Synthesis and applications of polyamine amino acid residues: Improving the bioactivity of an analgesic neuropeptide, neurotensin

Article abstract of DOI:10.1021/jm801481y

Conjugated polyamines are potential carriers for biothera- peutics targeting the central nervous system. We describe an efficient synthesis of a polyamine-based amino acid, lysine-trimethylene(diNo-syl)-spermine(triBoc) with Dde or Fmoc orthogonal protect

Full text of DOI:10.1021/jm801481y

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J. Med. Chem. 2009, 52, 1514–1517
Synthesis and Applications of Polyamine
Amino Acid Residues: Improving the
Bioactivity of an Analgesic Neuropeptide,
Neurotensin
Liuyin Zhang,† Hee-Kyoung Lee,† Timothy H. Pruess,‡
H. Steve White,‡ and Grzegorz Bulaj*,†
Department of Medicinal Chemistry and Department of
Pharmacology and Toxicology, College of Pharmacy, UniVersity of
Utah, Salt Lake City, Utah 84108
Figure 1. Design of polyamine-NT analogue. C-terminal fragments
of NT and Contulakin-G (shown in boxes) are critical for binding to
neurotensin receptors. In the truncated Contulakin-G analogue, a
naturally occurring glycoamino acid, was replaced with a polyamine-
based amino acid residue.
ReceiVed NoVember 25, 2008
Abstract: Conjugated polyamines are potential carriers for biothera-
peutics targeting the central nervous system. We describe an efficient
synthesis of a polyamine-based amino acid, lysine-trimethylene(diNo-
syl)-spermine(triBoc) with Dde or Fmoc orthogonal protecting groups.
This nonnatural amino acid was incorporated into a neurotensin
analogue using standard Fmoc-based protocols. The analogue main-
tained high affinity and agonist potency for neurotensin receptors and
exhibited dramatically improved analgesia in mice. Our work provides
a basis for use of polyamine amino acids in polypeptides.
in acetic acid writhing and hot plate tests.5 The spinal NTS1
was suggested to play an important role in reducing inflamma-
tory response in the formalin test, a model of persistent pain.6
The naturally occurring NT analogue, contulakin-G, containing
site-specific O-glycosylation, exhibited picomolar potency in a
rat model of inflammatory pain following intrathecal injection.7,8
Therefore, NT is a convenient model peptide for improving its
central nervous system (CNS) bioavailability because NT
analogues with improved BBB penetration might be expected
to produce enhanced antinociceptive effects in animals.9-11
Numerous strategies have been attempted to improve the BBB
permeability of NT analogues. These include backbone and/or
side chain modifications to cyclic and mimetic analogues.10,12-17
One of the modified NT(8-13) analogues that penetrates the
BBB, NT69L ((N-methyl-Arg)-Lys-Pro-(L-neo-Trp)-(tert-Leu)-
Leu), is a potent analgesic and it appeared a very useful
pharmacological tool to study the role of neurotensin receptors
in the CNS.6,11,18,19 In the research presented here, we synthe-
sized and characterized pharmacological properties of an NT
analogue containing lysine-CH2CH2CH2-spermine, with the
main hypothesis that the polyamine-modified compound will
exhibit improved analgesic properties.
Our design strategy was to introduce a polyamine amino acid
into the truncated NT/contulakin-G analogue (Figure 1). In both
NT and contulakin-G, the active fragment comprises the last
six amino acid residues (RRPYIL and KKPYIL, respectively).7
Replacements of Arg residues in NT(8-13) with many cationic
amino acids did not change receptor binding properties.10,16
Because contulakin-G contains a bulky moiety ꢀ-D-Gal-(1f3)-
R-D-GalNAc-(1f) disaccharide attached to Thr10 that was
apparently benign to the bioactivity of this NT analogue, we
designed the truncated contulakin-G analogue containing the
polyamine amino acid instead of glycoamino acid. Thus, our
lead compound, named polyamine-NT, was based on the
truncated contulakin-G analogue, cont-G(10-16), in which the
glycoamino acid was replaced by a polyamine amino acid.
Because natural biogenic polyamines almost exclusively
incorporate 1,3-diaminopropyl or 1,4-diaminobutyl units, the
spacer -CH2CH2CH2- between lysine and spermine was
selected to maintain the physicochemical properties of the side
chain for our construct. Structure 1 was designed to be
compatible with solid-phase peptide synthesis (SPPS, Scheme
1). Nosyl (ortho-nitrobenzenesulfonyl, Ns) strategy was used
to conjugate lysine and spermine moieties because of synthetic
accessibility, no undesired tertiary amine and/or quaternary
ammonium salt formation, and highly efficient alkylation with
Polyamines and polyamine-based compounds are ubiquitous
in nature; these compounds are also broadly applicable for drug
delivery, therapeutics, and engineering nanomaterials. Polyamine-
modified polypeptides exhibit a significant increase in penetra-
tion across the blood-nerve and blood-brain barriers (BBBa).1,2
Our recent study showed that a combination of cationization
and lipidization applied to truncated galanin analogues improved
their brain penetration, yielding very potent anticonvulsant
