spectrometer in-
struments in CDCl3, CD3OD and DMSO‑d6 with (CH3)4Si is an internal
standard for 1H NMR spectra and solvent signals as internal standard for
13C NMR spectra at ambient temperature. Chemical shifts (δ) are re-
ported in ppm. Coupling constants J were reported in Hz. NMR signal
splitting patterns were designated as follows: s - singlet, d - doublet, dd
-doublet of doublet, dt -doublet of triplet, td - triplet of doublet, t- triplet,
q-quartet, p-pentet, bs-broad singlet, m-multiplet, br-broad. For ESI-MS,
m/z values are reported in atomic mass units and recorded in Shimadzu
vulsant [15], antiallergic [16] and antifungal [17] agents.
most potent agonistic antigen of a natural killer T-cell receptor. Lee et al.
prepared a series of 1,2,3-triazole-containing -GalCer analogues in
which the lipid chain lengths were incrementally varied. Isosteric
replacement of the amide moiety of -GalCer with a triazole increased
the IL-4 versus IFN-γ bias of released cytokines [18]. The isosteric
replacement of amide with 1,2,3-triazole scaffold in -GalCer stimulated
higher levels of both IFN-γ and IL-4 cytokine expression in vitro
α-Galcer is the
α
α
α
compared to that of α-GalCer [19]. Mohamed R. Aouad et al. have re-
ported different 1,2,3-triazole conjugates with anti microbial [20–24],
anticancer[25–29] and antitubercular [30] properties. Some of the
1,2,3-triazole containing drugs and other bioactive compounds have
been depicted in Fig. 1.
1,3-Dipolar Huisgen cycloaddition of organic azides with alkynes has
been recognized as the most widely reported method for the construc-
tion of 1,2,3-triazole scaffold. Herein, we present the synthesis and in
vivo adjuvanticity of some novel 1,2,3-triazole tethered Pam3CAG-
Fig. 1. Some examples of 1,2,3-triazole containing drugs and bioactive molecules.
2

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
instrument.
peak at m/z 516 [M + Na+] further confirms the product formation.
This phenacyl ester was further subjected to cyclohexylidine depro-
tection by using 70% AcOH gave diol S4 which was confirmed by
2.1.1. Synthesis of new 1,2,3-triazolyl tethered Pam3CAG conjugates
Seven new Pam3CAG conjugates have been designed, comprising
different carbohydrates and simple aliphatic alcohols, tethered together
through a 1,2,3-triazol-4-yl methanamine spacer employing azide-
alkyne click chemistry approach (Fig. 2). Carbohydrate employed
include both furanosyl and pyranosyl entities lined to peptide at
anomeric or primary hydroxyl position.
1
absence of peaks related to cyclohexane in its H NMR and 13C NMR
spectra. This structure assignment was further confirmed with its
observed ESI-MS peak at m/z 436.452 [M + Na+]. This diol compound
S4 was subjected to dipalmitoylation by using palmitic acid under DIC/
DMAP condition to furnish protected Pam2Cys dilipidated compound
which is confirmed by its ESI-MS peak at m/z 912.45 [M + Na+] and the
proton signals corresponds to palmitoyl group in 1H NMR. To obtain
trilipidated acid compound 2, corresponding di-palmitoylated precursor
was subjected to N-Boc deprotection using 20% TFA/DCM followed by
coupling of free amine with palmitic acid under EDC.HCl/HOBt condi-
tion to yield protected tri-palmitoylated compound S5 which was sup-
ported by its 1H NMR signals at δ 7.90–7.60(5H) for aromatic protons, δ
2.30–1.25 ppm for –CH2– of three ester chains and δ 0.87 (t, J = 6.4 Hz,
9H,) for terminal –CH3 of lipid chains. Further assigned structure of S5
was confirmed by the presence of five signals ranging between δ
191.27–170.49 ppm corresponds to carbonyl carbons in 13C NMR
(signal at δ 191.27 corresponds to benzoyl carbon of the phenacyl group,
signals at δ173.40 and 173.31 corresponds to carbonyl carbons of two
palmitoyl esters, signal at δ 173.16 carbonyl of phenacyl ester and a
signal at δ 170.49 corresponds amide carbonyl) and δ134.15–127.78 for
aromatic carbons. Formation of the compound S5 is further evidenced
by its observed mass at 1050.7797 [M + Na]+. Phenacyl protected tri-
palmitoylated compound (S5) thus obtained was treated with acti-
vated Zn/AcOH to afford Pam3Cys-OH (2) [32] (Scheme 1) in good yield
which was confirmed by the absence of aromatic region in the 1H and
13C spectra, further evidenced by its ESI-MS peak at m/z 932.50 [M +
Na]+.
Synthesis of target conjugates 6a-g includes the preparation of three
key fragments viz., Pam3Cys-OH (2) the lipid head group mainly
responsible for TLR agonistic activity, Boc-Ala-Gly-propargyl 4 (dipep-
tide alkyne) and different hydrophilic azides (I-VII).
A lipopeptide backbone skeleton Pam3cys-OH mainly responsible for
the agonistic activity was synthesized in twelve steps [32]. Lipid head
group was synthesized starting from cyclohexylidine protected vicinal
diol of D-mannitol which was further subjected to oxidative cleavage to
give (R)-2,3-O-Cyclohexylidene-D-glyceraldehyde. The aldehyde formed
in the previous step was reduced to (R)-1,4-dioxaspiro[4.5]decan-2-
ylmethanol 1 by using NaBH4/methanol (Scheme1) [31]. Conversion of
aldehyde to corresponding alcohol 1 was confirmed by the appearance
of a new double doublet at δ 3.73 & 3.57 ppm corresponds to two pro-
tons in 1H NMR spectra and a mass peak observed at m/z 195.10 [M +
Na+] in ESI-MS. This hydroxyl compound was further treated with
iodine, triphenylphosphine and imidazole to afford the corresponding
iodo compound. This iodo compound later on reacted with commercial
N-Boc-L-Cys-methyl ester in presence of DIPEA in DMF to afford
orthogonally protected cysteine coupled protected glycerol moiety
which is confirmed by 1H, 13C NMR and ESI. S-alkylation of cysteine was
confirmed by the appearance of the peaks corresponding to both
cysteine (chemical shifts at δ 3.76 (s,3H), 1.45 (s, 9H) corresponds to
Synthetic pathway for another fragment Boc-Ala-Gly-Propargyl
dipeptide 4 is presented in scheme 2. Mixed anhydride coupling of
Boc-alanine and ethyl glycinate afforded ethyl ester of dipeptide 3 which
is confirmed by its observed mass at m/z 297.25 [M + Na+] in ESI-MS.
Further supported by the signals of ethyl at δ 4.08 (q, J = 7.1 Hz, 2H) for
two protons, 1.37 (s, 9H) for Boc methyl, 1.21–1.15 for three protons
1
methyl ester and -NHBoc groups respectively in H NMR spectra and
protected glyceryl moieties and an up field shift of methylene (–CH2-)
proton signal of gleceryl moiety. A mass peak at 412.30 [M + Na+] in
ESI-MS has finally confirmed the S-alkylation of cysteine. Cysteinyl
methyl ester was then hydrolyzed with LiOH in THF/water (1:1) to
afford corresponding carboxylic acid which was directly subjected to
phenacylation with phenacyl bromide/KF to yield phenacyl ester.
Appearance of the peaks at aromatic region (δ 7.89–7.49(5H).) and the
disappearance of the peak corresponds to methyl ester in 1H NMR
confirms the phenecyl protection of the carboxylic acid. This structure
assignment was supported by 13C NMR spectra in which observed sig-
nals at δ 191.78 for carbonyl of phenacyl, 170.67 carbonyl of ester,
from ethyl group & β –CH3 of alanine. This was further evidenced by its
13
–
–
C O
–
C signals at δ 173.16 for carbonyl of alanine, 169.60 for
glycine and 154.87 for carbonyl of Boc group. Compound 3 was sub-
jected to hydrolysis gave Boc-Ala-Gly-OH in good yield [33]. This was
used without further purification for the amide coupling with propargyl
amine to obtain required building block propargylated Boc-Ala-Gly-(4)
(Scheme 2). Compound 4 was confirmed by HRMS-MS peak at m/z
306.25 [M + Na]+. Further evidenced with observed peaks at δ 2.20 (t, J
–
–
155.50 (
C O of Boc), 134.29–127.80 for aromatic carbons. ESI-MS
–
HO
HO
OH
O
O
HO
HO
HO
HO
HO
H31
O
C15
O
6b
6a
O
O
HO
HO
OH
O
O
H31
C15
HO
HO
R
N
N
S
HO
6c
HO
O
OH
O
H
N
H
6d
H31
C15
N
N
H
N
N
H
O
OH
OH
O
O
6a-g
OH
HO
Lipid
Peptide
1,2,3-Triazole
HO
6f
6e
OH
methanamine linker
6g
Fig. 2. Design of target molecules.
3

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
OH
NHBoc
O
O
a
OH OH
c
b
S
O
OH
HO
OH
HO
O
O
OH OH
S4
1
D-Mannitol
S1
H31
C15
H31
C15
O
O
O
O
d
H31
NH
C15
H31
O
O
H31
O
NH
C15
H31
O
C15
O
S
O
C15
S
OH
O
O
O
O
S5
2
Pam
3Cys-OH
Scheme 1. Synthesis of Pam3Cys-OH.
NHBoc
O
NHBoc
O
e
NHBoc
O
O
f
g
H
H
OH
N
N
O
N
H
O
3
4
Boc-Alanine
H31
O
C15
O
O
O
H31
C15
S
O
O
H
N
H
H31
C15
N
N
H
N
H
O
O
5
Scheme 2. Synthesis of terminal alkyne-Pam3CAG conjugate.
