A new GLP-1 analogue with prolonged glucose-lowering activity in vivo via backbone-based modification at the N-terminus

Article abstract of DOI:10.1016/j.bmc.2016.01.036

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic hormone with wonderful glucose-lowering activity. However, its clinical use in type II diabetes is limited due to its rapid degradation at the N-terminus by dipeptidyl peptidase IV (DPP-IV). Among the N-terminal modifications of GLP-1, backbone-based modification was rarely reported. Herein, we employed two backbone-based strategies to modify the N-terminus of tGLP-1. Firstly, the amide N-methylated analogues 2-6 were designed and synthesized to make a full screening of the N-terminal amide bonds, and the loss of GLP-1 receptor (GLP-1R) activation indicated the importance of amide H-bonds. Secondly, with retaining the N-terminal amide H-bonds, the β-peptide replacement strategy was used and analogues 7-13 were synthesized. By two rounds of screening, analogue 10 was identified. Analogue 10 greatly improved the DPP-IV resistance with maintaining good GLP-1R activation in vitro, and showed approximately a 4-fold prolonged blood glucose-lowering activity in vivo in comparison with tGLP-1. This modification strategy will benefit the development of GLP-1-based anti-diabetic drugs.

Full text of DOI:10.1016/j.bmc.2016.01.036

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A new GLP-1 analogue with prolonged glucose-lowering activity
in vivo via backbone-based modification at the N-terminus
y
y
⇑
⇑
Xiaohui Bai , Youhong Niu , Jingjing Zhu, An-Qi Yang, Yan-Fen Wu , Xin-Shan Ye
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic hormone with wonderful glucose-
lowering activity. However, its clinical use in type II diabetes is limited due to its rapid degradation at
the N-terminus by dipeptidyl peptidase IV (DPP-IV). Among the N-terminal modifications of GLP-1, back-
bone-based modification was rarely reported. Herein, we employed two backbone-based strategies to
modify the N-terminus of tGLP-1. Firstly, the amide N-methylated analogues 2–6 were designed and syn-
thesized to make a full screening of the N-terminal amide bonds, and the loss of GLP-1 receptor (GLP-1R)
activation indicated the importance of amide H-bonds. Secondly, with retaining the N-terminal amide H-
bonds, the b-peptide replacement strategy was used and analogues 7–13 were synthesized. By two
rounds of screening, analogue 10 was identified. Analogue 10 greatly improved the DPP-IV resistance
with maintaining good GLP-1R activation in vitro, and showed approximately a 4-fold prolonged blood
glucose-lowering activity in vivo in comparison with tGLP-1. This modification strategy will benefit
the development of GLP-1-based anti-diabetic drugs.
Received 5 November 2015
Accepted 18 January 2016
Available online xxxx
Keywords:
GLP-1 analogues
N-terminus modifications of GLP-1
Amide N-methylation
b-Peptide
Drug design
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
blood glucose returns back to normal level, which effectively avoids
the hypoglycemia symptom.7–9 Moreover, GLP-1 can also reduce
Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotro-
pic hormone secreted by the gastrointestinal L cell mainly existing
in the ileum, colon and rectum.1,2 As blood glucose rises via food
intake or stimulation by neural and endocrine factors, GLP-1 will
be secreted and released rapidly into the plasma to reduce blood
glucose level, mainly by promoting insulin secretion and suppress-
ing glucagon secretion.3–5 To date, GLP-1 is considered to be the
most potent insulinotropic hormone and a candidate therapeutic
agent for type II diabetes.6 In comparison with other drugs, GLP-1
promotes the biosynthesis of insulin in a glucose-dependent pat-
tern: it is rapidly secreted at a high HbA1c level and ceases when
diabetes patients’ weight by slowing gastric emptying and sup-
pressing appetite. However, the majority of GLP-1 will be rapidly
degraded by dipeptidyl peptidase IV (DPP-IV)10–12 as well as a small
quantity will be cleaved by neutral endopeptidase (NEP) 24.11 at
multiple positions,13 leading to that its existing half-life in the
plasma is only 2 min, which largely limits GLP-1 as a therapeutic
agent (Fig. 1). To overcome this defect, considerable efforts have
been made in the past decades and two strategies seem to be acces-
sible: one is the use of small molecule DPP-IV inhibitors including
several clinically approved drugs such as Sitagliptin,14,15
Saxagliptin,16,17 Linagliptin,18,19 and Vildagliptin;20,21 The other is
the use of GLP-1 analogues, which are resistant to enzymatic degra-
dation through structural modifications that prevent protease
cleavage or increase plasma protein binding to reduce total plasma
clearance, including some clinical drugs in the market such as
Liraglutide,22,23 Dulaglutide,24,25 and Exenatide with 53% of
