Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Article abstract of DOI:10.1016/j.bmc.2019.05.005

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.

Full text of DOI:10.1016/j.bmc.2019.05.005

Accepted Manuscript
Identification and immunological evaluation of novel TLR2 agonists through
structure optimization of Pam3CSK4
Xinming Du, Jiawen Qian, Yujie Wang, Mingming Zhang, Yiwei Chu, Yingxia
Li
PII:
S0968-0896(19)30261-5
https://doi.org/10.1016/j.bmc.2019.05.005
BMC 14892
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 February 2019
26 April 2019
4 May 2019
Please cite this article as: Du, X., Qian, J., Wang, Y., Zhang, M., Chu, Y., Li, Y., Identification and immunological
evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.005
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Identification and immunological evaluation of novel TLR2 agonists
through structure optimization of Pam3CSK4
Xinming Dua, Jiawen Qianb, Yujie Wanga, Mingming Zhanga, 
,
Yiwei Chub, , Yingxia Lia, 
aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China.
bDepartment of Immunology, School of Basic Medical Sciences and Institute of Biomedical Sciences,
Fudan University, Shanghai, 200032, China.
ABSTRACT
Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2
agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no
TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective
agonists, a novel series of Pam3CSK4 derivatives were designed based on the crystal structure of
hTLR2-hTLR1-Pam3CSK4 complex, synthesized and evaluated for their immune-stimulatory
activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a
human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2
agonistic activity comparable to Pam3CSK4, which included: elevated IL-6 expression level (EC50
= 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of
DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and
35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.
Keywords: TLR2 agonists, Pam3CSK4, Structure optimization, TLR2/1, TLR2/6
 Corresponding author: For Y. L. Email: liyx417@fudan.edu.cn
 Corresponding author: For Y. C. Email: ywchu@shmu.edu.cn
 Corresponding author: For M. Z. Email: zmmworld@gmail.com
1

1. Introduction
Professional antigen-presenting cells (APCs) belonging to innate immune cells, such as
dendritic cells (DCs), rapidly recognize invading pathogens and initiate a cascade of adaptive
immunity.1, 2 The important factors that trigger these immune responses are Toll-like receptors
(TLRs) which are mainly present on the surface of APCs or other immune cells.3-5 TLRs are
pattern recognition receptors (PRRs) with the ability to recognize conserved pathogen-associated
molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), including
bacterial lipopeptides, lipopolysaccharides (LPS), microorganism nucleic acids and so on.6-9
Activation of DCs upon recognition of PAMPs by TLRs up-regulates the efficiency of antigen
presentation as well as the release of diverse cytokines, subsequently induces naive T cell
11
differentiation.10,
Thus, TLRs play an irreplaceable role in bridging innate and adaptive
immunity. There are 13 TLR family members identified so far.12 TLR1 – TLR10 are expressed in
both humans and mice, whereas TLR11 – TLR13 are only expressed in mice. A variety of
agonists targeting TLRs have been discovered in the light of the important role of TLRs in
immune regulation, some of them have been approved by FDA, such as Imiquimod (a TLR7
agonist, 3M Pharmaceuticals Inc.) which has been approved for treating actinic keratosis, genital
warts and nonmelanoma skin cancer.13-15
As a type I transmembrane protein, TLR2 recognizes a variety of bacteria, fungi, and other
pathogens by forming a heterodimer with TLR1 or TLR6.16 Subsequently, the activation of NF-κB
through myeloid differentiation factor 88 (MyD88) dependent pathway leads to cytokines
production.17 In addition to improving the immune response, TLR2 agonists can also inhibit
cancer development and induce tumors regression.18 Therefore, effective activation of TLR2 is
increasingly becoming the crucial direction of tumor immunotherapy.
Pam3CSK4 (TLR2/1 agonist) and Pam2CSK4 (TLR2/6 agonist) are derived from Escherichia
coli19-22 and Mycoplasma fermentans,23, 24 respectively. These two preclinical lipopeptides are
currently the most potent TLR2 agonists. However, high molecular weight and poor stability
limited their applications.25, 26 Therefore, several TLR2 agonists have been identified based on the
structural modification of Pam3CSK4 or Pam2CSK4 (Fig. 1). The primary structure-activity
relationship (SAR) studies for Pam3CSK4 showed that the length of two ester-bound fatty acid was
no fewer than twelve carbon atoms to activate hTLR2 and the length of amide bound fatty acid
2

was undemanding (series A).27 Besides, the amino acid residues of Pam3CSK4 are replaceable
because Pam3Cys-RPDRY and Pam3Cys-QPDRY have similar potency to Pam3CSK4 (series E).28
The agonistic activity improved about one-fold when the amide bound of Pam3CSK4 was
substituted by ureido, probably forming an additional hydrogen bond between Phe312 and the
nitrogen atom of urea (series C).29 Replacement of the ester-bound with carbamate resulted in a
Pam3CSK4 analogue SUP3 (compound D) which showed high stability in vivo and improved
potency on activation of T cells.26 Various glycosides were introduced in Pam3CSK4, and these
analogues revealed good potency to induce cytokine expression (series B).30 The structure
optimization of Pam2CSK4 showed that introduction of an amide group into the lipid moiety
resulted in decreased activity (series H).31 Novel Pam2CSK4 derivatives modified by a sugar
moiety were synthesized, and these derivatives were able to stimulate the maturation of DCs
(series G).32 Small molecular TLR2/6 agonist F with good agonistic activity and water solubility
had been identified.33-36 Monoacyl lipopeptides had been synthesized through thiol-ene coupling
reaction and the potential to induce CD80 response was detected (structure not shown).37 In
addition to traditional drug design, novel TLR2-dependent small molecule activators had been
identified through high-throughput screening (structure not shown).38-40
C
15H31
O
O
C
15H31
O
C14H29
C
15H31
HN
n
m
O
O
O
O
O
O
O
O
O
S
NH
O
O
NH
O
O
S
NH
H
S
N
K4
SK4
C15H31
carbohydrate
N
N
C
15H31
N
m
O
H
N
O
R
O
A
B
C
C15H31
O
H
C
15H31
O
N
O
C14H29
C15H31
NH
O
O
O
R
O
O
NH2
O
O
O
NH
O
O
S
S
SK4
S
SK4
N
O
H
C
14H29
N
GK3
C15H31
n
m
H
O
H
O
O
D
Pam CSK , R
Pam CSK , R
=palmitoyl
=H
3
4
2
4
OH
C15H31
OH
HO
C15H31
O
O
S
C15H31
NH
O
OH
O
O
O
C
O
O
O
O
S
15H31
H
H
HO
O
N
N
N
O
peptide
C
16H33
S
N
C
15H31
N
H
H
O
NH
O
O
NH2
E
G
O
F
Fig.1. Identification of novel TLR2 agonists based on optimization of Pam2CSK4 or Pam3CSK4.
Despite the intensive work on TLR2 agonists, there has been no breakthrough. This objective
is hardly realized because of various reasons: Firstly, the structure of hTLR2 and mTLR2 are
different, so that mTLR2 agonists may not be effective for human;41 Secondly, native TLR2
3

agonists derived from microorganisms are mostly pyrogens which may deem to be harmful to
human.42 Finally, it is necessary to determine whether compounds bind TLR2/1 or TLR2/6
because their different potential therapeutic benefits. However, most TLR2 agonists did not
perform this test.
Encouraged by the small molecular agonist F, we carried out the further structural
simplification of Pam3CSK4 based on the crystal structure of hTLR2-hTLR1-Pam3CSK4 complex
to discover drug-like TLR2 selective agonists. A novel series of Pam3CSK4 derivatives were
designed and synthesized. Furthermore, immunological evaluation of these compounds against
brain microglia cells (BV2), macrophage cell line (Raw 264.7), and phorbol 12-myristate 13-
acetate (PMA) differentiated human monocytic cells (THP-1) which all expressed TLR2 had been
done.
2. Results and discussion
2.1 Chemistry
2.1.1 Design
The crystal structure of hTLR2-hTLR1-Pam3CSK4 complex had been published in 2007 by
Lee.41 The two ester-bound lipid chains were inserted into a pocket in TLR2 and the remaining
amide bound lipid chain was located into a narrow channel in TLR1. The Ser peptide unit, as the
connection part, was situated in the narrow neck region of the heterodimer that played a pivotal
role in maintaining molecular active conformation. The lysine residues regulated physicochemical
properties of the molecule. Pam3CSK4 formed four hydrogen bonds with TLR2/1: between ester
oxygen and Phe349; between amide oxygen and Gln316; between backbone nitrogen of lysine and
Gly313; between side chain nitrogen of lysine and Asn294 (Fig. 2).
4

Connection part:
located in the
narrow neck
physicochemical property
regulation part
Activity regulation part:
binding to the TLR2/1
region of TLR2/1
F349
NH2
Q316
O
C15H31
C
15H31
NH
lipid chain
modification
C
15H31
O
O
O
S
NH
O
O
O
O
S
H
N
H

