Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides

Article abstract of DOI:10.1002/chem.201905279

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure–activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.

Full text of DOI:10.1002/chem.201905279

A Journal of
Accepted Article
Title: Optical Control of Cytokine Production Using Photoswitchable
Galactosylceramides
Authors: Dirk Trauner, Nina Hartrampf, Toshiyuki Seki, Andreas
Baumann, Philip Watson, Anja Hoffmann-Röder, Moriya Tsuji,
Nynke Veprek, and Belinda Hetzler
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To be cited as: Chem. Eur. J. 10.1002/chem.201905279
Link to VoR: http://dx.doi.org/10.1002/chem.201905279
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10.1002/chem.201905279
Chemistry - A European Journal
Accepted Article
Communications
Optical Control of Cytokine Production Using Photoswitchable
Galactosylceramides
Nina Hartrampf,[a] Toshiyuki Seki,[c],[d] Andreas Baumann,[a] Philip Watson,[a] Nynke A. Vepřek,[a],[b]
Belinda E. Hetzler, [b] Anja Hoffmann-Röder,[a] Moriya Tsuji,[c] Dirk Trauner*[a],[b]
Abstract: α-Galactosylceramides are glycosphingolipids that show
promise in cancer immunotherapy. After presentation by CD1d they
activate natural killer T cells (NKT), which results in the production of
a variety of pro-inflammatory and immunomodulatory cytokines. Here
we report the synthesis and biological evaluation of photochromic
derivatives of KRN-7000, the activity of which can be modulated with
light. Based on established structure-activity relationships, we
designed photoswitchable analogs of this glycolipid that control the
production of pro-inflammatory cytokines, such as IFN-γ. The
azobenzene derivative α-GalACer-4 proved to be more potent than
KRN-7000 itself when activated with 370 nm light. Photolipids of this
type could improve our mechanistic understanding of cytokine
production and could open new directions in photoimmunotherapy.
Cytokines themselves show great promise in cancer
immunotherapy, but their clinical application has been limited due
to off-target effects.[4] Methods to selectively activate cytokine
production or activation in the tumor microenvironment could
overcome some of these limitations. Indeed, several techniques
have been developed to individually address components of the
immune system with spatiotemporal control: Using their “tag and
remotely induce guided immune response strategy” (TRIGIR),
Esser-Kahn and co-workers demonstrated light-activation of
fibroblasts and dendritic cells.[5] Parasar and Chang developed a
photocaged histone deacetylase inhibitor to regulate cytokine
production on a transcriptional level and modulate inflammation.[6]
a)
HO
OH
HO
O
HN
O
OH
HO
KRN-7000 is a synthetic analog of the marine natural product
agelasphin-9b that was isolated from the Japanese sponge
Agelas mauritianius (Figure 1a).[1] It is a strong immunostimulant
and has been under investigation as an adjuvant in cancer
immunotherapy.[2] KRN-7000 activates natural killer T (NKT) cells
following presentation by the glycoprotein CD1d. After binding to
CD1d, the glycolipid forms a ternary complex with the T cell
receptor, which leads to the production of a variety of cytokines
that can have different downstream effects.[3] These include TH1-
type cytokines or pro-inflammatory cytokines, such as interferon-
γ (IFN-γ), and TH2-type cytokines or immunomodulatory cytokines,
such as interleukin 4 (IL-4). Numerous derivatives of KRN-7000
have been synthesized to map structure-activity relationships in
an attempt to modulate cytokine production.[2]
HO
O
OH
Agelasphin-9b
O
OH
HO
HO
O
HN
OH
HO
O
OH
KRN-7000
b)
N
OH
HO
O
N
O
HN
O
HO
OH
C14H29
OH
HO
HN
O
HO
HO
N
O
N
OH
α-GalACer-4
Cl
OH
HO
O
Cl
C14H29
OH
N
OH
O
N
α-GalACer-1
HO
HO
Cl
O
HN
Cl
OH
C14H29
OH
HO
HO
HO
O
N
O
HN
O
OH
N
[a]
[b]
Prof. Dr. D. Trauner, Nynke A. Vepřek, Belinda E. Hetzler
Department of Chemistry
New York University
100 Washington Square East, New York, NY 10003, (USA)
E-mail: dirk.trauner@nyu.edu
Dr. N. Hartrampf, Dr. A. Baumann, P. Watson, Nynke A. Vepřek,
