Chemical Synthesis and Self-Assembly of a Ladderane Phospholipid

Article abstract of DOI:10.1021/jacs.6b10706

Ladderane lipids produced by anammox bacteria constitute some of the most structurally fascinating yet poorly studied molecules among biological membrane lipids. Slow growth of the producing organism and the inherent difficulty of purifying complex lipid mixtures have prohibited isolation of useful amounts of natural ladderane lipids. We have devised a highly selective total synthesis of ladderane lipid tails and a full phosphatidylcholine to enable biophysical studies on chemically homogeneous samples of these molecules. Additionally, we report the first proof of absolute configuration of a natural ladderane.

Full text of DOI:10.1021/jacs.6b10706

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Communication
Chemical synthesis and self-assembly of a ladderane phospholipid
Jaron A M Mercer, Carolyn M. Cohen, Steven R. Shuken, Anna M. Wagner, Myles W. Smith, Frank R.
Moss, Matthew D Smith, Riku Vahala, Alejandro Gonzalez-Martinez, Steven G. Boxer, and Noah Z. Burns
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b10706 • Publication Date (Web): 21 Nov 2016
Downloaded from http://pubs.acs.org on November 23, 2016
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Chemical synthesis and self-assembly of a ladderane phos-
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Jaron A. M. Mercer,1 Carolyn M. Cohen,1 Steven R. Shuken, Anna M. Wagner,1 Myles W. Smith,1 1
Frank R. Moss III,1 Matthew D. Smith,1 Riku Vahala,2 Alejandro GonzalezꢀMartinez,2* Steven G. Boxꢀ
er,1* and Noah Z. Burns1*
1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
2Department of Built Environment, School of Engineering, Aalto University, Aalto, FIꢀ00076 Espoo, Finland.
*Correspondence to: alejandro.gonzalezmartinez@aalto.fi, sboxer@stanford.edu, nburns@stanford.edu
Supporting Information Placeholder
In order to circumvent these difficulties, we designed a
synthetic route that would allow us to access any ladꢀ
derane lipid.
ABSTRACT: Ladderane lipids produced by anammox
bacteria constitute some of the most structurally fasciꢀ
nating yet poorly studied molecules among biological
membrane lipids. Slow growth of the producing organꢀ
ism and the inherent difficulty of purifying complex
lipid mixtures have prohibited isolation of useful
amounts of natural ladderane lipids. We have devised a
highly selective total synthesis of ladderane lipid tails
and a full phosphatidylcholine in order to enable bioꢀ
physical studies on chemically homogeneous samples of
these molecules. Additionally, we report the first proof
of absolute configuration of a natural ladderane.
Ladderane phospholipids (e.g. 1, Scheme 1) isolated
from anammox enrichment cultures comprise a mixture
containing [5]ꢀladderane tails, as in acid 2, and [3]ꢀ
ladderane tails, as in alcohol 3; the nomenclature refers
to the number of cyclobutane rings at the terminus of the
chain.1,5 Typically, a [3]ꢀladderane is attached to the cenꢀ
tral snꢀ2 position by an ether linkage; [5]ꢀ and [3]ꢀ ladꢀ
derane lipids, as well as common straightꢀchain lipids,
are found etherꢀ or esterꢀlinked at the snꢀ1 position. Poꢀ
lar head groups at the snꢀ3 position include phosphochoꢀ
line, phosphoethanolamine, and phosphoglycerol.
