Design of stapled oxyntomodulin analogs containing functionalized biphenyl cross-linkers

Article abstract of DOI:10.1016/j.tet.2018.11.060

A panel of three lipid-modified, functionalized biphenyl cross-linkers (fBph) were synthesized and subsequently employed in the preparation of the stapled oxyntomodulin (OXM) analogs. In a luciferase-based reporter assay, these stapled OXM analogs showed varying degree of potency in activating GLP-1R and GCGR, presumably due to the disparate effect of the lipid chains on the local environment close to the ligand-receptor binding interface. In particular, the fBph-1 cross-linked peptide with the lipid chain attached to position-3 of the biphenyl cross-linker exhibited the highest dual agonist activity.

Full text of DOI:10.1016/j.tet.2018.11.060

Tetrahedron xxx (xxxx) xxx
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design of stapled oxyntomodulin analogs containing functionalized
biphenyl cross-linkers
,
,
,
, c, *
Yulin Tian a c, Huafei Zou b, Peng An a c, Zhihong Zhou b, Weijun Shen b **, Qing Lin a
a Department of Chemistry, State University of New York at Buffalo, Buffalo, NY, 14260, United States
b Department of Biology, Calibr at the Scripps Research Institute, 11119 North Torrey Pines Road, La Jolla, CA, 92037, United States
c Transira Therapeutics, 1576 Sweet Home Road, Baird Research Park, Amherst, NY, 14228, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A panel of three lipid-modified, functionalized biphenyl cross-linkers (fBph) were synthesized and
subsequently employed in the preparation of the stapled oxyntomodulin (OXM) analogs. In a luciferase-
based reporter assay, these stapled OXM analogs showed varying degree of potency in activating GLP-1R
and GCGR, presumably due to the disparate effect of the lipid chains on the local environment close to
the ligand-receptor binding interface. In particular, the fBph-1 cross-linked peptide with the lipid chain
attached to position-3 of the biphenyl cross-linker exhibited the highest dual agonist activity.
© 2018 Elsevier Ltd. All rights reserved.
Received 24 May 2018
Received in revised form
23 November 2018
Accepted 27 November 2018
Available online xxx
Keywords:
Oxyntomodulin
Cross-linker
GLP-1R
GCGR
Dual agonist
1. Introduction
bis(bromomethyl)biphenyl (Bph) and 6,60-bis(bromomethyl)-3,30-
bipyridine (Bpy), which react with peptides containing cysteines
Oxyntomodulin (OXM) is a natural 37-amino acid peptide hor-
mone derived from proglucagon, and regulates glucose meta-
bolism, insulin secretion, food intake, and energy expenditure [1,2].
As a GLP-1R/GCGR dual-agonist, OXM exhibits remarkable weight
loss and glucose lowering effects that are superior to the GLP-1R-
only agonists, making it an attractive therapeutic candidate for
the concurrent treatment of obesity and type 2 diabetes mellitus
(T2DM) [3]. However, the clinical application of OXM has not been
realized because OXM undergoes rapid proteolytic degradation and
renal clearance after administration, and thus requires more
frequent injections [4].
located at i and iþ7 positions to generate stapled peptides with
improved proteolytic stability and cell permeability [9e13]. To
extend this strategy to OXM, we designed a chimeric peptide, OXM-
7, by incorporating some of the key residues of GLP-1 into the
sequence along with two cysteines located at i, iþ7 positions [14].
We then employed Bph and Bpy to cross-link OXM-7 to give rise to
the stapled analogs, OXM-7-Bph and OXM-7-Bpy, respectively
(Fig. 1A). The stapled OXMs showed greater plasma stability and
sub-nanomolar dual agonist activities in activating GLP-1R and
GCGR in the cell-based assays. The computational modeling of
OXM-7-Bph in complex with the extracellular domain of GLP-1R
reveals a binding mode in which the Bph cross-linker reinforces
Covalent side-chain cross-linking of bioactive peptides (also
known as ‘peptide stapling’) to stabilize peptide
a-helical confor-
a-helical structure but projects away from the binding interface
mation has proven to be a valuable strategy to increase peptide
potency, stability and cell permeability [5e8]. Previously, we re-
ported the design of a series of biaryl cross-linkers including 4,40-
(Fig. 1B) [15]. Since the half-lives of these analogs were measured to
be ~13 h in mouse serum in vitro and ~1e2 h in vivo after injection
into mice, they are still not sufficiently long for once-weekly
administration in clinical setting.
As a well-known human serum albumin binding motif, the
PEGylated fatty acid has been used in modifying GLP-1 and other
peptides to extend their circulatory half-lives [16e20]. Herein, we
report the design and synthesis of three lipid-modified biphenyl
cross-linkers, and their use in preparing the stapled OXM analogs
* Corresponding author. Department of Chemistry, State University of New York
at Buffalo, Buffalo, NY, 14260, United States.
** Corresponding author. Department of Biology, Calibr at the Scripps Research
Institute, 11119 North Torrey Pines Road, La Jolla, CA 92037, United States.
E-mail addresses: wshen@calibr.org (W. Shen), qinglin@buffalo.edu (Q. Lin).
https://doi.org/10.1016/j.tet.2018.11.060
0040-4020/© 2018 Elsevier Ltd. All rights reserved.
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Y. Tian et al. / Tetrahedron xxx (xxxx) xxx
Fig. 1. (A) Scheme for preparing the Bph and Bpy cross-linked oxyntomodulin derivatives. (B) Structural model of the cross-linked oxyntomodulin, OXM-7-Bph, bound to the
extracellular domain of GLP-1R (PDB code: 3C59): green, GLP-1R; purple, OXM-7-Bph; cyan, Bph; red, the cysteine cross-linking sites.
with enhanced pharmacokinetic properties.
(1) in 94% yield [24]. After protecting the phenol with triflate (2),
the carboxylates were reduced to the benzyl alcohols (3) by DIBAL
followed by protection with TBS chloride to give compound 4.
Subsequent Sonogashira cross-coupling with N-Cbz-prop-2-yn-1-
amine (L1) led to compound 5 [25]. Deprotection of the Cbz
group together with reduction of alkyne to alkane under the hy-
drogenation condition afforded the propylamine intermediate,
which was then coupled with N-Cbz-2-(2-(2-aminoethoxy)ethoxy)
acetic acid (L2) to afford compound 6. Then, three successive
rounds of hydrogenationecoupling reactions were performed to
conjugate 6 with L2, L3, and L4 to give the fully protected com-
pound 9 in 66% yield over 6 steps. After removal of the TBS group,
treatment with PBr3 converted the benzyl alcohols into the benzyl
bromides and simultaneously hydrolyze the tert-butyl esters to
afford the final lipid-modified cross-linker 10 (fBph-1) in 63% yield
over two steps (Scheme 1).
For the synthesis of fBph-2 (20), the procedure was essentially
the same as fBph-1 except the order of Sonogashira cross-coupling
and DIBAL-mediated reduction was switched to increase the overall
reaction yield (Scheme 2). For the synthesis of fBph-3 (27), 2-(4-
bromophenyl)-2-hydroxyacetic acid was coupled with L5 to give
compound 21. Then, the Suzuki cross-coupling between 21 and (4-
(hydroxymethyl)phenyl) boronic acid was performed to generate
biphenyl 22 in 59% yield. The hydroxyl groups in 22 were subse-
quently protected with the TBS group to give compound 23. Then,
three successive rounds of hydrogenationecoupling reactions were
performed to conjugate 23 with L2, L3 and L4 to give rise to the
fully protected lipid-modified cross-linker 26. Subsequent depro-
tection followed by bromination with PBr3 gave the lipid-modified
cross-linker 27 (fBph-3) win 59% yield over two steps (Scheme 3).
For preparation of the cross-linked OXM peptides, the lipid-
modified cross-linker (fBph-1/2/3) was allowed to react with
OXM-7 in a mixed solvent containing 30 mM NH4HCO3 buffer/
CH3CN (3:2) at room temperature for three hours. The reaction
mixtures were purified by preparative HPLC to give the corre-
sponding cross-linked OXM peptides in 17e20% yield (last steps in
Schemes 1e3). The purity and identity of the stapled OXM peptides
were confirmed by analytical HPLC and mass spectrometry anal-
ysis, respectively (Fig. 3).
