Synthesis and anti-osteoporosis activity of novel Teriparatide glycosylation derivatives

Article abstract of DOI:10.1039/d0ra05136e

Osteoporosis is a metabolic bone disease that is characterized by low bone mass and micro-architectural deterioration of bones. The mechanism underlying this disease implicates an imbalance between bone resorption and bone remodeling. In 2002, the US Food and Drug Administration (FDA) approved Teriparatide for the treatment of osteoporosis, and so far, this compound is the only permitted osteoanabolic. However, as a structurally flexible linear peptide, this drug may be further optimized. In this study, we develop a series of novel N-acetyl glucosamine glycosylation derivatives of Teriparatide and examine their characteristics. Of the analyzed compounds, PTHG-9 exhibits enhanced helicity, greater protease stability, and increased osteoblast differentiation promoting ability compared with the original Teriparatide. Accordingly, PTHG-9 is suggested as a therapeutic candidate for postmenopausal osteoporosis (PMOP) and other related diseases. The successful development of an enhanced osteoporosis drug proves that the method proposed herein can be used to effectively enhance the chemical and biological properties of linear peptides with various biological functions.

Full text of DOI:10.1039/d0ra05136e

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Synthesis and anti-osteoporosis activity of novel
Teriparatide glycosylation derivatives†
Cite this: RSC Adv., 2020, 10, 25730
a
b
a
a
a
Nan Wang, ‡ Jingyang Li,‡ Hui Song, Chao Liu,
Honggang Hu,
c
a
Hongli Liao * and Wei Cong*
Osteoporosis is a metabolic bone disease that is characterized by low bone mass and micro-architectural
deterioration of bones. The mechanism underlying this disease implicates an imbalance between bone
resorption and bone remodeling. In 2002, the US Food and Drug Administration (FDA) approved
Teriparatide for the treatment of osteoporosis, and so far, this compound is the only permitted
osteoanabolic. However, as a structurally flexible linear peptide, this drug may be further optimized. In
this study, we develop a series of novel N-acetyl glucosamine glycosylation derivatives of Teriparatide
and examine their characteristics. Of the analyzed compounds, PTHG-9 exhibits enhanced helicity,
greater protease stability, and increased osteoblast differentiation promoting ability compared with the
original Teriparatide. Accordingly, PTHG-9 is suggested as a therapeutic candidate for postmenopausal
osteoporosis (PMOP) and other related diseases. The successful development of an enhanced
osteoporosis drug proves that the method proposed herein can be used to effectively enhance the
chemical and biological properties of linear peptides with various biological functions.
Received 11th June 2020
Accepted 1st July 2020
DOI: 10.1039/d0ra05136e
rsc.li/rsc-advances
phosphorous and calcium metabolisms, as well as in neuro-
muscular excitation and blood clotting. In a previous study, it
Introduction
Osteoporosis is dened as the systemic degradation of bone was shown that PTH1–84 has osteoanabolic activity, and that
micro-architecture due to low bone mineral density (BMD) and daily, subcutaneous, low-dose injections of this hormone could
1
0–12
poor bone quality. As such, this disease is associated with a high promote bone formation.
risk of fragility fractures. Indeed, more than 8.9 million cases
1
–3
Teriparatide (PTH1–34) is a human parathyroid hormone N-
of osteoporosis-stimulated fractures are reported yearly on terminal analogue of PTH1–84 that is created using genetic
a global level. Considering the great inuence and serious engineering recombination technology. This analogue retains
consequences of osteoporosis, this disease is classied by the the same properties as the natural hormone, and thus, it is
World Health Organization (WHO) as a major public health capable of binding to PTH/PTHrP receptors and activating
4
–6
concern.
To restore proper bone structure and treat osteoporosis, it is bone formation and increased bone density in osteoporosis
downstream osteoblast signalling, which leads to enhanced
10,11
important to re-establish the balance between bone resorption patients.
and bone formation. Numerous studies have investigated this by the U.S. Food and Drug Administration (FDA).
Today, PTH1–34 is the only osteoanabolic approved
13–15
However,
issue, and many therapeutic agents have been proposed, the osteoanabolic effectiveness of PTH1–34 may be further
including antiresorptives such as bisphosphonates, selective improved, especially considering that linear peptides such as
7
–9
estrogen receptor modulator (SERM), and salmon calcitonin.
