Neoglycolipids for Prolonging the Effects of Peptides: Self-Assembling Glucagon-like Peptide 1 Analogues with Albumin Binding Properties and Potent in Vivo Efficacy

Article abstract of DOI:10.1021/acs.molpharmaceut.6b00787

Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides.

Full text of DOI:10.1021/acs.molpharmaceut.6b00787

Article
pubs.acs.org/molecularpharmaceutics
Neoglycolipids for Prolonging the Effects of Peptides: Self-
Assembling Glucagon-like Peptide 1 Analogues with Albumin
Binding Properties and Potent in Vivo Efficacy
Søren B. van Witteloostuijn,†,‡ Karin Mannerstedt,† Pernille Wismann,†,§ Esben M. Bech,†,‡
Mikkel B. Thygesen,‡ Niels Vrang,† Jacob Jelsing,† Knud J. Jensen,*,‡ and Søren L. Pedersen*,†
†Gubra ApS, Hørsholm Kongevej 11B, 2970 Hørsholm, Denmark
‡Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
§Department of Clinical Medicine, Faculty of Health Science, University of Copenhagen, Blegdamsvej 9, 2100 København Ø,
Denmark
S
* Supporting Information
ABSTRACT: Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full
pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like
peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity.
Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin
binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues
was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both
decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general
method for modulating the properties of therapeutic peptides.
KEYWORDS: neoglycolipid, lipidation, glucagon-like peptide 1, glycolipid, half-life extension, biopharmaceutical, peptide
binding and activation.4,5 One of the most widely adapted
chemical modifications of biopharmaceutical peptides and
proteins has been the introduction of large poly(ethylene
glycol) (PEG) groups, a process that is referred to as
INTRODUCTION
■
During the past 30 years, biopharmaceuticals have become
increasingly important for the treatment of a variety of diseases,
including several cancer forms, inflammatory disease, diabetes,
PEGylation. Conjugation of large PEG polymers increases the
and obesity. The combined annual revenue of the more than
hydrodynamic radius of the modified peptide or protein,6
thereby rendering it less susceptible to renal clearance and
more resistant toward protease degradation.7,8 Despite the
commercial success of PEGylated biopharmaceuticals, the
potential safety concerns associated with the unnatural PEG
polymers remain a matter of debate.9,10 Moreover, most large
PEGs are not monodisperse, leading to polydisperse PEGylated
proteins. Finally, PEGylation significantly increases the
200 approved drug products has reached 140 billion USD.1
Due to their endogenous origin, biopharmaceuticals are usually
highly selective and potent compounds. However, since they
are based on native polypeptides, they suffer from rapid
proteolytic degradation and, in the case of peptides and smaller
proteins, susceptibility to renal clearance. This often results in
very short in vivo half-lives that are incompatible with a
meaningful dosing regimen.
To compensate for poor pharmacokinetic properties, a
variety of half-life extension techniques have been developed.2,3
Peptides and to a lesser extent proteins have been chemically
modified at their side-chains to introduce groups that modify
their pharmacokinetic properties without inhibiting receptor
Received: August 25, 2016
Revised: November 4, 2016
Accepted: December 1, 2016
Published: December 1, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.molpharmaceut.6b00787
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Figure 1. Synthesis of Fmoc-Muc-OH.
molecular mass of the target biopharmaceutical, which may
complicate formulation of high doses suitable for subcutaneous
(sc) administration.
and appetite.25 The physiology of GLP-1 makes this peptide a
very important target for the treatment of metabolic syndrome,
and several GLP-1 analogues are approved or in clinical
development for the treatment of type 2 diabetes as well as
obesity.26,27
We present the design and synthesis of novel neoglycolipids
based on an open-chain sugar amino acid as well as the
synthesis of a library of neoglycolipidated GLP-1 analogues. We
show that neoglycolipidation directs self-assembly of the
peptide conjugate and mediates binding to HSA. Compared
to native GLP-1, the neoglycolipidated GLP-1 analogues had
the same or even improved in vitro potency. This translates
into superior in vivo efficacy with respect to reducing acute
food intake and improving glucose homeostasis in lean mice.
Another technology that enhances the pharmacokinetic
properties of peptides and proteins is lipidation with long-
chain fatty acids. This approach has successfully been used by
Novo Nordisk in two long-acting insulins (insulin detemir and
insulin degludec) and two glucagon-like peptide 1 (GLP-1)
analogues (liraglutide and semaglutide).11−14 Half-life extension
arises from noncovalent anchoring of peptides and proteins to
the fatty acid binding sites on human serum albumin
(HSA).15−17 When bound to HSA, renal clearance is reduced
and proteolytic degradation is minimized due to steric effects.
Moreover, introduction of fatty acids can induce self-assembly
of the modified polypeptides,11,12,18 thereby effectively forming
an in vivo sc depot resulting in delayed absorption. However,
lipidation increases the overall lipophilicity of the native
polypeptide, and this may lead to solubility issues, especially
for very long fatty acids such as stearic acid and arachidic acid.
Incorporation of a hydrophilic spacer between the lipid and the
polypeptide has been used to compensate for the overall
decrease in water solubility. γ-L-Glutamic acid (γGlu) has been
used extensively and successfully for this purpose, both
alone12,13,19−23 and in combination with the short PEG-type
spacer 8-amino-3,6-dioxaoctanoic acid (O2Oc) such as in
semaglutide.14
We hypothesized that carbohydrates inserted between the
peptide side chain and the lipid moiety could modulate the
physicochemical properties of biopharmaceuticals in novel
ways. Carbohydrates are often hydrophilic due to the number
of hydroxyl groups, they have a large structural diversity, their
chemistry is very well-described, and many carbohydrates are
easily available. In general, short carbohydrates (mono- and
oligosaccharides) show a low degree of immunogenicity. Some
carbohydrates are used as excipients to prevent aggregation and
to increase the solubility of polypeptides. Furthermore,
glycolipids such as octyl β-D-glucopyranoside are widely used
detergents for the solubilization of integral membrane
proteins.24
A lipid−carbohydrate−peptide conjugate can also be viewed
as a peptide modified by a glycolipid; as a non-natural
glycolipid it is termed a neoglycolipid. We hypothesized that
covalently anchored neoglycolipids could constitute a new class
of half-life extending moieties for peptides and proteins,
allowing binding to albumin and directing oligomerization in
the nanoscale in the absence of albumin. Here, we focused on
open-chain monosaccharide derivatives.
To validate the effects of neoglycolipidation in a
biopharmaceutical context, we chose GLP-1 as the first target.
GLP-1 is a peptide hormone secreted from the gut in response
to food ingestion. In the periphery, GLP-1 actions include
amplification of pancreatic insulin secretion (the “incretin
effect”), inhibition of glucagon secretion, stimulation of beta
cell proliferation, and inhibition of gastric acid secretion,
intestinal motility, and gastric emptying (the “enterogastrone
effect”). In addition, GLP-1 acts centrally to inhibit food intake
RESULTS
■
Design and Synthesis of Neoglycolipidated GLP-1
Analogues. In the neoglycolipid design, we focused on
optimizing in vitro potency and physicochemical properties of
the target peptide while maintaining albumin binding and
ensuring in vivo efficacy. As glycoconstituent, we chose mucic
acid (1), a commercially available aldaric acid obtained by
oxidation of galactose. To incorporate mucic acid into peptides
by established solid-phase methods, the aldaric acid was
converted into an amino acid building block amenable to
Fmoc-based solid-phase peptide synthesis (SPPS). The first
step was protection of the hydroxyl groups of mucic acid by
acid-catalyzed acetylation to yield the corresponding tetraace-
tate (2) (Figure 1). The second step was incorporation of
Fmoc-protected ethylenediamine (Fmoc-EDA) to provide an
Fmoc-protected amino functionality. We first explored direct
coupling of Fmoc-EDA and mucic acid tetraacetate using
standard reagents for amide bond formation, but generation of
significant amounts of the disubstituted byproduct made this
approach unattractive. Instead, we synthesized the acid
chloride, which upon amidation with Fmoc-EDA provided
the monosubstituted sugar amino acid (3) (named Fmoc-Muc-
OH) in acceptable yield (Figure 1). This carbohydrate-based
amino acid is directly compatible with Fmoc-SPPS and can be
anchored to N-terminal or side-chain peptide amino groups by
an amide bond. Moreover, multiple Muc units can be coupled
sequentially, thereby providing neoglycolipids with carbohy-
drate moieties of different size.