Innovative synthetic efforts from Corey and Mascitti
resulted in a racemic synthesis of [5]ꢀladderanoic acid
(+/–)ꢀ2 and an enantioselective synthesis which utilized
preparative chiral HPLC to produce ca. 20 mg entꢀ2.6
Ladderane lipids, named for the ladderꢀlike polycyꢀ
clobutane motifs in their hydrophobic tails, are produced
by anaerobic ammonium oxidizing (anammox) bacteria
as a significant fraction of their membrane lipids.1 In a
large intracellular compartment called the anammoxoꢀ
some, anammox bacteria couple ammonium and nitrite
to produce dinitrogen as their principle source of enerꢀ
gy.2 Experimental and computational evidence suggests
that ladderane lipids form a densely packed membrane
around the anammoxosome and serve to limit the transꢀ
membrane diffusion of toxic and/or valuable metabolites
from anammox catabolism.3 Ladderane lipids likely imꢀ
part a number of previously unobserved characteristics
in membrane settings, but their biophysical characterizaꢀ
tion has been hampered by a lack of access to pure mateꢀ
rial.3a An axenic culture of anammox bacteria is not
available, and growth rates of enrichment cultures in
laboratory reactors are slow (doubling times on the order
of 1–2 weeks).4 Furthermore, anammox bacteria proꢀ
duce an array of ladderane and non ladderane lipids,
which have proven inseparable on a preparative scale.3a
Scheme 1. Retrosynthetic analysis of a natural lad-
derane phospholipid
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Scheme 2. Synthesis of a [5]-ladderanoic acid
Page 2 of 6
1
2
3
4
5
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8
Cl
H
H
H
H
H
H
H
H
H
H
H
1. Mn(TMP)Cl
NaOCl (40%)
1. MsCl (94%)
KOt-Bu
CuOTf
HO
HO
O
S
2. KOt-Bu
(91%)
2. Na2S;
H2O2 (93%)
3. SO2Cl2
(89%)
DMSO
(49%)
hν = 254 nm
C6H6, –4 ˚C
(42%)
H
H
8
H
5
9
4
12
Cu(MeCN)4PF6
(R)-DM-SEGPHOS
B2pin2, NaOt-Bu
bicyclohexene
>15 g per batch
[X-ray]
(95%, 90% ee)
>10 g produced
OTBS
1.
Li
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
pinB
H
H
H
H
H
H
H
5
HO
9
1. H2, Ra-Ni
HO
NBS, NaOMe
10
11
12
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16
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19
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60
O
2. CrO3/H2SO4
(86%, 2 steps)
2. HF•pyr
(88%, 2 steps)
H
2: [5]-ladderanoic acid
> 600 mg produced
14
13
No synthesis of a [3]ꢀladderane lipid has been reported.
Despite attempts towards its elucidation, the biosyntheꢀ
sis of ladderanes remains unsolved.7
small amounts of 4 could be observed in reactions conꢀ
ducted in nonꢀcoordinating heptane (entry 3). In a key
discovery, we found that the electrocyclic ring opening
of 5 to 10 is mediated by CuOTf even in the absence of
light, and that this undesired pathway could be slowed at
temperatures below 0 ºC (entries 4–6). Reactions perꢀ
formed in toluene – despite its high absorbance cutoff
(284 nm) – delivered 5 in an improved 28% yield (entry
7). Surprisingly, the reaction proceeds optimally in benꢀ
zene, which is frozen under these conditions (entries 8,
9).11 The [5]ꢀladderane pentacycle 4 can thus be obꢀ
tained in 42% isolated yield. That we see more than one
turnover of the CuOTf catalyst seems to indicate that
there is some degree of mobility in the solvent matrix.
No reaction occurs in the absence of catalyst. Despite its
moderate efficiency, this transformation is highly enaꢀ
bling: the entire pentacyclobutane core is assembled in
just five steps.
An outline of our retrosynthetic strategy is illustrated
in Scheme 1. For both 2 and 3 we planned to install the
linear alkyl component at a late stage, focusing first on
the polycyclic cores. We targeted the symmetric pentaꢀ
cycle 4, consisting of all of the cyclobutanes in the [5]ꢀ
ladderane core, as a key intermediate, which we hoped
to construct by dimerization of bicyclohex[2.2.0]ene 5.
For [3]ꢀladderanol 3, retrosynthetic installation of two
ketones and an alkene on the cyclohexane ring as in 6
enabled two key disconnections: a conjugate addition
reaction to install the linear alkyl chain, and an enoneꢀ
olefin [2+2] photocycloaddition between a benzoquiꢀ
none equivalent (7) and bicyclohexene 5.