2.2. Biology
= 2.5 Hz, 1H) for propargyl proton in 1H NMR and signals at δ 78.68,
70.78 for both alkyne carbons in 13C spectra. This dipeptide on further
treatment with 20% TFA/DCM and coupling with previously synthe-
sized lipid head group Pam3Cys-OH (2) afforded Pam3Cys-Ala-Gly-
Propargyl lipopeptide (Pam3-CAG-propargyl) 5 (Scheme 2) in moderate
yields. Pam3CAG-propargyl compound 5 was confirmed by the charac-
teristic signals of long chains and propargyl moiety. Presence of signals
at δ 0.82(t, J = 6.4 Hz, 9H) for terminal methyl groups, methylene
protons in the range of δ (2.33–1.20) and signal for β-methyl of alanine
at δ 1.35 (d, J = 7.2 Hz, 3H) confirmed the formation of 5. It is further
supported by the observed signals in 13C spectra at δ 79.04, 70.06 for
alkyne carbons, δ 33.98–21.17 for carbons of methylene of long chains,
and δ16.58 for β-CH3 alanine & δ 13.71 for terminal methyl of long
chains. Observed ESI-MS peak at 1097.8274 [M + Na]+ further evi-
denced the structure.
2.2.1. Ligand induced TLR2 activation by SEAP assay
The assay was performed as per the manufacturer’s instructions.
HEK-Blue™ hTLR2 cells (280,000 cells per ml) were harvested in HEK-
Blue™ selection medium containing normocin treated with the com-
pounds i.e., Pam3CysSK4, 6a, 6b, 6c, 6d, 6e, 6f and 6g (10 µg/ml) and the
plates were incubated at 37 ◦C in 5% CO2 for 24 h. Stimulation with a
TLR2 ligand activates NF-ĸB which induce the production of SEAP
(secreted embryonic alkaline phosphatase). The levels of SEAP can be
observed with naked eye and determined using a spectrometer at
620–655 nm.
2.2.2. Ex vivo activity
Required sugar azides were synthesized from corresponding per
acetylated sugars which was subjected to azidation by using TMS-N3/
2.2.2.1. Immunization. Female BALB/c mice (8–9 weeks) were pur-
chased from CSIR-CCMB, Hyderabad, All animal experiments were
approved by the local ethical committee and animals were kept in
accordance with CPCSEA guidelines. They were immunized with albu-
min, from chicken egg (Sigma-Aldrich, St. Louis, MO) 100 µg in 100 µl at
0th day and sacrificed on 7th day and the spleen were collected.
SnCl4 gives per acetylated anomeric (α/β) azides which further treated
with NaOMe/MeOH gives free hydroxyl azides (I-V) in quantitative
yields [34–37]. 3-azidopropane-1, 2-diol (VI) was prepared from glyc-
erol in good yield [38]. 2-azidoethanol (VII) was synthesized from
commercially available 2-bromoethanol and NaN3 at 80 ◦C in 90% yield
[39].
2.2.2.2. Cell preparation. Mice were sacrificed 7 days post injection;
spleens were harvested in PBS and processed on ice to single-cell
4

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
suspensions by frosted slides. The cell suspension was further passed
through cell strainer to remove the debris. Then they were centrifuged at
2000 rpm for 10 min and ACK lysis buffer was then added to lyse the
erythrocytes. PBS saline wash was given thrice to the cell pellet. Cells
were re-suspended in complete RPMI 1640 medium with 10% FBS.
Viable cell concentration was determined by trypan blue staining.
Again plates were washed 3 times with PBS Tween20 followed by the
addition of serially diluted serum sample in 1% BSA with 2 h incubation
at 37 ◦C. After 4 times washing, HRP goat anti-mouse IgG (1:3000) in 1%
BSA was added to plates and kept for 1 h incubation at RT. Plates were
washed with PBS Tween20 3 times. 100 μl of TMB substrate (TMB re-
agent A + B in equal volume) was added to each well and incubates in
dark at RT for about 15 min or till color development. The reaction was
2.2.2.3. Re-stimulation of splenocytes. 1 × 105 Splenocytes/well in 96-
well plates were cultured in complete RPMI medium with 10% FBS
and re-stimulated with 20 µg/mL of ovalbumin (Sigma-Aldrich, St.
Louis, MO). Cells were treated with 6 different (M1-6) compounds at a
stopped by adding stopping reagent i.e. 2 N H₂SO₄, 50
μl/well and
absorbance was measured at 450 nm by using multimode reader (Tecan,
Infinite pro).
concentration of 1, 10 and 100
μ
g/ml. Pam3CysSK4 (Vacci GradeTM
2.2.3.4. Spleen collection and splenocyte isolation. Spleen was collected
only from those groups which showed good IgG titer for further studies.
Mice were sacrificed and spleen was collected in chilled PBS. The flesh
was cleaned in PBS and spleen was homogenized in RPMI media (900 ml
autoclaved water, 1 packet of RPMI 1640 (HIMEDIA, Mumbai), 2.0 g
sodium bicarbonate, 10 ml antibiotic antimycotic solution 100x, 10 ml
InvivoGen, USA) was used as a control. Treated cells were incubated for
◦
48 h at 37 C in 5% CO2 incubator. Supernatants were harvested and
checked for cytokine expression. They were stored at 70 ◦C until further
analysis.
penicillin streptomycin 2x, 3.94 μl 2-mercaptoethanol, 100 ml FBS) on
2.2.2.4. Cytokine estimation – sandwich ELISA. Quantification of pro
inflammatory cytokines IL-6 and TNF-alpha from cell supernatant of
splenocytes treated with compounds at three different concentrations
was carried out by Opti Elisa Kit (BD) according to manufacturer’s
protocol. Briefly 1 × 105 cells/well were seeded in 12 well plates and
incubated at 37 ◦C with 5% CO₂ for 48 h. The supernatant was collected
from each well separately for cytokine estimation by coating ELISA
0.22
μm filter by applying pressure with syringe. The filtrate was
collected in 50 ml Tarson tube and centrifuged for 10 min at 2000 rpm at
◦
4 C. ACK (1 ml/spleen) treatment was given to the pellets for 1 min
30 s. PBS wash and then cell count.
2.2.3.5. Immunophenotyping. Splenocytes prepared in RPMI media from
immunized mice were taken for immunophenotyping. 3 × 105 cells
from each group were taken separately to FACS tubes and washed with
plates with purified anti-mouse antibody IL-6 and TNF-
bicarbonate-carbonate buffer and incubated at 4 ◦C overnight. Plates
were washed thrice with PBST 20 and blocked with 100 l of 1% BSA.
After 1 h incubation at RT, plates were again washed thrice with same
washing buffer. 100 l of serum sample was added along with serially
α diluted with
μ
staining buffer. Fc blocker was added 1 μl/tube and incubated for 15 min
at 4 ◦C. After incubation cells were washed twice with staining buffer.
Flurochrome characterized antibodies (FITC-A antiCD4 and APC-Cy7-A
μ
◦
diluted recombinant antibody (IL-6 and TNF-
α). Plates were incubated
antiCD8) were added to each tube and incubated for 30 min at 4 C.
for 3 h at 37 ◦C. Post incubation was followed by four times washing and
then addition of detection antibody prepared in BSA with 1 h incubation
at RT. After four times washing, HRP streptavidin (1:3000) in 1% BSA
Again tubes were centrifuged with staining buffer. Cells were collected
and resuspended in sheath fluid for analysis using BD FACS suit
software.
was added 100 μl each and kept for incubation for 30 min at RT. TMB
substrate was added after trice washing and kept for incubation till
development of color or for 15 min. The reaction was stopped by adding
2.2.3.6. Splenocyte proliferation. Splenocytes from each group of
selected immunized mice were seeded in 96 well plates with cell count
1 × 105 cells/well. The cells were restimulated by using HBsAg, LPS and
50 μl of 2 N H2SO4 and the absorbance was measured at 450 nm in ELISA
reader (Tecan, Infinite pro).
ConA with concentration 0.1, 10 and 2 μg/ml respectively. The plates
were incubated at 37 ◦C for 48 h having 5% CO₂. After the completion of
incubation, MTT reagent (5 mg/ml PBS) was added to the cells i.e. 20 l/
well and incubated for 3 h at 37 ◦C. The untransformed MTT was
2.2.3. In vivo activity studies
μ
2.2.3.1. Immunization methods. For detail study Balb/c female mice
were used, purchased from Palamur Biosciences Private Limited,
Hyderabad. All the experiments on animals were approved by the
Institutional Animal Ethics Committee (IAEC) of CSIRIICT for 168 nos of
Balb/c mice with IAEC approval number: IICT/IAEC/014/2019. 71
Balb/c female mice were immunized with Pam3Cys analogs i.e.6a, 6b,
removed (50 μl each) and was recovered by 50 μl of DMSO, followed by
15 min incubation at RT. The absorbance was measured at 630 nm and
% proliferation of the cells was calculated by keeping cell control as
reference [40].
