Development of N-acetylated dipalmitoyl-S-glyceryl cysteine analogs as efficient TLR2/TLR6 agonists

Article abstract of DOI:10.3390/molecules24193512

Cancer vaccine is a promising immunotherapeutic approach to train the immune system with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in cancer vaccines to mimic an infection process and amplify immune responses.

Full text of DOI:10.3390/molecules24193512

molecules
Article
Development of N-Acetylated Dipalmitoyl-S-Glyceryl
Cysteine Analogs as Efficient TLR2/TLR6 Agonists
Yang Zhou † , Abid H. Banday †, Victor J. Hruby and Minying Cai *
Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ 85721, USA;
yangzhou@email.arizona.edu (Y.Z.); abidrrl@gmail.com (A.H.B.); hruby@email.arizona.edu (V.J.H.)
* Correspondence: mcai@email.arizona.edu; Tel.: +1-520-621-8617
† These authors contributed equally.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Henry Mosberg, Tomi Sawyer and Carrie Haskell-Luevano
Received: 28 August 2019; Accepted: 25 September 2019; Published: 27 September 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Cancer vaccine is a promising immunotherapeutic approach to train the immune system
with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in
cancer vaccines to mimic an infection process and amplify immune responses. The Toll-like receptor 2
and 6 (TLR2/TLR6) agonist dipalmitoyl-S-glyceryl cysteine (Pam2Cys) was demonstrated as an ideal
candidate for synthetic vaccine adjuvants. However, the synthesis of Pam2Cys requires expensive
N-protected cysteine as a key reactant, which greatly limits its application as a synthetic vaccine
adjuvant in large-scaled studies. Here, we report the development of N-acetylated Pam2Cys analogs
as TLR2/TLR6 agonists. Instead of N-protected cysteine, the synthesis utilizes N-acetylcysteine to
bring down the synthetic costs. The N-acetylated Pam2Cys analogs were demonstrated to activate
TLR2/TLR6 in vitro. Moreover, molecular docking studies were performed to provide insights into
the molecular mechanism of how N-acetylated Pam2Cys analogs bind to TLR2/TLR6. Together,
these results suggest N-acetylated Pam2Cys analogs as inexpensive and promising synthetic vaccine
adjuvants to accelerate the development of cancer vaccines in the future.
Keywords: cancer vaccine; synthetic vaccine; adjuvant; Toll-like receptor; Pam2Cys; N-acetylated
Pam2Cys
1. Introduction
In the recent past, cellular immunotherapy has come up as one of the most suitable approaches for
treating major diseases, such as infection and cancer, due to its high selectivity. The Chimeric Antigen
Receptor T (CAR-T) therapy involves collecting T cells from patients and genetically modifying them to
recognize specific antigens that are only expressed on tumor cells [
parallel to CAR-T therapy, cancer vaccines and synthetic vaccines utilize isolated or synthesized
antigens to train the immune system to recognize tumors or pathogens [ ]. The antigens used in cancer
vaccines can be designed based on information collected from individual patients, thus opening up
opportunities for personalized cancer treatment [ ]. However, many isolated or synthetic antigens have
poor immunogenicity on their own [ ], and require adjuvants to help enhance the magnitude and
1]. As immunotherapy approaches
2
3
4
quality of immune responses specific to various antigens [5].
Adjuvants include liposomes, lipopeptides, single stranded DNA etc., that mimic a natural
infection to activate various immune components such as dendritic cells, macrophages and lymphocytes
to produce desired immunological effects [6]. As a result, vaccine adjuvants can substantially reduce
the number of requisite immunizations as well as the amount of antigen required [
7
]. The bacterial
cell wall constituents Pam2Cys and Pam3Cys, together with the synthetic analogue Pam2CSK4
(Figure 1), have been shown as successful vaccine adjuvants due to their ability to activate Toll-like
receptors (TLRs) [8–10]. Specifically, Pam2Cys was shown to be recognized by the toll-like receptor 2
Molecules 2019, 24, 3512; doi:10.3390/molecules24193512
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3512
2 of 10
(TLR2) and Toll-like receptor 6 (TLR6) heterodimers [11]. These adjuvants can induce TLR activation,
which further activates NF-kB pathway on both innate and adaptive immune system to induce cytokine
production and enhance immune responses against synthetic antigens [12].