The commercially available fatty acids palmitic acid (C16),
stearic acid (C18), and arachidic acid (C20) were used to
synthesize neoglycolipids containing fatty acids of different
lengths. Fatty acids are directly compatible with Fmoc-SPPS
and may be readily incorporated into peptides using standard
solid-phase conditions.
The target peptide of choice, GLP-1, circulates in two forms:
an amidated variant known as GLP-1(7−36)-NH2 (4) and a
Gly-extended variant termed GLP-1(7−37)-OH, with the
amidated form being predominant in humans.25 A slightly
modified form of GLP-1(7−37)-OH, termed [Arg34]GLP-
1(7−37)-OH (5), constitutes the peptide backbone of the well-
characterized GLP-1 analogue liraglutide (7), which in addition
B
DOI: 10.1021/acs.molpharmaceut.6b00787
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Figure 2. Architecture of neoglycolipidated peptides exemplified by compound 8 (neoglycolipid variant: C16-γGlu-Muc-). The lipid constituent is
marked in blue, γGlu is marked in red, the glyco constituent is marked in green, and the peptide backbone is marked in black (with Lys26 expanded
for clarity).
to the K34R mutation is modified at the side chain of Lys26
with a γGlu-spaced palmitic acid (C16-γGlu-).13 Lys26 in
peptide 5 was identified as an optimal lipidation site during the
development of liraglutide (7).28 Thus, we used this position
for neoglycolipidation. In addition to the carbohydrate and lipid
moieties, a γGlu unit was also included in the neoglycolipid
design, since the presence of this amino acid is known to have
positive effects on albumin binding. Importantly, this allowed
direct comparison with liraglutide (7), when studying the
impact of neoglycolipidation on peptide properties.
A series of neoglycolipidated peptides was prepared by
Fmoc-SPPS. C-terminal peptide acids were obtained by SPPS
on a TentaGel resin with a 4-alkoxybenzyl alcohol (PHB)
linker and the first Gly anchored as the ester. Site-specific
lipidation at the side chain of Lys26 was achieved by selective
removal of the orthogonal Alloc protecting group using
(PPh3)4Pd(0), followed by on-resin assembly of the neo-
glycolipids using standard coupling reagents. Neoglycolipidated
peptides containing acetyl-protected carbohydrate moieties
were released from the solid support, purified by preparative
RP-HPLC, and lyophilized. The acetyl protecting groups were
subsequently removed in solution using NaOCH3 in CH3OH,
and the final products were obtained by a second round of
preparative RP-HPLC (Figures 2, S1). All peptides were
analyzed by LC−MS. In total, six neoglycolipidated peptides
(compounds 8−13) were prepared (Table 1). In addition, the
des-γGlu version of liraglutide (6) and a version containing
O2Oc as part of the spacer (14) were synthesized as reference
compounds.
Table 1. Glycolipid Variant and Functional Screening Data
for the Library of Neoglycolipidated GLP-1 Analogues
a
EC50 (pM) GLP-
compd no.
glycolipid variant
n/a
1R
4 (GLP-1(7−36)-NH2)
5 ([Arg34]GLP-1(7−37)-OH) n/a
41.2 3.9
64.1 4.5
67.8 7.6
63.6 6.4
21.8 2.5
44.1 2.0
118.8 28.2
13.4 1.8
16.8 2.3
41.2 4.2
35.9 3.7
6
C16-
7 (liraglutide)
C16-γGlu-
8
C16-γGlu-Muc-
C18-γGlu-Muc-
C20-γGlu-Muc-
C16-γGlu-Muc-Muc-
C18-γGlu-Muc-Muc-
C20-γGlu-Muc-Muc-
C16-γGlu-O2Oc-
9
10
11
12
13
14
a
All lipidated and neoglycolipidated peptides were functionalized at
position Lys26 (bold) of [Arg34]GLP-1(7-37)-OH, H-HAEGTFTS-
DVSSYLEGQAAKEFIAWLVRGRG-OH. EC50 values are presented
as mean SEM derived from three independent experiments carried
out in triplicate (n = 3).
(C20-γGlu-Muc-, EC50 = 118.8
28.2 pM), all neo-
glycolipidated peptides were equipotent with or even more
potent than liraglutide (7, C16-γGlu-, EC50 = 63.6 6.4 pM),
suggesting that inclusion of an extra carbohydrate moiety as
part of the spacer gave optimal interaction between peptide and
receptor. A comparison of 8 (C16-γGlu-Muc-, EC50 = 21.8
2.5 pM) and 14 (C16-γGlu-O2Oc-, EC50 = 35.9 3.7 pM)
suggests that our carbohydrate-based spacer compared
favorably with the O2Oc moiety in terms of maintaining
compound potency.
Physicochemical Properties. Peptide Lipophilicity. In a
first attempt to assess the impact of neoglycolipidation on
peptide properties, the interaction between the neoglycolipi-
dated peptides and an analytical C18 column was studied by
UPLC. The presence of organic solvents ensured that the
peptides were evaluated on their monomeric form. Column
retention times (tR) for compounds 5−14 are listed in Table 2.
As expected, the interaction between the neoglycolipidated
peptides and the column material is primarily governed by the
length of the fatty acid, with longer fatty acids providing a
longer retention time (as exemplified by compounds 8−10 and
11−13, respectively). The pronounced difference in retention
time for 6 (C16, tR = 12.3 min) and liraglutide (7, C16-γGlu-,
tR = 7.7 min) illustrates the contribution of the γGlu spacer to
peptide lipophilicity. Inclusion of one Muc unit provides an
Functional Screening. To assess the impact of neo-
glycolipidation on functional activity, the in vitro potency of all
compounds was investigated using a human embryonic kidney
(HEK293) cell line expressing the EPAC cAMP-sensor and the
GLP-1 receptor (GLP-1R). The dose−response curves of all
compounds are shown in Figure 3, and the derived EC50 values
are listed in Table 1. All compounds were full GLP-1R agonists,
with EC50 values in the picomolar range. The most potent
compound was 11 (C16-γGlu-Muc-Muc-, EC50 = 13.4 1.8
pM), with potency gradually declining for 12 (C18-γGlu-Muc-
Muc-, EC50 = 16.8 2.3 pM) and 13 (C20-γGlu-Muc-Muc-,
EC50 = 41.2
4.2 pM). The same trend was observed for
compounds 8−10, suggesting that increasing fatty acid length
moderately interferes with receptor activation, possibly due to
steric effects. Moreover, compounds 11−13 were generally
more potent than compounds 8−10, suggesting that increasing
the distance between the peptide backbone and the lipid moiety
was beneficial for receptor activation. With the exception of 10
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Figure 3. In vitro dose−response relationship of compounds 4−14 on GLP-1R. Functional activity of all compounds was investigated using a
HEK293 cell line expressing the EPAC cAMP-sensor and the GLP-1R. (A) Compounds 4−6, liraglutide (7), and 14. (B) Compounds 8−10. (C)
Compounds 11−13. Data are presented as mean SEM of three independent experiments carried out in triplicate (n = 3).
a
dissolved in PBS (pH 7.4) until a saturated solution was
obtained. The samples were centrifuged, and the absorbance at
280 nm was measured and used to calculate the peptide
content of the supernatant, which was used as a measure of
maximal solubility (Table 2). The low solubility of 5 (1.4 mg
mL−1) indicates that the unmodified peptide backbone has a
low intrinsic solubility. A slight decrease in solubility is
observed upon incorporation of palmitic acid (6, C16-, 1.2
mg mL−1), whereas inclusion of a γGlu spacer potently
improves solubility (liraglutide, 7, C16-γGlu-, 2.6 mg mL−1).
