Silyl-protected propargyl glycine for multiple labeling of peptides by chemoselective silyl-deprotection

Article abstract of DOI:10.1016/j.tetlet.2021.153093

We synthesized Fmoc-propargyl glycine derivatives bearing different silyl protecting groups that can be readily introduced by using a standard solid-phase peptide coupling procedures. Taking advantage of the orthogonality between the different silyl prote

Full text of DOI:10.1016/j.tetlet.2021.153093

Tetrahedron Letters 73 (2021) 153093
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Silyl-protected propargyl glycine for multiple labeling of peptides by
chemoselective silyl-deprotection
a,c,
Naoki Kamo a, Gosuke Hayashi b, , Akimitsu Okamoto
⇑
⇑
a Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
b Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan
c Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized Fmoc-propargyl glycine derivatives bearing different silyl protecting groups that can be
readily introduced by using a standard solid-phase peptide coupling procedures. Taking advantage of the
orthogonality between the different silyl protecting groups, chemoselective incorporation of functional
molecules into a 19-mer peptide through click reactions was demonstrated.
Received 4 March 2021
Revised 8 April 2021
Accepted 14 April 2021
Available online 17 April 2021
Ó 2021 Elsevier Ltd. All rights reserved.
Keywords:
Silyl-protecting groups
Chemical protein synthesis
Chemoselective labeling
Site-specific chemical modification of peptides or proteins is an
essential technique for biological research and drug discovery [1].
Total chemical protein synthesis, which consists of solid-phase
peptide synthesis (SPPS) and peptide ligation, has become a
promising technology to obtain homogeneously modified proteins
[2,3]. Native chemical ligation [4], which utilizes the chemoselec-
tivity between a C-terminal thioester and an N-terminal Cys resi-
due, is the most widely employed peptide ligation technique to
condense divided peptide segments and form native amide bonds.
To synthesize proteins that do not contain Cys residues in the
sequences, free-radical desulfurization to convert Cys residues into
Ala residues has been exploited [5]. However, the radicals gener-
ated in the desulfurization often cause side reactions of functional
molecules such as alkyne moieties and fluorescent dyes that con-
steric hindrance, enabling to site-selective labeling of peptides by
copper-catalyzed azide–alkyne cycloaddition (CuAAC) [8].
However, these silyl-protected alkynes are generally
incorporated at Lys residues or N-terminal amino groups, and the
replacement of amino groups with hydrophobic silyl groups leads
to significant reduction in the water solubility of the peptides.
Moreover, we could not fine-tune the length of the linker when
functional molecules were introduced at side chains of Lys resi-
dues, which were not suitable for application in distance-depen-
dent analyses such as Förester resonance energy transfer (FRET)
assays.
Herein, we developed two Fmoc-propargylglycine derivatives
bearing dimethylethylsilyl (DMES)-protected or t-butyldimethylsi-
lyl (TBS)-protected alkynes [Fmoc-Pra(DMES)-OH or Fmoc-Pra
(TBS)-OH] that can be readily introduced by using standard cou-
pling SPPS. We prepared 19-mer peptide with these Fmoc-amino
acids, and site-selective CuAAC was preformed through the orthog-
onal deprotection of DMES and TBS groups (Scheme 1).
To synthesize Fmoc-Pra(DMES)-OH and Fmoc-Pra(TBS)-OH, 2-
tetrahydropyranyl (THP)-protected propargylalcohol (1) was used
as starting material (Scheme 2). The hydrogen at the terminal
alkyne was abstracted by n-BuLi in tetrahydrofuran (THF) at
À78 °C and then DMES or TBS chloride was added to introduce
each silyl group onto the terminal alkyne. Upon completion, the
THP group was removed under acidic conditions to afford com-
pounds 2a and 2b. Diphenylphosphate, a bulky leaving group,
was introduced at the hydroxy group of 2a and 2b in the presence
of 4-dimethylaminopyridine (4-DMAP) and pyridine to obtain
tain
p-extended conjugations [6,7], limiting the introduction of
functional molecules into chemically synthesized proteins.
To address this issue, we previously developed a method to
incorporate functional molecules into peptides or proteins site-
selectively by using alkynes protected with different silyl groups.
Silyl-protected alkynes tolerate the free-radical desulfurization
conditions and can be removed orthogonally based on differing
⇑
Corresponding authors at: Department of Chemistry and Biotechnology,
Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-
ku, Tokyo 113-8656, Japan (A. Okamoto).
E-mail addresses: hayashi@chembio.nagoya-u.ac.jp (G. Hayashi), okamoto@-
chembio.t.u-tokyo.ac.jp (A. Okamoto).
https://doi.org/10.1016/j.tetlet.2021.153093
0040-4039/Ó 2021 Elsevier Ltd. All rights reserved.