sequence identical to GLP-1.26,27
Abbreviations: GLP-1, glucagon-like peptide-1; DPP-IV, dipeptidyl peptidase IV;
NEP, neutral endopeptidase; His, histidine; Thr, threonine; Val, valine; GLP-1R, GLP-
1 receptor; Ala, alanine; Aha, 6-amino-hexanoic acid; DCM, dichloromethane; DMF,
dimethylformamide; EA, ethyl acetate; PE, petroleum ether; Me, methyl; Fmoc, 9-
fluorenylmethoxycarbonyl; AA, amino acid; HoAt, 1-hydroxy-7-azabenzotriazole;
NMM, 4-methylmorpholine; NMP, N-methyl-2-pyrrolidone; Trt, triphenylmethyl;
TFA, trifluoroacetic acid; EDT, 1,2-ethanedithiol; DIPEA, N,N-diisopropylethy-
GLP-1 is a polypeptide consisting of 30 or 31 amino acids with
two main occurring forms: a C-terminus amidated (GLP-1 (7–36)
amide, 75%) and a glycine-extended (GLP-1 (7–37), 25%), of which
the former has been proved to be the active form in the plasma
(Fig. 1).28 Notably, GLP-1 (7–36) amide (tGLP-1) is a proteolytic
product of preproglucagon gene (proglucagon (78–107) amide),
lamine;
HATU,
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate.
⇑
Corresponding authors. Tel.: +86 10 82805736; fax: +86 10 82802724.
E-mail addresses: wuyanfen@pku.edu.cn (Y.-F. Wu), xinshan@bjmu.edu.cn
(X.-S. Ye).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.01.036
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. Two existing forms of GLP-1. The long arrow indicates the DPP-IV cleaving position, and the short arrows indicate the NEP 24.11 cleaving positions.
so GLP-1 is named corresponding to proglucagon with its first his-
tidine at the N-terminus generally defined His.729 GLP-1 is specifi-
cally recognised by GLP-1R, which belongs to family B1 seven-
transmembrane G protein-coupled receptors. The most possible
ligand-receptor binding model is considered to be a two-step
mechanism: firstly, the C-terminus and middle domain of GLP-1
and Ala8 to improve resistance to DPP-IV, but GLP-1R activation
was reduced by 16-fold.50
To develop new long-lasting GLP-1 analogues for the treatment
of type II diabetes, and to explore the interacting conformation of
N-terminus in the GLP-1R activating process to some extent,
herein, we choose two backbone-based strategies to modify the
N-terminus of tGLP-1. One is N-methylation of amide bond, which
has significant influence on the function of protein or polypeptide,
including conformational modulation, prevention of hydrogen
bonds, improvement of receptor subtype selectivity, oral bioavail-
ability and cell permeability.51–55 The other is b-peptide substitu-
tion, which has been proved to be applicable to the development
of protein or peptide drugs with improved properties without
destroying the secondary structure and conformation.56–60
in a
-helix conformation from Thr13 to Val33 bind to the N-terminal
extracellular domain of GLP-1R;30–32 secondly, the N-terminus of
GLP-1 interacts with the transmembrane region and connecting
loops of GLP-1R, initiating the receptor-activating process.33–35
However, the N-terminal conformation of GLP-1 is still uncertain
because of the high specificity and flexibility during the rapid
interaction process,36 which makes the classic crystal structure-
based drug design inapplicable to modification at the N-terminus
of GLP-1. Besides, DPP-IV removes His7-Ala8 from the N-terminus
of tGLP-1 to yield inactive GLP-1 (9–36) amide in the plasma, indi-
cating that the N-terminus is also pivotal for DPP-IV cleavage as
well as GLP-1R activation. In recent years, many attempts on the
N-terminal modifications of GLP-1 have been made,6,37–46 aiming
at the development of GLP-1 analogues with improved DPP-IV
resistance and retained GLP-1R activation. Among these modifica-
tions, most of them were based on side chains, as the side chains of
these amino acids at the N-terminus are critical to GLP-1R activa-
tion according to the structure–activity characterisation.47–49
However, the backbone-based modification at the N-terminus of
GLP-1 has been less studied, probably due to the uncertain binding
conformation during the receptor activation process. Doyle group
once incorporated a 6-amino-hexanoic acid (Aha) between His7
2. Results and discussion
2.1. Chemistry
As shown in Schemes 1 and 2, Fmoc-N-Me-AA-OH and Fmoc-b-
AA-OH were synthesized.61 All peptides (Table 1) were synthesized
by the standard solid-phase peptide synthesis with Fmoc Rink
Amide-MBHA resin, Fmoc-AA-OH, HoAt, HATU and DIPEA as
reagents, DMF as solvent. For the coupling of some unnatural
amino acids, NMM instead of DIPEA, and NMP instead of DMF were
used. After cleavage from the resin, all peptides were purified by
preparative RP-HPLC. However, for the preparation of peptides 3,
5, 8, 10, 11, 12 and 13, after the HPLC purification, Sephadex G25
Scheme 1. General synthetic route of several Fmoc-N-Me-AA-OH. Reagents and conditions: (a) p-toluenesulfonic acid, paraformaldehyde, toluene, reflux, 3 h; (b)
trifluoroacetic acid, triethylsilane, CHC13, rt, overnight.
