Synthesis of truncated analogues of preptin-(1-16), and investigation of their ability to stimulate osteoblast proliferation

Article abstract of DOI:10.1016/j.bmc.2014.05.026

Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the β-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1-16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1-16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1-16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1-8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1-8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.

Full text of DOI:10.1016/j.bmc.2014.05.026

Bioorganic & Medicinal Chemistry 22 (2014) 3565–3572
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of truncated analogues of preptin-(1–16), and investigation
of their ability to stimulate osteoblast proliferation
c
c
Renata Kowalczyk a,b,d, Sung H. Yang a,b,d, Margaret A. Brimble a,d, , Karen E. Callon , Maureen Watson ,
⇑
Young-Eun Park c, Jillian Cornish c,d
a The School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand
b The School of Biological Sciences, University of Auckland, 3 Symonds St, Auckland 1010, New Zealand
c Department of Medicine, University of Auckland, Private Bag 92019, Auckland 1010, New Zealand
d Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1010, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the b-pancreatic cells
and is active in fuel metabolism. We have previously established that a shorter fragment of preptin,
namely preptin-(1–16), stimulates bone growth by proliferation and increasing the survival rate of oste-
oblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-
(1–16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large
size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest
preptin analogue that retains or even increases the bone anabolic activity as compared to the parent
preptin-(1–16) peptide. Truncations were made in a methodical manner from both the N-terminus
and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity
was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified,
ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylgly-
cine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin
(1–8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and
enhances bone nodule formation. Preptin (1–8) is a useful lead compound for the development of orally
active therapeutics for the treatment of osteoporosis.
Received 5 March 2014
Revised 5 May 2014
Accepted 13 May 2014
Available online 20 May 2014
Keywords:
Preptin
Peptidomimetics
Osteoporosis
Ring closing metathesis (RCM)
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
bone-resorbing cells called osteoclasts. During osteoporosis, an
imbalance between bone resorption and bone formation leads to
The bone disease osteoporosis (literally meaning ‘porous bone’),
is characterized by reduced bone mass and disruption of bone mic-
roarchitecture, leading to weakened bones and increased risk of
fractures.1 A major health issue worldwide, osteoporosis affects
millions of people globally, predominantly postmenopausal
women. Estimates suggest one in three women and one in five
men over the age of fifty worldwide will sustain an osteoporotic
fracture, leading to a significant burden on both the individual
and society.1 Partly due to the increased longevity of the popula-
tion, and rise in expectation of increased physical activity in the
elderly, the number of fractures is expected to rise in the near
future.2
the development of fragile bone tissue. Most current osteoporotic
therapies are anti-resorptive, inhibiting bone loss by reducing
osteoclast activity.3 Unfortunately, anti-resorptive therapy cannot
restore bone mass or bone structure already lost due to increased
bone remodelling. Therapies acting on osteoblasts are therefore
highly attractive, but the only such treatment currently in use is
human parathyroid hormone (PTH) and its N-terminal fragment,
teriparatide (PTH 1–34).2 Unfortunately, PTH therapy is limited
due to its high cost, the need for daily subcutaneous injections,
and side effects including hypercalcemia and a possible increased
risk of osteosarcoma.4 Considerable demand exists for a new ana-
bolic drug to treat osteoporosis.
Healthy bone is maintained throughout life by a well-coordi-
nated balance between bone-forming cells called osteoblasts and
Preptin is a 34-residue polypeptide hormone secreted from the
pancreatic b-cells that corresponds to the Asp(69)-Leu(102)
fragment of proIGF-II E (Fig. 1).5 It was isolated from cultured
pancreatic b-cells, along with insulin and amylin. All three
hormones are involved in glucose metabolism, and preptin may
⇑
Corresponding author. Tel.: +64 9 373 7599x88259; fax: +64 9 3737422.
E-mail address: m.brimble@auckland.ac.nz (M.A. Brimble).
http://dx.doi.org/10.1016/j.bmc.2014.05.026
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3566
R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
Mouse:
34
5
10
15
20
1
25
30
Asp-Val-Ser-Thr-Ser-Gln-Ala-Val-Leu-Pro-Asp-Asp-Phe-Pro-Arg-Tyr-Pro-Val-Gly-Lys-Phe-Phe-Gln-Tyr-Asp-Thr-Trp-Arg-Gln-Ser-Ala-Gly-Arg-Leu
Rat:
PREPTIN-(1-16)
34
1
5
10
15
20
25
30
Asp-Val-Ser-Thr-Ser-Gln-Ala-Val-Leu-Pro-Asp-Asp-Phe-Pro-Arg-Tyr-Pro-Val-Gly-Lys-Phe-Phe-Lys-Phe-Asp-Thr-Trp-Arg-Gln-Ser-Ala-Gly-Arg-Leu
PREPTIN-(1-16)
Human:
34
5
10
15
20
1
25
30
Asp-Val-Ser-Thr-Pro-Pro-Thr-Val-Leu-Pro-Asp-Asn-Phe-Pro-Arg-Tyr-Pro-Val-Gly-Lys-Phe-Phe-Gln-Tyr-Asp-Thr-Trp-Lys-Gln-Ser-Thr-Gln-Arg-Leu
PREPTIN-(1-16)
Figure 1. Comparison of primary structures of mouse, rat, and human preptin.
be a physiological amplifier of glucose-mediated insulin secretion.6
Rat preptin-(1–34) and analogues in which Phe-21 was substituted
by various unnatural amino acid residues, stimulate insulin secre-
tion in the presence and absence of glucose.7
truncated analogues containing modifications of the N- and C-ter-
mini (5–28). A ring closing metathesis (RCM) technique was used
to synthesize a hydrocarbon linked cyclic peptide 29 in the search
of an enzymatically more stable therapeutic to treat osteoporosis
(Fig. 2).17,18 All analogues with the exception of 11 were evaluated
for their proliferative activity in vitro using primary rat osteoblasts.
