Total Synthesis of an Immunogenic Trehalose Phospholipid from Salmonella Typhi and Elucidation of Its sn-Regiochemistry by Mass Spectrometry

Article abstract of DOI:10.1021/acs.orglett.9b01725

Diphosphatidyltrehalose (diPT) is an immunogenic glycolipid, recently isolated from Salmonella Typhi. Despite rigorous structure elucidation, the sn-position of the acyl chains on the glycerol backbone had not been unequivocally established. A stereoselective synthesis of diPT and its regioisomer is reported herein. Using a hybrid MS³ approach combining collisional dissociation and ultraviolet photodissociation mass spectrometry for analysis of the regioisomers and natural diPT, the regiochemistry of the acyl chains of this abundant immunostimulatory glycolipid was established.

Full text of DOI:10.1021/acs.orglett.9b01725

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Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis of an Immunogenic Trehalose Phospholipid from
Salmonella Typhi and Elucidation of Its sn-Regiochemistry by Mass
Spectrometry
Vivek K. Mishra,† Jeffrey Buter,† Molly S. Blevins,‡ Martin D. Witte,† Ildiko Van Rhijn,§,∥
D. Branch Moody,∥ Jennifer S. Brodbelt,‡ and Adriaan J. Minnaard*,†
†Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
‡Department of Chemistry, University of Texas, Austin, Texas 78712, United States
§Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The
Netherlands
∥Department of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: Diphosphatidyltrehalose (diPT) is an immunogenic glycolipid, recently isolated from Salmonella Typhi. Despite
rigorous structure elucidation, the sn-position of the acyl chains on the glycerol backbone had not been unequivocally
established. A stereoselective synthesis of diPT and its regioisomer is reported herein. Using a hybrid MS3 approach combining
collisional dissociation and ultraviolet photodissociation mass spectrometry for analysis of the regioisomers and natural diPT,
the regiochemistry of the acyl chains of this abundant immunostimulatory glycolipid was established.
design (“immune response booster”) in vaccine development,
including lipid A and trehalose dibehenate.13,14
ipids function as the key structural components of cellular
Lmembranes and have specific biological roles in regulating
cell response.1 For example, many lipids of bacterial origin
trigger immune responses by binding innate receptors of the
immune system. Perhaps the most studied molecule of bacterial
pathogens is lipopolysaccharide (LPS),2 a virulence factor3
embedded in the outer cell membrane of Gram-negative
bacteria.4 A major constituent of LPS is lipid A (2, Figure
1),5−7 which acts as a potent stimulant (pg/mL)8 that triggers
cytokine release by ligating to Toll-like receptor 4 (TLR4).9
Another important lipid that stimulates the immune system is
trehalose dimycolate (TDM, 4), which is also known as the cord
factor and is abundantly present in the cell wall of Mycobacterium
tuberculosis. TDM binds to the macrophage-inducible Ca2+-
dependent C-type lectin10 (Mincle) receptor,11,12 a carbohy-
drate binding protein, leading to macrophage activation and
cytokine release. Lipid A and TDM thus stimulate the innate
immune response. Based on detailed analysis of the aspects of
the structure needed for immune response, both natural
molecules have served as important scaffolds for adjuvant
Recently, the first members of a new class of lipids were
discovered, which consist of trehalose attached to a phosphatidic
acid: phosphatidyltrehalose (PT, 3) and diphosphatidyltreha-
lose (diPT, 1). These glycolipids were isolated from the typhoid
fever-causing, Gram-negative bacterium Salmonella Typhi.15 PT
and diPT bear notable structural resemblance to both TDM and
LPS (Figure 1). The presence of phosphate moieties makes PT
and diPT somewhat more closely resemble lipid A, whereas
trehalose and a cyclopropyl-containing alkyl chain are congruent
with TDM. The strong structural analogies of these newly
discovered molecules with TDM led to immunological studies,
which showed that diPT is similarly potent as a Mincle ligand
and thus can serve as a new candidate for adjuvant develop-
ment.15
The overall structure of PT and diPT (Figure 1) was
elucidated using collisional mass spectrometry and NMR
Received: May 16, 2019
© XXXX American Chemical Society
A
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Figure 1. Structure of lipid A, phosphatidyltrehalose, diphosphatidyltrehalose, and trehalose dimycolate.