Inclusion of Muc or O2Oc did not increase solubility beyond
2.6 mg mL−1.
Size Exclusion Chromatography. As molecular self-
assembly may affect solubility properties, and since it has
been reported that some lipidated peptides form soluble
oligomers, the oligomeric states of the neoglycolipidated
peptides at concentrations of maximal solubility were
investigated. The apparent molecular weights of compounds
5−14 were determined by size exclusion chromatography
(SEC) using a Superdex 75 column. Figure 4 depicts the
retention volumes (VR) of compounds 5−14, and the derived
apparent molecular weights and oligomeric states (m) are listed
in Table 3. Rewardingly, with the exception of 5 and 6, all
compounds eluted as single peaks, indicating the presence of
uniform particle sizes. The retention volumes of the two peaks
observed for 5 indicated the presence of two species with
apparent molecular weights of 1.9 and 5.3 kDa, respectively
(corresponding to m = 0.6 and m = 1.6, respectively), indicating
a monomer−dimer distribution. The single peaks observed for
compounds liraglutide (7) and 8−14 all have retention
Table 2. Physicochemical Properties of Compounds 5−14
tR
max. solubility
compd no.
glycolipid variant
n/a
C16-
(min)
(mg mL−1
)
5
6
2.7
12.3
7.7
1.4
1.2
2.6
2.6
1.6
2.6
nd
7 (liraglutide) C16-γGlu-
8
C16-γGlu-Muc-
6.1
9
C18-γGlu-Muc-
10.3
21.8
5.9
10
11
12
13
14
C20-γGlu-Muc-
C16-γGlu-Muc-Muc-
C18-γGlu-Muc-Muc-
C20-γGlu-Muc-Muc-
C16-γGlu-O2Oc-
10.4
21.8
7.0
1.1
1.0
2.4
a
Estimate of hydrophilicity is provided as the maximal solubility in
PBS (pH 7.4). Estimate of hydrophobicity is provided as retention
time following isocratic elution from an analytical C18 column.
additional decrease in retention time (8, C16-γGlu-Muc-, tR =
6.1 min), whereas incorporation of a second Muc unit seems to
have almost no additional effect (11, C16-γGlu-Muc-Muc-, tR =
5.9 min). A comparison of 8 and 14 (C16-γGlu-O2Oc-, tR = 7.0
min) suggests that the O2Oc moiety is more lipophilic than
Muc, emphasizing the hydrophilic nature of the carbohydrate-
based building block. The very short retention time of 5 (tR =
2.7 min) may be attributed to a very low lipophilicity of the
peptide backbone itself.
Peptide Solubility. The effect of neoglycolipidation on
peptide solubility was estimated by the maximal solubility of
compounds 5−14 under aqueous conditions. Compounds were
Figure 4. Estimation of apparent molecular weight of compounds 5−14 by SEC. VR of MW standards is indicated by vertical dashed lines. (A)
Compounds 5, 6, liraglutide (7), and 14. (B) Compounds 8−10. (C) Compounds 11−13.
D
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Table 3. Retention Volume, Apparent Molecular Weight, and Oligomeric State of Compounds 5−14 and Hydrodynamic Radius
a
and Polydispersity Index of Compounds 7−10
SEC
DLS
compd no.
glycolipid variant
n/a
VR (mL)
app MW (kDa)
1.9/5.3
m
rH (nm)
nd
PI
5
6
16.4/14.0
8.0/6.1
9.1
0.6/1.6
b
nd
nd
b
C16-
nd
nd
nd
7 (liraglutide)
C16-γGlu-
43.4
43.4
47.2
53.7
11.6
10.9
11.8
13.3
8.7
9.6 0.56
7.7 0.17
23.2 1.5
7.8 0.28
nd
0.21 0.01
8
C16-γGlu-Muc-
C18-γGlu-Muc-
C20-γGlu-Muc-
C16-γGlu-Muc-Muc-
C18-γGlu-Muc-Muc-
C20-γGlu-Muc-Muc-
C16-γGlu-O2Oc-
9.1
0.29 0.02
9
8.9
0.20 0.008
10
11
12
13
14
8.6
0.24 0.008
b
c
9.5
36.5
nd
nd
nd
nd
8.9
8.7
9.4
47.2
53.7
38.1
11.1
12.6
9.8
nd
nd
nd
a
Apparent molecular weight and oligomeric state were determined by SEC using a linear calibration curve, whereas hydrodynamic radius was
determined by DLS and reported as the value derived from the data fit generated by second-order cumulant analysis. The values of hydrodynamic
b
c
radius and the polydispersity index are presented as mean SEM, n = 4−8. Peaks eluted outside the range of the calibration curve. Sample
concentration was below maximal solubility.
Figure 5. Sensorgrams showing interaction between immobilized HSA and compounds 5 and 7−14. Analytes were injected for 150 s, followed by
injection of running buffer for 180 s. Data are presented as mean SEM of triplicate injections from a single representative experiment. Data fits by
nonlinear regression are depicted as dotted lines.
volumes of 8.6−9.5 mL, corresponding to apparent molecular
weights of approximately 35−55 kDa (m = 8.7−13.3),
suggesting that these compounds assemble into oligomeric
structures containing between 9 and 13 monomer subunits,
respectively. Interestingly, compared to lipidation with the C16-
γGlu- motif, neoglycolipidation seems to direct formation of
slightly smaller oligomers, as indicated by, e.g., the oligomeric
state of liraglutide (7), 8, and 11 (m = 11.16, m = 10.9, and m =
8.7, respectively). In addition, increasing fatty acid length
results in formation of larger oligomeric structures, as evident
by the oligomeric state of, e.g., compounds 11−13 (Figure 4C,
m = 8.7 for C16, m = 11.1 for C18, and m = 12.6 for C20,
respectively).
E
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Dynamic Light Scattering. In addition to the apparent
molecular weight derived from SEC, the hydrodynamic radius
(rH) was determined for liraglutide (7) and a subset of the
neoglycolipidated peptides (compounds 8−10) by dynamic
light scattering (DLS) (Table 3). For all four compounds, data
fitting using a regularization algorithm indicated the presence of
monodisperse solutions (data not shown), allowing rH to be
determined using second-order cumulant analysis. All com-
pounds exhibited narrow size distributions, as indicated by low
polydispersity indexes (PI). The reported values of rH are
consistent with oligomer formation.29 The similar hydro-
dynamic radii of liraglutide (7), 8, and 10 (rH = 9.6 nm, rH =
7.7 nm, and rH = 7.8 nm, respectively) reflects the similarity of
the oligomeric states (m = 11.6, m = 10.9, and m = 13.3,
respectively) derived from SEC. The significantly greater
hydrodynamic radius of 9 (rH = 23.2 nm) may be attributed
to the presence of a small amount of very large oligomers,
which are detectable only by DLS and not by SEC.
Albumin Binding. In order to estimate the albumin binding
propensity of the neoglycolipidated peptides, we investigated
the interaction between immobilized HSA and neoglycolipi-
dated peptides in solution using surface plasmon resonance
(SPR) based detection on a Biacore X100 instrument. HSA was
immobilized on Biacore CM5 chips, and analyte solutions
containing peptides 5 and 7−14 were passed over the chip
surface. Under conditions of total internal reflection, the
changes in SPR response were measured during association and
dissociation of the analytes. Figure 5 shows representative
sensorgrams obtained for compounds 5 and 7−14. Under the
chosen assay conditions, all peptides exhibited reversible
albumin binding, as indicated by the increase and decrease in
response units (RU) during sample and buffer injections,
respectively (Figure 5). Peptide 5 displayed very rapid
association and dissociation kinetics with a calculated
equilibrium dissociation constant, KD, of 28.5 μM, suggesting
that the unmodified peptide backbone interacts with HSA in a
highly dynamic manner. Peptides 7−14 also displayed fast
association kinetics. However, the dissociation rates for these
compounds were much lower than that of 5, indicative of a
vastly enhanced affinity toward albumin. Saturation of the chip
surface for these compounds was apparent at 25 μM or in some
cases even at 5 μM. At analyte concentrations of 1 μM or lower,
the association phases of compounds 7−14 displayed delayed,
sigmoidal onsets of surface interaction, thus precluding
straightforward analyses at low analyte concentrations (Figure
5). Variations in flow rate or level of immobilized HSA did not
improve the data (data not shown). Due to the inadequate
binding curves obtained at 1 μM, binding analyses had to be
based on analyte concentration of 5, 25, and 50 μM, at or close
to saturating conditions. Association kinetics at these analyte
concentrations were too fast for proper estimation of the
corresponding association rate constants, ka, likely leading to
gross underestimation. On the other hand, saturating
conditions should not affect the dissociation rate constants,
kd. The release of analyte from the immobilized ligand, i.e.,
albumin, in the dissociation phase is in most kinetic models
described by a simple monoexponential function. Thus, we
found that the kd values could be used as a simple measure for
evaluating albumin binding of neoglycolipidated peptides.
