Labeling of polyprenylated macromolecules using mild Ene reactions

Article abstract of DOI:10.1016/j.tet.2020.131917

Protein posttranslational modifications play pivotal roles in a wide range of functions such as gene expression regulation, differentiation, cell migration, various human diseases and so on. Polyprenylated group are a lipid modification only found in the Ras super family protein. It was demonstrated that farnesyl, geranylgeranyl groups are covalently attached into sulfur atom on cysteine of Caxx motif, playing essential roles in anchoring on plasma membrane and signal transduction etc. We first reported a straightforward chemical approach to labelling of polyprenylated macromolecules. Our method mainly relied on a novel fluorination/Ene reaction, exhibiting application potential in the study of proteome-wide mapping of protein prenylation target.

Full text of DOI:10.1016/j.tet.2020.131917

Tetrahedron 81 (2021) 131917
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Labeling of polyprenylated macromolecules using mild Ene reactions
Jingwen Zhang, Jizhong Zhao, Li Wang, Hongmei Hu, Sheng Wang, Ping Yu, Rui Wang*
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein posttranslational modifications play pivotal roles in a wide range of functions such as gene
expression regulation, differentiation, cell migration, various human diseases and so on. Polyprenylated
group are a lipid modification only found in the Ras super family protein. It was demonstrated that
farnesyl, geranylgeranyl groups are covalently attached into sulfur atom on cysteine of Caxx motif,
playing essential roles in anchoring on plasma membrane and signal transduction etc. We first reported a
straightforward chemical approach to labelling of polyprenylated macromolecules. Our method mainly
relied on a novel fluorination/Ene reaction, exhibiting application potential in the study of proteome-
wide mapping of protein prenylation target.
Received 19 November 2020
Received in revised form
12 December 2020
Accepted 28 December 2020
Available online 6 January 2021
Keywords:
Prenyl
Fluorination
Ene
© 2020 Elsevier Ltd. All rights reserved.
Labeling
Geranylated
1. Introduction
(including geranylgeranyl and farnesyl) modifications in Ras family
proteins are highly conserved, playing diverse roles in varieties of
Covalent modifications on histone tails have been well identi-
fied, involving in the regulation of a wide range of physiological
process in living cell [1]. Protein posttranslational modifications
include methylation [2a], acetylation [2b], phosphorylation [2c],
sulfation [2d], N-myristylation [2e], S-palmitoylation [2f], pre-
nylation [2g] and many others [3]. Two types of protein post-
translational modifications by lipids have been documented,
namely, palmitoylation and prenylation. The prenyl groups are
covalently attached to cysteine residues of a subset of Ras super-
family proteins in eukaryotic cells (this polyprenylated modifica-
tions also found on nucleic acids termed geranyl) [9]. This poly-
prenylated modifications have demonstrated to be localized into a
carboxyl-terminal Caax motif, whereas C represents cysteine, and a
represents aliphatic amino acid and x is any amino acid. In 1978,
Fujino and co-workers first reported the prenylated protein in fungi
[4]. In 1988, this lipid-modification have been found in mammalian
cells in Lamin B by Glomset and coworkers [5]. Recently, genome-
wide analysis of protein posttranslational modifications of the
human cell has revealed that there are more than 600 potentially
prenylatable proteins [6]. Prenylations were facilitated by three
types of eukaryotic protein prenyl transferases, namely FTase (far-
nesyl transferases) and GGTase (geranylgeranyl). Polyprenylated
fields, for instance, signaling transduction pathway are activated by
prenylation via selectively anchoring cell membrane and lipid side
chain [7]. It has been noted that prenylated proteins are involved in
a wide range of human diseases such as precocious aging, malig-
nant disorders osteoporosis and up to 30% of cancers are associated
with protein prenylation.
While evolving technologies for global profiling of gene
expression regulated by polyprenylated modification have ach-
ieved significantly, technique for selective and direct enrichment of
prenylation proteomic with reduced complexity remains elusive.
The efficiency of current methods is very dependent on the reaction
specificity in virtue of competitive nonspecific interaction in the
complicated endogenous environment. In order to form a covalent
adduct based on the intrinsic property of polyprenylated group(s),
unique chemical reaction with fast kinetics and high specificity
deserve to be explored. Proteomic investigations of polyprenylated
protein have been reported in recent years [7], in which the
metabolic labelling using prenylated analogues was carried out to
selectively tag prenylated proteins with a clickable handle (i.e.,
azide or alkyne). These modified proteins are further modified by
bioorthogonal click reactions, adding either a fluorophore for gel-
based proteomic or a biotin moiety for enrichment of poly-
prenylated proteins (route 1, Fig. 1) [7a-7b]. However, chemical
modifications on prenyl functionalities may lead to the structure
perturbation, critical prenylation information may be incomplete
or missing. Therefore, exploration of molecular tools for selective
* Corresponding author.
E-mail address: ez_rwang@hust.edu.cn (R. Wang).
https://doi.org/10.1016/j.tet.2020.131917
0040-4020/© 2020 Elsevier Ltd. All rights reserved.