Figure 2. DiPT retrosynthesis.
spectroscopy. Two features remained unknown or uncertain,
however. Whereas it is reasonable to assume that the
stereochemistry of the phosphatidic acid matches the known
stereochemistry of bacterial sn-glycerol-3-phosphate16 and that
of the cyclopropyl function is (9R,10S) as found in Escherichia
coli,17 a species closely related to Salmonella, neither have been
formally established.17,18 In addition, the regiochemistry of the
acyl chains is unknown. A second rationale for determining the
complete structure of phosphatidyl trehalose is the desire to
study these compounds in immunological assays, which requires
synthetic material that is entirely free from immunologically
active impurities. Third, large amounts of compound are needed
for human in vitro and animal in vivo studies of glycolipid
function. Therefore, we undertook the total synthesis of diPT.
From the outset, it was clear though that several regioisomers
had to be prepared to establish the structure of the natural
compound. The synthesis described herein is the first one of this
new class of compounds and sets the stage for a new adjuvant
design based on this novel molecular scaffold.
DiPT features two identical phosphatidic acid residues
anchored to the C6-positions of trehalose. Although C2-
symmetric, synthesis by dimerization was not desired as the
glycosidic bond in trehalose is particularly difficult to
construct.19 In addition, trehalose itself is readily available and
was therefore chosen as the starting material (Figure 2).
We explored both a phosphate and a phosphoramidite
coupling strategy. The first strategy was used in the synthesis of
β-mannosylphosphomycoketide,20 an antigen of Mycobacterium
tuberculosis, and synthesis of acceptors21 for glycosyl trans-
ferases. The phosphoramidite coupling22,23 is an approach used
B
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extensively in phosphorylated protein and nucleotide syn-
thesis.24−26
Scheme 2. Synthesis of Diacylglycerol 6
For the regio- and stereoselective synthesis of the
diacylglycerol, we planned to capitalize on our earlier work in
which we developed a catalytic method of epoxide ring opening
that provides flexibility in the introduction of the fatty acid
residues, minimizing acyl shift.27 Given the unknown
regiochemistry of the acyl residues, we planned the synthesis
of both regioisomers.
In our first strategy, we attempted to prepare the phosphatidic
acid of 6 to activate and couple it to trehalose. Indeed, glycidol,
upon reaction with dibenzyl phosphorochloridate, produced the
phosphorylated glycidol (74%), but epoxide ring opening under
Jacobsen28 and basic (KOH) conditions with palmitic acid did
not lead to product formation. On the other hand, when
diglyceride 6 was treated with 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (CEP-Cl, 8) in basic medium (Hunig’s
̈
base), an inseparable mixture of regioisomers (sn-3/sn-2 = 4/1)
was formed in moderate yield (46%) due to extensive acyl shift.
We discontinued this approach and decided to place the
phosphoramidite units on the trehalose building block, which
therefore needed a suitable protecting group. We briefly
explored persilylated (TMS)6-trehalose29 that was treated with
CEP-Cl to produce the corresponding persilylated bisphosphor-
amidite. However, this compound was unstable under the
required phosphoramidite coupling conditions. As an alternative
approach, we decided to make perbenzyl ether 12, according to a
literature procedure,30 which was subsequently treated with
CEP-Cl (8) to give bisphosphoramidite 5 in good yield (Scheme
1).
not suitable here as it leads to ring opening of the cyclopropane.
The analytical data of (R,S)-9 and its enantiomer were in
agreement with that of previously reported synthetic dihy-
drosterculic acid.31,35
Next, silyl-protected glycidol 10 (Scheme 2) was subjected to
epoxide ring opening by employing Jacobsen’s catalyst with
palmitic acid 11 to give acyl glycerol 17.28 Subsequently, 17 was
reacted with (R,S)-9 under Steglich conditions to produce
protected diglyceride 18. Diacylglycerols are prone to acyl
migration,36 but as previously reported, BF3·CH3CN-mediated
deprotection of 18 under carefully controlled conditions
produced advanced intermediate 6, without NMR-detectable
migration.
Scheme 1. Bisphosphoramidite 5 Synthesis
With all building blocks in hand, a careful study of the
phosphoramidite coupling was performed using dipalmitoyl
glycerol, leading to straight-chain diPT4 (see Supporting
Information) and trehalose bisphosphoramidite 5. The progress
of the reaction was monitored by proton-decoupled 31P NMR.