2.2.4. DC activation (CD86 and MHC II)
6c, 6d, 6e, 6f and 6g each 1 mg/10
standard (20 g/dose) and HbsAg (1
munization i.e. 1 mg/ml each. Immunization was done to different
μ
l. Along with this Pam3CysSK4
The EasySep™ Pan Dendritic Cell Isolation Kit, (MACS Miltenyi
Biotec-Germany) mouse, is a fast enrichment kit for untouched isolation
of all dendritic cell subpopulations. Isolation assay was performed as per
the manufacturer’s instructions. Briefly, cells were prepared (1 ×
108 cells/ml) in the range of 0.25–2.0 ml and sample was added to 5.0
μ
μg/dose) was also used for im-
groups with varying concentration i.e. 10, 20, 30 μg/dose. The mice
were provided with booster dose on 14th day and were sacrificed on
28th day of immunization.
ml tubes. Enrichment cocktail (50 μl/ml) was added to the cells, which
was mixed well and incubated at 2–8 ◦C for 15 min. The cells were then
washed with the recommended medium and centrifuged at 300g for 10
min. Supernatant was discarded and was resuspended in the original
volume of 0.25–2.0 ml with recommended medium. This was followed
2.2.3.2. Serum collection. To measure the count of antibody against that
particular antigen, retro-orbital blood was collected separately from
each group on 28th day. The blood was centrifuged at 2000 rpm for 20
min at RT. Serum was carefully collected in eppendorf tubes and stored
at 40 ◦C for estimation of IgG titer.
by adding the Biotin selection cocktail (100 μl/ml) to the cells, mixed
well and was incubated at 2–8 ◦C for 10 min. After which, the magnetic
particles were vortexed and 75 μl/ml of it was added to the cells, which
was then mixed and incubated at 2–8 ◦C for 10 min. Recommended
medium was added to the sample, mixed gently by pipetting up and
down. The tubes, without lid was inserted into the magnet and incu-
bated at RT for 5 min. The magnet was lifted and at once inverted the
magnet with tube, pouring the entire enriched cell suspension into a new
2.2.3.3. IgG titer – indirect ELISA. ELISA plates (HIMEDIA, Mumbai)
were coated with HbsAg (1 μg/ml) diluted in bicarbonate-carbonate
buffer having pH 9.4 and incubated overnight at 4 ◦C. Thrice plates
were washed with PBS Tween20 (pH 7.4) and blocked with 1% BSA
(100 μl/well) to prohibit non-specific binding with 1 h incubation at RT.
5

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
tube. The above mentioned step was repeated one more time and the
isolated cells were ready for use.
(36.82 mg, 0.186 mmol) in water:ter-butanol (5 ml, 1:1) mixture at room
temperature for 8 to12hrs which furnished the desired products 6a-g in
excellent yields. All the compounds synthesized were subjected to pu-
rification by column chromatography. The novel 1,2,3-triazole conju-
gates 6a-g were fully characterized based on their 1H and 13C nuclear
magnetic resonance (NMR) and mass spectrometry (MS) spectra. The
structures of all the compounds were in agreement with their spectro-
scopic data. The formation of the triazoles 6a-g was evidenced by their
1H NMR spectra, which revealed the absence of the terminal acetylenic
proton confirming its involvement in the cycloaddition reaction.
The structure of the representative compound 6a was established on
the basis of its NMR and mass spectral analysis. In the 1H NMR spectra of
6a, the existence of triazolyl proton at δ 7.94 ppm (s, 1H) and anomeric
sugar proton of glucose at δ 5.52 ppm (d, J = 9.1 Hz, 1H, H-1) confirms
the formation of 1,4-disubstituted 1,2,3-triazole compound. Further, the
presence of signal at δ 1.39 (d, J = 7.1 Hz, 3H) corresponds to β-methyl
of alanine and the presence of signals corresponds to long chain terminal
methyl protons at 0.85 (t, J = 6.5 Hz, 9H) in 1H NMR supports the
product formation. All glucose protons and other deshielded protons
observed in the range of δ (5.17–3.52) ppm at usual chemical shifts. This
was further evidenced by its 13C spectra wherein all the signals observed
at their usual chemical shifts i.e. δ 144.45, 121.55 characteristic peaks
for triazole carbons. Ester long chains and peptidewere confirmed by
Then cells were treated with standard Pam3CysSK4 and compounds
6e at 20 μg concentration each for 24 h. Then cells were stained with
CD86 and MHC II markers to estimate the ability of compounds to
activate DCs.
2.3. Computational studies
The crystal structure of high resolution TLR1–TLR2 heterodimer
with a tri-acylated lipopeptide (Pam3CysSK4) was downloaded from
Protein Data Bank (PDB ID: 2Z7X). The resolution is 2.10 Å. The docking
study with the native ligand (tri-acylated lipopeptide) and the 6a-g
molecules was performed using Schrondinger suite 2016 software. All
the crystallographic water molecules and other small molecules were
removed. Hydrogen atoms were added using OLPS. Then the docking
protocol file was generated using the Glide-Dock module using SP and
XP mode. The active site was detected using the ligand bound protein-
ligand complex and was used for the detection of the gridbox with box
size of 25, 30, 28. Docking was carried out in both SP and XP mode using
the same gridbox. The active site was confirmed with the reference of
the ligand, which was prepared using the Ligprep option. The native
ligand for docking was extracted from the complex structure retrieved
from Protein Data Bank. The molecular structures of 6a-g were prepared
using Chemdraw 3D software and initially all the prepared molecules
were minimized using the MMFF protocol. Three conformations with
the top ranking scores (top 3 conformers) for molecules 6a-g were saved
for further study. Generation of an accurate complex structure for 6a-g
of the synthesized ligands showed activity but due to large hydrophobic
pockets the docking in an existing (rigid) structure of the receptor in-
duces higher standard deviation from the experimental data. Thus,
rescue of false negatives (poorly scored true binders) in virtual screening
experiments, where instead of screening against a single conformation of
the receptor, additional conformations obtained with the induced fit
protocol are used. We performed docking of the designed ligands with
TLR1, TLR2 and TLR1-TLR2. The results were concluded by opting
LIAISION calculations (A Liaison simulation combines a molecular-
mechanics calculation with experimental data to build a model
scoring function used to correlate or to predict ligand-protein binding
free energies) and MM/PBSA calculation (The binding free energy (ΔG)
of the complex is calculated using molecular mechanics/Pois-
son–Boltzmann surface area (MM/ PBSA) approach).
–
–
–
C
–
–
C
carbonyl region δ 175.04 for
O of ester, 173.55 for
O of ester,
–
–
–
173.43 for NH
for carbonyl carbons of peptide. Observed peaks of β-methyl of alanine
at δ 16.04, -CH- of alanine, glycine, triazole-CH2– & -CH of cysteine at
C O of palmitoyl amide and 171.69, 171.32, 169.92
–
α
α
δ 52.37,42.33,42.20, 49.73 respectively, terminal methyl of long chains
at δ 13.38 and all –CH2– of long chains in the range of δ (33.62–22.16)
further confirmed presence of peptide and long chains. D-glucose car-
bons were observed at δ 87.72 (C-1), 78.95(C-5), 72.27(C-3), 69.91(C-
4), 68.83(C-2) and 60.62(-C-6) which further proves the conjugation
of sugar with lipid head group through 1,2,3-triazole linker. The pres-
ence of signal at m/z 1280.9145 [M + H+] in ESI-HRMS finally confirms
the product formation of 6a.
A focused library of seven novel 1,2,3-triazole-tethered Pam3CAG
conjugates have been synthesized by employing the click chemistry
approach and the final compounds are depicted in Table 1.
3.2. Biological activity
3.2.1. TLR specificity assay
Novel Pam3CAG triazolyl conjugates were subjected to in vitro
screening to evaluate the possible modulation in TLR2 specificity, po-
tency, and binding affinity after introduction of the hydrophilic moieties
derived from various monosaccharide and aliphatic alcohols. The novel
analogs of Pam3CAG were subjected to in vitro assay to evaluate their
ligand induced TLR2 signal activation on HEK blue TLR2 cell lines and
found that all analogues stimulated TLR2 in tune with the standard TLR2
ligand Pam3CysSK4. The results obtained were depicted in Fig. 3.
3. Results
3.1. Chemistry
A focused library of seven highly regioselective 1, 4-disubstituted
1,2,3-triazole products was prepared by using click chemistry protocol
(Scheme 3). Cycloaddition of compound 5 (100 mg, 0.093 mmol) and
various hydrophilic azides (I-VII, 0.111 mmol) were carried out in the
presence of CuSO4.5H20 (47 mg, 0.186 mmol) and sodium ascorbate
H31
O
H31
O
C15
C15
O
O
O
O
O
O
H31
H31
C15
C15
+
R-N
,
(I-VII)
3
R
S
S
O
O
O
O
CuSO4.
Na-ascorbate(2.0
5
H
N
eq.)
eq.)
2O(2.0
H
H
N
H
H
N
N
H31
H31
C15
C15
N
N
N
H
N
H
N
N
N
H
H
ter-butanol:water(1:1),
RT,8-12hrs
O
O
O
O
6a-g
5
R= Hexose and Pentose sugars, Aliphatic alcohols
Scheme 3. Synthesis of Pam3CAG based triazole analogues (6a-g).
6

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
Table 1
1,2,3-triazole-tethered Pam3CAG conjugates.
Sr. No
Alkyne (Compound 5)
Azides R-N3 (I-VII)
I
Pam3CAG conjugate (6a-g)
Yield (%)
93
1
2
3
4
91
88
96
II
III
IV
5
6
98
96
V
VI
7
93
VII
3.2.2. Cytotoxicity assay
All new Pam3CAG conjugates were tested for their possible cyto-
toxicity on splenocytes and the observed % proliferation of immune cells
after treatment with different doses of these analogs was quite impres-
sive (Fig. 4), indicative of their nontoxic nature. Both B- lymphocyte and
T-lymphocyte proliferation was quantified after re stimulation with LPS
and ConA.