Scheme 2. General synthetic route of several Fmoc-beta-AA-OH. Reagents and conditions: (a) thionyl chloride (SOC12), (Trimethylsilyl)diazomethane, CH2C12, 55–60 °C, 3 h;
b) CH3COOAg, dioxane:H2O = 4:1, 50–80 °C, 2 h.
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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Table 1
Serial numbers and sequential structures of all peptides with different types of substitution (X)
No.
Sequential structure
X
GLP-1
N-methylation
1
2
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
HXEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
3
4
5
6
HA X GTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
HAE X TFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
HAEG X FTSDVSSYLEGQAAKEFIAWLVKGR-NH2
HAEGT X TSDVSSYLEGQAAKEFIAWLVKGR-NH2
7
8
H X EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
HAXGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
9
HAE X TFTSDVSSYLEGQAAKEF1AWLVKGR-NH2
HX EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
H X EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
10
11
b-Peptide
12
13
H X EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
H X EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Cyclopropyl substitution
Table 2
Characterisation of peptides 1–13 HPLC
Peptide
Retention time/min (HPLC)
Molecular mass (MALDI-TOF)
Found
Purity
Calculated
1
2
3
4
5
6
7
8
14.611a
14.702a
14.612a
14.289a
14.721a
14.127a
14.536a
14.669a
14.566a
13.751b
14.005b
13.997b
13.842b
3295.66
3309.68
3309.68
3309.68
3309.68
3309.68
3309.68
3309.68
3309.68
3295.66
3309.68
3335.69
3307.66
3318.6252 [M+Na]+
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
>98%
3332.4775 [M+Na]+
3332.5276 [M+Na]+
3332.6460 [M+Na]+
3332.5447 [M+Na]+
3332.2703 [M+Na]+
3332.2549 [M+Na]+
3332.6384 [M+Na]+
3332.7061 [M+Na]+
3296.3513 [M+H]+
3310.2490 [M+H]+
3336.3916 [M+H+
3308.3230 [M+H]+
9
10
11
12
13
Conditions: 20–80% acetonitrile in H2O containing 0.1% trifluoroacetic acid over 30 min, flow rate of 1 ml/min.
a
Tested with agilent technologies 1200 series.
Tested with agilent technologies 1260 infinity.
b
must be used repeatedly to remove some short peptide impurities.
The peptide purity was tested by analytical RP-HPLC and the
molecular mass was confirmed by MALDI-TOF mass spectrum.
All results were presented in Table 2 and the Supplementary data.
2.2. Biological activities
2.2.1. N-Methylation of amide bonds
In order to explore the significance of amide H-bonds at the N-
terminus of tGLP-1 without a-helical conformation, we employed
N-methylation to make a full screening of all sites. Analogues 2–
6 were chemically synthesized, and their resistance to DPP-IV
degradation and GLP-1R activation were tested in vitro.