Initially, instead of a phosphite intermediate, we observed acyl
migration when solid dicyanoimidazole (DCI) was used as the
catalyst for the reaction. Surprisingly, using a DCI solution (1 M,
in CH3CN), no NMR-detectable acyl migration was observed.
Complete consumption of starting material was observed within
1 h, and the produced intermediate was oxidized in situ into the
corresponding phosphate 24 with tBuOOH. We found that an
excess of tBuOOH led to oxidation of the benzyl ethers into the
corresponding benzoyl esters, as confirmed by HRMS. The issue
was solved using just a slight excess of tBuOOH (2.5 equiv) at 0
°C. The optimized conditions were employed successfully to
couple 5 and 6 to get phosphate 19 in an acceptable yield (67%,
Scheme 3). The final deprotections were delicate, and we
observed a clean elimination of the cyanoethyl groups in 19 by
employing DBU for 5 min to produce phosphate 20. Finally,
hydrogenolysis with a combination of Pd/C and Pd(OH)2/C
(2:3) in a CH2Cl2/MeOH/water mixture produced the desired
product diPT (1, called “diPT1” from here on) in excellent yield.
Using the synthetic sequence outlined, two analogues of diPT1
(1) were synthesized in good overall yields by replacing (R,S)-9
with (S,R)-9 to provide diPT2 (31). We switched the
dihydrosterculic acid position (from sn-2 to sn-1) in the
phosphatidic acid, leading to diPT3 (38).
Next, the synthesis of diglyceride 6 commenced with the
preparation of the cis-cyclopropyl fatty acid. A versatile method
developed by Williams et al., based on rhodium-catalyzed
cyclopropenation,31 was used with some modifications to
prepare 9 and its enantiomer. In short, intermediate 14,
obtained from octyne in four steps (Scheme 2), was subjected
to oxidation with Dess−Martin periodinane to produce
aldehyde 15, which was further used in a Wittig reaction to
give alkene 16 in 86% yield (Z/E = 8/2).32,33 Reduction of 16 to
the desired fatty acid (R,S)-9 (87%) was effected by diimide, in
situ generated from H2N-NH2·H2O and a flavin catalyst
reported earlier.34 Transition-metal-catalyzed hydrogenation is
With diPT1 1, its stereoisomer 31, its regioisomer 38, and the
1
natural isolate in hand, their H and 13C resonances could be
compared (Tables S1 and S2). DiPT1 1 has a very close match
C
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differences are resonances appearing in 1 (δ, 1.82−1.66 ppm)
and in natural diPT (δ, 2.37−2.12 ppm), which are attributed to
unidentifiable impurities. The NMR data showed a strong
correspondence between diPT1 1 and natural diPT. The sn-
position of the acyl chains on each glycerol backbone, however,
could not be determined nor could the cyclopropane ring in the
acyl chains be localized. To solve this structural puzzle, we
resorted to ultraviolet photodissociation MS (UVPD-MS),37−39
a method that has proven useful for detailed structural
characterization of complex lipids.
Scheme 3. DiPT1 Synthesis End Game
Unlike traditional collisional activation methods, UVPD
affords direct localization of subtle structural elements in lipids
including double bonds and cyclopropane rings.37,38,40 For
example, UVPD allows the positions of cyclopropane rings in
lipids to be pinpointed via generation of a diagnostic pair of
fragment ions spaced 14 Da apart.40 Here, this method was
applied for analysis of diPT lipids in negative ion mode. Negative
mode MS1 spectra for all diPT species are provided in Figure S1,
allowing confirmation of chemical formula via high-resolution
high-mass accuracy data. As shown in Figure 3a−d, cyclo-
propane localization in all three synthetic diPT species as well as
the natural diPT lipid is possible via UVPD of the doubly
deprotonated precursor [M − 2H]2− of m/z 812. Cyclopropane
is determined to reside at C9 as counted from the acyl chain
carbonyl carbon, as illustrated by the fragments in green font in
the map in Figure 3e and observed as diagnostic ions of m/z 756
and m/z 763 in Figure 3a−c, corresponding to a difference of 14
Da.
