H-Phosphonate Synthesis and Biological Evaluation of an Immunomodulatory Phosphoglycolipid from Thermophilic Bacteria

Article abstract of DOI:10.1021/acs.orglett.0c00487

The synthesis of a library of bacterial phosphoglycolipid, PGL-1, is described. Key features of the synthesis include regioselective esterification of the primary alcohol of the diacylglycerol moiety and an H-phosphonate method to install the phosphate in PGL-1 in comparison with earlier reported procedures. A representative set of PGL-1 analogues was prepared and evaluated for their biological activities. Results showed that the immunological activity of PGL-1 is dependent on the chain lengths of the fatty acids.

Full text of DOI:10.1021/acs.orglett.0c00487

pubs.acs.org/OrgLett
Letter
H‑Phosphonate Synthesis and Biological Evaluation of an
Immunomodulatory Phosphoglycolipid from Thermophilic Bacteria
Chin Heng Gan, Hadhi Wijaya, Lan-Hui Li, Chih-Feng Wei, Yi-Jen Peng, Shih-Hsiung Wu,*
Kuo-Feng Hua,* and Yulin Lam*
Cite This: https://dx.doi.org/10.1021/acs.orglett.0c00487
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The synthesis of a library of bacterial phosphoglycolipid, PGL-1, is described. Key features of the synthesis include
regioselective esterification of the primary alcohol of the diacylglycerol moiety and an H-phosphonate method to install the
phosphate in PGL-1 in comparison with earlier reported procedures. A representative set of PGL-1 analogues was prepared and
evaluated for their biological activities. Results showed that the immunological activity of PGL-1 is dependent on the chain lengths of
the fatty acids.
negative thermophilic bacteria.4 Biological studies of the PGL-
ipopolysaccharide, a major constituent of the outer leaflet
Lof the Gram-negative bacterial outer membrane, is an 1/PGL-2 mixtures showed that PGL-1 stimulates the release of
prointerleukin-1β (proIL-1β) in human THP-1 monocytes,
while PGL-2 partially inhibits PGL-1-mediated proIL-1β
production in the PGL-1/PGL-2 mixture.5 In addition, we
found that the PGL-1 obtained from different species of
thermophilic bacteria have different lengths of fatty acids
attached to the glycerol moiety and showed different potencies
in the PGL-mediated proIL-1β production.4 This implied that
the length of the fatty acid in PGL-1 plays an important role in
its immunomodulatory properties.
endotoxin that elicits strong pro-inflammatory response in the
host during infection.1 However, Gram-negative thermophilic
bacteria do not contain lipopolysaccharides but have high
amounts of glycoglycerolipids and phosphoglycolipids (PGLs)
which maintain the organism’s biological functions under
extreme environmental conditions.2,3
In our earlier studies, we have isolated and elucidated two
new forms of PGLs, namely, PGL-1 and PGL-2 (Figure 1),
from the Thermus and Meiothermus strains of the Gram-
To determine how the lengths of the fatty acids affect the
biological function, PGL-1 analogues containing different fatty
acid chain lengths are required. However, the PGLs isolated
from the thermophilic bacterial strains could not be separated
by general chromatographic methods.4 To address this
problem, two synthetic routes were published earlier by Lin6
and Fujimoto7 (Figure 2). Both strategies installed the
phosphate via a phosphoramidite whose reagents and
intermediates were reported to be hydrolytically unstable.8−10
Received: February 6, 2020
Figure 1. Structures of isolated PGL-1 and PGL-2.
https://dx.doi.org/10.1021/acs.orglett.0c00487
© XXXX American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

Organic Letters
pubs.acs.org/OrgLett
Letter
Figure 2. Synthetic route toward PGL-1 reported by (a) Fujimoto et al. and (b) Lin et al.
Scheme 1. Retrosynthesis of PGL-1 and Its Analogues
Scheme 3. Synthesis of Diacylglycerol 4
Scheme 4. Synthesis of PGL-1 Analogues 1
Scheme 2. Synthesis and O-Glycosylation of Glyceramide 3
isomerism on the immunomodulatory properties, we sought a
simpler and shorter route toward the synthesis.
A retrosynthetic analysis of PGL-1 is shown in Scheme 1.