The parathyroid hormone (PTH1–84) secreted by the para- poor metabolic stability, poor membrane permeability, and
PTH1–34 are generally characterized by low oral bioavailability,
16
thyroid glands has been suggested as an effective agent for the rapid clearance. Moreover, linear peptides are usually highly
treatment of osteoporosis, as it is implicated in the regulation of unstructured, resulting in a low intrinsic secondary structure in
solution, which constitutes a major problem for target
17
binding.
a
Institute of Translational Medicine, Shanghai University, Shanghai, China. E-mail:
Previously, we had reported that glycosylation can improve
the properties and functions of peptides. Specically, glycosyl-
ation increases peptide hydrophilicity and oral bioavailability,
forces peptides to adopt certain conformations and/or stabilizes
the existing conformations, and enhances peptide resistance to
viviencong@shu.edu.cn
b
Department of Pediatric Respiratory Medicine, Xinhua Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai, China
c
School of Pharmacy, Chengdu Medical College, Chengdu, China. E-mail: liaohl213@
1
†
1
‡
26.com
Electronic supplementary information (ESI) available. See DOI: proteolytic cleavage.18,19 The osteoprotegerin (OPG) peptidomi-
0.1039/d0ra05136e
metic and N-acetyl glucosamine glycosylation strategy designed
Nan Wang and Jingyang Li contributes equally to this work.
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Scheme 2 Synthetic route of PTHG-2. Reagents and conditions: (a)
1
0% hydrazine hydrate/DMF, r.t., overnight; (b) TFA/EDT/TIPS/water
(95 : 2 : 2 : 1, v/v/v/v), r.t., 2 h, 25%; the resin-bound peptides were
protected on side chains at asterisk sites. The following protecting
groups for amino acid side chains were used: tert-butyl (tBu; for Glu
and Ser), 2,2,4,6,7-pentamethyldihydrobenzo-furane-5-sulfonyl (pbf;
for Arg), tert-butyloxycarbonyl (tBoc; for Lys and His) and trityl (Trt; for
Asn).
Fig. 1 Structures of Teriparatide (PTHG-1) and its N-acetyl glucos-
amine glycosylation derivatives (PTHG-2–9).
by our group leads to more stable secondary structures and
stronger chymotrypsin resistance, resulting in greater osteo-
2
0
clast inhibitory activity. Based on these results, we believe that
glycosylation can also be an effective strategy for improving the
chemical and biological properties of Teriparatide. In this
study, by replacing all the asparagine and serine residues of
Teriparatide one by one with the pre-made O-link and N-link N-
acetyl glucosamine amino acid building blocks, we synthesize
N-acetyl glucosamine glycosylation derivatives of Teriparatide
21–23
23
Asn(Ac GlcNAc)-OH (4)
and Fmoc-Ser(Ac GlcNAc)-OH (6).
3
3
To prepare compound 4, N-acetyl-b-D-glucosamine (1) was
selected as starting material, and it was reacted with acetyl
chloride to obtain the acetyl-protected chloroside intermediate.
This intermediate was then reacted with sodium azide, and the
two-step reaction was catalyzed by tetrabutylammonium iodide
(
(
phase transfer catalyst). The yield of the resulting product
compound 2) was 82.4%. Subsequently, the fully protected
(
Fig. 1B, 4), and we demonstrate that these derivatives exhibit
a more stable secondary structure conformation, increased
protease resistance, and higher anti-osteoporosis activity,
compared with the original compound.
glycoamino acid 3 was formed at 71.2% yield by coupling the
reduced azide group of compound 2 with Fmoc-Asp-OtBu
through HOBt/DIC. Finally, compound 3 was deprotected
using a mixture of TFA and DCM, and the resulting building
block 4 was obtained in 95.3% yield (Scheme 1A).
Results and discussion
The Teriparatide glycosylation derivatives were synthesized
using the key glycoamino acid building blocks, Fmoc-
Scheme 1 Synthetic route of Fmoc-Asn(Ac
Fmoc-Ser(Ac GlcNAc)-OH (6). Reagents and conditions: (a) (i) acetyl
chloride, r.t., 4 days; (ii) NaN
steps; (b) (i) H
3
GlcNAc)-OH (4) and
3
, Bu
3 4
NI, DCM/water, r.t., 2 h, 82.4% in 2 Fig. 2 (A) CD spectra of the glycosylation derivatives in 50.0% TFE
, Pd/C, MeOH, r.t., overnight; (ii) Fmoc-Asp-OtBu, HOBt, aqueous solution at 20 C. (B) Proteolytic stability of glycosylation
ꢀ
2
DIC, DMF, r.t., overnight, 71.2% in 2 steps; (c) DCM/TFA (3 : 1, v/v), r.t., derivatives under a-chymotrypsin treatment. Data points are displayed
ꢀ
2
h, 95.3%; (d) (i) 4 A˚ molecular sieves, BF
3
$Et
2
O, DCM, 0 C, 12 h; (ii) as the mean value SEM of duplicate independent experiments. The
Et
3
N, Fmoc-Ser-OH, DCM/MeCN (1 : 2, v/v), r.t., 3 days, 32% in 2 steps. percent residual peptide was monitored via HPLC.
