Membrane-interacting properties of the functionalised fatty acid moiety of muraymycin antibiotics

Article abstract of DOI:10.1039/c4md00526k

Functional insights into bioactive natural products with medicinal potential are often hindered by their structural complexity. We herein report a simplified model system to investigate the functional significance of a structural motif of biologically potent muraymycin antibiotics of the A-series. These compounds have a highly unusual ω-guanidinylated fatty acid moiety, which has been proposed to mediate membrane penetration, thus enabling the interaction of A-series muraymycins with their intracellular target MraY. Our assay was based on a synthetic conjugate of this fatty acid structure with a negatively charged fluorophore lacking membrane permeability. Using this conjugate, immobilised giant unilamellar lipid vesicles and confocal laser scanning fluorescence microscopy, we demonstrated that the attachment of the ω-N-hydroxy-guanidinyl fatty acid unit led to an enhanced uptake of the fluorophore into the vesicles. This represents the first experimental evidence of this unusual structural motif's functional relevance for the parent natural product, which may support the future design of novel muraymycin analogues.

Full text of DOI:10.1039/c4md00526k

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Membrane-interacting properties of the
functionalised fatty acid moiety of muraymycin
antibiotics†
Cite this: Med. Chem. Commun.,
2015, 6, 879
a
a
a
ab
*
Oliver Ries, Christian Carnarius, Claudia Steinem and Christian Ducho
Functional insights into bioactive natural products with medicinal potential are often hindered by their
structural complexity. We herein report a simplified model system to investigate the functional significance
of a structural motif of biologically potent muraymycin antibiotics of the A-series. These compounds have
a highly unusual ω-guanidinylated fatty acid moiety, which has been proposed to mediate membrane pen-
etration, thus enabling the interaction of A-series muraymycins with their intracellular target MraY. Our
assay was based on a synthetic conjugate of this fatty acid structure with a negatively charged fluorophore
lacking membrane permeability. Using this conjugate, immobilised giant unilamellar lipid vesicles and con-
focal laser scanning fluorescence microscopy, we demonstrated that the attachment of the ω-N-hydroxy-
guanidinyl fatty acid unit led to an enhanced uptake of the fluorophore into the vesicles. This represents
the first experimental evidence of this unusual structural motif's functional relevance for the parent natural
product, which may support the future design of novel muraymycin analogues.
Received 18th November 2014,
Accepted 6th March 2015
DOI: 10.1039/c4md00526k
www.rsc.org/medchemcomm
via inhibition of the bacterial membrane protein MraY, a key
Introduction
enzyme in the intracellular part of peptidoglycan
Bacterial strains with resistances towards established antibi-
otics continue to emerge. However, only very few new anti-
bacterial agents have been developed in recent years.1,2 It is
therefore highly desirable to identify novel antimicrobial com-
pounds displaying new or yet unexploited modes of action.
Starting with the development of the penicillins in the 1940s,
natural products and their analogues have served as clinically
useful antibiotic agents. Thus, one may assume that naturally
occurring antibiotics will probably play a key role in overcom-
ing the current lack of new antibacterials for clinical use. This
will include the identification of such compounds, their total
synthesis for structure–activity relationship (SAR) studies, and
also their functional elucidation. Due to the structural com-
plexity of many natural products though, their functional
analysis is often not trivial and requires time-consuming
multi-step syntheses of suitable molecular probes.
formation.5–9 As the active site of MraY is located at the cyto-
solic side of the bacterial membrane, MraY inhibitors need
to be able to penetrate the bacterial cell wall and the plasma
membrane.
Streptomyces-produced
muraymycins
(e.g.
muraymycins A1 1 to A5 5, B5 6, C4 7 and D2 8, Fig. 1) repre-
sent one subclass of nucleoside antibiotics. They show prom-
ising activity against Staphylococcus aureus and Enterococcus
strains via inhibition of MraY.10
Some SAR data have already been reported for
muraymycins and their analogues.11–15 However, a structural
feature of muraymycins which has only found limited atten-
tion so far is the presence of ω-guanidinylated fatty acid moie-
ties in the biologically most potent muraymycins of the A-
series. These fatty acid units are attached to the muraymycin
backbone via esterification of a 3-hydroxy-L-leucine motif.