The key synthetic considerations are (i) to protect the
acetamide on C-2 of the glucosamine fragment as an azide to
facilitate α-selective glycosylation, (ii) to provide a more
concise synthesis of the glyceramide, and (iii) to install the
phosphate via bench-stable H-phosphonate intermediates, a
method used by Seeberger and co-workers to synthesize
phosphatidyl mannosides11 and subsequently by Larsen,12,13
Guo,14 and Hung15 to synthesize phosphatidylinositols. Based
on these considerations, three building blocks 2−4 (Scheme 1)
were designed. Compound 2 was synthesized from D-
glucosamine according to a procedure reported by Ren et
al.,16 while compounds 3 and 4 were both prepared from (S)-
Moreover, the synthesis of the glyceramide prior to
phosphorylation required multiple synthetic steps to accom-
plish. In our efforts to obtain well-defined PGL-1 and its
analogues to investigate the effects of chain length and
B
https://dx.doi.org/10.1021/acs.orglett.0c00487
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Table 1. List of PGL-1 Analogues 1 Synthesized
cpd
R1
R2
R3
1a
1b
1c
1d
1e
CH3(CH2)14-
CH3(CH2)15-
(CH3)2CH(CH2)12-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)16-
(CH3)2CH(CH2)18-
(CH3)2CH(CH2)14-
(CH3)2CH(CH2)12-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)11-
CH3(CH2)14-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)13-
CH3(CH2)14-
(CH3)2CH(CH2)12-
CH3(CH2)14-
(CH3)2CH(CH2)10-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)13-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
CH3(CH2)14-
CH3(CH2)12-
CH3(CH2)13-
CH3(CH2)15-
CH3(CH2)12-
a
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
CH3(CH2)14-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)10-
(CH3)2CH(CH2)12-
(CH3)2CH(CH2)11-
(CH3)2CH(CH2)11-
1s
a
R-isomer.
Figure 4. (a, b) J774A.1 macrophages were incubated for 0.5 h with
1e or vehicle (0.1% DMSO) followed by incubation for 6 h with 1
μg/mL of LPS. The levels of IL-6 (a) and TNF-α (b) in the
supernatants were measured by ELISA. (c) J-Blue NF-κB reporter
cells were incubated for 0.5 h with 1e or vehicle (0.1% DMSO)
followed by incubation for 24 h with 1 μg/mL of LPS or vehicle
(0.1% PBS). NF-κB activity was analyzed by an NF-κB reporter assay.
(d) J774A.1 macrophages were incubated for 0.5 h with 15 μg/mL of
1e or vehicle (0.1% DMSO) followed by incubation for 2 or 4 h with
1 μg/mL of LPS. The levels of IL-6 mRNA were analyzed by RT-
qPCR. The data were expressed as mean SD; n = 3.
Figure 3. Effect of 1 on (a) IL-6 production and (b) TNF-α
production in LPS-activated macrophages.
solketal. By applying appropriate functional group trans-
formations to the free hydroxyl group on (S)-solketal, we
could either perform amide coupling as in the case of 3 or
release the other two hydroxyl groups for acylation with fatty
acids R1COOH and R2COOH to give 4.
The synthesis of glyceramide 3 is depicted in Scheme 2. (S)-
Solketal was oxidized with TEMPO/BAIB to yield 5 which
then underwent amide coupling, using CDI, with the
respective alkylamine to provide 6. Subsequent deprotection
of the acetal afforded glyceramide 3 which was then O-
glycosylated with 2. The O-glycosylation of 2 is highly selective
for the primary alcohol with only minor secondary O-
glycosylated product being observed. Selectivity for the α-
glycoside was 2:1 over the β-glycoside (both the α- and β-
glycosides could be purified out separately by column
chromatography). Although the glycosylation yield is com-
paratively lower than those reported by Lin6 and Fujimoto,7
C
https://dx.doi.org/10.1021/acs.orglett.0c00487
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
this approach nevertheless provided 8 in higher overall yield
(15% over 4 steps as compared to those reported by Lin (10%
over 15 steps) and Fujimoto (4% over 9 steps)). Subsequent
conversion of the azide on 7 to acetamide using thioacetic acid
gave 8 in 64−70% yield.
from the NF-κB reporter assay (Figure 4c) indicated that LPS
significantly increased the NF-κB activity compared to control
J774A.1 macrophages, and this effect was further enhanced by
1e in a dose-dependent manner. It is noteworthy that 1e alone
did not alter the NF-κB activity in control cells. This suggests
that 1e increases LPS-induced TNF-α production in macro-
phages possibly through enhancing NF-κB activity.
