Self-Adjuvanting Cancer Vaccines from Conjugation-Ready Lipid A Analogues and Synthetic Long Peptides

Article abstract of DOI:10.1021/acs.jmedchem.0c00851

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.

Full text of DOI:10.1021/acs.jmedchem.0c00851

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Self#adjuvanting cancer vaccines from conjugation#
ready lipid A analogues and synthetic long peptides
Niels Reintjens, Elena Tondini, Ana R. de Jong, Nico J. Meeuwenoord, Fabrizio Chiodo, Evert Peterse,
Herman S. Overkleeft, Dmitri V. Filippov, Gijsbert A. van der Marel, Ferry Ossendorp, and Jeroen D. C. Codée
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00851 • Publication Date (Web): 22 Sep 2020
Downloaded from pubs.acs.org on September 27, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 51
Journal of Medicinal Chemistry
1
2
3
Self-adjuvanting cancer vaccines from conjugation-ready lipid A
analogues and synthetic long peptides
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Niels R. M. Reintjens,†,‡ Elena Tondini,†,§ Ana R. de Jong, Nico J. Meeuwenoord, Fabrizio Chiodo,
‡
‡
‡,∥
‡
‡
‡
‡
Evert Peterse, Herman S. Overkleeft, Dmitri V. Filippov, Gijsbert A. van der Marel, Ferry
§
,*
‡,*
Ossendorp, and Jeroen D. C. Codée
‡Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC
Leiden, The Netherlands
§Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden
University, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
∥Amsterdam UMC, Vrije Universiteit Amsterdam, Dept. of Molecular Cell Biology and Immunology,
Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands
†
These authors contributed equally.
KEYWORDS: Toll-Like receptor ligand; conjugate vaccine; adjuvant; cancer vaccine; lipid A
ABSTRACT
Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating
agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR)
ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-
ready TLR4-ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-
vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 51
ligand as well as the connection of it to the peptide antigen. Different linker systems and connection
modes to a model peptide were explored and in vitro evaluation of the conjugates showed them to be
powerful immune-activating agents, significantly more effective than the separate components.
Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine
modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation and
in initiating specific CD8+ T cell-mediated killing of antigen-loaded target cells in vivo. Synthetic
TLR4-ligands thus show great promise in potentiating the conjugate vaccine platform for application in
cancer vaccination.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
. INTRODUCTION
Immunotherapy has become a powerful strategy to combat cancer. Significant advances have been made
in the activation of anti-tumor T cell immunity, including the development of immune checkpoint
1
2
blockade antibodies , chimeric antigen receptor T cells (CAR T cells) and vaccination strategies, in
which the immune system is trained to recognize cancer neoantigens.3,4 To optimally direct an immune
reaction against cancer via vaccination, adjuvants are used to activate antigen-presenting cells, such as
5
dendritic cells (DCs) and macrophages. DCs express pathogen recognition receptors (PRRs) , through
which they recognize invading pathogens and initiate an immune response, which eventually leads to the
6
priming of T cells. Pathogen associated molecular patterns (PAMPs) are ligands for these PRRs and can
be used as molecular-adjuvants. Molecular-adjuvants are well-defined single molecule immunostimulants
that act directly on the innate immune system to enhance the adaptive immune response against antigens.
Many well-defined PAMPs have been explored over the years, and the most extensively targeted PRR-
7
8
families are the Toll-like receptors (TLRs) , C-type lectins , and Nucleotide-binding Oligomerization
Domain (NOD)-like receptors9,10. To further improve vaccine activity, the antigen and adjuvants have
been combined in covalent constructs, delivering “self-adjuvanting” vaccine candidates.11,12 In the
immune system, the stimulation of different TLRs can activate distinct signaling cascades and thereby
support the generation of polarized types of immune reactions. Hence, targeting of distinct TLRs in
2
ACS Paragon Plus Environment

Page 3 of 51
Journal of Medicinal Chemistry
vaccination influences the nature of the adaptive immune response induced.13,14 Several TLR
agonists11,15,16 have been conjugated to antigenic peptides (often synthetic long peptides, SLPs), including
ligands for TLR2,17–22 TLR723,24 and TLR9,20,25,26 yielding vaccine modalities with improved activity with
respect to their non-conjugated counterparts. Lipid A (Figure 1A), a conserved component of the bacterial
cell wall, is one of the most potent immune-stimulating agents known to date and it activates the innate
immune system through binding with TLR4. The high toxicity of lipid A makes it unsuitable for safe use
in humans, but monophosphoryl lipid A (MPLA, Figure 1A), a lipid A derivative in which the anomeric
phosphate has been removed, has proven its effectiveness as an adjuvant in various approved vaccines.27–
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9
It has also been used recently in conjugates in which it was covalently attached to a tumor-associated
carbohydrate antigen (TACA) or a synthetic bacterial glycan.30–34 The latter conjugate was able to elicit
34
a robust IgG antibody response in mice, critical for effective anti-bacterial vaccination. MPLA thus
represents a very attractive PAMP to be explored in SLP conjugates, targeting cancer epitopes. The
physical properties and challenging synthesis of lipid A derivatives, however, limit its accessibility.35–37
Because of its potent immunostimulating activity, many mimics of MPLA have been developed and the
class of aminoalkyl glucosamine 4-phosphates (AGPs) has been especially promising.38–41 AGPs have
been shown to be efficacious adjuvants and to be clinically safe, resulting in their use in a Hepatitis B
4
2
38
vaccine. CRX-527 (Figure 1A) has been established as one of the most potent AGPs.
We here introduce conjugation-ready derivatives of CRX-527 for application in the development of
adjuvant-SLP vaccines conjugates. We have established a robust synthetic route to generate linker-
equipped CRX-527 analogues and used these in the assembly of SLP conjugates. The self-adjuvanting
SLPs carrying this TLR4-ligand are capable of mobilizing a strong T cell immune response against the
incorporated antigen and are capable of promoting effective and specific killing of target cells expressing
the antigen in vivo.
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 51
OH
OH
A
O
P
O
OH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
O
O
P
O
O
O
O
P
COO
NH
O
O
O
O
O
O
O
O
HO
HO
O
O
NH
O
O
P
NH
O
O
O
NH
O
O
O
O
O
O
O
OH
O
O
O
NH
NH
O
O
O
O
O
O
O
O
HO
O
HO
O
O
O
O
O
O
HO
HO
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Lipid A
MPLA
CRX-527 (1)
B
Linker
COO
R
X
O
P
X = O or NH
O
O
O
O
O
O
O
NH
NH
O
O
O
A
N
H
O
O
O
O
O
O
O
O
B
C
O
O
O
O
Linker =
O
O
N
H
O
O
O
*
S
N
N
H
O
R = Acetyl or DEVA K (=DEVSGLEQLESIINFEKLAAAAAK)
5
1
2
3
X = OH
X = O
X = O
CRX527
5 X = O Linker = C R = DEVA5K (N-terminus) CRX527
O
TEG
DEVA5K
H
N
Linker = A R = Ac CRX527
Linker = B R = Ac CRX527
O
O
Alkyl
6 X = NH Linker = C R = DEVA K (N-terminus) CRX527
TEG DEVA K
5
5
TEG
TEG
7 X = O Linker = C R = DEVA K (C-terminus) DEVA K
TEG
TEG
CRX527
CRX527
5
5
O
H
N
H
N
4 X = NH Linker = B R = Ac CRX527
8 X = NH Linker = C R = DEVA K (C-terminus) DEVA K
5
5
Figure 1. (A) Representative structures of lipid A of E. coli and MPLA of Salmonella enterica serotype
minnesota Re 595; Structure of CRX-527 (1). (B) Structures of CRX-527 derivatives 2-4 and CRX-527
conjugates 5-8. The DEVA K peptide in the conjugates carries the SIINFEKL epitope in its sequence.
5
2
. RESULTS AND DISCUSSION
.1 Synthesis of the ligands and conjugates. In the development of the conjugation-ready CRX-527-
2
derivates we set out to probe both the influence of the nature of the linker and the mode of connectivity
of the linker to the TLR4-ligand, CRX-527 (See Figure 1B). In lipopolysaccharides bacterial O-antigens
are connected to a lipid A anchor through the C6-position of the glucosamine-C4-phosphate residue and
from the crystal structure of lipid A in complex to the TLR4-MD2 complex, it is apparent that this position
4
ACS Paragon Plus Environment