O
O
O
O
O
O
O
O
14
14
S
OH
S
OH
14
O
O
NH2
HN
14
14
1
2
O
NH2
O
NH2
O
NH2
O
O
O
H
H
H
N
O
S
14
N
N
N
H
OH
N
OH
O
H
O
O
O
14
3
NH2
NH2
Figure 1. Structures of Pam2Cys (1), Pam3Cys (2) and Pam2CysSK4 (3).
Although the synthesis and many structural-activity relationship (SAR) studies of Pam2Cys and
its analogues have been described [13
synthetic vaccines adjuvants is still difficult due to its high cost. All previously reported synthetic
methods involve costly synthetic strategies of orthogonal protection-deprotection techniques [13 15].
To ensure that the primary amine group on cysteine does not participate in any reactions during the
synthesis, it has to be protected with either Boc [14 15] or Fmoc [13] protecting groups, which are cost
–17], efficient large-scale synthesis of Pam2Cys analogues as
–
,
inefficient for large scale synthesis. For the receptor-ligand interactions, an X-ray crystal structure
of Pam2Cys bound to TLR2/TLR6 dimer suggests that the primary amine of the Cys residue does
not have a major contribution to the ligand-receptor interactions through any hydrogen bonding
interactions [11]. In addition, introducing N-acetylation to Pam2Cys analogs was shown to have
minimal impact on the compound’s ability to induce immune responses [18].
In this research, novel synthetic pathways were developed using the inexpensive
reactant N-acetylcysteine to synthesize N-acetyl Pam2Cys analogs, which can avoid orthogonal
protection-deprotection steps and greatly reduce the costs for the synthesis of vaccine adjuvants.
The ability of N-acetyl lipopeptides to activate TLR2/TLR6 signaling were examined in an NF-kB
activation assay in comparison with the commercially available synthetic vaccine adjuvant candidate
Pam2CysSK4 [8]. Molecular docking studies were performed to simulate the ligand-receptor interactions
between N-acetyl Pam2Cys and TLR2/TLR6. Our results confirmed that N-acetyl Pam2Cys analogs
can cause TLR2/TLR6 activation, and thus are promising candidates for cancer vaccine and synthetic
vaccine adjuvants.
2. Results
2.1. Synthesis
Scheme 1 shows the novel synthetic design, which uses N-acetylcysteine instead of N-protected
cysteine to avoid orthogonal protection-deprotection steps and lower synthetic costs. 1-hydroxyl and
2-hydroxyl groups on glycerol (
The hydroxyl group on compound
on N-acetylcysteine ( ) reacts with compound
groups were first exposed by removing the protecting group with AcOH to yield compound
coupled with fatty acids of different lengths to form the final products of AHB 1–4 (Scheme 1, Table 1).
The diastereomers were separated when they were first encountered in the synthesis i.e., from
to . Product used for further synthesis was diastereomerically pure. No further stereochemical
1
) were first protected by cyclohexanone (
was charged by p-TsCl ( ) to form compound
, yielding the thioether compound . The two hydroxyl
, and then
2
) to form compound
3.
3
4
5. The thiol group
6
5
7
8
6
7
7

Molecules 2019, 24, 3512
3 of 10
complexity is observed thereafter. The AHB1-SK4 was further synthesized using standard Fmoc
solid-phase peptide synthesis (Table 1).
Scheme 1. Synthesis of N-acetyl diacyl-S-glyceryl cysteine analogs.
Table 1. Structures of various lapidated N-acetyl cysteine analogs analyzed for their TLR2/TLR6
agonist activity.
Entry
Nature of R
Nature of R0
Yield (%)
AHB1
AHB2
AHB3
AHB4
AHB1-SK4
CH3-(CH2)16
CH3-(CH2)14
CH3-(CH2)12
CH3-(CH2)10
CH3-(CH2)14
-
-
-
-
-
-H
-H
-H
-H
-SK4
87
83
78
79
54
2.2. In Vitro TLR2/6 Activation
It was previously reported that the acyl chain lengths of Pam2Cys analogs can affect the TLR
activation [15]. To optimize the ability of N-acetyl lipopeptides to activate TLR2/TLR6, different
analogs with varying diacyl chain lengths (12, 14, 16 or 18 carbons, Table 1) were synthesized. To test

Molecules 2019, 24, 3512
4 of 10
their abilities to activate TLR2/TLR6, TLR2 and TLR6 as well as an ELAM-SEAP reporter gene were
transfected into HEK293 cells. Cells were treated with indicated compounds at 1 M for 6 h, and the
µ
TLR2/TLR6 activation was tested by an NF-kB based activation assay. Specifically, TLR activation
leads to the activation of the transcription factor NF-kB, which is recognized by the ELAM promoter to
initiate the transcription/translation of the SEAP reporter. The SEAP protein is then quantified due
to its ability to catalyze the hydrolysis of p-Nitrophenyl phosphate producing a yellow end product.