Muraymycins of the B-series carry shorter and
unfunctionalised fatty acids, while the fatty acid unit is miss-
ing in muraymycins of the C- and D-series. As a general rule,
the A-series muraymycins show the best antibacterial activi-
ties, followed by the B-series congeners, while C- and D-series
muraymycins are pronouncedly less active.10 These SAR find-
ings might indicate (i) that the presence of the fatty acid moi-
ety mediates membrane penetration of the otherwise polar
muraymycin scaffold14 (and therefore cellular uptake of the
acylated congeners) and (ii) that ω-guanidinylated fatty acids,
despite the polarity of the ω-functionality, provide an even
more pronounced membrane-penetrating effect.
Nucleoside antibiotics represent a class of natural prod-
ucts targeting cell wall biosynthesis.3,4 Antibacterially active
nucleoside antibiotics interfere with peptidoglycan assembly
a Georg-August-University Göttingen, Department of Chemistry, Institute of
Organic and Biomolecular Chemistry, Tammannstr. 2, 37 077 Göttingen,
Germany. E-mail: christian.ducho@uni-saarland.de
b Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal
Chemistry, Campus C2 3, 66 123 Saarbrücken, Germany
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of synthesised compounds. See DOI: 10.1039/c4md00526k
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Fig. 1 Selected naturally occurring muraymycin nucleoside antibiotics 1–8.
The most obvious strategy to experimentally study this
hypothesis would be to synthesise fluorescently labelled ana-
logues of O-acylated muraymycins and to test them for mem-
brane interaction and permeability e.g. with lipid vesicles.
However, an attached fluorophore would potentially alter the
molecular properties of muraymycins to a significant degree.
Furthermore, the total synthesis of muraymycin-derived fluo-
rescent probes would be time-consuming. We therefore
decided to design a pronouncedly simplified model system to
assay the fatty acid moiety of muraymycin A1 1 (residue X1,
Fig. 1) for its ability to mediate membrane interaction and
penetration. The goal was to study potential accumulation at
the membrane interface and a possible increase of mem-
brane permeability.
exhibits large photostability. It was envisioned to convert a
commercially available azide-labelled derivative 9 of AF488
with the propargyl ester 10 of the fatty acid moiety of
muraymycin A1 1 (subsequently named “lipid side chain”,
LSC, Fig. 2B). Using the copper-catalysed version of the
Huisgen alkyne–azide cycloaddition (“click”-chemistry),16–18
this should furnish AF488 LSC conjugate 11. It can then be
tested if the presence of the LSC unit makes the AF488 moi-
ety membrane-permeable, i.e. if fluorescence can be detected
within lipid vesicles upon treatment with 11 (Fig. 2A). In
order to ensure that the linker unit, i.e. the 1,4-substituted
triazole, does not influence the low membrane permeability
of the fluorescent dye, acetyl derivative 12 (“AF488 acetate”)
lacking the full-length LSC motif was envisioned to serve as a
reference (Fig. 2B).
Results and discussion
Experimental design
Synthesis of fluorescent probes
The concept of the employed assay is depicted in Fig. 2. The
fluorescent dye AlexaFluor 488 (AF488) was chosen as it
For the synthesis of AlexaFluor 488 LSC conjugate 11,
propargyl ester 10 had to be prepared first (Scheme 1).
Fig. 2 A: Simplified assay system to test the influence of the fatty acid moiety of muraymycin A1 1 (LSC) on membrane-penetrating properties of
an attached structure lacking membrane permeability (schematic representation). B: AlexaFluor 488 derivatives 11 and 12 conceived for the assay.
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Scheme 1 Synthesis of AlexaFluor 488 derivatives 11 and 12.