Page 5 of 51
Journal of Medicinal Chemistry
4
3
is exposed from the complex. The C6-position of the glucosamine-C4-phosphate can thus be used for
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
conjugation purposes. Indeed, previous work on anti-bacterial MPLA conjugate vaccines has shown that
3
4
the adjuvant can be modified at this position without compromising adjuvant activity. We explored two
types of linkers at the C6-position of CRX-527: a hydrophobic alkyl linker (A) and a hydrophilic
triethylene glycol (TEG) linker (B). These linkers were connected to CRX-527 through an ester bond,34
or via a more stable amide bond. To connect the ligands to the SLPs, the linkers were equipped with a
maleimide, to allow for a thiol-ene conjugation to the sulfhydryl functionalized SLP. We used the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0
ovalbumin derived SLP, DEVSGLEQLESIINFEKLAAAAAK (DEVA K) as a model antigen. Herein,
5
the MHC-I epitope SIINFEKL is embedded in a longer peptide motif to ensure that the peptide will have
to undergo proteasomal processing to produce the minimal epitope. The target compounds generated for
this study are depicted in Figure 1 and include CRX-527 (1), ester-linker CRX-527 2 and 3, amide-linker
CRX-527 4 as well as the conjugates 5 and 6 having the CRX-527 ligand at the N-terminus of the peptide
and conjugates 7 and 8, with the ligand at the C-terminus of the SLP. To obtain CRX-527 derivatives 1-
4
, building blocks 16a/b were required and the assembly of these key intermediates was accomplished as
4
1
depicted in Figure 2A. Based on the synthesis route to CRX-527 developed by Johnson and co-workers ,
we assembled glucosaminyl serine building block 11 from glucosamine donor 9 and serine 10.
Condensation of 9 and 10 under the influence of boron trifluoride etherate proceeded in a completely β-
selective manner to give a mixture of the desired product and unreacted donor 9. The mixture could be
separated after hydrogenolysis of the benzyl ester giving acid 11 in 63% yield on 170 mmol scale. Next,
all acetyls were removed, before the benzyl ester was re-installed using phase transfer conditions to deliver
triol 12. To enable the introduction of the chiral lipid tail we masked the C4- and C6-hydroxyl groups in
1
2 with a silylidene ketal. This protecting group strategy proved to be crucial as the use of a C6-O-tert-
4
1
butyldimethylsilyl (TBDMS) group as previously reported, led to an intractable mixture when the lipid
tails were attached. As lipid A analogues bearing fewer lipid tails may have a different immunological
4
4
response , the purity of the ligand is of utmost importance. Next, the Troc-protecting groups were
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 51
removed from both amine groups, after which an N,N,O-triacetylation event using fatty acid 14
Supporting information Scheme 1) and EDC∙MeI and catalytic DMAP (0.03 eq.) delivered compound
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
15. On a 9.5 mmol scale this intermediate was obtained in 57% over two steps. The silylidene ketal was
removed, and then the primary alcohol was selectively protected with a TBDMS group, and the phosphate
triester was installed at the C4-OH. Desilylation provided key building block 16a on a multi-gram scale.
The alcohol in 16a was transformed into the corresponding primary azide using Mitsunobu conditions
delivering 16b.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
With building blocks 16a/b available in sufficient amounts, attention was directed to the assembly of
ligands 1-4, having either an alkyl or triethylene glycol (TEG) linker (Figure 2B). Debenzylation of 16a
using Pd/C gave the original CRX-527 (1). Elongation of 16a with the N-acetylated linkers 17 or 18,
under the influence of EDC∙MeI and DMAP, furnished the fully protected linker-CRX-527 compounds
that were subjected to a hydrogenation reaction to obtain ligands 2 and 3. In contrast to the findings of
Guo and coworkers, in their synthesis of linker functionalized MPLA derivatives, where the C6-ester
34
bond was found unstable, no hydrolysis of esters 2 and 3 was observed. Ligand 4 was obtained from
azide 16b by zinc mediated reduction, condensation with linker 18 and subsequent hydrogenation.
Next, the synthesis of the CRX-527-peptide conjugates 5-8 was undertaken (Figure 2C). Based on the
immunological evaluation of the ligands 1-4 (vide infra, Figure 3) the TEG linker was used for the
assembly of the peptide antigen conjugates. First, 16a and azido linker 19 were conjugated under the
agency of EDC. Reduction of the azide and benzyl esters was then followed by the introduction of the
maleimide functionality using sulfo-N-succinimidyl 4-maleimidobutyrate to give conjugation-ready
CRX-527 20a. The amide congener of this compound was assembled from 16b in an analogous manner.
The DEVA K-peptides with a thiol function at the N-terminus (21) or the C-terminus (22) were assembled
5
using a semi-automated solid phase peptide synthesis protocol and purified to homogeneity by RP-HPLC
(See Supporting information for full synthetic details). The conjugation of the ligand and the peptide-
antigen required significant optimization because of the physical properties of the ligand and the peptides.
6
ACS Paragon Plus Environment

Page 7 of 51
Journal of Medicinal Chemistry
We found that the thiol-maleimide coupling could be achieved by dissolving 21 or 22 in DMF/H O (4/1
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
v/v) followed by the addition of a solution of maleimide 20a or 20b in CHCl . After shaking for two days,
3
LC-MS analysis confirmed the full conversion of the maleimide and the conjugates were purified by C18
column chromatography (See Supporting information for details). Figure 2D shows the analysis of the
conjugation of maleimide 20a with an excess of thiol 22, providing conjugate 7 (Figure 2D, left panel),
which was purified by HPLC to provide the pure conjugate (Figure 2D, middle panel). The integrity and
purity of the synthesized N-terminus conjugates 5 and 6, and C-terminus conjugates 7 and 8 were
ascertained by LC-MS analysis and MALDI-TOF MS (See Figure 2D, right panel for the MALDI-TOF
MS spectrum of 7).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.2. In vitro activity. Immunological evaluation of TLR4-ligands 1-4 and conjugates 5-8 was performed
by first assessing their ability to induce maturation of dendritic cells and to present antigen to T cells in
vitro. Binding of the lipid A to the TLR4/MD-2 complex triggers the production of inflammatory
cytokines and maturation of the DCs. Production of the subunit IL-12p40 of the pro-inflammatory
cytokine IL-12 is a marker of this activation. We first analyzed if the addition of the peptide would affect
the interaction with TLR4 and impede the activity of the ligand. To probe activation of the DCs by the
CRX-527-ligands and conjugates, murine DCs were stimulated for 24h with the different compounds and
the amount of IL-12p40 in the supernatant was measured. First, the effect of the different linkers
(compounds 2-4) on the activity of the CRX-527 ligand was evaluated. As can be seen in Figure 3A,
stimulation of the DCs with the CRX-527 ligand 1 induces strong IL-12p40 secretion and its activity is
higher than the commercially available TLR4-ligand MPLA. The addition of an ester-alkyl linker (2)
significantly decreased the ability of the ligand to induce DC-activation. However, the ester- or amide-
TEG linker functionalized ligands mostly preserve the induction of IL-12p40, as shown for compounds 3
and 4. For these two ligands,
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
ACS Paragon Plus Environment

Page 9 of 51
Journal of Medicinal Chemistry
Figure 2. (A) Synthesis of building blocks 16a/b. Reagents and conditions: a) i. BF ·OEt , DCM, 0°C to
3
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rt; ii. H , Pd/C, THF, 63% over two steps; b) i. NH OH, MeOH; ii. BnBr, TBAB, DCM/NaHCO (aq.
2
4
3
sat.), 79% over two steps; c) (tBu) Si(OTf) , DMF, -40°C, 94%; d) i. Zn dust, AcOH; ii.) 14, EDC·MeI,
2
2
DMAP, DCM, 57% over two steps. e) i. HF·Et N. THF, 0°C, 92%; ii. TBDMSCl, pyridine, 88%; iii.
3
dibenzyl N,N-diisopropylphosphoramidite, tetrazole, DCM, 0°, 1h; iv. 3-chloroperbenzoic acid, quant.
over two steps; v. TFA, DCM, 84%. f) PPh , DEAD, DPPA, THF, 67%; (B) Synthesis of TLR4-ligands
3
1
-4. Reagents and conditions: g) H , Pd/C, THF, 1: 89%; h) i. 17, EDC∙MeI, DMAP, DCE, 88%; ii. H ,
2
2
Pd/C, THF, 2: 56%; i) i. 18, EDC∙MeI, DMAP, DCE, 74%; ii. H , Pd/C, THF, 3: 66%; j) i. Zn, NH Cl,
2
4
DCM/MeOH/H O; ii. 18, EDC∙MeI, DMAP, DCE, 40% over two steps; iii. H , Pd/C, THF, 4: 61%; (C)
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Assembly of conjugates 5-8. Reagents and conditions: k) i. 19, EDC∙MeI, DMAP, DCE, 80%; ii. H2,
Pd/C, THF, 77%; iii. sulfo-N-succinimidyl 4-maleimidobutyrate sodium salt, Et N, DCM, 20a: 84%. l) i.
3
Zn, NH Cl, DCM/MeOH/H O; ii. 19, EDC∙MeI, DMAP, DCE, 56% over two steps; iii. sulfo-N-
4
2
succinimidyl 4-maleimidobutyrate sodium salt, Et N, DCE, 20b: 81%; m) 21, DMF/CHCl /H O, 48h, 5:
3
3
2
5
2%, 6: 54%; n) 22, DMF/CHCl /H O, 48h, 7: 57%, 8: 42%. (D) LC-MS trace of crude and purified C-
3 2
terminus conjugate 7, and MALDI analysis of 7.
the activity was comparable to the unmodified ligand 1 up to 1.56 nM, and slightly reduced at the lowest
concentrations. However, substantial levels of IL-12p40 could still be detected at these concentrations.
Possibly, the hydrophobic nature of the alkyl linker of compound 2 induces a different configuration of
4
3
the ligand that affects binding to the MD-2/TLR4 pocket, preventing activation of the signaling cascade .
The DC-activating capacity of compounds 1, 3 and 4 indicates that functionalization at the C6-position
with a hydrophilic linker does not inhibit binding of the ligand to the receptor.
Next, the DEVA K peptide conjugates 5-8 were evaluated for their ability to induce IL-12p40 in DCs
5
(Figure 3B). We analyzed whether conjugation of the peptide via the ester- (compounds 5 and 7) or the
amide-TEG linker (compounds 6 and 8) could differently modulate the activity of the conjugates.
Moreover, we investigated whether conjugation of the ligand to the N- or C-terminus of the peptide could
also influence activity. Interestingly, we found that even if ligands 3 and 4 do not display any differences
in activity (Figures 3A and 3B), their respective peptide conjugates show differential potencies (Figure
3
B). Specifically, the ester conjugates 5 and 7 induce high levels of IL-12p40, similarly to the ligands 3
and 4, while the amide conjugates 6 and 8, display overall lower levels of IL-12p40 production. No
difference in activity was observed between the N- or C-terminal DEVA K conjugates. The activity of
5
the ester conjugates 5 and 7 titrates faster than the free ligands, 3 and 4, as can be observed at the lowest
concentrations.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 51
These data show that conjugation of CRX-527 to a long peptide via different linkers results in
immunologically active compounds and has therefore potential for vaccination. Importantly, the efficacy
of peptide vaccines relies on the ability of DCs to take up the peptide and process it to enable surface
presentation of the epitope on MHC molecules. Recognition of the antigen-MHC complex by the T cell
receptor and simultaneous co-stimulation by mature DCs then results in the initiation of a T cell
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
response. The uptake and processing of the conjugates 5-8 was evaluated in an antigen presentation
assay using the T cell hybridoma reporter line B3Z, which is specific for the SIINFEKL epitope contained
in the DEVA K peptide. The B3Z cell line possesses a T cell receptor, specific for the SIINFEKL epitope,
5
which controls the expression of the beta-galactosidase reporter gene. Recognition of the SIINFEKL
epitope induces the expression of the enzyme, which can subsequently be detected through a colorimetric
reaction caused by the conversion of a substrate. The efficiency in antigen presentation of the conjugates
was compared to free peptide 23 and to a mixture of peptide 23 and CRX-527 ligand 1. Figure 3C shows
that incubation of DCs with the ester conjugates 5 and 7, leads to similar levels of T cell activation as the
free peptide and the mixture of the peptide and CRX-527 1. Therefore, conjugation of the peptide to the
CRX-527 ligand does not affect its uptake and processing, both after N-terminus and C-terminus
conjugation. Notably, incubation with the amide conjugates 6 and 8 resulted in slightly enhanced antigen
presentation. In this case, the N-terminus conjugate 6 displayed higher antigen presentation and
consequent T cell activation than its C-terminal counterpart. It is possible that hydrolysis takes place for
the ester conjugates 5 and 7 prior to uptake in the DCs, leading to diminished uptake of the peptide moiety
(as compared to their amide counterparts) and resulting in lower antigen presentation. Importantly, this
readout system is not influenced by the co-stimulatory signals provided by mature DCs, and only reports
whether uptake and processing occur in DCs.
To summarize, in vitro evaluation of the conjugates revealed that the ester conjugates display higher
potency in inducing DC maturation, while the amide conjugates are presented more efficiently. Therefore,
1
0
ACS Paragon Plus Environment