2. Results and discussion
In designing functionalized cross-linkers for stapling with OXM-
7, we decided to choose a long-chain lipid comprised of a PEG
linker, glutamic acid, and C18 fatty diacid (octadecanedioic acid),
which was reported to afford significantly improved half-life and
glucose tolerance when attached to GLP-1 and excedin-4 (Fig. 2)
[18,19]. To identify optimal attachment site, the lipid chain was
introduced at three different positions of the Bph cross-linker: (i)
position-3 on the biphenyl ring; (ii) position-2 on the biphenyl
ring; and (iii) the benzylic position. The resulting cross-linkers were
termed as fBph-1, fBph-2 and fBph-3, respectively.
Three parallel routes were chosen for the synthesis of the lipid-
modified Bph cross-linkers (Schemes 1e3). In general, benzyl
bromides were converted from benzyl alcohols in the final step
because of their high reactivity. The Pd-catalyzed cross-coupling
reaction was used to construct the biphenyl scaffold, and the lipid
chain was attached through a series of condensation reactions.
Several protecting groups were employed during the synthesis,
including tert-butyldimethylsilyl (TBS) for the hydroxyl group,
carboxybenzyl (Cbz) for the amino group, and tert-butyl for the
carboxylic acid group. Five lipid chain building blocks, N-Cbz-prop-
2-yn-1-amine (L1), N-Cbz-2-(2-(2-aminoethoxy)ethoxy)acetic acid
(L2), N-Cbz-O-tert-butyl-glutamic acid (L3), octadecanedioic acid
mono-tert-butyl ester (L4) and 2,2'-(ethane-1,2-diylbis(oxy))
diethanamine mono-Cbz carbamate (L5) were either commercially
available or synthesized following the reported procedures
[21e23].
For the synthesis of fBph-1 (Scheme 1), the Suzuki cross-
coupling between methyl 4-bromo-2-hydroxybenzoate and (4-
(methoxycarbonyl)phenyl)boronic acid was carried out to produce
the biphenyl core bearing two carboxylates and one phenol group
With the stapled OXM peptides in hand, we assessed their
agonist activity toward the GLP-1R and GCGR using a luciferase
reporter assay in HEK293 cells stably expressing human GLP-1R or
GCGR and a cAMP response element (CRE)-driven luciferase
Fig. 2. Lipid-modified functionalized Bph cross-linkers.
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3
Scheme 1. Synthesis of OXM-7-fBph-1.
reporter (Fig. 4) [19,20]. The previously reported dual agonists,
3. Conclusion
OXM-7-Bph and OXM-7-Bpy, were used as positive controls. All
three fBph-stapled OXM-7 peptides showed decreased potency
compared to the Bph- or Bpy-cross-linked peptides, particularly
against GCGR (Table 1), which was also observed in the literature
[26]. We hypothesize that the long lipid chain may affect the
conformation of the biphenyl cross-linker to varying degree, which
in turn affects local conformation of the middle region of the OXM
peptide and subsequently the preferred binding mode towards
GLP-1R and GCGR. OXM-7-fBph-1 with the lipid chain attached at
position-3 of the biphenyl scaffold showed the highest agonist
activity against both GLP-1R and GCGR (Table 1), suggesting that
the lipid attachment to this position produces smallest perturba-
tion to the local conformation of the OXM peptide. In contrast,
OXM-7-fBph-2 with lipid chain attached to position-2 showed
significant loss of potency against both GLP-1R and GCGR, sug-
gesting that the lipid chain at the ortho-position may dramatically
alter the orientation of the biphenyl ring and as a result the local
conformation of the OXM peptide. When the lipid chain was
introduced at the benzylic position in OXM-7-fBph-3, only 2-fold
lower agonist activities were observed compared to OXM-7-fBph-
1 (Table 1). We then compared the stability of OXM-7-fBph-1 in
fresh mouse serum to that of parent peptide using the GLP-1R
activation assay, and found that indeed the fatty diacid modified,
crosslinked OXM peptide, OXM-7-fBph-1, exhibited longer half-life
than the parent crosslinked peptide, OXM-7-Bph (Fig. 5).
In conclusion, we have designed and synthesized three lipid-
chain modified, functionalized biphenyl cross-linkers, and
employed them to prepare three stapled oxyntomodulin analogs
containing cysteines at i, iþ7 positions. The purity and identity of
the stapled OXM peptides were confirmed by HPLC and mass
spectrometric analyses, respectively. In the receptor activation
assay, the stapled OXM analog with the lipid chain attached at
position-3 of the biphenyl cross-linker showed the most potent
dual agonist activity against GLP-1R and GCGR, most likely due to
the smallest perturbation of the lipid chain to the local conforma-
tion of the OXM peptide. Our initial stability assay confirmed that
the fatty diacid attachment extends serum half-life of the cross-
linked OXM peptide. We are currently in the process of character-
izing the in vivo pharmacokinetic properties of these lipid-
modified crosslinked OXM analogs.
4. Experimental section
4.1. General information
Solvents and chemicals were purchased from commercial
sources and used directly without further purification. Flash chro-
matography was performed with SiliCycle P60 silica gel (40e63
mm,
60 Å). 1H and 13C NMR spectra were recorded with Varian Mercury-
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Scheme 2. Synthesis of OXM-7-fBph-2.
300 or Inova-500 MHz spectrometer. Chemical shifts were reported
in ppm using either TMS or deuterated solvents as internal stan-
dards (TMS, 0.00; CDCl3, 7.26; CD3OD, 3.31; DMSO‑d6, 2.50). Mul-
tiplicity was reported as follows: s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet, brs ¼ broad. 13C NMR spectra were
recorded at 75 MHz or 126 MHz, and chemical shifts were reported
in ppm using deuterated solvents as internal standards (CDCl3).
Electrospray LC-MS analysis was performed using a Finnigan LCQ
Advantage IonTrap mass spectrometry coupled with a Surveyor
(23 mg, 0.1 mmol), PPh3 (26 mg, 0.1 mmol) and K2CO3 (829 mg,
6 mmol). The mixture was stirred at room temperature under argon
protection for 12 h. The solution was neutralized with 1 N HCl and
the aqueous phase was extracted with EtOAc (5 mL ꢀ 3). The
combined organic layer was wash with H2O, brine, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified by silica gel flash column chromatography (hexanes/
EtOAc ¼ 5:1) to afford compound 1 (537 mg, 94%). Yellow solid; 1H
NMR (500 MHz, CDCl3)
d
10.82 (s, 1H), 8.12 (d, J ¼ 8.2 Hz, 2H), 7.92
HPLC system. Peptides were purified using
a
Gilson semi-
(d, J ¼ 8.3 Hz,1H), 7.68 (d, J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 1.3 Hz, 1H), 7.15
(dd, J ¼ 8.3, 1.3 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H); ESI-MS calcd for
preparative reverse-phase HPLC system equipped with Phenom-
enex C18 column with a flow rate of 5 mL/min and a gradient of
5e95% acetonitrile/H2O while monitoring at 220 and 254 nm.
Analytical HPLC was performed using Kinetex C18 column
(250 ꢀ 4.6 mm) with a flow rate of 1.0 mL/min and a gradient of
5e95% acetonitrile/H2O over 15 min with UVevis detection wave-
length set at 220 and 254 nm. The intact peptide masses were
derived through deconvoluting charge ladders using ProMass
software (Thermo Scientific). High resolution mass spectrometry
was performed on a Bruker solariX XR Fourier transform ion
cyclotron resonance mass spectrometer (FT-ICR-MS).
C
16H15O5 287.1 [M þ Hþ], found 287.1.