3.2.3. Th1 and Th2 cytokines
Cytokine estimation was done by sandwich ELISA. Bar representa-
tion in Fig. 5 shows that these analogs of Pam3CAG help immune cells to
release IL-6 and TNF-
α cytokines, thus representing both Th1 and Th2
response. Th1 covers TNF-
α
cytokines whereas Th2 response covers IL-6.
Fig. 3. Human TLR-2 SEAP assay. HEK-Blue™ hTLR2 cells were accelerated
with agonists: Pam3CysSK4, 6a, 6b,6c, 6d, 6e, 6f and 6g (10 µg/ml). After 24 h
incubation SEAP activity was determined using QUANTI-Blue™ reading OD at
655 nm.
3.2.4. Antigen specific IgG titer
Post antigen challenge serum IgG estimation was done by indirect
ELISA. From the Figure it is clear that 6e and 6f analog of Pam3CAG at
concentration 20 and 30 μg/dose respectively showed higher HBsAg
antigen specific IgG titer against the standard TLR2 agonist
Fig. 4. Cytotoxicity effect of Pam3CysSK4 (20 µg) and 6a, 6b, 6c, 6d, 6e, 6f, 6g at 1, 10, 100 µg each on spleen cells. After 48 h incubation the OD was observed at
630 nm.
7

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
Fig. 5. Serum cytokine estimation @450 nm. BALB/c mice were immunized with ova albuminin Ag (100 µg) restimulated with Pam3CysSK4 (20 µg) and analogs 6a,
6b, 6c, 6d, 6e, 6f at 1, 10, 100 µg each.
Pam3CysSK4, as compared to other test compounds. However, most of
the analogues exhibited equally better IgG response compared to anti-
gen alone groups (Fig. 6). Thus, the compounds which showed good IgG
response were taken forward for further studies.
Pam3Cys quantification of CD4 and CD8 markers was done by using flow
cytometer. To quantify the markers, splenocytes so isolated were labeled
with flurochrome antibodies i.e. FITC-A antiCD4 and APC-Cy7-A
antiCD8. Lymphocytes were gated and markers were estimated
proving that these analogs have significantly given both humoral and
cell mediated immunity. Data shown in Fig. 7 clearly mentioned the % of
CD4 cells which are responsible for humoral or antibody-dependent
3.2.5. Immunophenotyping
To check the immune response evoked by the 6e and 6f analogs of
Fig. 6. Total Ig G mesrement from the treated mice sera. BALB/c mice were immunized with HBS Ag (1 µg) alone or in combination with Pam3CysSK4 (20 µg), 6a,
6b,6c, 6d, 6e, 6f, 6g at 10, 20, 30 µg each @450 nm.
8

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
Fig. 7. T cell stimulation by cell surface markers. BALB/c mice were immunized with HBS Ag (1 µg) alone or in combination with Pam3CysSK4 (20 µg), 6e, 6f at 10,
20, 30 µg each. Staining of spleen cells (3X105cells/FACS tubes) with T cell surface marker FITC-A antiCD4 and APC-Cy7-A antiCD8.
immunity thus activating Th cells and antigen presenting cells. Apart
from CD4 cells, CD8 cells were also quantified which are responsible for
cell mediated immunity. Both the groups were more and the less
maintained at the same level thus giving a clear correlation of antigen
cross presentation.
designed ligand set its has been found that in 6a-d showed interactions
Tyr335 with triazolyl centre, Gly313, Asp, 327, leu 330, lys437, phe
325, proline 352 and 6e-f Asp327 triazolyl, val311, gln316, leu350,
phe349 hydrogen bond. The table IFD docking represents three stage of
the docking algorithm used for the study. (i) surface docking of mono-
chain insertion in TLR1; (ii) Di-leg insertion in TLR2 and; (iii) three
leg insertion in TLR2-TLR1 complex. Though the results obtained are
deviated from their biological behaviour Figs. 10 and 11.
3.2.6. Splenocyte proliferation by MTT assay
Splenocyte proliferation was done by MTT assay. The cells were
restimulated with HBsAg, LPS and ConA. After 48 h incubation MTT
reagent was added for the quantification of proliferation of splenocyte.
The results displayed in Fig. 8 shows that 6e and 6f group shows potent
proliferation when compared to that of standards (Pam3CysSK4 and
HBsAg).
Binding affinities prediction for of the full series PamCys3SK4 ana-
logues was obtained explaining the TLR specificity assay (RMSD). By
comparison, the best model obtained where the two charged ligands (6a
and 6g) were omitted is only somewhat better with a RMSD of 0.872
kcal/mol. This is probably a result of the knowledge of the initial docked
position from the crystal structures. Using this technique, we also
investigated different combinations of protein charged states and elec-
trostatic cutoffs. The best comparison was obtained using a charged
residue shell of 9.6 Å around the ligand in combination with a 15 Å
residue-based electrostatic cutoff. The robustness of the results is well-
illustrated by the 1.355 kcal/mol RMSD obtained using the leave-one-
out cross validation technique. This indicates that the fit is not biased
by any particular ligand. It is well evidenced that 6e and 6f shoed
ꢀ 1143.20 and ꢀ 1138.64 kcal/mol. Therefore to inspect further we
performed docking with TLR1 and TLR2 individually and the
3.2.7. DC activation- surface marker estimation
From the data it is clear that compound 6e has elicited significant
MHC II expression when compared to that of the standard Pam3CysSK4
(Fig. 9).
3.3. In silico studies
The docking showed that all the ligands are effectively with TLR1-
TLR2 complex as it is clearly evidented from their XP score. The resi-
dues 256, 258, 261, 266, 269–70, 273–74, 279, 282–84, 289, 293–95
(hydrogen bonding sites) and 304–29, 331–41, 343, 346–52, 355, 359,
367, 370–71, 376 (as hydrphobic pocket) were selected for the Prime
Refinement stage. Especially Hydrogen bond forming amino acid resi-
dues were considered to be most crucial for the docking. Total 2279
binding poses were analyzed for the best ligand 6e, 6f and 6d out of the
TLR1ꢀ TLR2
TLR1
TLR2
ΔΔG
ΔG
calculated by substarcting the ΔG
+ΔG
from
binding
binding
binding
TLR1ꢀ TLR2
it was found that 6e and 6f showed favourabilty for the
binding
foramation of the stabilised complex.
The MM/PBSA studies disclosed that 6e and 6f interacted TLR1-
TLR2 complex with ΔGbinding ꢀ 3764.79 and ꢀ 4469.43 kcal/mol
9

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
Fig. 7. (continued).
hydrophilic alcohols with Pam3CAG is an interesting option to modulate
the overall HLB of the conjugate, to make them suitable for use as ad-
juvants with subunit antigens. Azide-alkyne click chemistry is an
attractive and convenient tool to tether together both these entities
through a triazolyl spacer, enabling high yields of the desired conjugates
with complete regioselectivity. A focused library of seven novel conju-
gates of Pam3CAG were synthesized and tested for their immunoge-
nicity as vaccine adjuvants against HBsAg.
First of all, the focused library was subjected for mechanistic studies
to check the target specificity against TLR-2. When compared to stan-
dard Pam3CysSK4, it was found that all the compounds were acting as a
potential ligand for TLR-2. In ex vivo studies, the foremost thing was to
check the cytotoxity of the compounds. The mice were immunized with
Fig. 8. % proliferation of splenocytes @630 nm. BALB/c mice were immunized
with HBS Ag (1 µg) alone or in combination with Pam3CysSK4 (20 µg), 6e, 6f at
10, 20, 30 µg each. The cells were re-stimulate with HBS Ag, LPS and CONA at
0.1, 10 and 2 µg/ml respectively.
ovaalbumin (100 μg) on 0th day and finally sacrifices on 7th day for
spleen collection. The splenocytes were restimulated with ova albu-
minin alone or in combination with Pam3CysSK4, analogs (6a, 6b, 6c, 6d,
6e, 6f), LPS and CON A. The results derived after 48 h incubation proves
that these compounds are non-toxic in nature, interestingly these com-
pounds were proliferating splenocytes. Splenocyte proliferation was
further conforms by cytokine estimation. Both Th1 and Th2 cytokines
were estimated from the cell culture supernatant. Previous studies in-
though the values are lower than the standard ligand which showed very
high ΔGbinding of ꢀ 1054 kcal/mol due to the high number of hydrogen
bonding site offering more stable Protein-ligand complex.
4. Discussion
dicates that IL-6 and TNF-α are the signature cytokines for Pam3CysSK4,
Even though commercially available standard TLR2 agonists like
Pam3CysSK4 show potential adjuvant activity, their actual usefulness
with various unconjugated antigens in vaccine formulations has been
severely limited which is most likely attributed to its polycationic na-
ture. Even though other TLR2 agonist Pam3CAG is known to be prom-
ising ligand that induce DC maturation and B cell proliferation in vitro, it
is more suitable for liposomal formulations as evidenced in case of
so were considered for study [41]. The cytokine profile of the test
compounds at different doses is given in Fig. 5 which is indicative of
their capability to induce both IL6 and TNF-α, as modulator of Th1/Th2
cytokine expression to varying levels. IL-6 being a pro-inflammatory
cytokine, helps in inflammation along with maturation of B-cells.