Page 11 of 51
Journal of Medicinal Chemistry
the combined action of co-stimulation, induced by the triggering of the TLR4 and antigen presentation to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CD8+ T cells was evaluated in an in vivo immunization study.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The TLR4-ligands and the cognate conjugates induce IL-12 production and T cell activation in
vitro. (A) The D1 dendritic cell line was stimulated for 24 hours with the synthetic compounds 1-4 and
the induction of DC maturation was analyzed by measuring IL-12p40 production. MPLA was included
as a control for TLR4 stimulation (B) The activity of the conjugates 5-8 together with their reference
ligands 3 and 4 was analyzed by measuring IL-12p40 production. Unconjugated peptide was included as
negative control. (C) Antigen uptake and presentation was measured by incubation of compound-pulsed
D1 with the SIINFEKL-specific hybridoma T cell line B3Z. B3Z activation was determined by
colorimetric reaction of the CPRG reporter enzyme and measurement of absorbance.
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 51
2
.3. In vivo activity. Having established that the conjugates maintained the capacity to activate DCs
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and to induce antigen presentation, the conjugated vaccines were compared for their ability to induce de
novo T cell responses in vivo. Mice were injected intradermally with 5 nmol each of conjugates 5-8, or a
mixture of peptide 23 and TLR4-ligand 1, and the presence of SIINFEKL-specific T cell responses was
monitored in blood via SIINFEKL-Kb tetramer staining. Analysis of blood after the first vaccine injection
demonstrated the successful induction of SIINFEKL-specific T cell responses in all groups vaccinated
with the CRX-527 conjugates. No significant differences could be distinguished between the groups
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Supporting information Figure 1A). Two weeks after the first injection, mice were boosted with the same
formulations and the SIINFEKL-specific responses were measured in blood 7 days later. As shown in
+
Figure 4A and B, the strongest induction of SIINFEKL-specific CD8 T cells was detected in the group
that received the N-terminal ester conjugate 5. Overall, the two N-terminal conjugates 5 and 6 displayed
higher T cell induction than their C-terminal counterpart conjugates 7 and 8. T cell responses were
detectable also in mice vaccinated with a mixture of CRX-527 and peptide, however, these responses
displayed a higher spread than all conjugate groups. One day later, the spleens and lymph nodes draining
the vaccination site were harvested to analyze the presence and the phenotype of the SIINFEKL-specific
responses in these organs. Analysis of T cell responses in the spleen displayed a similar trend to that
observed in blood (Supporting information Figure 1B). However, in the inguinal lymph nodes (Figure
4
C) a higher percentage of SIINFEKL-specific T cell responses was detected for the N-terminal
conjugates 5 and 6. In this organ, mice vaccinated with the C-terminus conjugates 7 and 8 or the mixture
display low T cell responses. Next, we investigated whether the phenotype of the SIINFEKL-specific T
cells induced by the N-terminal conjugates 5 and 6 was different compared to the group that was
vaccinated with the mixture of TLR4-ligand 1 and peptide 23. The induction of differentiation into
+
low
memory CD127 /KLRG1 responses is a marker for T cell quality, which is associated with improved
46
functions and tumor clearance. This phenotype was pronounced in the groups vaccinated with the
conjugates 5 and 6 and was clearly less present in the group that was vaccinated with the mixture (Figure
1
2
ACS Paragon Plus Environment

Page 13 of 51
Journal of Medicinal Chemistry
4
D). Within the T cell memory population, two further subsets can be distinguished based on the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
expression of CD62L, a surface protein that, when present, determines homing at lymphoid tissues, rather
than circulation in the blood vessels and tissues. High expression of CD62L defines central memory T
cells, while lower expression of this surface
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. The conjugates induce SIINFEKL-specific T cell responses with an effector memory phenotype
in vivo. (A) Representative plots of SIINFEKL-Kb tetramer positive T cells in blood of vaccinated mice.
(B) Percentage of SIINFEKL-specific T cell responses measured by tetramer staining in blood at day 21
after booster vaccination. Every dot represents a single animal. (C) At day 22, inguinal lymph nodes were
harvested and the presence of SIINFEKL-specific T cells was measured by tetramer staining in all groups.
Statistical significance was determined by Kruskal-Wallis test followed by multiple comparison rank test
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 51
and Dunn’s correction, * p< 0.05. (D and E) The phenotype of SIINFEKL-specific T cells was
characterized in the higher-responding groups in spleen and lymph nodes by analyzing the expression of
the surface markers KLRG1, CD127 (D) and CD62L (E) by flow cytometry. Statistical significance was
determined by two-way ANOVA followed by multiple comparison and Tukey correction, ** p< 0.01 ***
p< 0.001, **** p< 0.0001.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
protein determines effector memory T cells. This last subset recirculates in tissues and can exert
immediate effector functions upon antigen reencounter. We observed significantly higher differentiation
into effector memory T cells when mice were immunized with the conjugated vaccines 5 and 6 rather than
the mixture (Figure 4E). It has been shown that the promotion of differentiation into effector memory T
cells rather than short-lived effector cells is dependent on optimal priming conditions, such as the presence
of helper T cells46–48 and proper co-stimulatory signals49,50. These data indicate that conjugation of the
TLR4-adjuvant and peptide represents an effective strategy to achieve an increased effector
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
memory T cell phenotype, which is an important hallmark for effective vaccination.51–53
Finally, we investigated the functionality of the induced T cell responses upon vaccination with
conjugate 6 in an in vivo cytotoxicity assay. Mice were immunized with the N-terminus conjugate 6, and
the kinetics of the T cell response was followed in blood by SIINFEKL-Kb tetramer staining (Figure 5A).
After 14 days a boost was administered, and one week later animals were intravenously injected with cells
loaded with either SIINFEKL peptide or an irrelevant peptide, to measure the ability of the induced T
cells to specifically kill SIINFEKL-loaded cells. Prior to injection, the two groups of target cells were
differentially labelled with the fluorescent dye CFSE, to be able to distinguish the two populations during
later analysis by flow cytometry. After 18 hours, the spleens were harvested and the killing of the two
peptide-loaded populations was determined in naïve and vaccinated mice via flow cytometric analysis.
(Figure 5A and B). As expected, naïve mice displayed similar relative frequencies of the two CFSE-
labelled populations. On the contrary, specific killing of the SIINFEKL-loaded target cells was observed
in the vaccinated mice. Notably, four out of five mice that were vaccinated with conjugate 6 displayed >
+
9
0% killing. The killing degree reflected the levels of specific CD8 T cells present, as detected in the
inguinal lymph nodes by tetramer staining (Figure 5C).
1
4
ACS Paragon Plus Environment

