Synthesis of diacylglycerol analogs bearing photoaffinity tags for labelling mammalian diacylglycerol kinase

Article abstract of DOI:10.1039/c4ra16730a

Signaling lipids such as diacylglycerol (DAG), phosphatidic acid, and the phosphatidylinositol polyphosphates are site-specific ligands for protein binding partners. Herein, we report the apotheoses of our initial approach to the development of diverse probes which are vital for understanding lipid-protein interactions. When incorporated into liposomes, these probes reduce mammalian diacylglycerol kinase's (DGK) enzymatic activity in a concentration, time, and light dependent manner. This journal is

Full text of DOI:10.1039/c4ra16730a

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Synthesis of diacylglycerol analogs bearing
photoaffinity tags for labelling mammalian
diacylglycerol kinase†
Cite this: RSC Adv., 2015, 5, 25457
Received 19th December 2014
Accepted 2nd March 2015
*
Sammy Eni Eni, Meng Rowland and Michael D. Best
DOI: 10.1039/c4ra16730a
www.rsc.org/advances
Signaling lipids such as diacylglycerol (DAG), phosphatidic acid, and
Elucidation of the molecular level details of protein–lipid
the phosphatidylinositol polyphosphates are site-specific ligands for binding interactions has proven to be a challenging task, due in
protein binding partners. Herein, we report the apotheoses of our part to the complex nature of the membrane environment as
initial approach to the development of diverse probes which are vital well as the protein targets, which oen possess multiple
for understanding lipid–protein interactions. When incorporated into binding domains that display differential binding properties
liposomes, these probes reduce mammalian diacylglycerol kinase's towards different lipids. For example, the most prominent
(DGK) enzymatic activity in a concentration, time, and light dependent family of DAG-binding proteins is the protein kinase Cs,9 which
manner.
includes multiple isozymes possessing C1 binding domains
that exhibit varying DAG-binding affinity.10,11 To overcome the
challenges of studying protein–lipid binding interactions, there
has been substantial interest in the development of function-
alized lipid probes that are effective for the characterization of
protein binding properties, which are typically synthetic lipid
probes bearing appended reporter tags.9,12–15 Probes bearing
photoaffinity tags are of particularly interest as they allow for
covalent cross-linking to protein targets, which enables studies
such as the mapping of protein binding domains and discovery
of protein targets.16,17 In examples involving photoaffinity-
labeled lipid probes, Nemoz and co-workers developed PA
probes bearing phenylazide photoaffinity lables within the acyl
chains that was used in the covalent labeling of type 4 cAMP-
phosphodiesterases (PDE4).16 In addition, a range of photo-
activatable PIPn probes have been developed and applied to
characterize binding interactions.8,18–20
Previously, we reported a modular approach to the produc-
tion of derivatized DAG probes17 that allowed for the efficient
incorporation of a range of reporter groups in the nal step of
synthesis using click chemistry. This included the production of
an analog bearing a benzophenone tag linked at the sn-1 posi-
tion of the headgroup for photoaffinity labeling of DAG-binding
proteins. Following synthesis, each of these probes was found to
retain PKC-binding using an in vitro assay, indicating that
modication of the lipid headgroup, but away from the hydroxyl
group, was tolerated by protein targets. Headgroup derivatiza-
tion was specically targeted to enhance labeling by placing the
tag in close proximity to bound peripheral proteins. While this
initial approach was effective for producing active probes, the
synthesis was complicated by signicant problems with acyl
A small family of unique lipids present within plasma and
organelle membranes act as critical regulatory biomolecules
that control a variety of critical cellular pathways.1,2 Although a
number of mechanisms exist for the regulation of protein
function by lipids, a common motif involves the actions of
lipids such as diacylglycerol (DAG),3,4 phosphatidic acid,5,6 and
the phosphatidylinositol polyphosphates (PIPns)2 as site-
specic ligands for protein binding partners. These binding
interactions anchor peripheral proteins on the surfaces of
cellular membranes and modulate the function of the bound
protein either directly or through interaction with other
proteins at the membrane interface. In addition, the generation
of these signalling lipids is strictly regulated in a spatial and
temporal manner, as they exist at low physiological concentra-
tions, but become upregulated in response to biological
processes, and thus their presentation within the cell explicitly
controls the localization of receptors.7 As a result of the prom-
inence of protein–lipid binding events as the basic interactions
that feed information into critical cellular pathways, aberrant
lipid activities oen correlate to disabling diseases. For
instance, the activities of DAG have been linked to a litany of
noteworthy pathophysiological events including cancer and
diabetes.8
Department of Chemistry, University of Tennessee, 1420 Circle Dr., Knoxville, TN
37996-1600, USA. E-mail: senieni@vols.utk.edu
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra16730a
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chain migration that was only circumvented through careful options available for probe synthesis and reporter group
protecting group manipulations. In addition, since cross- introduction.