Having identified 5 as a key building block for the
synthesis of both ladderane tails, we first developed a
procedure to obtain this strained olefin in large quantiꢀ
ties.8 Our optimized route commences with diol 8
(Scheme 2), which is commercially available or easily
accessible from the photocycloadduct of ethylene and
maleic anhydride (see Supporting Information). In three
steps we reached αꢀchlorosulfoxide 9 as an inconsequenꢀ
tial mixture of diastereomers. Treatment of 9 with exꢀ
cess potassium tertꢀbutoxide effected an atypical sulfoxꢀ
ide Ramberg–Bäcklund olefination providing, after disꢀ
tillation, over 15 grams of 5 per batch. The use of a sulꢀ
foxide in place of the typical sulfone precursor was cruꢀ
cial for high yields in this transformation. The observaꢀ
tion that the sulfoxide variant of the Ramberg–Bäcklund
reaction excels in constructing strained cyclobutenes
was first made by Weinges in the early 1980s, but this
procedure has seen no use in synthesis.9
Table 1. Optimization of bicyclohexene [2+2]a
In order to accomplish the conversion of 5 to [5]ꢀ
ladderane core 4, we hoped to employ a procedure deꢀ
veloped by Salomon and Kochi for the dimerization of
strained, but otherwise unactivated, olefins.10 Unfortuꢀ
nately, irradiation of THF or ether solutions of 5 with
254 nm light in the presence of catalytic copper(I) triꢀ
flate (CuOTf) resulted exclusively in the formation of
1,3ꢀcyclohexadiene (10) and a mixture of hexatriene
isomers (11) (Table 1, entries 1 and 2). Encouragingly,
aReactions were conducted on 1 mmol scale in 1 mL inꢀ
dicated solvent. Yields calculated by comparison to H
b
1
NMR internal standard. cOnly 10.
Tetramesitylporphyrinatomanganese(III)
chlorideꢀ
catalyzed C–H chlorination according to Groves’ proceꢀ
dure12 was uniquely capable of functionalizing 4,
providing 40% of an intermediate chloroꢀladderane
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Scheme 3. Synthesis of a [3]-ladderanol.
1
2
3
4
OH
O
O
O
O
OPMB
H
H
H
H
H
H
H
H
H
H
H
H
H
H
hν = 350 nm
Br
Br
Br
Br
Br
Br
pyridine
CrO3/H2SO4
(82%)
ZnI
PMBO
8
0 ºC
6
H
(78%)
(73%, 2 steps)
5
6
7
8
OH
O
O
(–)-15
17
19
16
(99% ee)
18
(88% ee)
[X-ray]
> 4 g produced
1. DDQ
2. DIBAL–H
H
5
H2NNH2
NH2
1. Crabtree's
cat., H2
(99%)
9
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HO
10
11
12
13
14
15
16
17
18
19
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60
Cu-TMEDA
PMBO
8
PMBO
8
1,4-CHD
(59%, 2 steps)
2. DDQ (89%)
H
H
H
3: [3]-ladderanol
> 600 mg produced
22
21
20
[X-ray]
H2N
along with 18% recovered 4. This is the first report of
termediates 18 and 19 (as the crystalline derivative 20,
Scheme 3) were confirmed by Xꢀray crystallography. All
that remained to elaborate 19 to a [3]ꢀladderane was the
complete reduction of the sixꢀmembered ring. While
attempting a double Wolff–Kishner reduction on bisꢀ
hydrazone 21 (Scheme 3), we discovered instead the
formation of a trace amount of 1,3ꢀcyclohexadiene 22,
arising from an apparent 2ꢀelectron oxidation and reꢀ
lease of two molecules of dinitrogen. After extensive
optimization we arrived at commercial CuꢀTMEDA as
an effective oxidant and 1,4ꢀcyclohexadiene as an H
atom source for this transformation. The resulting diene
22 underwent diastereoselective hydrogenation under
action of Crabtree’s catalyst. Finally, cleavage of the
PMB ether completed our synthesis of [3]ꢀladderanol 3.
this chemistry on a cyclobutane. Subsequent elimination
with potassium tertꢀbutoxide delivered olefin 12
(Scheme 2). Desymmetrization of 12 was accomplished
by enantioselective hydroboration with a recently disꢀ
closed copper–DMꢀSEGPHOS catalyst,13 delivering
pinacolboronic ester 13 in 95% yield and 90% ee. We
employed a Zweifel olefination to install the 8ꢀcarbon
alkyl chain.14 Following deprotection of the primary
alcohol, we obtained alkene 14 in 88% yield over two
steps. Hydrogenation with RaꢀNi yielded a crystalline
[5]ꢀladderanol intermediate (Xꢀray: Supporting Inforꢀ
mation). Subsequent Jones oxidation completed our synꢀ
thesis of the [5]ꢀladderanoic acid 2.