Synthesis of the diacylglycerol fragment 4 started with the
benzylation of (S)-solketal (Scheme 3). Since the fatty acid in
the secondary position is labile to 1,2-acyl migration under
acidic/basic conditions,17 the benzyl moiety was chosen as the
protecting group of the primary alcohol. Subsequent removal
of the isopropylidene acetal gave compound 9 in 90% yield.
To attach different fatty acids on the two hydroxyl groups of
9, we attempted a regioselective esterification of the primary
alcohol using the DCC/DMAP system. However, the reaction
was only moderately selective for the primary ester. Attempts
to selectively esterify the primary alcohol using other reaction
conditions (Mukaiyama reagent, oxyma/EDC, B(OH)3, Zn-
(OTf)2, PPh3/DIAD, PPh3/I2, CDI, Fe(acac)3, DCC/2,6-
lutidine) resulted in either very low yields or no desired
product. Further optimization eventually showed that the
TBTU/base (base: TEA, DIEA,18 DBU, K2CO3, NMM, and
DABCO) system is highly selective for the primary alcohol
(>90%) with TBTU/NMM giving 10 with the best yield and
selectivity (yield: 73−85%; selectivity of 1°:2° = 99:1). A
second esterification with DCC/DMAP gave 11 (yield: 70−
91%), following which debenzylation using 10% Pd/C in
ethanol with 5% acetic acid provided the diacylglycerol 4 in
quantitative yield.
To investigate the mechanism of action of 1e on the
inhibition of IL-6 production, Western blots were performed.
The results showed that while LPS significantly increased
phosphorylation levels of ERK1/2, JNK1/2, and p38, treat-
ment of the same LPS-activated macrophages with 1e did not
alter the phosphorylation levels (Supporting Information,
Figure S2), implying that 1e-mediated modulation of IL-6
and TNF-α did not occur via the activation of mitogen-
activated protein kinases. Western blot results (Supporting
Information, Figure S3) also showed that treatment with 1e
did not change the expression levels of the autophagy markers
LC3, p62, and ATG5, indicating that 1e did not alter
autophagy induction either. Hence we decided to examine
the effects of 1e on the IL-6 mRNA expression in LPS-
activated macrophages using RT-qPCR. The results obtained
(Figure 4d) showed that treatment with LPS significantly
increased the IL-6 mRNA expression, but this expression was
reduced in the presence of 1e, suggesting a correlation between
1e treatment and IL-6 mRNA expression. Further experiments
are presently underway to elucidate the detailed mechanism of
action.
In summary, a shorter synthesis of the PGL-1 analogue 1 has
been developed. Immunomodulatory studies show that the
activity of 1 is dependent on the chain lengths of the three fatty
acid tails. Preliminary studies to elucidate the mechanism of
action suggest that the immunomodulatory activity of 1 could
proceed via NF-κB activation.
Treatment of 4 with PCl3 in the presence of imidazole and
TEA at 0 °C11 gave the H-phosphonate intermediate 12 which
was stable at room temperature, thus allowing easy handling
and installing of the same H-phosphonate intermediate on
different analogues of 8 (Scheme 4). Phosphorylation to
compound 13 was achieved under microwave conditions using
trichloroacetonitirile19 as an activator. Amberlyst-15 ion-
exchange resin was then used to remove all the TEAH+ ions
prior to hydrogenation with 10% Pd/C catalyst to yield 1 in
the acid form. A total of 19 PGL-1 analogues were synthesized
(Table 1).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c00487.
To investigate the immunomodulatory properties of 1,
macrophages were treated with the respective compounds and
then activated with LPS. The levels of IL-6 and TNF-α
produced were quantified using ELISA. Results obtained
showed that most of the compounds reduced IL-6 production
(Figure 3a) and increased TNF-α production in LPS-activated
macrophages (Figure 3b), indicating that 1 exhibits immuno-
modulatory activities in macrophages. Among the PGL-1
derivatives, compounds 1b, 1e, and 1g exhibited stronger
inhibition of IL-6 production and larger induced TNF-α
production in LPS-activated macrophages and were thus
further evaluated for their cytotoxicities against J774A.1
macrophages (Supporting Information, Figure S1). The results
obtained showed that all three compounds reduced the
viability of J774A.1 macrophages in a dose-dependent manner,
with 1e being the least cytotoxic. Thus, compound 1e was used
in subsequent biological studies.