Prof. Dr. A. Hoffmann-Röder, Prof. Dr. D. Trauner
Department of Chemistry
Ludwig-Maximilians-Universität München
Butenandtstrasse 5–13, 81377 München (Germany)
Dr. T. Seki, Prof. Dr. M. Tsuji
The Aaron Diamond AIDS Research Center
Affiliate of the Rockefeller University
OH
HO
α-GalRACer-4
O
C14H29
OH
N
F
O
N
HO
HO
α-GalACer-3
O
HN
OH
O
HO
O
F
C14H29
HN
HO
HO
OH
α-6F'-GalACer-4
N
O
N
OH
Cl
HO
O
Cl
C14H29
OH
N
F
O
N
HO
HO
Cl
[c]
[d]
O
α-6F'-GalACer-1
HN
Cl
OH
C14H29
HO
O
OH
α-6F'-GalRACer-4
455 First Avenue, 7th Floor, New York, NY 10016 (USA)
Dr. T. Seki
Department of Obstetrics and Gynecology
The Jikei University School of Medicine
3- 25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105 (Japan)
Figure 1. a) marine natural product agelasphin 9b and the synthetic derivative
KRN-7000 b) Photochromic KRN-7000 derivatives synthesized.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201905279
Chemistry - A European Journal
Communications
stage introduction of the FAAzo side-chains and thereby for the
rapid synthesis of multiple derivatives (Figure 2).[10] Ceramide
building block 2 was obtained from commercially available
phytosphingosine (1) through standard protection and
deprotection chemistry.[11] For the galactose building block 5, b-
D-galactose pentaacetate (3) was thiolated at the anomeric center
and fully deprotected under Zemplén conditions.[12] Selective
reprotection of the hydroxyl groups at C4 and C6 as a benzylidene
In photopharmacology, biological function is controlled with
synthetic
light-responsive
molecules.[7]
These
include
azobenzene derivatives that change their molecular configuration
upon irradiation and can thermally relax to their more stable form
in the absence of light. Both the isomerization wavelegths and the
thermal relaxation rates depend on the substitution pattern of the
photoswitch. The change in conformation and polarity can result
in a difference in binding affinity to the target receptor and efficacy. acetal then afforded galactose acetal 4.[12a] Benzyl protection of
This enables control over cellular signaling with the high
spatiotemporal precision of light.
the remaining two hydroxyl groups followed by thioacetal
hydrolysis and activation of anomeric hydroxy group as the
trichloroacetimidate furnished glycosyl donor 5.[13] For a-6F’-
GalACer derivatives the fluorinated analog was synthesized
following an established procedure.[14] The ensuing steps were
identical for all derivatives and are only described for the
synthesis of a-GalACer-4. TMSOTf-promoted glycosylation of
ceramide building block 2 with trichloroacetimidate 5 afforded
glycoside 6 with excellent a-selectivity. The protected glycoside 6
was then hydrogenated using Pd/C and re-protected with acetic
We reasoned that the optical control of NKT-cell activation with
photoswitchable immunostimulants could allow for the
spatiotemporal control of cytokine production, providing
a
powerful method to study NKT cell immunobiology. Therefore, we
decided to endow KRN-7000 with a photoswitch. In 2015, we had
introduced a series of photoswitchable fatty acids (FAAzos), that
incorporate an azobenzene within the fatty acid chain.[8] We
demonstrated that FAAzos in their trans-form resemble saturated
fatty acids, whereas in their cis-form they behave more like highly
unsaturated fatty acids, such as arachidonic acid.[9] As a new
direction in photopharmacology we now describe an approach to
immunotherapy based on photolipids.
anhydride. The resultant acetyl-protected glycoside
7 was
deprotected and a FAAzo (e.g. FAAzo-4)[9] attached to the
glycoside via amide coupling. Final global deprotection then
yielded a-GalACer-4.
We designed two sets of KRN-7000 analogs with different
carbohydrate headgroups: the four a-galactosyl ceramides a-
GalACer-1, a-GalACer-3, a-GalACer-4 and a-GalRACer-4 as
well as the three 6F’-galactose-based ceramides a-6F’-GalACer-
1, a-6F’-GalACer-4 and a-6F’-GalRACer-4 (Figure 1b). They
differ in the position and substitution pattern of the azobenzene
switch in the acyl side chain.