4.2.2. Dimethyl 3-(((trifluoromethyl)sulfonyl)oxy)-[1,10-biphenyl]-
4,40-dicarboxylate (2)
To a solution of 1 (440 mg, 1.5 mmol) in dichloromethane
(10 mL) was added pyridine (593 mg, 7.5 mmol) and Tf2O (650 mg,
2.3 mmol) at 0 ꢁC. The mixture was stirred at room temperature for
12 h, and then concentrated. The residue was dissolved in EtOAc
(10 mL), washed with 1 N HCl, brine, dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by silica gel
flash column chromatography (hexanes/EtOAc ¼ 4:1) to afford
compound 2 (614 mg, 98%). Colorless oil; 1H NMR (300 MHz, CDCl3)
4.2. Synthesis of OXM-7-fBph-1
4.2.1. Dimethyl 3-hydroxy-[1,10-biphenyl]-4,40-dicarboxylate (1)
To a solution of methyl 4-bromo-2-hydroxybenzoate (463 mg,
2 mmol) in (CH3OCH2)2/H2O (10 mL, 1:1) was added (4-(methox-
ycarbonyl)phenyl)boronic acid (432 mg, 2.4 mmol), Pd(OAc)2
d
8.22e8.11 (m, 3H), 7.71 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.68e7.61 (m, 2H),
7.51 (d, J ¼ 1.5 Hz, 1H), 4.00 (s, 3H), 3.96 (s, 3H); ESI-MS calcd for
C
17H14F3O7S 419.0 [M þ Hþ], found 419.1.
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Scheme 3. Synthesis of OXM-7-fBph-3.
4.2.3. 4,40-Bis(hydroxymethyl)-[1,10-biphenyl]-3-yl
trifluoromethanesulfonate (3)
(hexanes/EtOAc ¼ 3:1) to afford compound
Colorless oil; 1H NMR (300 MHz, CDCl3)
7.61 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.56e7.48 (m, 2H), 7.44e7.40 (m, 3H),
4.86 (s, 2H), 4.79 (s, 2H), 0.96 (s, 18H), 0.13 (d, J ¼ 4.5 Hz, 12H); ESI-
MS calcd for C27H41F3O5SSi2Na 613.2 [M þ Naþ], found 613.1.
4
(754 mg, 87%).
d
7.69 (d, J ¼ 8.1 Hz, 1H),
To a solution of 2 (614 mg, 1.5 mmol) in Et2O (10 mL) was added
1.2 M DIBAL in toluene (5 mL, 6 mmol) at ꢂ78 ꢁC. The mixture was
stirred at room temperature for 3 h. Then 10% potassium sodium
tartrate solution was added, and the mixture was stirred for
another 1 h. The organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified by silica gel flash column chromatography (hexanes/
EtOAc ¼ 2:1) to afford compound 3 (530 mg, 99% yield). White
4.2.5. Benzyl (3-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-3-yl)prop-2-yn-1-yl)carbamate (5)
solid; 1H NMR (300 MHz, CDCl3)
d 7.71e7.60 (m, 2H), 7.60e7.53 (m,
To a solution of 4 (605 mg, 1 mmol) in DMSO (8 mL) was added
N-Cbz-prop-2-yn-1-amine (L1) (291 mg, 1.5 mmol), Pd(OAc)2
(7 mg, 0.03 mmol), PPh3 (32 mg, 0.12 mmol), K3PO4 (255 mg,
1.2 mmol). The mixture was stirred at 80 ꢁC under argon protection
for 16 h. Then H2O was added and the aqueous phase was extracted
with Et2O (4 mL ꢀ 3). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered and concentrated. The
residue was purified by silica gel flash column chromatography
2H), 7.51e7.45 (m, 3H), 4.84 (d, J ¼ 5.6 Hz, 2H), 4.77 (d, J ¼ 4.8 Hz,
2H); ESI-MS calcd for C15H13F3O5SNa 385.0 [M þ Naþ], found 385.0.
4.2.4. 4,40-Bis(((tert-butyldimethylsilyl)oxy)methyl)-[1,10-
biphenyl]-3-yl trifluoromethanesulfonate (4)
To a solution of 3 (530 mg, 1.5 mmol) in DMF (8 mL) was added
imidazole (89 mg, 11.8 mmol) and TBSCl (888 mg, 5.9 mmol) at 0 ꢁC.
The mixture was stirred at room temperature for 1 h, saturated
NH4Cl was added and the aqueous phase was extracted with EtOAc
(4 mL ꢀ 3). The combined organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated. The res-
idue was purified by silica gel flash column chromatography
(hexanes/EtOAc ¼ 4:1) to afford compound
5 (220 mg, 34%).
Colorless oil; 1H NMR (500 MHz, CDCl3)
d
7.61 (s, 1H), 7.57 (s, 2H),
7.53 (d, J ¼ 8.1 Hz, 2H), 7.39e7.29 (m, 7H), 5.15 (s, 2H), 5.01 (s, 1H),
4.87 (s, 2H), 4.78 (s, 2H), 4.28 (s, 2H), 0.97 (s, 18H), 0.12 (s, 12H); ESI-
MS calcd for C37H51NO4Si2Na 652.3 [M þ Naþ], found 652.3.
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Y. Tian et al. / Tetrahedron xxx (xxxx) xxx
Fig. 3. Characterization of the purity and identity of the stapled OXM peptides. (A) HPLC traces of the lipid-modified Bph cross-linked oxyntomodulin analogs. (B) Deconvoluted
mass spectrum of OXM-7-fBph-1. Calcd mass for C241H367N59O70S3 5306.6, found 5306.3. (C) Deconvoluted mass spectrum of OXM-7-fBph-2. Calcd mass for C241H367N59O70S3
5306.6, found 5306.3. (D) Deconvoluted mass spectrum of OXM-7-fBph-3. Calcd mass for C239H363N59O70S3 5278.6, found 5278.2.
Fig. 4. Agonist activity of the cross-linked oxyntomodulin analogs against GLP-1R (A) and GCGR (B) in a cell-based luciferase reporter assay.
4.2.6. Benzyl (2-(2-(2-((3-(4,40-bis(((tert-butyldimethylsilyl)oxy)
methyl)-[1,10-biphenyl]-3-yl)propyl)amino)-2-oxoethoxy)ethoxy)
ethyl)carbamate (6)
To a solution of 5 (200 mg, 0.32 mmol) in MeOH (5 mL) was
added Pd/C (40 mg, 20%). The mixture was stirred at room tem-
perature under H2 for 1 h, then filtered and concentrated to afford
crude amine (118 mg, 74% yield) as colorless oil, which was directly
used for the next step. To a solution of crude amine (118 mg,
0.24 mmol) in DMF (3 mL) was added N-Cbz-2-(2-(2-aminoethoxy)
ethoxy)acetic acid (L2) (86 mg, 0.29 mmol), HATU (110 mg,
0.29 mmol) and DIPEA (124 mg, 0.96 mmol). The mixture was
stirred at room temperature for 1 h. Then H2O was added and the
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4.2.8. tert-Butyl 26-(((benzyloxy)carbonyl)amino)-1-(4,40-
bis(((tert-butyldimethylsilyl)oxy)methyl)-[1,10-biphenyl]-3-yl)-
5,14,23-trioxo-7,10,16,19-tetraoxa-4,13,22-triazaheptacosan-27-
oate (8)
Table 1
Agonist activity of the cross-linked oxyntomodulin analogs in the activation of GLP-
1R and GCGR.
Name
GLP-1R EC50 (nM)
GCGR EC50 (nM)
To a solution of 7 (92 mg, 0.1 mmol) in MeOH (2 mL) was added
Pd/C (18 mg, 20%). The mixture was stirred at room temperature
under H2 for 1 h, filtered and concentrated to afford crude amine
(74 mg, 94% yield) as colorless oil, which was directly used for the
next step. To a solution of crude amine (68 mg, 0.09 mmol) in DMF
(1 mL) was added N-Cbz-O-tert-butyl-glutamic acid (L3) (35 mg,
0.1 mmol), HATU (38 mg, 0.1 mmol) and DIPEA (44 mg, 0.34 mmol).
The mixture was stirred at room temperature for 1 h. Then H2O was
added and the aqueous phase was extracted with EtOAc (2 mL ꢀ 3).
The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified by silica gel flash column chromatography (DCM/
MeOH ¼ 15:1) to afford compound 8 (95 mg, 99%). Colorless oil; 1H
OMX-7-Bph
OMX-7-Bpy
OMX-7-fBph-1
OMX-7-fBph-2
OMX-7-fBph-3
0.143
0.162
2.498
36.71
5.560
0.163
0.186
24.47
>80
44.44
NMR (500 MHz, CDCl3)
d
7.55 (d, J ¼ 8.1 Hz, 2H), 7.49e7.40 (m, 2H),
7.40e7.35 (m, 3H), 7.34e7.29 (m, 5H), 7.05 (s, 1H), 6.82 (s, 1H), 6.50
(s, 1H), 5.08 (s, 2H), 4.78 (s, 2H), 4.76 (s, 2H), 4.22e4.19 (m, 1H), 3.98
(s, 2H), 3.96 (s, 2H), 3.70e3.29 (m, 20H), 2.71 (t, J ¼ 7.9 Hz, 2H),
2.30e2.10 (m, 2H),1.93e1.88 (m, 2H),1.56 (s, 9H), 0.95 (d, J ¼ 4.4 Hz,
18H), 0.12 (s, 12H); ESI-MS calcd for C58H92N4O13Si2Na 1131.6
[M þ Naþ], found 1131.5.