Next, a hybrid collisional activation/UVPD-MS3 method was
employed as previously described for determination of sn-
configuration.37 In brief, sodium-adducted diPT species were
fragmented using higher-energy collisional dissociation (HCD)
for generation of five-membered cyclic sodium-adducted species
of m/z 585 via loss of the headgroup, followed by subsequent
activation of the product by UVPD. As displayed in Figure 4a,
diPT1 1 yields mainly a product of m/z 319 upon hybrid HCD/
with the natural material. The anomeric protons appear as a
doublet in 1 (δ, 5.08 ppm, J = 3.8 Hz) and natural diPT (δ, 5.08
ppm, J = 3.7 Hz), whereas these are observed as an apparent
singlet in 31 (δ, 5.10 ppm) and 38 (δ, 5.06 ppm). Apart from this
difference, the diastereotopic signals of the cyclopropyl group
have a similar chemical shift (δ, −0.32 ppm) in 1 and natural
diPT, which differs by 0.04 ppm in 31 and 38. The only minor
Figure 3. Negative mode UVPD mass spectra of [M − 2H]2− (m/z 812) for (a) diPT1, (b) diPT2, (c) diPT3, and (d) natural diPT. (e) Representative
fragmentation of diPT3.
D
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occurring diPT, its stereoisomer 31, its regioisomer 38, and the
availability of a robust synthetic route can now be used to
determine a structure−activity relationship for activation of
Mincle. Moreover, the synthesis allows for the design of other
novel adjuvants which can greatly facilitate vaccine develop-
ment.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b01725.
Experimental data, NMR, and MS (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: a.j.minnaard@rug.nl.
ORCID
Martin D. Witte: 0000-0003-4660-2974
Adriaan J. Minnaard: 0000-0002-5966-1300
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Figure 4. UVPD mass spectra of [M − 2H]2− (m/z 812) for (a) diPT1,
(b) diPT3, and (c) natural diPT in negative mode with (d,e) fragment
ion maps.
■
We thank P. van der Meulen, Dr. J. Kemmink, and R. Sneep
(University of Groningen) for assistance on analyses. Funding
from the NIH (R01 AI116604, J.S.B.) and (GM103655,
M.S.B.), the Welch Foundation (F-1155, J.S.B.), and the UT
System for support of the UT System Proteomics Core Facility
Network is gratefully acknowledged. The Dutch Science
Foundation NWO is acknowledged for funding.
UVPD activation, with a lower abundance product of m/z 331.
These two fragment ions are attributed to photolytic cleavage
across the dioxolane ring, resulting in sodium-adducted fatty
acid allyl esters of each sn-1 fatty acid chain. Cyclic structures for
the product ion of m/z 585 and the UVPD fragment ions of m/z
319 and m/z 331 are shown in Figure 4d,e. In contrast to this
fragmentation pattern, the ion of m/z 585 of diPT3 38 produces
mainly an ion of m/z 331 upon UVPD, with a low-abundance
ion of m/z 319. Natural diPT exclusively produces an ion of m/z
319, suggesting that the sn-configuration of natural diPT aligns
with the sn-configuration of synthetic diPT1. These results
indicate that natural diPT contains the 16:0 acyl chain at the sn-1
fatty acid position and the 17:1(C9Δ) acyl chain at the sn-2 fatty
acid position. The data also suggest that both diPT1 and diPT3
contain low-abundance impurities of the opposite sn-isomer
which were not detected using NMR analysis. These impurities
are suspected to have arisen in the epoxide opening (10 + 11) or
the DCI-mediated phosphate coupling (5 + 6). Examples of
positive mode MS1 and MS/MS spectra for diPT samples are
provided in Figures S2 and S3. In summary, UVPD-MS
provided characterization of key structural elements including
cyclopropane localization and sn-determination, i.e., regiochem-
istry, for both synthetic and natural diPT species.
In conclusion, the synthesis of diphosphatidyltrehalose (diPT,
1), an immunogenic trehalose phospholipid from Salmonella
Typhi, has been achieved. The synthesis of 1 (diPT1) and its
regioisomer 38 (diPT3) allowed the determination of the fatty
acid regiochemistry in natural diPT. It is shown that UVPD-MS
is able to solve this long-standing problem of fatty acid
regiochemistry by comparing the hybrid MS3 HCD/UVPD
fragmentation patterns of natural and synthetic compounds.
Additionally, using this state-of-the-art technique, we deter-
mined the cyclopropyl group to be in the 9,10-position of the
acyl chain as previously inferred. Access to synthetic, naturally
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DOI: 10.1021/acs.orglett.9b01725
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