Starting from erucic acid 13, epoxidation and alkaline epox-
ide opening gave 13,14-dihydroxy behenic acid 14 as a mix-
ture of stereoisomers in 94% yield. Esterification with
propargyl alcohol by carbodiimide activation then furnished
propargylic ester 15 in 52% yield. Subsequent periodate cleav-
age afforded aldehyde 16, i.e. an ω-functionalized tridecanoic
acid propargyl ester, in 85% yield. Aldehyde 16 was then
employed in a sequence of oxime formation (product 17,
92% yield) and reduction to give N-alkyl hydroxylamine 18
(66% yield for the reduction step). As part of our synthetic
studies on muraymycins and their analogues,19–26 we have
systematically investigated the preparation of N-alkyl-N-
hydroxy-guanidines by guanidinylation27 of N-alkyl hydroxyl-
amine precursors.28 A protecting group-free method using
guanidinylation reagent 19 turned out to be the best method
for this transformation. Consequently, treatment of 18 with
19 gave the desired N-alkyl-N-hydroxy-guanidine 10 with high
conversion, but due to difficult purification of the amphi-
philic product, in a moderate yield of 47% (Scheme 1).
by mass spectrometry as the amount was insufficient for
NMR spectroscopy. AF488 acetate 12 was prepared in an anal-
ogous manner using propargyl acetate 20 as the alkyne com-
ponent for the “click” reaction (Scheme 1), and both target
compounds 11 and 12 were thus available for the aforemen-
tioned fluorescence-based assay.
Membrane partitioning assay
The influence of AlexaFluor 488 LSC conjugate 11 on lipid
membranes was investigated by means of fluorescence
microscopy based on giant unilamellar vesicles (GUVs). GUVs
composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), fixed to a silicon substrate via biotin–avidin interac-
tion, were incubated with conjugate 11 and confocal laser
scanning microscopy images were taken. The fluorescence
image depicted in Fig. 3 clearly shows that conjugate 11
Unexpectedly, the subsequent copper-catalysed “click”
reaction towards conjugate 11 turned out to be non-trivial.
First attempts under standard conditions only gave low con-
versions, which we speculate to be a consequence of the
amphiphilic nature of alkyne 10. Following a thorough varia-
tion of the reaction conditions, the reaction could be signifi-
cantly improved by reductive in situ-generation of the
copperIJI) catalyst, application of the solvent system water/
DMF/methanol and addition of the detergent Triton X-100.
However, due to the high costs for azide-labelled AF488 deriv-
ative 9, the reaction could only be performed on the sub-mg
scale, thus making an unambiguous determination of the
yield difficult. Product 11 could be obtained after HPLC puri-
fication, and the identity of the product could be confirmed
Fig. 3 A: Fluorescence image of GUVs composed of POPC after
incubation with 0.8 μM AF488 LSC conjugate 11. The compound
accumulates in the lipidic phase of the GUVs and is partially also
localised inside the GUVs. B: Fluorescence image of POPC-GUVs after
incubation with 0.8 μM AF488 acetate 12. AF488 acetate 12 does not
accumulate at the membrane interface, but it is found in some of the
GUVs. Scale bars (white boxes): 20 μm, POPC = 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine.
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Fig. 4 A: Fluorescence image showing an individual GUV composed
of POPC/POPE (7 : 3) with increased fluorescence intensity at the
membrane interface and fluorescence intensity inside the GUV after
incubation with AF488 LSC conjugate 11. B: Line profile along the
white line shown in A clearly demonstrating the fourfold increase in
fluorescence intensity at the lipid membrane. POPC = 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine, POPE
sn-glycero-3-phosphoethanolamine.
= 1-palmitoyl-2-oleoyl-
accumulates in the lipid membrane (also see Fig. 4). Part of
the GUVs is also filled with the conjugate 11. As a control,
the same experiment was performed with AF488 acetate 12
lacking the fatty acid moiety. In this case, no enrichment of
the dye in the lipid membrane was observed. To quantita-
tively analyse the number of GUVs which were filled with the
fluorescent conjugate, we defined a GUV as dye-filled if F/F0
> 0.5, with F being the intensity of the GUV interior and F0
being the background intensity. 24% of the POPC GUVs were
filled with AF488 LSC conjugate 11 after 40 min and only 6%
after incubation with AF488 acetate 12 (with the entire distri-
bution for F/F0 > 0.5 being taken into account, Fig. 5A). It
should be noted that values F/F0 > 1.0 could be ruled out as
this would have required an active transport process (equilib-
rium is reached for F/F0 = 1.0). Active transport processes
need external sources of energy (e.g. an electrochemical gra-
dient or light coupling) though, and these were absent in the
employed assay.
