Design and synthesis of fluorescent galactolipid probes

Article abstract of DOI:10.1016/j.tetlet.2014.04.059

Fluorescent galactolipid analogues (1, 1a, and 2) have been synthesized with a pyrene group attached to the amino-terminal of a hexanoyl chain bound to an otherwise normal galactolipid structure. The synthetic lipids are obtained from peracetyl galactose by a general and versatile procedure based on the trichloroacetimidate methodology. The intense violet fluorescence (376 and 395 nm) and good photostability of pyrene make these compounds highly suitable as probes to study galactolipid metabolism. As proof of concept, we report that compound 2 is a valid tool to detect galactolipase activity in enzymatic preparations of potato tubers.

Full text of DOI:10.1016/j.tetlet.2014.04.059

Accepted Manuscript
Design and Synthesis of Fluorescent Galactolipid Probes
Emiliano Manzo, Giuliana D’Ippolito, Dario Pagano, Francesco Tinto, Angelo
Fontana
PII:
S0040-4039(14)00671-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.04.059
TETL 44521
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2014
15 April 2014
16 April 2014
Please cite this article as: Manzo, E., D’Ippolito, G., Pagano, D., Tinto, F., Fontana, A., Design and Synthesis of
Fluorescent Galactolipid Probes, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.04.059
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Design and Sinthesis of Fluorescent Galactolipid Probes
Emiliano Manzo*, Giuliana Dippolito, Dario Pagano, Francesco Tinto and Angelo Fontana
O
O
OH
R
O
O
OH
HO
HO
O
O
O
OH
H
N
OH
OH
O
O
HO
HO
COOH
H2N
O
OH
COOH
Compound 2
OH

Design and Synthesis of Fluorescent Galactolipid Probes
Emiliano Manzo,* Giuliana D’Ippolito, Dario Pagano, Francesco Tinto and Angelo Fontana
Consiglio Nazionale delle Ricerche- Istituto di Chimica Biomolecolare, Via Campi Flegrei,
34 -80078-I Napoli- Italy
Corresponding author
Email: emanzo@icb.cnr.it
Phone: +39 081 8675310
Fax.: +39 081 8041770
2

Abstract
Fluorescent galactolipid analogues (1, 1a and 2) have been synthesized with a pyrene
group attached to the amino-terminal of a hexanoyl chain bound to an otherwise normal
galactolipid structure. The synthetic lipids are obtained from peracetyl galactose by a
general and versatile procedure based on the trichloroacetimidate methodology. The
intense violet fluorescence (376 and 395 nm) and good photostability of pyrene make
these compounds highly suitable as probes to study galactolipid metabolism. As proof of
concept, we report that compound 2 is a valid tool to detect galactolipase activity in
enzymatic preparations of potato tubers.
Keywords
Glycoglycerolipids, Fluorescence, Chemical Probes, Lipid Metabolism
3

Galactose-containing glycerolipids, commonly named galactolipids, are main components
of chloroplast membranes but can be also present in non-photosynthetic tissues, such as
potato tuber or apple. In shortage of phosphate, galactolipids can also substitute
phospholipids in a process that has acquired growing consideration as the major
mechanism of cell-membrane lipid homeostasis in plants.1 The two most common
galactolipids are mono- and di-galactosyldiacylglycerol (MGDG and DGDG) that basically
differ for the presence of one or two sugars linked to glycerol. Both classes are
characterized by uncommon levels of polyunsaturated fatty acids that in certain algae can
account for more than 80% of total fatty acids. Recent studies have demonstrated that
galactolipids play an important role in not only the organization of photosynthetic
membranes, but also in their photosynthetic activities.2 Furthermore, these lipids are
crucial precursors of eco-physiological mediators, including jasmonate and volatile organic
deterrents, in plants3 and algae.4 Galactolipids are a primary source of galactose and
polyunsaturated fatty acids (PUFAs) in the human diet5 and also exhibit COX mediated
anti-inflammatory activity6 and inhibitory effects on tumor promoter-induced Epstein-Barr
virus (EBV) activation.7
Despite the growing body of evidence about the biological role, there is a general
deficiency of cellular and molecular tools devised to investigate metabolism and functional
role of galactolipids. Recently we have reported a novel synthetic approach for the
preparation of galacto- and sulfoglyco-lipids.8 The method is of general application and
can be used to prepare natural or syntetic variants containing a regiospecific distribution of
polyunsaturated fatty acids. In the present work we aim at describing the synthesis and the
properties of pyrene- containing β-galactosyl-diacylglycerols that were specifically
designed as fluorescent substitutes of natural galactolipids in functional and structural
studies. As demonstration of the potential application of these molecules in studies on lipid
4

metabolism and signaling, we also report the use of the fluorescent compound 2 to monitor
galactolipase activity in raw preparations of potato tubers.9
O
O
O
O
NH
N
H
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
OH
1a
1
O
O
O
O
NH
O
O
O
HO
HO
OH
OH
2
Figure 1. Structures of compounds 1, 1a and 2.
Pyrene (342 nm excitation wavelength and 376 nm emission wavelength) is one of the
most common fluorophores and has been widely used as a fluorescent label in research
on lipid metabolism.10 Our choice to use pyreneacetic acid (3) as the fluorescent group to
tag β-galactosyl-acylglycerols was due to chemical and biochemical considerations
including high extinction coefficient with stable fluorescence, chemical and biological
inertness, compatibility with biological systems and stability during chemical synthesis. In
order to link the fluorescent tag to glycerol, 1-pyreneacetic acid (3) was functionalized by
introducing a six carbon atom spacer (Scheme 1).
5