N-acetyl lipopeptide with 18 carbons in the acyl chain (AHB-1) was shown to have the highest level
of TLR2 and TLR6 activation (Figure 2A). To compare N-acetyl lipopeptides with the commercially
available standard compound Pam2CSK4 [8] in the ability to activate the TLR2/TLR6, AHB-1 was
conjugated with the short peptide SKKKK. The ability of AHB-1SK4 and Pam2CSK4 to activate TLR2/6
were tested in the NF-kB based activation assay. The results indicated that AHB-1SK4 was able to
achieve 67% of the TLR2/TLR6 activation comparing to Pam2CysSK4 at 1 µM concentration (Figure 2B),
suggesting that AHB-1 analogs can be effectively used as vaccine adjuvants through activating TLR2/6.
Figure 2. TLR2 and TLR6 activation by N-acetyl Pam2Cys and analogues. HEK293 cells were transfected
with plasmids encoding TLR2, TLR6 and the ELAM-SEAP reporter. After 24 h, cells were treated with
1
µ
M of indicated compounds for 6h. SEAP activities were measured by spectrophometer (OD412).
The data shown are representative of three independent experiments. ( ) TLR2 and TLR6 activation
) Comparison of TLR2 and TLR6 activation via AHB-1SK4
A
studies using synthetic compounds AHBs; (
B
and Pam2CysSK4.
2.3. Docking Studies
To understand the effect of the N-acetyl group on lipopeptide binding to TLR2 and TLR6, in-silico
molecular docking experiments were performed. The structure of Pam2CysSK4 bound to active TLR2
and TLR6 was previously determined [11] and was retrieved from Protein Data Bank (3A79). Since there
are more than 50 rotatable bonds in Pam2CysSK4, which lead to inaccuracy in Glide flexible docking [19],
the four lysine residues which do not interact with the receptor [20] were deleted and the diacyl chains
were trimmed to six carbons to form the model structure of Cap2CysSer and N-acetyl Cap2CysSer
molecules. The Cap2CysSer fits well into the binding pocket (Figure 3), with a Glide docking score
of
−
8.149. With N-acetyl Cap2CysSer, a docking score of 10.025 was achieved, suggesting that the
−
extra N-acetyl group does not abolish the interactions between lipopeptides and TLR2/6. What is more
interesting, is that comparing to the original position of Cap2CysS, the N-acetyl Cap2CysS was found
to be inserted deeper into the binding pocket (Figure 3). Our docking results suggest that the extra
N-acetyl group can be tolerated during binding of N-acetyl Pam2Cys analogs to TLR2/6.

Molecules 2019, 24, 3512
5 of 10
Figure 3. Comparison of the best flexible docking result (lowest docking score) of N-acetyl Cap2Cys
(in blue) with the original conformation of the Cap2Cys (in red). Ligands are inserted into the binding
pocket from up right to bottom left. The N-acetyl Cap2Cys is much more embedded into the binding
pocket comparing to the position of Cap2Cys.
3. Discussion
Though Pam2Cys analogs are widely considered as promising candidates for synthetic vaccine
adjuvants [ ], application in large scales can be limited by its relatively high synthetic costs.
8
Here, we provide novel synthetic pathways to synthesize N-acetyl Pam2Cys analogs with inexpensive
materials and avoiding extra orthogonal protection-deprotection steps. Our bioassay results confirmed
that the N-acetyl Pam2Cys analogs can effectively activate TLR2/6, which is consistent with previous
findings that an extra N-acetyl group did not produce any substantial difference in the ability of
an Pam2CSK4 analog to induce CD80 expression [18]. Moreover, our docking results suggest that the
extra N-acetyl group can be tolerated during ligand-receptor interactions. Given that the reported
synthetic procedure of N-acetyl lipopeptides can effectively reduce the production cost, it is promising
that N-acetyl lipopeptides will serve as adjuvants for synthetic vaccines in the future.