The lead compounds identified (9, and 20) were further tested for
their ability to form bone nodules. This is the first study that
enables identification of the shortest biologically active preptin-
(1–16)-derived peptide with potential to treat osteoporosis as an
agent delivered orally.
We have recently demonstrated a stimulatory effect of rat prep-
tin on bone growth6 and have established that preptin-(1–34) acts
on osteoblast-like cells to stimulate proliferation, differentiation
and survival in vitro and in vivo.6 It was observed that proliferative
effect of preptin is mediated through a G protein-coupled receptor
activating Gi-dependent phosphorylation of p42/44 MAP kinases.6
Interestingly, a human condition of high bone mass phenotype has
been observed when increased circulating levels of a pro-IGF-II
peptide complexed with IGF-binding protein-2 occur in patients
with chronic hepatitis C infection. Additionally, Li et al.8 have
recently shown that osteoporotic men have lower circulating levels
of preptin than healthy individuals. These results clearly demon-
strate the osteogenic nature of preptin in humans. However, little
is known about the mechanism of action of preptin and its influ-
ence on bone metabolism and the preptin receptor is still
unknown. Liu et al.9 have shown that connective tissue growth fac-
tor (CTGF) mediates preptin-induced proliferation and differentia-
tion of human osteoblasts. CTGF plays an important role in skeletal
development stimulating osteoblastic cells for proliferation and
differentiation. These findings suggest that preptin might be a very
promising new pharmaceutical lead for the anabolic treatment of
osteoporosis.
We have observed that a shorter rat preptin fragment, namely
preptin-(1–16), is still anabolic to osteoblasts in both in vitro and
in vivo studies, and unlike full-length preptin has no activity in
the pancreas thus it does not affect carbohydrate metabolism
(Fig. 1).10 This smaller peptide is a more attractive candidate for
pharmaceutical development and can be used as a model peptide
for the creation of simpler but more orally active analogues.
To the best of our knowledge no reports have been documented
in the literature on the study of preptin and preptin analogues as
drug candidates to treat osteoporosis. Therefore, the aim of this
study was to investigate whether N- and C-modified analogues of
rat preptin-(1–16) and its truncated analogues, retain or increase
anabolic activity on bone growth compared to the parent 16-amino
acid residue peptide. Due to our on-going interest in the design and
synthesis of peptidomimetics of biologically active peptides we
were also interested to see if the lead peptide identified during
the study could be used to afford more stable and thus more attrac-
tive pharmaceutical candidates.11–16
2. Results and discussion
2.1. Synthesis of preptin-(1–16) (1), N-acetylated preptin-(1–16)
(2), C-amidated preptin-(1–16) (3), and N-acetylated and
C-amidated preptin-(1–16) (4)
As depicted in Scheme 1 Fmoc Solid Phase Peptide Synthesis
(SPPS) was employed for the preparation of rat preptin-(1–16)
(1), N-acetylated preptin-(1–16) (2), C-amidated preptin-(1–16)
(3), and N-acetylated and C-amidated preptin-(1–16) (4). In order
to obtain a C-terminal acid for peptides 1 and 2, the growing pep-
tide chain was attached to the solid support via 4-(hydroxy-
methyl)phenoxypropionic acid linker (HMPP). In order to afford
the C-terminal amide in preptin-(1–16) analogues 3 and 4, Rink
amide linker was employed to anchor the growing peptide chain
to the resin.