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However, building block 6 was generated by direct glycosyl- and thus, their protease stability is greater than that of the other
ation between 1,3,4,6-tetra-O-acetyl-N-acetyl-b-D-glucosamine peptides. These results suggest that the 16- and 30-asparagine
(
5) and Fmoc-Ser-OH, with successive BF $Et O and Et N residues play extremely signicant roles in the process of Ter-
3 2 3
treatment (Scheme 1B).
iparatide protease degradation.
With the key glycoamino acid building blocks in hand, the
The alkaline phosphatase (ALP) activity of Teriparatide
Teriparatide glycosylation derivatives were synthesized. For (PTHG-1) and its N-acetyl glucosamine glycosylation derivatives
example, PTHG-2 was synthesized by incorporating building (PTHG-2–9) was also analysed. For this purpose, MC3T3-E1 cells
block 6 into the peptide backbone via standard Fmoc SPPS, were treated with different peptides at a concentration of 50 nM,
0
0
using O-(7-azabenzotriazol-1-yl)-N,N,N ,N -tetramethyluronium followed by spectrophotometric and optical density measure-
hexauorophosphate (HATU) as a coupling reagent (Scheme 2). ments (405 nm). As shown in Fig. 3, the PTHG-1 group pre-
The acetyl protection group of the resulting on-resin fully pro- sented statistically signicant ALP activity, which means that
tected peptide 7 was subsequently deprotected using hydrazine PTHG-1 is capable of promoting osteoblast differentiation, as
hydrate DMF solution, and on-resin intermediate 8 was previously reported in the literature. Compared with PTHG-1,
produced. Aer acidic cleavage and global deprotection, the the promoting activities of PTHG-2, PTHG-4, and PTHG-9
crude glycopeptide was obtained (Scheme 2), and then it was were 1.18-, 1.12-, and 1.3-times greater, respectively. However,
puried by preparative high-performance liquid chromatog- only the stimulation effect of PTHG-9 was statistically signi-
raphy. The yield of the pure PTHG-2 product was found to be cant. Overall, the results indicate that both protease resistance
25.0%.
and conformation stability are important factors contributing
The secondary structures of the synthesized glycopeptides to an enhanced osteoblast differentiation ability of Teriparatide
were analysed by circular dichroism (CD). As shown in Fig. 2A, and its glycosylation derivatives. This explains the improved
the helicity values of the Teriparatide derivatives ranged activity of PTHG-9 compared with PTHG-1.
between 26.1% and 39.2%, compared with 26.1% for the orig-
inal Teriparatide (PTHG-1). This conrms that N-acetyl glucos-
amine glycosylation can improve the helicity of peptides.
Conclusions
Among the investigated compounds, PTHG-6, PTHG-8, and A novel series of Teriparatide glycosylation derivatives have
PTHG-9 showed the highest degrees of helicity (39.2%, 36.7%, been designed and successfully synthesized, with reasonable
and 36.1%, respectively), with values that are 1.50-, 1.40-, and yield, via the standard Fmoc SPPS strategy using pre-made
1.38-fold greater than that of PTHG-1, respectively.
glycoamino acid building blocks. In vitro data suggest that the
To assess the protease stability of Teriparatide (PTHG-1) and analogue PTHG-9 has improved helicity, greater protease
its derivatives, a-chymotrypsin-mediated degradation tests were stability, and increased osteoblast differentiation promoting
performed, and the compounds were monitored by HPLC. As ability compared with the original Teriparatide. Therefore,
shown in Fig. 2B, 50% of PTHG-1 remained aer 60 minutes of PTHG-9 has great potential for use in the development of novel
protease exposure, compared with 25–75% for the synthesized therapeutics for postmenopausal osteoporosis. More impor-
glycopeptides. Among the investigated compounds, only PTHG- tantly, the derivatization method proposed herein constitutes
5
, PTHG-7, and PTHG-9 had half-lives longer than 60 minutes, a useful strategy for improving the chemical and biological
properties of linear peptides with various biological functions.