Page 15 of 51
Journal of Medicinal Chemistry
To conclude, in vivo evaluation of the CRX-527-peptide conjugates shows that conjugates 5-8 are
1
2
effective in initiating antigen-specific T cell responses. In particular, the N-terminus conjugates could
raise higher responses than their C-terminal counterparts. Phenotypic and functional analysis of these
responses revealed that CRX-527 conjugate 6 was capable of raising an adequate protective immune
response, effectively killing cells presenting the antigen against which the vaccination was directed and
underscoring the potential of these new conjugates for anti-cancer immunotherapy.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Immunization with CRX-527 conjugate 6 results in efficient specific killing of peptide-loaded
target cells. Naïve C57BL/6 mice (n=5) were injected intradermally with 5 nmol of CRX-527 conjugate.
(A) After booster vaccination, mice were injected with differentially CFSE-labelled target cells. 18 hours
after injection, spleens were analyzed for the presence of the two CFSE-labelled target populations. (B)
Calculated specific-killing of SIINFEKL-loaded cells. (C) The amount of SIINFEKL-specific T cells was
determined in the lymph nodes by tetramer staining.
3. CONCLUSIONS
Adjuvant-antigen conjugates are promising agents for cancer immunotherapy. Well-defined molecular
adjuvants are essential to stimulate relevant immune subsets and generate the most appropriate type of
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 51
immunity against distinct tumor types. MPLA is one of the most potent innate immune-stimulating agents,
which is currently used as an adjuvant in vaccines, but the application of this TLR4-ligand in adjuvant-
antigen constructs is hampered by its challenging synthesis. CRX-527 is a potent MPLA analogue and
we have here disclosed an expeditious synthesis of conjugation ready derivatives of this immune-
stimulating agent and demonstrated the preparation of TLR4-ligand-peptide antigen conjugates for the
first time. The assembly of the conjugation-ready ligand critically depended on the protecting group
strategy and the use of a silylidene ketal in the glucosaminyl serine proved crucial for the efficient
introduction of the lipid tails. The developed route of synthesis is high-yielding and could be executed on
a multigram scale to allow the generation of several peptide conjugates. Different linker systems and
connection modes were probed to conjugate the TLR4-ligand to a synthetic long peptide antigen. In vitro
evaluation of the conjugates showed that the attachment of a lipophilic linker at the C6 of CRX-527
abrogates the activity of the ligand. The use of a hydrophilic glycol-based linker provided conjugates
which could induce strong DC maturation, and allowed effective antigen processing and presentation. In
vivo evaluation of the conjugates demonstrated the efficacy of the vaccine modalities in priming de novo
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
CD8 T cell responses. Conjugation of the TLR4-ligand at the N-terminus of the peptide stimulated the
best induction of T cell responses, promoting differentiation into effector memory T cell responses.
Finally, it was shown that the CRX-527-peptide, in which the TLR4-ligand was conjugated to the N-
terminus of the SLP through an amide linked spacer, was a potent inducer of antigen-specific effector
+
CD8 T cell responses in vivo. Overall, we have developed a platform to potentiate synthetic peptide
vaccines with a potent and well-defined TLR4-ligand, a powerful addition to the toolbox available to
generate self-adjuvanting vaccines. CRX-527 conjugates hold great promise for the development of anti-
cancer SLP-vaccines and the availability of the conjugation-ready ligand and chemistry to fuse the ligand
to peptide antigens will enable the generation of conjugates bearing other peptide epitopes, such as defined
1
6
ACS Paragon Plus Environment

Page 17 of 51
Journal of Medicinal Chemistry
oncoviral antigens or cancer neoantigens. The generated conjugation-ready CRX-527 may also find
application in the generation of well-defined anti-bacterial, viral or fungal vaccines.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4. EXPERIMENTAL SECTION
4
.1. Materials and Methods All reagents were of commercial grade and used as received unless stated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
otherwise. Reaction solvents were of analytical grade and when used under anhydrous conditions stored
over flame-dried 3Å molecular sieves. All moisture and oxygen sensitive reactions were performed under
an argon atmosphere. Column chromatography was performed on silica gel (Screening Devices BV, 40-
6
3 µm, 60 Å). For TLC analysis, pre-coated silica gel aluminum sheets (Merck, silica gel 60, F254) were
used with detection by UV-absorption (254/366 nm) where applicable. Compounds were visualized on
TLC by UV absorption (245 nm), or by staining with one of the following TLC stain solutions:
(
NH ) Mo O ·H O (25 g/L), (NH ) Ce(SO ) ·2H O (10 g/L) and 10% H SO in H O; bromocresol (0.4
4 6 7 24 2 4 4 4 4 2 2 4 2
g/L) in EtOH; KMnO (7.5 g/L), K CO (50 g/L) in H O. Staining was followed by charring at ~150°C.
4
2
3
2
1
13
31
H, C and P NMR spectra were recorded on a Bruker AV-300 (300/75 MHz), AV-400 (400/100 MHz)
spectrometer, a Bruker AV-500 Ultrashield (500/126 MHz) spectrometer, a Bruker AV-600 (600/151
MHz) or a Bruker AV-850 (850/214 MHz) and all individual signals were assigned using 2D-NMR
spectroscopy. Chemical shifts are given in ppm (δ) relative to TMS (0 ppm) in CDCl or via the solvent
3
residual peak. Coupling constants (J) are given in Hz. LC-MS analysis were done on an Agilent
Technologies 1260 Infinity system with a C18 Gemini 3 µm, C18, 110 Å, 50 x 4.6 mm column or a Vydac
2
19TP 5 µm Diphenyl, 150 x 4.6 mm column with a flow of 1, 0.8 or 0.7 ml/min. Absorbance was
measured at 214 nm and 256 nm and an Agilent Technologies 6120 Quadrupole mass spectrometer was
used as detector. Peptides, TLR2-ligand and conjugate were purified with a Gilson GX-281 preparative
HPLC with a Gemini-NX 5u, C18, 110 Å, 250 x 10.0 mm column or a Vydac 219TP 5 µm Diphenyl, 250
x 10 mm column. Peptide fragments were synthesized with automated solid phase peptide synthesis on
an Applied Biosystems 433A Peptide Synthesizer. Optical rotations were measured on an Anton Paar
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 51
Modular Circular Polarimeter MCP 100/150. High resolution mass spectra were recorded on a Synapt
G2-Si or a Q Exactive HF Orbitrap equipped with an electron spray ion source positive mode. Mass
analysis of the TLR4-ligands and TLR4-ligand conjugates was performed on an Ultraflextreme MALDI-
TOF or a 15T MALDI-FT-ICR MS system. Infrared spectra were recorded on a Perkin Elmer Spectrum
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
FT-IR. Unprotected lipid A derivatives were dissolved in a mixture of CDCl /MeOD 5/1 v/v for NMR
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
analysis. DC activation and B3Z assay results were analysed with GraphPad Prism version 7.00 for
Windows, GraphPad Software. Purity of all compounds is > 95% as determined by NMR or LC-MS
analysis. FA = fatty acid.
4
.1.1. Automated solid phase synthesis general experimental information. The automated solid-
phase peptide synthesis was performed on a 250 μmol scale on a Protein Technologies Tribute-UV IR
Peptide Synthesizer applying Fmoc based protocol starting from Tentagel S RAM resin (loading 0.22
mmol/g). The synthesis was continued with Fmoc-amino acids specific for each peptide. The consecutive
steps performed in each cycle for HCTU chemistry on 250 μmol scale: 1) Deprotection of the Fmoc-group
with 20% piperidine in DMF for 10 min; 2) DMF wash; 3) Coupling of the appropriate amino acid using
a four-fold excess. Generally, the Fmoc amino acid (1.0 mmol) was dissolved in 0.2 M HCTU in DMF
(5 mL), the resulting solution was transferred to the reaction vessel followed by 2 mL of 1.0 M DIPEA in
DMF to initiate the coupling. The reaction vessel was then shaken for 30 min at 50°C; 4) DMF wash; 5)
capping with 10% Ac O in 0.1 M DIPEA in DMF; 6) DMF wash; 7) DCM wash. Aliquots of resin of the
2
obtained sequences were checked on an analytical Agilent Technologies 1260 Infinity system with a
Gemini 3 µm, C18, 110 Å, 50 x 4.6 mm column or a Vydac 219TP 5 µm Diphenyl, 150 x 4.6 mm column
with a 1 ml/min flow. The Fmoc amino acids applied in the synthesis were: Fmoc-Ala-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-
Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(MMT)-OH, Fmoc-Phe-OH, Fmoc-Ser(OtBu)-
OH Fmoc-Val-OH.
1
8
ACS Paragon Plus Environment