As displayed in Figure 2 and Table 3, GLP-1R activation of ana-
logue 2 (EC50 = 23.7 3.0 pM) merely decreased a little when com-
pared with tGLP-1 (EC50 = 5.3 1.5 pM), whereas its resistance to
DPP-IV degradation (t1/2 = 0.83 0.01 h) was unexpectedly weaker
than tGLP-1 (t1/2 = 2.65 0.12 h). This could be explained by the
fact that DPP-IV also recognises His7-Pro,8 whose structure is very
Figure 2. DPP-IV reaction with tGLP-1, 2, 3, 4, 5, 6. The remaining of peptides was
calculated at 0, 1, 2, 4, 8 and 12 h. All experiments were performed triplicate and
repeated three times (n = 3).
activity decreased severely (EC50 >1000 pM), similar to analogues
4, 5 and 6 (EC50 >1000 pM). Moreover, analogues 4, 5, 6 changed
little on the DPP-IV resistance in comparison with tGLP-1, mainly
close to His7-(N-Me)-Ala.8 Although analogue
resistant to DPP-IV degradation (t1/2 >96 h), its GLP-1R activation
3 was highly
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 3
was not improved in comparison with tGLP-1. Analogue 8 showed
The results of DPP-IV reaction and GLP-1R activation
higher resistance to DPP-IV (t1/2 ꢀ 96 h), but it could hardly acti-
vate GLP-1R (EC50 >1000 pM). However, analogue 7 was not only
highly resistant to DPP-IV degradation (t1/2 >96 h), but also capable
of activating GLP-1R. In spite of its higher EC50 (188.6 14.0 pM)
than that of tGLP-1 (5.3 1.5 pM), the data of analogue 7 indicated
a promising site for further modification to improve GLP-1R activa-
tion, with the anticipated DPP-IV resistance kept.
Peptide
DPP-IV reaction (t1/2a/h)
GLP-1R activationc (EC50a/pM)
tGLP-l(l)
2
3
4
5
6
7
8
2.65 0.12
0.83 0.01
>96b
2.42 0.22
2.70 0.17
3.46 + 0.06
>96
5.3 1.5
23.7 3.0
>1000
>1000
>1000
>1000
188.6 14.0
>1000
63.6 9.2
32.9 3.9
20.9 2.2
76.6 9.0
28.4 3.7
Based on the structure of analogue 7 (Table 1), we intended to
explore the feasibility of further modification at the
a-C and b-C
ꢀ96
9
2.39 0.09
>96
atoms of b-AA in peptide 7. Thus, analogues 10, 11, 12 were syn-
thesized. Peptide 13 was designed and synthesized as the less-car-
bon analogue of 12, in which the side chain of Ala8 was substituted
10
11
12
13
>96
>96
>96
by cyclopropyl group and
a-Me-Ala is well known as a DPP-IV
resistant replacement for Ala.8,42 The biological activities of ana-
logues 10–13 were tested in vitro (Table 3). To our expectation,
no reduction of analogues 10–13 was observed after incubating
them with DPP-IV for 96 h, and their GLP-1R activation ability
was improved greatly in comparison with analogue 7. When com-
pared with tGLP-1, all analogues 10, 11, 12 and 13 significantly
enhanced the resistance to DPP-IV degradation while maintaining
a retained level of GLP-1R activation, so we further tested their
blood glucose-lowering activities in vivo using glucose tolerance
testing (GTT) assay.62
a
Data of t1/2 and EC50 are given as mean SD. All the experiments were per-
formed triplicate and repeated three times (n = 3).
b
Reaction time was prolonged to 96 h without finding out any reduction of
peptide 3.
Forty minutes prior to the first intraperitoneal glucose injection,
saline (control), tGLP-1 or tGLP-1 analogues were intraperitoneally
administrated to the Kunming mice (n = 4). After 310 min when
blood glucose of all groups returned back to normal level, the sec-
ond intraperitoneal glucose injection was performed. Blood
glucose values were measured at 0, 40, 55, 80, 115, 160, 310,
325, 350, 385, 430 min and the results are shown in Figures 4
and 5.
Compared with the control group, tGLP-1 reduced the blood
glucose concentration during the first GTT, but had no effect during
the second GTT because of the total degradation in vivo. As for ana-
logues 10, 11, 12 and 13, they all decreased blood glucose level
greater than tGLP-1 during the first GTT (Fig. 5A and B) despite
of their lower GLP-1R activation than tGLP-1. This could be
explained by their longer half-life in the plasma. However, during
the second GTT, similarly to tGLP-1, analogue 12 lost the blood glu-
cose-lowering activity (Fig. 5C and D). Although analogue 11 still
had some effects with lower average area value of blood glucose
(AUC) than tGLP-1 (Fig. 5D), it did not decrease the peak value of
blood glucose at 325 min (Fig. 5C). Analogue 13 decreased the
blood glucose values slightly during the second GTT. Fortunately,
analogue 10 lowered the blood glucose values all the time during
the second GTT, including both the peak value and AUC of blood
glucose. Viewing from the structure of analogue 10 (Table 1), there
Figure 3. DPP-IV reaction with tGLP-1, 7, 8, 9. The remaining of peptides was
calculated at 0, 1, 2, 4, 8, 12, 24, 48, 72 and 96 h. All experiments were performed
triplicate and repeated three times (n = 3).
because the enhanced steric hindrance merely by N-methylation
was insufficient to affect the interaction with DPP-IV, especially
for analogues 5 and 6, modified sites of which are a little far from
the DPP-IV cleaving site.