Our initial focus was to develop reliable synthetic conditions
that allow the rapid generation of a library of analogues of the
preptin-(1–16) peptide. Use of microwave irradiation reduces the
times for SPPS affording improved synthetic yields19 hence it was
adopted in the present work. Preptin-(1–16) contains three aspar-
tic acid residues (Asp-1, Asp-11, and Asp-12) in its sequence that
can potentially undergo aspartimide formation when using piper-
a
idine for N -Fmoc protecting group removal under microwave con-
ditions.20,21 It was therefore decided to use a milder deprotection
base, namely piperazine (5% in DMF).19,21,22 However, formation
of aspartimide by-products was not reported during the synthesis
of preptin-(1–34) and its analogues when the synthesis was per-
formed at room temperature using 20% piperidine in DMF.7
HBTU is one of the most commonly used coupling reagents23
and we anticipated its use for the current study.24,25 Although cou-
pling of Arg-15 was challenging when HBTU was employed as the
coupling reagent for the synthesis of preptin-(1–34) for the synthe-
sis of shorter fragments of preptin using microwave-enhanced
We herein report the synthesis of preptin-(1–16) (1), its
N-acetylated, and C-amidated analogues (2–4) and a family of

R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
3567
Truncated analogues of preptin-(1-16) at C-terminus (5-16)
Truncated analogues of preptin-(1-16) at N-terminus (17-28)
and stapled analogue 29
FPRY-NH2 (19)
7
( )
DVST-NH2
FPRY (17)
(preptin-(13-16))
5
DVST (
)
18
)
Ac-FPRY (
6
)
Ac-DVST (
preptin-(1-4)
(
)
20
( )
Ac-FPRY-NH2
8
Ac-DVST-NH2 ( )
(29)
Ac-Agl-FPRY-Agl-NH2
Ac-DVST-SQAV-LPDD-FPRY (2)
11
)
DVST-SQAV-NH2
(
23
)
LPDD-FPRY-NH2
(
1
DVST-SQAV-LPDD-FPRY ( )
(preptin-(1-16))
9
DVST-SQAV ( )
preptin-(1-8)
LPDD-FPRY (21)
(preptin-(9-16))
10
Ac-DVST-SQAV (
)
22
Ac-LPDD-FPRY (
)
(
)
3
( )
DVST-SQAV-LPDD-FPRY-NH2
12
)
Ac-DVST-SQAV-NH2
(
Ac-LPDD-FPRY-NH2 (24)
4
(
Ac-DVST-SQAV-LPDD-FPRY-NH2
)
15
27
( )
DVST-SQAV-LPDD-NH2
(
)
SQAV-LPDD-FPRY-NH2
13
25
SQAV-LPDD-FPRY ( )
DVST-SQAV-LPDD (
)
Ac-SQAV-LPDD-FPRY (26)
Ac-DVST-SQAV-LPDD (14)
preptin-(1-12)
preptin-(5-16)
)
(
)
(
Ac-DVST-SQAV-LPDD-NH2 (16)
Ac-SQAV-LPDD-FPRY-NH2 (28)
Figure 2. Preptin-(1–16) (1) and its analogues (2–29) synthesised during the study.
RINK AMIDE
LINKER
N
H
Fmoc-Tyr(tBu)
HMPP LINKER
N
H
Fmoc
In house prepared26,27
PS
PS
(
)
In house prepared26,27
3
MW Fmoc SPPS
Repeat step 1 and 2 until desired
peptide sequenceis assempled
(1) Deprotect Fmoc (a)
(2) Fmoc-AA coupling (b) (4) Repeat step 1
PG PG
DVST-SGAV-LPDD-FPRY
PG PG PG PG
PG
PG PG
PG
PG
PG
RINK AMIDE
LINKER
HMPP LINKER
N
H
DVST-SGAV-LPDD-FPRY
PG PG
N
H
PG
PG
PS
PS
(1) Cleave from resin and remove
protecting groups (c)
(1) N-Acetylation (d)
(1) Cleave from resin and remove
protecting groups (c)
(1) N-Acetylation (d)
(2) Cleave from resin and remove
(2) Cleave from resin and remove
protecting groups (c)
protecting groups (c)
DVST-SGAV-LPDD-FPRY (1)
3
)
Ac-DVST-SGAV-LPDD-FPRY (2)
DVST-SGAV-LPDD-FPRY-NH2
(
Ac-DVST-SGAV-LPDD-FPRY-NH2 (4)
and in a similar fashion:
and in a similar fashion:
6, 10, 14, 18, 22, 26
and in a similar fashion:
7 11 15 19 23 27
, , , , ,
and in a similar fashion:
8, 12, 16, 20, 24, 28
5 9 13 17 21 25
,
,
,
,
,
Reagents and conditions:
(a) 5% piperazine, DMF, (compounds 1-4) and 5% piperazine, 0.1 M 6-Cl-HOBt, DMF, (compounds 5-28), 62 W, 75 °C, 1 x 0.5 min + 1 x 3 min;
(b) HBTU, iPr2EtN, DMF, 25 W, 75 °C, 5 min (all Fmoc-AAexcept Fmoc-Arg(Pbf)) and (i) 30 min, rt (ii) 25W, 72 °C, 5 min (Fmoc-Arg(Pbf));
(c) TFA, iPr3SiH, DODT, H2O, rt, 2 h; (d) 20% Ac2O, DMF, rt, 2 x 20 min.
Scheme 1. Synthesis of preptin-(1–16) (1), its analogues 2–4 and truncated analogues at the C-terminus (5–16) and at the N-terminus (17–28).
conditions it was anticipated that HBTU/iPr2EtN would be a
suitable coupling reagent.
of side chain protecting groups which was performed at room tem-
perature. Pleasingly, the expected product 1 was afforded as the
major product (as measured by RP-HPLC at 210 nm) and contained
only one aspartimide (18 Da less) as a minor impurity (22%). The
site of aspartimide formation was not determined.