4. Materials and Methods
4.1. Synthesis of (1,4-Dioxa-spiro[4,5]dec-2-yl)-methanol (3)
OH
O
O
To a solution of glycerol 1 (5.0 g, 54.34 mmol) and cyclohexanone 2 (6.39 g, 65.16 mmol) in
◦
n-hexane (54.25 mL) was added conc. H2SO4 (3 mL) drop wise at 0 C over a period of 15 min.
The reaction mixture was slowly allowed to attain the room temperature and stirred for further
12 h. After completion of the reaction, as monitored by TLC, upper Hexane layer was separated.
Then, powdered K2CO3 (1.41 g, 8.8 mmol) was charged for trapping the traces of acid that might be

Molecules 2019, 24, 3512
6 of 10
present in the organic layer. Finally, hexane layer was evaporated under vacuum to yield the crude
product (10.0 g). The crude mixture was subjected to vacuum distillation to afford pure product 3
(8.41 g, 48.86 mmol, 89.9% yield) as a colorless syrupy liquid.
1H-NMR (CDCl3, 200 MHz):
δ 1.41–1.64 (m,10 H), 3.55–3.61 (m, 1 H), 3.76–3.82 (m, 2 H), 3.99–4.07
(m, 1 H), 4.21–4.26 (m, 1 H). ESI-MS: 173 (M + H+).
4.2. Synthesis of Toluene-4-sulfonic acid 1,4-dioxaspiro[4,5]dec-2-yl-methyl ester (5)
Compound
3 (8.41 g, 48.86 mmol) was dissolved in dry pyridine (35 mL) and immersed into
an ice bath under nitrogen atmosphere. p-toluenesulfonyl chloride 4 (9.32 g, 48.0 mmol) was added
slowly over a period of 20 min. to it and then the reaction mixture was slowly allowed to attain the
room temperature and stirred for further 12 h. After completion of the reaction, as monitored by TLC,
the reaction mixture was poured on to the crushed ice. The desired cyclohexanone protected glyceryl
tosylate was obtained in its crude form after repeated extraction with water: EtOAc (4–25 mL).
The crude product was purified using silica gel (100–200 mesh) chromatography to afford pure product
5as a colorless solid (10.95 g, 33.6 mmol, 70% yield).
1H-NMR (CDCl3, 200 MHz):
m, 3 H), 4.23–4.32 (m, 1 H), 7.37 (d, 2 H, J = 8.09 Hz), 7.81 (d, 2 H, J = 8.29 Hz). ESI-MS: 349 (M + Na+).
δ 1.38–1.54 (m, 10 H), 2.46 (s, 3 H), 3.73–3.80 (m, 1 H), 3.92–4.12
(
4.3. Synthesis of 2-Acetyl amino-3-(1,4-dioxa-spiro[4,5]dec-2-ylmethyl sulfanyl)-propionic acid (7)
To the stirred suspension of N-acetyl cysteine 6 (5.47 g, 33.6 mmol) in methanolic
KOH (2.81 g, 49.0 mmol in 40 mL methanol) solution, was added toluene-4-sulfonic acid
1,4-dioxa-spiro[4,5]dec-2-yl-methyl ester 5 (10.95 g, 33.6 mmol) under nitrogen at room temperature.
Reaction mixture was heated at reflux for 8–10 h. After completion of the reaction, as monitored
by TLC, reaction mixture was filtered off (potassium tosylate was precipitated during the course of
reaction) and was acidified to 2 pH using 2 N HCl. Organic compound was extracted with DCM
(3
×
100 mL) and Ethyl acetate (2
×
100 mL). The combined organic layers were dried over sodium
sulphate and evaporated under vacuum to give the crude product which was purified on flash column
chromatogram (silica gel as stationary phase, DCM: MeOH as mobile phase) to yield the product 7 in
its pure form (8.2 g, 25.86 mmol, 77% yield).