Table 4 lists the observed saturation response levels (Rmax) as
well as calculated kd values for compounds 5 and 7−14. The
values of ka,app and KD,app are reported in Table S1.
Table 4. Saturation Response Levels (Rmax) and Dissociation
Rate Constants (kd) Obtained for the Interaction of
Neoglycolipidated Peptides with Immobilized HSA
a
compd no.
glycolipid variant
n/a
Rmax (RU)
kd (s−1
)
5
nd
0.1664 0.0024
0.0056 0.00040
0.0094 0.0027
0.0024 0.00035
0.0019 0.00015
0.0081 0.0018
0.0020 0.000014
0.00089 0.00062
0.026 0.0005
7 (liraglutide)
C16-γGlu-
250
400
320
150
400
300
120
260
8
C16-γGlu-Muc-
C18-γGlu-Muc-
C20-γGlu-Muc-
C16-γGlu-Muc-Muc-
C18-γGlu-Muc-Muc-
C20-γGlu-Muc-Muc-
C16-γGlu-O2Oc-
9
10
11
12
13
14
a
Data are presented as mean SEM of triplicate injections of two
independent measurements (n = 2) conducted on the same chip.
In Vivo Pharmacology. Acute Effect on Glucose
Tolerance. Since one of the most important pharmacological
effects of GLP-1 analogues is their ability to decrease blood
glucose levels and improve glycemic control, we evaluated the
glucose lowering effects of compounds 8−10 in lean mice
during an oral glucose tolerance test (OGTT) using liraglutide
(7) as positive control (Figure 6). Compounds were injected
45 min prior to the oral glucose load (t = 0), and blood glucose
levels were measured at 0, 30, 60, 90, and 120 min after
administration of the glucose load. Administration of neo-
glycolipidated peptides 8−10 improved glucose tolerance by
50−62% (assessed as total AUC during OGTT, p < 0.001 vs
vehicle), while administration of liraglutide (7) provided a 54%
improvement (p < 0.001 vs vehicle).
Acute Effect on Food Intake. In addition to the beneficial
effects on glucose homeostasis, administration of liraglutide and
other GLP-1 analogues is also known to decrease food intake in
rodents as well as humans. As such, we also investigated the
anorectic effect of compounds 8, 9, 11, and 12 in lean mice
(Figure 7). Compounds were administered as a single dose, and
food intake was measured for 18 h postdosing using a fully
automated HerdsMan-2 (HM-2) food intake monitoring
system, which enables advanced synchronous real-time
monitoring of the feeding behavior of individual animals.
Administration of neoglycolipidated peptides 8, 9, 11, and 12
potently reduced cumulative food intake by 47−55% (p < 0.001
versus vehicle), whereas liraglutide (7) decreased food intake
by 45% (p < 0.001 versus vehicle).
DISCUSSION
■
In the present study, we describe the development and
characterization of neoglycolipidated GLP-1 analogues. The
neoglycolipidation motif was designed to modulate peptide
properties by inducing molecular self-assembly and prolonging
plasma half-life via noncovalent albumin binding, while
minimizing the decrease in solubility that is often observed
with current lipidation strategies. Rewardingly, neoglycolipida-
tion of GLP-1 occurred without compromising bioactivity, as
the in vitro potency and in vivo efficacy were sustained.
Importantly, neoglycolipidation decreased lipophilicity and
provided noncovalent oligomerization. Neoglycolipidation
represents a novel principle for directing self-assembly of
therapeutic peptides. This feature is significant, since self-
assembly has been suggested as one of the main reasons for the
delayed absorption of liraglutide and insulin degludec and is
F
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Figure 6. Evaluation of the glucose lowering potential of liraglutide (7) and compounds 8−10 in an OGTT. Compounds were administered (sc 50
nmol kg−1) at t = −45 min, and glucose was dosed (per oral, 2 g/kg) at t = 0. Mice were semifasted for 5 h before the glucose load. (A) Glucose
time−response curve (−45 to 120 min). (B) Area under the curve (AUC0−120). Data presented as mean SEM, n = 7. Statistical analysis: one-way
ANOVA with Bonferroni’s post test against vehicle (***p < 0.001 versus vehicle).
Figure 7. Cumulated food intake following acute single-dose administration (sc, 50 nmol kg−1) of liraglutide (7) and compounds 8, 9, 11, and 12 to
lean mice. Food intake was monitored for 18 h postdosing using a fully automated HM-2 system. For each group, data was obtained from two
independent studies and pooled to increase statistical power. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test (***p <
0.001 versus vehicle). Data presented as mean SEM, n = 8−17.
recognized as an important contributor to the prolonged half-
life observed with these lipidated biopharmaceuticals.12,18
In a first attempt to evaluate the impact of neoglycolipidation
on the physicochemical properties of peptides, the lipophilicity
of the neoglycolipidated peptides was estimated. Although this
parameter is primarily influenced by fatty acid length,
incorporation of a carbohydrate-based amino acid did provide
a decrease in lipophilicity, in terms of a shorter column
retention time. In contrast to our initial expectations, this
decrease in lipophilicity did not translate into improved
solubility, as the inclusion of a carbohydrate moiety in the
spacer did not increase the maximal aqueous solubility beyond
that of liraglutide (7). The exact reason for this remains
unknown, but the negative charge of the γGlu unit is known to
contribute significantly to the solubility of other lipidated
peptides.20,23,28 As alternative to Muc, a carbohydrate moiety
derived from e.g. glucuronic acid or sialic acid can be
envisioned. Both of these species carry negative charges, and
this may improve solubility even further.
nature of the neoglycolipidated peptides makes it likely that
they self-assemble into micelle-like structures; however, this has
not yet been studied. A similar assembly has been suggested for
liraglutide oligomers.29
The oligomeric state of liraglutide has been determined using
complementary techniques. Using analytical ultracentrifugation,
Steensgaard et al. showed that at pH 8.0 liraglutide assembles
into an aggregate with molecular weight of 25.9 kDa,
corresponding to a heptameric structure (m = 6.9). Heptamer
formation was observed in the concentration range of 0.001−
1.2 mM, suggesting that the critical micelle concentration
(CMC) is ≤1 μM.18 Wang et al. used a combination of DLS
and static light scattering (SLS) to investigate liraglutide self-
assembly as a function of pH. In 20 mM sodium phosphate
buffer at pH 7.4, DLS revealed that liraglutide undergoes
oligomer formation in a well-defined manner, as indicated by a
narrow size distribution. The rH of the oligomers was 2.8 0.1
nm. Using SLS, the molecular weight of the oligomers was
calculated to 30 2 kDa, which corresponds to an octamer (m
= 8.0 0.4).29 SEC revealed a well-defined oligomerization of
liraglutide, as indicated by the presence of a single narrow peak.