Generally it is secreted at sites of acute and chronic inflammation
inducing transcriptional inflammatory response via IL-6 receptors
Pam3CAG-PEG conjugates. Undoubtedly, suitable analogues of Pam3
-
(CD126). TNF-α, an inflammatory cytokine is generally produced by the
CAG fine tuned HLB, suitable for in vivo application with exogenous
action of macrophages or monocytes at the site of acute inflammation.
subunit antigen such as HBsAg is highly needed. Covalently conjugating
10

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
6e
Fig. 9. DC stimulation by cell surface markers. BALB/c mice were sacrificed and splenocytes isolated were treated with Pam3CysSK4 and compounds 6e at 20
concentration each for 24 h. Staining of spleen cells (3 × 105 cells/FACS tubes) with cell surface marker FITC-A anti MHC II and APC-A anti CD86.
μg
Fig. 10. Crystal structure of TLR1-TLR2 with Pam3CysSK4 and depiction of legs of the ligand with TLR1-TLR2 complex.
TNF-
α
exhibit a disparate dimension of signaling actions within cells
Estimation of humoral response by ELISA revealed dose dependent
which ultimately leads to necrosis or apoptosis.
enhancement in IgG response and amongst all the test compounds, only
Encouraging results from ex vivo analysis made us to take it further in
vivo analysis. Mice were immunized in different groups with varying
concentrations of test compounds along with HBsAg antigen using
standard adjuvant Pam3CysSK4 as reference standard. All the test groups
(HBsAg, Pam3CysSK4, 6a, 6b, 6c, 6d, 6e, 6f and 6g) were analyzed for
HBsAg specific IgG response. IgG is a dominating class of immuno-
globulin found in blood serum covering approximately 75% of total
serum immunoglulins. The production of IgG indicates the secondary
immune response against the antigen. Thus IgG response is a principal
tool for evaluating the quality of host immune response to a vaccine.
two analogues 6e and 6f shown significant response when compared
with standard adjuvant. Thus compound 6f at concentration 20 μg and
6e at concentration 30
μg exhibited almost equal response when
compared to that of standard adjuvant Pam3CysSK4.
Immunophenotyping of splenocytes upon treatment with varying
doses of test compound 6e and 6f exhibited dose dependent improve-
ment in CD4 and CD8 populations compared to standard adjuvant
Pam3CysSK4 or antigen (HBsAg alone) with compound 6e enhancing
CD8+ population markedly at 20
μg concentration (2e, 27.55% >
Pam3CysSK4 + HBsAg, 20.99% > HBsAg, 13.39%)which confirm that
11

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
Fig. 11. The IFD results obtained and their superimpoition (6d and 6f).
predominant cell mediated response, even though the corresponding
tethered Pam3CAG conjugates exhibited their capability to potentiate
both humoral and cell mediated immune response, demonstrating their
usefulness as vaccine adjuvants.
CD4+ response was slightly lower compared to standard adjuvant
Pam3CysSK4 (Pam3CysSK4 + HBsAg,25.77 > 2e, 23.91% at 30 μg). CD8
cells contribute to adaptive immunity, crucial for surmounting cytotoxic
T-lymphocyte response against intracellular pathogens like viral in-
fections whereas, CD4 are the surface markers present on Th cells which
stimulates antigen presenting cells like B-cells, macrophages including
CD8 T lymphocytes, thus coordinating the adaptive immune response.
Enhanced antibody response upon compound (6e) treatment along with
6. Experimental section
6.1. Chemistry
6.1.1. Synthesis of (R)-3-((S)-3-oxo-3-(2-oxo-2-phenylethoxy)- 2-palmi-
tamidopropylthio) propane-1,2-diyldipalmitate (S5)
HBsAg at 20 μg concentration reveal that the observed stimulation of
CD4 is sufficient enough to evoke effective humoral immunity. Thus
bothCD4 and CD8 play crucial role in inducing quality immune system
required for pathogen clearance. Ex vivo estimation of MHC class-II
expression on isolated DCs,upon compound treatment, indicated sig-
The diol compound S4 [32] (3.01 g, 7.28 mmol) was dissolved in dry
THF and palmitic acid (5.59 g, 21.8 mmol), DIC (3.6 g, 27.9 mmol),
DMAP (0.35 g, 2.9 mmol), were added one after another under inert
atmosphere. This reaction mixture was allowed to stirr for 4 h at room
temperature. After complete consumption of starting material solvent
was evaporated under reduced pressure to furnish semi solid residue
which was used further without purification. This obtained residue (6 g,
6.74 mmol) was dissolved in 20% TFA: CH2Cl2 and stirred at room
temperature for 40 min. After completion of starting material excess
solvent was evaporated by rota vacuo. Residue co-evaporated with
hexane, then subjected to azeotropic evaporation with toluene and the
residue obtained was used in the next step. Resulting amine compound
(5.32 g, 6.7 mmol) was dissolved in dry THF and the solution was added
to previously stirred solution of the palmitic acid (2.05 g, 8.04 mmol),
DIC (1.03 g, 8.04 mmol), HOBt (1.23 g, 8.04 mmol) in dry THF. After
addition, reaction mixture was stirred at room temperature for 5 h. After
completion of starting material reaction mixture was diluted with ethyl
acetate, washed with water (50 ml), satd NaHCO3 (50 ml). The aqueous
layer extracted with ethyl acetate (20 ml). The combined organic layers
were dried over anhydrous Na2SO4 and the solvent was evaporated
under vacuum to afford the impure residue. The residue was purified by
column chromatography from using 35% Ethyl acetate/hexane as eluent
to give c ompound S5 as light yellow solid (4.9 g, 60% over three steps).
1H NMR (300 MHz, CDCl3) δ 7.90 (d, J = 7.5 Hz, 2H,aromatic), 7.62 (t,
J = 7.4 Hz, 1H,aromatic), 7.49 (t, J = 7.6 Hz, 2H,aromatic), 6.43 (dd, J
= 18.2, 7.6 Hz, 1H,–NH), 5.50 (d, J = 16.3 Hz, 1H,phenacyl-CH2-), 5.35
(d, J = 16.4 Hz, 4H,Phenacyl-CH2-), 5.26 – 5.13 (m, 1H,–CH-O ester),
nificant enhancement in MHC-II expression (3.82%, 20
μg of 6e)
compared to standard adjuvant (Pam3CysSK4 + HBsAg, 2.77%).
LPS and CON-A are standard mitogens for B and T cell proliferation.
When selected groups administered with test compounds in different
doses, were restimulated with HBsAg, LPS and CON-A, the % prolifer-
ation of cells in MTT assay showed potential proliferation of B and T cells
without any cytotoxicity. A, but test compound 6e (at 30
20
μg) and 6f (at
μ
g) showed significant proliferation. Both B-cell proliferation and T-
cell proliferation is essential to fight any kind viral and bacterial in-
fections. B-cells not only produce antibodies against that particular
pathogen but also retain the memory for further defense from the
particular pathogen.
5. Conclusion
Our TLR-2 functional bioassay determined that novel Pam3CAG an-
alogues presented here had human TLR-2 agonistic activity comparable
to those of the reference compound Pam3CysSK4. The novel Pam3CAG
conjugates, show activities of different magnitude indicating the mod-
ulation of immune response by different substitution with varieties of
sugars and alcohols. Based on the immune response derived, it could be
inferred that the number of hydroxyl groups present on the scaffold on
substitution “R” is playing a significant role rather than the cyclic or
acyclic nature of the substitution. Thus, the substituent with two and
three hydroxyl groups exhibited optimal immune response compared to
substituent with lesser (one hydroxyl) or more than three hydroxyls.
Compounds 6e and 6f demonstrated significant enhancement of OVA
and HBsAg specific IgG response. Both the analogues showed marked
improvements in CD4 and CD8 surface markers expression, a hall mark
of enhanced Th and cell mediated immunity, indicating their usefulness
in vaccines against intracellular pathogenesis. Thus the new triazole
4.96 (dd, J = 12.1, 6.4 Hz, 1H,α-CH-of cys), 4.35 (dd, J = 11.7, 3.4 Hz,
1H, –CH2-O ester), 4.16 (dd, J = 11.7, 3.4 Hz, 1H, –CH2-O ester), 3.27
(dd, J = 13.9, 4.8 Hz, 1H,-S-CH2-), 3.12 (dd, J = 14.0, 6.5 Hz, 1H,-S-CH2-
), 2.81 (d, J = 6.4 Hz, 2H, –CH2-S-), 2.30 (dt, J = 16.1, 7.9 Hz, 6H,
–
–
–
α
-CH -C O-), 1.61 (m, 1.70–1.53, 6H, β-CH -C O), 1.25 (bs, 76H,
–
2 2
–CH2-lipid chains), 0.87 (t, J = 6.4 Hz, 9H, 3-CH3 lipid chains); 13C NMR
–
–
–
–
–
–
C
(101 MHz, CDCl3) δ 191.27 (
C
O phenacyl ring), 173.40 (
palmitoyl ester),173.16(
C
O
O
–
–
–
–
–
palmitoyl ester), 173.31(
C
–
O
12

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
–
–
phenacyl ester), 170.49(NH
C
–
O palmitoyl amide), 134.15(aro-
adding saturated citric acid. Organic layer was separated and aqueous
layer was extracted (3 × 30) with ethyl acetate. Combined organic layer
was dried over Na2SO4 and concentrated under reduced pressure to give
crude Boc-Ala-Gly-OH. This acid compound (9.48 g, 36.44, 100%) was
directly used for next reaction. EDC.HCl (10.44 g, 54.68 mmol) and
HOBt (3.69 g, 27.34 mmol) were added to a solution of crude acid
compound (4.74 g, 18.22 mmol) dissolved in 100 ml dry THF. Resulting
mixture was stirred for 30 min at ambient temperature. After 30 min of
stirring, reaction kept at 0 ◦C and propargyl amine (2.80 ml, 43.72
mmol), DIPEA (12.59 ml, 72.88 mmol) in 20 ml dry THF were added
slowly to the above reaction mixture. Reaction was allowed to stir at
ambient temperature for 10 hrs. After completion of reaction, water (50
ml) and EtOAc (50 ml) were added. Aqueous layer was separated and
organic layer was washed with brine (25 ml) and sat NaHCO3 (30 ml).