Page 19 of 51
Journal of Medicinal Chemistry
4
.1.2. General procedure for cleavage from the resin, deprotection and purification. 30 μmol resin
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was washed with DMF, DCM and dried after the last synthesis step followed by a treatment for 180
minutes with 0.6 mL cleavage cocktail of 95% TFA, 2.5% TIS and 2.5% H O. The suspension was
2
filtered, the resin was washed with 0.6 mL of the cleavage cocktail, and the combined TFA solutions were
added dropwise to cold Et O and stored at -20°C overnight. The obtained suspension of the product in
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Et O was centrifuged, Et O was removed and the precipitant was dissolved in CH CN/H O/tBuOH (1/1/1
2
2
3
2
v/v/v) or DMSO/CH CN/H O/tBuOH (3/1/1/1 v/v/v/v). Purification was performed on a Gilson GX-281
3
2
preparative RP-HPLC with a Gemini-NX 5u, C18, 110 Å, 250 x 10.0 mm column or a Vydac 219TP 5
µm Diphenyl, 250 x 10 mm column.
4.1.3. General purification method for CRX-527-O-conjugates. A C18 column was washed
subsequently with CH CN, MeOH, DCM/MeOH (1/1 v/v), MeOH, CH CN, CH CN/H O, H O. The
3
3
3
2
2
reaction mixture was added on the column and the Eppendorf was rinsed with a mixture of
CH CN/tBuOH/MilliQ H O (1/1/1 v/v/v, 0.50 mL), which was also added on the C18 column. The
3
2
column was subsequently flushed with 6 mL of the follow solvent systems: H O, CH CN/H O (1/1 v/v),
2
3
2
CH CN, DMSO, CH CN/tBuOH/MilliQ H O (1/1/1 v/v/v) and collected in Eppendorfs containing 1.0
3
3
2
mL of each solvent system. The column was then flush with MeOH (6.0 mL), followed by DCM/MeOH
1/1 v/v, 6.0 mL), which were collected in separate flasks, concentrated in vacuo at 35°C and lyophilized
by dissolving in CH CN/tBuOH/MilliQ H O (1/1/1 v/v/v), yielding the conjugate as a white solid. *For
(
3
2
the amide conjugates, H O + 0.1% TFA was used instead of H O.
2
2
4.1.4. MALDI-TOF measurements. MALDI-TOF measurements: 1 μL of a DMSO solution of the
compound was spotted on a 384-MTP target plate (Bruker Daltonics, Bremen, Germany) and air-dried.
Subsequently 1 μL of 2,5-dihydroxybenzoic acid (2,5-DHB; Bruker Daltonics) matrix (20 mg/mL in
ACN/water; 50:50 (v/v)) was applied on the plate and the spots were left to dry prior MALDI-TOF
analysis. An Ultraflextreme MALDI-TOF (Bruker Daltonics), equipped with Smartbeam-II laser was
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 51
used to measure the samples in reflectron positive ion mode. The MALDI-TOF was calibrated using a
peptide calibration standard prior to measurement.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4.2. Synthesis and Characterizations. The synthesis and characterizations for compounds 14, 17, 18,
and 18, together with the characterizations of the intermediates is described in the supplementary
information.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
.2.1. Acetyl 3,4,6-tri-O-acetyl-2-N-trichloroethoxycarbonyl-α/β-D-glucopyranoside (9). NaHCO (144
3
g, 1.65 mol, 3.0 eq.) and 2,2,2-trichloroethoxycarbonyl chloride (93 mL, 0.68 mol, 1.2 eq.) were added to
a solution of D-glucosamine·HCl (0.12 kg, 0.55 mol, 1.0 eq.) in H O (1.1 L). The reaction was stirred
2
vigorously at room temperature overnight, after which the resulting white suspension was filtered and the
residue was washed with cold H O. The white solid was co-evaporated with toluene (3x) before dissolving
2
in pyridine (0.60 L). The reaction mixture was cooled to 0°C and Ac O (0.30 L, 3.2 mol, 5.8 eq.) was
2
added. The reaction mixture was allowed to warm-up to room temperature and stirred overnight. The
reaction mixture was cooled to 0°C, quenched by the addition of H O and subsequently diluted with
2
EtOAc. The organic layer was washed several times with 1 M HCl, dried over MgSO , filtered and
4
concentrated in vacuo. TLC analysis showed no full conversion, therefore the oil was dissolved in pyridine
(
0.60 L) and cooled to 0°C. Ac O (0.45 L, 4.8 mol, 8.7 eq.) was added and after 30 minutes the mixture
2
was allowed to warm-up to room temperature. After 2.5 hours TLC analysis showed full conversion. The
reaction was quenched by the addition of MeOH and concentrated in vacuo. Co-evaporation with toluene
(
3x) gave compound 9 (189 g, 362 mmol, 66%), which was used without further purification. R : 0.20
f
1
(7/3 pentane/EtOAc); H NMR (CDCl , 400 MHz, HH-COSY, HSQC): δ 6.16 (d, 1H, J = 3.8 Hz, H-1),
3
5
.43 (d, 1H, J = 9.5 Hz, NH), 5.27 – 5.17 (m, 1H, H-3), 5.13 (t, 1H, J = 9.9 Hz, H-4), 4.76 (d, 1H, J =
2.1 Hz, CHH Troc), 4.56 (d, 1H, J = 12.1 Hz, CHH Troc), 4.23 – 4.11 (m, 2H, H-2, CHH-6), 4.02 – 3.94
1
1
3
(m, 2H, H-5, CHH-6), 2.13 (s, 3H, CH Ac), 2.02 (s, 3H, CH Ac), 1.98 – 1.96 (m, 6H, 2x CH Ac); C-
3
3
3
APT NMR (CDCl , 101 MHz, HSQC): δ 171.2, 170.7, 169.2, 168.7 (C=O Ac), 154.1 (C=O Troc), 95.3
3
2
0
ACS Paragon Plus Environment

Page 21 of 51
Journal of Medicinal Chemistry
(
C Troc), 90.4 (C-1), 74.6 (CH Troc), 70.3 (C-3), 69.6 (C-5), 67.6 (C-4), 61.5 (CH -6), 53.1 (C-2), 20.9,
q
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
2
0.7, 20.6, 20.5 (CH Ac); FT-IR (neat, cm ): 3329, 2958, 2258, 2126, 1742, 1536, 1432, 1368, 1212,
3
1172, 1141, 1123, 1095, 1080, 1031, 1012, 952, 910, 820, 728, 681, 648, 599, 568, 526, 475; HRMS:
+
[M+Na] calcd. for C H Cl NO Na: 544.0151, found 544.0159.
1
7
22
3
11
4
.2.2. Benzyl N-trichloroethoxycarbonyl-L-serinate (10). L-Serine (49.6 g, 472 mmol, 1.0 eq.) was
dissolved in a mixture of CCl /benzyl alcohol (1/1 v/v, 0.46 L). p-Toluenesulfonic acid (96.6 g, 508 mmol,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
1
.1 eq.) was added and the white suspension was heated to 100°C using a Dean-Stark apparatus. After
stirring overnight, a clear solution was obtained, which was cooled down to room temperature before
concentrating in vacuo. The residue was dissolved in DCM and washed with sat. aq. NaHCO (3x). The
3
organic layer was extracted with 1 M HCl (3x) and the combined aqueous layers were concentrated in
vacuo. Co-evaporation with toluene yielded the intermediate as a white solid (46.6 g, 201 mmol), which
was dissolved in DCM (1.0 L) under an argon atmosphere. Succinimidyl-2,2,2-trichloroethyl carbonate54
(61.5 g, 212 mmol, 1.05 eq.) was added to the reaction mixture, followed by the addition of Et N (42 mL,
3
0
.30 mol, 1.5 eq.) under a flow of argon. After 1 hour, TLC analysis showed complete conversion of the
starting material and the reaction mixture was washed with 1 M HCl (1x) and H O (1x). The aqueous
2
layers were extracted with DCM (1x) and the combined organic layers were dried over MgSO , filtered
4
and concentrated in vacuo. Purification by column chromatography (20100% EtOAc in pentane)
+
yielded the title compound (69.4 g, 187 mmol, 40% over two steps). HRMS: [M+H] calcd. for
5
4
C H O NCl : 370.00103, found 370.00105. Analytic data were in agreement with reported data.
13 15
5
3
4.2.3.
N-trichloroethoxycarbonyl-O-[3,4,6-tri-O-acetyl-2-N-trichloroethoxycarbonyl-β-D-
glucopyranosyl]-L-serine (11). Compounds 9 (88.9 g, 170 mol, 1.0 eq.) and 10 (69.3 g, 187 mmol, 1.1
eq.) were co-evaporated with toluene (2x) under an argon atmosphere and dissolved in DCM (0.28 L).
The mixture was cooled to 0°C, followed by the slow addition of BF ·OEt (42 mL, 0.34 mol, 2.0 eq.).
3
2
The mixture was allowed to warm-up to room temperature and stirred for an additional 48 hours. The
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 51
mixture was quenched with Et N and washed with sat. aq. NaHCO (1x). The aqueous layer was extracted
3
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with DCM (3x) and the combined organic layers were dried over MgSO , filtered and concentrated in
4
vacuo. Purification by column chromatography (20100% EtOAc in pentane) gave an oil (117 g), which
was a mixture of unreacted donor 9 and benzyl N-trichloroethoxycarbonyl-O-[3,4,6-tri-O-acetyl-2-N-
trichloroethoxycarbonyl-β-D-glucopyranosyl]-L-serinate. After co-evaporating with toluene (3x) under
an argon atmosphere, the oil was dissolved in THF (1.2 L), followed by the addition of Pd/C (10%, 11.7
g). The black suspension was purged with argon for 15 minutes, followed by purging with H2(g) and after
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5 minutes a H -filled balloon was applied. After stirring at room temperature overnight, the mixture
2(g)
was filtered over a Whatmann-filter and concentrated in vacuo. Purification by column chromatography
(10100% acetone in pentane) yielded the title compound (79.7 g, 107 mmol, 63% yield over two steps)
25
and unreacted donor 11 (18.5 g, 35.4 mmol). R : 0.13 (9/1 DCM/MeOH); [훼] +11.3° (c = 0.23, CHCl );
f
D
3
1H NMR (MeOD, 400 MHz, HH-COSY, HSQC): δ 5.31 – 5.21 (m, 1H, H-3), 4.98 (t, 1H, J = 9.7 Hz,
H-4), 4.86 (d, 1H, J = 5.1 Hz, CHH Troc), 4.83 – 4.74 (m, 2H, 2x CHH Troc), 4.74 – 4.68 (m, 2H, H-1,
CHH Troc), 4.46 – 4.38 (m, 1H, CH serine), 4.31 (dd, 1H, J = 12.4, 4.6 Hz, CHH-6), 4.25 – 4.07 (m, 2H,
CHH-6, CHH serine), 3.95 (dd, 1H, J = 10.5, 3.9 Hz, CHH serine), 3.81 (ddd, 1H, J = 10.0, 4.3, 2.3 Hz,
H-5), 3.58 (dd, 1H, J = 10.4, 8.6 Hz, H-2), 2.07 (s, 3H, CH Ac), 2.00 (s, 3H, CH Ac), 1.97 (s, 3H, CH
3
3
3
13
Ac); C-APT NMR (MeOD, 101 MHz, HSQC): δ 172.5, 172.4, 171.7 (C=O Ac), 171.3 (C=O serine),
56.7, 156.5 (C=O Troc), 101.9 (C-1), 97.1 (C Troc), 75.7, 75.4 (CH Troc), 73.7 (C-3), 73.0 (C-5), 70.2
1
q
2
-1
(C-4), 70.1 (CH serine), 63.1 (CH -6), 57.1 (C-2), 55.7 (CH serine), 20.6 (CH Ac); FT-IR (neat, cm ):
2
2
3
+
3
340, 2958, 1744, 1532, 1369, 1232, 1170, 1102, 1048, 819, 769, 734, 569; HRMS: [M+Na] calcd. for
C H Cl N O Na: 762.9407, found 762.9416.
2
1
26
6
2
14
4
.2.4. Benzyl N-trichloroethoxycarbonyl-O-[2-N-trichloroethoxycarbonyl-β-D-glucopyranosyl]-L-
serinate (12). Compound 11 (79.6 g, 107 mmol, 1.0 eq.) was dissolved in MeOH (1.1 L) and NH OH
4
(13.4 M, 73.5 mL, 985 mmol, 9.2 eq.) was added. After two days stirring at room temperature, TLC
2
2
ACS Paragon Plus Environment

