Late-stage lipidation of fully elaborated tryptophan-containing peptides for improved pharmacokinetics

Article abstract of DOI:10.1016/j.tetlet.2017.02.031

The late-stage modification of native peptides to alter and/or enhance their properties and functions is attractive but formidably challenging. Peptide lipidation is one of the effective strategies to overcome short half-life and rapid clearance. Herein, we report a late-stage installation of a fatty acid lipid onto fully elaborated peptides, using glucagon as an example, through regio- and chemoselective functionalization of tryptophan with high potency and remarkable in vivo half-life extension.

Full text of DOI:10.1016/j.tetlet.2017.02.031

Tetrahedron Letters 58 (2017) 1219–1222
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Late-stage lipidation of fully elaborated tryptophan-containing peptides
for improved pharmacokinetics
a
b
b
a
Chunhui Huang a, , Cannon B. Wille , Huaibing He , Vijay Bhasker Gangula Reddy , Ravi P. Nargund ,
⇑
Songnian Lin a, Anandan Palani a
a Discovery Chemistry Department, MRL, Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Pharmacokinetics Pharmacodynamics & Drug Metabolism, MRL, Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The late-stage modification of native peptides to alter and/or enhance their properties and functions is
attractive but formidably challenging. Peptide lipidation is one of the effective strategies to overcome
short half-life and rapid clearance. Herein, we report a late-stage installation of a fatty acid lipid onto fully
elaborated peptides, using glucagon as an example, through regio- and chemoselective functionalization
of tryptophan with high potency and remarkable in vivo half-life extension.
Received 10 January 2017
Revised 3 February 2017
Accepted 9 February 2017
Available online 13 February 2017
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
Late-stage functionalization
Lipidation
Peptides
Incretin
Glucagon
Introduction
group can be introduced through late-stage functionalization of a
peptide, which allows for further site-specific manipulation.
Peptide drugs enjoy great intrinsic advantages over other chem-
istry modalities due to their superior efficacy, high selectivity, and
low toxicity.1 However, most naturally occurring peptides are
quickly degraded in serum and rapidly cleared from the body.
The proteolytic instability and short circulating half-life signifi-
cantly limit their use as effective therapeutics. Lipidation is one
of the chemical modification methods which have been proved
to be highly useful and practical for improving the physicochemi-
cal, pharmacological and even biological properties of peptides.2–4
The typical lipidation method is through acylation of peptide
lysine side chain with long-chain, saturated fatty acid lipids.2,5,6
It usually requires de novo synthesis of peptides and different
amino protecting groups are implemented if the peptide has mul-
tiple lysine residues. Other methods such as cysteine S-alkyla-
tion,7,8 serine O-esterification9 were also reported. However, the
ability to modify selected amino acid (AA) side chains for lipidation
in fully elaborated peptides is still very limited.2,10 The desired
late-stage, site-specific lipidation approach would obviate the need
for the protection/deprotection steps and the need for engineering
peptides with limited synthetic handles or the use of unnatural
amino acids. In order to achieve that, an orthogonal functional
The glucagon-related peptides, including glucagon, GLP-1,
GLP-2, GIP, oxyntomodulin (OXM), and their synthetic versions of
dual11 and/or triple12 receptor agonist peptides, have been exten-
sively studied for the use of diabetes treatment. Lipidation plat-
form through acylation of lysine residue is currently the gold
standard chemical approach for engineering long-acting peptide
analogs.5,6 This technology has an established track record for
pharmacokinetic improvement in commercialized products, such
as once-daily GLP-1R agonist Liraglutide,13 insulin Levemir,6 insu-
lin Degludec,14,15 as well as a phase 3 clinical candidate, once-
weekly GLP-1RA Semaglutide.16 Both Liraglutide and Semaglutide
are lipidated in position 20 (the N-terminal His counted as position
1) of GLP-1 (7–36) amide. In order to achieve site-specific monoa-
cylation, substitution of Lys28 to Arg was applied in both cases. It
should be noted that other positions were also reported for lipid
attachment through Lys acylation or Cys alkylation in
GLP-1RAs.11,12,17 However, all of them require different levels of
sequence alteration and/or functional group protection strategy.
Sequence alignment and analysis of glucagon-related peptides
revealed that tryptophan (Trp) at position 25 is highly conserved
across the panel and only one single Trp residue is incorporated
in each peptide (Fig. 1a). We envisioned that it could be an
ideal setting for late-stage, site-specific functionalization of such
fully elaborated peptides. Although challenging, Trp-selective
⇑
Corresponding author.
E-mail address: chunhui.huang@merck.com (C. Huang).
http://dx.doi.org/10.1016/j.tetlet.2017.02.031
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