1H-NMR (CDCl3, 200 MHz):
δ 1.40–1.61 (m, 10 H), 2.05 (s, 3 H), 2.66–2.91 (m, 3 H), 3.29–3.31
(
m, 1 H), 3.66–3.70 (m, 1 H), 4.03–4.11 (m, 1 H), 4.23–4.27 (m, 1 H), 4.59–4.63 (m, 1 H). ESI-MS:
340 (M + Na+).
4.4. Synthesis of 2-Acetyl amino-3-(2,3-dihydroxy-propyl sulfanyl)-propionic acid (8)

Molecules 2019, 24, 3512
7 of 10
Compound 7 (8.2 g, 25.86 mmol) was dissolved in 75% aqueous acetic acid (20 mL) and heated to
reflux for 3 h. After completion of the reaction, as monitored by TLC, the mixture was evaporated under
vacuum to yield the crude product. The crude mass was purified through chromatography (Silica gel,
300–400 mesh) using chloroform: methanol gradient as the eluant to yield the pure compound 8 (5.80 g,
24.5 mmol, 94.6% yield).
1H-NMR (CDCl3, 200 MHz):
δ 2.05 (s, 3 H), 2.68–2.93 (m, 3 H), 3.29–3.35 (m, 1 H), 3.68–3.76
(m, 1 H), 4.03–4.08 (m, 1 H), 4.27–4.30 (m, 1 H), 4.57–4.66 (m, 1 H). ESI-MS: 260 (M + Na+).
4.5. Synthesis of N-Acetyl Pam2Cys and Analogs (AHB-1–4)
To the stirred solution of compound 8 (1 g, 4.20 mmol, 1 equation), in TFA (Trifluoroacetic
acid) (30 mL) was added palmitoyl chloride (2.45 g, 8.40 mmol, 2 eq.) slowly under nitrogen at room
temperature. Reaction mixture was stirred for 30 min. After the completion of the reaction, as monitored
by TLC, reaction mixture was dried under vacuum to yield the crude product (3.5 g), which was purified
by flash chromatography (silica gel, 200–400 mesh) using chloroform and methanol gradient as eluant
to afford N-Acetyl Pam2Cys (AHB2) in its pure form (2.69 g, 3.77mmol, 89.8% yield). The assignment
of NMR peaks is given as under for AHB1 as the assignment will be same for all other compounds and
only the number of methylene protons in the fatty chain will change. (AHB2–4 do not show carbon
numbers.) C1–17 in both the fatty chains are the same, thus we have given the same number to all such
carbons in both the chains. Their protons and carbons have exactly the same chemical shift values.
Hexadecanoic acid 2-(2-acetylamino-2-carboxy-ethylsulfanyl)-1-octadecanoyl-oxy methyl–ethyl
ester (N-Acetyl Str2Cys) (AHB1):
[α]2D5
−
15.2 (c 0.10, CHCl3), 1H-NMR (CDCl3, 400 MHz): 0.87–0.90 (t, 6H) [6Hs on two C1],
1.24–1.45 (m, 56H) [Protons on C2–15], 1.58–1.63 (m, 4H) [Protons on C16], 2.12 (s, 3H) [Protons on
C27], 2.30–2.36 (m, 4H) [Protons on C17], 2.75 (m, 2H) [Protons on C21], 3.06–3.15 (m, 2H) [Protons on
C22], 4.12–4.17 (m, 1H) [Proton on C19], 4.34–4.40 (m, 1H) [Next Proton on C19], 4.82 (m, 1H) [Protons
on C23], 5.18 (m, 1H) [Protons on C20], 6.86–6.92 (m, 1H) [-NH proton] (Total assigned protons 82,
Carboxyl proton out of range). 13C-NMR (CDCl3, 400 MHz): 14.12 [C1], 22.68 [C2], 24.88 [C26], 29.36,
29.48, 29.61[C3-C16 and C22], 31.90 [C17], 52.18 [C21], 52.51 [C23], 63.72 [C19], 70.25 [C20], 171.74
[C18], 172.58 [C27], 173.52 [C25], 173.66 [C24]. IR (KBr, cm−1): 3014, 2921, 2360, 1741, 1703, 1658, 1530,
1345, 1215, 1167, 964, 756. ESI-MS: 792 (M + Na+). Anal. Calcd. for C44H83NO7S, C, 68.61; H, 10.86;
N, 1.82. Found C, 68.64; H, 10.83; N, 1.86.