Organic layer was dried over Na2SO4 and concentrated under reduced
pressure. Crude product 4 was purified by column chromatography
using 2%CH3OH:CHCl3 as eluent. Yield: 85% (8.81 g) White solid MP-
149–151 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.02 (bs, 1H,–NH), 6.83 (bs,
matic), 133.89(aromatic), 128.99(aromatic), 127.78(aromatic), 70.31
(–CH- ester), 66.78(
cysteine), 36.40(–CH2-S-), 34.65(-S-CH2-), 34.34(
amide), 34.12( -CH2- of palmitoyl ester), 32.98(
α
-CH2-phenacyl), 63.63(–CH2-O-ester), 51.84(
α-CH-
α
α
-CH2- of palmitoyl
-CH2- of palmitoyl
α
ester), 31.96, 29.74, 29.56, 29.40, 29.18, 25.56, 24.96, 24.91, 22.72,
(31.96 to 22.72 methylene carbons of lipids), 14.15(–CH3 of lipids);
LRMS: Calculated for C62H109NO8S 1027.78; Found: 1050.7797 [M +
Na]+.
6.1.2. Synthesis (S)-3-((R)-2,3-bis(palmitoyloxy)propylthio)-2-
palmitamidopropanoic acid (2)
Compound S5 (4.9 g, 4.7 mmol) was dissolved in glacial in acetic
acid (50 ml) and activated Zn (4.95 g, 76.2 mmol) was added to it at
room temperature. Reaction mixture was stirred for 2 h at same tem-
perature. After complete consumption of the starting material, the re-
action mass was filtered through celite. The filtrate was evaporated
under reduced pressure to afford crude product. This crude product was
subjected to purification by column chromatography, using 50–80%
EtOAc/Hexane as eluent to afford pure compound 2 (3.89 g, 90%) as
white solid, mp 63–65 ◦C.1H NMR (400 MHz, CDCl3) δ 6.58 (d, 7.2 Hz,
1H,–NH), 5.15 (qd, J = 6.4, 3.5 Hz, 1H,–CH-O ester), 4.77 (ddd, J =
1H,–NH), 5.06 (d, J = 5.8 Hz, 1H,–NH), 4.14 – 3.89 (m, 5H, α-CH of
–
–
C ONHCH -of gly), 2.20 (t, J = 2.5 Hz, 1H,alkyne-
–
ala,
α
-CH2 and
CH), 1.45 (s, 9H,-Boc), 1.38 2(d, J = 7.1 Hz, 3H,–CH3 of ala);13C NMR
–
–
–
(100 MHz, CDCl ) 174.28(C O of ala), 169.26(C O of gly), 156.13
–
3
–
15.8, 11.9, 5.8 Hz, 1H,
α
-CH Cys), 4.38–4.31 (m, 1H, –CH2-O ester),
(C O of Boc), 79.92(>C< of Boc), 78.68(alkyne -C-), 70.78(alkyne-CH)
–
4.17–4.08 (m, 1H, –CH2-O ester), 3.19 – 2.99 (m, 2H,-S-CH2- cys), 2.80 –
50.40(α-CH of ala), 42.06(α-CH2 of gly), 29.20, 28.33, 27.75(Boc-CH3),
2.68 (m, 2H, –CH2-S-), 2.35 – 2.24 (m, 6H,
α
-CH2 of lipid chains), 1.69 –
16.93(–CH3 of ala); HRMS (ESIMS) calcd. for C13H21N3O4 [M + Na]+:
calcd. m/z 306.1430; found: 306.1435
1.52 (m, 6H,β-CH2 of lipid chains), 1.25 (bs, 72H, –CH2- lipid chains),
0.87 (t, J = 6.8 Hz, 9H, 3-CH3 lipid chains); 13C NMR (126 MHz, CDCl3)
–
–
–
–
–
–
–
–
δ 174.49(
C
O of acid), 174.29(
C
O ester), 173.64(
C
O ester),
6.1.5. Synthesis of (9R,12S,16R)-9-methyl-5,8,11-trioxo-12-palmitamido-
14-thia-4,7,10-triazaheptadec-1-yne-16,17-diyl dipalmitate (5)
–
–
–
172.70(
C O amide) 70.24(–CH-O ester), 63.70(–CH -O ester), 52.10
–
2
(
(
α
α
-CH Cys), 51.93(–CH2-S-), 36.35(
α
-CH2 amide), 36.31(-S-CH2-), 34.37
To a solution of Compound 2 (Pam3Cys-OH, 5 g, 5.49 mmol) in 50 ml
dry THF under N2 atmosphere were added EDC.HCl (1.57 g, 8.23 mmol)
and HOBt (1.11 g, 8.23 mmol). This reaction mixture was allowed to stir
for 30 min at RT. Meanwhile compound 4 (0.605 g, 11 mmol) was
treated with 20%TFA: DCM (20 ml) for 40 min and this reaction mixture
concentrated and treated with DIPEA (3.82 ml, 21.96 mmol) at 0 ◦C in
dry THF. This was added to the above reaction mixture of acid at 0 0C.
The resulting reaction mixture was allowed to stir overnight. After the
completion of reaction, CH2Cl2 (50 ml) was added to it. The organic
layer was washed with water (2 × 50 ml) and saturated NaHCO3 (2 × 50
ml), dried over Na2SO4 and organic solvent was removed under vacuum.
The crude residue was purified by column chromatography (2% MeOH:
Chloroform) to obtain required compound 5 in good yield (4.14 g, 70%)
-CH2- ester), 34.13(α-CH2- ester), 32.97, 31.96, 29.75, 29.70, 29.57,
29.40, 29.34, 29.17, 25.57, 24.96, 24.90, 22.72, (32.97 to 22.72 –CH2-
of lipid chains) 14.14(–CH3 of lipids); LRMS: Calculated for
C
54H103NO7S 909.7455; Found: 932.50 [M + H]+.
6.1.3. Synthesis of (R)-tert-butyl (1-oxo-1-((2-oxo-2-(prop-2-yn-1-
ylamino)ethyl)amino)propan-2-yl)carbamate (3)
Compound 3 (Boc-Ala-Gly-OEt) was synthesized from Boc-Alanine
and ethyl glycinate. EDC.HCl (30 g, 158 mmol) and HOBt (21.42 g,
158 mmol) were added to a solution of Boc-Ala-OH (25 g, 132.0 mmol)
dissolved in dry DMF. This reaction mixture was stirred for 15 min then
HCl.NH2-Gly-OEt (18.5 g, 132.0 mmol) and DIPEA (29 ml, 264.0 mmol)
were added. The mixture was stirred at room temperature for 8 h. After
complete consumption of starting material, reaction was quenched with
addition of ice cold water. Ethyl acetate was added to this solution and
extracted with it. The organic layer was washed with 5% sodium bi-
carbonate and water. The organic layer was dried with anhydrous so-
dium sulfate, was concentrated to dryness to get an oily residue of
compound 3. This was purified by column chromatography (2:98
CH3OH:CHCl3) (25.37 g, 70% yield). 1H NMR (400 MHz, DMSO‑d6) δ
8.16 (t, J = 5.8 Hz, 1H,Gly-NH), 6.92 (d, J = 7.6 Hz, 1H,Ala-NH), 4.08
as white solid. mp 145.0 C; 1H NMR (300 MHz, CDCl3 + CD3OD) δ
◦
5.18–5.09 (m, 1H, –CH-O ester), 4.47 (t, J = 6.9 Hz, 1H, α-CH-cys),
4.39–4.20 (m, 2H, α-CH-ala,CH2-O ester), 4.08 (dd, J = 12.0, 6.5 Hz, 1H,
–CH2-O ester), 3.95 (dd, J = 4.1, 2.4 Hz, 2H, –CH2-gly), 3.83 (d, J = 13.1
Hz, 2H, –CH2-propargyl), 2.93 (dd, J = 13.5, 6.5 Hz, 1H,-S-CH2–), 2.80
(dd, J = 13.8, 7.2 Hz, 1H, -S-CH2–), 2.75 – 2.64 (m, 2H, –CH2-S-), 2.33 –
2.14 (m, 7H, alkyne-CH, α-CH2 lipid chains), 1.64–1.45 (m, 6H,β-CH2-
lipid chains), 1.35 (d, J = 7.2 Hz, 3H, –CH3 alanine), 1.20 (bs, 70H,
–CH2-lipid chains), 0.82(t, J = 6.4 Hz, 9H,3-CH3 lipid chains); 13C NMR
–
–
–
–
C
–
C
(q, J = 7.1 Hz, 2H,–OCH2CH3), 3.99 (dd, J = 14.4, 7.1 Hz, 1H,
α
-H of
(75 MHz, CDCl3 + CD3OD) δ 174.46(
O ester), 173.61(
O
–
ala), 3.81 (ddd, J = 37.3, 17.4, 5.9 Hz, 2H,-glycine-CH2),1.37 (s, 9H,-
ester), 173.54(–NHC=O,palmitoyl) 172.85(–NHC=O peptide), 171.10
(–NHC=O peptide), 169.06(–NHC=O peptide), 79.04(alkyne C), 70.95
(–CH-O ester), 70.06(terminal C-alkyne), 63.58(–CH2-O ester), 52.30
Boc), 1.21–1.15 (m, 6H,–OCH2CH3 and –CH3 of Ala), 13C NMR (75
–
–
–
–
MHz, DMSO‑d ) δ 173.16 (>C O ala), 169.60(>C O gly), 154.87(–CO
6
Boc), 79.05(>C < of Boc, 77.88, 60.23(O-CH2CH3), 49.36(
α
-CH of ala),
(
α
-CH-cysteine), 49.60(
α
-CH-alanine), 42.18(
α
-CH-glycine), 35.89
-CH2 of
40.56(
α
-CH2 of gly), 28.08(3 –CH3 of t-butyl), 18.07(β-CH3 ala), 13.93
(–CH2-S-), 34.06(S-CH2–), 33.98(
α
-CH2 of NHCOpalm), 33.83(α
(O-CH2CH3); LRMS: Calculated for C12H22N2O5 274.3134; Found:
COOpalm), 32.46, 31.65, 29.41(–NHCH2-propargyl), 29.25, 29.08,
28.86, 28.55, 27.51, 25.31, 24.67, 24.60, 22.39, 21.17, (32.46 to
21.17,–CH2– lipid chains), 16.58(β-CH3 alanine), 13.71(–CH3 lipid
chains); HRMS (ESIMS) calcd. for C62H114N4O8S [M + Na]+: calcd. m/z
1097.8255; found: 1097.8274
297.25 [M + Na]+.