J. Zhang, J. Zhao, L. Wang et al.
Tetrahedron 81 (2021) 131917
resonance, would facilitate deep understanding of genome-wide
protein prenylation. Application potential for the studies of
genome-wide polyprenylated proteins, especially associated with
various cancer disease are also underway.
To explore the feasibility of the approach, the dynamic property
of a model reaction with polyprenylated functionality has been
implemented. The reaction of geranyl acetate with Selectfluor re-
agent was performed in a mixture of PBS/acetonitrile at ambient
temperature. The reaction proceeded smoothly, forming the
desired products (1a and 1x, which means several fluorine atoms
attached) in 5 min (Fig. 2A, top). It has been proposed that this
process was enabled by the so-called Ene-reaction [8]. Notably,
several point(s) in allylic positions of geranyl acetate have been
identified to be attached with fluorine atom(s) (Supplementary
Material, S1eS6). Crude or purified products have been proven to
be attached with several fluorine atoms, which was confirmed by
high-resolution liquid chromatography-mass spectrometry and
NMR studies (S1eS6, Supplementary Material). The results hinted
that polyprenylated substrate was able to be selectively modified
with fluorine atom(s) under the mild reaction condition by use of
commercially available Selectfluor reagent. The fluorination pro-
ceeded in a superfast manner, exhibiting significant application
potential in the study of labeling and detection of polyprenylated
proteins. As mentioned, method used for distinguishing farnesyl
and geranylgeranyl substrates by use of chemical tool remain
Fig. 1. Strategies for genome-wide analysis of prenylated proteins. Traditional ap-
proaches relied mainly on the derivation of prenyl functionalities with the so-called
handles (i.e., azido, propargyl) for the subsequent biorthogonal reaction, biotin
group thus are able to be introduced for further enrichment. Our strategy focuses on
the chemical reactivity of prenylated functionality, providing straightforward route for
labelling or enrichment studies. Route 1 include: Step 1, synthesis of prenylated de-
rivative; Step 2, evaluation of the recognition efficiency by corresponding enzyme;
Step 3, utilization of biorthogonal reaction to label or enrich target proteins. This study
use fluorination/fluorescent PTAD to label polyprenylated proteins.
and direct detection and quantification of prenylated proteins
would be great beneficial for early diagnosis study of Ras related
cancers, such as lung cancer, rectal cancer and pancreatic cancer
[7d,2g].
Considering the protein abundances varying across a wide dy-
namic range in cells and probably prenylations are sub stoichio-
metric (approximate 0.4% in tRNA, compared to 0.4e0.6% of methyl
in proteome), sensitive enrichment tool for capturing poly-
prenylated group along with mass spectrometry-based proteomics
profiling is in high demand. Global analysis of posttranslational
polyprenyl modifications could largely be restricted by the syn-
thesis of the polyprenyl analogues, recognition efficiency of prenyl
transferases and beyond. We aim to develop a chemical probe
based on polyprenylated group’s functionality and usher it as a
surrogate to profile polyprenylated protein in vitro and in vivo. On
the basis of our preliminary results, we reported here a straight-
forward and chemical approach for detection of polyprenylated
macromolecules via the so-called fluorination/Ene reaction (Fig. 1).
In view of the unique nature of prenyl functionality, we explored
and developed a practical [2,3]-rearrangement process using
Selectfluor/4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) de-
rivatives to introduce a fluorine or fluorescence/biotinated groups
to the target macromolecules under mild condition. Given the
importance of polyprenylated groups in Ras relevant proteins, our
approach was able to omit a complicated synthesis of prenyl ana-
logues process and further substrate recognition by prenyl
transferase.
To address these challenges, we reported herein a chemically
fluorination/Ene reaction of polyprenylated macromolecules to
facilitate the detection of polyprenylated protein, thus paving the
way in the extensive study of protein (or nucleic acid) prenylation
(route 2, Fig. 1). The process was mainly relied a fluorination/Ene
reaction depending on the reaction activity of the prenyl func-
tionality, which can be carried out in a physiological condition.
Moreover, intensive studies of prenylated molecules, enabled by
our Ene reaction along with the fluorine-19 nuclear magnetic
Fig. 2. Investigations of model reaction of fluorination of geranyl acetate. (A) Proposed
reaction mechanism of the fluorination of geranyl acetate (1) with Selectfluor. (B)
Proton NMR monitoring of the model reaction. The conversion of the reaction was
calculated by use of internal standard tertbutyl alcohol. The conversion reached up to
90% in as soon as 5 min (J, red color). (C) Isolated products (1a and 1b) and the
calculated conversion results. Over 90% conversion in 5 min was observed under the
physiological condition. NC represents negative control. Three duplicates were run.
2