C
15H31
N
SK4
N
Lys-Lys
NH2
C15H31
O
H
O
O
O
O
HO
35a
G313
N294
Pam3CSK4
ester group
modification
C
14H29
HN
C
15H31
lipid chains and
amide acid
modification
C15H31
amide group
modification
O
O
NH
O
O
S
NH
O
O
S
NH
C
16H33
S
S
K
K
K
K
C
16H33
S K K K K
m
C14H29
SK4
X
N
O
N
X
N
H
H
H
O
O
O
35f
35d: X = O, m = 1
35e: X = N, m = 1
35g: X = O, m = 0
35b:
35c:
X = CH2
X = O
Fig.2. Design of novel TLR2 agonists from Pam3CSK4 (hydrogen bond was presented by yellow).
Inspection of crystal structure indicated that the one ester-bound did not form hydrogen bond
in TLR2 and previous SAR studies of the two ester bound lipid chains revealed that removal of
one ester bound lipid chain which forming hydrogen bond did not show a stimulatory activity.43
Thus, compound 35a was designed with only one ester-bound lipid chain involved in hydrogen
bonding. Besides, ester group was liable to be hydrolyzed by esterase26 so that bioisosterism was
applied to replace ester group with stable groups such as amide, carbamate, and ureido. We
thereby designed compounds 35b, 35c, and 35e. Moreover, compound 35d with different acyl
chain compared to 35c was designed to investigate the effect of length on the activity. Crystal
structure showed that the fourth lysine residue did not participate in H-bond interaction, therefore,
compound 35g with three lysine residues was designed. Guided by the co-crystal structure, an
additional hydrogen bond may be formed through substitution of the α-CH2 of the fatty acid amide
of Pam3CSK4 with an NH as described in the literature.29 Therefore, compound 35f with ureido
group was designed (Fig. 2). All the compounds were evaluated for their agonist activity in BV2
cells and PMA-differentiated THP-1 cells.
2.1.2 Synthesis
Synthesis of target compounds involved two key intermediates: lipophilic acyl chains and
hydrophilic short peptides. General synthetic route to key intermediate 14a was outlined in
5

Scheme 1. The starting material L-cystine reacted with di-tert-butyl dicarbonate to afford 1.
Subsequent protection of the carboxyl with benzyl bromide gave 2. Then, treatment of 2 with tri-
n-butylphosphine resulted in the formation of 3. Reaction of 3 with 2-Iodoethanol in the presence
of potassium carbonate gave 4. Subsequent reaction with palmitoyl chloride through esterification
provided 5. Upon removal of the protecting group with CF3CO2H, the corresponding product 6
was treated with palmitoyl chloride in dichloromethane to give 7. Subsequent reaction with
Pd(OH)2 in the presence of HCO2NH4 provided the key intermediate 14a.
Boc
Boc
O
HN
O
HN
O
NH2
O
c
S
OH
a
b
S
O
Ph
HO
Boc
S
S
OH
Ph
O
S
HO
S
NH
O
NH
O
NH2
Boc
1
2
Boc
Boc
Boc
O
HN
HN
HN
f
d
e
S
O
Ph
HS
O
Ph
S
O
Ph
C15H31
O
HO
O
O
3
O
5
4
C15H31
NH
C15H31
NH
O
O
S
O
NH2
O
O
S
h
g
OH
S
O
Ph
O
Ph
C
15H31
O
C15H31
O
C15H31
O
O
O
O
14a
7
6
Scheme 1. Reagents and Conditions: (a) (Boc)2O, TEA, H2O, 0 °C to r.t., overnight; (b) BnBr, K2CO3, DMF, r.t.,
10 h, 83.2%; (c) Bu3P, THF, r.t., 0.5 h; H2O, r.t., 1 h, 95.1%; (d) 2-Iodoethanol, K2CO3, acetone, r.t., overnight,
92%; (e) Palmitoyl chloride, DIPEA, anhydrous DCM, 0 °C to r.t., overnight, 79.6%; (f) TFA, DCM, 0 °C, 2 h; (g)
Palmitoyl chloride, DIPEA, anhydrous DCM, 0 °C to r.t., overnight, 83.1%; (h) Pd(OH)2, HCO2NH4, MeOH,
reflux, 1 h.
The synthesis of intermediate 14b – 14d were shown in Scheme 2. Intermediate 4 was briefly
treated with methanesulfonyl chloride to afford 8, which followed by convertion into 9 with
sodium azide. Compound 9 subsequently reacted with triphenylphosphine to form 10 through
Staudinger reaction. This compound was treated with the corresponding acyl chlorides to give 11b
– 11d, which further reacted with CF3CO2H in dry dichloromethane to give 12b – 12d.
Subsequent acylation of the amino group with palmitoyl chloride gave 13b – 13d. These
compounds were then reacted with Pd(OH)2 in the presence of HCO2NH4 to afford key
intermediates 14b – 14d.
6

Boc
Boc
Boc
HN
Boc
HN
HN
HN
O
S
O
Ph
O
c
a
b
S
O
Ph
S
O
Ph
H2N
S
S
O
Ph
HO
N3
O
O
g
O
O
O
10
8
9
4
C
15H31
Boc
O
NH2
O
O
HN
O
O
S
NH
d
e
f
S
O
Ph
S
O
Ph
O
Ph
R
N
H
R
N
H
R
N
H
O
O
12b - 12d
13b - 13d
11b - 11d
C
15H31
NH
C
15H31
11b 12b 13b 14b
,
,
,
:
:
R=
R=
O
O
S
O
O
OH
11c 12c 13c 14c
,
,
,
:
C14H29
R
N
H
O
11d 12d 13d 14d
,
,
,
14b - 14d
R=
C16H33
Scheme 2. Reagents and Conditions: (a) Methanesulfonyl chloride, DIPEA, anhydrous DCM, 0 °C to r.t.,
overnight; (b) NaN3, DMF, 70 °C, 8 h, 79.0%; (c) Ph3P, THF, H2O, 70 °C, 4 h, 75.1%; (d) Acid chloride, DIPEA,
anhydrous DCM, 0 °C to r.t., overnight, 61.5-91.9%; (e) TFA, DCM, 0 °C, 2 h; (f) Palmitoyl chloride, DIPEA,
anhydrous DCM, 0 °C to r.t., overnight, 68.6-78.3%; (g) Pd(OH)2, HCO2NH4, MeOH, reflux, 1 h.
The synthesis of intermediate 14e was described in Scheme 3. To access 16, 1-
Isocyanatohexadecane 15, which was prepared from hexadecan-1-amine by carbonylation,
underwent an acylation with 10 in dry dichloromethane. Then, 16 was used as a starting material
to synthesize 14e by deprotection, acylation, and debenzylation as mentioned earlier.
Boc
O
NH2
O
O
HN
a
10,
16H33
b
C
c
C
16H33
C16H33
S
O
Ph
C
16H33
S
O
Ph
O
C
N
H2N
N
N
N
N
H
H
H
H
17
16
O
15
C
15H31
C
15H31
NH
O
O
NH
O
O
e
d
C16H33
S
OH
C
16H33
S
O
Ph
N
N
H
N
N
H
H
H
14e
O
O
18
Scheme 3. Reagents and Conditions: (a) triphosgene, anhydrous Tol, reflux, 6 h; (b) Et3N, anhydrous DCM, 0 °C
to r.t., overnight, 78.3%; (c) TFA, DCM, 0 °C, 2 h; (d) Palmitoyl chloride, DIPEA, anhydrous DCM, 0 °C to r.t.,
overnight, 80.4%; (e) 20% Pd(OH)2, HCO2NH4, MeOH, reflux, 1 h.
The intermediate 14f was obtained through carbonylation, acylation, and debenzylation using
1-tetradecylamine as starting material (shown in Scheme 4).
C14H29
C
14H29
HN
HN
O
O
NH
O
O
NH
C
16H33
S
OH
c
C16H33
S
O
Ph
C14H29
12d
,
b
a
C14H29
O
N
O
N
H2N
O
C
N
H
H
O
14f
O
19
20
Scheme 4. Reagents and Conditions: (a) Triphosgene, anhydrous toluene, reflux, 6 h; (b) Et3N, anhydrous DCM,
0 °C to r.t., overnight, 80.6%; (c) 20% Pd(OH)2, HCO2NH4, MeOH, reflux, 1 h.
Key intermediates 30 and 32, as short peptide parts shared by the target compounds, were
7