Based on the results, we learned that these amide bonds in
Ala8–Glu,9 Glu9–Gly,10 Gly10–Thr11 and Thr11–Phe12 are crucial to
the N-terminal interaction of GLP-1 with GLP-1R during the GLP-
1R activating process, as the N-methylation of amide bonds can
both block the H-bonds and alter the conformation.
are no extra substitutions at the a-C and b-C atoms of b-AA, indi-
cating that the further modification may be probably infeasible.
Therefore, analogue 10 was found to possess the best prolonged
blood glucose-lowering activity in vivo.
2.2.2. b-Peptide replacement
In consideration of retaining the amide H-bonds, the b-peptide
modification with the Fmoc-AA-OH replaced by the corresponding
Fmoc-b-AA-OH using the solid-phase peptide synthesis was
employed. To find out the best site for further modification,
tGLP-1 analogues 7, 8, and 9 were synthesized to make a screening
of N-terminus (Table 1). Based on the fact that the N-methylation
at positions far from the DPP-IV cleaving site has little effect on
DPP-IV resistance, we only chose to modify Ala,8 Glu,9 and Gly,10
which are very close to the enzymatic cleaving site. The results
of DPP-IV reaction and GLP-1R activation in vitro were shown in
Figure 3 and Table 3.
To measure the quantitative level of prolonged blood glucose-
lowering activity of analogue 10 in comparison with tGLP-1, we
calculated the peak value and AUC ratios of tGLP-1 or analogue
10 with the control. As displayed in Table 4, analogue 10 showed
a lower AUC ratio (81%) during the second GTT than that of
tGLP-1 (91%) during the first GTT, indicating a stronger continuous
blood glucose-lowering activity of analogue 10 during 310–
430 min than that of tGLP-1 during 40–160 min. In addition, the
peak value ratio of analogue 10 (84%) at 325 min with the control
group was similar to that of tGLP-1 (84%) at 80 min, indicating
approximately a 4-fold prolonged blood glucose-lowering activity
of analogue 10 compared to tGLP-1.
Despite that analogue 9 maintained GLP-1R activation to some
degree (EC50 = 63.6 9.2 pM), its resistance to DPP-IV degradation
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 4. Results of glucose tolerance testing. X axis indicates time (min), Y axis indicates blood glucose value (mmol/L). At 0 min, saline (control), tGLP-1 or analogue (10–
13) was intraperitoneally (ip) injected to normal Kunming mice. At 40 min, glucose was firstly ip injected to mice. At 310 min, glucose was secondly ip injected to mice. The
blood glucose value was measured at 0, 40, 55, 80, 115, 160, 310, 325, 350, 385 and 430 min.
propionic acid were purchased from Sigma–Aldrich. (1S,2S)-
Fmoc-Acpc was purchased from Matrix Scientific. 1-(Fmoc-
amino)cyclopropanecarboxylic acid was purchased from Accela
ChemBio Co. Ltd (Shanghai, China). DCM and DMF specific for
solid-phase peptide synthesis were purchased from Bo Maijie
Technology Co. Ltd (Beijing, China). Acetonitrile for HPLC grade
was purchased from Fisher Scientific. DPP-IV was purchased from
R&D System. The cAMP dynamic kit 2 was purchased from Cisbio
(Bedford, MA). The other reagents were purchased from Alfa Aesar
and Sigma–Aldrich. All reagents were obtained from commercial
suppliers. For purification, Waters 2545 preparative RP-HPLC sys-
tem was used. In the analysis of purity, peptides 1–9 were tested
with Agilent Technologies 1200 series and peptides 10–13 were
tested with Agilent Technologies 1260 Infinity. For testing molecu-
lar mass, AB Sciex TOF/TOFTM Series ExplorerTM 72115 was used. For
glucose tolerance testing assay, Kunming mice (male, 9 weeks old)
were purchased from the Department of Laboratory Animal
Science (Peking University Health Science Center, China). All labo-
ratory animal experiments were performed according to the
national guidelines and approved by the Institutional Animal Care
and Use Committee of Peking University. The blood glucose was
monitored with ACCU-CHEK Performa Nano Blood Glucose Meter
(Roche).