Using a calibration curve derived from well-defined
molecular weight standards, we calculated the size of the
liraglutide oligomers to 43.4 kDa, corresponding to a 12-mer
(m = 11.6) at pH 7.4. The reason for the discrepancy between
this value and previously reported values has not yet been fully
Another important solubility driver is the ability of lipidated
peptides to undergo self-assembly and form oligomers that
remain in solution. This has previously been shown for both
insulin degludec12 and liraglutide.18 Using SEC, we demon-
strate the ability of neoglycolipidated peptides to form soluble
oligomers in a size range of 35−55 kDa, corresponding to
oligomers containing 9−13 peptide subunits. The amphiphilic
G
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clarified, but Wang et al. showed that the degree of
oligomerization is strongly dependent on the pH value of the
surrounding buffer (with m increasing from 8.0 0.4 at pH 7.4
to 12.0 0.6 at pH 6.7),29 indicating that minute differences in
the chemical environment of the different experimental setups
could account for the different observations. This notion is
supported by biophysical studies conducted by Clodfelter et al.
on an octanoylated GLP-1 analogue, which illustrated that the
oligomerization process was strongly solvent, temperature, and
time dependent.30
Although albumin binding via lipidation is a well-established
method for prolonging peptide half-life in vivo, a robust in vitro
assay able to directly measure albumin affinity of lipidated
peptides in a screening setup has proven difficult to develop.
Kurtzhals et al. investigated albumin binding of insulin detemir
and other lipidated insulin analogues using an ultrafiltration-
based assay with albumin immobilized to an agarose matrix,15
and Plum et al. developed an optimized equilibrium dialysis
method and used it to determine the free fraction of liraglutide
in the presence of albumin.31 However, the requirement for
radiolabeled ligands for both of these assays makes them less
feasible for screening purposes. In a recent publication, Lau et
al. estimated albumin binding of lipidated GLP-1 analogues by
measuring binding to the GLP-1 receptor in the presence of
albumin, using the ratio of IC50 values obtained with 0% and
2% HSA, respectively, as a surrogate of albumin affinity.14 This
indirect assay is more appropriate for a screening setup, but is
not suitable for ligands with a strong affinity for both albumin
and the corresponding receptor.
In contrast, SPR-based techniques such as Biacore require no
labeling and provide a direct measure of the interaction
between ligand and albumin. A drawback associated with this
method is the need for immobilization of albumin, which may
cause inadvertent steric occlusion of binding sites. However,
this effect is compensated by the fact that immobilization is
performed randomly at surface-exposed lysine residues, and
that albumin contains multiple fatty acid binding sites. Using a
Biacore assay for albumin binding, we observed reversible
association and dissociation phases for liraglutide (7) and
peptides 8−14 (Figure 5). However, binding analyses were
complicated by apparent sigmoidal association curves at low
analyte concentrations, as well as rapid onsets of binding
saturation, thus precluding straightforward determination of
binding constants (KD). This could be due to an unspecific
component in the binding interactions, in which some lipidated
peptides may be irreversibly bound to surfaces in the
instrument. Nonetheless, the albumin binding propensity of
the neoglycolipidated peptides could be evaluated from the
determined values of kd and Rmax at the higher concentrations.
Clearly, the most important factor governing albumin
binding kinetics is the length of the fatty acid moieties. This
may be observed directly from the sensorgrams, e.g., by
comparison of Figures 5D−F, and is also reflected in the
reported kd values, indicating stronger albumin affinities for
peptides containing longer fatty acids (Table 4). Interestingly, it
seems that increasing fatty acid length decreases not only
dissociation rates but also the corresponding association rates,
suggesting that the length of the fatty acid impacts both the way
the analyte approaches and binds the immobilized HSA and its
subsequent release from HSA.
that the neoglycolipidated peptides undergo self-assembly, a
possible explanation could be that peptide oligomers are
present in the samples, resulting in a competing monomer−
oligomer equilibrium during the association phase that
apparently secludes the interaction between neoglycolipidated
peptides and immobilized albumin.
Interestingly, the observed Rmax values correlate closely with
the determined oligomeric state of compounds liraglutide (7)
and 8−14, given in Table 3. For example, the saturation
response of compound 13, carrying a C20 fatty acid and
displaying an oligomeric state of 12.0, amounted to only half
that of compound 11 carrying a C16 fatty acid with an
oligomeric state of 8.4 (Figures 5I and 5G, respectively).
Importantly, neoglycolipidated peptides carrying one or two
Muc units in the spacer, e.g., compounds 8 and 11, respectively,
provided a more pronounced interaction with albumin, as
judged by the higher Rmax relative to liraglutide (7), while
maintaining similar dissociation rate constants. While the
additional Muc unit in compound 11 relative to 8 seems to
have little effect on the binding kinetics (Figure 5G versus 5D),
it is interesting that the hydrophilic nature of the Muc unit
relative to the oligoethylene glycol O2Oc moiety of compound
14 is responsible for the enhancement of albumin interaction.
Functional screening of the neoglycolipidated GLP-1
analogues revealed that all compounds were full GLP-1R
agonists and had maintained potency in the picomolar range,
suggesting that neoglycolipidation did not interfere with
receptor activation. Although lipidation of GLP-1 at Lys26 is
known to be well-tolerated, the molecular composition of the
spacer must be carefully designed to avoid disrupting the
receptor interaction.13,14,28 Rewardingly, inclusion of a mucic
acid based moiety in the neoglycolipid design did not harm
GLP-1R agonism, and the maintained in vitro potency
translated into pronounced in vivo efficacy, as indicated by
the improvement of glucose tolerance and potent reduction of
food intake following acute administration of neoglycolipidated
GLP-1 analogues. This validated our choice of Lys26 for
lipidation.
The data on acute food intake (Figure 7) clearly demonstrate
that the duration of action of compounds 8, 9, 11, and 12
equals that of liraglutide (7), suggesting that the half-life of the
neoglycolipidated GLP-1 analogues resembles that of liraglutide
and is compatible with once-daily dosing in man. Thus, the
desired extension of the function of neoglycolipidated peptides
was achieved and we did not determine the PK profile of the
neoglycolipidated peptides separately.
CONCLUSION
■
In summary, we have described the design of a novel
neoglycolipid motif based on an open-chain sugar amino acid
and the synthesis of an ensemble of neoglycolipidated GLP-1
analogues. Neoglycolipidation reduces peptide lipophilicity,
maintains solubility, and mediates albumin binding. Introduc-
tion of the neoglycolipid motif directs formation of soluble
peptide oligomers of variable size, depending on the chemical
composition of the neoglycolipid. Neoglycolipidation of GLP-1
resulted in maintained or even improved in vitro potency
compared to native GLP-1, which translated into potent in vivo
efficacy. Acute administration of neoglycolipidated GLP-1
analogues improved glucose tolerance in lean mice, and the
neoglycolipidated peptides exhibited a duration of action
similar to that of liraglutide in terms of reducing food intake.
Surprisingly, we did not observe similar values of Rmax for
neoglycolipidated peptides with approximately identical molec-
ular mass, for example, for compounds 8−10 or 11−13. Given
H
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We suggest neoglycolipidation as a novel principle for
modulating the properties of therapeutic peptides.
flask followed by heating at 60 °C for 3 h. After the reaction
mixture was cooled to room temperature, H2O (20 mL) was
added and the mixture was stirred at 0 °C for 1 h. The product
was filtered, washed with H2O (20 mL), and dried to give a
EXPERIMENTAL SECTION
■
1
white solid (6.80 g, 17.99 mmol, 76%): H NMR (300 MHz,
General Procedures. Peptides were purified by preparative
RP-HPLC on a C18 column (Gemini-NX 5μ, 110 Å, 100 ×
21.2 mm, Phenomenex) using a Waters 150 LC system
equipped with a UV detector and a solvent system consisting of
buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in
CH3CN). Crude acetylated peptides were eluted from the
column using a 10−60% linear gradient of buffer B (0−27 min,
10−60%; 27−30 min, 60−100%; flow rate, 30 mL min−1).