synthesized as described in Scheme 5 and 6 according to the procedure described in the literature
with some changes.44 The reaction of commercially available Fmoc-Lys(Boc)-OH with benzyl
bromide in N,N-dimethylformamide led to the formation of the corresponding product 21. After
removing the Fmoc protecting group by 20% piperidine, the corresponding product 22 was treated
with Fmoc-Lys(Boc)-OH in the presence of HATU and N,N-diisopropylethylamine in
dichloromethane to give 23. Then condensation of commercially available Fmoc-O-tert-Butyl-L-
serine with 28 afforded 29, which subsequently transformed to key intermediate 30 using 20%
piperidine. For the synthesis of 32, the intermediate 26 was condensed with Fmoc-Lys(Boc)-OH
to give 31 using HATU/DIPEA, which was then deprotected in the presence of 20% piperidine to
give the key intermediate 32.
Boc
Boc
NH
HN
Boc
Boc
Boc
NH
NH
NH
O
O
H
N
H2N
O
Ph
a
O
Ph
b
b
c
N
Fmoc
N
H
O
Ph
H
Fmoc
O
Ph
O
Fmoc
H2N
O
N
N
COOH
H
H
O
O
24
Boc
23
21
22
HN
HN
Boc
Boc
Boc
Boc
Boc
NH
O
NH
O
Boc
Boc
NH
NH
O
NH
NH
H
N
H
N
O
b
H
N
c
c
H
O
Ph
Fmoc
O
Ph
Fmoc
N
N
N
O
Ph
N
N
H
H2N
N
H
H
H
O
O
H
O
O
O
O
26
27
25
HN
HN
NH
O
HN
Boc
Boc
HN
NH
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
NH
NH
O
NH
NH
O
O
O
O
O
O
H
N
H
b
b
H
d
H
N
H
N
O
Ph
H2N
N
O
Ph
N
O
Ph
HN
N
N
H
N
H
N
H2N
N
N
Fmoc
H
H
H
H
O
O
O
O
O
O
O
O
HN
HN
HN
HN
30
HN
HN
28
29
Boc
Boc
Boc
Boc
Boc
Boc
Scheme 5. Reagents and Conditions: (a) BnBr, K2CO3, DMF, r.t., overnight, 96.2%; (b) 20% piperidine, DMF, r.t.,
2 h; (c) Fmoc-L-Lys(Boc)-OH, HATU, DIPEA, DCM, 0 °C to r.t., 4 h, 80.6-83.3%; (d) HATU, DIPEA, Fmoc-O-
tert-Butyl-L-serine, DCM, 0 °C to r.t., 4 h, 72.1%.
Boc
Boc
Boc
Boc
Boc
Boc
NH
O
NH
O
NH
O
NH
O
NH
NH
O
H
N
H
N
H
N
H
N
b
H2N
O
Ph
a
O
Ph
O
Ph
N
N
Fmoc
N
N
H2N
N
H
H
H
H
H
O
O
O
O
O
O
O
O
HN
32
HN
31
26
HN
Boc
Boc
Boc
Scheme 6. Reagents and Conditions: (a) HATU, DIPEA, Fmoc-O-tert-Butyl- L-serine, DCM, 0 °C to r.t., 4 h,
78.5%; (b) 20% piperidine, DMF, r.t., 2 h.
8

The targeted compounds 35a – 35g were synthesized in three steps as shown in Scheme 7
according to the procedure described in the literature with some changes.44 Condensation of
intermediates 14a – 14f with intermediate 30 or 32 afforded 33a – 33g in the presence of
HOBT/EDCI/DIPEA, which was subsequently transformed to carboxylic acids 34a – 34g using
Pd(OH)2 in the presence of HCO2NH4. Deprotections of tert-butoxycarbonyl and tert-butyl with
HCl/dioxane provided target compounds 35a – 35g.
Boc
Boc
Boc
Boc
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
a,
30 32
H
N
H
N
H
N
H
N
or
Y
X
O
m
Ph
b
Y
X
OH
m
CnH2n+1
S
N
N
N
CnH2n+1
S
N
N
N
Intermediate 14a-14f
H
H
H
H
H
H
O
O
NH
O
O
O
O
NH
O
O
Z
Z
C
14H29
C
14H29
HN
HN
HN
HN
Boc
Boc
Boc
Boc
33a-33g
34a - 34g
NH2
O
NH2
OH
33a, 34a, 35a : X=O; Y=CH2; Z=CH2; m=1; n=14
33b, 34b, 35b : X=NH; Y=CH2; Z=CH2; m=1; n=14
O
O
H
H
Y
X
N
N
N
H
OH
m
CnH2n+1
S
N
N
H
c
H
O
O
NH
O
O
O
33c, 34c, 35c : X=NH; Y=O; Z=CH2; m=1; n=14
33d, 34d, 35d : X=NH; Y=O; Z=CH2; m=1; n=16
33e, 34e, 35e : X=NH; Y=NH; Z=CH2; m=1; n=16
33f, 34f, 35f : X=NH; Y=O; Z=NH; m=1; n=16
33g, 34g, 35g : X=NH; Y=O; Z=CH2; m=0; n=16
Z
C
14H29
NH2
NH2
35a - 35g
Scheme 7. Reagents and Conditions: (a) HOBT, EDCI, DIPEA, anhydrous DCM, 0 °C to r.t., 6 h, 52.1-71.9%; (b)
Pd(OH)2, HCO2NH4, MeOH, reflux, 1 h; (c) HCl/dioxane, 0 °C, 2 h, 32.7-52.3%.
2.2 Biological activity
2.2.1 mIL-6 ELISA assay in BV2 cell line
All designed compounds were evaluated for their agonist activity against BV2 cells from
mice using enzyme-linked immunosorbent assay (ELISA) based on the secretion levels of
inflammatory cytokines IL-6. In this evaluation, Pam3CSK4 (Invivogen) was selected as a positive
control and the BV2 cells were incubated with three concentrations of compounds (0.5, 1, 2 μM)
for 24 h (Fig. 3). The compound 35a with only one ester lipid chain exhibited moderate agonistic
activity in a dose-dependent manner, which showed about one-fold decrease in activity compared
to Pam3CSK4 at 2 μM. Although the removal of one ester lipid chain partially reduced the
immunological activities, the assays revealed that two ester chains of Pam3CSK4 were not
necessary to exist simultaneously. In addition, compound 35c with carbamate displayed much
better activity than 35a, and this result showed that the ester group is not strictly required for the
activity. Compared with 35c, compound 35d with hexadecyl ester showed a slight increase in
9

activity similar to Pam3CSK4. Moreover, compound 35g, which have three lysine residues,
showed lower activity than 35d. Unexpectedly, compound 35b in which amide substituted for
ester, compound 35e in which urea substituted for carbamate, and compound 35f in which amide
bond replaced by urea proved to be detrimental for the activity and the cause of significant
declining activity were perhaps due to the changes in molecular conformation. Taken together, we
found that 35a showed moderate agonistic activity and 35c, 35d exhibited similar potency
compared with Pam3CSK4.
600
* *
0.5 μM
1 μM
2 μM
n.s.
400
200
0
Pam3 35a
35b
35c
35d
35e
35g
35f Control
Compounds
Fig.3. Cytokine IL-6 induction by the designed compounds 35a – 35g in BV2 cells. All data represented the mean
± SD of three independent experiments. **p < 0.01, “n.s.” meaned statistically non-significant.
2.2.2 mTLRs selectivity assay
We used murine bone marrow-derived macrophage (BMDM), which derived from TLR2
knock-out and wild type mice, to explore the TLRs selectivity of the active compounds 35a, 35c,
and 35d. The expression of TLR2 in TLR2-/- and WT BMDM cells were detected by flow
cytometry analysis and the result showed that BMDM cells which derived from TLR2-/- mice
hardly expressed TLR2 (Fig. 4). In addition, LPS (TLR4 agonist), Poly I:C (TLR3 agonist), and
ODN (TLR7 and TLR8 agonist) can both activate TLR2-/- BMDM cells in a dose-dependent
manner (Fig. S1). Subsequently, IL-6 (Fig. 5A), IL-12 (Fig. 5B), and TNF-α (Fig. 5C) production
were detected after stimulated with two concentrations of compounds (0.1, 1 μM). Pam3CSK4 was
chosen as the positive control. The result showed that the TLR2-/- BMDM was unable to respond
to the stimulation of all three compounds, while all the compounds stimulated different levels of
cytokine production in WT BMDM. Among these three compounds, 35d had the most potent
10

agonistic activity at 1 μM. Therefore, the designed agonists 35a, 35c, and 35d stimulated murine
macrophages through TLR2-dependent manner.
A
Control
B
WT
TLR2-/-
**
Control TLR2-/- WT
TLR2
Fig.4. Flow cytometry analysis of BMDM cells which derived from TLR2-/- mice and wild type mice for TLR2'
expression (A) and the statistic calculation is shown in (B). All data represented the mean ± SD of three
independent experiments. **P < 0.01 vs TLR2-/- group.
* *
* *
1600
A
n.s.
WT
TLR2-/-
###
###
###
1200
###
###
###
###
800
400
0
Control 1μM 0.1μM 1μM 0.1μM 1μM 0.1μM 1μM
Pam
35d
35a
35c
B
1800
n.s.
WT
TLR2-/-
###
n.s.
###
###
###
###
###
1200
600
0
###
Control 1μM 0.1μM 1μM 0.1μM 1μM 0.1μM 1μM
Pam
35d
35a
35c
11