To investigate the dose response of 1e on IL-6 and TNF-α
in mouse macrophages, macrophages were treated with varying
concentrations of 1e and then activated with LPS. The levels of
IL-6 and TNF-α produced were quantified using ELISA.
Results showed that 1e dose-dependently reduced IL-6
production (Figure 4a) and increased TNF-α production in
LPS-activated macrophages (Figure 4b). In addition, results
Experimental procedures, characterization of the prod-
ucts, and cytotoxicity data of compounds 1b, 1e, and 1g
against J774A.1 macrophages (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Shih-Hsiung Wu − Institute of Biological Chemistry, Academia
Sinica, Taipei 115, Taiwan; Email: shwu@gate.sinica.edu.tw
Kuo-Feng Hua − Department of Biotechnology and Animal
Science, National Ilan University, Yilan County 260, Taiwan;
Department of Pathology, Tri-Service General Hospital,
National Defense Medical Center, Taipei 11490, Taiwan;
Department of Medical Research, China Medical University
Hospital, China Medical University, Taichung 40402, Taiwan;
Email: kuofenghua@gmail.com
Yulin Lam − Department of Chemistry, National University of
Singapore, Singapore 117543 Singapore; orcid.org/0000-
0003-0984-9492; Email: chmlamyl@nus.edu.sg
Authors
Chin Heng Gan − Department of Chemistry, National
University of Singapore, Singapore 117543 Singapore;
orcid.org/0000-0003-1625-5430
D
https://dx.doi.org/10.1021/acs.orglett.0c00487
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
(11) Liu, X.; Stocker, B. L.; Seeberger, P. H. Total synthesis of
phosphatidylinositol mannosides of Mycobacterium tuberculosis. J.
Am. Chem. Soc. 2006, 128 (11), 3638−3648.
Hadhi Wijaya − Department of Chemistry, National University
of Singapore, Singapore 117543 Singapore
Lan-Hui Li − Department of Laboratory Medicine, Linsen,
Chinese Medicine and Kunming Branch, Taipei City Hospital,
Taipei 10844, Taiwan; Department of Pathology, Tri-Service
General Hospital, National Defense Medical Center, Taipei
11490, Taiwan
Chih-Feng Wei − Department of Pathology, Tri-Service General
Hospital, National Defense Medical Center, Taipei 11490,
Taiwan
Yi-Jen Peng − Department of Pathology, Tri-Service General
Hospital, National Defense Medical Center, Taipei 11490,
Taiwan
(12) Rankin, G. M.; Compton, B. J.; Johnston, K. A.; Hayman, C.
M.; Painter, G. F.; Larsen, D. S. Synthesis and mass spectral
characterization of Mycobacterial phosphatidylinositol and its
dimannosides. J. Org. Chem. 2012, 77 (16), 6743−6759.
(13) Lee, A. M. M.; Painter, G. F.; Compton, B. J.; Larsen, D. S.
Resolution of orthogonally protected myo-inositols with Novozym
435 providing an enantioconvergent pathway to Ac2PIM1. J. Org.
Chem. 2014, 79 (22), 10916−10931.
(14) Johnson, C. L.; Guo, Z. Synthesis of novel, fluorescently tagged
analogs of glycosylphosphatidylinositol (GPI) anchors. J. Carbohydr.
Chem. 2013, 32 (5), 301−323.
(15) Patil, P. S.; Cheng, T.−J. R.; Zulueta, M. M. L.; Yang, S.-T.;
Lico, L. S.; Hung, S.-C. Total synthesis of tetraacylated
phosphatidylinositol hexamannoside and evaluation of its immuno-
modulatory activity. Nat. Commun. 2015, 6 (1), 7239.
(16) Ren, L.; Zhang, J.; Ma, H.; Sun, L.; Zhang, X.; Yu, G.; Guan,
H.; Wang, W.; Li, C. Synthesis and anti-influenza A virus activity of 6’-
amino-6’-deoxy-glucoglycerolipids analogs. Mar. Drugs 2016, 14 (6),
No. 116.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c00487
Notes
The authors declare no competing financial interest.