H
N
O
CO2H
CO2H
1. DCC, DMAP
in CH2Cl2
H2N
COOMe
2. Na2CO3
MeOH/H2O 3:1
4
5 (70%)
3
Scheme 1. Synthesis of the fluorescent-armed tag 6-pyreneacetamido-hexanoic acid
(PAH) (5).
In a convergent synthetic strategy (Scheme 2), 6-pyreneacetamido-hexanoic acid (PAH)
(5) was then condensed to 2’,3’,4’,6’-tetracetylated-monogalactosyl glycerol (10) by DCC.
This latter compound was prepared by trichloroacetimidate-mediated condensation of
peracetyl D-galactose with glycerol acetonide,8,11 followed by selective hydrolysis of the
isopropylidene by zinc nitrate hexahydrate in acetonitrile.8-12 Notably, equimolar amounts
of reagents gave only introduction of the armed-pyrenyl substituent at the primary alcohol
of glycerol, as indicated by long-range NMR-experiments that showed for 11 a diagnostic
correlation between the carboxylic ester at δ 173.1 with the down-shifted methylene
protons (δ 4.13) of glycerol. In the 1H-NMR spectrum of compound 11, anomeric signals of
two diastereoisomers at δ 4.50 and 4.48 were distinguishable in a ratio of 1:1 due to use of
racemic glycerol acetonide in the coupling reaction. Successive reaction of 3-O-[2’,3’,4’,6’-
tetracetyl-β-galactose]-1-[6’’-pyreneacetamido-hexanoyl] glycerol (11) with equimolar
concentration of arachidonic acid (AA) or linoleic acid (LA) in the presence of DCC gave
derivatives 12 and 13 that were converted to the target product 1-[6’’-pyreneacetamido-
hexanoyl]-2-arachidonoyl-3-O-β-galactosyl glycerol (1) and 1-[6’’-pyreneacetamido-
hexanoyl]-2-linoleoyl-3-O-β-galactosyl glycerol (1a) after selective removal of acetyl
groups by hydrazine hydrate (Scheme 2). Similarly, the regioisomeric product 2, 1-
6

arachidonoyl-2-[6’’-pyreneacetamido-hexanoyl]-3-O-β-galactosyl glycerol, was obtained
from 10 by sequential condensation of arachidonic acid and 6-pyreneacetamido-hexanoic
acid (5) as reported in Scheme 2.
Trichloroacetonitrile
DBU/CH2Cl2/0°C
O
OH
OH
O
OAc
OAc
Benzylamine
THF
O
OH
Ac2O/pyr
HO
HO
AcO
AcO
AcO
AcO
OAc
OH
OAc
OAc
6 (92%)
7 (74%)
O
O
OH
CH2Cl2/BF3OEt2
-10°C
O
O
O
O
O
OH
O
O
CCl3
AcO
Zn(NO3)2
AcO
AcO
AcO
AcO
CH3CN
50°C
AcO
OAc
OAc
OH
O
OAc
OAc
OAc
OAc
O
9 (81%)
8 (79%)
10 (76%)
1.2 eq PAH or AA
DCC, DMAP
in CH2Cl2
OR2
OR2
OH
OR1
O
OR1
O
O
O
O
OR1
O
1.2 eq PAH,
AA or LA
6'
HO
AcO
AcO
AcO
AcO
NH2NH2
1'
3
1
4'
HO
OH
OAc
OAc
DCC, DMAP
in CH2Cl2
85%EtOH
45°C
OH
OAc
OAc
11. R1 = PAH (93%)
14. R1 = AA (92%)
1. R1 = PAH; R2 = AA (23%)
1a. R1 = PAH; R2 = LA (23%)
2. R1 = AA; R2 = PAH (25%)
12. R1 = PAH; R2 = AA (93%)
13. R1 = PAH; R2 = LA (93%)
15. R1 = AA; R2 = PAH (93%)
Scheme 2. Synthesis of fluorescent β-galactolipids 1, 1a and 2. PAH= 6-pyreneacetamido-
hexanoic acid; AA= arachidonic acid; LA= linoleic acid.
In organic solvents and buffer solutions (pH =7.2), probes 1, 1a and 2 exhibited
fluorescent spectra similar to that of pyrene with a major excitation peak at 342 nm and
two emission bands at 376 and 395 nm (Figure 2). The fluorescent emission could be
easily monitored on TLC plates with a detection limit that was 1 nmol by visualization over
a 365 nm UV lamp. Emission intensity remained stable during preparation and
development of TLC plate, as well as the fluorescent spot was still detectable after 30 min
from the analysis.
7