1600
1200
800
400
0
C
WT
TLR2-/-
###
* *
* * *
###
###
###
###
###
###
Control 1μM 0.1μM 1μM 0.1μM 1μM 0.1μM 1μM
Pam 35d 35a
35c
Fig.5. Cytokine IL-6 (A), IL-12 (B), and TNF-α (C) induction by the compounds 35d, 35a, and 35c in BMDM
cells which derived from TLR2-/- mice and wild type mice, respectively. All data represented the mean ± SD of
three independent experiments. ###P < 0.001 vs TLR2-/- group, respectively. ***p < 0.001, **p < 0.01. “n.s.”
meaned statistically non-significant.
2.2.3 Downstream NF-κB signaling pathway evaluation
When TLR2 recognizes PAMPs, adaptor proteins MAL and MYD88 are then recruited by
Toll-interleukin-1 resistance (TIR) domain, which triggers the activation of interleukin-1 receptor-
associated kinase 4 (IRAK4) followed by IRAK1 and NF-κB, respectively.14 Hence, we used a
classical chemical tool called triptolide which was a known efficient NF-κB inhibitor to
investigate the signaling pathway of 35d-triggered activation.45 Different concentrations of
triptolide used in this assay ensured the cell viability above 90% through cell counting kit-8
(CCK-8) assay (data not shown). We found that the triptolide dose-dependently inhibited 35d-
induced IL-6 production in BV2 cells (Fig. 6A). In addition, we used western blot analysis to
further investigate the activation of p-IκBα after stimulation of BV2 cells for 12 hours by
compound 35d and Pam3CSK4 was selected as a positive control.46 The result showed that 35d
increased IκBα phosphorylation in a dose-dependent manner without significantly affecting the
protein expression levels of IκBα, which further indicated that 35d functioned via the NF-κB
signaling pathway (Fig. 6B).
12

500
400
300
200
100
0
A
1 μM 35d + Triptolide
35d
2 μM
Pam3CSK4 35d
0.2 μM 0.2 μM
B
Control
0
5
15
20
10
Triptolide / nM
Fig.6. Compound 35d activated downstream NF-κB signaling pathway. (A) Triptolide inhibited the 35d-induced
NF-κB activation in BV2 cells in a dose-dependent manner. (B) Phosphorylated IκBα and total IκBα were
analyzed by western blotting. β-actin was used as the loading control. All data represented the mean ± SD of three
independent experiments.
2.2.4 EC50 evaluation in Raw 264.7 cells
We tested the half-maximal effective concentration (EC50) of the compound 35d through
ELISA assay toward Raw 264.7 cells which overexpressed TLR2 (Fig. 7). The result showed that
35d activates the IL-6 production in Raw 264.7 cells with an EC50 of 83.08 ± 5.94 nM.
1200
35d (μM)
Pam3CSK4 (μM)
800
400
0
0
0.02 0.04 0.08 0.1 0.3 0.5 0.8 1.0 1.5 0.5
Concentrations
Fig.7. Compound 35d induced IL-6 production in Raw 264.7 macrophage cells with an EC50 of 83.08 ± 5.94 nM.
All data represented the mean ± SD of three independent experiments.
2.2.5 Quantitative RT-PCR assay
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to
investigate the effects on the expression of mRNA levels for NF-κB pathway downstream
cytokines TNF-α (Fig. 8A) and IL-6 (Fig. 8B) after treatment of 35d, 35a, 35c and Pam3CSK4,
respectively. The results indicated that TNF-α and IL-6 mRNA levels were potently increased in
13

the presence of 0.1 μM designed compounds. Notably, the degree of activation was similar to
Pam3CSK4 in the presence of 1 μM 35d. All the three compounds had dose-dependent activity
based on the mRNA levels of TNF-α and IL-6. In addition, the above phenomenon was not
observed in the TLR2 knock-out group.
n.s.
8
6
4
2
0
A
WT
TLR2-/-
n.s.
#
##
#
###
##
#
##
Control 1μM 0.1μM 1μM 0.1μM 1μM 0.1μM 1μM
Pam
35d
35a
35c
80
B
WT
TLR2-/-
**
n.s.
60
40
20
0
##
#
#
###
##
##
##
Control 1μM 0.1μM 1μM 0.1μM 1μM 0.1μM 1μM
Pam
35d
35a
35c
Fig.8. TNF-α (A) and IL-6 (B) mRNA induction by the compounds 35d, 35a, and 35c in BMDM cells which
derived from TLR2-/- mice and wild type mice, respectively. The data meant the changes of gene expression levels
(fold change) relative to the expression levels of controls which treated with dimethyl sulfoxide. All data
represented the mean ± SD of three independent experiments. #P < 0.05, ##P < 0.01, ###P < 0.001 vs TLR2-/- group,
respectively. **p < 0.01, “n.s.” meaned statistically non-significant.
2.2.6 Flow cytometric analysis
The up-regulation of costimulatory molecules upon TLRs activation was a significant symbol
of the functional maturation of DCs. Efficient presentation of antigen was required for mature
DCs, which was essential for the activation of T cell and initiation of adaptive immunity.11 To
investigate whether designed compounds affected the expression of costimulatory molecules on
DCs, we used compound 35d to stimulate DCs in vitro at a concentration of 100 ng/mL and
14

conjugated with antibodies to costimulatory molecules and MHC class II (Fig. 9A). Pam3CSK4
was chosen as the positive control. Flow cytometric analysis showed that 35d effectively induced
up-regulation of CD80, CD86 and MHC class II on DCs, demonstrating that 35d induced
maturation of DCs similar to Pam3CSK4 in vitro (Fig. 9B). However, the above phenomenon was
not observed in the TLR2 knock-out group.
A
Pam3CSK4
35d
Control
B
WT
TLR2-/-
4
3
WT
TLR2-/-
***
CD86
**
***
***
**
2
1
**
CD80
0
Control
35d
Pam
CD86
35d
Pam
CD80
35d
Pam
MHCII
MHCII
Fig.9. Flow cytometry analysis of the 35d-induced DCs for antigen-presenting function-related markers (A) and
the statistic calculation is shown in (B). All data represented the mean ± SD of three independent experiments. **P
< 0.01, ***P < 0.001 vs TLR2-/- group, respectively.
2.2.7 hIL-6 ELISA assay in PMA-differentiated THP-1 cell line
Because of the differences between hTLR2 and mTLR2, we evaluated the agonistic activity
of all designed compounds toward PMA-differentiated THP-1 cells that expressed the hTLR2. In
these ELISA evaluations, Pam3CSK4 was selected as a positive control and the cells were
incubated with three concentrations of synthetic compounds (0.5, 1, 2.5 μM) for 24 h (Fig. 10).
According to these data, 35a and 35g exhibited moderate agonistic activity and 35b as well as 35e
were inactive on hTLR2 which were consistent with the potency on mTLR2. Notably, for
compound 35c, almost inactivity was observed in human cells, but it contrarily activated the
mouse cells. Furthermore, 35d which had two more carbons in carbamate bound lipid chains than
35c showed moderate agonistic activity on hTLR2. These results demonstrated that hTLR2 and
mTLR2 were different, specifically the lipid-binding pocket in mouse TLR2 is shallower.41 To our
surprise, compound 35f, which could not stimulate the mTLR2 at all, exhibited apparent agonistic
15

activity on hTLR2 in a dose-dependent manner.
900
0.5 μM
1 μM
2.5 μM
600
300
0
Pam3
35a
35b
35c
35d
35e
35g
35f Control
Compounds
Fig.10. Cytokine hIL-6 induction by the designed compounds 35a – 35g in PMA-differentiated THP-1 cells. All
data represented the mean ± SD of three independent experiments.
2.2.8 hTLR2/1 or hTLR2/6 agonists assay
The hTLRs selectivity of compounds 35a, 35d, and 35f was tested by using hTLRs
antibodies (Invivogen), where Pam3CSK4 was selected as a positive control (Fig. 11). The results
showed that the activation of 35a, 35d, and Pam3CSK4 can be restrained by either anti-hTLR2-IgA
or anti-hTLR1-IgG. Thus, 35a and 35d were TLR2/1 agonists. Interestingly, compound 35f as a
TLR2/6 agonist was obtained just by changing some atoms of 35d based on the phenomenon that
the function of 35f can be inhibited by either anti-hTLR2-IgA or anti-hTLR6-IgG in a dose-
dependent manner. We further investigate the selectivity of 35a and 35d by TLR2/1 selective
inhibitor CU-CPT22, which inhibited TLR2/1 without affecting other TLRs such as TLR2/6,
TLR3, TLR4, and TLR7.47 We found that the CU-CPT22 dose-dependently inhibited 35a and
35d-induced IL-6 production in BV2 cells (Fig. 12), which further indicated that 35a and 35d
were TLR2/1 agonists.
16