N. Kamo, G. Hayashi and A. Okamoto
Tetrahedron Letters 73 (2021) 153093
(6a) and Fmoc-Pra(TBS)-OH (6b). Compounds 6a and 6b were syn-
thesized in 45% and 34%, respectively, over five reaction steps, and
the gram-scale synthesis of each compound was accomplished.
Next, peptide 7 was also prepared to perform chemoselective
labeling on a peptide utilizing 6a and 6b (Scheme 3). After the
incorporation of compound 6b on rink amide resin using 1-[bis
(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexaflu-
orophosphate (HBTU) as a condensation reagent, the peptide chain
was extended by using an automated peptide synthesizer. Com-
pound 6a was then coupled, and the peptide was cleaved with tri-
fluoroacetic acid (TFA) cocktail (92.5% TFA, 5% triisopropylsilane,
2.5% 1,3-dimethoxybenzene). The peptide was recovered by ether
precipitation and the crude peptide was purified by high-perfor-
mance liquid chromatography (HPLC); however, we observed the
removal of DMES or TBS group during cleavage from the resin
(Fig. S2), indicating that optimization of the cleavage conditions
was required.
To check the stability of silyl-protected alkynes under desulfu-
rization conditions, the Cys residue in peptide 7 was converted into
an Ala residue through free-radical desulfurization (Scheme 3).
Quantitative conversion into the desired Ala residue was observed
after 2 h reaction without side reactions on silyl-protected alkynes,
and peptide 8 was isolated by HPLC purification in 60% yield
(Fig. S3).
Scheme 1. General strategy of this study.
Scheme 2. Synthetic strategy of Fmoc-Pra(DMES)-OH (6a) and Fmoc-Pra(TBS)-OH
(6b).
compounds 3a and 3b. To achieve enantioselective synthesis, we
employed the Schollkopf reagent [9]. First, the proton with the
higher acidity of the Schollkopf reagent was abstracted with n-BuLi
in THF at À78 °C, and the mixture was added to the solution con-
taining compound 3a or 3b bearing diphenylphosphate. The steric
hindrance between the isopropyl group of the Schollkopf reagent
and the diphenylphosphate led to the diastereoselective reaction
(Scheme S1). While compound 4b with TBS-protected alkyne was
obtained without any byproducts, we observed a small amount
of the diastereomer of compound 4a with DMES-protected alkyne
(Fig. S1), suggesting that the steric repulsion between the Schol-
lkopf reagent and silyl groups might affect the diastereoselectivity.
Compounds 4a and 4b were hydrolyzed under acidic conditions
and each compound was mixed with N-(9-fluorenylmethoxycar-
bonyloxy)succinimide (Fmoc-OSu) in the presence of NaHCO3 to
protect the amino group with the Fmoc group to afford compounds
5a and 5b. Finally, the methyl ester moiety was hydrolyzed under
basic conditions in the presence of CaCl2, which prevented degra-
dation of the Fmoc groups by reducing the concentration of the
hydroxyl ions [10] to afford final products Fmoc-Pra(DMES)-OH
Scheme 3. Strategy of free-radical desulfurization and dual incorporation of small
molecules into a peptide. Desulfurization condition: peptide 7 (1.5 mM), TCEP
(250 mM), GSH (50 mM), VA-044 (5 mM) in denaturing solution (6 M Gn-HCl, 0.2 M
NaH2PO4 pH 6–7), 37 °C. Simultaneous DMES removal and click reaction condition:
peptide 8 (1 mM), 2-azidoacetic acid (2 mM), CuSO4 (1.5 mM), THPTA (5 mM),
sodium ascorbic acid (20 mM), KF (4 mM), amino guanidine (20 mM) in H2O/t-
BuOH (7:3), 37 °C. TBS removal condition: peptide 9 (1 mM), KF (15 mM) and
AgNO3 (15 mM) in H2O/t-BuOH (7:3), 37 °C. Click reaction condition: peptide
(0.5 mM), azido-(PEG)3-NH2 (2 mM), CuSO4 (2 mM), THPTA (5 mM), sodium
ascorbic acid (20 mM) and amino guanidine (20 mM) in H2O/t-BuOH (7:3), 37 °C.
2