Hexadecanoic acid 2-(2-acetylamino-2-carboxy-ethylsulfanyl)-1-hexadecanoyl-oxy methyl–ethyl
ester (N-Acetyl Pam2Cys) (AHB2):
Specific rotation: [α]D25
1.24–1.31 (m, 48H), 1.58–1.66 (m, 4H), 2.14 (s, 3H), 2.33–2.43 (m, 4H), 2.78 (d, 2H, J = 6.37 Hz), 3.10–3.17
d, 2H, J = 54 Hz), 4.16–4.26 (m, 2H), 4.79 (m, 1H), 5.16 (m, 1H), 6.78–6.82 (m, 1H). 13C-NMR (CDCl3,
−
21.2 (c 0.10, CHCl3), 1H-NMR (CDCl3, 400 MHz):
δ 0.87–0.92 (m, 6H),
(

Molecules 2019, 24, 3512
8 of 10
400 MHz): δ14.13, 22.70, 22.80, 24.71, 24.87, 29.12, 29.31, 29.38, 29.52, 29.71, 31.93, 32.93, 33.97, 34.11,
34.40, 52.04, 52.22, 63.72, 70.23, 70.27, 171.48, 172.99, 173.62, 173.69. IR (KBr, cm−1): 3013, 2921, 2851,
2360, 1741, 1703, 1658, 1530, 1464, 1452, 1374, 1345, 1215, 1167, 964, 755. ESI-MS: 713 (M + H+).
Anal. Calcd. for C40H75NO7S, C, 62.28; H, 10.59; N, 1.96. Found C, 62.31; H, 10.55; N, 1.93.
Hexadecanoic acid 2-(2-acetylamino-2-carboxy-ethylsulfanyl)-1-tetradecanoyl-oxy methyl–ethyl
ester (N-Acetyl Myr2Cys) (AHB3):
[α]2D5
1.56–1.63 (m, 4H), 2.13 (s, 3H), 2.30–2.36 (m, 4H), 2.76 (m, 2H), 3.06–3.16 (m, 2H), 4.13–4.17 (m, 1H),
4.33–4.40 (m, 1H), 4.81 (m, 1H), 5.17 (m, 1H), 6.86–6.91 (m, 1H). 13C-NMR (CDCl3, 400 MHz):
14.08,
−
27.7 (c 0.10, CHCl3), 1H-NMR (CDCl3, 400 MHz):
δ 0.86–0.92 (t, 6H), 1.22–1.33 (m, 40H),
δ
22.66, 24.88, 29.36, 29.48, 29.61, 31.90, 52.18, 52.51, 63.72, 70.23, 70.27, 171.74, 172.52, 173.54, 173.67.
IR (KBr, cm−1): 3012, 2921, 2851, 2360, 1741, 1701, 1658, 1530, 1464, 1452, 1376, 1345, 1215, 1167, 964,
754. ESI-MS: 680 (M + Na+), Anal. Calcd. for C36H67NO7S, C, 65.71; H, 10.26; N, 2.13. Found C, 65.82;
H, 10.28; N, 2.10.
Hexadecanoic acid 2-(2-acetylamino-2-carboxy-ethylsulfanyl)-1-dodecanoyl-oxy methyl ethyl
ester (N-Acetyl Lau2Cys) (AHB4):
[α]2D5
1.59–1.65 (m, 4H), 2.12 (s, 3H), 2.30–2.36 (m, 4H), 2.75 (m, 2H), 3.06–3.15 (m, 2H), 4.12–4.17 (m, 1H),
4.34–4.40 (m, 1H), 4.82 (m, 1H), 5.18 (m, 1H), 6.86–6.90 (m, 1H). 13C-NMR (CDCl3, 400 MHz):
14.09,
−
31.3 (c 0.10, CHCl3), 1H-NMR (CDCl3, 400 MHz):
δ 0.88–0.91 (t, 6H), 1.24–1.35 (m, 32H),
δ
22.68, 24.88, 29.36, 29.48, 29.61, 31.90, 52.18, 52.51, 63.72, 70.24, 70.27, 171.74, 172.58, 173.54, 173.68.