Deacetylated peptides were eluted from the column using a
30−50% linear gradient of buffer B (0−1 min, 10%; 1−2 min,
10−30%; 2−27 min, 30−50%; 27−30 min, 50−100%; 30 mL
min−1). RP-HPLC fractions were quantified by LC−MS using a
Waters XEVO SQD instrument equipped with a C18 analytical
column (Acquity UPLC BEH Amide column, 1.7 μm, 130 Å,
2.1 mm × 100 mm, Waters) and a solvent system consisting of
buffer C (95% H2O, 5% CH3CN, 0.1% HCOOH) and buffer D
(95% CH3CN, 5% H2O, 0.1% HCOOH). Peptides were eluted
from the column using a linear gradient of buffer D (0−6 min,
0−100%; flow, 0.5 mL min−1). NMR spectra were recorded on
CH3OH-d4) δ 5.70 (s, 2H), 5.15 (s, 2H), 2.15 (s, 6H), 2.05 (s,
6H); 13C NMR (75 MHz, CH3OH-d4) δ 171.65, 170.55,
170.12, 70.74, 69.60, 20.40, 20.32.
Synthesis of (2S,3R,4S,5R)-6-((2-(N-Fmoc)aminoethyl)-
amino)-2,3,4,5-tetraacetoxy-6-oxohexanoic Acid (Fmoc-
Muc-OH, 3). Mucic acid tetraacetate (2, 1.0 g, 2.64 mmol) and
SOCl2 (30 mL) were heated to reflux for 5 h. The reaction
mixture was cooled down and evaporated to give the acid
chloride as a pale yellow solid. The product was used without
further purification. The acid chloride (2.64 mmol) was
dissolved in THF (10 mL), followed by addition of DIPEA
(2 mL). Fmoc-EDA·HCl (0.42 g, 1.32 mmol) in THF (10 mL)
were added dropwise, and the reaction mixture was stirred at
room temperature overnight. The solvent was evaporated and
the residue redissolved in CH2Cl2 (50 mL), washed with 1 M
HCl (2 × 20 mL) and brine (30 mL), dried with Na2SO4, and
evaporated. The product was precipitated by dissolving the
residue in a small amount of CH3OH (1 mL) and thereafter
adding Et2O (50 mL) during stirring. The mixture was left in
the fridge overnight, and the solid was filtered and dried to give
1
a Bruker 300 (300 MHz for H, 75 MHz for 13C) equipped
with a BBO probe using either CH3OH-d4 or CH3CN-d3 as
solvent. Chemical shifts are reported in parts per million (ppm)
(δ relative to tetramethylsilane (TMS)). Statistical analysis, data
fitting, and data plotting were performed using GraphPad
software (GraphPad Prism version 5.04 for Windows, San
Diego, CA, USA).
1
a white solid (0.669 g, 1.04 mmol, 79%): H NMR (CH3CN-
d3) δ 7.84 (d, J = 7.3 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.43 (t,
J = 7.5 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.06 (s, 1H), 5.76 (s,
1H), 5.56 (s, 2H), 5.14 (s, 1H), 5.06 (s, 1H), 4.34 (d, J = 6.7
Hz, 2H), 4.23 (t, J = 6.8 Hz, 2H), 3.27−3.08 (m, 4H), 2.14 (s,
3H), 2.08 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H); 13C NMR
(CH3CN-d3) δ 170.84, 170.14, 168.39, 167.82, 157.89, 145.19,
142.14, 128.69, 128.12, 126.14, 120.97, 71.91, 70.34, 69.43,
68.84, 67.13, 48.10, 40.98, 40.52, 21.02, 20.69, 20.60; ESI-MS
m/z calculated for C31H34N2O13 [M + H]+ 643.61, found
643.4.
Materials. Liraglutide (7) was purchased as Victoza (6 mg
mL−1 formulation) and either used as such (for in vivo studies)
or purified by preparative RP-HPLC prior to use (for functional
screening and studies of physicochemical properties). Fmoc-
protected amino acids, ethyl (hydroxyimino)cyanoacetate
(OxymaPure), N,N′-diisopropylcarbodiimide (DIC), piperi-
dine, N,N-diisopropylethylamine (DIPEA), TFA, N-methyl-2-
pyrrolidone (NMP), and DMF were purchased from Iris
Biotech GmbH (Marktredwitz, Germany). Mucic acid,
concentrated H2SO4, SOCl2, THF, CH2Cl2, palmitic acid
(C16), stearic acid (C18), arachidic acid (C20), TES, Et2O,
CH3CN, NaOCH3 (0.5 M) in CH3OH, CH3COOH,
HCOOH, (PPh3)4Pd(0), borane dimethylamine complex, 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
HSA (fatty acid free), MgCl2, CaCl2, BSA, and cell dissociation
solution (nonenzymatic) were purchased from Sigma-Aldrich
(Denmark). Zeocin and geneticin were purchased from
Invitrogen (Thermo Fisher Scientific, Denmark). Growth
medium (Dulbecco’s modified Eagle medium (DMEM) with
glutaMAX-I and 4.5 g/L D-glucose), fetal bovine serum (FBS),
penicillin and streptomycin, geneticin (G418), PBS (pH 7.4),
PBS without CaCl2 and MgCl2, and Hanks balanced salt
solution (HBSS) were purchased from Gibco (Denmark). 384-
Well format black sterile microplates were purchased from BD
Falcon (USA). Excitation filter CFP 430/24 (X430), emission
filters FITC 535/25 (M535), and CFP 470/24 (M470) as well
as a CFP/YFP D450_515 single mirror were purchased and
placed in an Envision 2104 Multilabel Reader (PerkinElmer,
USA). Milli-Q water (Merck Millipore) was used for all
experiments
Peptide Synthesis. The [Arg34,Lys26(All)]GLP-1(7−37)-
OH peptide backbone was prepared by automated peptide
synthesis on a Syro II peptide synthesizer (MultiSynTech,
Witten, Germany; Biotage AB, Uppsala, Sweden) by Fmoc-
SPPS. Peptide synthesis was conducted on a 0.1 mmol scale
using Fmoc Gly TentaGel S PHB resin (0.24 mmol g−1, Rapp
Polymere GmbH, Tuebingen, Germany) as solid support. All
amino acids were incorporated as standard Fmoc amino acids,
except His1 and Phe6-Thr7, which were incorporated as Boc-
His(Trt)-OH and as the pseudoproline Fmoc-L-Phe-L-Thr-
[PSI(Me,Me)Pro]-OH, respectively. Side-chain protecting
groups for Fmoc amino acids were tert-butyl (tBu, for Glu,
Asp, Ser, Thr, Tyr), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl (Pbf, for Arg), and trityl (Trt, for Asn, Gln, His). Fmoc
amino acids (4 equiv) were coupled using DIC (3.8 equiv) and
OxymaPure (4 equiv) in DMF for 2 × 2 h at room
temperature. Nα-Fmoc deprotections were performed using
20% piperidine in NMP for 3 min, followed by 20% piperidine
in NMP for 22 min. Resins were washed with 2 × NMP, 2 ×
CH2Cl2, and 2 × NMP. Following completion of the backbone
sequence, the resin was washed with anhydrous CH2Cl2 and
the Alloc protecting group at Lys26 was selectively removed
using (PPh3)4Pd(0) (1 equiv) and borane dimethylamine
complex (1.2 equiv) in anhydrous CH2Cl2 for 2 h at room
temperature. The neoglycolipids were then assembled on the
partially deprotected peptidyl-resin. Fmoc-Muc-OH (4 equiv),
Fmoc-Glu-OtBu (4 equiv), Fmoc-O2Oc-OH (4 equiv), C16 (4
Synthesis of Mucic Acid Tetraacetate (2).32 Acetic
anhydride (35 mL) and concentrated H2SO4 (5 drops) were
added to mucic acid (1, 5.0 g, 23.8 mmol) in a round-bottom
I
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a
Table 5. Analytical Data for Compounds 4−14
molecular mass (Da)
obsd
compd no.