Figure 2. Excitation (black) and emission (green) spectra of 7.4 mM 3-O-β-galactosyl-1-
[6”-pyreneacetamido-hexanoyl]-2-arachidonoyl-glycerol (1) in MeOH. Spectra of probes 1a
and 2 are identical.
Potato tubers contain galactolipid-hydrolyzing enzymes that are commonly referred to as
patatins.13 These proteins that belong to the class of lipolytic acyl hydrolases (LAHs)
account for up to 40% of total proteins in the tubers and their activity is sufficient to rapidly
hydrolyze native and model molecules of phospholipids and galactolipids.9 For these
reasons, we decided to test the hydrolysis of compounds 1 and 2 by potato homogenates
in order to verify if the pyrenyl fluorophore affected LAH recognition of β-galactosyl
diacylglycerols. Crude preparations of potato tuber proteins were carried out by
precipitation with 40-70% ammonium sulfate followed by dialysis.14
8

1
Figure 3. Hydrolysis of fluorescent probe 2 by potato tuber homogenates. A) H-NMR
(CDCl3) of compound 2; B) SiO2-TLC (CHCl3/MeOH/water 65:25:4) analysis of the reaction
after 1h. Compound 2 Rf = 0.75; Compound 17 Rf = 0.60. C) 1H-NMR (CD3OD) of product
(17) recovered after enzymatic reaction.
LAH activity of this preparation was first tested on natural MGDG isolated from the diatom
Skeletonema marinoi according to ref. 4a, verifying the hydrolysis of complex lipids by
GCMS on the corresponding methyl esters (data not shown).
Then the potato tuber homogenates were incubated with 200 µg of the fluorescent probes
1 and 2. After 1 hour of incubation, TLC analysis showed the almost complete
disappearance of probe 2 with formation of a fluorescent spot at lower Rf (Figure 3),
whereas probe 1 remained unaltered (not shown). To verify these results, the incubation
9

buffers were extracted with diethyl ether and the resulting organic fractions were
methylated by CH2N2. After purification on silica gel, compound 1 was recovered almost
completely (82% recovery). On the other hand, the incubation of probe 2 resulted in a
15:85 mixture of compounds 16 and 17, as proved by signal of H-2 of the acylated
glycerol of 16 together with the signals of 17 in the spectra of the reaction mixture
(Supporting Information). Both products derived by selective hydrolysis of arachidonic
acid from the primary position of glycerol in compound 2. It is worth noting that the 1-acyl
derivative 17 is the product of the thermodinamically-favored intramolecular migration of
the PAH from the secondary to the primary position of glycerol. These data were fully
corroborated by GC-MS analysis of the extracts that revealed significant release of
arachidonic acid only from compound 2 (Supporting Material). Even if compound 1 and 2
were in diasteroisomeric form, the LAH activity did not show diasterospecificity during the
incubation. Nevertheless, we cannot rule out that the two diasteroisomers may be
recognized in a different way in other assays.
O
O
NH
O
HO
O
NH
OH
O
O
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
OH
17
16
Figure 4. Structures of compounds 16 and 17.
In this study we have reported the preparation and the characterization of three fluorescent
galactolipid analogues (1, 1a and 2) that are designed to study galactolipid metabolism. In
particular, compound 2 proved to be very effective to assess the hydrolytic activity of
10

patatins, members of a wide class of enzymes that play a fundamental role in lipid
remodeling and plant signalling.15 The TLC assays were very quick to perform and
visualization of the fluorescent probes over a UV lamp was straightforward and readily
usable for the study of biosynthesis, tailoring and relocation of MGDG and DGDG. On the
other hand, the fluorescent galactolipid 1 was only marginally hydrolyzed by the potato
homogenates thus suggesting that the large pyrene group at position sn-1 of glycerol both
hampers a productive interaction with the hydrolytic enzyme(s) and lends to these
molecules a biochemical stability that can be potentially used in fluorescence microscopy
or DGDG biosynthesis. Galactolipid analogues like 1 and 2 are particularly attractive from
a synthetic point of view since the PAH residue (a) can be easily introduced by DCC
condensation, (b) is compatible with the use of polyunsaturated and monounsaturated fatty
acids and (c) is stable under the very mild hydrolytic removal of the acetyl groups that are
necessary for the trichloroacetimidate methodology of coupling. In this view, the synthetic
strategy here reported can be readily used to prepare a wide variety of reliable and
sensitive lipid probes.
Acknowledgements
Authors thank Mrs. D. Melck for NMR spectra, Mr. C. Iodice for spectrophotometric
measurements and Mr. M. Zampa for mass spectra analysis. The project financed by the
ERDF and FdR subsidies of the National Operational Programme for Research and
Competitiveness 2007-2013 and FAR funds of the Italian Ministry of Education, University
and Research, Project "Antigens and Adjuvants for Vaccines and Immunotherapy -
Identification of novel natural compounds with adjuvant and immunomodulatory activity
from marine microalgae (PON01-00117).
Supporting data
11

Supplementary data associated with this article can be found, in the online version, at
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