Page 23 of 51
Journal of Medicinal Chemistry
analysis showed complete conversion of the starting material. The reaction mixture was concentrated in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
vacuo and co-evaporated with toluene. The obtained oil was dissolved in a DCM/sat. aq. NaHCO mixture
3
(1/1 v/v, 2.6 L), after which tetrabutylammonium bromide (34.9 g, 108 mmol, 1.0 eq.) and benzyl bromide
(64 mL, 0.54 mol, 5.0 eq.) were added. The reaction mixture was stirred overnight. The layers were
separated and the aqueous layer was extracted with CHCl (2x) and DCM (1x). The combined organic
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
layers were dried over MgSO , filtered and concentrated in vacuo. Purification by column
4
chromatography (20100% EtOAc in pentane, then 20% MeOH in EtOAc) afforded compound 12 (44.2
25
1
g, 65.5 mmol, 79% yield over two steps). R : 0.49 (9/1 DCM/MeOH); [훼] -15.2° (c = 0.48, MeOH); H
f
D
NMR (MeOD, 400 MHz, HH-COSY, HSQC): δ 7.41 – 7.28 (m, 5H, Ar), 5.25 – 5.12 (m, 2H, CH Bn),
2
4
.85 (d, 1H, J = 12.2 Hz, CHH Troc), 4.79 – 4.69 (m, 3H, CHH Troc, CH Troc), 4.49 (t, 1H, J = 4.4 Hz,
2
CH serine), 4.45 (d, 1H, J = 8.2 Hz, H-1), 4.24 (dd, 1H, J = 10.2, 5.2 Hz, CHH serine), 3.93 – 3.83 (m,
H, CHH serine, CHH-6), 3.67 (dd, 1H, J = 11.8, 5.5 Hz, CHH-6), 3.45 (dd, 1H, J = 10.2, 8.2 Hz, H-3),
2
1
3
3.41 – 3.33 (m, 1H, H-2), 3.31 – 3.21 (m, 2H, H-4, H-5); C-APT NMR (MeOD, 101 MHz, HSQC): δ
1
71.2 (C=O serine), 157.1, 156.5 (C=O Troc), 137.0 (C Ar), 129.6, 129.3, 129.1 (Ar), 102.8 (C-1), 96.8
q
(
C Troc), 78.0 (C-5), 75.6 (CH Troc), 75.5 (C-3), 72.0 (C-4), 69.6 (CH serine), 68.2 (CH Bn), 62.7
q 2 2 2
-1
(CH -6), 58.9 (C-2), 56.2 (CH serine); FT-IR (neat, cm ): 3423, 2955, 2487, 1729, 1431, 1332, 1293,
2
+
1
173, 1060, 820, 731, 569; HRMS: [M+Na] calcd. for C H Cl N O Na: 726.9560, found 726.9576.
2
2
26
6
2
11
4
.2.5.
Benzyl
N-trichloroethoxycarbonyl-O-[4,6-O-di-tert-butylsilylidene-2-N-
trichloroethoxycarbonyl-β-D-glucopyranosyl]-L-serinate (13). A solution of compound 12 (2.01 g, 2.84
mmol, 1.0 eq.) in DMF (14 mL) was cooled to -40°C. Di-tert-butylsilanediyl-bistriflate (0.92 mL, 3.1
mmol, 1.1 eq.) was added drop-wise. After one hour, the reaction was allowed to warm-up to room
temperature and stirred overnight. The reaction mixture was quenched by the addition of pyridine (1.6
mL, 19.9 mmol, 7.0 eq.). The mixture was diluted with Et O and the organic layer was washed with H O
2
2
(
1x) and sat. aq. NaHCO (3x), dried over Na SO , filtered and concentrated in vacuo. After purification
3 2 4
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 51
by column chromatography (23% acetone in DCM), the title compound (2.07 g, 2.44 mmol, 86%) was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
5
1
obtained as a white foam. R : 0.60 (1/1 pentane/Et O); [훼] -24.0° (c = 0.86, CHCl ); H NMR (CDCl ,
f
2
D
3
3
400 MHz, HH-COSY, HSQC): δ 7.38 – 7.28 (m, 5H, Ar), 6.30 (s, 1H, NH serine), 5.80 (d, 1H, J = 7.7
Hz, NH GlcN), 5.23 – 5.12 (m, 2H, CH Bn), 4.81 – 4.65 (m, 5H, H-1, 2x CH Troc), 4.53 (dt, 1H, J =
2
2
7
.8, 3.4 Hz, CH serine), 4.25 (dd, 1H, J = 10.3, 3.3 Hz, CHH serine), 4.14 (dd, 1H, J = 10.1, 5.0 Hz, CHH-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6), 3.89 – 3.80 (m, 2H, CHH-6, CHH serine), 3.80 – 3.72 (m, 1H, H-3), 3.67 (t, 1H, J = 8.9 Hz, H-4), 3.42
–
3
3.34 (m, 1H, H-5), 3.34 – 3.25 (m, 1H, H-2), 3.22 (br, 1H, OH), 1.04 (s, 9H, 3x CH tBu), 0.97 (s, 9H,
3
1
3
x CH tBu); C-APT NMR (CDCl , 101 MHz, HSQC): δ 169.3 (C=O serine), 154.6, 154.5 (C=O Troc),
3
3
135.1 (C Ar), 128.6, 128.5, 128.2 (Ar), 100.8 (C-1), 95.5, 95.4 (C Troc), 77.4 (C-4), 74.6 (CH Troc),
q
q
2
7
2
1
3.5 (C-3), 70.3 (C-5), 68.9 (CH serine), 67.6 (CH Bn), 66.0 (CH -6), 57.4 (H-2), 54.5 (CH serine),
2 2 2
-1
7.4, 27.0 (CH tBu), 22.6, 19.9 (C tBu); FT-IR (neat, cm ): 3340, 2935, 2886, 2860, 1730, 1523, 1473,
3
q
387, 1365, 1336, 1243, 1201, 1160, 1076, 1009, 943, 909, 826, 765, 730, 697, 653, 618, 569, 476;
+
HRMS: [M+Na] calcd. for C H Cl N O SiNa: 867.0581, found 867.0599.
3
0
42
6
2
11
4
.2.6. Benzyl N-[(R)-3-(decanoyloxy)tetradecanoyl]-O-[4,6-O-di-tert-butylsilylidene-2-N-[(R)-3-
(decanoyloxy)tetradecanoyl]-3-O-[(R)-3-(decanoyloxy)tetradecanoyl]-β-D-glucopyranosyl]-L-serinate
(15). To a solution of compound 13 (2.55 g, 3.00 mmol, 1.0 eq.) in THF (30 mL) was added activated
zinc (4.0 g, 61 mmol, 20 eq.) and AcOH (0.69 mL, 12 mmol, 4.0 eq. ) under an argon atmosphere. The
suspension was stirred for 25 minutes and the mixture was subsequently sonicated for 5 min. The mixture
was stirred again for 25 min, followed by sonicating for 5 minutes. TLC and LC-MS analysis showed
complete conversion of the starting material. The suspension was filtered over a Whatmann filter and the
residue was washed with DCM and EtOAc. The combined filtrates were concentrated in vacuo, co-
evaporated with toluene (3x) and the obtained solid was dissolved in EtOAc. The solution was
subsequently washed with 0.1 M HCl (1x), sat. aq. NaHCO (1x) and brine (1x). The organic layer was
3
dried over Na SO , filtered and concentrated in vacuo. A mixture of the obtained yellow oil and acid 14
2
4
2
4
ACS Paragon Plus Environment