N. Kamo, G. Hayashi and A. Okamoto
Tetrahedron Letters 73 (2021) 153093
In conclusion, we synthesized Fmoc-Pra(DMES)-OH and Fmoc-
Pra(TBS)-OH, which are compatible with Fmoc-SPPS. The chemos-
elective labeling of a peptide was accomplished by using the
orthogonality based on the different steric bulk of the silyl-protect-
ing groups. We envision that these Fmoc-amino acids bearing silyl-
protected alkynes can be applied to produce a high diversity of
peptides and proteins that will facilitate drug discovery.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
Fig. 1. Reaction tracking of dual labeling on the model peptide using the
orthogonality between the DMES and TBS groups. HPLC peaks were monitored at
220 nm in a linear gradient of water/acetonitrile containing 0.1% TFA with 5C18-AR-
ⅠⅠ. (A) Simultaneous DMES deprotection and click reaction with 2-azidoacetic acid.
Gradient: 20–50% for 30 min. (B) TBS deprotection, followed by click reaction with
azido-(PEG)3-NH2. 9*= TBS-deprotected 9. # = Click reagents. Gradient: 15–45% for
30 min.
This work was supported by Japan Society for the Promotion of
Science (JSPS) KAKENHI 18H03931, 18H05504, and 19K22245 (A.
O.), 18K05313, 19H05287, and 20H04704 and JST PRESTO
JPMJPR19K6 (G.H.). N.K. was supported by a Research Fellow of
the Japan Society for the Promotion of Science 19J13608.
Appendix A. Supplementary data
Site-selective modification that leveraged the orthogonality
between the DMES and TBS groups was then performed. DMES
removal with potassium fluoride (KF) and CuAAC with 2-azi-
doacetic acid were conducted in a one-pot manner. Notably, the
deprotection of the DMES group was accelerated in the presence
of Cu(I) cation, without affecting the TBS group [8,11]. HPLC anal-
ysis showed the quantitative conversion of peptide 8 into peptide 9
after 2 h reaction at 37 °C, and the desired product was isolated in
69% yield (Fig. 1A, S4). Peptide 9 was then treated with 15 equiv. of
KF and 15 equiv. of AgNO3 to remove the TBS group (Fig. 1B). After
completion of the deprotection, sodium chloride was added to the
reaction mixture to trap the silver ion via precipitation of AgCl salt,
and the supernatant and precipitates were separated. To the super-
natant were added azido-(PEG)3-NH2 and other reagents for
CuAAC. After 1 h of reaction at 37 °C, the HPLC data showed a
new peak derived from peptide 10 (Fig. 1B), which was identified
by matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF MS) and isolated in 47% yield (Fig. S5).
The chemoselective incorporation of small molecules into a pep-
tide was demonstrated using newly synthesized amino acids bear-
ing different silyl-protected alkynes.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153093.
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