3. Conclusion
To find new long-lasting GLP-1 analogues for the treatment of
type II diabetes, two backbone-based modifications at the N-termi-
nus of tGLP-1 were reported. The first strategy was the N-methyla-
tion of amide bonds, however, the loss of GLP-1R activation
indicated the significance of amide H-bonds to the binding confor-
mation of N-terminus during GLP-1R activating process. In consid-
eration of retaining the amide H-bonds, the b-peptide replacement
strategy was employed and a promising long-lasting tGLP-1 ana-
logue 10 was successfully identified. Peptide 10 showed not only
to be highly resistant to DPP-IV degradation but also to maintain
GLP-1R activation. More importantly, peptide 10 showed approxi-
mately a 4-fold prolonged blood glucose-lowering activity in vivo
when compared with tGLP-1. The disclosed modification strategy
will benefit the discovery of more prolonged GLP-1 analogues for
the potential treatment of type II diabetes.
4. Experimental section
4.1. Materials
Fmoc-protected amino acids (Fmoc-AA-OH) were purchased
from Aladdin reagent (Shanghai, China). Fmoc-Rink Amide-MBHA
resin and Fmoc-b-Ala-OH were purchased from GL Biochem
(Shanghai) Ltd. Fmoc-N-Me-Glu(OtBu)-OH, N-Fmoc-L-b-homoglu-
tamic acid 6-tert-butyl ester, and (S)-3-(Fmoc-amino)-2-methyl-
4.1.1. 9-Fluorenylmethoxycarbonyl-protected N-methylated
alanine (Fmoc-N-Me-Ala-OH)
Paraformaldehyde (1.92 g, 64.0 mmol) and p-toluenesulfonic
acid (0.1920 g, 0.12 mmol) were added to
a solution of
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 5. Peak value of blood glucose and average area under the curve (AUC) during the first GTT (GTT-1) and the second GTT (GTT-2). (A) Peak value of blood glucose at
80 min during GTT-1. (B) AUC from 40 to 160 min during GTT-1. (C) Peak value of blood glucose at 325 min during GTT-2. (D) AUC from 310 to 430 min during GTT-2. Data are
shown as mean SD, *P <0.05 vs tGLP-1.
Table 4
4.1.2. 9-Fluorenylmethoxycarbonyl-protected N-methylated
glycine (Fmoc-N-Me-Gly-OH)
The peak value and AUC ratios of tGLP-1 or analogue 10 with the control
Following the procedure described in the preparation of Fmoc-
N-Me-Ala-OH, this compound was prepared from Fmoc-Gly-OH
(3.00 g, 10.1 mmol) to give the white solid product (2.29 g, 73%
yield). 1H NMR (400 MHz, CDCl3): (isomer I) d = 7.76–7.24 (8H,
m), 4.45–4.41 (2H, m), 4.27 (1H, t, J = 6.9 Hz), 4.10 (2H, s), 3.02
(3H, s); (isomer II) d = 7.76–7.24 (8H, m), 4.45–4.41 (2H, m), 4.19
(1H, t, J = 6.1 Hz), 3.91 (2H, s), 2.98 (3H, s).
GTT-1
Peak value
GTT-2
Peak value
AUC (%)
AUC (%)
(80 min) (%)
(325 min) (%)
Control
tGLP-1
Analogue 10
100
84
61
100
91
63
100
98
84
100
98
81
4.1.3. 9-Fluorenylmethoxycarbonyl-protected N-methylated
phenylalanine (Fmoc-N-Me-Phe-OH)
Fmoc-Ala-OH (3.00 g, 9.6 mmol) in toluene (150 mL). The mixture
was refluxed for 3 h with Dean–Stark apparatus. After cooled to
room temperature, the resultant mixture was washed with satu-
rated NaHCO3, dried over anhydrous Na2SO4, filtered and concen-
trated under reduced pressure. After recrystallization with ethyl
acetate and petroleum ether, the intermediate was obtained
without further purification.