Page 25 of 51
Journal of Medicinal Chemistry
(
5.39 g, 13.5 mmol, 4.5 eq.) was co-evaporated with toluene (1x) and dissolved in DCM (30 mL) under
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
an argon atmosphere. EDC·MeI (4.01 g, 13.5 mmol, 4.5 eq.) and DMAP (11 mg, 90 µmol, 0.03 eq.) were
added and the reaction mixture was stirred 4 hours, after which the mixture was concentrated in vacuo.
Several purifications by column chromatography (220% EtOAc in DCM + 0.1% Et N and 010%
3
acetone in DCM + 0.1% Et N) gave compound 15 (3.07 g, 1.87 mmol, 62% over two steps) as a white
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
1
foam. R : 0.58 (95/5 DCM/acetone); [훼] -15.4° (c = 0.50, CHCl ); H NMR (CDCl , 500 MHz, HH-
f
D
3
3
COSY, HSQC): δ 7.37 – 7.27 (m, 5H, Ar), 7.01 (d, 1H, J = 7.8 Hz, NH serine), 6.27 (d, 1H, J = 8.3 Hz,
NH GlcN), 5.22 – 5.05 (m, 5H, 3x CH FA, CH Bn), 5.06 – 4.98 (m, 1H, H-3), 4.72 – 4.66 (m, 2H, H-1,
2
CH serine), 4.21 (dd, 1H, J = 10.7, 3.0 Hz, CHH serine), 4.14 (dd, 1H, J = 10.2, 5.0 Hz, CHH serine),
3
–
.88 – 3.77 (m, 3H, H-4, CHH-6, CHH serine), 3.73 – 3.65 (m, 1H, H-2), 3.44 – 3.37 (m, 1H, H-5), 2.67
2.20 (m, 12H, 6x CH FA), 1.71 – 1.50 (m, 12H, 6x CH FA), 1.40 – 1.17 (m, 90H, 45x CH FA), 1.02
2
2
2
1
3
(s, 9H, 3x CH tBu), 0.94 (s, 9H, 3x CH tBu), 0.87 (t, 18H, J = 6.7 Hz, 6x CH FA); C-APT NMR
3 3 3
(CDCl , 126 MHz, HSQC): δ 173.9, 173.8, 173.3, 170.6, 170.3, 170.2 (C=O FA), 169.5 (C=O serine),
3
1
5
35.5 (C Ar), 128.6, 128.4, 128.1 (Ar), 101.6 (C-1), 75.1 (C-4), 74.4 (C-3), 71.5, 71.5 (CH FA), 70.8 (C-
q
), 70.1 (CH FA), 68.8 (CH serine), 67.3 (CH Bn), 66.3 (CH -6), 54.7 (C-2), 52.8 (CH serine), 42.2,
2
2
2
41.3, 39.2, 34.7, 34.6, 34.6, 34.5, 34.0, 32.1, 32.0, 32.0, 29.9, 29.8, 29.8, 29.8, 29.7, 29.7, 29.7, 29.6, 29.6,
9.6, 29.5, 29.5, 29.5, 29.4, 29.4, 29.3 (CH FA), 27.5, 27.0 (CH tBu), 25.5, 25.4, 25.2, 25.2, 25.1, 22.8
2
2
3
-1
(CH FA), 22.7, 20.0 (C tBu), 14.2 (CH FA); FT-IR (neat, cm ): 3285, 3068, 2956, 2923, 2854, 1734,
2 q 3
1652, 1540, 1450, 1466, 1378, 1364, 1246, 1173, 1075, 1030, 1011, 837, 827, 769, 723, 696, 652, 581,
+
4
63; HRMS: [M+H] calcd. for C H N O Si: 1638,2549, found 1638.2493.
9
6
173
2
16
4
.2.7. Benzyl N-[(R)-3-(decanoyloxy)tetradecanoyl]-O-[4-O-bis(benzyloxy)phosphoryl-2-N-[(R)-3-
decanoyloxy)tetradecanoyl]-3-O-[(R)-3-(decanoyloxy)tetradecanoyl]-β-D-glucopyranosyl]-L-serinate
(
(16a). Compound 15 (1.92 g, 1.17 mmol, 1.0 eq.) was dissolved in THF (12 mL) under an argon
atmosphere and cooled to 0°C. HF·Et N (0.58 mL, 3.6 mmol, 3.0 eq.) was added and the reaction mixture
3
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 51
was stirred for 1.5 h, after which TLC analysis showed complete conversion of the starting material. The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reaction was quenched with sat. aq. NaHCO , diluted with EtOAc and washed with brine (1x). The organic
3
layer was dried over MgSO , filtered and concentrated in vacuo. Purification by column chromatography
4
(04% MeOH in DCM) gave the diol intermediate (1.61 g, 1.08 mmol, 92%). TBDMSCl (290 mg, 1.92
mmol, 1.5 eq.) was added to a solution of the previously obtained diol (1.81 g, 1.21 mmol, 1.0 eq) in
pyridine (8.0 mL). After stirring at room temperature for 3 hours, TLC analysis showed complete
conversion of the starting material. The reaction mixture was diluted with EtOAc, washed with 1 M HCl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2x), sat. aq. NaHCO (2x), dried over MgSO , filtered and concentrated in vacuo. Purification by column
3 4
chromatography (520% EtOAc in toluene) afforded the intermediate (1.71 g, 1.06 mmol, 88%), which
was co-evaporated with toluene (2x) under an argon atmosphere and dissolved in dry DCM (18 mL).
Dibenzyl diisopropylaminephosphoramidite (0.70 mL, 1.9 mmol, 1.8 eq.) and tetrazole (186 mg, 2.65
mmol, 2.5 eq.) were added. After stirring for 35 minutes, the reaction mixture was cooled to 0°C, followed
by the addition of m-CPBA (0.74 g, 3.0 mmol, 2.8 eq.). After 40 minutes, TLC analysis showed complete
conversion into the phosphate. The reaction was diluted with aq. sat. NaHCO and extracted with DCM
3
(
3x). The combined organic layers were dried over Na SO , filtered and concentrated in vacuo.
2 4
Purification by column chromatography (020% EtOAc in toluene) and several size exclusions
DCM/MeOH: 1/1) gave the phosphate intermediate in quantitative yield (2.00 g). TFA (0.81 mL, 11
(
mmol, 10 eq.) was added to a solution of the previously obtained phosphate (2.00 g, 1.06 mmol, 1.0 eq.)
in DCM (21 mL) at 0°C. After 20 minutes, the resulting yellow solution was allowed to warm-up to room
temperature and stirred for an additional 3 hours. TLC analysis showed complete conversion and the
reaction was quenched with aq. sat. NaHCO at 0°C. The reaction mixture was further diluted with H O
3
2
and extracted with DCM (3x). The combined organic layers were dried over Na SO , filtered and
2
4
concentrated in vacuo. After purification by column chromatography (1050% EtOAc in toluene),
compound 16a (1.57 g, 0.893 mmol, 84%) was obtained as a white foam. R : 0.70 (1/1 pentane/EtOAc);
f
2
6
ACS Paragon Plus Environment