6.1.4. Synthesis of compound 4
Lithium hydroxide (1.312 g, 54.66 mmol) was added portion wise to
a solution of compound 3 (10 g, 36.44 mmol) in MeOH: Water (60 ml, v/
v) at 0 ◦C. Then reaction mixture was allowed to stir at RT for 2hrs. After
complete consumption of starting material, reaction mixture was diluted
with EtOAc (30 ml) and water (20 ml). Basic PH was adjusted upto 3 by
6.1.6. General procedure for the synthesis of 1, 2, 3-triazole tethered
hydroxy-Pam3CAG hybrid conjugates (6a-g)
Compound 5 (100 mg, 0.093 mmol) and polyhydroxyl azides I-VII
13

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
(1.2 eq, 0.111 mmol) were suspended in ter-butanol and water (5 m l,
1:1, v/v) mixture. To the above stirred solution CuSO4⋅5H2O (47 mg,
0.186 mmol) and sodium ascorbate (36.82 mg, 0.186 mmol) were added
at ambient temperature. Resulting heterogeneous mixture was allowed
to stir vigorously for 8–12 h at same temperature. TLC analysis indicated
complete consumption of the reactants, solvent was removed under
reduced pressure and crude was diluted with EtOAc (30 ml) and water
(20 ml). Organic layer was separated and aqueous layer was extracted
with ethyl acetate (2 × 30). Combined organic layers were washed with
brine and dried over an.Na2SO4. Organic layers were concentrated
under vacuum to give crude product which was purified by column
chromatography techniques using 5% CH3OH:CHCl3 as eluent to get
corresponding 1,2,3-triazolyl Pam3Cys-Ala-Gly hybrid conjugates in
quantitative yields (90 to 98%) as white solids.
28.39, 25.01, 24.19, 21.91(28.96 to 21.91,–CH2– lipid chains), 15.78
(β-CH3 ala), 13.00(3-CH3 lipid chains); HRMS (ESI-MS) calcd. for
C68H125N7O13S [M + H]+: calcd. m/z 1280.9134; found: 1280.9126
6.1.6.3. (7R,10S,14R)-7-methyl-3,6,9-trioxo-10-palmitamido-1-(1-
((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)-1H-1,2,3-triazol-4-yl)-12-thia-2,5,8-triazapentadecane-14,15-
diyl dipalmitate (6c). Compound 6c was prepared from compound 5
(100 mg, 0.093 mmol) and 1-azido-β-D-galactose III (23 mg, 0.111
mmol) according to the given general procedure in good yield as a white
solid. (104 mg, 88%). Melting point: 168-1700C; 1H NMR (300 MHz,
–
CDCl + CD OD) δ 8.01 (s, 1H, CH triazole), 5.49 (d, J = 9.2 Hz, 1H,H-
–
3
3
1 Gal), 5.24–5.09 (m, 1H,–CH-O ester), 4.52–4.32 (m, 4H,
-CH-ala and –CH2-triazole), 4.28 – 3.96 (m, 4H, –CH2-O ester and
–CH2-gly), 3.92 – 3.62 (m, 6H,H-2,H-3,H-4,H-5,H-6 and H-6), 3.04 –
α-CH cys,
α
6.1.6.1. (7R,10S,14R)-7-methyl-3,6,9-trioxo-10-palmitamido-1-(1-
((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)-1H-1,2,3-triazol-4-yl)-12-thia-2,5,8-triazapentadecane-14,15-
diyl dipalmitate (6a). Compound 6a was prepared from compound 5
(100 mg, 0.093 mmol) and 1-azido-β-D-glucose I (23 mg, 0.111 mmol)
according to the above general procedure in good yield. (93%, 110 mg)
2.64 (m, 4H,-S-CH2– and –CH2-S-), 2.45 – 2.10 (m, 6H,α-CH2– lipid
chains), 1.67–1.52 (m, 6H,β-CH2-lipid chains), 1.48 – 1.08 (m, 75H,β-
CH3 ala and –CH2-lipid chains), 0.86 (t, J = 6.5 Hz, 9H,–CH3 lipid
chains); 13C NMR (101 MHz, CDCl3 + CD3OD) δ 174.68(
O ester),
–
–
C
–
–
–
173.54(
C O ester), 173.42(–NHCO-palm), 173.21(–NHCO-peptide),
–
–
–
–
–
171.32(–NHCO-peptide), 144.35( C<,triazole ring), 121.34( CH,
White solid; Melting point: 190–193 ◦C; 1H NMR (300 MHz, CDCl3
+
triazole ring), 88.32(C-1 Gal), 77.78(C-5 Gal), 73.46(–CH-O ester),
69.99(C-3 Gal), 69.77(C-4 Gal), 68.55(–CH2-O ester), 63.47(C-2 Gal),
CD3OD) δ 7.94 (s, 1H,=CH triazole), 5.52 (d, J = 9.1 Hz, 1H,H-1 glu),
5.17–5.07 (m, 1H,–CH-O ester), 4.50–4.31 (m, 4H,
α
-CH-cys,
α
-CH-ala
60.84(C-6 Gal), 52.14(
33.87(S-CH2–), 33.65(
palm), 31.46(
α
-CH- cys), 49.69(
α
-CH- ala), 35.57(CH2-S),
and –CH2-triazole), 4.28 – 4.07 (m, 3H, –CH2-Oester and –CH-glycine),
3.93 – 3.68 (m, 5H,H-6,H-6,H-5,H-3 of glu,–CH-gly), 3.57–3.52 (m, 2H,
H-2, H-4 glu), 2.97 – 2.68 (m, 4H, –CH2-S- and –S-CH2–), 2.37 – 2.05 (m,
α
-CH2– of NHCOpalm), 32.13(α
-CH2– of -COO-
α
-CH2– of –COOpalm), 29.23, 29.19, 29.10, 28.95, 28.89,
28.68, 25.22, 24.50, 24.44, 22.19(29.23 to 221.19, –CH2-lipid chain),
16.13(–CH3 ala), 13.42(–CH3 lipid chains); HRMS (ESI-MS) calcd. for
6H,α-CH2– lipid chains), 1.66–1.48 (m, 6H, β-CH2– lipid chains), 1.39
(d, J = 7.1 Hz, 3H,β-CH3 ala), 1.34–1.15 (m, 72H,–CH2– lipid chains),
C
68H125N7O13S [M + H]+: calcd. m/z 1280.9134; found: 1280.9175
0.85 (t, J = 6.5 Hz, 9H,–CH3 lipid chains); 13C NMR (101 MHz, CDCl3 +
–
–
–
–
C
–
C
CD3OD)
δ
175.04(
O
ester), 173.55(
O ester), 173.43
–
6.1.6.4. (7R,10S,14R)-7-methyl-3,6,9-trioxo-10-palmitamido-1-(1-
(((2R,3R,4S,5R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methyl)-
1H-1,2,3-triazol-4-yl)-12-thia-2,5,8-triazapentadecane-14,15-diyl dipalmi-
tate (6d). Compound 6d can be prepared from compound 5 (100 mg,
0.093 mmol) and 6-azido-D-galactose IV (23 mg, 0.111 mmol) according
to the above general procedure to afford the compound in good yield as
–
–
–
–
– –
C
(palmNH
C
O), 171.69 (–NH-CO), 171.32( NH
O),169.92
–
–
–
–
– –
NH C O), 144.45(>C = of triazole ring), 121.55( CH– triazole
–
(
ring), 87.72 (C-1 glu), 78.95(C-5 glu), 76.35(–CH-ester), 72.27(C-3 glu),
69.91(C-4 glu), 68.83(C-2- glu), 63.24 (–CH2O-ester), 60.62(-C-6 glu),
52.37(α-CH– cys), 49.73(α-CH– ala), 42.33(–CH2-gly),42.20(–CH2-tri-
a white solid (114 mg, 96%). Melting Point:179-1810C; 1H NMR (300
azole), 35.31(CH2-S), 33.83(S-CH2–), 33.62(α-CH2– of NHCOpalm),
–
31.43 α-CH2– of COOpalm, 29.19, 28.85, 28.63, 25.19, 24.40, 22.16
MHz, CDCl3 + CD OD) δ 7.73 (s, 1H, CH triazole), 5.10 (s,1H,H-1
–
3
(29.19 to 22.16, –CH2-lipid chains) 16.04(–CH3 of ala), 13.38(3-CH3
lipid chains); HRMS (ESI-MS) calcd. for C68H125N7O13S [M + H]+:
calcd. m/z 1280.9134; found: 1280.9145
galactose), 4.37–4.00 (m,8H, –CH-O ester, α-CH-cys, α-CH-ala, –CH2-
triazole,1H-CH2-Oester, –CH2-gly) 3.94–3.50 (m, 6H, 1H of –CH2O
ester,H-6,H-6,H-5,H-2,H-3 galactose), 3.46–3.40 (m, C-4 Gal),