Further optimization of PTHG-9 and more in-depth biological
evaluation are ongoing in our laboratory, and the results will be
disclosed in due course.
Experimental section
General information
All reagents were purchased from Acros, Sigma-Aldrich, Alfa
Aesar and Adamas. Amino acids were commercial available
from GL Biochem Shanghai Co. Ltd. All solvents used were
bought from Sinopharm Chemical Reagent Co. Ltd. Dichloro-
methane (DCM) and N,N-dimethylformamide (DMF) were
2
distilled over calcium hydride (CaH ) under argon atmosphere
˚
and stored in ask containing 4 A molecular sieves. All reac-
tions vessels were oven-dried before use. Reactions were
monitored by thin-layer chromatography (TLC) and visualized
by UV analyzer (254 nm), ninhydrin and/or phosphomolybdic
acid. Peptides were analyzed and puried by reverse phase
Fig. 3 Activity of the glycosylation derivatives at 50 nM concentrations
as assessed by alkaline phosphatase (ALP) activity in MC3T3-E1 cells.
Data represent the mean ꢃ SEM from three independent experiments:
HPLC. A C18 analytic column (Shimadzu Shim-pack VP-ODS,
ꢂ1
4
.6 ꢁ 250 mm, 5 mm particle size, ow rate 1 mL min ) was
*
p < 0.01 as compared with PTHG-1.
used for analytical RP-HPLC, and a C18 column (Shimadzu
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Shim-pack PRC-ODS, 50 ꢁ 250 mm, 15 mm particle size, ow column chromatography (100 : 1–10 : 1, DCM/MeOH) to give 3
ꢂ1
1
rate 10 mL min ) was used for preparative RP-HPLC. The as a white powder (14.10 g, 71.2%, 2 steps). H-NMR (600 MHz,
solvents systems were buffer A (0.1% TFA in CH CN) and buffer CDCl ): d 7.77 (d, J ¼ 6 Hz, 2H), 7.62 (d, J ¼ 6 Hz, 2H), 7.41 (t, J ¼
3
3
B (0.1% TFA in water). Data were recorded and analyzed using 12 Hz, 2H), 7.32 (t, J ¼ 12 Hz, 2H), 7.23 (d, J ¼ 6 Hz, 1H), 6.18 (d, J
the soware system LC Solution. High resolution mass spectra ¼ 6 Hz, 1H), 5.98 (d, J ¼ 6 Hz, 1H), 5.14–5.09 (m, 3H), 4.55 (s,
(
HR-MS) were measured on a Waters Xevo G2 QTOF mass 1H), 4.44–4.43 (m, 1H), 4.33–4.31 (m, 2H), 4.24 (t, J ¼ 12 Hz, 1H),
1 13
spectrometer. H- and C-NMR spectrum was recorded on 4.16–4.13 (m, 1H), 4.08 (d, J ¼ 6 Hz, 1H), 3.78–3.76 (m, 1H);
a Bruker Avance 300 MHz instrument. Chemical shis (d) were 2.88–2.85 (m, 1H); 2.74–2.71 (m, 1H); 2.54 (s, 2H); 2.09–2.06 (m,
1
13
13
3
reported relative to TMS (0 ppm) for H- and C-NMR spectra. 9H); 1.98 (s, 3H); 1.47 (s, 9H). C-NMR (600 MHz, CDCl ):
The coupling constants (J) were displayed in Hertz (Hz) and the d 171.91, 171.47, 170.60, 170.15, 169.45, 168.74, 155.64, 143.42,
splitting patterns were dened as follows: singlet (s); broad 143.31, 140.78, 127.20, 126.57, 124.69, 119.47, 81.75, 79.73,
singlet (s, br); doublet (d); doublet of doublet (dd); triplet (t); 73.07, 72.40, 67.11, 66.66, 61.17, 53.00, 50.51, 46.64, 37.48,
quartet (q); multiplet (m).
45 3 13
27.40, 22.53, 20.19, 20.07. ESI-MS m/z calcd for C37H N O
+
739.2952; found [M + H] 740.19.