Trifluoroacetic acid (48.0 mL) and triethylsilane (4.60 mL,
28.8 mmol) were added to a solution of above intermediate in
CHCl3 (25 mL). The resultant mixture was stirred at room temper-
ature overnight. After concentration, the residue was dissolved in
CH2Cl2 and concentrated again; after repeated for three times,
the oil liquid was turned to white solid. Then the white solid was
dissolved in diethyl ether, concentrated, washed three times with
petroleum ether. The residue was purified by flash-column chro-
matography on silica gel, to yield the white solid product (2.51 g,
80% yield). 1H NMR (400 MHz, CDCl3): (isomer I) d = 7.77–7.29
(8H, m), 4.91 (1H, dd, J = 14.2, 6.8 Hz), 4.51–4.37 (2H, m), 4.27
(1H, t, J = 6.7 Hz), 2.91 (3H, s), 1.46 (3H, d, 7.4 Hz); (isomer II)
d = 7.77–7.29 (8H, m), 4.62 (1H, dd, J = 14.1, 6.9 Hz), 4.51–4.37
(2H, m), 4.22 (1H, t, J = 6.4 Hz), 2.90 (3H, s), 1.37 (3H, d, J = 6.9 Hz).
Following the procedure described in the preparation of Fmoc-
N-Me-Ala-OH, this compound was prepared from Fmoc-Phe-OH
(3.00 g, 7.7 mmol) to give the white solid product (2.39 g, 77%
yield). 1H NMR (400 MHz, CDCl3): (isomer I) d = 7.75–6.95 (13H,
m), 4.95 (1H, dd, J = 10.8, 4.7 Hz), 4.39–4.31 (2H, m), 4.20 (1H, t,
J = 7.0 Hz), 3.40 (1H, dd, J = 14.5, 4.6 Hz), 3.15–3.09 (1H, m), 2.80
(3H, s); (isomer II) d = 7.75–6.95 (13H, m), 4.62–4.52 (2H, m),
4.39–4.31 (2H, m), 4.13 (1H, t, J = 5.2 Hz), 3.15–3.09 (1H, m), 2.79
(3H, s).
4.1.4. (S)-3-(9-Fluorenylmethoxycarbonyl-amino)butanoic acid
(Fmoc-Ala(CH2)-OH)
To a solution of Fmoc-Ala-OH (2.00 g, 6.4 mmol) in CH2Cl2 was
added thionyl chloride (7.00 mL, 64.2 mmol). The mixture was
refluxed under argon for 3 h at 55 to 60 °C, and then concentrated
under reduced pressure. The residue was dissolved in anhydrous
CH2Cl2 and added dropwise to the solution of (trimethylsilyl)dia-
zomethane (9.63 mL, 19.3 mmol) in CH2Cl2 at ꢁ20 to ꢁ15 °C under
argon protection. After stirred for 5 h, dilute acetic acid was added
to the reactant solution. Then the mixture was washed with brine,
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X. Bai et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
5% Na2CO3 solution and brine again, dried over anhydrous Na2SO4,
filtered and concentrated. The intermediate was then purified by
flash-column chromatography on silica gel.
CO2 at 37 °C in DMEM (Gibco 31053), added with 10% FBS,
2 mmol/L -glutamine, 50 g/mL streptomycin, 50 units/mL peni-
L
l
cillin, and 20 mmol/L HEPES. The two-step protocol of the cAMP
dynamic kit 2 from Cisbio (Bedford, MA) was chosen to perform
the assay: cells and compounds were plated in 384-well white
microplate (Greiner, 784075) and incubated for 30 min, then
cAMP-d2 conjugate and cryptate conjugate in lysis buffer were
added and incubated for another 60 min. Fluorescence data was
read with FlexStation 3 Molecular Devices and the curve was plot-
ted by fluorescence value versus peptide concentration. EC50 was
measured with GraphPad Prism 6.
To the solution of above intermediate in dioxane and H2O (4:1,
v/v) was added a catalytic amount of CH3COOAg. After stirred for
2 h at 50 to 80 °C, the resultant mixture was filtered, concentrated,
dissolved in 5% Na2CO3 solution and extracted with ethyl acetate.
The organic layer was washed twice with brine, dried over anhy-
drous Na2SO4, filtered and concentrated. The residue was then
purified by flash-column chromatography on silica gel, yielding
the product (0.68 g, 33% yield over two steps). 1H NMR
(400 MHz, CDCl3): d = 7.76–7.29 (8H, m), 5.19 (1H, br s), 4.39
(2H, br s), 4.22–4.13 (2H, m), 2.60 (2H, br s), 1.26 (3H, d, J = 6.0 Hz).