(17) Fureby, A. M.; Virto, C.; Aldercreutz, P.; Mattiasson, B. Acyl
group migrations in 2-monoolein. Biocatal. Biotransform. 1996, 14 (2),
89−111.
(18) Twibanire, J. D.; Grindley, T. B. Efficient and controllably
selective preparation of esters using uranium-based coupling agents.
Org. Lett. 2011, 13 (12), 2988−2991.
ACKNOWLEDGMENTS
■
The authors thank the Ministry of Education (Singapore)
(MoE Tier 2: R-143-000-589-112) for the financial support
and NUS for a PhD scholarship (to C.H.G. and H.W.).
(19) Shih, H.-W.; Chen, K.-T.; Cheng, W.-C. One-pot synthesis of
phosphate diesters and phosphonate monoesters via a combination of
microwave-CCl3CN-pyridine coupling conditions. Tetrahedron Lett.
2012, 53 (2), 243−246.
REFERENCES
■
(1) Wu, D.; Fujio, M.; Wong, C. H. Glycolipids as immunostimulat-
ing agents. Bioorg. Med. Chem. 2008, 16 (3), 1073−1083.
(2) Forterre, P.; Bouthier de la Tour, C.; Philippe, H.; Duguet, M.
Reverse gyrase from hyperthermophiles: porbable transfer of a
thermoadaptation trait from archaea to bacteria. Trends Genet.
2000, 16, 152−154.
(3) Lesley, S. A.; Kuhn, P.; Godzik, A.; Deacon, A. M.; Mathews, I.;
Kreusch, A.; Spraggon, G.; Klock, H. E.; McMullan, D.; Shin, T.;
Vincent, J.; Robb, A.; Brinen, L. S.; Miller, M. D.; McPhillips, T. M.;
Miller, M. A.; Scheibe, D.; Canaves, J. M.; Guda, C.; Jarpszewski, L.;
Selby, T. L.; Elsliger, M.-A.; Wooley, J.; Taylor, S. S.; Hodgson, K. O.;
Wilson, I. A.; Schultz, P. G.; Stevens, R. C. Structural genomics of the
Thermotaga maritima proteome implemented in a high-throughput
structure determination pipeline. Proc. Natl. Acad. Sci. U. S. A. 2002,
99, 11664−11669.
(4) Yang, Y. L.; Yang, F. L.; Jao, S. C.; Chen, M. Y.; Tsay, S. S.; Zou,
W.; Wu, S. H. Structural elucidation of phosphoglycolipids from
strains of the bacterial thermophiles Thermus and Meiothermus. J.
Lipid Res. 2006, 47, 1823−1832.
(5) Yang, F. L.; Hua, K. F.; Yang, Y. L.; Zou, W.; Chen, Y. P.; Liang,
S. M.; Hsu, H. Y.; Wu, S. H. TLR-independent induction of human
monocyte IL-1 by phosphoglycolipids from thermophilic bacteria.
Glycoconjugate J. 2008, 25 (5), 427−439.
(6) Lin, H. J.; Adak, A. K.; Reddy, L. V.; Wu, S. H.; Lin, C. C. Total
synthesis of an immunomodulatory phosphoglycolipid from thermo-
philic bacteria. Chem. - Eur. J. 2013, 19 (24), 7989−7998.
(7) Fujimoto, Y.; Mitsunobe, K.; Fujiwara, S.; Mori, M.; Hashimoto,
M.; Suda, Y.; Kusumoto, S.; Fukase, K. Synthesis and biological
activity of phosphoglycolipids from Thermus thermophilus. Org.
Biomol. Chem. 2013, 11 (30), 5034−5041.
(8) Crossey, K.; Hardacre, C.; Migaud, M. E. Nucleoside
phosphitylation using ionic liquid stabilised phosphorodiamidites
and mechanochemistry. Chem. Commun. 2012, 48 (98), 11969−
11971.
(9) Ellington, A.; Pollard, J. D., Jr. Introduction to the synthesis and
purification of oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 2000,
00 (1), A.3C.1−A.3C.22.
̈
(10) Nurminen, E.; Lonnberg, H. Mechanisms of the substitution
reactions of phosphoramidites and their congeners. J. Phys. Org. Chem.
2004, 17 (1), 1−17.
E
https://dx.doi.org/10.1021/acs.orglett.0c00487
Org. Lett. XXXX, XXX, XXX−XXX