tR (min)
purity (%)
calcd
[M + H]2+
[M + H]3+
[M + H]4+
[M + H]5+
660.8
4
3.5
3.5
4.5
4.4
4.4
4.4
4.6
4.3
4.4
4.6
4.4
95
95
99
99
99
99
99
99
99
99
99
3297.7
3383.7
3622.2
3751.3
3985.6
4013.5
4041.6
4219.7
4247.8
4275.8
3896.4
1649.8
1693.4
1812.4
1876.5
1100.3
1129.0
1208.4
1251.3
1329.9
1338.9
1348.5
1408.0
1417.2
1426.5
1299.6
825.8
847.1
5
6
906.6
7
938.8
751.3
798.1
8
997.4
9
2008.3
2022.2
2111.7
1004.6
1011.6
1056.3
1063.2
1070.3
975.1
10
11
12
13
14
850.7
856.5
1948.9
a
Retention time and purity are derived from analytical RP-HPLC using a C18 column. Calcd, calculated; obsd, observed.
equiv), C18 (4 equiv), and C20 (4 equiv) were coupled by
hand using DIC (3.8 equiv) and OxymaPure (4 equiv) in DMF
for 2 × 4−16 h at room temperature. Nα-Fmoc deprotections
were performed using 20% piperidine in NMP for 5 min,
followed by 20% piperidine in NMP for 25 min. Resins were
washed with 3 × NMP, 3 × CH2Cl2, and 3 × NMP. Peptide
release from the solid support and removal of acid-labile side-
chain protecting groups was performed by treatment with
TFA:TES:H2O cleavage mixture (95:2.5:2.5, 5 mL) for 2 h.
The resins were washed with additional TFA (5 mL), and the
crude peptides were precipitated using Et2O (40 mL) followed
by centrifugation for 5 min at 4400 rpm. The crude peptides
were redissolved in DMF and purified by preparative RP-
HPLC. RP-HPLC fractions were analyzed by LC−MS, and
fractions containing the acetylated peptides were collected and
lyophilized to provide the acetylated peptides. Removal of the
Ac protecting groups was accomplished by dissolving the
peptides in CH3OH to a concentration of approximately 1 mg
mL−1 and adding NaOCH3 (0.5 M) in CH3OH (corresponding
to 1% of the total volume). The reaction mixture was stirred for
30 min at room temperature and then quenched by addition of
CH3COOH (corresponding to 2% of the total volume). The
reaction mixture was evaporated to dryness, redissolved in a
mixture of CH3COOH, H2O, and CH3CN (25:37.5:37.5), and
purified by preparative RP-HPLC. RP-HPLC fractions were
quantified by LC−MS, and fractions containing deacetylated
peptide were collected and lyophilized to provide the final
product. Analytical data are presented in Table 5.
Functional Screening. A HEK293 cell line with a stable
expression of the EPAC cAMP-sensor as well as the GLP-1
receptor was utilized for functional assay. The cell line utilizes
mCerulean and mCitrine attached to the EPAC protein as
fluorescence resonance energy transfer (FRET) pair and was
previously described by Mathiesen et al.33 Cells were kept at 37
°C in DMEM growth medium supplemented with 10% heat-
inactivated FBS and 1% penicillin−streptomycin in a
humidified 5% CO2 incubator. Furthermore, zeocin (0.05 mg
mL−1) and geneticin (0.5 mg mL−1) were added to the growth
medium to maintain selection pressure. On the day of
measurement, cells were detached using nonenzymatic cell
dissociation solution, spun down, and resuspended in assay
buffer (HBSS with HEPES (20 mM) supplemented with CaCl2
(1 mM), MgCl2 (1 mM), and 0.1% albumin, pH 7.4) to a cell
concentration of 105 cells mL−1. Cells were then transferred to
black 384-well microplates, where ligands were added in the
indicated concentrations. Immediately afterwards, increases in
cAMP levels were measured as increases in the mCerulean/
mCitrine emission ratio using an Envision 2104 Multilabel
Reader, with measurement starting from 1 min and continuing
for 60 min after ligand addition. The energy donor mCerulean
was excited using an optical filter CFP-430 (X430) and a CFP/
YFP (D450_515) single mirror. Subsequently, emission from
both the donor mCerulean and the acceptor mCitrine was
measured using YFP-535 (M535) and CFP-470 (M470) filters.
The 470 nm/535 nm emission ratio was plotted as a function
of time. At the time point of maximum cAMP levels
(approximately 30 min after addition of ligands), dose−
response curves were generated and ligand EC50 values were
calculated.
Assessment of Peptide Lipophilicity. Peptides were
dissolved in a 1:1 mixture of H2O and CH3CN and analyzed by
analytical RP-UPLC coupled to ESI-MS (Acquity UPLC/SQ,
Waters) using a C18 column (Acquity UPLC BEH Amide
column, 1.7 μm, 130 Å, 2.1 mm × 100 mm, Waters) and a
solvent system consisting of buffer C and buffer D. Peptides
were eluted from the column by isocratic elution with buffer D
(0−1 min, 0−42%; 1−20 min, 42%; 20−20.5 min, 42−100%;
20.5−22.5 min, 100%; flow, 0.5 mL min−1).
Assessment of Peptide Solubility. Peptides were
dissolved in 1 mL of PBS (pH 7.4) until a saturated solution
was obtained. Samples (n = 1 per peptide) were sonicated for 1
h, and the pH value was readjusted to 7.4 using aqueous NaOH
(0.1 M). Samples were stored overnight at room temperature
and centrifuged for 15 min at 4400 rpm, and the absorbance at
280 nm (A280) of the supernatant was measured using a
NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA).
Molar concentration was calculated from the Beer−Lambert
law (l = 1 cm, ε280 = 6970 M−1 cm−1 for all compounds). The
supernatants were subsequently analyzed by SEC and DLS
within 24 h.
Size Exclusion Chromatography. Samples (100 μL) were
̈
size-separated by FPLC (AKTAPurifier100, GE Healthcare) on
a calibrated Superdex 75 10/300 column (Pharmacia, Uppsala,
Sweden) at 10 °C using PBS (pH 7.4) as running buffer and a
flow rate of 0.45 mL min−1. Column eluate was monitored at
280 nm using an UV detector. Molecular weight standards were
purchased from GE Healthcare (aprotinin, ribonuclease,
carbonic anhydrase, and ovalbumin) and Sigma-Aldrich
J
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(vitamin B12) or produced in-house (PYY3−36). Apparent
molecular weight was calculated from molecular weight
standards using a linear calibration curve generated from a
plot of retention volume versus log(MW) (Figure S2). For
clarity, peak height was normalized to 1 for all compounds.
Dynamic Light Scattering. Samples (300−500 μL) were
filtered through a 0.2 μm syringe filter (Whatman Anotop Plus,
GE Healthcare, Chicago, IL, USA) and placed in a quartz
cuvette. Samples were analyzed using an instrument consisting
of a BI-200SM goniometer, a BI-APD Avalanche photodiode
detector, a BI-9000AT digital autocorrelator, and a 632.8 nm
HeNe laser with a power of 35 mW (Brookhaven Instruments
Corp., NY, USA). All measurements were obtained at 25 °C as
4−8 acquisitions of 60 s. Data were analyzed using software
provided by Brookhaven Instruments (9KDSLW). A regulari-
zation algorithm was used to establish the presence of a
monomodal size distribution, and cumulant analysis was used
to estimate the average hydrodynamic radius (rH) of the
particles via the Stokes−Einstein equation. The reported values
of rH are derived from the data fit generated by second-order
cumulant analysis.
Albumin Binding. SPR experiments were conducted on a
Biacore X100 optical biosensor equipped with a CM5 chip
(Biacore AB, GE Healthcare, Uppsala, Sweden). All buffers and
reagents were filtered through a 0.2 μm filter and degassed by
sonication for at least 30 min prior to use. HSA (fatty acid free)
was immobilized by amine-coupling chemistry according to
Rich et al.34 The CM5 chip was docked and primed 5−10 times
with running buffer (PBS, pH 7.4). The chip was activated by
injecting a 1:1 mixture of 3-(N,N-dimethylamino)propyl-N-
ethylcarbodiimide (EDC, 0.4 M) and N-hydroxysuccinimide
(NHS, 0.1 M) in PBS buffer pH 7.4 to both flow cells 1 and 2
for 2 × 7 min at 25 °C at a flow rate of 10 μL min−1.