4.4. Glucose tolerance testing (GTT) assay
4.1.5. General procedure of solid-phase peptide synthesis and
HPLC purification of peptides 1–13
Blood glucose-lowering activities of tGLP-1 analogues were
tested with Kunming mice (male, 9 weeks old). After an overnight
fast, glucose tolerance testing assay was performed. After
intraperitoneal injection of saline (control), tGLP-1 or tGLP-1 ana-
logues (25 nmol/kg) at 0 min, glucose (ip, 2 g/kg) was adminis-
trated to mice (n = 4/group) at 40 min. Blood was collected from
the tail vein at 55 min, 80 min, 115 min, 160 min and blood glucose
values were measured with ACCU-CHEK Performa Nano Blood Glu-
cose Meter (Roche). Then glucose (ip, 2 g/kg) was administrated
secondly to the mice at 310 min. Blood was collected from the tail
vein at 325 min, 350 min, 385 min, 430 min and blood glucose val-
ues were measured. The curve was plotted by blood glucose value
versus time with GraphPad Prism 6. The average area under the
curve (AUC) was calculated with DAS 2.1.1 and statistical analysis
was conducted with SPSS 13.0.
tGLP-1 and its analogues 2–13 were all synthesized using gen-
eral solid-phase peptide synthesis of N-Fmoc/tBu chemistry.63
Fmoc Rink Amide-MBHA resin (0.1 mmol) was added to a 25 ml
peptide synthetic vessel and swollen with DMF for 40 min. After
deprotected by 25% piperidine in DMF, a solution of Fmoc-AA-OH
(0.4 mmol), HATU (0.4 mmol), HoAt (0.4 mmol) and DIPEA
(0.8 mmol) in DMF was added to the vessel. After reacted for 1 h,
the resin was washed three times with DMF and three times with
CH2Cl2, then qualitative ninhydrin testing was performed to mon-
itor whether some free amino groups still existed on the resin or
not. If not, the resin was washed three times with DMF again
and repeated the procedures of deprotection and coupling. For
the coupling of some unnatural amino acids, NMM instead of
DIPEA and NMP instead of DMF were used. Besides, the reaction
time was prolonged to 4 h. Following the final deprotection of N-
terminus, the target peptide was cleaved from resin with Reagent
K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2 h at
room temperature. After filtration, the residue solution was con-
centrated, precipitated with cold diethyl ether and centrifuged
for three times. The residue was dissolved in water and purified
by Waters 2545 preparative RP-HPLC system. Sephadex G-25 was
used for the further purification to remove some short peptide
impurities. The molecular mass of the target peptide was con-
firmed by MALDI-TOF. The purity of peptide was tested with ana-
lytical RP-HPLC, and the conditions were as follows: a linear
gradient of 20% mobile phase A and 80% mobile phase B to 80%
mobile phase A and 20% mobile phase B (A: acetonitrile containing
0.1% TFA; B: H2O containing 0.1% TFA) in 30 min, at a flow rate of
1 mL/minute with UV detection at 214 nm.
Acknowledgments
We gratefully acknowledge Xun Huang (Division of Antitumor
Pharmacology, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences) for providing the HEK293 cells stably
expressing GLP-1 receptor. This work was financially supported
by the grants (2012CB822100, 2013CB910700) from the Ministry
of Science and Technology of China, and the National Natural
Science Foundation of China (21232002, 81573274).
Supplementary data
Supplementary data (compound characterization and purity
data, as well as biological assay data) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmc.2016.01.036.
4.2. The stability of analogues against recombinant human DPP-
IV
References and notes
tGLP-1 or tGLP-1 analogues 2–13 (20
DPP-IV (0.33 g/mL) at 37 °C in 100 nM HEPES buffer containing 1
nM EDTA-2Na (pH 7.5) and 0.05% Tween 80 (60 L). At 1, 2, 4, 8,
12, 24, 48, 72 or 96 h, guanidine hydrochloride (8 M, 240 L) was
added to terminate the reaction. After centrifugation for 20 min,
supernate (100 L) was obtained to subject to RP-HPLC analysis.
The conditions were as follows: a linear gradient of 20% mobile
phase A and 80% mobile phase B to 80% mobile phase A and 20%
mobile phase B (A: acetonitrile containing 0.1% TFA; B: H2O contain-
ing 0.1% TFA) in 30 min, at a flow rate of 1 mL/minute with UV
detection at 214 nm. The remaining of peptides (%) was plotted ver-
sus time and half-life was determined with Graphpad Prism 6.
lM) was incubated with
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