‘In house’ prepared aminomethyl polystyrene resin (AM-PS)
a
functionalised with either an N -Fmoc-protected tyrosine residue
attached to the HMPP linker (for peptides 1 and 2) or Rink amide
linker (for analogue 3 and 4)26,27 was used for SPPS that was car-
ried out using a CEM Liberty 1 peptide synthesiser. The final step
for the synthesis of native preptin-(1–16) (1) was TFA mediated
cleavage of the resin-bound peptide 1 with concomitant removal
a
Initial N-acetylation of Fmoc-deprotected N amino group of
Asp-1 of resin-bound peptide 2 (Scheme 1) was required before
final treatment with TFA to afford crude N-acetylated preptin-
(1–16) analogue (2). RP-HPLC analysis of the crude mixture

3568
R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
revealed that the expected product 2 was present as the major
product, and similar to the synthesis of the parent peptide 1 it only
contained one aspartimide as a minor by-product (23%). Table 1
summarises RP-HPLC and MS analysis for the peptides 1–4 (Sup-
porting information). These results demonstrated that use of a
milder base (piperazine) than piperidine under microwave condi-
tions was not sufficient to suppress aspartimide formation.
Subsequent synthesis of C-amidated preptin-(1–16) (3), and
N-acetylated and C-amidated preptin-(1–16) (4) using Rink amide
linker following the same synthetic route as for 1 and 2 afforded
crude peptides 3 and 4 in good purity (55%, and 63%, respectively)
contaminated with an aspartimide by-product (26%, Scheme 1).
The presence of the bulkier Rink amide linker used for the synthe-
sis of C-amidated analogues (3), and (4) had no effect on asparti-
mide formation compared to the HMPP linker used for the
synthesis of preptin-(1–16) (1) and N-acetylated preptin-(1–16)
(2). The rate of aspartimide formation depends on the nature
of the neighbouring residues to Asp on both the N-terminus and
C-terminus and we were interested to see if the type of the linker
influenced formation of this by-product.20
Pleasingly, subsequent RP-HPLC purification enabled separation
of the desired products 1–4 from the aspartimide by-product to
afford peptides 1–4 in excellent >98% purity (Table 1, Supporting
information). However, further optimisation of the Fmoc deprotec-
tion conditions may be required in the future. It is anticipated that
formation of the undesired aspartimide by-product can be further
prevented by addition of an acid additive (hydroxybenzotriazole-
based) to the piperazine-based deprotection mixture as previously
described.19,21 Given that we successfully separated the desired
product in quantities that were sufficient for subsequent biological
assays, further optimisation of the synthesis of 1–4 was not under-
taken in this study.
(1) with purities ranging from 72% (analogue 12) to 97% (analogue
6). Similar to DVST-NH2 (7), its N-acetylated derivative 8 proved
difficult to retain on the analytical RP-HPLC column thus the purity
of crude peptide 8 was not calculated and the presence of the
desired product 8 was confirmed using ESI-MS analysis (m/z
462.2) [M+H]+ requires 461.46.
Peptides 5–16 were subjected to final RP-HPLC purification to
afford the C-terminus truncated analogues of preptin-(1–16) (1)
in excellent purity ranging from 93% to 99% (Table 1, Supporting
information) ready for subsequent biological evaluation. It was
unexpected to find that preptin-(1–8)-NH2 (11) was poorly soluble
in solvents used for the subsequent bioassay (methanol, DMSO,
H2O) therefore it was decided to withdraw analogue 11 from fur-
ther study. The reason for insolubility of 11 is unknown.
2.3. Synthesis of N-terminus truncated analogues of preptin-(1–
16) (17–28)
Initial synthesis of analogues of preptin-(1–16) truncated at the
C-terminus afforded eleven shorter peptides (5–10, and 12–16)
that were assessed for their anabolic bone activity in vitro. Subse-
quent preparation of analogues that were truncated at the N-ter-
minus, namely preptin-(13–16) (17), preptin-(9–16) (21),
preptin-(5–16) (25), their N-acetylated analogues (18, 22, 26), as
well as C-amidated variants (19, 23, 27), and both N-acetylated
and C-amidated analogues (20, 24, 28) was next undertaken
(Fig. 2).
Microwave-enhanced Fmoc SPPS using the synthetic conditions
summarised in Scheme 1 successfully delivered the desired prod-
ucts 17, 21, 25 and their C-amidated derivatives (19, 23, 27),
respectively. The crude peptides were obtained with purities vary-
ing from 84% to 94% (Table 1, Supporting information). Resin-
a
bound and N -terminally deprotected peptides 18, 20, 22, 24, 26,
2.2. Synthesis of C-terminus truncated analogues of preptin-(1–
16) (5–16)
28 were treated with acetic anhydride before TFA mediated resin
cleavage to afford N-acetylated preptin-(13–16) (18), preptin-(9–
16) (22), preptin-(5–16) (26), and their C-amidated analogues
(20, 24, and 28), respectively, with crude purities >81% (Table 1,
Supporting information). Subsequent RP-HPLC purification of trun-
cated N-terminus analogues of preptin-(1–16) gave products 17–
28 with purities >98% with the exception of Ac-SQAV-LPDD-FPRY
(26) and Ac-SQAV-LPDD-FPRY-NH2 (28) (96% and 94%, respec-
tively) ready for subsequent biological assessment.