J. Zhang, J. Zhao, L. Wang et al.
Tetrahedron 81 (2021) 131917
elusive (Chart 3, SI) [7a]. To explore our method’s practicality, we
performed the competition reaction of geranyl acetate/ger-
anylgeranyl alcohol with Selectfluor in a mixture of acetonitrile and
PBS buffer. We noticed that the former substrate seemed to proceed
in a more rapid manner. (Fig. 1).
although light modifications may be incomplete and ignored due to
the sensitive of the NMR and mass spectrums. In a word, we proven
herein a simple addition of the Selectfluor reagent into BSA in vitro
would facilitate the detection of the polyprenylated protein by 19
NMR spectroscopy and mass spectrometry.
F
Although the Ene reactions of nitroso derivatives with preny-
lated (Fig. 3B)substrates have been reported [9], there is no such
fluorination/Ene reaction carried out in aqueous phase or utilized in
genome-wide analysis of protein prenylation. With these in mind,
we next sought to investigate whether our method can be utilized
in the studies of detection of macromolecules, i.e., peptide or pro-
teins. Bovine serum albumin (BSA) was selected as a model. First,
the 4-nitrophenyl geranyl formate (4, Fig. 3A) was synthesized in
linear two-steps, which was identified by NMR and mass spec-
trometry. To testify the viability of our method, this intermediate
(4) was used to in situ modify the BSA (fraction V, 5). The gerany-
lated BSA was shown to be a very narrow band (Fig. 3B, Lane 3) after
the attachments of this hydrophobic group into the various lysines
(and other amino acids) of BSA at different sites. Next, the fluori-
nation with Selectfluor reagent was performed, which was
analyzed by the gel electrophoresis assays. Upon observation of tiny
newly-generated band in the gel electrophoresis assay (Fig. 3B,
Lane 4), we aimed to verify whether our fluoridation can be utilized
in the study of detection of polyprenylated BSA via 19F NMR
experiment. Delightedly, a fluorine NMR signal at ꢀ179.99 ppm
(Fig. 3C) was indeed observed, which was consistent with reported
data [8b]. To further illustrate our hypothesis, we also sought to run
the mass spectrometry of this crude samples (Table S1 and S2). It
was demonstrated that K187 of BSA was successfully attached with
the geranyl group, followed by the selective attachment with one
and two fluoride atoms (m/z 198 and m/z 216, red color in Fig. 3D).
Moreover, three additional sites (K273, K275 and K413 in red color,
respectively) have further been identified to be anchored with
geranyl groups and further attachment with fluorine atom(s). It was
suggested that our fluorination was a rather random process based
on the crystal structure of BSA (Fig. 3D and Fig. S10), modifications
were identified at varied sites of lysines in BSA (Fig. 3D, red parts),
Similar results for polyprenylated peptides have also been
identified (Supplementary Material, Schemes S2 and S3, S10).
Either by employing organic synthesis (peptide I, Gly-Cys (SStBu)-
Met-Gly-Leu-Pro-Cys (geranyl), Supplementary Material, Scheme
S2, Fig. S13), or from other source (peptide II, Phe-Gly-Gly-Gly-
Lys-Lys-Lys-Ser-Lys-Thr-Lys-Cys (farnesyl), Supplementary Mate-
rial, S15), we performed the fluorination reaction based on the
established method. The prenylated intermediate peptide I and
peptide II were both isolated and identified by fluorine-19 NMR.
Mass identification of the fluoride peptide II remains elusive, since
it has reported that the geranyl/farnesyl motifs are liable to
decompose [10] (unreported results). Nevertheless, the fluorine
NMR along with mass spectrum results indicated that the target
fluoride peptides were proven to be formed, indicating the appli-
cation potential by use of our fluorination.
Eventually, the application of the polyprenylated group
involving Ene reaction in the study of live cell labelling was
investigated. In addition to fluorination process, we adopted the
Ene reaction using PTAD derivatives considering that fluorination is