To investigate the influence of the lipid mixture on the
dye distribution, we used a POPC/POPE IJ1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphoethanolamine, 7 : 3) mixture to form
GUVs better resembling the composition of bacterial mem-
branes. Also for POPC/POPE membranes, an increased fluo-
rescence intensity in the membrane interface as a result of
accumulation of AF488 LSC conjugate 11 was found, leading
to an almost four times larger fluorescence intensity at the
membrane compared to the bulk solution (Fig. 4). 14% of the
GUVs were filled with AF488 LSC conjugate 11, but only 4%
in case of AF488 acetate 12 (Fig. 5B). Distribution patterns
were similar to those found for the POPC GUVs (vide supra,
also see Fig. 5A).
Fig. 5 A: Statistical analysis of dye-filled POPC GUVs after the addition
of AF488 LSC conjugate 11 and AF488 acetate 12. 24% of the GUVs (n
= 410) were filled with AF488 LSC conjugate 11, while only 6% (n =
283) were filled with AF488 acetate 12. B: For POPC/POPE (7 : 3) GUVs
incubated with 11 or 12, 14% of the GUVs were filled with AF488 LSC
conjugate 11 (n = 246) and 4% with AF488 acetate 12 (n = 230). POPC
=
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,
POPE
=
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine.
the membrane interface, which can disturb the integrity of
the bilayer. From these results, we conclude that muraymycin
membrane partitioning and cell entry is probably facilitated
by the ω-guanidinylated lipophilic side chain.
The role of guanidine moieties in membrane penetration
and thus in cellular uptake processes has been widely investi-
gated and discussed.29 Arginine-rich cell-penetrating peptides
have been extensively studied,30–33 and guanidinylated cat-
ionic lipids were reported to be effective transfection
agents.34 Guanidinylated dendrimers were found to display
pronounced interactions with liposomal phospholipid mem-
branes,35 and conjugates of aminoglycoside antibiotics with
guanidinylated cationic lipids showed antibacterial activity.36
However, such oligo-guanidinylated systems are probably
prone to cooperative or multivalency effects, and it is there-
fore quite remarkable that a single ω-guanidinylated fatty
acid motif (as in A-series muraymycins or in AlexaFluor 488
LSC conjugate 11) can enable membrane interaction and
penetration as reported herein. It should be taken into
account though that the N-hydroxylated guanidine moiety of
A-series muraymycins and conjugate 11 is likely to have
reduced basicity in comparison to non-hydroxylated guanidines.
Based on the simplified model system, we were thus able
to provide evidence that the fatty acid moiety of A-series
muraymycins probably contributes to the accumulation of
these compounds at a lipid membrane. An increased number
of GUVs were filled with the lipidated fluorescent dye, which
might be a result of the accumulation of the compound at
Conclusions
In summary, we have developed an efficient and facile
method to study the interaction of a naturally occurring
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structural motif with lipid membranes. Using this assay, we
were able to demonstrate that the ω-functionalised fatty acid
moiety found in some muraymycin nucleoside antibiotics of
the A-series can mediate membrane accumulation and even
membrane penetration of a fluorescent dye. This represents
the first experimental evidence of this unusual structural
motif's functional relevance for the parent natural product.
While there is no close structural relation of the employed
fluorophore and the natural product scaffold, the relative
ease of our method will allow to test a significant number of
fatty acid motifs for their potential to mediate membrane
accumulation and penetration. This will be important to elu-
cidate the relevance of the ω-substituent as well as of the
length of the lipophilic moiety, i.e. the supposed interplay of
polar ω-functionalisation and lipophilicity. Based on the
results reported herein, we will therefore synthesise and
investigate varying fatty acid units found in the muraymycin
family as well as non-natural congeners with respect to dif-
ferent chain lengths and functional groups in the ω-position
(e.g. amino-substituted and unfunctionalised analogues).
Selected fatty acid structures will then be used for the syn-
thesis of muraymycin analogues with potentially improved
cellular uptake and thus also with enhanced biological activ-
ities. Overall, it is anticipated that these results will enable
the design of muraymycin analogues with optimised biologi-
cal profiles. This strategy might also have the potential to be
employed within SAR studies on other lipidated natural
products,37–41 and it might generally stimulate the functional
analysis of medicinally relevant natural products based on
simplified, chemically more tractable model systems.