In order to identify the minimum length of the peptide that still
retains or increases the activity of the parent preptin-(1–16) 1,
C-terminal truncated analogues were initially prepared. Thus,
synthesis of preptin-(1–4) (5), preptin-(1–8) (9), preptin-(1–12)
(13), their derivatives acetylated at the N-terminus (6, 10, 14),
amidated at the C-terminus (7, 11, 15) and, both N-amidated and
C-acetylated (8, 12, 16), was undertaken following the synthetic
method previously used for peptides 1–4 (Scheme 1). Due to
the observed formation of an aspartimide by-product during
the synthesis of preptin-(1–16) (1) and its analogues 2–4, 5%
piperazine solution in DMF with 0.1 M 6-Cl-HOBt as additive
2.4. In vitro proliferation effect of synthesised analogues on
primary rat osteoblasts
Preptin-(1–16) (1) and synthetic analogues 2–10 and 12–28
were assessed for their proliferation of bone activity in vitro using
cultures of foetal rat osteoblasts as previously described.28 The
effects of the compounds tested were evaluated by measuring
the [3H]-thymidine incorporation levels over the last 6 h of the
incubation period (24 h). Pleasingly, three out of the twenty six
preptin-(1–16) analogues tested, namely, Ac-preptin-(1–16)-NH2
(4), preptin-(1–8) (9), and Ac-preptin-(13–16)-NH2 (20), together
with the intact preptin-(1–16) (1) showed a significant increase
in thymidine incorporation levels (a 5% significance level was used
throughout) (Fig. 3). The rest of the peptides studied (2, 3, 5–8, 10,
12–19, 21–28) had no significant effect on osteoblast proliferation
(data not shown).
Ac-preptin-(1–16)-NH2 (4) has the same length as native prep-
tin-(1–16) (1), thus our focus was only directed at the smaller ana-
logues 9 and 20. Interestingly, these two smaller peptides are both
derived from two distinct preptin-(1–16) sites, N-terminal (ana-
logue 9), and C-terminal (analogue 20), suggesting that the internal
region of the preptin-(1–16) (fragment 9–12) is not important for
bone activity. These much shorter peptide analogues are attractive
a
was used to effect N -Fmoc-protecting group removal thereby
minimising potential aspartimide formation.21
Once the desired peptide sequences for analogues 5, 9, 13, and
their C-amidated analogues (7, 11, and 15, respectively) were
assembled, peptides were detached from resin (TFA) affording pep-
tides with crude purity greater than 80%. The purity of the crude
analogue DVST-NH2 (7) could not be determined due to difficulties
experienced in retaining peptide 7 on the analytical column under
the conditions used for RP-HPLC. However, ESI-MS analysis of
crude analogue 7 confirmed that the desired product was formed
as the major product (m/z 420.2) [M+H]+ requires 420.44. Pleas-
ingly, no presence of an aspartimide by-product was detected. A
summary of the chromatographic and mass spectrometry data
for compounds (5–16) is given in Table 1 (Supporting information).
a
Fully assembled, resin-bound and N -Fmoc-deprotected trun-
cated analogues (6, 8, 10, 12, 14, and 16) were initially treated with
acetic anhydride and the resin was subsequently cleaved using TFA
to afford crude N-acetylated (6, 10, and 14), and both, N-acetylated
and C-amidated (8, 12, and 16) shorter analogues of preptin-(1–16)

R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
3569
modifications to peptides in order to deliver compounds with
improved pharmacokinetic profile.29 Peptide cyclization using ring
closing metathesis generates stable carbon–carbon bonds and is
often used in the drug discovery area to increase metabolic stabil-
ity, improve peptide activity, and stabilise peptide conforma-
tion.30–32
A RCM technique has been successfully used in our laboratory
to afford biologically active analogues of amylin-(1–8) therefore,
we decided to use this technique in the current study.12 Our focus
was to synthesise a macrocyclic analogue of the shortest, 4-amino
acid active peptide, Ac-preptin-(13–16)-NH2 (20). To minimise dis-
ruption to the primary sequence of this short analogue it was
decided to introduce allylglycine (Agl) residues required for subse-
quent RCM at both the N- and the C-terminus of the peptide, acet-
a
ylate the N -terminus and amidate C-terminus. Attention thus
focused on the synthesis of the RCM analogue 29 (Scheme 2).
With this idea in mind, the synthesis of the resin-bound linear
RCM precursor 30 was undertaken starting from in house prepared
aminomethyl polystyrene resin modified with Rink amide linker
using manual Fmoc SPPS at room temperature.26,27 20% piperidine
Figure 3. Osteoblast activity of the preptin-(1–16) (1) and analogues tested
showing the significance level (4, 9, and 20). Data are expressed as a ratio of
treatment to control, mean SEM; ⁄significantly different from control (p <0.05).
a
in DMF was used to remove the N -Fmoc protecting group, and
targets for further drug development as they have greater potential
to afford orally available agents than preptin-(1–16). However, the
poor pharmacokinetic profile of these natural peptides requires
further chemical modifications to generate more stable and more
bioavailable compounds. We therefore decided to initially attempt
to optimise the structure of the shortest active preptin-(1–16) frag-
ment, Ac-preptin-(13–16)-NH2 (20).