not feasible with conventional fluorescent labeling [11]. In order to
introduce the polyprenylated (geranylgeranyl, gege) functionality
onto live cells, the antibody cetuximab was chosen as a substrate
for the incorporation of geranylgeranyl functionality and further
installation gege-cetuximab for specifically targeting antigens on
HeLa cells through the specific binding to EGFR on the HeLa cells
surface (Fig. 4B). Upon addition of our well-designed FITC-PTAD
reagent (Fig. 4B, dash frame), we observed that the cells with the
gege-cetuximab on the cell surface was able to be labelled with
green fluorescence by using FITC. It has been demonstrated that
intense green fluorescence was observed on the cell surface upon
addition of FITC-PTAD, although the difference was minimal (in the
presence or absence of gege-treatment, Supplementary Material,
Fig. S22), partly due to the geranylgeranyl functionality bearing no
fluorescence and HeLa cells itself bind extensively with this FITC-
PTAD reagent in a nonspecific manner (Supplementary Material,
Fig. S22). In addition, control experiment using FITC-PTAD to
incubate with HeLa cells without gege-cetuximab treatment
showed tiny fluorescence signal (Supplementary Material, Fig. S22,
right lane). Taken together, our HeLa cells labelling assays
confirmed the successful of the Ene reaction of geranylgeranyl
functionality with our well-designed FITC-PTAD reagent. The
capability of control chemical ligation in living systems with tem-
poral resolution will pave the way to various biological applications
of this polyprenylated-involving bioorthogonal click reaction.
2. Conclusions
In summary, we reported here a method using the fluoridation/
Ene reaction to introduce a fluorine atom or fluorescent/biotinated
functionalities into the macromolecules (proteins and peptides).
The reactions have been shown to proceed smoothly in a superfast
manner (5 min). We demonstrated that the polyprenylated BSA or
peptides (I and II) can be further modified by fluorination following
our established protocol. In regard for application in living cell, we
modified the cetuximab with geranyl functionality, which was
facilitated by the cetuximab-EGFR selective binding, the fluorescent
labelling of HeLa cells was demonstrated to be efficient. Method
applied to investigate the genome-wide protein prenylation remain
elusive and tedious, our approach exhibits potential in the genome-
wide study of protein prenylation. While evolving applications of
Fig. 3. Selective detection of geranyl-modified BSA by fluorination. (A) Reaction con-
ditions: (a) potassium carbonate (3 eq.), MeOH, r. t., 12 h, 70%. (b) DIPEA, CH2Cl2, r. t.,
12 h, 90%. (c) PBS/CH3CN, r. t., 30 min. (d) Selectfluor (100 eq.), PBS/CH3CN, r. t., 30 min.
Identify of the fluoride BSA by SDS-PAGE and 19F Nuclear Magnetic Resonance. (B) Gel
electrophoresis analysis: BSA lane is the sample (5) used herein. BSA-Ge is the sample
(5) treated with excess of geranylated reagent (4). BSA-Ge-F is the sample (6) treated
with excess of Selectfluor reagent. Only tiny band (55 kDa) are observed. (C) The 19F
NMR of samples (7) shown ꢀ177.07 ppm fluorinated peak. (D) Structural analysis of
BSA (chain A form, PDB 3v03), Red color residues indicated the fluorinated geranyl-
modified sites and sequence were also depicted (Supplementary Material, Fig. S3).
3

J. Zhang, J. Zhao, L. Wang et al.
Tetrahedron 81 (2021) 131917
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
R. Wang thanks the starting fund from Huazhong University of
Science and Technology for financial support. One-Hundred-
Talents Youth Program of Hubei Province is appreciated. NSFC
(21904044) is appreciated for financial support. We thank Prof.
Chaomei Xiong for NMR experiment and Prof. Lingkui Meng for
mass spectrum.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2020.131917.
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