Diode Array Detector (DAD) and a LiChroCart™ column (4 ×
125 mm) containing reversed phase silica gel Purospher™
RP18e (5 μm) purchased from VWR. 300 MHz-1H and 75
MHz-, 76 MHz-, and 126 MHz-13C NMR spectra were recorded
on Varian MERCURY 300, UNITY 300 and INOVA 500 spectro-
meters. All 13C NMR spectra are 1H-decoupled. All spectra
were recorded at room temperature except of samples in
DMSO-d6 (standard 35 °C) and where indicated otherwise
and were referenced internally to solvent reference frequen-
cies wherever possible. Chemical shifts (δ) are quoted in
ppm, and coupling constants (J) are reported in Hz. Assign-
ment of signals was carried out using 1H,1H-COSY, HSQC
and HMBC spectra obtained on the spectrometers mentioned
above. Low resolution ESI mass spectrometry was performed
on a Varian MAT 311 A spectrometer operating in positive or
negative ionisation mode. High resolution (HR) ESI mass
spectrometry was carried out on a Bruker microTOF spectro-
meter or a Bruker 7T FTICR APEX IV spectrometer. Melting
points (mp) were measured on a Büchi instrument and are
not corrected. Infrared spectroscopy (IR) was performed on a
Jasco FT/IR-4100 spectrometer equipped with an integrated
ATR unit (GladiATR™, PIKE Technologies). Wavenumbers (ν)
are quoted in cm−1. UV spectroscopy was carried out on a
Jasco V-630 spectrometer.
Propargyl 13-IJN-hydroxyguanidino)-tridecanoate 10. 1H-
Pyrazole-1-carboxamidine hydrochloride 19 (72 mg, 0.49
mmol) and NEt3 (0.14 mL, 0.10 g, 1.00 mmol) were added to
a solution of N-alkyl hydroxylamine 18 (100 mg, 0.353 mmol)
in DMF (2 mL) and the reaction mixture was stirred at rt for
15 h. The solvent was evaporated under reduced pressure
and the resultant residue was dissolved in sat. NH4Cl solu-
tion (5 mL). The aqueous solution was extracted with Et2O (3
× 10 mL) and the combined organics were dried over Na2SO4
and evaporated under reduced pressure. The resultant crude
product was purified by column chromatography IJCH2Cl2–
MeOH, 9 : 1 → 1 : 1) to give 10 (53 mg, 47%) as a colourless
solid; mp 77 °C; TLC Rf 0.28 IJCH2Cl2–MeOH, 4 : 1); IR (ATR)
νmax/cm−1 2920, 2848, 1728, 1626, 1468, 1166 and 1105; δH
(300 MHz, DMSO-d6) 1.09–1.36 (16 H, m, 4-H2-11-H2), 1.41–
1.65 (4 H, m, 3-H2, 12-H2), 2.30 (2 H, t, J 7.3, 2-H2), 3.50 (1 H,
d, J 2.5, 3′-H), 3.54 (2 H, t, J 7.0, 13-H2), 4.66 (2 H, d, J 2.5, 1′-
H2) and 7.61 (4 H, br s, NH, OH); δC (76 MHz, DMSO-d6)
24.3, 25.7, 25.7, 28.2, 28.5, 28.7, 28.8, 28.9, 33.1, 50.7, 51.4,
77.4, 78.4, 157.7 and 172.1; m/z (ESI+) 326.2 (M + H+); m/z
(HR-ESI+) 326.2438 (M + H+, C17H32N3O3 requires 326.2438).
AlexaFluor 488 lipid side chain conjugate 11. A solution of
azide-labelled AlexaFluor 488 9 (50 mM in DMF, 2.0 μL, 0.10
μmol 9), a solution of lipid side chain propargyl ester 10 (50
mM in MeOH, 8.0 μL, 0.40 μmol 10), a solution of sodium
L-ascorbate (20 mM in water, 4.0 μL, 80 nmol sodium
L-ascorbate), a solution of CuSO4 (10 mM in water, 4.0 μL, 40
nmol CuSO4), MeOH (6 μL) and Triton X-100 (10% in water,
48 μL) were mixed and stirred in a ThermoMixer (50 °C, 200
rpm) for 15 h. The reaction mixture was then directly purified
by preparative HPLC (MeCN–water (+0.01% TFA), 15 : 85 →
50 : 50, 1.0 mL min−1). Product-containing fractions were
Experimental
Synthesis
General methods. Azide-labelled AlexaFluor 488 was
obtained from Life Technologies. All other chemicals were
purchased from standard suppliers. SiO2-supported NaIO4
was prepared according to an established procedure.42,43
Reactions involving oxygen and/or moisture sensitive
reagents were carried out under an atmosphere of argon
using anhydrous solvents. Anhydrous solvents were obtained
in the following manner: CH2Cl2 was dried over CaH2 and
distilled, pyridine was dried over CaH2 and distilled, DMF
was dried over activated molecular sieves (4 Å) and degassed.