2.5 μM compound + anti-hTLR2-IgA
2.5 μM compound + anti-hTLR1-IgG
2.5 μM compound + anti-hTLR6-IgG
500
400
300
200
100
0
35a
35d
35f
Pam
35f
Anti-hTLR antibody
Anti-hTLR antibody
(2.5 μg/mL)
(5 μg/mL)
Fig.11. Cytokine hIL-6 induction by the compounds 35a, 35d, and 35f in PMA-differentiated THP-1 cells which
incubated with different anti-hTLR antibodies for 2 hours in advance. All data represented the mean ± SD of three
independent experiments.
600
2 μM 35d + CUCPT 22
2 μM 35a + CUCPT 22
400
200
0
0
0.1
0.5
1
5
10
CUCPT 22 / μM
Fig.12. CU-CPT22 inhibited the 35a and 35d-induced secretion of IL-6 in BV2 cells in a dose-dependent manner.
All data represented the mean ± SD of three independent experiments.
2.2.9 In vitro cytotoxicity studies
The toxicity of active compounds 35a, 35c, 35d, 35f, and 35g was evaluated by a CCK-8
colorimetric method (Fig. S2). The CCK-8 data showed that these compounds had no cytotoxicity
at a concentration up to 5 μM, indicating that the activity was not related to the toxicity of
compounds.
3. Conclusion
In conclusion, we identified novel mice or human-specific TLR2-selective agonists which
17

have simpler structures and lower molecular weights compared with Pam3CSK4. In vitro
evaluation suggested that some compounds showed moderate to excellent potency. Among them,
compound 35d showed good potency similar to Pam3CSK4 as follows: activated production of IL-
6 (EC50 = 83.08 ± 5.94 nM), up-regulated expression of TNF-α and IL-6 mRNA and promoted
maturation of DCs through the NF-κB signaling pathway. In addition, we found that 35c and 35f
were agonists of mTLR2 and hTLR2, respectively, and 35d, 35a could both activate mTLR2 and
hTLR2. Further research showed that 35d and 35a selectively activate hTLR2/1, while 35f
selectively activate hTLR2/6. Moreover, all active compounds showed no cytotoxicity. Further
studies on the structural optimization of compound 35d are ongoing, we will choose active
compounds to evaluate their agonist activity against lymphocytes cells such as T cells in vivo and
in vitro and the results will be reported in due course.
4. Experiment
4.1 Chemistry
All chemical reagents and solvents were reagent grade which purchased from companies
such as TCI, InvitroGen etc. Target compounds were confirmed by NMR, LC-MS and high
resolution mass spectrometry (HRMS). NMR spectra were recorded with a 600 MHz or 400 MHz
Bruker spectrometer. HRMS was tested by AB 5600+Q TOF. Compounds were purified by flash
column chromatography and all reactions were monitored by thinlayer chromatography (TLC)
which both performed on silica gel (200–300 mesh, Qingdao, China). The purity of all the tested
compounds was analyzed using a SHIMADZU 20AD HPLC system and were conformed to be >
95% (Table S1).
4.1.1 (2R, 2'R)-3,3'-disulfanediylbis(2-((tert-butoxycarbonyl)amino)propanoic acid) (1)
To a solution of L-cystine (1.00 g, 4.16 mmol) in H2O (15 mL) was added TEA (1.73 mL,
12.49 mmol), di-tert- butyl pyrocarbonate (2.73 g, 12.49 mmol). The reaction mixture was stirred
for 0.5 h at 0 °C then warmed to room temperature and stirred overnight. After completion of the
reaction that monitored by thin-layer chromatography (TLC), ethyl acetate (150 mL) was added
and the mixture was extracted with ethyl acetate twice. The combined extracts were washed with
18

brine (50 mL × 2) and dried over Na2SO4. After filtration, the solvent was removed under vacuum
to obtain compound 1 as a white solid without further purification. ESI-MS: [M-H]- m/z 439.0.
4.1.2 (2R,2'R)-dibenzyl 3,3'-disulfanediylbis(2-((tert-butoxycarbonyl)amino)propanoate) (2)
To a solution of compound 1 (1.82 g, 4.14 mmol) in DMF (20 mL) was added benzyl
bromide (1.10 mL, 9.15 mmol), potassium carbonate (1.43 g, 10.40 mmol) and the reaction
mixture was stirred at room temperature for 10 h. Poured the reaction solution to ice-water (200
mL) and the precipitate was filtered, washed with water, dried to afford crude compound. The
crude compound was then purified by column chromatography on silica (10% EA/PE) to give
1
compound 2 as a white solid (2.14 g, yield 83.2%). H NMR (400 MHz, CDCl3) δ 7.35 (s, 10H),
5.43 – 5.38 (m, 2H), 5.17 (s, 4H), 4.65 – 4.58 (m, 2H), 3.15 – 3.09 (m, 4H), 1.43 (s, 18H). 13C
NMR (150 MHz, CDCl3) δ 170.37, 155.25, 135.24, 128.82, 128.77, 128.59, 80.44, 67.68, 54.96,
28.42, 27.52. ESI-MS: [M+Na]+ m/z 643.2.
4.1.3 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-mercaptopropanoate (3)
To a solution of compound 2 (0.05 g, 0.08 mmol) in THF (1 mL) was added tri-n-
butylphosphine (0.02 mL, 0.09 mmol) and stirred for 30 min at room temperature. Then H2O (0.01
mL, 0.40 mmol) was added, the reaction solution was stirred for 1 h at room temperature. After
completion of the reaction, water (50 mL) and ethyl acetate (50 mL) were added and the mixture
was extracted with ethyl acetate twice. The combined extracts were washed with water (20 mL × 2)
and brine (20 mL × 2), and dried over Na2SO4. After filtration, the solvent was removed under
vacuum to obtain the crude compound. The crude compound was then purified by column
chromatography on silica (10% EA/PE) to give compound 3 as a white solid (0.05 g, yield 95.1%).
1H NMR (400 MHz, CDCl3) δ 7.37 (s, 5H), 5.46 – 5.41 (m, 1H), 5.27 – 5.14 (m, 2H), 4.67 – 4.62
(m, 1H), 3.06 – 2.89 (m, 2H), 1.63 – 1.59 (m, 1H), 1.44 (s, 9H). 13C NMR (150 MHz, CDCl3) δ
170.37, 155.26, 135.27, 128.81, 128.76, 128.58, 80.42, 67.66, 55.00, 28.42, 27.49. ESI-MS:
[M+Na]+ m/z 333.8.
4.1.4 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-hydroxyethyl)thio)propanoate (4)
19

To a solution of compound 3 (2.00 g, 6.43 mmol) in acetone (20 mL) was added potassium
carbonate (1.33 g, 9.64 mmol), 2-iodoethanol (0.60 mL, 7.72 mmol) and stirred overnight at room
temperature. After completion of the reaction, water (150 mL) and ethyl acetate (150 mL) were
added and the mixture was extracted with ethyl acetate twice. The combined extracts were washed
with water (50 mL × 2) and brine (50 mL × 2), and dried over Na2SO4. After filtration, the solvent
was removed under vacuum to obtain the crude compound. The crude compound was then
purified by column chromatography on silica (60% EA/PE) to give compound 4 as a yellow oil
(2.10 g, yield 92.0%). 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 5H), 5.51 – 5.47 (m, 1H), 5.30 – 5.26
(m, 1H), 5.24 – 5.12 (m, 2H), 4.60 – 4.54 (m, 1H), 3.69 – 3.63 (m, 2H), 3.02 – 2.90 (m, 2H), 2.68
– 2.64 (m, 2H), 1.43 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 170.96, 155.46, 135.12, 128.76,
128.72, 128.62, 80.45, 67.63, 60.76, 53.77, 53.56, 36.29, 34.91, 28.39. ESI-MS: [M+Na]+ m/z
378.2.
4.1.5 (R)-2-((3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)ethyl palmitate (5)
To a solution of compound 4 (1.00 g, 2.82 mmol) in anhydrous DCM (15 mL) was added
DIPEA (0.54 mL, 3.10 mmol), palmitoyl chloride (0.85 g, 3.10 mmol). The reaction mixture was
stirred at 0 °C for 1 h, then warmed to room temperature and stirred overnight. After completion
of the reaction, water (100 mL) and DCM (100 mL) were added and the mixture was extracted
with DCM twice. The combined extracts were washed with water (40 mL × 2) and brine (40 mL ×
2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the
crude compound. The crude compound was then purified by column chromatography on silica (5%
EA/PE) to give compound 5 as a yellow oil (1.33 g, yield 79.6%). 1H NMR (400 MHz, CDCl3) δ
7.36 (s, 5H), 5.41 – 5.35 (m, 1H), 5.24 – 5.14 (m, 2H), 4.62 – 4.55 (m, 1H), 4.15 (t, J = 6.4 Hz,
2H), 3.07 – 2.96 (m, 2H), 2.69 (t, J = 6.5 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 1.62 – 1.59 (m, 2H),
1.44 (s, 9H), 1.28 – 1.23 (m, 24H), 0.87 (t, J = 6.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.71,
170.95, 155.26, 135.18, 128.77, 128.71, 128.61, 67.64, 63.18, 53.58, 34.83, 34.29, 32.06, 31.31,
29.83, 29.80, 29.76, 29.61, 29.50, 29.41, 29.28, 28.42, 25.02, 22.83, 14.27. ESI-MS: [M+H]+ m/z
594.3.
4.1.6 (R)-2-((2-amino-3-(benzyloxy)-3-oxopropyl)thio)ethyl palmitate (6)
20