IR (KBr, cm−1): 3014, 2921, 2851, 2360, 1741, 1703, 1658, 1530, 1464, 1452, 1376, 1345, 1215, 1167, 964,
756. ESI-MS: 601 (M + H+). Anal. Calcd. for C32H59NO7S, C, 63.86; H, 9.88; N, 2.33. Found C, 63.85; H,
9.83; N, 2.37.
4.6. Cell Culture
HEK293 cells were grown in the MEM medium (Minimum Essential Medium) (Gaithersburg,
MD, USA) with 1sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum
(Sigma-Aldrich, MO, USA) and 1% Pen-Strep (Sigma-Aldrich, MO, USA) at 37 ◦C in 5% CO2 incubator.
4.7. NF-kB Activation Assay
HEK293 cells (5
(Invivogen, San Diego, CA, USA) and 0.3
the FuGENE 6 transfection kit (Roche Diagnostics, Mannheim, Germany). The cells were seeded into
96-well plates 6 h after transfection. After 24 h, the cells were treated with lipopeptides (1 M) and the
medium was collected 6 h after stimulation. SEAP activity was measure using SEAP reporter assay kit
×
106) were transiently transfected with 5.7
g of TLR2/TLR6 expression vector (Invivogen, CA) using
µg of ELAM-SEAP reporter gene
µ
µ
(Invivogen, CA) and µQuant Microplate Reader (BioTek, Winooski, VT, USA) at OD412
.
4.8. Molecular Docking
The structure of the Pam2CSK4 bound to active TLR2 and TLR6 were retrieved from the 2.9Å
crystal structure (PDB: 3A79) [20]. The TLR2/TLR6 structure was prepared by protein preparation

Molecules 2019, 24, 3512
9 of 10
wizards in Maestro with the force field of OPLS 2005. The ligand binding pocket was defined in the
prepared receptor structure to generate a receptor grid by Glide receptor grid generation. The scaling
factor was set to 1.0 and the partial charge cutoff was set to 0.25. The four lysine residues and
10 methylenes in each diacyl chain were deleted from the Pam2CSK4 structure to create the model
of Cap2CS molecule. An acetyl group was added to the amide group of cysteine to create the model
for N-acetyl Cap2CS. The structures of Cap2CS and N-acetyl Cap2CS were optimized using ligand
preparation before docking. The ligand structures were docked into the receptor grid using Glide
docking in flexible docking mode. The scaling factor was 0.8 and the partial charge cutoff was 0.15.
The poses with the lowest docking score for each ligand were used for comparison. Poses were
rejected if the Coulomb-vdW energy were greater than 0 kcal/mol. Extra precision (XP) was used in
docking experiments.
Author Contributions: Conceptualization, A.H.B.; synthesis, A.H.B.; bioassays, Y.Z.; docking studies, Y.Z.;
writing, Y.Z and A.H.B.; experimental design, writing and funding acquisition, M.C. and V.J.H.
Funding: This work was funded in part by the US Public Health Service, NIH NIDA, Grant RO1 DA 13449.
Acknowledgments: A.B. thanks UGC, India for the award of Raman postdoctoral fellowship under Singh–Obama
21st century knowledge initiative. The authors dedicate this paper to Victor J. Hruby in honor of his 80th birthday
and would like to thank him for his extraordinary guidance.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Kingwell, K. CAR T therapies drive into new terrain. Nat. Rev. Drug Discov. 2017, 16, 301–304. [CrossRef]
[PubMed]
Temizoz, B.; Kuroda, E.; Ishii, K.J. Vaccine adjuvants as potential cancer immunotherapeutics. Int. Immunol.
2016, 28, 329–338. [CrossRef] [PubMed]
Ott, P.A.; Hu, Z.; Keskin, D.B.; Shukla, S.A.; Sun, J.; Bozym, D.J.; Zhang, W.; Luoma, A.; Giobbie-Hurder, A.;
Peter, L.; et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 2017, 547,
217–221. [CrossRef] [PubMed]
4.
5.
6.
7.
8.