Our synthesis of [3]ꢀladderanol 3 (Scheme 3) centered
on the use of chiral dibromobenzoquinone 16 both as a
[2+2] partner for a highꢀyielding photocycloaddition
with bicyclohexene 5 and as a means to relay stereoꢀ
chemistry into the [3]ꢀladderane core. Enantioenriched
16 is easily accessible from known lipaseꢀresolved diꢀ
bromodiol (–)ꢀ15 following oxidation.15 Both π faces of
16 are equivalent, and photocycloaddition with 5 from
either side provides the same enantiomer of dibromoꢀ
diketone 17. Upon treatment with pyridine, 17 underꢀ
goes selective elimination via proton abstraction from
the convex face to form vinyl bromide 18 with slight
erosion of enantiopurity. Enabled by the C2ꢀsymmetry of
16, this twoꢀstep sequence effectively achieves an enanꢀ
tioselective [2+2] cycloaddition to desymmetrized 18.
Table 2. Enantiodivergent coupling strategiesa
b
aReactions conducted with 80% ee 18. Yields reflect
c
isolated yields after silica chromatography. Enantiomeric
Initial attempts to install the linear alkyl chain by
Negishi cross coupling were accompanied by significant
loss of enantiopurity (80% ee 18 to +30% ee 19, Table 2,
entry 1). We reasoned that two pathways might be comꢀ
peting: (1) palladiumꢀcatalyzed cross coupling at the site
of bromine substitution and (2) uncatalyzed addition of
the alkylzinc iodide reagent at the unsubstituted posiꢀ
tion, followed by elimination of HBr.16 Indeed, treatꢀ
ment of 18 with an alkylzinc iodide reagent in the abꢀ
sence of catalyst delivered 19 with inversion of the subꢀ
stitution pattern (entry 2). Alternatively, replacing the
nucleophilic zinc reagent with a potassium trifluoroboꢀ
rate salt resulted in an enantioretentive Suzuki cross
coupling (entry 3). The absolute configurations of inꢀ
excess determined by chiral HPLC.
The glycerol stereocenter of ladderane phospholipids
has been established to be (R), as drawn in 1, which is
consistent with other prokaryotic membrane phospholipꢀ
ids.17 However, the absolute stereochemistry of the ladꢀ
derane tails has remained unknown. With the goal of
completing this assignment, we conducted a lipid extracꢀ
tion of biomass from an anammox enrichment culture
grown over four months in a laboratoryꢀscale bioreacꢀ
tor.18 Cognizant that [3]ꢀladderane glycerol diol 23
(Scheme 4, top) is one of the most readily separable
components of ladderane lipid mixtures,1,5 we treated
our crude extracts directly with lithium aluminum hyꢀ
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Page 4 of 6
Scheme 4. Absolute configuration of [3]-ladderane and the synthesis of a ladderane phospholipid.
1
2
3
4
5
HO
OH
1. NaH (91%)
1. extraction
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
RO
2. LiAlH4
(0.025%)
[ca. 20 mg]
TrO
OTr
OH
24
2. HCl (71%)
89% ee
23
6
7
8
R = H: 3
R = Ms: 25
natural [α]D24 = +14.1
synthetic [α]D24 = +12.4
MsCl
(97%)
80 g granular biomass
9
O
O
H
H
H
H
H
H
H
H
H
1. NaH, 25 (60%)
2. HCl (86%)
3. EDC, 2 (74%)
4. DDQ (89%)
OTr
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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gentle hydration
37 to 23 ºC
O
P
PMBO
Me3N
O
O
OH
O
5.
H
H H H
O
O
Cl
P
O
26
O
1
6. Me3N (50%, 2 steps)
Tm hydrated bilayers = 9.3 ºC
H
H H
giant unilamellar vesicles
dride to reduce all ester and phosphoester linkages and
enrich for 23. From 80 grams of dried biomass we obꢀ
tained a sufficient amount of 23 for an optical rotation
([α]D24 = +14.1). Synthetic 23 prepared via alkylation of
glycerol 24 with [3]ꢀladderanol mesylate 25 was also
the biophysical properties of ladderane phospholipids.
Efforts to validate the hypothesis that ladderanes prevent
diffusion of small anammox metabolites are underway.
The synthetic route presented here will allow efficient
access to a range of natural and nonꢀnatural ladderane
phospholipids, enabling full exploration of the structural
and functional space around these fascinating molecules.
24
found to be dextrorotatory ([α]D = +12.4 at 88% ee),
establishing the natural configuration of 23 as drawn.