(
2R,3S,4R,5R,6R)-5-Acetamido-2-(acetoxymethyl)-6-azidotetra-
hydro-2H-pyran-3,4-diyl diacetate (2)
N
(
(
2
-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N
(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-
acetoxymethyl)tetrahydro-2H-pyran-2-yl)asparagines (4)
4
-
To N-acetyl-b-D-glucosamine (25.0 g, 113.1 mmol) was added
acetyl chloride (50 mL, 707.0 mmol), dropwise over 15 min at
ꢀ
0
C. The reaction mixture was stirred vigorously at room 3 (10.0 g, 13.53 mmol) was dissolved in TFA/DCM (1 : 1, 50.00
temperature for 4 day. This mixture was diluted with DCM mL) and stirred for 2 hours at room temperature. The reaction
100.00 mL) and saturated NaHCO (100 mL) aqueous solution. mixture was concentrated in vacuo to yield 4 as a white powder
(
3
The organic phase was separated, washed with saturated (8.77 g, 95.3%) which was used directly in SPPS without further
1
NaHCO3 and brine (3 ꢁ 100.00 mL), dried over Na SO and purication. H-NMR (600 MHz, DMSO): d 8.58 (d, J ¼ 6 Hz, 1H),
2
4
concentrated, and used without further purication. A mixture of 7.88 (d, J ¼ 6 Hz, 3H), 7.70 (d, J ¼ 6 Hz, 2H), 7.50 (d, J ¼ 6 Hz,
commercially available NaN (8.82 g, 135.70 mmol) and tetrabu- 1H), 7.41 (t, J ¼ 12 Hz, 2H), 7.32 (t, J ¼ 12 Hz, 2H), 5.17 (t, J ¼
3
tylammonium iodide (16.68 g, 45.23 mmol) in DCM/water (1 : 1, 12 Hz, 1H), 5.10 (t, J ¼ 12 Hz, 1H), 4.82 (t, J ¼ 12 Hz, 1H), 4.38–
2
00.00 mL) was stirred for 2 hours at room temperature. The 4.37 (m, 1H), 4.28–4.17 (m, 4H), 3.95–3.87 (m, 7H), 2.67–2.64
1
3
organic layer was separated, washed with brine (3 ꢁ 100.00 mL), (m, 1H), 1.99–1.96 (m, 7H), 1.90 (s, 3H), 1.72 (s, 3H). C-NMR
dried over Na SO , concentrated and puried by column chro- (600 MHz, DMSO): d 172.91, 170.00, 169.77, 169.46, 169.28,
2
4
matography (20 : 1–1 : 1, petro ether/EtOAc) to give 2 as a white 155.80, 143.76, 140.59, 127.60, 127.05, 125.22, 120.07, 78.05,
1
powder (13.80 g, 82.4%, 2 steps). H-NMR (600 MHz, CDCl ): 73.33, 72.25, 68.36, 65.67, 61.80, 52.09, 49.96, 46.56, 36.82,
3
d 5.81 (d, J ¼ 6 Hz, 1H), 5.27 (t, J ¼ 12 Hz, 1H), 5.12 (t, J ¼ 12 Hz, 22.55, 20.49, 20.36, 20.33. ESI-MS m/z calcd for C H N O
3
3
37 3 13
+
ꢂ
1
1
H), 4.79 (d, J ¼ 6 Hz, 1H), 4.28–4.27 (m, 1H), 4.20 (d, J ¼ 6 Hz, 683.2326; found [M + Na] 706.54, [M ꢂ H] 682.39.
H), 3.96–3.91 (m, 1H), 3.83–3.81 (m, 1H), 2.12 (s, 3H), 2.05 (d,
1
3
J ¼ 6 Hz, 6H), 2.00 (s, 3H). C-NMR (600 MHz, CDCl
3
): d 170.48,
L-Serine,N-[(9H-luoren-9-ylmethoxy)carbonyl]-O-(2,3,4,6-tetra-
O-acetyl-b-D-galactopyranosyl) (6)
1
2
3
70.17, 169.94, 168.77, 87.91, 73.48, 71.65, 67.58, 61.36, 53.67,
2.73, 20.21, 20.12, 20.07. ESI-MS m/z calcd for C14
72.1281; found [M + H] 373.29.