Fig. 5. Comparison of the in vitro stability of the lipid-modified OXM-7-fBph-1 vs.
OXM-7-Bph in fresh mouse serum. The concentrations of the remaining intact OXM
peptides in serum were determined using the GLP-1R activation assay.
4.2.9. tert-Butyl 1-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-3-yl)-26-(tert-butoxycarbonyl)-5,14,23,28-tetraoxo-
7,10,16,19-tetraoxa-4,13,22,27-tetraazapentatetracontan-45-oate
(9)
aqueous phase was extracted with EtOAc (2 mL ꢀ 3). The combined
organic layer was washed with brine, dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by silica gel
flash column chromatography (hexanes/EtOAc ¼ 1:2) to afford
compound 6 (100 mg, 54% yield). Colorless oil; 1H NMR (300 MHz,
To a solution of 8 (95 mg, 0.09 mmol) in MeOH (2 mL) was added
Pd/C (19 mg, 20%). The mixture was stirred at room temperature
under H2 for 1 h, then filtered and concentrated to afford crude
amine (66 mg, 79% yield) as colorless oil, which was directly used
for the next step. To a solution of crude amine (65 mg, 0.07 mmol)
in DMF (1 mL) was added octadecanedioic acid mono-tert-butyl
ester (L4) (30 mg, 0.08 mmol), HATU (30 mg, 0.08 mmol) and DIPEA
(26 mg, 0.2 mmol). The mixture was stirred at room temperature
for 1 h. Then H2O was added and the aqueous phase was extracted
with EtOAc (2 mL ꢀ 3). The combined organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated.
The residue was purified by silica gel flash column chromatography
(DCM/MeOH ¼ 15:1) to afford compound 9 (82 mg, 92% yield).
CDCl3)
d
7.55 (d, J ¼ 8.1 Hz, 2H), 7.48e7.41 (m, 2H), 7.41e7.35 (m,
3H), 7.34e7.30 (m, 5H), 6.79 (s, 1H), 5.07 (s, 2H), 4.78 (s, 2H), 4.76 (s,
2H), 3.97 (s, 2H), 3.61 (dd, J ¼ 11.0, 5.5 Hz, 4H), 3.53 (t, J ¼ 5.1 Hz,
2H), 3.42e3.31 (m, 4H), 2.73e2.64 (m, 2H), 1.94e1.81 (m, 2H), 0.95
(d, J ¼ 3.2 Hz, 18H), 0.12 (s, 12H); ESI-MS calcd for C43H66N2O7Si2Na
801.4 [M þ Naþ], found 801.4.
4.2.7. Benzyl (21-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-3-yl)-8,17-dioxo-3,6,12,15-tetraoxa-9,18-
diazahenicosyl)carbamate (7)
Colorless oil; 1H NMR (500 MHz, CDCl3)
d
7.55 (d, J ¼ 8.0 Hz, 2H),
7.49e7.41 (m, 2H), 7.40e7.35 (m, 3H), 7.05 (s, 1H), 6.85 (s, 1H), 6.75
(s, 1H), 6.49 (d, J ¼ 7.2 Hz, 1H), 4.78 (s, 2H), 4.76 (s, 2H), 4.45e4.37
(m, 1H), 3.99 (s, 2H), 3.97 (s, 2H), 3.71e3.31 (m, 20H), 2.72 (t,
J ¼ 7.8 Hz, 2H), 2.32e2.24 (m, 2H), 2.19 (t, J ¼ 7.4 Hz, 2H), 1.97e1.83
(m, 2H), 1.65e1.59 (m, 6H), 1.45 (s, 9H), 1.44 (s, 9H), 1.37e1.16 (m,
24H), 0.95 (d, J ¼ 4.2 Hz,18H), 0.12 (s, 12H); ESI-MS calcd for
To a solution of 6 (80 mg, 0.1 mmol) in MeOH (2 mL) was added
Pd/C (16 mg, 20%). The mixture was stirred at room temperature
under H2 for 1 h, then filtered and concentrated to afford crude
amine (64 mg, 99%) as colorless oil, which was directly used for the
next step. To a solution of crude amine (64 mg, 0.1 mmol) in DMF
(2 mL) was added N-Cbz-2-(2-(2-aminoethoxy)ethoxy)acetic acid
(L2) (36 mg, 0.12 mmol), HATU (46 mg, 0.12 mmol) and DIPEA
(52 mg, 0.4 mmol). The mixture was stirred at room temperature
for 1 h. Then H2O was added and the aqueous phase was extracted
with EtOAc (2 mL ꢀ 3). The combined organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated.
The residue was purified by silica gel flash column chromatography
(DCM/MeOH ¼ 15:1) to afford compound 7 (92 mg, 99% yield).
C
72H126N4O14Si2Na 1349.9 [M þ Naþ], found 1349.8.
4.2.10. 1-(4,40-Bis(bromomethyl)-[1,10-biphenyl]-3-yl)-26-carboxy-
5,14,23,28-tetraoxo-7,10,16,19-tetraoxa-4,13,22,27-
tetraazapentatetracontan-45-oic acid (10, fBph-1)
To a solution of 9 (69 mg, 0.05 mmol) in THF (1 mL) was added
1 M TBAF in THF (0.13 mL, 0.13 mmol) at 0 ꢁC. The mixture was
stirred at room temperature for 2 h. Then H2O was added and the
aqueous phase was extracted with EtOAc (2 mL ꢀ 3). The combined
organic layer was washed with brine, dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by silica gel
flash column chromatography (DCM/MeOH ¼ 10:1) to afford
alcohol compound (40 mg, 70% yield) as colorless oil. To a solution
of alcohol (40 mg, 0.036 mmol) in DCM (1 mL) was added PBr3
(49 mg, 0.18 mmol) at 0 ꢁC. The mixture was stirred at room tem-
perature for 2 h, then H2O was added．The organic layer was
Colorless oil; 1H NMR (500 MHz, CDCl3)
d
7.55 (d, J ¼ 7.9 Hz, 2H),
7.47 (d, J ¼ 7.7 Hz,1H), 7.42 (d, J ¼ 7.3 Hz,1H), 7.38 (d, J ¼ 6.7 Hz, 3H),
7.34e7.28 (m, 5H), 7.13 (s, 1H), 6.77 (s, 1H), 5.51 (s, 1H), 5.08 (s, 2H),
4.78 (s, 2H), 4.76 (s, 2H), 3.95 (s, 4H), 3.60e3.50 (m, 12H), 3.48e3.41
(m, 2H), 3.41e3.30 (m, 4H), 2.75e2.62 (m, 2H), 1.93e1.80 (m, 2H),
0.95 (d, J ¼ 4.6 Hz, 18H), 0.12 (s, 12H); ESI-MS calcd for
C
49H77N3O10Si2Na 946.5 [M þ Naþ], found 946.4.
Please cite this article as: Y. Tian et al., Design of stapled oxyntomodulin analogs containing functionalized biphenyl cross-linkers, Tetrahedron,
https://doi.org/10.1016/j.tet.2018.11.060

8
Y. Tian et al. / Tetrahedron xxx (xxxx) xxx
washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated to afford compound 10 (fBph-1) (36 mg, 90% yield).
NMR (500 MHz, CDCl3) d 7.57e7.44 (m, 3H), 7.42e7.29 (m, 9H), 5.11
(s, 2H), 4.82 (s, 1H), 4.70 (s, 2H), 4.69 (s, 2H), 4.07 (d, J ¼ 4.4 Hz, 2H);
White solid; 1H NMR (500 MHz, CDCl3)
d
7.55 (d, J ¼ 8.0 Hz, 2H),
ESI-MS calcd for C25H23NO4Na 424.2 [M þ Naþ], found 424.2.