1220
C. Huang et al. / Tetrahedron Letters 58 (2017) 1219–1222
H
N
C'
1a-4a
N'
1 eq. AuCl(SMe2)
3 eq. TIPS-EBX
2% TFA, MeCN
RT, 16 h
TIPS
I
O
H
N
O
TIPS
(TIPS-EBX)
1b-4b
Scheme 1. A representative reaction scheme of Trp alkynylation in glucagon-
related peptides.
Table 1
Trp alkynylation of glucagon-related peptides.
Entry
Peptidea
Yield, %b
1
2
3
4
GCG (1a)
GLP-1 (2a)
OXM (3a)
GLP-1/GIP coagonist (4a)
44
33
25
38
Fig. 1. (a) Sequences of glucagon (GCG), GLP-1, oxyntomodulin (OXM), and
GLP-1/GIP dual agonist.11 C-terminal amidation for GLP-1, and GLP-1/GIP dual
agonist. X = 2-aminoisobutyric acid (or Aib). Trp is conserved across the panel and
highlighted in red. (b) X-ray crystal structure of Semaglutide peptide in complex
with GLP-1 receptor extracellular domain (PDB code: 4ZGM). The Trp25 is
highlighted with stick structure, whose C2 position pointing away from the
GLP-1R ECD.
a
See Fig. 1a for amino acid sequence.
Reaction conditions: peptide (1 equiv), TIPS-EBX (3 equiv), AuCl(SMe2) (1
equiv), MeCN with <2% TFA/HOAc, RT, 16 h. The low isolated yields were attributed
to the incomplete reaction conversion and purification loss on RP-HPLC C8 column.
b
functionalization in peptides and proteins has been described. A
rhodium carbenoid approach reported by Francis and coworkers,
although giving a mixture of both N1 and C2 substituted products,
is still useful for Trp-containing protein labeling.18 More recently, a
complete regio- and chemoselective Trp functionalization in pep-
tides and proteins has also been reported.19,20 This elegant method
allows for direct installation of an orthogonal handle (i.e., an
alkyne) at C2 position of Trp in the peptides. Since alkyne-azide
cycloaddition ‘‘click” chemistry is the most widely used bioconju-
gation strategy for manipulating the properties of large
molecules,22 we thought this sequential Trp C2 alkynylation/click
reaction offers a unique suite of late-stage lipidation of native pep-
tides for pharmacokinetic (PK) improvement.
Based on the X-ray crystal structure of Semaglutide in complex
with GLP-1R extracellular domain (Fig. 1b),16 the C2 position of Trp
is solvent exposed and pointing away from the receptor protein.
Therefore, grafting a fatty acid lipid on C2-position of Trp in gluca-
gon-related peptides would potentially have minimal negative
impact on its biological activity, yet very likely with improved PK
profiles.
Remarkably, reactive amino acid residues (such as His, Ser, Phe,
Try, Lys, Thr and Met) were all tolerated. Interestingly, although
Waser reagent is a hypervalent iodine oxidant, the Met residue
in GCG peptide was intact, with no trace amount of overoxidized
product observed during the reaction. With this result, we were
encouraged to test the alkynylation reactions of the aforemen-
tioned glucagon-related peptides in the Fig. 1a panel, which all suf-
fer from the high clearance and short half-lives with regular
formulation.24 Therefore, GLP-1 peptide 2a, bearing a 48% homol-
ogy to GCG peptide, transformed to Trp-alkynylated product 2b
in 33% yield (entry 2). Oxyntomodulin (OXM, 3a), a naturally
occurring dual agonist binding both the GLP-1 receptor and the
glucagon receptor, contains the full amino acid sequence of GCG
followed by extra an 8 AAs extension at C-terminus. The alkynyla-
tion of OXM only resulted in 25% isolated yield of product 3b (entry
3). Another reported dual agonist 4a11 with activation on both the
GLP-1 receptor and the GIP receptor went on alkynylation reaction
to give 38% yield of desired product 4b (entry 4). This material was
analyzed by MS/MS, confirming that the TIPS ethynyl group ends
up on Trp residue in the peptide.
In order to enhance the PK properties of those peptides, we fur-
ther reacted the alkynylated model peptide GCG 1b with the fatty
Results and discussion
acid lipid 5, containing PEG2PEG2cEC18-OH unit, an established
With this in mind, we chose native glucagon (GCG, 1a) peptide
as our model peptide because of its short half-life, around 4–7 min
in human plasma.23 Under slightly modified conditions,20,21 the
initial trial reaction of 1a and Waser reagent (TIPS-EBX, 3 equiv)
in the presence of AuCl(SMe2) (1 equiv) catalyst at room tempera-
ture gave a full conversion of starting material to the desired pro-
duct 1b based on LCMS analysis (see Scheme 1). It was isolated in
44% yield with reverse-phase HPLC C8 column (Table 1, entry 1).
long-chain, saturated lipid for once-weekly administration of
GLP-1R agonists.16 Initial desilylation of the TIPS ethynyl GCG pep-
tide 1b with polymer-supported fluoride (10X) led to no reaction.
Nevertheless, the treatment of 1b with TBAF immediately gave
the desilylated product ethynyl GCG, which further reacted with
azido-containing fatty acid lipid 5 under CuAAC condition to give
the Trp25-lipidated GCG peptide 1c in 37% yield over 2 steps
(Scheme 2).