To a solution of compound 5 (1.00 g, 1.69 mmol) in anhydrous DCM (16 mL) was added
TFA (4 mL) and the reaction mixture was stirred for 2 h at 0 °C. After completion of the reaction,
saturated sodium bicarbonate solution (100 mL) and DCM (100 mL) were added and the mixture
was extracted with DCM twice. The combined extracts were washed with water (30 mL × 2) and
brine (30 mL × 2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum
to obtain the crude compound as a white solid. The product 6 was used for the next step without
further purification. ESI-MS: [M+H]+ m/z 494.3.
4.1.7 (R)-2-((3-(benzyloxy)-3-oxo-2-palmitamidopropyl)thio)ethyl palmitate (7)
To a solution of compound 6 (1.00 g, 2.03 mmol) in anhydrous DCM (15 mL) was added
DIPEA (0.39 mL, 2.23 mmol), palmitoyl chloride (0.61 g, 2.23 mmol). The reaction mixture was
stirred at 0 °C for 1 h, then warmed to room temperature and stirred overnight. After completion
of the reaction, water (100 mL) and DCM (100 mL) were added and the mixture was extracted
with DCM twice. The combined extracts were washed with water (40 mL × 2) and brine (40 mL ×
2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the
crude compound. The crude compound was then purified by column chromatography on silica (30%
EA/PE) to give compound 7 as a white solid (1.23 g, yield 83.1%). 1H NMR (400 MHz, CDCl3) δ
7.36 (s, 5H), 6.33 – 6.27 (m, 1H), 5.25 – 5.13 (m, 2H), 4.91 – 4.85 (m, 1H), 4.13 (t, J = 6.5 Hz,
2H), 3.13 – 3.05 (m, 1H), 3.05 – 2.96 (m, 1H), 2.66 (t, J = 6.6 Hz, 2H), 2.31 – 2.21 (m, 4H), 1.67
– 1.53 (m, 4H), 1.29 – 1.22 (m, 48H), 0.88 (t, J = 6.5 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ
173.76, 173.19, 170.81, 135.07, 128.80, 128.77, 128.64, 67.76, 63.10, 52.02, 36.64, 34.62, 34.29,
32.07, 31.29, 29.84, 29.80, 29.63, 29.50, 29.43, 29.28, 25.67, 25.03, 22.83, 14.27. ESI-MS:
[M+H]+ m/z 732.8.
4.1.8 (R)-2-palmitamido-3-((2-(palmitoyloxy)ethyl)thio)propanoic acid (14a)
To a solution of compound 7 (0.50 g, 0.68 mmol) in methanol (15 mL) was added
ammonium formate (0.11 g, 1.87 mmol) and 20% Pd(OH)2 (0.25 g). The resulting mixture was
refluxed for 1 h. The catalyst was filtered through celite and the filtercake was washed with
methanol (10 mL x 2). After filtration, the solvent was removed under vacuum to obtain the crude
compound as a white solid, the product 14a was used for the next step without further purification.
21

ESI-MS: [M-H]- m/z 640.3.
4.1.9 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-((methylsulfonyl)oxy)ethyl)thio)
propanoate (8)
To a solution of compound 4 (2.00 g, 5.63 mmol) in anhydrous DCM (15 mL) was added
DIPEA (1.18 mL, 6.76 mmol), methanesulfonyl chloride (0.52 mL, 6.76mmol). The reaction
mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred overnight. After
completion of the reaction, water (100 mL) and DCM (100 mL) were added and the mixture was
extracted with DCM twice. The combined extracts were washed with water (40 mL × 2) and brine
(40 mL × 2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum to
obtain the crude compound 8 as a yellow oil. The product 8 was used for the next step without
further purification. ESI-MS: [M+H]+ m/z 434.3.
4.1.10 (R)-benzyl 3-((2-azidoethyl)thio)-2-((tert-butoxycarbonyl)amino)propanoate (9)
To a solution of compound 8 (0.50 g, 1.15 mmol) in DMF (30 mL) was added NaN3 (0.38 g,
5.77 mmol) and the reaction mixture was stirred at 70 °C for 8 h. After completion of the reaction,
water (50 mL) and ethyl acetate (50 mL) were added and the mixture was extracted with ethyl
acetate twice. The combined extracts were washed with water (20 mL × 2) and brine (20 mL × 2),
and dried over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the
crude compound. The crude compound was then purified by column chromatography on silica (10%
EA/PE) to give compound 9 as a yellow oil (0.35 g, yield 79.0%). 1H NMR (400 MHz, CDCl3) δ
7.36 (s, 5H), 5.45 – 5.39 (m, 1H), 5.26 – 5.14 (m, 2H), 4.61 – 4.55 (m, 1H), 3.37 (t, J = 5.7 Hz,
2H), 3.09 – 2.97 (m, 2H), 2.64 (t, J = 6.4 Hz, 2H), 1.44 (s, 9H). 13C NMR (150 MHz, CDCl3) δ
135.10, 128.75, 128.71, 128.62, 80.40, 67.66, 53.63, 51.05, 34.80, 32.03, 28.38. ESI-MS:
[M+Na]+ m/z 403.2.
4.1.11 (R)-benzyl 3-((2-aminoethyl)thio)-2-((tert-butoxycarbonyl)amino)propanoate (10)
To a solution of compound 9 (0.70 g, 1.84 mmol) in THF (20 mL) was added Ph3P (0.72 g,
2.75 mmol) and the reaction mixture was stirred at 70 °C for 2 h. Then, H2O (5 mL) was added,
and the reaction solution was stirred at 70 °C for 2 h. After completion of the reaction, water (50
22

mL) and ethyl acetate (50 mL) were added and the mixture was extracted with ethyl acetate twice.
The combined extracts were washed with water (20 mL × 2) and brine (20 mL × 2), and dried
over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the crude
compound. The crude compound was then purified by column chromatography on silica (2%
1
MeOH/DCM) to give compound 10 as a colorless oil (0.49 g, yield 75.1%). H NMR (400 MHz,
CDCl3) δ 7.36 (s, 5H), 5.67 – 5.59 (m, 1H), 5.26 – 5.11 (m, 2H), 4.62 – 4.53 (m, 1H), 3.00 – 2.92
(m, 2H), 2.79 (t, J = 5.7 Hz, 2H), 2.58 – 2.53 (m, 2H), 1.60 – 1.52 (m, 2H), 1.43 (s, 9H). ESI-MS:
[M+H]+ m/z 355.4.
4.1.12 General procedure for the synthesis of 11b – 11d
To a solution of compound 10 (1.00 g, 2.82 mmol) in anhydrous DCM (15 mL) was added
DIPEA (0.54 mL, 3.10 mmol) and corresponding acyl chlorides (3.10 mmol). The reaction
mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred overnight. After
completion of the reaction, water (100 mL) and DCM (100 mL) were added and the mixture was
extracted with DCM twice. The combined extracts were washed with water (40 mL × 2) and brine
(40 mL × 2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum to
obtain crude compound. The crude compound was then purified by column chromatography on
silica to give compounds 11b – 11d.
4.1.12.1 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-palmitamidoethyl)thio)propanoate (11b)
1
40% EA/PE, white solid, yield 61.5%. H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H), 6.12 –
6.04 (m, 1H), 5.40 – 5.34 (m, 1H), 5.23 – 5.14 (m, 2H), 4.58 – 4.50 (m, 1H), 3.43 – 3.31 (m, 2H),
3.01 – 2.94 (m, 1H), 2.93 – 2.86 (m, 1H), 2.61 (t, J = 5.5 Hz, 2H), 2.15 (t, J = 6.3 Hz, 2H), 1.64 –
1.57 (m, 2H), 1.44 (s, 9H), 1.27 – 1.22 (m, 24H), 0.87 (t, J = 5.4 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 173.44, 170.92, 155.44, 135.15, 128.81, 128.77, 128.63, 80.48, 67.68, 53.80, 53.55,
38.29, 36.84, 34.89, 33.14, 32.05, 29.82, 29.79, 29.64, 29.49, 28.43, 25.85, 22.82, 14.24. ESI-MS:
[M+H]+ m/z 593.4.
4.1.12.2 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-(((tetradecyloxy)carbonyl) amino)ethyl)
thio)propanoate (11c)
23