linking is affected by the presentation of the photoaffinity tag,
Based on these designs, the targets of the current work
which is difficult to predict, it is benecial to produce multiple include triazole-linked benzophenone analog 2, amide-linked
probes with varying tag displays. Thus, herein we report a benzophenone probe 3, and amide-linked diazirine 4, for
strategy for the production of multiple photoactivatable DAG which the synthetic routes are depicted in Scheme 2. The route
probes with varying photoaffinity tag presentation that commenced with diethyl-L-tartrate (5), for which the diol was
promotes protein–lipid interactions and circumvents our protected through acetonide formation to 6, followed by
previously encountered acyl migration enigma.
reduction to bis-hydroxymethyl compound 7. Next mono-
In photoaffinity labeling, the presentation ofthe cross-linking tosylation to 8 was followed by azide displacement to 9.
group is critical for successful labeling. As an example of the Protection of the free hydroxyl group of 9 as a para-methoxy-
ramications of tag placement, in the development of a PI(3,4,5) benzyl (PMB) ether to 10 was then followed by acetonide
P3 activity probe for proteomic identication of protein targets, deprotection to produce diol 11. At this point, two hexadecyl
we found that the linker length between the lipid headgroup and chains were introduced through the Williamson ether synthesis
the photoaffinity label signicantly affected labeling.21 Since at what will eventually become the sn-2 and sn-3 positions of the
ideal tag presentation is difficult to predict, it is oen advisable subsequent probes to generate 12. Finally, PMB deprotection
to generate multiple probes with varying tag display to discern was performed to set up late stage reporter tag introduction
the optimal approach. With the current strategy, in order to onto azido diether-DAG scaffold 13.
produce multiple photoaffinity probes with varying tag presen-
From precursor 13, tert-butyloxy (Boc) carbamate was added
tation, we took advantage of the versatile nature of the azide under hydrogen to 14. The Boc group was then deprotected,
group to append photoactivatable groups using different linkage followed by amide bond coupling to append the benzophenone
strategies. Specically, the azide group was either directly func- of probe 3. Additionally, we sought to further decrease the size
tionalized via click chemistry or reduced to an amine for amide of the photoaffinity label by instead introducing a diminutive
bond coupling, which would yield distinct linker lengths and diazirine group. To do so, diazirine-carboxylic acid was
geometries. In addition, different photoaffinity moieties were synthesized and then coupled to the DAG scaffold to produce
added, which would be expected to provide disparate results in probe 4.
cross-linking. In particular, we sought to decrease the size of the
DGKs are interfacial enzymes, catalyzing reactions at the
added cross-linking group to maximize the ability to t within two-dimensional interface between the membrane and the
the connes of protein binding domains which led us to the aqueous phase. Eukaryotic DGKs have been implicated in a
synthesis of probed 1–4 in Scheme 1.
number of physiological roles and human diseases. Very little is
In the current studies, we additionally sought to overcome known about the structure of the catalytic domain, especially
the issue of sn-2 to sn-3 acyl chain migration in the generation of the lipid substrate binding site.
DAG probes. Such migration in acylglycerols, which occurs
A photoaffinity labeling approach was carried out to identify
under both acidic and basic conditions, generally complicates the lipid binding site due to covalent attachment by the probes.
both synthesis and biological analysis.22 To circumvent this As observed from Fig. 1, radiometric DGK activity assay was
issue, the current DAG probe targets contain hydrophobic tails used to determine the enzyme's activity with probe 4 incorpo-
linked via ether bonds to lock these groups into place. This rated in liposomes in different concentrations pertaining to
attachment was chosen due to prior evidence that ether-linked probe concentration, light exposure, and temperature. The
DAG analogs retain protein binding properties. Ether linkage results showed that probe 1 is used as a substrate to DGK and
also has the added benets of blocking enzymatic removal of binds to DGK covalently as concentration of DGK increases
acyl chains, thus decreasing the routes by which the probes will which is also proportionate to time and light exposure. DGK
be modied in biological samples. Finally, removal of the labile
ester groups from these structures also expands the synthetic
Scheme 1 Photoaffinity probes 1–4.
25458 | RSC Adv., 2015, 5, 25457–25461
Scheme 2 Synthesis of DAG probes 1–4.
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Fig. 1 DGK activity assay with probe 4 showing reduction of its activity
in a concentration, temperature, and light dependent fashion.
activity studies were carried out with probe 1 which shows it
deactivated DGK activity but not monotonically as probes 4 and
3 as shown in Fig. 2.
The results in Fig. 1 and 2 indicate a substantial difference in
the activities of these probes to DGK. In these regard, we
propose that the different photoaffinity tags might play a role in
this variation in activity. Benzophenone, because of its bulky
moiety and also requiring a high photo-activation energy, could
account for its lower reactivity with DGK compared to the dia-
zirine moiety which is smaller and requires a low photo-energy
of activation.
The enzymatic activity of DGK was also measured on lipo-
somes containing compound 3 which demonstrated that DGK's
Michaelis constant (Km) for compound 3 must be high (Fig. 3A).
To further verify the cross-linking of the probes to the active
site of DGK, probe 4 was used in which the band containing
probe 4 and cross-linked DGK was evaluated using in-gel trypsin
digestion following SDS-PAGE.