C. Huang et al. / Tetrahedron Letters 58 (2017) 1219–1222
1221
H
N
TIPS
S
Q
G
T
F
T
S
D
Y
S
K
1b
Y
L
D
S
R
R
A
Q
D
F
V
Q
N
H
L M N T
H
O
1) TBAF
2) CuSO4, NaAsc
5
N3-PEG2PEG2γEC18-OH (
)
(37% isolated over 2 steps)
O
O
H
H
H
O
N
O
N
N
N
H
O
O
N
N
HO
O
O
CO2H
N
S
Q
G
T
F
T
S
D
Y
S
K
Y
L
D
S
R
R
A
Q
D
F
V
Q
N
H
L M N T
H
O
1c
Scheme 2. Late-stage lipidation of GCG peptide, involving the steps of desilylation with TBAF and conjugation by CuAAC. N3-PEG2PEG2
c
EC18-OH (5): (S)-1-azido-22-
carboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid.
The activity of the lipidated GCG 1c remains potent against the
glucagon receptor with EC50 = 0.24 0.04 nM (n = 2). Although less
potent compared with the native GCG (EC50 = 0.012 nM), the lipi-
dated GCG 1c could potentially be a very attractive option for the
treatment of Type 2 diabetes patients with obesity when co-dosing
with long-acting GLP-1RAs in a reasonable combination as well as
for the treatment of hypoglycemia.
Next, we examined whether the lipidation strategy
translates into better pharmacokinetic profiles. The PK properties
of the lipidated GCG 1c were evaluated in male Wistar Han
naïve rats (n = 3 per group) following i.v. or s.c. administration
(Fig. 2). Following i.v. administration, 1c exhibited low
systemic plasma clearance of 0.27 mL/min/kg with elimination
half-life of 6.8 h. The volume of distribution was 0.14 L/kg. The sub-
cutaneous bioavailability of 1c was 55% (Table 2). This remarkable
extension of half-life (compared to native GCG) was presumably
attributed to the strong albumin binding and reduced clearance,
even without sequence modification to protect toward DPP-IV
degradation.
Finally, parathyroid hormone (PTH) belongs to the same B-fam-
ily G protein-coupled receptors (GPCRs) as glucagon-related pep-
tides. The bioactive portion of this hormone is PTH (1–34) with a
product Teriparatide already in the market for the treatment of
osteoporosis. However, its half-life is only 4 min. The only reported
long-acting version of this hormone is Fc fusion protein, which
showed to treat hypoparathyroidism more effectively and for a
longer period of time than PTH in rats and monkeys.25 By examin-
ing the amino acid sequence of PTH (1–34), only one single Trp is
incorporated in the sequence and the molecular model of hPTH (1–
34) binding to its receptor indicating Trp C2 position has no direct
interaction with receptor and is solvent exposed.26 Hence, hPTH
(1–34) is a perfect setting for chemical late-stage lipidation to
improve pharmacokinetics. Thus, hPTH (1–34) (sequence:
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF) was treated with
TIPS-EBX and gold catalyst to afford TIPS ethynyl PTH (28% yield),
which underwent desilylation with TBAF (52% yield) and followed
by click reaction with azido-PEG2PEG2
cEC18-OH (5) to furnish the
desired lipidated PTH (see supporting information).
Fig. 2. Plasma concentration vs. time profiles of lipidated GCG 1c in rats following IV (A) and SC (B) administration. GCG 1c was formulated in mannitol buffer (5% mannitol,
6 mM sodium phosphate, pH 8) and dosed IV or SC at 1 mL/kg, n = 3 rats/group. Concentrations of GCG 1c were determined by LCMS assay.
Table 2
Pharmacokinetics of GCG 1c in male Wistar Han naïve rats following IV and SC dosing.a
Dose
(mg/kg)
AUC0-1
(mMꢀh)
1.5 0.2
AUC0–1
(mM h)
CLp
(mL/min/kg)
Vdss
(L/kg)
T1/2
(h)
Route
IV
0.1
0.27 0.03
Cmax
(nM)
0.14 0.00
6.8 0.7
F
(%)
55
Dose
(mg/kg)
0.2
Tmax
(h)
7
Route
SC
1.6 0.5
80 22
a
GCG 1c was formulated as a solution and administered in rats as described above. Plasma concentrations of GCG 1c were determined by LCMS assay. Values are
means SD.

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C. Huang et al. / Tetrahedron Letters 58 (2017) 1219–1222
Conclusion
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Supplementary data associated with this article can be found, in
theonlineversion, at http://dx.doi.org/10.1016/j.tetlet.2017.02.031.