7.46 (d, J ¼ 8.0 Hz, 2H), 7.44e7.37 (m, 3H), 7.17 (s, 1H), 7.12 (s, 1H),
7.04 (s, 1H), 4.59 (s, 2H), 4.55 (s, 2H), 4.52e4.47 (m, 1H), 4.08 (s, 2H),
4.02 (s, 2H), 3.76e3.31 (m, 18H), 2.88e2.78 (m, 2H), 2.53e2.33 (m,
2H), 2.32 (t, J ¼ 7.4 Hz, 2H), 2.23 (t, J ¼ 7.5 Hz, 2H), 2.18e2.04 (m,
4.3.5. Benzyl (3-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-2-yl)prop-2-yn-1-yl)carbamate (15)
Compound 15 was prepared using the same procedure as that
2H), 2.05e1.97 (m, 2H), 1.68e1.55 (m, 4H), 1.28e1.22 (m, 24H); 13
C
described for compound 4. Yield: 93% (287 mg); Brown oil; 1H NMR
NMR (75 MHz, CDCl3)
d
177.48,174.72,173.92,173.47,170.84,170.64,
(500 MHz, CDCl3)
d
7.51 (d, J ¼ 7.6 Hz, 2H), 7.45 (s, 1H), 7.38e7.29
141.18, 140.86, 140.45, 137.17, 134.67, 131.27, 129.55, 128.30, 127.45,
125.35, 70.79, 70.43, 70.41, 70.25, 70.16, 69.75, 69.73, 69.47, 52.21,
39.58, 38.89, 38.63, 36.26, 33.95, 33.24, 32.30, 31.56, 30.47, 29.68,
29.64, 29.58, 29.50, 29.43, 29.35, 29.30, 29.27, 29.23, 29.14, 29.06,
28.95, 28.22, 25.60, 24.74, 22.63; HRMS (ESI) calcd for
(m, 9H), 5.12 (s, 2H), 4.79 (s, 1H), 4.76 (s, 2H), 4.73 (s, 2H), 4.10 (d,
J ¼ 4.3 Hz, 2H), 0.96 (d, J ¼ 1.1 Hz, 18H), 0.12 (d, J ¼ 4.8 Hz, 12H); ESI-
MS calcd for C37H51NO4Si2Na 652.3 [M þ Naþ], found 652.3.
4.3.6. Benzyl (2-(2-(2-((3-(4,40-bis(((tert-butyldimethylsilyl)oxy)
methyl)-[1,10-biphenyl]-2-yl)propyl)amino)-2-oxoethoxy)ethoxy)
ethyl)carbamate (16)
C
52H81Br2N4O12 1113.4197 [M þ Hþ], found 1113.4212.
4.2.11. OXM-7-fBph-1
Compound 16 was prepared using the same procedure as that
described for compound 6. Yield: 67% for 2 steps (240 mg); Yellow
To a solution of OXM-7 peptide (10 mg, 0.0023 mmol) in 30 mM
NH4HCO3 buffer (0.5 mL) was added a solution of fBph-1 (2.5 mg,
0.0023 mmol) in CH3CN (0.5 mL). The mixture was stirred at room
temperature for 3 h. Under ice cooling, acetic acid was subse-
quently added dropwise to pH 5. Crude cross-linked peptide was
purified by preparative reverse phase HPLC (RP-HPLC) to afford the
title compound as a white powder after lyophilization (2.0 mg, 17%
yield). Calcd mass for C241H367N59O70S3 5306.6 [M], ESI-MS m/z
found 885.4 [Mþ6H]6þ, 1062.3 [Mþ5H]5þ, 1327.5 [Mþ4H]4þ, 1769.9
[Mþ3H]3þ; Deconvoluted mass found 5306.3.
oil; 1H NMR (500 MHz, CDCl3)
d 7.37e7.28 (m, 5H), 7.25e7.17 (m,
6H), 7.15 (d, J ¼ 8.1 Hz, 1H), 6.60 (s, 1H), 5.07 (s, 2H), 4.78 (s, 2H),
4.75 (s, 2H), 3.90 (s, 2H), 3.60e3.54 (m, 4H), 3.54e3.46 (m, 2H),
3.39e3.29 (m, 2H), 3.18e3.08 (m, 2H), 2.66e2.56 (m, 2H), 1.72e1.60
(m, 2H), 0.96 (d, J ¼ 3.0 Hz, 18H), 0.13 (d, J ¼ 3.4 Hz, 12H); ESI-MS
calcd for C43H66N2O7Si2Na 801.4 [M þ Naþ], found 801.4.
4.3.7. Benzyl (21-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-2-yl)-8,17-dioxo-3,6,12,15-tetraoxa-9,18-
diazahenicosyl)carbamate (17)
4.3. Synthesis of OXM-7-fBph-2
Compound 17 was prepared using the same procedure as that
described for compound 7. Yield: 96% for 2 steps (151 mg); Yellow
4.3.1. Dimethyl 2-hydroxy-[1,10-biphenyl]-4,40-dicarboxylate (11)
Compound 11 was prepared using the same procedure as that
described for compound 1. Yield: 51% (220 mg); Yellow solid; 1H
oil; 1H NMR (500 MHz, CDCl3)
d 7.38e7.27 (m, 6H), 7.23 (d,
J ¼ 8.0 Hz, 2H), 7.22e7.17 (m, 2H), 7.15 (d, J ¼ 8.2 Hz, 2H), 6.59 (s,
1H), 5.08 (s, 2H), 4.78 (s, 2H), 4.75 (s, 2H), 3.95 (s, 2H), 3.88 (s, 2H),
3.68e3.32 (m, 16H), 3.15 (q, J ¼ 6.8 Hz, 2H), 2.67e2.55 (m, 2H),
1.73e1.62 (m, 2H), 0.96 (d, J ¼ 3.3 Hz, 18H), 0.13 (d, J ¼ 3.6 Hz, 12H);
ESI-MS calcd for C49H77N3O10Si2Na 946.5 [M þ Naþ], found 946.4.
NMR (500 MHz, CDCl3)
d
8.16 (d, J ¼ 8.1 Hz, 2H), 7.69 (dd, J ¼ 7.9,
1.1 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 7.9 Hz,
1H), 3.96 (s, 3H), 3.94 (s, 3H); ESI-MS calcd for C16H15O5 287.1
[M þ Hþ], found 287.1.
4.3.8. tert-Butyl 26-(((benzyloxy)carbonyl)amino)-1-(4,40-
bis(((tert-butyldimethylsilyl)oxy)methyl)-[1,10-biphenyl]-2-yl)-
5,14,23-trioxo-7,10,16,19-tetraoxa-4,13,22-triazaheptacosan-27-
oate (18)
4.3.2. Dimethyl 2-(((trifluoromethyl)sulfonyl)oxy)-[1,10-biphenyl]-
4,40-dicarboxylate (12)
Compound 12 was prepared using the same procedure as that
described for compound 2. Yield: 85% (373 mg); White solid; 1H
Compound 18 was prepared using the same procedure as that
described for compound 8. Yield: 92% for 2 steps (176 mg); Yellow
NMR (300 MHz, CDCl3)
d 8.17e8.03 (m, 3H), 8.06 (s, 1H), 7.60e7.55
(m, 3H), 3.99 (s, 3H), 3.96 (s, 3H); ESI-MS calcd for C17H14F3O7S
oil; 1H NMR (500 MHz, CDCl3)
d 7.38e7.28 (m, 7H), 7.24 (d,
419.0 [M þ Hþ], found 419.1.
J ¼ 7.9 Hz, 2H), 7.23e7.19 (m, 2H), 7.15 (d, J ¼ 8.2 Hz,1H), 7.05 (s, 1H),
6.64 (s, 1H), 6.55 (s, 1H), 5.08 (s, 2H), 4.79 (s, 2H), 4.75 (s, 2H),
4.24e4.16 (m,1H), 3.97 (s, 2H), 3.91 (s, 2H), 3.68e3.34 (m,18H), 3.15
(t, J ¼ 6.6 Hz, 2H), 2.68e2.55 (m, 2H), 2.33e2.23 (m, 2H), 2.22e2.11
(m, 1H), 2.00e1.87 (m, 1H), 1.68 (s, 9H), 0.96 (d, J ¼ 3.6 Hz, 18H), 0.13
(d, J ¼ 4.1 Hz, 12H); ESI-MS calcd for C58H92N4O13Si2Na 1131.6
[M þ Naþ], found 1131.5.