Page 27 of 51
Journal of Medicinal Chemistry
2
5
1
[
훼] +2.2° (c = 0.33, CHCl ); H NMR (CDCl , 400 MHz, HH-COSY, HSQC): δ 7.38 – 7.24 (m, 15H,
D
3
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ar), 7.19 – 7.08 (m, 1H, NH serine), 6.57 (d, 1H, J = 7.7 Hz, NH GlcN), 5.36 (t, 1H, J = 9.7 Hz, H-3),
5.23 – 5.07 (m, 5H, 3x CH FA, CH CO Bn), 5.05 – 4.88 (m, 5H, H-1, 2x CH dibenzyl phosphate), 4.76
2
2
2
–
4.67 (m, 1H, CH serine), 4.44 (q, 1H, J = 9.3 Hz, H-4), 4.28 – 4.18 (m, 1H, CHH serine), 3.93 – 3.84
(
m, 1H, CHH serine), 3.82 – 3.71 (m, 3H, CH -6, OH), 3.60 – 3.51 (m, 1H, H-2), 3.37 (d, 1H, J = 9.7 Hz,
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H-5), 2.69 (dd, 1H, J = 14.7, 6.1 Hz, CHH FA), 2.54 (dd, 1H, J = 14.7, 5.8 Hz, CHH FA), 2.43 – 2.23 (m,
8
9
H, 4x CH FA), 2.20 (t, 2H, J = 7.5 Hz, CH FA), 1.74 – 1.48 (m, 12H, 6x CH FA), 1.48 – 1.06 (m,
2 2 2
1
3
0H, 45x CH FA), 0.94 – 0.80 (m, 18H, 6x CH FA); C-APT NMR (CDCl , 101 MHz, HSQC): δ
2
3
3
173.5, 173.4, 173.1, 170.6, 170.1, 169.7 (C=O FA), 169.4 (C=O serine), 135.3, 135.2, 135.2, 135.1, 135.1
C Bn), 128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8 (Ar), 100.4 (C-
(
q
1
), 74.7, 74.7 (C-5), 73.1, 73.0 (C-4), 72.3, 72.2 (C-3), 71.2, 70.8 (CH FA), 69.9, 69.9 (CH dibenzyl
2
phosphate), 69.8 (CH FA), 68.7 (CH serine), 67.0 (CH CO Bn), 60.3 (CH -6), 55.1 (C-2), 52.8 (CH
2
2
2
2
serine), 41.4, 41.0, 39.0, 34.4, 34.3, 34.2, 31.8, 31.8, 29.6, 29.6, 29.6, 29.5, 29.5, 29.5, 29.5, 29.4, 29.4,
9.4, 29.3, 29.3, 29.2, 29.2, 29.2, 29.1, 29.1, 29.1, 25.2, 25.2, 25.0, 24.9, 24.9, 22.6, 22.6 (CH FA), 14.0
2
2
3
1
-1
(
CH FA); P-APT NMR (CDCl , 162 MHz, HMBC): δ -0.05; FT-IR (neat, cm ): 3317, 3066, 2956,
3
3
2923, 2853, 1733, 1654, 1640, 1541, 1499, 1466, 1456, 1379, 1238, 1166, 1128, 1106, 1080, 1034, 1016,
+
9
14, 736, 696, 602, 531, 498; HRMS: [M+H] calcd. for C H N O P: 1758,2130, found 1758.2065.
1
02 170
2
19
4
.2.8.
(decanoyloxy)tetradecanoyl]-3-O-[(R)-3-(decanoyloxy)tetradecanoyl]-β-D-glucopyranosyl]-L-serine
16b). After co-evaporating with toluene (2x), compound 16a (82 mg, 47 µmol, 1.0 eq.) was dissolved in
N-[(R)-3-(decanoyloxy)tetradecanoyl]-O-[6-azide-4-O-phosphoryl-2-N-[(R)-3-
(
THF under an argon atmosphere. PPh (48 mg, 0.18 mmol, 3.9 eq.) was added and the reaction mixture
3
was cooled to -20 °C. DEAD (15 µL, 96 µmol, 2.0 eq.) and DPPA (20.5 µL, 96 µmol, 2.0 eq.) were added
subsequently and the stirring was continued for 1 hour, followed by the addition of DEAD (15 µL, 96
µmol, 2.0 eq.) and DPPA (20.5 µL, 96 µmol, 2.0 eq.). After stirring for 1 hour at -20°C, the reaction
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 51
mixture was slowly warmed-up to room temperature overnight. The mixture was concentrated in vacuo.
Purification by column chromatography (1030% EtOAc in pentane) afforded the title compound (56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mg, 31 µmol, 66%). R : 0.52 (7/3 pentane/EtOAc); H NMR (CDCl , 400 MHz, HH-COSY, HSQC): δ
f
3
7
.38 – 7.24 (m, 15H, Ar), 7.02 (d, 1H, J = 8.1 Hz, NH serine), 6.44 (d, 1H, J = 7.4 Hz, NH GlcN), 5.34
(
dd, 1H, J = 10.5, 8.9 Hz, H-3), 5.21 – 5.12 (m, 5H, 3x CH FA, CH CO Bn), 5.03 (d, 1H, J = 8.2 Hz,
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H-1), 5.00 – 4.94 (m, 2H, 2x CHH Bn), 4.91 (dd, 2H, J = 7.9, 3.2 Hz, 2x CHH Bn), 4.78 – 4.70 (m, 1H,
CH serine), 4.32 – 4.21 (m, 2H, H-4, CHH serine), 3.85 (dd, 1H, J = 11.2, 2.8 Hz, CHH serine), 3.55 –
3
1
.48 (m, 1H, H-5), 3.46 – 3.37 (m, 2H, H-2, CHH-6), 3.28 (dd, 1H, J = 13.4, 6.2 Hz, CHH-6), 2.67 (dd,
H, J = 14.9, 6.2 Hz, CHH FA), 2.55 – 2.44 (m, 2H, 2x CHH FA), 2.41 – 2.19 (m, 9H, CHH FA, 4x CH2
FA), 1.71 – 1.43 (m, 12H, 6x CH FA), 1.37 – 1.14 (m, 90H, 45x CH FA), 0.93 – 0.81 (m, 18H, 6x CH
3
2
2
13
FA); C-APT NMR (CDCl , 101 MHz, HSQC): δ 173.8, 173.7, 171.1, 170.3, 170.0 (C=O FA), 169.5
3
(
C=O serine), 135.5, 135.5, 135.4 (C Bn), 128.9, 128.8, 128.8, 128.7, 128.6, 128.4, 128.3, 128.3, 128.2
q
(Ar), 100.3 (C-1), 74.5, 74.5 (C-5), 74.0, 73.9 (C-4), 72.3, 72.3 (C-3), 71.4, 71.0, 70.3 (CH FA), 70.0,
0.0, 69.9, 69.9 (CH dibenzyl phosphate), 69.2 (CH serine), 67.3 (CH CO Bn), 55.8 (C-2), 52.8 (CH
7
2
2
2
2
serine), 50.7 (CH N ), 41.6, 41.2, 39.8, 34.7, 34.6, 34.6, 34.6, 32.0, 32.0, 29.9, 29.8, 29.8, 29.8, 29.7,
2
3
2
9.7, 29.6, 29.6, 29.5, 29.5, 29.5, 29.4, 29.4, 29.4, 29.3, 25.5, 25.4, 25.3, 25.2, 25.2, 25.1, 22.8, 22.8 (CH2
3
1
-1
FA), 14.2 (CH FA); P-APT NMR (CDCl , 162 MHz, HMBC): δ -1.03; FT-IR (neat, cm ): 3276, 2992,
3
3
2
853, 2361, 2102, 1733, 1654, 1543, 1498, 1457, 1274, 1248, 1171, 1108, 1010, 904, 734, 696, 601, 506;
+
HRMS: [M+H] calcd. for C H N O P: 1783.22059, found 1783.22059.
102 169
5
18
4.2.9.
N-[(R)-3-(decanoyloxy)tetradecanoyl]-O-[4-O-phosphoryl-2-N-[(R)-3-
(
decanoyloxy)tetradecanoyl]-3-O-[(R)-3-(decanoyloxy)tetradecanoyl]-β-D-glucopyranosyl]-L-serine
1). After co-evaporating with toluene (3x) under an argon atmosphere, compound 16a (21.7 mg, 12.3
(
µmol, 1.0 eq.) was dissolved in THF (1.0 mL), followed by the addition of Pd/C (10%, 21 mg). A H -
2
(g)
filled balloon was applied on the obtained black suspension. After stirring at room temperature for 3 hours,
2
8
ACS Paragon Plus Environment

Page 29 of 51
Journal of Medicinal Chemistry
the mixture was filtered over a Whatmann filter. The filter was washed with DCM, followed by the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
addition of Et N (3.4 µL, 24 µmol, 2.0 eq.). After mixing for 5 minutes, the clear solution was
3
concentrated in vacuo and purified by size exclusion (DCM/MeOH: 1/1). Lyophilization gave compound
1
1
5
4
–
(18.2 mg, 12.2 µmol, 99%) as a white solid. H NMR (CDCl , 850 MHz, HH-COSY, HSQC) δ 5.20 –
3
.15 (m, 1H, CH FA), 5.14 – 5.07 (m, 3H, H-3, 2x CH FA), 4.54 – 4.49 (m, 2H, H-1, CH serine), 4.19 –
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.08 (m, 2H, H-4, CHH serine), 3.88 (d, J = 13.1 Hz, 1H, CHH-6), 3.84 – 3.78 (m, 1H, CHH serine), 3.72
3.66 (m, 2H, H-2, CHH-6), 3.28 (d, J = 9.8 Hz, 1H, H-5), 2.63 – 2.45 (m, 4H, 2x CH FA), 2.40 (dd, J
2
=
14.7, 7.3 Hz, 1H, CHH FA), 2.29 (dd, J = 14.7, 5.7 Hz, 1H, CHH FA), 2.28 – 2.20 (m, 6H, 3x CH FA),
2
1
.60 – 1.47 (m, 12H, 6x CH FA), 1.30 – 1.15 (m, 90H, 45x CH FA), 0.85 – 0.81 (m, 18H, 6x CH FA);
2 2 3
1
3
C NMR (CDCl , 214 MHz, HSQC) δ 173.8, 173.8, 173.7, 170.8, 170.6, 170.5 (C=O), 100.7 (C-1), 75.3
3
(
C-5), 73.5 (C-3), 71.1, 70.8 (CH FA), 70.3 (C-4), 70.1 (CH FA), 69.2 (CH serine), 59.9 (CH -6), 54.1
2
(C-2), 52.6 (CH serine), 41.0, 40.5, 38.8, 34.4, 34.4, 34.2, 34.1, 34.0, 31.8, 31.8, 31.8, 31.8, 29.6, 29.6,
29.6, 29.6, 29.6, 29.6, 29.5, 29.5, 29.5, 29.4, 29.4, 29.4, 29.4, 29.3, 29.3, 29.3, 29.3, 29.2, 29.2, 29.2, 29.2,
31
2
2
9.1, 29.1, 29.1, 25.2, 25.1, 25.1, 24.9, 24.9, 22.6, 22.5, 22.5 (CH FA), 13.9 (CH FA); P NMR (CDCl ,
2
3
3
+
02 MHz, HMBC) δ 2.40; HRMS: [M+H] calcd. for C H N O P: 1488.0721, found 1488.0725.
8
1
152
2
19
4
.2.10.
decanoyloxy)tetradecanoyl]-3-O-[(R)-3-(decanoyloxy)tetradecanoyl]-6-O-(11-acetamidoundecanoyl)-
β-D-glucopyranosyl]-L-serine (2). Compound 16a (57.6 mg, 32.8 µmol, 1.0 eq) and acid 17 (20.8 mg,
5.5 µmol, 2.6 eq.) were co-evaporated twice with toluene under an argon atmosphere before being
dissolved in dry DCE (0.5 ml). The solution was cooled to 0°C, followed by the addition of EDC·MeI
20.8 mg, 68.6 µmol, 2.1 eq.) and DMAP (5.3 mg, 43 µmol, 1.3 eq.). The obtained yellow suspension
N-[(R)-3-(decanoyloxy)tetradecanoyl]-O-[4-O-phosphoryl-2-N-[(R)-3-
(
8
(
was allowed to warm-up to room temperature and was stirred overnight. The white suspension was diluted
with aq. sat. NaHCO and extracted with DCM (2x). The combined organic layers were dried over
3
Na SO , filtered and concentrated in vacuo. Purification by column chromatography (1060% EtOAc in
2
4
2
9
ACS Paragon Plus Environment