H N O
20 4 8
To a solution of 1,3,4,6-tetra-O-acetyl-N-acetyl-b-D-glucosamine
5, 1.08 g, 4.74 mmol) and molecular sieve (1.08 g) in DCM (100
+
(
ꢀ
mL) was added BF
3
$Et
2
O (1 mL, 6.26 mmol) at 0 C. Then the
(
2R,3S,4R,5R,6R)-6-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)
amino)-4-(tert-butoxy)-4-oxobutanamido)-5-acetamido-2-
acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (3)
reaction was stirred at room temperature for 12 h. Et
.4 mmol) was added to the mixture and stirred for 10 min. A
mixture of commercially available Fmoc-Ser-OH (1.2 g, 3.67
3
N (0.4 mL,
5
(
To a solution of 4 (10.00 g, 26.88 mmol) in MeOH (200.00 mL) mmol) in DCM/MeCN (1 : 2, 15 mL) was added to the reaction
was added Pd/C catalyst (1.00 g). Then the mixture was stirred and stirred for 3 days at room temperature. Then the reaction
overnight at room temperature under hydrogen. Aer the Pd/C was ltrated and washed with NaHCO (3 ꢁ 100.00 mL) and
3
catalyst was ltered and the MeOH was removed under vacuum, brine (3 ꢁ 100.00 mL). The organic phase was dried over
2 4
the residue was used without further purication. To a solution Na SO , ltered, concentrated and puried by column chro-
of the intermediate in DMF (50.00 mL) was added Fmoc-Asp- matography (100 : 1–10 : 1, DCM/MeOH) to give 6 as a white
1
OtBu (12.14 g, 29.56 mmol), HOBt (3.99 g, 29.56 mmol) and powder (14.1 g, 32%, 2 step). H-NMR (600 MHz, DMSO): d 9.32–
DCC (3.99 g, 29.56 mmol) mixture of DCM/DMF (4 : 1, 200.00 9.31 (m, 3H), 9.16–9.14 (m, 2H), 8.84 (t, J ¼ 12 Hz, 2H), 8.76–8.71
mL) solution. Then DIC (3.72 g, 29.56 mmol) was added to the (m, 3H), 6.51 (t, J ¼ 12 Hz, 1H), 6.27 (t, J ¼ 12 Hz, 1H), 6.13 (d, J ¼
solution. The reaction was stirred overnight at room tempera- 6 Hz, 1H), 5.71–5.61 (m, 6H), 5.44–5.39 (m, 3H), 5.20–5.11 (m,
1
3
ture. Then, the reaction was ltrated and the ltrate was washed 2H), 3.43 (s, 3H), 3.39 (s, 3H), 3.33 (s, 3H), 3.15 (s, 3H). C-NMR
successively with 1 M HCl (3 ꢁ 100.00 mL), saturated NaHCO (600 MHz, DMSO): d 171.5, 170.03, 169.61, 169.52, 169.23,
3 ꢁ 100.00 mL) and brine (3 ꢁ 100.00 mL). The organic phase 155.91, 143.39, 140.69, 127.63, 127.06, 125.24, 125.19, 120.09,
was dried over Na SO , ltered, concentrated and puried by 72.49, 70.82, 68.45, 65.83, 61.75, 54.02, 53.02, 46.56, 22.55,
3
(
2
4
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2
0.45, 20.37, 20.31. ESI-MS m/z calcd for C32
H
36
N
2
O
13 656.2217;
PTHG-4. PTHG-4 was obtained as a lyophilized white powder
(168 mg, 26% yield according to initial resin load, 99.1% purity).
ꢂ
found [M ꢂ H] 654.24.
HR-MS m/z calcd for C189
H
309
N
58
4+
O
54
S
2
4319.2263; found [M +
3
+
5+
3
8
H] ¼ 1440.7514; [M + 4H] ¼ 1080.8165; [M + 5H]
¼
6
+
64.8541; [M + 6H] ¼ 720.8795.
PTHG-5. PTHG-5 was obtained as a lyophilized white powder
181 mg, 28% yield according to initial resin load, 99.5% purity).
General procedures for the Fmoc solid phase peptide
synthesis
(
The amino acid residues were attached to the resin with a single HR-MS m/z calcd for C189H309N O S 4319.2263; found [M +
58
4+
54 2
3
+
5+
coupling procedure. All peptides were synthesized with a scale 3H] ¼ 1440.7524; [M + 4H] ¼ 1080.8166; [M + 5H]
¼
6
+
of 0.15 mmol.
864.8556; [M + 6H] ¼ 720.8808.
(a) Standard pre-activation of resin protocol: the resin was
PTHG-6. PTHG-6 was obtained as a lyophilized white powder
swollen in DCM/DMF mixture solvent for 10 minutes.
(176 mg, 27% yield according to initial resin load, 99.3% purity).