1
10% EA/PE, white solid, yield 82.9%. H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H), 5.42 –
5.36 (m, 1H), 5.24 – 5.13 (m, 2H), 4.60 – 4.52 (m, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.33 – 3.24 (m,
2H), 3.03 – 2.96 (m, 1H), 2.96 – 2.89 (m, 1H), 2.59 (t, J = 5.9 Hz, 2H), 1.61 – 1.55 (m, 2H), 1.44
(s, 9H), 1.29 – 1.21 (m, 22H), 0.87 (t, J = 6.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.93,
156.80, 155.34, 135.19, 128.79, 128.74, 128.65, 80.43, 67.65, 65.31, 53.64, 53.55, 40.02, 34.60,
33.21, 32.05, 29.79, 29.73, 29.69, 29.49, 29.44, 29.16, 28.43, 26.00, 22.82, 14.24. ESI-MS:
[M+H]+ m/z 595.2.
4.1.12.3 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-(((hexadecyloxy)carbonyl)amino)ethyl)
thio)propanoate (11d)
1
10% EA/PE, white solid, yield 90.3%. H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H), 5.43 –
5.33 (m, 1H), 5.27 – 5.12 (m, 2H), 5.11 – 5.03 (m, 1H), 4.61 – 4.52 (m, 1H), 4.03 (t, J = 6.1 Hz,
2H), 3.34 – 3.22 (m, 2H), 3.06 – 2.88 (m, 2H), 2.65 – 2.54 (m, 2H), 1.62 (t, J = 11.1 Hz, 4H), 1.44
(s, 9H), 1.26 – 1.23 (m, 26H), 0.87 (t, J = 5.9 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.94,
156.80, 155.34, 135.16, 128.80, 128.76, 128.67, 80.44, 67.66, 65.31, 53.60, 39.97, 34.58, 33.20,
32.06, 29.83, 29.80, 29.74, 29.70, 29.50, 29.45, 29.15, 28.42, 26.00, 22.83, 14.27. ESI-MS:
[M+H]+ m/z 623.4.
4.1.13 General procedure for the synthesis of 12b – 12d
The compounds 12b – 12d were synthesized following the preparation protocol of compound 6.
The products 12b – 12d were used for the next step without further purification.
4.1.14 General procedure for the synthesis of 13b – 13d
The compounds 13b – 13d were synthesized following the preparation protocol of compound
7. The crude compounds were then purified by column chromatography on silica to give
compounds 13b – 13d.
4.1.14.1 (R)-benzyl 2-palmitamido-3-((2-palmitamidoethyl)thio)propanoate (13b)
1
0.5% MeOH/DCM, white solid, yield 73.7%. H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H),
6.49 – 6.35 (m, 1H), 6.15 – 6.03 (m, 1H), 5.29 – 5.11 (m, 2H), 4.92 – 4.78 (m, 1H), 3.45 – 3.29 (m,
24

2H), 3.06 – 2.97 (m, 1H), 2.95 – 2.85 (m, 1H), 2.60 (t, J = 5.8 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H),
2.16 (t, J = 7.4 Hz, 2H), 1.67 – 1.55 (m, 4H), 1.30 – 1.21 (m, 48H), 0.87 (t, J = 5.8 Hz, 6H). 13C
NMR (150 MHz, CDCl3) δ 173.64, 173.50, 170.85, 135.08, 128.83, 128.64, 67.78, 53.55, 52.21,
38.47, 36.82, 36.65, 34.67, 32.97, 32.06, 29.84, 29.80, 29.50, 25.86, 25.70, 22.82, 14.24. ESI-MS:
[M+H]+ m/z 731.8.
4.1.14.2 (R)-benzyl 2-palmitamido-3-((2-(((tetradecyloxy)carbonyl)amino)ethyl)thio)propanoate
(13c)
0.5% MeOH/DCM, white solid, yield 78.3%. 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H),
6.43 – 6.37 (m, 1H), 5.25 – 5.13 (m, 2H), 5.05 – 4.99 (m, 1H), 4.90 – 4.83 (m, 1H), 4.02 (t, J = 6.2
Hz, 2H), 3.30 – 3.22 (m, 2H), 3.05 – 2.99 (m, 1H), 2.98 – 2.92 (m, 1H), 2.59 – 2.53 (m, 2H), 2.24
(t, J = 7.5 Hz, 2H), 1.66 – 1.54 (m, 6H), 1.27 – 1.21 (m, 44H), 0.87 (t, J = 5.5 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 173.26, 170.84, 156.86, 135.12, 128.82, 128.67, 67.75, 65.36, 53.55, 52.10,
40.12, 36.63, 34.34, 33.15, 32.06, 29.83, 29.80, 29.49, 29.17, 26.01, 25.67, 22.83, 14.25. ESI-MS:
[M+H]+ m/z 733.6.
4.1.14.3 (R)-benzyl 3-((2-(((hexadecyloxy)carbonyl)amino)ethyl)thio)-2-palmitamidopropanoate
(13d)
0.5% MeOH/DCM, white solid, yield 71.9%. 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H),
6.43 – 6.36 (m, 1H), 5.25 – 5.13 (m, 2H), 5.01 (t, J = 5.8 Hz, 1H), 4.91 – 4.83 (m, 1H), 4.03 (t, J =
6.7 Hz, 2H), 3.31 – 3.22 (m, 2H), 3.07 – 2.99 (m, 1H), 2.99 – 2.91 (m, 1H), 2.57 (t, J = 6.2 Hz,
2H), 2.24 (t, J = 7.6 Hz, 2H), 1.67 – 1.55 (m, 4H), 1.28 – 1.22 (m, 50H), 0.87 (t, J = 6.7 Hz, 6H).
13C NMR (150 MHz, CDCl3) δ 173.25, 170.84, 156.86, 135.12, 128.81, 128.67, 67.74, 65.36,
53.55, 52.09, 40.12, 36.63, 34.34, 33.15, 32.06, 29.83, 29.80, 29.49, 29.17, 26.01, 25.67, 22.82,
14.25. ESI-MS: [M+H]+ m/z 761.6.
4.1.15 General procedure for the synthesis of 14b – 14d
Following the general synthesis method of 14a, using compounds 13b – 13d (1.32 mmol)
instead of compound 7, gave crude compounds as white solid and the products 14b – 14d were
used for the next step without further purification.
25

4.1.16 1-Isocyanatohexadecane (15)
To a solution of triphosgene (0.14 g, 0.47 mmol) in anhydrous toluene (5 mL) was added
hexadecan-1-amine (0.23 g, 0.94 mmol) in anhydrous toluene drop-wise and the resulting mixture
was stirred at reflux temperature for 6 h. The solvent was evaporated to obtain the crude
compound as a colorless oil and the product 15 was used for the next step without further
purification.
4.1.17 (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-((2-(3-hexadecylureido)ethyl)thio)propanoate
(16)
To a solution of compound 10 (0.40 g, 1.13 mmol) in anhydrous DCM (15 mL) was added
Et3N (0.66 mL, 4.76 mmol) and product 15 from the previous step. The reaction mixture was
stirred at 0 °C for 1 h, then warmed to room temperature and stirred overnight. After completion
of the reaction, water (100 mL) and DCM (100 mL) were added and the mixture was extracted
with DCM twice. The combined extracts were washed with water (40 mL × 2) and brine (40 mL ×
2), and dried over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the
crude compound. The crude compound was then purified by column chromatography on silica (1%
MeOH/DCM) to give compound 16 as a yellow solid (0.46 g, yield 78.3%). 1H NMR (400 MHz,
CDCl3) δ 7.36 (s, 5H), 5.37 (d, J = 8.3 Hz, 1H), 5.24 – 5.21 (m, 1H), 5.20 – 5.15 (m, 2H), 4.70 –
4.63 (m, 1H), 4.57 – 4.48 (m, 1H), 3.49 – 3.33 (m, 1H), 3.33 – 3.20 (m, 1H), 3.19 – 3.07 (m, 2H),
3.02 – 2.89 (m, 1H), 2.89 – 2.79 (m, 1H), 2.78 – 2.66 (m, 1H), 2.65 – 2.48 (m, 1H), 1.76 (s, 2H),
1.44 (s, 9H), 1.30 – 1.18 (m, 26H), 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.98,
158.25, 155.71, 135.13, 128.83, 128.78, 128.62, 80.63, 67.71, 53.98, 40.64, 39.38, 35.64, 34.36,
32.06, 30.39, 29.84, 29.74, 29.50, 28.46, 27.07, 22.83, 14.25. ESI-MS: [M+H]+ m/z 622.0,
[M+Na]+ m/z 643.8.
4.1.18 (R)-benzyl 2-amino-3-((2-(3-hexadecylureido)ethyl)thio)propanoate (17)
The compound 17 was synthesized following the preparation protocol of compound 6. The
product 17 was used for the next step without further purification.
26