All other solvents were of technical quality and distilled prior
to their use, and deionised water was used throughout. Col-
umn chromatography was carried out on silica gel 60 (0.040–
0.063 mm, 230–400 mesh ASTM, VWR) under flash condi-
tions. TLC was performed on aluminium plates precoated
with silica gel 60 F254 (VWR). Visualisation of the spots was
carried out using UV light (254 nm) and/or staining under
heating (KMnO4 staining solution: 1 g KMnO4, 6 g K2CO3
and 1.5 mL 1.25 M NaOH solution, all dissolved in 100 mL
H2O). Preparative HPLC was performed on a VWR-Hitachi
system equipped with an L-2300 pump, an L-2200
autosampler, an L-2300 column oven (25 °C), an L-2455
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pooled, and the solvent was removed with an Eppendorf Con-
centrator 5301 to give 11 (estimated from UV-Vis data: 9.0
nmol) as a red solid; preparative HPLC tR 13.3 min; m/z
(ESI+) 984.3 (M + H+) and 1006.3 (M + Na+); m/z (HR-ESI+)
984.3594 (M + H+, C44H58N9O13S2 requires 984.3590).
640; δH (300 MHz, DMSO-d6) 0.84 (3 H, t, J 6.6, 22-H3), 1.12–
1.46 (32 H, m, 4-H2-12-H2, 15-H2-21-H2), 1.47–1.63 (2 H, m,
3-H2), 2.28 (2 H, t, J 7.4, 2-H2), 3.13 (1 H, t, J 2.4, 3′-H), 3.14–
3.27 (2 H, m, 13-H, 14-H), 3.89 (2 H, d, J 4.0, OH) and 4.61 (2
H, d, J 2.4, 1′-H2); δC (76 MHz, DMSO-d6) 13.6, 22.0, 25.3,
28.3, 28.5, 28.6, 28.7, 28.9, 28.9, 28.9, 29.0, 29.1, 31.2, 32.6,
24.2, 33.1, 51.1, 73.1, 77.8, 78.5 and 171.7; m/z (ESI+) 433.4
(M + Na+); m/z (HR-ESI+) 433.3288 (M + Na+, C25H46NaO4
requires 433.3288).
Propargyl 13-oxotridecanoate 16. To a solution of diol 15
(857 mg, 2.09 mmol) in CH2Cl2 (20 mL), SiO2-supported
NaIO4 (ref. 42 and 43) (0.610 mmol g−1, 5.14 g, 3.14 mmol
NaIO4) was added. The resultant suspension was stirred at rt
for 1 h and then filtered. The filtrate was evaporated under
reduced pressure. The resultant crude product was purified
by column chromatography (petroleum ether–EtOAc, 6 : 1) to
give 16 (473 mg, 85%) as a colourless oil; TLC Rf 0.29 (petro-
leum ether–EtOAc, 6 : 1); IR (ATR) νmax/cm−1 2924, 2853, 1738,
1724, 1157, 1106, 1025, 997 and 666; δH (300 MHz, DMSO-d6)
1.15–1.60 (18 H, m, 3-H2-11-H2), 2.32 (2 H, t, J 7.3, 2-H2), 2.40
(2 H, td, J 7.2, J 1.6, 12-H2), 3.47 (1 H, t, J 2.2, 3′-H), 4.67 (2
H, d, J 2.2, 1′-H2) and 9.66 (1 H, t, J 1.6, 13-H); δC (76 MHz,
DMSO-d6) 24.2, 28.2, 28.4, 28.5, 28.5, 28.6, 28.7, 28.7, 28.8,
33.1, 42.9, 51.3, 77.2, 78.4, 172.0 and 203.3; m/z (ESI+) 289.2
(M + Na+); m/z (HR-ESI+) 289.1778 (M + Na+, C16H26NaO3
requires 289.1774).