BocHN
HO
OBz
C14H29
NH2
OH
a - d
HO
C14H29
OBz
OH
2
1
Ph
Ph
O
O
O
O
OAc
O
AcO
h - j
e - g
O
O
BnO
SPh
HO
AcO
OAc
Ph
BnO
O
NH
CCl3
OH
OAc
4
5
3
O
O
OAc
AcO
O
O
NHBoc
OBz
NHBoc
OBz
n - p
l, m
k
BnO
AcO
α-GalACer-4
2 + 5
BnO
AcO
O
O
C14H29
C14H29
OBz
OBz
6
7
Figure 2. Synthesis of the KRN-7000 derivative a-GalACer-4. a) Boc2O, THF,
r.t., 12 h; b) TrCl, DMAP, pyridine, 80 °C, 12 h (83% over 2 steps); c) BzCl,
DMAP, pyridine, r.t., 12 h (93%); d) p-TSA, CH2Cl2, MeOH, r.t., 4 h (91%); e)
PhSH, BF3・OEt2, CH2Cl2, 0 °C → r.t., 12 h; f) NaOMe, MeOH, 60 °C, 2 h; g)
PhCH(OMe)2, p-TSA, MeCN, r.t., 3.5 h (90% over 3 steps); h) BnBr, NaH, DMF,
0 °C → r.t., 12 h (85%); i) NBS, acetone, H2O, 0 °C, 1 h (89%); j) CCl3CN, DBU,
CH2Cl2, r.t., 2 h (98%); k) TMSOTf, Et2O/THF, 4Å MS, -30 °C, 3.5 h (46%); l)
10% Pd/C, H2, EtOH/EtOAc, r.t., 12 h; m) Ac2O, DMAP, pyridine, r.t., 12 h (99%
over 2 steps); n) TFA, DCM, 0 °C, 2 h; o) FAAzo-4, HBTU, NMM, CH2Cl2, r.t.,
12 h (92% over 2 steps); p) NaOMe, MeOH, r.t., 12 h (86%). Ac2O = acetic
anhydride, Boc2O = di-tert-butyl dicarbonate, BnBr = benzyl bromide, BzCl =
benzoyl chloride, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DMAP = 4-
dimethylaminopyridine, HBTU = (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate, NBS
= N-bromosuccinimide, NMM = N-
methylmorpholine, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TMSOTf
= Trimethylsilyl trifluoromethanesulfonate, TrCl = triphenylmethyl chloride, p-
TSA = p-toluenesulfonic acid.
Figure 3. a) Light-dependent reversible isomerization of trans-a-GalACer-1 and
cis-a-GalACer-1; b) Light-dependent reversible isomerization of trans-a-
GalACer-4 and cis-a-GalACer-4; c) Absorption of a-GalACer-1 (30 µM in
DMSO) in the dark-adapted state and at 370 nm and 440 nm; d) Absorption of
A modular synthesis strategy based on previous work by Tsuji,
Wong and co-workers was developed, which allowed for the late-
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10.1002/chem.201905279
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Communications
a-GalACer-4 (30 µM in DMSO) in the dark-adapted state and at 370 nm and
440 nm; e) reversible isomerization of a-GalACer-1 (30 µM in DMSO) over
multiple switching cycles; f) reversible isomerization of a-GalACer-4 (30 µM in
DMSO) over multiple switching cycles; g) thermal relaxation of a-GalACer-1 (30
µM in DMSO at 37 °C); h) thermal relaxation of a-GalACer-1 (30 µM in DMSO
at 37 °C).
Figure 4. a) IFN-γ secretion by human NKT cell line when stimulated by 10-100
ng/mL of indicated glycolipids was measured after 16 h of culture. Illumination
with 370 nm leads to an 6-fold increase in IFN-γ production for 10 ng/mL a-
GalACer-4. b) IL-4 secretion by human NKT cell line when stimulated by 10-100
ng/mL of indicated glycolipids was measured after 16 h of culture. Illumination
with 370 nm leads to a 4-fold increase in IL-4 production for 10 ng/mL a-
GalACer-4. Results are expressed as relative activities as mean of triplicate
assays. Representative data from one of two experiments are shown.