Immobilization was conducted by injecting fatty acid free HSA
(30 μg/mL) dissolved in NaOAc buffer (10 mM, pH 5.2) to
flow cell 2 for 2 × 7 min at 25 °C at a flow rate of 10 μL min−1.
After immobilization, the chip was blocked and washed by
injection of ethanolamine (1 M) to flow cells 1 and 2 for 2 × 3
min at 25 °C at a flow rate of 30 μL/min, followed by injection
of NaOH (50 mM) to flow cells 1 and 2 for 3 × 12 s at 25 °C
at a flow rate of 30 μL min−1. The procedure resulted in
immobilization of HSA on flow cell 2 corresponding to
approximately 8300 RU. Binding analysis was performed using
PBS (pH 7.4) containing 3% DMSO as running buffer. For
each compound, a sample series containing 50, 25, and 5 μM
samples was prepared by making a 50 μM stock solution
followed by dilution with running buffer to avoid bulk shift
effects. All compounds were analyzed twice (n = 2), with all
samples being injected in triplicate with the lowest concen-
tration being analyzed first to prevent carryover effects. All
samples were injected through both flow cells 1 and 2 to allow
double referencing (i.e., subtraction of the inherent SPR
response from the reference surface as well as the running
buffer). Each sample cycle consisted of an association phase
(sample injection for 150 s at 30 μL min−1), a dissociation
phase (running buffer injection for 180 s at 30 μL min−1), and a
regeneration step (injection of 50 mM NaOH for 4 s at 30 μL
min−1). After each sample series, a blank injection was included
in order to visualize any compound carryover. Data was
analyzed by nonlinear regression using the Association then
Dissociation model (constraints: HotNM = data set constant
Data plot represents mean SEM of a single representative
experiment with triplicate injections. The reported values of kd,
ka,app and KD,app are reported as mean SEM, n = 2.
Animal Welfare. Animal experiments were conducted in
accordance with internationally accepted principles for the care
and use of laboratory animals and were covered by a personal
license issued for Jacob Jelsing (permit number: 2013-15-2934-
00784, approved by the Danish Committee for Animal
Research). Mice (male NMRI mice, each weighing approx-
imately 30 g) were obtained from Taconic (Lille Skensved,
Denmark). The mice were housed in groups of four in a light-
controlled, temperature-controlled, and humidity-controlled
room (a 12/12 light−darkness cycle, lights on at 2:00 a.m.;
22 2 °C; 50% relative humidity). During experiments, the
mice had ad libitum access to regular chow diet (Altromin
1324, Brogaarden A/S, Lynge, Denmark) and domestic quality
tap water.
Acute Effect on Glucose Tolerance. The effect on
glucose homeostasis of compounds 8−10 was evaluated in
comparison with liraglutide and vehicle (5 mL kg−1, consisting
of PBS (pH 7.4) containing 0.1% BSA in an oral glucose
tolerance test (OGTT). Male NMRI mice (each weighing 25−
30 g) were acclimatized for a minimum of 5 days. Prior to
entering the experiment, animals are randomized into treat-
ment groups according to body weight. On the day of
experiment, animals were fasted for 3 h prior to compound
administration (sc, 50 nmol kg−1) at t = −45 min. At t = 0,
animals are given an oral glucose load (2 g kg−1; dose volume
10 mL kg−1; glucose solution 200 mg mL−1, Fresenius Kabi,
Sweden). Blood samples were drawn from the tail vein at time
points −45, 0, 15, 30, 60, and 120 min, collected into 10 μL
heparinized glass capillary tubes, and immediately suspended in
buffer (0.5 mL of glucose/lactate system solution, EKF-
diagnostics, Germany) and analyzed for glucose on the test
day using a BIOSEN c-Line glucose meter (EKF-diagnostics,
Germany) according to the manufacturer’s instructions.
Statistical analysis was performed using one-way analysis of
variance (ANOVA) followed by Bonferroni’s post test against
vehicle (p < 0.05 for statistical significance). Data are presented
as mean SEM, n = 7.
Acute Effect on Food Intake. Food intake was measured
using a fully automated food intake monitoring system (HM-2;
MBRose ApS, Faaborg, Denmark) allowing for real-time
monitoring of food intake behavior of individual animals as
described in details by Axel et al.35 Upon arrival, the mice were
transferred to the HM-2 system, acclimatized to their new
environment for a minimum of 5 days, and handled daily to
accustom them to the experimental paradigm. Mice were
uniquely identified with sc implantable microchips (Pet ID
Microchip, E-vet, Haderslev, Denmark) to allow identification
of each individual upon entry and exit from the food channel.
On the day of dosing, animals were randomized into treatment
groups according to body weight, and the acute anorectic effect
of compounds 8, 9, 11, and 12 was evaluated in comparison
with liraglutide and vehicle (5 mL kg−1, consisting of PBS (pH
7.4) containing 0.1% BSA. Mice were dosed sc in the lower
back (dose, 50 nmol kg−1; dose volume, 5 mL kg−1) in the
afternoon just prior to lights out, and food intake data were
collected for 18 h post dosing. Data were obtained from two
independent studies. The first study contained the following
treatment groups: vehicle (n = 6), liraglutide (n = 8), 8 (n = 6),
9 (n = 7), and 12 (n = 8). The second study contained the
following groups: vehicle (n = 9), 8 (n = 9), 9 (n = 9), 11 (n =
(=column title); kon = Shared value for all data sets; koff
=
Shared value for all data sets; Time0 = Constant equal to 145).
K
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9), and 12 (n = 9). All data from the two studies were pooled
prior to analysis. Statistical analysis was performed using a one-
way ANOVA followed by Dunnett’s multiple comparison test
(p < 0.05 for statistical significance). Data are presented as
mean SEM, n = 8−17.
solid-phase peptide synthesis; SLS, static light scattering; VR,
retention volume
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ASSOCIATED CONTENT
* Supporting Information
■
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The Supporting Information is available free of charge on the
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ceut.6b00787.
Synthesis strategy for neoglycolipidated peptides, SEC
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: kjj@chem.ku.dk. Phone: +4535332430.
*E-mail: slp@gubra.dk. Phone: +4531522650.
ORCID
Knud J. Jensen: 0000-0003-3525-5452
Author Contributions
S.B.v.W., K.M., P.W., and E.M.B. performed the experiments.
S.B.v.W., K.M., M.B.T., N.V., J.J., K.J.J., and S.L.P discussed the
data. S.B.v.W., M.B.T., K.J.J., and S.L.P. wrote the manuscript.
■
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The authors declare the following competing financial
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ACKNOWLEDGMENTS
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Osborne are thanked for providing the facilities for conducting
the functional assays. The authors wish to acknowledge the
Innovation Fund Denmark for cofinancing PhD stipends to
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ABBREVIATIONS USED
■
ANOVA, analysis of variance; C16, palmitic acid; C18, stearic
acid; C20, arachidic acid; DIC, N,N′-diisopropylcarbodiimide;
DIPEA, N,N-diisopropylethylamine; DLS, dynamic light
scattering; DMEM, Dulbecco’s modified Eagle medium; EDA,
ethylenediamine; EDC, 3-(N,N-dimethylamino)propyl-N-ethyl-
carbodiimide; ESI-MS, electrospray ionization mass spectrom-
etry; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethylox-
ycarbonyl; FRET, fluorescence resonance energy transfer;
HBSS, Hanks balanced salt solution; HEPES, 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid; HM-2, HerdsMan-2;
HSA, human serum albumin; ka, association rate constant; kd,
dissociation rate constant; KD, equilibrium dissociation
constant; m, oligomeric state; NHS, N-hydroxysuccinimide;
NMP, N-methyl-2-pyrrolidone; OGTT, oral glucose tolerance
test; OxymaPure, ethyl (hydroxyimino)cyanoacetate; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PI, poly-
dispersity index; RU, response unit; SEC, size exclusion
chromatography; SPR, surface plasmon resonance; SPPS,
L
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