Propargyl 13-hydroxyiminotridecanoate 17. A solution of
aldehyde 16 (359 mg, 1.35 mmol) and hydroxylamine hydro-
chloride (470 mg, 6.76 mmol) in EtOH (5 mL) and pyridine (5
mL) was stirred in the presence of molecular sieve (3 Å) at rt
for 46 h. The reaction mixture was filtered through a short
pad of celite and the solvent of the filtrate was evaporated
under reduced pressure. The resultant crude product was
purified by column chromatography (petroleum ether–EtOAc,
4 : 1) to give 17 (349 mg, 92%) as a mixture of E/Z-isomers as
a colourless solid; TLC Rf 0.22 (petroleum ether–EtOAc, 4 : 1);
δH (300 MHz, C6D6) 1.05–1.31 (16 H, m, 4-H2-11-H2), 1.43–
1.61 (2 H, m, 3-H2), 2.01 (1 H, t, J 2.4, 3′-H), 2.06 (2 H, t, J 7.4,
2-H2), 2.31 (2 H, td, J 7.1, J 5.4, 12-H2), 4.44 (2 H, d, J 2.4, 1′-
H2), 6.51 (1 H, t, J 5.4, 13-H) and 8.94 (1 H, br s, OH); δC (126
MHz, C6D6) 25.2, 25.4, 26.4, 29.4, 29.7, 29.7, 29.8, 29.9, 29.9,
30.0, 34.1, 51.6, 74.8, 78.4, 152.2 and 172.2; m/z (ESI+) 304.2
(M + Na+); m/z (HR-ESI+) 304.1884 (M + Na+, C16H27NNaO3
requires 304.1883).
Propargyl 13-hydroxyaminotridecanoate 18. NaBH3CN
(1.18 g, 18.8 mmol) and freshly prepared HCl in MeOH (1 M)
were added alternately and portionwise to a solution of oxime
17 (500 mg, 1.78 mmol) and methyl orange (small amount,
indicator) in i-PrOH (25 mL) until the reaction mixture
retained a pink color. The reaction mixture was stirred at rt
for 24 h and then neutralised with NEt3 and evaporated
under reduced pressure. The resultant residue was dissolved
in EtOAc (50 mL), washed with sat. NaHCO3 (2 × 50 mL) and
brine (1 × 50 mL), dried over Na2SO4 and evaporated under
reduced pressure. The resultant crude product was purified
by column chromatography (EtOAc) to give 18 (334 mg, 66%)
AlexaFluor 488 acetyl conjugate 12. A solution of azide-
labelled AlexaFluor 488 9 (5 mM in DMF, 20 μL, 0.10 μmol
9), a solution of propargyl acetate 20 (1 M in MeOH, 14 μL,
14 μmol 20), a solution of sodium L-ascorbate (20 mM in
water, 10 μL, 0.20 μmol sodium L-ascorbate), a solution of
CuSO4 (10 mM in water, 10 μL, 0.10 μmol CuSO4) and Triton
X-100 (10% in water, 48 μL) were mixed and stirred in a
ThermoMixer (10 °C, 200 rpm) for 17 h. The reaction mixture
was then directly purified by preparative HPLC (MeCN–water
(+0.01% TFA), 15 : 85 → 50 : 50, 1.0 mL min−1). Product-
containing fractions were pooled, and the solvent was
removed with an Eppendorf Concentrator 5301 to give 12
(estimated from UV-Vis data: 32 nmol) as a red solid; prepar-
ative HPLC tR 6.8 min; m/z (ESI−) 755.1 (M − H+); m/z (HR-
ESI−) 755.1446 (M − H+, C32H31N6O12S2 requires 755.1447).
13,14-Dihydroxybehenic acid 14. A solution of H2O2 (30%
in water, 15.0 mL, 16.7 g, 147 mmol H2O2) in formic acid (50
mL) was added dropwise to a suspension of erucic acid 13
(5.00 g, 14.8 mmol) in formic acid (50 mL) and stirred at rt
for 23 h. Excess H2O2 was destroyed with Na2SO3 (negative
iodine starch test) and the solvent was evaporated under
reduced pressure. The resultant residue was dissolved in
KOH solution (1 M, 100 mL) and heated under reflux for 4 h.