4.3.3. Dimethyl 2-(3-(((benzyloxy)carbonyl)amino)prop-1-yn-1-
yl)-[1,10-biphenyl]-4,40-dicarboxylate (13)
To a solution of 12 (490 mg, 1.2 mmol) in CH3CN (10 mL) was
added N-Cbz-prop-2-yn-1-amine (L1) (443 mg, 2.3 mmol),
PdCl2(PPh3)2 (82 mg, 0.12 mmol), CuI (22 mg, 0.12 mmol), PPh3
(61 mg, 0.23 mmol), Et3N (607 mg, 6 mmol). The mixture was stir-
red at 75 ꢁC under argon protection for 2 h, and then concentrated.
The residue was purified by silica gel flash column chromatography
(hexanes/EtOAc ¼ 2:1) to afford compound 13 (357 mg, 67% yield).
4.3.9. tert-Butyl 1-(4,40-bis(((tert-butyldimethylsilyl)oxy)methyl)-
[1,10-biphenyl]-2-yl)-26-(tert-butoxycarbonyl)-5,14,23,28-tetraoxo-
7,10,16,19-tetraoxa-4,13,22,27-tetraazapentatetracontan-45-oate
(19)
Yellow solid; 1H NMR (500 MHz, CDCl3)
d 8.21 (s, 1H), 8.10 (d,
J ¼ 7.9 Hz, 2H), 8.04 (d, J ¼ 8.1 Hz, 1H), 7.64 (d, J ¼ 7.7 Hz, 2H), 7.45
(d, J ¼ 8.1 Hz, 1H), 7.39e7.29 (m, 5H), 5.13 (s, 2H), 4.86 (s, 1H), 4.10
(d, J ¼ 5.0 Hz, 2H), 3.95 (s, 3H), 3.92 (s, 3H); ESI-MS calcd for
Compound 26 was prepared using the same procedure as that
described for compound 9. Yield: 68% for 2 steps (128 mg); Yellow
oil; 1H NMR (500 MHz, CDCl3)
d
7.35 (d, J ¼ 7.5 Hz, 2H), 7.24 (d,
C
27H23NO6Na 480.2 [M þ Naþ], found 480.1.
J ¼ 8.0 Hz, 2H), 7.20 (d, J ¼ 6.5 Hz, 2H), 7.15 (d, J ¼ 8.5 Hz, 1 H), 7.06
(s, 1H), 6.78 (s, 1H), 6.65 (s, 1H), 6.51 (d, J ¼ 7.2 Hz, 1H), 4.79 (s, 2H),
4.76 (s, 2H), 4.43e4.35 (m, 1H), 3.98 (s, 2H), 3.92 (s, 2H), 3.68e3.41
(m, 20H), 3.20e3.13 (m, 2H), 2.63 (t, J ¼ 7.8 Hz, 2H), 2.30e2.22 (m,
2H), 2.20 (t, J ¼ 7.5 Hz, 4H), 1.71e1.52 (m, 4H), 1.45 (s, 9H), 1.44 (s,
9H), 1.34e1.21 (m, 24H), 0.96 (d, J ¼ 3.5 Hz, 18H), 0.13 (d, J ¼ 4.5 Hz,
4.3.4. Benzyl (3-(4,40-bis(hydroxymethyl)-[1,10-biphenyl]-2-yl)
prop-2-yn-1-yl)carbamate (14)
Compound 14 was prepared using the same procedure as that
described for compound 3. Yield: 63% (195 mg); White solid; 1H
Please cite this article as: Y. Tian et al., Design of stapled oxyntomodulin analogs containing functionalized biphenyl cross-linkers, Tetrahedron,
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Y. Tian et al. / Tetrahedron xxx (xxxx) xxx
9
12H); ESI-MS calcd for C72H126N4O14Si2Na 1349.9 [M þ Naþ], found
4.4.3. Benzyl (5-(4'-(((tert-butyldimethylsilyl)oxy)methyl)-[1,10-
biphenyl]-4-yl)-2,2,3,3-tetramethyl-6-oxo-4,10,13-trioxa-7-aza-3-
silapentadecan-15-yl)carbamate (23)
1349.8.
Compound 23 was prepared using the same procedure as that
4.3.10. 1-(4,40-Bis(bromomethyl)-[1,10-biphenyl]-2-yl)-26-carboxy-
5,14,23,28-tetraoxo-7,10,16,19-tetraoxa-4,13,22,27-
tetraazapentatetracontan-45-oic acid (20, fBph-2)
Compound 20 was prepared using the same procedure as that
described for compound 10. Yield: 64% for 2 steps (50 mg); White
described for compound 4. Yield: 99% (127 mg); Color oil; 1H NMR
(500 MHz, CDCl3)
d 7.56e7.48 (m, 6H), 7.39e7.29 (m, 7H), 7.22 (s,
1H), 5.23 (s, 1H), 5.12 (s, 1H), 5.10 (s, 2H), 4.78 (s, 2H), 3.61e3.49 (m,
8H), 3.43e3.30 (m, 4H), 0.95 (d, J ¼ 5.0 Hz, 18H), 0.11 (d, J ¼ 4.9 Hz,
12H); ESI-MS calcd for C41H63N2O7Si2 751.4 [M þ Hþ], found 751.1.
Solid; 1H NMR (500 MHz, CDCl3)
d
7.44 (d, J ¼ 7.9 Hz, 2H), 7.32 (s,
4.4.4. Benzyl (5-(4'-(((tert-butyldimethylsilyl)oxy)methyl)-[1,10-
biphenyl]-4-yl)-2,2,3,3-tetramethyl-6,17-dioxo-4,10,13,19,22-
pentaoxa-7,16-diaza-3-silatetracosan-24-yl)carbamate (24)
Compound 24 was prepared using the same procedure as that
described for compound 7. Yield: 97% for 2 steps (140 mg); Light
1H), 7.30e7.18 (m, 3H), 7.15 (d, J ¼ 7.8 Hz, 1H), 6.85 (s, 1H), 4.56 (s,
2H), 4.52 (s, 2H), 4.50e4.44 (m, 1H), 4.02 (s, 2H), 3.99 (s, 2H),
3.71e3.34 (m, 16H), 3.19 (q, J ¼ 6.5 Hz, 2H), 2.66e2.56 (m, 2H),
2.53e2.37 (m, 2H), 2.32 (t, J ¼ 7.4 Hz, 2H), 2.25 (t, J ¼ 7.5 Hz, 2H),
2.22e2.10 (m, 1H), 2.10e1.99 (m, 1H), 1.76e1.67 (m, 2H), 1.67e1.53
(m, 4H), 1.36e1.16 (m, 24H); 13C NMR (75 MHz, CDCl3)
d 177.51,
yellow oil; 1H NMR (500 MHz, CDCl3)
d 7.56e7.48 (m, 6H),
174.70, 173.87, 173.54, 170.80, 170.41, 141.23, 141.16, 139.23, 137.21,
136.64, 130.63, 129.83, 129.49, 128.96, 126.74, 70.78, 70.72, 70.43,
70.31, 70.23, 70.10, 69.69, 69.49, 52.19, 39.55, 38.69, 38.60, 36.27,
34.02, 33.41, 33.31, 32.32, 31.56, 30.77, 30.19, 29.67, 29.43, 29.39,
29.37, 29.33, 29.26, 29.22, 29.20, 29.12, 29.00, 28.17, 25.62, 24.78,
22.63; HRMS (ESI) calcd for C52H81Br2N4O12 1113.4197 [M þ Hþ],
found 1113.4384.
7.40e7.29 (m, 7H), 7.19 (s, 1H), 5.54 (s, 1H), 5.12 (s, 1H), 5.10 (s, 2H),
4.78 (s, 2H), 3.98 (s, 2H), 3.67e3.43 (m, 16H), 3.42e3.29 (m, 4H),
0.95 (d, J ¼ 3.4 Hz, 18H), 0.11 (d, J ¼ 4.3 Hz, 12H); ESI-MS calcd for
C
47H73N3O10Si2Na 918.5 [M þ Naþ], found 918.5.