(
b) Standard Fmoc-deprotection protocol: aer treatment HR-MS m/z calcd for C191H311N O S 4361.2369; found [M +
58
4+
55 2
3
+
5+
with 20% piperidine in DMF solution (15 minutes twice) the 3H] ¼ 1454.7605; [M + 4H] ¼ 1091.3238; [M + 5H]
¼
6
+
7+
resin was washed with DMF (ꢁ5), DCM (ꢁ5), and DMF (ꢁ5).
c) Standard coupling of amino acids protocol: aer pre-
873.2618; [M + 6H] ¼ 727.8863; [M + 7H] ¼ 624.0468.
(
PTHG-7. PTHG-7 was obtained as a lyophilized white powder
activation of 4.0 equivalents of Fmoc-protected amino acid in (161 mg, 25% yield according to initial resin load, 99.2% purity).
DMF for 5 minutes using 3.8 equivalents of HCTU and 8.0 HR-MS m/z calcd for C189H309N O S 4319.2263; found [M +
58
4+
54 2
3
+
5+
equivalents of DIPEA, the solution was added to the resin. Aer 3H] ¼ 1440.7519; [M + 4H] ¼ 1080.8170; [M + 5H]
¼
6
+
3
0 minutes, the resin was washed with DMF (ꢁ5), DCM (ꢁ5), 864.8542; [M + 6H] ¼ 720.8797.
and DMF (ꢁ5). The coupling reaction was monitored with the
PTHG-8. PTHG-8 was obtained as a lyophilized white powder
ninhydrin test.
(183 mg, 28% yield according to initial resin load, 99.6% purity).
(
d) Standard acetylation protocol: a solution of acetic anhy- HR-MS m/z calcd for C191H311N O S 4361.2369; found [M +
58
4+
55 2
3
+
5+
dride and pyridine (1 : 1, v/v) was added to the resin. Aer 20 3H] ¼ 1454.7607; [M + 4H] ¼ 1091.3232; [M + 5H]
¼
6
+
minutes, the resin was washed with DMF (ꢁ5), DCM (ꢁ5), and 873.2612; [M + 6H] ¼ 727.8855.
DMF (ꢁ5).
PTHG-9. PTHG-9 was obtained as a lyophilized white powder
e) Standard stapling protocol: aer removal of N-terminus (176 mg, 26% yield according to initial resin load, 99.1% purity).
Fmoc and acetylation successively, 3.0 equivalents of 1st HR-MS m/z calcd for C197H322N O S 4522.3057; found [M +
(
59
4+
59 2
3
+
5+
Grubbs' reagent in dry dichloroethane solution was added to 3H] ¼ 1508.1172; [M + 4H] ¼ 1131.3393; [M + 5H]
¼
6
+
the resin. Aer overnight reaction, the resin was washed with 905.2731; [M + 6H] ¼ 754.5621.
DMF (ꢁ5), DCM (ꢁ5), and DMF (ꢁ5).
(
f) Standard cleavage protocol: the cleavage cocktail Protease stability experiment
(
TFA : TIPS : phenol : H
2
O ¼ 88 : 5 : 5 : 2, v/v/v/v) was added to
Puried peptides was dissolved in DMSO to a nal concentration
of 1 mM as solution A. a-chymotrypsin was dissolved in PBS
the resin. Aer stirring for 2 hours, the cleavage cocktail was
collected. The solution was bubbled with argon for concentra-
tion and the chilled diethyl ether was added to precipitate the
crude peptides. The peptide suspensions were centrifuged for 3
minutes at 3000 rpm and then the clear solution was decanted.
The step of precipitation, centrifugation and decantation
operations was repeated three times. The resulting white resi-
buffer (pH ¼ 7.4, containing 2 mM CaCl
2
) to a nal concentration
of 0.5 ng mL as solution B. 50 mL solution A and 1950 mL
solution B were mixed. Aer 0, 1, 2, 4, 8 and 12 hours' incubation
ꢂ1
ꢀ
at 37 C, the percent residual peptide was monitored by HPLC.
CD spectroscopy study
dues were dissolved in CH
RP-HPLC.
3
CN/water, analyzed and puried by
CD measurements were recorded on a JASCO J-820 spec-
tropolarimeter (JASCO Corp., Ltd). Peptides were dissolved in
PTHG-1. PTHG-1 was obtained as a lyophilized white powder
ꢂ1
5
0.0% TFE aqueous solution with a concentration of 0.1 mg mL
.
(154 mg, 25% yield according to initial resin load, 99.3% purity).
ꢀ
UV spectra were recorded at 20 C in a quartz cell of 10.0 mm path
HR-MS m/z calcd for C181
H
296
N
57
O
+
49
S
4H]
2
4116.1469; found
24,25
3
+
4+
length. Percent helicity (a) was calculated by the follow eqn (1).