The reaction mixture was then acidified with HCl (2 M, 100
mL) and the aqueous layer was extracted with EtOAc (3 × 100
mL). The combined organics were dried over Na2SO4 and the
solvent was evaporated under reduced pressure to give 14
(5.17 g, 94%) as a colourless solid; mp 110 °C; IR (ATR)
νmax/cm−1 3332, 3254, 2912, 2846, 1703, 1467, 720 and 656;
δH (300 MHz, DMSO-d6) 0.68 (3 H, t, J 6.7, 22-H3), 1.15–1.33
(28 H, m, 4-H2-11-H2, 16-H2-21-H2), 1.34–1.43 (4 H, m, 12-H2,
15-H2), 1.44–1.57 (2 H, m, 3-H2), 2.18 (2 H, t, J 7.3, 2-H2),
3.15–3.25 (2 H, m, 13-H, 14-H) and 4.06 (2 H, br s, OH); δC
(75 MHz, DMSO-d6) 13.8, 22.0, 28.5, 28.6, 28.7, 28.8, 28.9,
28.9, 29.0, 29.0, 29.2, 31.2, 24.4, 25.5, 32.3, 33.6, 73.0 and
174.3; m/z (ESI+) 373.3 (M + H+) and 395.4 (M + Na+); m/z
(HR-ESI+) 373.3311 (M + H+, C22H45O4 requires 373.3312).
Propargyl 13,14-dihydroxybehenoate 15. To a solution of
carboxylic acid 14 (1.71 g, 4.56 mmol), propargylic alcohol
(5.30 mL, 91.7 mmol) and 4-IJdimethylamino)-pyridine
(DMAP, 59 mg, 0.48 mmol) in CH2Cl2 (100 mL), dicyclohexyl
carbodiimide (DCC, 1.22 g, 5.91 mmol) was added. The reac-
tion mixture was stirred at rt for 20 h and then diluted with
sat. NH4Cl solution (90 mL). The aqueous layer was extracted
with CH2Cl2 (2 × 90 mL). The combined organics were
washed with water (1 × 90 mL), dried over Na2SO4 and evapo-
rated under reduced pressure. The resultant crude product
was purified by column chromatography (petroleum ether–
EtOAc, 3 : 1) to give 15 (980 mg, 52%) as a colourless solid;
mp 64 °C; TLC Rf 0.36 (petroleum ether–EtOAc, 2 : 1); IR
(ATR) νmax/cm−1 3300, 2913, 2846, 1740, 1467, 1170, 721 and
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as a colourless solid; mp 62 °C; TLC Rf 0.35 (EtOAc); IR (ATR)
νmax/cm−1 3299, 2916, 2848, 1740, 1464, 1390, 1276, 1225,
1199 and 1175; δH (300 MHz, CDCl3) 1.18–1.38 (16 H, m,
4-H2-11-H2), 1.46–1.69 (4 H, m, 3-H2, 12-H2), 2.34 (2 H, t, J
7.5, 2-H2), 2.46 (1 H, t, J 2.5, 3′-H), 2.92 (2 H, t, J 7.3, 13-H2),
4.66 (2 H, d, J 2.5, 1′-H2) and 5.83 (2 H, br s, NH, OH); δC
(126 MHz, CDCl3) 24.8, 26.7, 27.1, 29.1, 29.1, 29.2, 29.4, 29.5,
29.5, 29.6, 34.0, 51.7, 53.6, 74.7, 77.8 and 172.8; m/z (ESI+)
284.2 (M + H+) and 306.2 (M + Na+); m/z (HR-ESI+) 284.2223
(M + H+, C16H30NO3 requires 284.2220).
and 563 nm, respectively. Emission was detected at λem
495–538 nm and 633–690 nm, respectively.
=
Acknowledgements
The authors thank the Deutsche Forschungsgemeinschaft
(DFG, SFB 803 “Functionality controlled by organization in
and between membranes”) and the Fonds der Chemischen
Industrie (FCI, Sachkostenzuschuss) for financial support.
Notes and references
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