4.1.19 (R)-benzyl 3-((2-(3-hexadecylureido)ethyl)thio)-2-palmitamidopropanoate (18)
The compound 18 was synthesized following the preparation protocol of compound 7. The
crude compound was then purified by column chromatography on silica (0.3% MeOH/DCM) to
give compound 18 as a white solid, yield 80.4%. 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 5H), 6.57
– 6.35 (m, 1H), 5.44 – 5.30 (m, 1H), 5.24 – 5.13 (m, 2H), 5.04 – 4.89 (m, 1H), 4.88 – 4.77 (m,
1H), 3.44 – 3.33 (m, 1H), 3.31 – 3.21 (m, 1H), 3.18 – 3.06 (m, 2H), 3.03 – 2.92 (m, 1H), 2.90 –
2.81 (m, 1H), 2.75 – 2.67 (m, 1H), 2.62 – 2.52 (m, 1H), 2.26 (t, J = 7.6 Hz, 2H), 1.66 – 1.58 (m,
2H), 1.49 – 1.42 (m, 2H), 1.28 – 1.22 (m, 50H), 0.87 (t, J = 6.3 Hz, 6H). 13C NMR (150 MHz,
CDCl3) δ 173.80, 170.89, 158.43, 135.06, 128.84, 128.59, 67.80, 52.52, 40.62, 39.56, 36.67, 35.33,
32.07, 30.44, 29.85, 29.80, 29.66, 29.50, 29.39, 27.10, 25.70, 22.83, 14.25. ESI-MS: [M+H]+ m/z
760.6.
4.1.20 (R)-3-((2-(3-hexadecylureido)ethyl)thio)-2-palmitamidopropanoic acid (14e)
The compound 14e was synthesized following the preparation protocol of compound 14a.
The product 14e were used for the next step without further purification. ESI-MS: [M+H]+ m/z
670.4.
4.1.21 1-Isocyanatotetradecane (19)
The compound 19 was synthesized following the preparation protocol of compound 15. The
product 19 was used for the next step without further purification.
4.1.22 (R)-benzyl 3-((2-(((hexadecyloxy)carbonyl)amino)ethyl)thio)-2-(3-tetradecylureido)
propanoate (20)
The compound 20 was synthesized following the preparation protocol of compound 16. The
crude compound was then purified by column chromatography on silica (0.5% MeOH/DCM) to
give compound 20 as a white solid, yield 80.6%. 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 5H), 5.83
– 5.68 (m, 1H), 5.17 (q, J = 12.4 Hz, 2H), 5.09 – 5.03 (m, 1H), 4.88 – 4.82 (m, 1H), 4.03 (t, J =
6.4 Hz, 2H), 3.34 – 3.25 (m, 2H), 3.21 – 3.13 (m, 2H), 3.09 – 3.00 (m, 1H), 2.98 – 2.90 (m, 1H),
2.60 (t, J = 5.9 Hz, 2H), 1.63 – 1.55 (m, 2H), 1.49 – 1.43 (m, 2H), 1.28 – 1.23 (m, 46H), 0.87 (t, J
= 6.3 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 171.74, 157.61, 157.27, 135.42, 128.73, 128.58,
27

128.51, 67.44, 65.54, 53.50, 40.60, 35.37, 33.18, 32.06, 30.33, 29.84, 29.72, 29.50, 29.15, 27.08,
26.02, 22.82, 14.24. ESI-MS: [M+H]+ m/z 762.6.
4.1.23 (R)-3-((2-(((hexadecyloxy)carbonyl)amino)ethyl)thio)-2-(3-tetradecylureido)propanoic
acid (14f)
The compound 14f was synthesized following the preparation protocol of compound 14a.
The product 14f were used for the next step without further purification. ESI-MS: [M+H]+ m/z
672.5.
4.1.24 (S)-benzyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)
hexanoate (21)
To a solution of Fmoc-Lys(Boc)-OH (2.50 g, 5.34 mmol) in DMF (100 mL) was added
potassium carbonate (0.90 g, 6.40 mmol) and benzyl bromide (0.76 mL, 6.40 mmol), and the
resulting mixture was stirred overnight at room temperature. After completion of the reaction,
water (150 mL) and ethyl acetate (150 mL) were added and the mixture was extracted with ethyl
acetate twice. The combined extracts were washed with water (50 mL × 2) and brine (50 mL × 2),
and dried over Na2SO4. After filtration, the solvent was removed under vacuum to obtain the
crude compound. The crude compound was then purified by column chromatography on silica
(1.6% MeOH/DCM) to give compound 21 as a white solid (2.87 g, yield 96.2%). 1H NMR (400
MHz, CDCl3) δ 7.77 (d, J = 7.2 Hz, 2H), 7.60 (d, J = 7.1 Hz, 2H), 7.45 – 7.28 (m, 9H), 5.47 –
5.41 (m, 1H), 5.25 – 5.12 (m, 2H), 4.56 – 4.49 (m, 1H), 4.45 – 4.34 (m, 2H), 4.26 – 4.17 (m, 1H),
3.11 – 2.98 (m, 2H), 1.92 – 1.80 (m, 1H), 1.77 – 1.62 (m, 3H), 1.43 (s, 9H), 1.38 – 1.23 (m, 2H).
13C NMR (150 MHz, CDCl3) δ 172.43, 156.18, 144.07, 141.45, 135.42, 128.78, 128.66, 128.50,
127.84, 127.21, 125.24, 120.11, 79.32, 67.32, 67.17, 53.94, 53.55, 47.32, 40.19, 32.29, 29.72,
28.56, 22.44. ESI-MS: [M+H]+ m/z 559.3.
4.1.25 (S)-benzyl 2-amino-6-((tert-butoxycarbonyl)amino)hexanoate (22)
To a solution of compound 21 (1.00 g, 1.79 mmol) in 20% of piperidine in DMF (30 mL)
was stirred at room temperature for 2 h. After completion of the reaction, the solvent was
evaporated to obtain the crude compound 22 as a white solid. The product 22 was used for the
28

next step without further purification. ESI-MS: [M+H]+ m/z 337.2.
4.1.26 (S)-benzyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)
amino)hexanamido)-6-((tert-butoxycarbonyl)amino)hexanoate (23)
To a solution of Fmoc-Lys(Boc)-OH (1.00 g, 2.13 mmol) in anhydrous DCM was added
HATU (0.97 g, 2.56 mmol) and DIPEA (0.45 mL, 2.56 mmol) at 0 °C under nitrogen atmosphere.
After 30 minutes stirring, compound 22 (0.86 g, 2.56 mmol) was added to the reaction mixture
and reaction was allowed to warm to room temperature and stirred for 4 h. After completion of the
reaction, water (150 mL) and DCM (150 mL) were added and the mixture was extracted with
DCM twice. The combined extracts were washed with water (50 mL × 2) and brine (50 mL × 2),
and dried over Na2SO4. After filtration, the organic solvent was removed under vacuum to obtain
the crude compound. The crude compound was then purified by column chromatography on silica
(1.2% MeOH/DCM) to give compound 23 as a white solid (1.40 g, yield 83.3%). 1H NMR (400
MHz, CDCl3) δ 7.62 (d, J = 7.2 Hz, 2H), 7.48 – 7.42 (m, 2H), 7.30 – 7.12 (m, 10H), 6.63 – 6.46
(m, 1H), 5.54 – 5.38 (m, 1H), 5.02 (q, J = 33.3, 10.9 Hz, 2H), 4.65 – 4.39 (m, 3H), 4.29 – 4.16 (m,
2H), 4.12 – 4.00 (m, 2H), 3.03 – 2.80 (m, 4H), 1.78 – 1.66 (m, 2H), 1.60 – 1.46 (m, 2H), 1.33 –
1.23 (m, 24H), 1.19 – 1.05 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 172.07, 171.88, 156.35,
156.29, 144.00, 143.88, 141.42, 135.36, 128.77, 128.68, 128.55, 127.86, 127.24, 125.27, 120.12,
79.32, 67.38, 67.27, 54.76, 52.31, 47.24, 40.04, 39.90, 32.21, 31.59, 29.59, 29.51 , 28.56, 22.38.
ESI-MS: [M+Na]+ m/z 809.4.
4.1.27
(S)-benzyl
2-((S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-6-((tert-
butoxycarbonyl)amino)hexanoate (24)
The compound 24 was synthesized following the preparation protocol of compound 22. The
product 24 were used for the next step without further purification. ESI-MS: [M+Na]+ m/z 587.4.
4.1.28 (10S,13S,16S)-benzyl 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-13,16-bis(4-((tert-
butoxycarbonyl)amino)butyl)-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-
oate (25)
The compound 25 was synthesized following the preparation protocol of compound 23. The
29