½qꢄ
(1)
[
[
M
+
3H]
¼
1373.0696; [M
¼
1030.0539;
5
+
M + 5H] ¼ 824.2456.
2
22
a ¼
ꢁ 100%
PTHG-2. PTHG-2 was obtained as a lyophilized white powder
½
qꢄmax
(161 mg, 25% yield according to initial resin load, 99.2% purity).
[
q]222 is the molar ellipiticity of 222 nm; [q]max ¼ (ꢂ44000 + 250T)
HR-MS m/z calcd for C189
3
8
H
309
N
58
4+
O
54
S
2
4319.2263; found [M +
ꢀ
3
+
5+
(1 ꢂ k/n), k ¼ 4, n is the numbers of amino acids and T ¼ 20 C.
H] ¼ 1440.7517; [M + 4H] ¼ 1080.8169; [M + 5H]
¼
6
+
7+
64.8535; [M + 6H] ¼ 720.8793; [M + 7H] ¼ 618.1847.
Alkaline phosphatase assay
PTHG-3. PTHG-3 was obtained as a lyophilized white powder
(
181 mg, 28% yield according to initial resin load, 99.4% purity). At 70–80% conuency mice calverial osteoblast were trypsinized
3
HR-MS m/z calcd for C189H309N O S 4319.2263; found [M + and 2 ꢁ 10 cells per well were seeded onto 96-well plates for
58
4+
54 2
3
+
5+
3
8
H] ¼ 1440.7529; [M + 4H] ¼ 1080.8223; [M + 5H]
¼
alkaline phosphatase (ALP) measurement. Cells were treated
6
+
7+
64.8594; [M + 6H] ¼ 721.0512; [M + 7H] ¼ 618.0464.
with different concentration of test compound or vehicle for
25734 | RSC Adv., 2020, 10, 25730–25735
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Paper
RSC Advances
4
1
8 h in a-MEM supplemented with 10% charcoal treated FCS,
0 mM b-glycerophosphate, 50 mg mL ascorbic acid, and 1%
8 B. C. Silva and J. P. Bilezikian, Annu. Rev. Med., 2011, 62, 307–
322.
ꢂ1
penicillin/streptomycin (osteoblast differentiation medium). At
the end of incubation period, total ALP activity was measured
9 A. Wysocki, M. Butler, T. Shamliyan and R. L. Kane, Ann.
Intern. Med., 2011, 155, 680–W213.
using p-nitrophenyl phosphate (PNPP) as substrate and absor- 10 P. Sun, M. Wang and G.-Y. Yin, Biochem. Biophys. Res.
bance was read at 405 nm.
Commun., 2020, 525, 850–856.
11 L. L. Ding, F. Wen, H. Wang, D. H. Wang, Q. Liu, Y. X. Mo,
X. Tan, M. Qiu and J. X. Hu, Osteoporosis Int., 2020, 31,
Conflicts of interest
9
61–971.
12 M. E. Kraenzlin and C. Meier, Nat. Rev. Endocrinol., 2011, 7,
47–656.
3 H. Chtioui, F. Lamine and R. Daghfous, N. Engl. J. Med.,
011, 364, 1081–1082.
There are no conicts of interest to declare.
6
1
Acknowledgements
2
We thank LetPub for its linguistic assistance during the prep-
aration of this manuscript. We are grateful to the Key R & D
Projects of Science and Technology Department of Sichuan
Province (2019YFS0097), the Science and Technology Project of
Nantong, China (MS22019026), the National Natural Science
Foundation of China (No. 91849129, No. 81703526 and No.
1
1
4 V. Heath, N. Engl. J. Med., 2010, 363, 2396–2405.
5 A. Farooki, M. Fornier and M. Girotra, N. Engl. J. Med., 2007,
357, 2410–2411.
1
6 R. Mourtada, H. D. Herce, D. J. Yin, J. A. Moroco, T. E. Wales,
J. R. Engen and L. D. Walensky, Nat. Biotechnol., 2019, 37,
1
186–1197.
7 Y. S. Tan, D. P. Lane and C. S. Verma, Drug Discovery Today,
016, 21, 1642–1653.
41806088) and instrumental analysis centre of Second Military
1
1
Medical University for NMR spectroscopic and mass spectro-
metric analyses.
2
8 Y. Zou, Q. Zhao, C. Zhang, L. Wang, W. Li, X. Li, Q. Wu and
H. Hu, J. Pept. Sci., 2015, 21, 586–592.
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