Natural [5]ꢀladderane alcohol from our extracts was
inseparable from the corresponding [3]ꢀladderane alcoꢀ
hol and other straightꢀchain lipid alcohols. To the best of
our knowledge natural [5]ꢀladderane, first characterized
as its methyl ester, has never been completely separated
from contaminating [3]ꢀladderane methyl ester,1,5a makꢀ
ing synthesis the only way to obtain pure [5]ꢀladderane
lipids. We have assumed by analogy to 3 that the conꢀ
figuration of natural 2 is as drawn (levorotatory).
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website.
Experimental procedures, characterization, and spectral
data (PDF). Cif files (PDF).
AUTHOR INFORMATION
Corresponding Author
More than 20 mg of natural ladderane phosphocholine
lipid 1 was readily prepared by a short sequence
(Scheme 4, bottom). Alkylation of the enantiopure glycꢀ
erol derivative 26 with mesylate 25 installed a [3]ꢀ
ladderane at the snꢀ2 position. Removal of the trityl
group and esterification with 2 installed a [5]ꢀladderane
at the snꢀ1 position. Removal of the PMB group and
installation of the phosphocholine head group completed
the route to 1.
alejandro.gonzalezmartinez@aalto.fi,
sboxer@stanford.edu, nburns@stanford.edu
Funding Sources
This work was supported by Stanford University, the Terꢀ
man foundation, the National Institutes of Health
(GM069630 & GM118044 to SGB), the National Science
Foundation (graduate fellowships to JAMM, CMC, SRS,
and MDS), and the Center for Molecular Analysis and Deꢀ
sign (graduate fellowship to FRM).
Despite the crystallinity of multiple synthetic intermeꢀ
diates towards 2 and 3, preliminary evidence indicates
that pure 1 forms fluid hydrated bilayers at ambient
temperature. Differential scanning calorimetry of aqueꢀ
ous dispersions of 1 revealed a single transition at 9.3
ºC. Glassꢀsupported bilayers, a common model for bioꢀ
logical membranes, of 1 showed full fluorescence reꢀ
covery after photobleaching, indicating free lateral moꢀ
bility within the bilayer (see Supporting Information).
Upon gentle hydration, 1 selfꢀassembled into giant
unilamellar vesicles (Scheme 4, bottom and Supporting
Information) with uniform incorporation of a fluoresꢀ
cently labeled lipid (0.1% Texas Red DHPE). The disꢀ
covery that pure 1 selfꢀassembles under conditions
standard for natural acyclic phosphocholine lipids sugꢀ
gests that conventional techniques can be used to probe
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We are grateful to K. V. Pedram and A. H. Valentine for
experimental assistance, to Dr. A. Oliver (University of
Notre Dame) and Dr. S. Teat (Lawrence Berkeley National
Laboratories) for Xꢀray crystallographic analysis, to Dr. S.
Lynch (Stanford University) for assistance with NMR specꢀ
troscopy, and to Prof. J. Groves (Princeton University) for
helpful discussion. Prof. M. V. MartinezꢀToledo (Universiꢀ
ty of Granada) is acknowledged for microbiological assisꢀ
tance.
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(6) (a) Mascitti, V.; Corey, E. J. J. Am. Chem. Soc. 2004, 126,
15664–15665. (b) Mascitti, V.; Corey, E. J. J. Am. Chem. Soc. 2006,
128, 3118–3119.
(7) (a) Rattray, J. E.; Strous, M.; Op den Camp, H. J. M.; Schouten,
S.; Jetten, M. S. M.; Sinninghe Damsté, J. S. Biol. Direct 2009, 4, 8.
(b) Javidpour, P.; Deutsch, S.; Mutalik, V. K; Hillson, N. J.; Petzold,
C. J.; Keasling, J. D.; Beller, H. R. PLoS ONE 2016, 11, e0151087.
(8) An existing route to 5, though expeditious, was deemed ill suitꢀ
ed for scales larger than 1 gram: McDonald, R. N.; Reineke, C. E. J.
Org. Chem. 1967, 32, 1878–1887.
(9) Weinges, K.; Sipos, W.; Klein, J.; Deuter, J.; Irngartinger, H.
Chem. Ber. 1987, 120, 5–9.
(10) Salomon, R. G.; Kochi, J. K. J. Am. Chem. Soc. 1974, 96,
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documented: (a) Beukers, R.; Berends, W. Biochim. Biophys. Acta.
1960, 41, 550–551. (b) Beukers, R.; Berends, W. Biochim. Biophys.
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