Following their synthesis, the photophysical and thermal
properties of our photolipids were characterized (Figure 3).[9] As
depicted for a-GalACer-4 and a-GalACer-1, the dark state or
trans-a-GalACer changes its configuration upon illumination with
370 nm light to its cis-isomer. The compound can be switched
over multiple cycles and is configurationally stable when left in the
dark for an extended period of time.
The structural difference between a-GalACer-1 and a-GalACer-4
lies in the relative position of the azobenzene unit in the acyl side
chain. Our results suggest that the spacer chain length of a-
GalACer-1 was too short to interact with the aromatic residues in
the CD1d hydrophobic groove, whereas a-GalACer-4 appears to
be a better fit.[10] When comparing cytokine production of a-
GalACer-4 at 10 ng/mL concentration we observe that IFN-γ
production is increased 6-fold whereas IL-4 production is only
increased 4-fold.
With several photoswitchable galactosyl ceramide derivatives in
hand, we evaluated their ability to activate human Vα24+ NKT
cells. To this end, we measured IFN-γ production relative to KRN-
7000 in ELISA assays in a light-dependent way (Figure 4a).
Among the derivatives tested, a-GalACer-1 and a-GalACer-4
emerged as our lead compounds. At a galactosyl ceramide
concentration of 10 ng/mL both derivatives gave lower cytokine
production than KRN-7000 itself. Upon illumination with UV light,
the IFN-γ secretion of cells treated with a-GalACer-1 increased,
albeit only to a low level. However, when cells were treated with
a-GalACer-4, illumination with 370 nm resulted in a significant
increase in IFN-γ production.
In summary we have introduced a highly potent photoswitchable
immunomodulator, a-GalACer-4. It elicits very low cytokine
production in its trans configuration but triggers significant
cytokine secretion following irradiation. a-GalACer-4 is therefore
a valuable tool to investigate spatiotemporal activation of cytokine
production. Upon UV irradiation, the production of both TH1 and
TH2-type-cytokines is robustly enhanced. Our future attempts will
focus on the design and synthesis of a next generation
photochromic galactosyl ceramides, which might allow for
selective control of TH1- over TH2-response.
Next, we investigated the effect of a-GalACer-1 and a-GalACer-
4 on IL-4 production (Figure 4b). Treatment of a human NKT cell
line that expresses an invariant Vα24 T-cell receptor (TCR) with
a-GalACer-1 in the dark led to a slightly lower IL-4 secretion
compared with exposure to KRN-7000. Illumination with UV light
only led to a small increase in cytokine production. At low a-
GalACer-4 concentration, cytokine production was similar to the
one observed with KRN-7000. Upon irradiation with 370 nm light,
IL-4 production was significantly increased, surpassing the effect
of the reference compound.
Acknowledgements
We acknowledge the Deutsche Telekom Foundation (Ph.D.
fellowship to N.H.), the LMUMentoring program (fellowship N.H.),
the German Academic Scholarship Foundation (Ph.D. Fellowship
to N.A.V), the New York University (MacCracken Fellowship to B.
E. H.) as well as the Deutsche Forschungsgemeinschaft (SFB
749 and CIPSM) for generous funding. This work was partially
supported by the National Institutes of Health Grant AI070258 (to
M.T.).
Keywords: cancer immunotherapy • photopharmacology •
azobenzenes • galactosyl ceramides • natural killer T cells
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Chemistry - A European Journal
Communications
Entry for the Table of Contents
COMMUNICATION
A new direction in
photoimmunotherapy. The
synthesis and biological
evaluation of photochromic
derivatives of the α-
Nina Hartrampf, Toshiyuki Seki,
Andreas Baumann, Philip Watson,
Anja Hoffmann-Röder, Morija
Tsuji, Dirk Trauner*
Page No. – Page No.
galactosylceramide KRN-7000
are reported. The photolipid α-
GalACer-4 proved to be more
potent than KRN-7000 itself when
activated with 370 nm light.
Optical Control of Cytokine
Production Using
Photoswitchable
Galactosylceramides
This article is protected by copyright. All rights reserved.