amino acid couplings were performed using HCTU with iPr2EtN
as a base. Successive steps of Fmoc deprotection and couplings of
a
N -Fmoc-protected amino acids followed by final N-acetylation
a
of the free N -amino group of N-terminal allylglycine (acetic anhy-
dride in NMP) afforded Ac-Agl-Phe-Pro-Arg(Pbf)-Tyr(tBu)-Agl-Rink
amide-PS peptidyl resin 30. Subsequent TFA mediated cleavage of
an aliquot of resin 30 followed by LCMS analysis confirmed the
presence of the desired RCM precursor ready for subsequent mac-
rocyclization. Hoveyda–Grubbs’ II catalyst (30 mol % of catalyst,
CH2Cl2/DMF (4:1) MW, 100 °C, 2 h) was used to effect the on-resin
ring closing metathesis reaction, followed by removal of the mac-
rocyclic peptide from the resin (TFA/iPr3SiH/H2O/DODT).12 Pleas-
ingly, good conversion to the desired RCM product as an
inseparable mixture of cis and trans isomers was observed as
2.5. Synthesis and osteoblast proliferation activity of
macrocyclic analogue 29
Bioactive peptides comprising natural amino acids in their
sequence exhibit a high degree of flexibility and are susceptible
to protease degradation which hampers their use as therapeutics.29
These major obstacles make them unsuitable for drug develop-
ment. A peptidomimetic toolbox offers many types of structural
revealed by RP-HPLC analysis (Scheme 2, Rtcis
12.27 min,
or trans
26%, Rtcis or trans 12.59 min, 59%). Further purification of the crude
Scheme 2. Synthesis of RCM macrocyclic analogue of preptin-(1–16) (29).

3570
R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
Figure 4. Effect of preptin-(1–8) (9) and Ac-preptin-(13–16)-NH2 (20) on formation of bone nodules in primary cultures of rat osteoblast cells over a period of 3 weeks.
Cultures were stained for mineral using Von Kossa stain, and area of mineralized bone nodules were quantified. A shows nodule formation and B shows % of mineralised area.
product by RP-HPLC was performed affording peptide 29, as a mix-
ture of cis and trans isomers ready for biological analysis.
differentiation was studied using a bone nodule formation assay
with dexamethasone used as a positive control. The appropriate
peptides (9, or 20) were added to the confluent cells suspended
in media supplemented with osteoblast differentiation substances
Macrocyclic analogue 29 was subsequently tested for its ability
to stimulate the proliferative activity of primary rat osteoblasts.
Similar to previous analysis of preptin-(1–16) (1) and its analogues
2–10, and 12–28, [3H]-thymidine incorporation was measured and
compared to control. It was disappointing to see however, that no
significant activity to stimulate osteoblasts was observed. It is dif-
ficult to account for this lack of activity. The altered primary struc-
ture of 29 with the presence of non-natural allylglycine residues,
and the more constrained peptide conformation introduced by
the macrocyclic ring in RCM analogue 29 compared to the linear
peptide 20 could account for the lack of activity. Additionally, the
fact that the sequence of both linear analogue 20, and macrocyclic
analogue 29 comprise only four out of the sixteen amino acids in
preptin-(1–16) may also be a contributing factor. Interestingly,
proliferation activity was observed for the truncated, natural
Ac-preptin-(13–16)-NH2 (20) but no osteoblast stimulation effect
was observed for the macrocyclic peptide 29. We therefore decided
to further investigate the biological activity of the linear analogues,
preptin-(1–8) (9) and Ac-preptin-(13–16)-NH2 (20) using a bone
nodule formation assay, before further structural modifications
were undertaken. NMR analysis of macrocyclic analogue 29 to
establish the cis/trans isomeric ratio was not performed due to
the lack of activity observed in the osteoblast proliferation assay.
(L-ascorbic acid-2-phosphate and b-glycerophosphate). After 21
days, the cells were fixed, stained by Von Kossa stain, and the min-
eralized areas were quantified using Bioquant image analysis soft-
ware. The process of bone nodule formation relies on the activity of
differentiated osteoblasts to deposit and mineralise bone matrix.
We were pleased to see that preptin-(1–8) (9) significantly stimu-
lated formation of bone nodules and was able to increase the per-
centage of mineralised area in primary rat osteoblasts at 10ꢀ9
M
concentration. However, the shorter preptin-(1–16) analogue,
Ac-preptin-(13–16)-NH2 (20) did not demonstrate a significant effect
on bone nodule formation and mineralisation at concentration of
10ꢀ9 M and 10ꢀ10 M (Fig. 4). These results have further established
the importance of the N-terminal region of native preptin-(1–16)
that is most likely responsible for the observed anabolic effect on
osteoblasts. Future structure-activity optimisation studies will
therefore be focused on the key preptin-(1–8) fragment.
3. Conclusions
Our goal to identify the shortest analogues of the biologically
active native preptin-(1–16) peptide that retain bone-forming
activity was informed by the successful synthesis of a library of
twenty eight analogues of preptin-(1–16) and their in vitro assess-
ment on primary rat osteoblasts. We have established the impor-
tance of the N-terminal region of preptin-(1–16) to stimulate
osteoblasts. This is the first study of this kind that has enabled
the successful identification of the shortest preptin analogue (9),
namely preptin-(1–8), that retained bone activity in both
2.6. Effect of preptin-(1–8) (9) and Ac-preptin-(13–16)-NH2 (20)
on formation of bone nodules in primary rat osteoblast cells
In order to further demonstrate the anabolic activity of both the
lead peptides identified (9 and 20), the effect of both preptin-(1–8)
(9), and Ac-preptin-(13–16)-NH2 (20), on primary rat osteoblasts

R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
3571
proliferative and differentiation studies thus proving that its ana-
bolic action increases the number of bone forming cells as well
as the function of the cells. This 8-amino acid peptide is much
smaller than the parent 16-mer molecule and is therefore an inter-
esting target to develop new chemical entities for the oral treat-
ment of osteoporosis. The design and synthesis of chemically
modified preptin-(1–8) (9) analogues to further improve activity
and/or stability is currently being undertaken in our lab and the
results of this study will be reported in due course.