Skwarczynski, M.; Toth, I. Peptide-based synthetic vaccines. Chem. Sci. 2016, 7, 842–854. [CrossRef]
[PubMed]
Leroux-Roels, G. Unmet needs in modern vaccinology: Adjuvants to improve the immune response. Vaccine
2010, 28 (Suppl. 3), C25–C36. [CrossRef]
Gavin, A.L.; Hoebe, K.; Duong, B.; Ota, T.; Martin, C.; Beutler, B.; Nemazee, D. Adjuvant-enhanced antibody
responses in the absence of toll-like receptor signaling. Science 2006, 314, 1936–1938. [CrossRef] [PubMed]
Schijns, V.E. Immunological concepts of vaccine adjuvant activity. Curr. Opin. Immunol. 2000, 12, 456–463.
[CrossRef]
Jackson, D.C.; Lau, Y.F.; Le, T.; Suhrbier, A.; Deliyannis, G.; Cheers, C.; Smith, C.; Zeng, W.; Brown, L.E.
A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes
antibody or cytotoxic T cell responses. Proc. Natl. Acad. Sci. USA 2004, 101, 15440–15445. [CrossRef]
[PubMed]
9.
Aliprantis, A.O.; Yang, R.B.; Mark, M.R.; Suggett, S.; Devaux, B.; Radolf, J.D.; Klimpel, G.R.; Godowski, P.;
Zychlinsky, A. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science
1999, 285, 736–739. [CrossRef] [PubMed]
10. Moyle, P.M.; Toth, I. Self-adjuvanting lipopeptide vaccines. Curr. Med. Chem. 2008, 15, 506–516. [CrossRef]
[PubMed]
11. Kang, J.Y.; Nan, X.; Jin, M.S.; Youn, S.J.; Ryu, Y.H.; Mah, S.; Han, S.H.; Lee, H.; Paik, S.G.; Lee, J.O. Recognition
of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 2009, 31, 873–884.
[CrossRef] [PubMed]
12. Iwasaki, A.; Medzhitov, R. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 2004
5, 987–995. [CrossRef] [PubMed]
,

Molecules 2019, 24, 3512
10 of 10
13. Metzger, J.W.; Wiesmuller, K.H.; Jung, G. Synthesis of N alpha-Fmoc protected derivatives of
S-(2,3-dihydroxypropyl)-cysteine and their application in peptide synthesis. Int. J. Pept. Protein Res.
1991, 38, 545–554. [CrossRef] [PubMed]
14. Wu, W.; Li, R.; Malladi, S.S.; Warshakoon, H.J.; Kimbrell, M.R.; Amolins, M.W.; Ukani, R.; Datta, A.;
David, S.A. Structure-activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides.
J. Med. Chem. 2010, 53, 3198–3213. [CrossRef] [PubMed]
15. Agnihotri, G.; Crall, B.M.; Lewis, T.C.; Day, T.P.; Balakrishna, R.; Warshakoon, H.J.; Malladi, S.S.; David, S.A.
Structure-activity relationships in toll-like receptor 2-agonists leading to simplified monoacyl lipopeptides.
J. Med. Chem. 2011, 54, 8148–8160. [CrossRef] [PubMed]
16. Zeng, W.; Eriksson, E.; Chua, B.; Grollo, L.; Jackson, D.C. Structural requirement for the agonist activity of
the TLR2 ligand Pam2Cys. Amino Acids 2010, 39, 471–480. [CrossRef] [PubMed]
17. Azuma, M.; Sawahata, R.; Akao, Y.; Ebihara, T.; Yamazaki, S.; Matsumoto, M.; Hashimoto, M.; Fukase, K.;
Fujimoto, Y.; Seya, T. The peptide sequence of diacyl lipopeptides determines dendritic cell TLR2-mediated
NK activation. PLoS ONE 2010, 5. [CrossRef] [PubMed]
18. Wright, T.H.; Brooks, A.E.S.; Didsbury, A.J.; McIntosh, J.D.; Burkert, K.; Yeung, H.; Williams, G.M.;
Dunbar, P.R.; Brimble, M.A. An Improved Method for the Synthesis of Lipopeptide TLR2-Agonists Using
Click Chemistry. Synlett 2013, 24, 1835–1841. [CrossRef]
19. Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.;
Shelley, M.; Perry, J.K.; et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739–1749. [CrossRef] [PubMed]
20. Jin, M.S.; Kim, S.E.; Heo, J.Y.; Lee, M.E.; Kim, H.M.; Paik, S.G.; Lee, H.; Lee, J.O. Crystal structure of
the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 2007, 130, 1071–1082.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).