Fig. 3 DGK activity assay with probe 3 showing reduction of activity of
DGK batches (601 E and 227 A).
these peptides, ARPGPGPGPGPER and IPCTSVAPSLVRVP-
VAHCFGPR were found in the cross-linked sample, but
APGPAAAPGHSFR was not, even though it was the most abun-
dant in the control sample. If peptides were covalently modied
with compound 4, they will be disproportionately absent from
the cross-linked sample, either because the mass of the cross-
linked peptide had been shied and thus no longer appeared
as the unmodied mass, or because the cross-linked peptide
remained in the gel matrix.
Two such disproportionately absent peptides in these
samples were found and, even more encouragingly, both
coincided with regions of the protein predicted by primary
sequence homology to include DAG-binding sites as depicted
in Fig. 4.
Certain adjacent tryptic peptides were disproportionately
absent from the cross-linked sample as compared to the control
sample. For example, ARPGPGPGPGPER was found six times in
the control peptide, APGPAAAPGHSFR was found eleven times
and IPCTSVAPSLVRVPVAHCFGPR was found three times. Of
Detected peptides are highlighted in yellow. Peptides
disproportionately absent in the sample with compound 4 are
marked in red. The three C1 domains and the catalytic domain
of DGK, which are predicted to contain DAG-binding sites, are
shaded in blue and green, respectively. Oxidized sites are
marked in bright green. In addition to the sequencing, tandem
MS was carried out on probe 4 that cross-linked DGK which
produced a product ion whose molecular weight is consistent
with loss of either alkyl chain as shown in Fig. 5. Cross-linking
efficiency depended on the length of the linker between the
photoactivatable cross-linker and the DAG-like moiety in the
probe: probes with longer linkers are able to access the active
site and be used as substrates. DAG does not protect DGK from
Fig. 2 DGK activity assay with probe 1.
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proximity of the benzophenone was found to be very vital for
successful protein labeling. This further circumvented the
problem of the photoaffinity tag being buried in the membrane
core if a longer linker is used. Our results strongly indicated that
shorter linkers enhanced labeling to peripheral proteins
compared to longer linkers and also that small and easily photo-
activated moieties like diazirine bind DGK readily than bulkier
moieties like benzophenone.
Consequently, our synthesis of DAG probes 3 and 4 was
geared towards the placement of the benzophenone close to the
head group in the aqueous interface with a short hydrophobic
moiety which proved potent in deactivating DGK activity as
envisioned. Also included in these new probes 2, 3 and 4 unlike
probe 1 is the azide tag as our secondary handle. This azide
allows for selective detection and purication of proteins that
have been successfully labeled by the probe. The azide group is
specically chosen due to its diminutive size, robust properties,
and bio-orthogonal reactivity through the 1,3 dipolar cycload-
dition (click chemistry).13
Fig. 4 Amino acid coverage of in-gel trypsin-digested DGK cross-
linked with compound 4 with certain peptides disproportionately
absent in the cross-linked sample as compared to control sample.
Conclusion
Herein, we report the apotheoses of our initial approach to the
development of the diverse probes that are potent as function-
alized derivatives of DAG. Probes 2 and 3 are composed of a
benzophenone as the photoactivable tag while probe 4 has
diazirine. These two unique tags on the DAG probes are
essential for their correlation in reactivity pertaining to cross-
linking of peripheral proteins. Assay studies indicate that
these probes aer incorporation on a liposome, reduce diacyl-
glycerol kinase (DGK) activity in a concentration, time, and UV-
dependent manner. This dependence varies among the probes
and is observed considerably only when the linker connecting
the photoaffinity moiety and the DAG-like moiety on the probe
is sufficiently short (3 and 4), small and easily photo-activated.
This therefore indicates that these DAG probes possess variable
Fig. 5 Tandem MS of probe 4 cross-linking DGK.
cross-linking, possibly because DAG may be enhancing DGK properties for the labelling of the DGK enzyme. Our future goal
binding to probe-containing liposomes. is to use and apply these probes in other complex proteome
When cross-linked DGK is subjected to LC/MS/MS, certain systems and even live cells, in order to characterize their
peptides are disproportionately missing from the cross-linked interactions with peripheral proteins especially those upregu-
DGK as compared to DGK irradiated in the absence of photo- lated in cancer cells.
affinity probe. These missing peptides coincided with regions of
the protein predicted by primary sequence homology to contain
DAG-binding sites, and are candidates for sites of photoaffinity
Notes and references
cross-linking. We were unable to nd any peptides shied by
the total mass of the probe following cross-linking, but the
probe fragments during tandem mass spectrometry. The pho-
toactivable tags, benzophenone as well as diazirine, were pre-
sented at the aqueous interface to enforce proximity to bound
proteins for easy cross-linking aer irradiation.
The tag placement also greatly inuences the accessibility for
modication, in which presentation on the surface of the
membrane is also rewarding. We have pursued a variety of
schemes which introduces tags as a Y-shaped lysine linker.12
This method allowed us to vary the length of the linker between
the head group and the photoactivable tag as this length has
been known to affect the magnitude of cross-linking. The
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