4.4.5. tert-Butyl 29-(((benzyloxy)carbonyl)amino)-5-(4'-(((tert-
butyldimethylsilyl)oxy)methyl)-[1,10-biphenyl]-4-yl)-2,2,3,3-
tetramethyl-6,17,26-trioxo-4,10,13,19,22-pentaoxa-7,16,25-triaza-
3-silatriacontan-30-oate (25)
4.3.11. OXM-7-fBph-2
Compound 25 was prepared using the same procedure as that
described for compound 8. Yield: 77% for 2 steps (110 mg); Color-
Peptide OXM-7-fBph-2 was prepared using the same procedure
as that described for OXM-7-fBph-1. Yield: 17% (2 mg); White
solid; Calcd mass for C241H367N59O70S3 5306.6 [M], ESI-MS m/z
found 885.4 [Mþ6H]6þ, 1062.3 [Mþ5H]5þ, 1327.6 [Mþ4H]4þ, 1769.9
[Mþ3H]3þ; Deconvoluted mass found 5306.3.
less oil; 1H NMR (500 MHz, CDCl3)
d 7.57e7.47 (m, 6H), 7.40e7.28
(m, 7H), 7.23 (s, 1H), 7.09 (s, 1H), 6.49 (s, 1H), 5.73 (d, J ¼ 7.5 Hz, 1H),
5.12 (s, 1H), 5.09 (s, 2H), 4.78 (s, 2H), 4.25e4.17 (m, 1H), 4.00 (s, 2H),
3.67e3.46 (m, 16H), 3.45e3.30 (m, 4H), 2.31e2.23 (m, 2H),
2.23e2.11 (m, 1H), 1.99e1.88 (m, 1H), 1.45 (s, 9H), 0.95 (d, J ¼ 2.3 Hz,
18H), 0.12 (d, J ¼ 2.9 Hz, 12H); ESI-MS calcd for C56H88N4O13Si2Na
1103.6 [M þ Naþ], found 1103.5.
4.4. Synthesis of OXM-7-fBph-3
4.4.1. Benzyl (2-(2-(2-(2-(4-bromophenyl)-2-hydroxyacetamido)
4.4.6. tert-Butyl 29-(tert-butoxycarbonyl)-5-(4'-(((tert-
butyldimethylsilyl)oxy)methyl)-[1,10-biphenyl]-4-yl)-2,2,3,3-
tetramethyl-6,17,26,31-tetraoxo-4,10,13,19,22-pentaoxa-7,16,25,30-
tetraaza-3-silatritriacontan-33-oate (26)
ethoxy)ethoxy)ethyl)carbamate (21)
To
a solution of 2-(4-bromophenyl)-2-hydroxyacetic acid
(200 mg, 0.87 mmol) in DMF (6 mL) was added 2,2'-(ethane-1,2-
diylbis(oxy))diethanamine mono-Cbz carbamate (L5) (282 mg,
1 mmol), HATU (329 mg, 0.87 mmol) and DIPEA (225 mg,
1.74 mmol). The mixture was stirred at room temperature for 1 h.
Then H2O was added and the aqueous phase was extracted with
EtOAc (4 mL ꢀ 3). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered and concentrated. The
residue was purified by silica gel flash column chromatography
(DCM/MeOH ¼ 20:1) to afford compound 21 (280 mg, 65% yield).
Compound 26 was prepared using the same procedure as that
described for compound 9. Yield: 57% for 2 steps (43 mg); Colorless
oil; 1H NMR (500 MHz, CDCl3)
d 7.57e7.52 (m, 4H), 7.50 (d,
J ¼ 8.2 Hz, 2H), 7.38 (d, J ¼ 8.3 Hz, 2H), 7.24 (s, 1H), 7.10 (s, 1H), 6.72
(s, 1H), 6.56e6.50 (m, 1H), 5.12 (s, 1H), 4.78 (s, 2H), 4.45e4.37 (m,
1H), 4.00 (s, 2H), 3.70e3.65 (m, 2H), 3.64e3.47 (m, 14H), 3.46e3.31
(m, 4H), 2.31e2.24 (m, 2H), 2.23e2.17 (m, 4H), 2.16e2.09 (m, 1H),
1.95e1.86 (m, 1H), 1.64e1.54 (m, 4H), 1.45 (s, 9H), 1.44 (s, 9H),
1.32e1.22 (m, 24H), 0.96 (s, 18H), 0.12 (s, 12H); ESI-MS calcd for
Light yellow oil; 1H NMR (500 MHz, CDCl3)
d 7.50e7.27 (m, 9H),
6.82 (s, 1H), 5.28 (s, 1H), 5.12e5.03 (m, 3H), 3.66e3.44 (m, 9H),
3.43e3.31 (m, 4H); ESI-MS calcd for C22H28BrN2O6 495.1 [M þ Hþ],
found 495.0.
C
70H122N4O14Si2Na 1321.9 [M þ Naþ], found 1321.9.
4.4.7. 1-Bromo-1-(4'-(bromomethyl)-[1,10-biphenyl]-4-yl)-25-
carboxy-2,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-
tetraazanonacosan-29-oic acid (27, fBph-3)
4.4.2. Benzyl (2-(2-(2-(2-hydroxy-2-(4'-(hydroxymethyl)-[1,10-
biphenyl]-4-yl)acetamido)ethoxy)ethoxy)ethyl)carbamate (22)
To a solution of 21 (180 mg, 0.36 mmol) in MeOH (5 mL) was
added (4-(hydroxymethyl)phenyl)boronic acid (78 mg, 0.51 mmol),
PdCl2(CH3CN)2 (1 mg, 0.004 mmol), and Na2CO3 (76 mg,
0.72 mmol). The mixture was stirred at room temperature under
argon protection for 2 h, then concentrated. The residue was puri-
fied by silica gel flash column chromatography (DCM/
MeOH ¼ 15:1) to afford compound 22 (110 mg, 59% yield). Colorless
Compound 27 was prepared using the same procedure as that
described for compound 10. Yield: 59% for 2 steps (20 mg); White
Solid; 1H NMR (500 MHz, CDCl3)
d 7.60e7.49 (m, 6H), 7.49e7.40 (m,
2H), 7.22 (s, 1H), 7.16 (s, 1H), 6.16 (s, 1H), 5.73 (d, J ¼ 7.5 Hz, 1H), 5.55
(s, 1H), 4.54 (s, 2H), 4.51e4.45 (m, 1H), 4.04 (s, 2H), 3.70e3.27 (m,
20H), 2.45e2.36 (m, 2H), 2.35e2.28 (m, 4H), 2.18e2.10 (m, 1H),
2.09e1.98 (m, 1H), 1.68e1.55 (m, 4H), 1.38e1.16 (m, 24H); 13C NMR
(126 MHz, CDCl3)
d 174.52, 173.78, 173.66, 170.87, 168.81, 167.59,
oil; 1H NMR (500 MHz, CDCl3)
d
7.58e7.50 (m, 4H), 7.44 (dd,
137.25, 137.10, 136.62, 129.58, 129.55, 129.00, 127.48, 127.45, 70.85,
70.50, 70.21, 70.14, 70.07, 69.74, 69.59, 69.36, 50.35, 40.10, 39.55,
38.75, 36.31, 34.00, 33.90, 33.17, 32.45, 29.69, 29.64, 29.35, 29.29,
29.21, 29.17, 29.14, 29.08, 29.04, 29.02, 28.91, 25.59, 24.74, 24.12,
J ¼ 19.0, 7.5 Hz, 4H), 7.37e7.27 (m, 5H), 6.83 (s, 1H), 5.13e4.99 (m,
3H), 4.73 (s, 2H), 3.60e3.45 (m, 10H), 3.44e3.28 (m, 4H); ESI-MS
calcd for C29H34N2O7Na 545.2 [M þ Naþ], found 545.3.
Please cite this article as: Y. Tian et al., Design of stapled oxyntomodulin analogs containing functionalized biphenyl cross-linkers, Tetrahedron,
https://doi.org/10.1016/j.tet.2018.11.060

10
Y. Tian et al. / Tetrahedron xxx (xxxx) xxx
19.76; HRMS (ESI) calcd for C50H77Br2N4O12 1085.3884 [M þ Hþ],
were added and incubation was continued at room temperature for
10 min before luminescence acquisition using a luminometer
(Envision). The measurements were performed in duplicate and the
average concentrations were plotted using GraphPad Prism 6
software (GraphPad, San Diego, CA).
found 1085.4043.