2.97–2.55 (m, 4H,-S-CH2– and –CH2-S-), 2.35–2.08 (m, 6H,α-CH2-lipid
6.1.6.2. (7R,10S,14R)-7-methyl-3,6,9-trioxo-10-palmitamido-1-(1-
((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)-1H-1,2,3-triazol-4-yl)-12-thia-2,5,8-triazapentadecane-14,15-
diyl dipalmitate (6b). Compound 6b was synthesized from compound 5
chains), 1.53 (bs, 6H, β-CH2-lipid chains), 1.44 – 1.10 (m, 75H,β-CH3 ala
and –CH2– lipid chains), 0.79 (t, J = 5.6 Hz, 9H,–CH3 lipid chains); 13
C
–
– –
–
NMR (125 MHz, CDCl3 + CD3OD) δ 175.09(
C
O ester), 175.03
–
–
–
–
–
C
(
O ester), 173.62(NH-COpalm), 173.55( NH
C
O), 171.83
–
(100 mg, 0.093 mmol) and 1-azido-
α
-D-mannose II (23 mg, 0.111 mmol)
(–NHC=O pept),169.80(–NHC=O pept), 169.77(–NHC=O pept), 144.42
–
–
–
–
–
( C < triazole ring), 123.68( C H triazole ring), 123.42( CH tri-
– –
according to the above general procedure in excellent yield (91%, 108
mg) as white solid. Melting Point: 176–1780 C; 1H NMR (300 MHz,
azole ring), 96.74(C-1 Gal), 92.35(C-1 Gal), 73.02(–CH-Oester), 71.81
◦
CDCl3 + CD3OD)δ 7.94 (s, 1H,=CH triazole)), 5.52 (d, J = 9.1 Hz, 1H,H-
(C-2 Gal), 70.03(C-4 Gal), 69.54(C-4 Gal), 68.80(C-3 Gal), 68.67(C-3
1 man), 5.18–5.08 (m, 1H,–CH-Oester), 4.49–4.31 (m, 4H,
α-CH-cys,
Gal), 68.37(–CH2-O ester), 63.33(C-5 Gal), 52.41(C-6 Gal), 51.00(
α-CH-
α
-CH-ala and –CH2-triazole), 4.27 – 4.10 (m, 3H, –CH2-O ester,–CH-gly),
cys), 50.71(
ring), 35.55(–CH2-S-), 34.48(
33.78(
α
-CH- cys), 49.93(
α
-CH- ala), 42.58(–CH2-gly, –CH2-triazole
-CH2 of –NHCOpalm)), 33.99(-S-CH2–),
-CH2 of –COOpalm), 29.35, 29.22,
3.92 – 3.69 (m, 5H,–CH-gly, H-5, H-6, H-3, H-6 of man), 3.61–3.50(m,
2H, H-2 man and H-4 man), 2.96 – 2.66 (m, 4H,-S-CH2– and –CH2-S-),
α
α
-CH2 of –COOpalm), 31.58(
α
2.36 – 2.06 (m, 6H,
α-CH2– of lipid chains), 1.66–1.47 (m, 6H,β-CH2–
29.02, 28.79, 25.33, 24.55, 22.32 (29.35 to 22.32,–CH2– lipid chains)
16.23(β-CH3 ala), 13.62(–CH3 of lipid chains); HRMS (ESI-MS) calcd.
for C68H125N7O13S [M + H]+: calcd. m/z 1280.9134; found: 1280.9172
lipid chains), 1.39 (d, J = 7.1 Hz, 3H,β-CH3 ala), 1.23 (s, 74H,–CH2–
lipid chains), 0.85 (t, J = 6.5 Hz, 9H,–CH3 lipid chains); 13C NMR (101
–
–
–
–
–
MHz, CDCl3 + CD OD) δ 174.81(C O ester), 173.44(
C O ester),
173.28(–NHCO pal3m), 173.10(–NHCO- peptide), 171.37(–NHCO- pep-
6.1.6.5. (7R,10S,14R)-1-(1-((2S,3S,4R,5S)-3,4-dihydroxy-5-(hydrox-
ymethyl)tetrahydrofuran-2-yl)-1H-1,2,3-triazol-4-yl)-7-methyl-3,6,9-tri-
oxo-10-palmitamido-12-thia-2,5,8-triazapentadecane-14,15-diyl dipalmi-
tate (6e). Compound 6e was prepared from compound 5 (100 mg,
0.093 mmol) and 1-azido-ribose V (20 mg,0.111 mmol) according to the
general procedure in excellent yield as white solid (114 mg, 98% yield),
Melting point: 178-1800C; 1H NMR (400 MHz, CDCl3 + CD3OD) δ 7.83
–
–
–
C H,
tide), 169.68(–NHCO- peptide), 144.86(>C = triazole), 122.27(
triazole) 86.39(C-1 man), 76.18(C-5 man), 70.48(CH-O ester), 69.79(C-
3man), 68.08(C-4 man), 66.54(CH2-O ester),63.06(C-2man), 60.64 (C-6
man), 52.32(α-CH– cys), 49.41(α-CH– ala), 42.01(–CH2-triazole & gly),
35.09(CH2-S-), 33.54(S-CH2–), 33.34(
α-CH2– of NHCOpalm), 31.32
(
α
-CH2– of COOpalm), 31.20( -CH2– of COOpalm), 28.96, 28.84, 28.63,
α
14

T.B. Mhamane et al.
Bioorganic Chemistry 111 (2021) 104838
–
(s, 1H, CH triazole), 5.73 (s, 1H, H-1 rib), 5.21–5.04 (m, 1H, –CH-O
33.41(
α
-CH2– of ester), 32.78(
α
-CH2– ester), 31.41, 31.27, 29.02, 28.69,
–
ester), 4.56–4.07 (m, 6H,
α
-CH-cys,
α
-CH-ala,–CH2 of triazole and –CH2-
28.45, 25.05, 24.26, 21.98(31.41 to 21.98, –CH2-lipid chains), 15.78
(β-CH3 ala), 13.08(–CH3 lipid chains); (ESIMS) calcd. for C64H119N7O9S
[M + Na]+: calcd. m/z 1184.8690; found: 1184.8698
Oester), 4.06 – 3.73 (m, 6H, –CH2– gly, H-2,H-3,H-4,H-6, H-6),
2.98–2.62 (m, 4H,-S-CH2– and –CH2-S-), 2.40 – 1.96 (m, 6H,α-CH2-lipid
chains), 1.66–1.47 (m, 6H,β-CH2-lipid chains), 1.46 – 1.05 (m, 75H,β-
CH3 ala and –CH2– lipid chains), 0.89–0.80 (m, 9H,–CH3 lipid chains);
Declaration of Competing Interest
13C NMR (100 MHz, CDCl3 + CD3OD) δ 175.01(
O ester), 174.64
–
–
C
–
–
–
–
C
–
–
C
(
O ester), 173.46(
O, –NHCO palm), 173.33(–NHCO- pept),
–
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
171.67(–NHCO- pept), 85.39(C-1 rib), 70.40(C-4 rib), 69.91(–CH-O
ester) 69.37(C-3 rib), 66.24(C-2 rib), 65.06(–CH2-O ester), 63.19(C-5
rib), 52.42(α-CH-Cys), 52.03(α-CH-ala), 49.72(–CH2-gly), 49.66
(–CH2-triazole), 35.47(–CH2-S), 35.25(
α-CH2– -NHCOpalm)), 33.76(-S-
Acknowledgement
CH2), 33.55(α-CH2-COO ester), 31.38(α-CH2-COO ester), 29.14, 28.80,
28.58, 25.14, 24.35, 22.10(29.14 to 22.10,–CH2– lipid chains), 15.96
(β-CH3 ala), 13.29(–CH3 lipid chains); HRMS (ESIMS) calcd. for
TM and SS thank CSIR and DST for the award of fellowships. Authors
thank DST, New Delhi for the financial support through Grant-in-aid
project GAP0774. CSIR-IICT Manuscript No: IICT/Pubs./2019/365
C
67H123N7O12S [M + H]+: calcd. m/z 1250.9028; found: 1250.8999
6.1.6.6. (7R,10S,14R)-1-(1-((S)-2,3-dihydroxypropyl)-1H-1,2,3-triazol-
4-yl)-7-methyl-3,6,9-trioxo-10-palmitamido-12-thia-2,5,8-tri-
Appendix A. Supplementary material
azapentadecane-14,15-diyl dipalmitate (6f). Compound 6f was prepared
from compound 5 (100 mg, 0.093 mmol) and (S)-3-azido-1,2-propane
diol VI (13 mg, 0.111 mmol) according to the above general procedure
to yield the title compound in good yield (105 mg, 96% yield). White
Solid, Melting Point: 169–171 ◦C; 1H NMR (400 MHz, CDCl3 + CD3OD)
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104838.
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δ 7.93 (s, 1H, CH,triazole ring), 5.33–5.25 (m, 1H,–CH-O ester),
–
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–
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175.09(-C=Oester),
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α
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–
–
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16