72 °C. Fmoc protecting group was removed using 5% piperazine
in DMF (compounds 1–4) and 5% piperazine with 0.1 M 6-Cl-HOBt
in DMF (compounds 5–28). A 30 s deprotection cycle was followed
by a second deprotection for 3 min at 62 W and maximum temper-
ature of 75 °C.
Acetylation was performed by treatment of the resin with acetic
anhydride (20% in DMF) at room temperature (2 ꢁ 20 min), or by
using acetic anhydride (0.5 M in NMP), iPr2EtN (0.125 M in NMP)
and a catalytic quantity of HOBt in NMP (room temperature,
2 ꢁ 5 min).
A fritted glass reaction vessel was used for the manual synthesis
of 29 (0.25 mmol scale). The Fmoc protecting group was deprotec-
ted with 20% piperidine solution in DMF (1 ꢁ 5 min, 1 ꢁ 15 min).
The resin was washed with DMF (5 ꢁ 5 mL), and Fmoc-AA coupling
4. General methods
4.1. Chemistry
a
was performed. N -Fmoc-protected amino acid (4.0 equiv) was
4.1.1. Materials
dissolved in DMF, HCTU (3.8 equiv) was added and mixture shaken
until dissolved. The solution was transferred to the reaction vessel
and shaken for 2 min, followed by the addition of iPr2EtN
(8.0 equiv). The mixture was shaken for 45 min, filtered, and
washed with DMF (3 ꢁ 5 mL).
All reagents were purchased as reagent grade and used without
further purification. O-(Benzotriazol-1-yl)-N,N,N0,N00-tetramethyl-
uronium hexafluorophosphate (HBTU), O-(6-chlorobenzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HCTU),
4-[(R,S)-a-[1-(9H-floren-9-yl)]methoxycarbonylamino]-2,4-dime-
The resulting peptides were cleaved from the resin with simul-
taneous side chain protecting group removal by treatment with
TFA/iPr3SiH/H2O/DODT (v/v/v/v; 94/1/2.5/2.5), for 2 h at room
temperature. The crude peptides were precipitated and triturated
with cold diethyl ether, isolated (centrifugation), dissolved in 20%
acetonitrile (aq) containing 0.1% TFA and lyophilized.
thoxy]phenoxyacetic acid (Rink linker), and Fmoc-amino acids
were purchased from GL Biochem (Shanghai, China). Fmoc-amino
acids were supplied with the following side chain protection:
Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Arg(Pbf).
Piperazine, N,N-diisopropylethylamine (iPrNEt), N,N0-diisopropyl-
carbodiimide (DIC), 3,6-dioxa-1,8-octane-dithiol (DODT), formic
acid, 1-methyl-2-pyrrolidinone (NMP), 1,3-bis-(2,4,6-trimethyl-
phenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylm-
ethylene)ruthenium (Hoveyda–Grubbs’ II catalyst), and
triisopropylsilane (iPr3SiH) were purchased from Sigma–Aldrich
(Sydney, Australia). N,N-Dimethylformamide (DMF), acetonitrile
(ACN), and hydrochloric acid (HCl), were supplied from Scharlau
(Barcelona, Spain). Dichloromethane (CH2Cl2) was purchased from
ECP Limited. Trifluoroacetic acid (TFA) was purchased from Halo-
carbon (River Edge, New Jersey), 6-chloro-1-hydroxybenzotriazole
(6-Cl-HOBt) was purchased from Aapptec (Louisville, Kentucky),
and Fmoc-AA-OCH2PhOCH2CH2CO2H (Fmoc-AA-HMPP) were pur-
chased from PolyPeptide Group (Strasbourg, France). Dimethyl
sulfoxide (DMSO) was purchased from Romil Ltd (Cambridge,
United Kingdom). Aminomethyl polystyrene resin was synthesised
‘in house’ as previously described.26,27
Analytical reverse phase high-performance liquid chromatogra-
phy (RP-HPLC) was performed using either a Dionex P680 or Dio-
nex Ultimate U3000 system (flow rate of 1 mL/min), using
Waters XTerraÒ column (MS C18, 150 mm ꢁ 4.6 mm; 5
lm) using
gradient systems as indicated in the Supporting information.
The solvent system used was A (0.1% TFA in H2O) and B (0.1%
TFA in acetonitrile) with detection at 210 nm, 254 nm, and
280 nm. The ratio of products was determined by integration of
spectra recorded at 210 nm. A Hewlett Packard (HP) 1100MSD
mass spectrometer using ESI in the positive mode spectrometer
was used for ESI-MS analysis (positive mode). Peptide purification
was performed using a Waters 600E system using a semiprepara-
tive Phenomenex Gemini C18, 250 mm ꢁ 10 mm; 5
lm column or
m column. Gradient
Phenomenex Luna C8, 250 mm ꢁ 10 mm; 5
l
systems were adjusted according to the elution profiles and peak
profiles obtained from the analytical RP-HPLC chromatograms.