4.4.8. OXM-7-fBph-3
Peptide OXM-7-fBph-3 was prepared using the same procedure
as that described for OXM-7-fBph-1. Yield: 20% (2.4 mg); White
solid; Calcd mass for C239H363N59O70S3 5278.6 [M], ESI-MS m/z
found 880.7 [Mþ6H]6þ,1056.6 [Mþ5H]5þ,1320.5 [Mþ4H]4þ,1760.4
[Mþ3H]3þ; Deconvoluted mass found 5278.2.
Acknowledgment
This work was supported by the National Institutes of Health
(R01GM085092 to Q.L.; R43DK117740 to Y.T. and Q.L.) and the
Technology Accelerator Fund from Research Foundation for the
State University of New York (to Q.L.).
4.5. Generation of CRE-Luc stable cell line overexpressing GLP-1R or
GCGR
HEK293 cells were infected with lentivirus encoding firefly
luciferase gene under the control of cAMP responsive element
(CRE) promoter (Qiagen, The Netherlands) and then were selected
References
[1] D.J. Drucker, Nat. Clin. Pract. Endocrinol. Metabol. 1 (2005) 22e31.
[2] X. Du, J.R. Kosinski, J. Lao, X. Shen, A. Petrov, G.G. Chicchi, G.J. Eiermann,
A. Pocai, Am. J. Physiol. Endocrinol. Metab. 303 (2012) E265eE271.
[3] A. Pocai, Mol. Metabol. 3 (2014) 241e251.
[4] K. Fosgerau, T. Hoffmann, Drug Discov. Today 20 (2015) 122e128.
[5] V. Azzarito, K. Long, N.S. Murphy, A. Wilson, J. Nat. Chem. 5 (2013) 161e173.
[6] L.D. Walensky, G.H. Bird, J. Med. Chem. 57 (2014) 6275e6288.
[7] P.M. Cromm, J. Spiegel, T.N. Grossmann, ACS Chem. Biol. 10 (2015)
1362e1375.
using 1 mg/mL puromycin (Life Technologies, Carlsbad) for 1 week.
The surviving cells (referred to as CRE-HEK293) were expanded and
then transfected with a G418 selective mammalian expression
plasmid encoding human GLP-1R or GCGR. In brief, GLP-1R or GCGR
plasmid was transfected into CRE-HEK293 cells using Lipofect-
amine 2000 and selected with 400 mg/mL Geneticin (Life Technol-
ogies, Carlsbad, CA). Single colony stable cell line overexpressing
CRE-luciferase and GLP-1R (HEK293-GLP-1R-CRE) or GCGR
(HEK293-GCGR-CRE) was then established for in vitro activity assay.
[8] D.P. Fairlie, A. Dantas de Araujo, Biopolymers 106 (2016) 843e852.
[9] A. Muppidi, X. Li, J. Chen, Q. Lin, Bioorg. Med. Chem. Lett 21 (2011)
7412e7415.
[10] A. Muppidi, Z. Wang, X. Li, J. Chen, Q. Lin, Chem. Commun. 47 (2011)
9396e9398.
[11] A. Muppidi, K. Doi, S. Edwardraja, E.J. Drake, A.M. Gulick, H.G. Wang, Q. Lin,
J. Am. Chem. Soc. 134 (2012) 14734e14737.
4.6. In vitro GLP-1R or GCGR activation reporter assay
HEK293-GLP-1R-CRE cells or HEK293-GCGR-CRE cells were
seeded in 384-well plates at a density of 5000 cells per well and
cultured for 18 h in DMEM with 10% FBS at 37 ꢁC and 5% CO2. Cells
were treated with peptides in a dose dependent manner for 16 h,
and receptor activation was reported by luminescence intensities,
using One-Glo (Promega, WI) luciferase reagent following manu-
facturer's instruction. The EC50 of each peptide was determined
using GraphPad Prism 6 software (GraphPad, San Diego, CA). The
assay was performed in triplicate, and the results were obtained
with three independent experiments.
[12] A. Muppidi, K. Doi, C.P. Ramil, H.G. Wang, Q. Lin, Tetrahedron 70 (2014)
7740e7745.
[13] A. Muppidi, H. Zhang, F. Curreli, N. Li, A.K. Debnath, Q. Lin, Bioorg. Med. Chem.
Lett 24 (2014) 1748e1751.
[14] A. Muppidi, H. Zou, P.Y. Yang, E. Chao, L. Sherwood, V. Nunez, A.K. Woods,
P.G. Schultz, Q. Lin, W. Shen, ACS Chem. Biol. 11 (2016) 324e328.
[15] S. Runge, H. Thogersen, K. Madsen, J. Lau, R. Rudolph, J. Biol. Chem. 283 (2008)
11340e11347.
[16] C.Q. Pan, J.M. Buxton, S.L. Yung, I. Tom, L. Yang, H. Chen, M. MacDougall,
A. Bell, T.H. Claus, K.B. Clairmont, J.P. Whelan, J. Biol. Chem. 281 (2006)
12506e12515.
[17] B.P. Ward, N.L. Ottaway, D. Perez-Tilve, D. Ma, V.M. Gelfanov, M.H. Tschop,
R.D. Dimarchi, Mol. Metabol. 2 (2013) 468e479.
[18] J. Lau, P. Bloch, L. Schaffer, I. Pettersson, J. Spetzler, J. Kofoed, K. Madsen,
L.B. Knudsen, J. McGuire, D.B. Steensgaard, H.M. Strauss, D.X. Gram,
S.M. Knudsen, F.S. Nielsen, P. Thygesen, S. Reedtz-Runge, T. Kruse, J. Med.
Chem. 58 (2015) 7370e7380.
[19] P.Y. Yang, H. Zou, E. Chao, L. Sherwood, V. Nunez, M. Keeney, E. Ghartey-
Tagoe, Z. Ding, H. Quirino, X. Luo, G. Welzel, G. Chen, P. Singh, A.K. Woods,
P.G. Schultz, W. Shen, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 4140e4145.
[20] P.Y. Yang, H. Zou, C. Lee, A. Muppidi, E. Chao, Q. Fu, X. Luo, D. Wang,
P.G. Schultz, W. Shen, J. Med. Chem. 61 (2018) 3218e3223.
[21] H. Teller, M. Corbet, L. Mantilli, G. Gopakumar, R. Goddard, W. Thiel,
A. Furstner, J. Am. Chem. Soc. 134 (2012) 15331e15342.
[22] M. Adamczyk, J.R. Fishpaugh, M. Thiruvazhi, Org. Prep. Proced. Int. 34 (2002)
326e331.
4.7. In vitro serum stability
To determine serum stability, peptides were incubated with
fresh mouse serum at 37 ꢁC. The incubations were initiated with the
final peptide concentration of 10 mM. Aliquots of 20 ml mixture were
taken at 0 min, 10 min, 30 min, 1 h, 8 h, 24 h and 48 h, and the
amount of intact peptides was analyzed by the CRE reporter assay.
Briefly, HEK293 cells expressing the GLP-1R were seeded overnight
in a white 384-well plate at 5000 cells per well in 20
medium supplemented with 10% FBS in a humidified 37 ꢁC, 5% CO2
incubator. Then, 5
l peptide (prepared as 5 ꢀ solution in OPTI-
MEM) were added to each well and incubated at 37 ꢁC. The
following day, 10 l/well Bright-Glo Luciferase reagent (Promega)
ml DMEM
[23] M. Keller, S. Teng, G. Bernhardt, A. Buschauer, ChemMedChem 4 (2009)
1733e1745.
[24] G. Filippini, M. Nappi, P. Melchiorre, Tetrahedron 71 (2015) 4535e4542.
[25] R.H. Vekariya, R. Liu, J. Aube, Org. Lett. 16 (2014) 1844e1847.
[26] C.S. Fishburn, J. Pharmaceut. Sci. 97 (2008) 4167e4183.
m
m
Please cite this article as: Y. Tian et al., Design of stapled oxyntomodulin analogs containing functionalized biphenyl cross-linkers, Tetrahedron,
https://doi.org/10.1016/j.tet.2018.11.060