Fractions were collected, analysed by either RP-HPLC or ESI-MS,
pooled and lyophilised 3 times from 10 mM aq HCl.
4.1.2. Peptide synthesis, purification and analysis
For the synthesis of peptides affording a C-terminal acid (1, 2, 5,
6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26), AM-PS resin was initially
swollen in CH2Cl2 (30 min) and subsequently reacted with
Fmoc-AA-OCH2PhOCH2CH2CO2H (2.0 equiv), and DIC (2.0 equiv)
in CH2Cl2 (2.0 mL) for 2 h at room temperature.27 The Kaiser test
was negative.33 For the synthesis of peptides containing C-terminal
amide (3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 29), AM-PS
resin was initially swollen in DMF (30 min) and subsequently
reacted with Rink linker (5.0 equiv), DIC (5.0 equiv), and 6-Cl-HOBt
(5.0 equiv) in DMF (2.0 mL) for 2 h at room temperature.27 The Kai-
ser test was negative.33 Fmoc SPPS was then performed on a Liberty
1 Microwave Peptide Synthesiser (CEM Corporation, Mathews, NC)
on a 0.1 mmol scale using the Fmoc/tBu strategy. All amino acid
couplings were performed as single coupling cycles, with the
exception of Fmoc-Arg(Pbf) where a double coupling cycle was per-
formed as part of a synthetic protocol recommended by CEM
Microwave Technology. All Fmoc-AA couplings were performed
using Fmoc-AA (5.0 equiv, 0.2 M), HBTU (4.5 equiv, 0.45 M), and
iPrNEt (10 equiv, 2 M) in DMF, for 5 min, at 25 W and maximum
temperature of 75 °C, except Fmoc-Arg(Pbf) that was initially cou-
pled for 25 min at room temperature which was followed by the
second coupling for 3 min, at 25 W and maximum temperature of
4.2. Biology
4.2.1. Bone growth activity assays (proliferation)
Osteoblasts were isolated from 20-day fetal rat calvariae, as
previously described.28 Briefly, calvariae were excised and the
frontal and parietal bones, free of suture and periosteal tissue, were
collected. The calvariae were sequentially digested using collage-
nase (Sigma) and the cells from third and fourth digests were col-
lected, pooled and washed. Cells were grown in T75 flasks in 10%
FBS/Dulbecco’s modified eagle medium (DMEM)(Invitrogen) and
5
lg/mL
L-ascorbic acid 2-phosphate (Sigma) for 2 days and then
changed to 10% FBS/MEM (Invitrogen)/ 5
l
g/mL -ascorbic acid 2-
L
phosphate and grown to 90% confluency. Cells were then seeded
into 24 well plates at 2.5 ꢁ 104 cells/well in 5% FBS/MEM 5
lg/mL
L-ascorbic acid 2-phosphate for 24 h. Cells were growth-arrested
in 0.1% bovine serum albumin (BSA) (ICP, Auckland, New
Zealand)/5 lg/mL L-ascorbic acid 2-phosphate for 24 h. Cells
were pulsed with [3H] thymidine 6 h before the end of the exper-
imental incubation. The experiments were then terminated and
[3H]-thymidine incorporation assessed, as a measurement of
cell growth. Each of the analogues was screened at 3 different

3572
R. Kowalczyk et al. / Bioorg. Med. Chem. 22 (2014) 3565–3572
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Endocrinol. Metab. 2007, 292, E117.
concentrations. There were 6 wells in each group and each exper-
iment was repeated 3 or 4 times. All treatments were compared to
a vehicle control; data were analyzed using analysis of variance
with post-hoc Dunnett’s tests for significant main effect. A 5%
significance level was used throughout.
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4.2.2. Bone growth activity assays (differentiation)
Osteoblasts were isolated from 20 day old fetal rat calvariae, as
described above. Primary rat osteoblast cells were plated in 6-well
tissue culture dishes, at a density of 5 ꢁ 104 cells/well, in Minimum
Essential Medium Alpha Medium (
with 5 g/mL
fluent (approximately 3 days after plating), media were changed
to MEM/15% fetal bovine serum supplemented with 50 g/mL
-ascorbic acid-2-phosphate and 10 mM b-glycerophosphate, and
aMEM)/10% fetal bovine serum
l
L-ascorbic acid-2-phosphate. When cells were con-
a
l
L
test substances were added. These supplemented media were
changed twice weekly and test substances were replaced. After
21 days, the cells were fixed in 10% neutral buffered formalin,
rinsed with distiled water, and the cultures stained using Von Kos-
sa stain. Quantification of mineralized areas was performed using
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Acknowledgements
We thank the University of Auckland Thematic Research Initia-
tive, Biopharma Sector Development and Freemasons Roskill Foun-
dation Postdoctoral Fellowship for financial support of this work
(R.K.).
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Supplementary data
Supplementary data (Experimental data for the synthesis of
peptides 1–29, RP-HPLC and ESI-MS traces) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2014.05.026.
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References and notes
1. Hernlund, E.; Svedbom, A.; Ivergård, M.; Compston, J.; Cooper, C.; Stenmark, J.;
McCloskey, E. V.; Jönsson, B.; Kanis, J. A. Arch. Osteoporos. 2013, 8, 1.