compounds.3 Furthermore, cell penetrating peptides and peptidic
vectors that transfer conjugated cargos into the nervous system
contain a large number of cationic amino acid residues. Although
the mechanism by which cationized peptides can penetrate
biological membranes is not fully understood, several potential
pathways, including internalization and adsorptive-mediated
endocytosis (AME), have been discussed.4 Despite a potential
of improving penetration of bioactive peptides into the nervous
system by cationization, no reports exist on employing polyamine-
based amino acids into neuropeptides.
To investigate how the structural addition of polyamines to
the active moiety could improve pharmacological properties of
neuroactive peptides, we selected neurotensin (NT) as the model
peptide. NT is an endogenous neuropeptide that produces potent
analgesic activity mediated spinally and/or supraspinally by NT
receptors.5,6 Two subtypes of NT receptors, NTS1 and NTS2,
have been demonstrated to mediate the various antinociceptive
effects, including stress-induced antinociception, or analgesia
* To whom correspondence should be addressed. Phone: 801-581-4629.
Fax: (801) 581-7087. E-mail: bulaj@pharm.utah.edu. Address: Department
of Medicinal Chemistry, College of Pharmacy, University of Utah, 421
Wakara Way, Suite 360, Salt Lake City, Utah 84108.
†
Department of Medicinal Chemistry.
Department of Pharmacology and Toxicology.
Abbreviations:AME,absorptive-mediatedendocytosis;BBB,blood-brain
‡
a
barrier; CNS, central nervous system; Dde, 1-(4,4-dimethyl-2,6-dioxacy-
clohexylidene)ethyl; DIPEA, N,N-diisopropylethylamine; ip, intraperitoneal;
Ns, ortho-nitrobenzenesulfonyl; NT, neurotensin; NTS1, neurotensin recep-
tor subtype 1; NTS2, neurotensin receptor subtype 2; PyBop, (benzotriazol-
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; SPPS, solid-phase
peptide synthesis.
10.1021/jm801481y CCC: $40.75
2009 American Chemical Society
Published on Web 02/23/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1515
Scheme 1. Synthesis of Polyamine Amino Acidsa
a Reagents and conditions: (a) CF3COOEt, -78 to 4 °C; Boc2O, 4 °C; K2CO3, MeOH/H2O (51%); (b) 2-Nitrobenzenesulfonyl chloride, Et3N, 0 °C; (c)
3-Bromo-1-propanol, Cs2CO3, 50 °C (76% from 2 to 4); (d) MeSO2Cl; DIPEA, 0 °C; (e) NaI, acetone, reflux (74%); (f) Alloc-Lys(Ns)-OAll, Cs2CO3, 50
°C (72%); (g) Dde-Lys(Ns)-OAll, Cs2CO3, 50 °C (64%); (h) Pd(PPh3)4, pyrrolidine (48%); (i) FmocOSu, NaHCO3 (76%); (j) Pd(PPh3)4, pyrrolidine (87%).
Figure 2. Pharmacological properties of polyamine-NT. (A) Competitive binding assay for the human NTS1 (membrane preparations containing
the recombinant NTS1 used in the binding assays were derived from HEK-293T cells). (B) Agonist activity determined by stimulation of intracellular
Ca2+ mobilization in HEK-293T cells expressing human NTS1. (C) Analgesic activity in the formalin pain assay in mice (ip, 4 mg/kg, injected 1 h
prior to formalin injection in a paw). Paw-licking (expressed in seconds) was monitored continuously for 2 min every 5 min. Increased duration of
licking between 0-5 min reflects the phase I response (acute pain), whereas that between 15-30 min is defined as the phase II response (inflammatory
pain).
RX or ROH.20 Ns strategy had been successfully applied to the
synthesis of polyamine toxins such as spider venom HO-416b21
and philanthotoxin-343.20
with catalytic Pd(PPh3)4 in the presence of pyrrolidine gave
low yields (48%). Other scavengers such as morpholine24 or
barbituric acid25 produced similar or lower yields. Carrasco’s
one-pot strategy for Alloc/allyl removal followed by Fmoc
protection without purification gave only 10% yield.26
Compound 2 was prepared by selective trifluoroacetyl
monoprotection of spermine, followed by Boc derivatization
of the remaining amino groups. Subsequent hydrolysis of the
trifluoroacetyl functionality was accomplished with metha-
nolic K2CO3, which gave better results than other published
methods (NaOH/H2O/MeOH or MeOH/NH4OH).22,23 Nosy-
lation of 2 gave 3, which yielded 4 after alkylation with
3-bromo-1-propanol (overall 76% yield for the two steps).
Mitsunobu coupling of 4 with Alloc-Lys(Ns)-OAll to form
6a directly gave low yields and inseparable byproduct. The
problem was circumvented via 5, which was prepared
conventionally in good yield by mesylation of 4 followed
by displacement with iodide.
Alloc-Lys(Ns)-OAll was synthesized smoothly from Fmoc-
Lys-OAll in three steps (see Supporting Information).
However, attempts to synthesize NR-Fmoc protected 1a by
way of 5 and Alloc-Lys(Ns)-OAll gave mixed results.
Although alkylation of 5 with Alloc-Lys(Ns)-OAll proceeded
well, simultaneous deprotection of both Alloc and allyl groups
Because of relatively low yields of the above methods, 1b
with Dde protection at NR-position of polyamine acid was
designed. To this end, Dde-Lys(Ns)-OAll was synthesized in
four steps from H-Lys(Boc)-OH: NR-Dde protection, allyl ester
formation, Nε-Boc deprotection, and Nε-nosylation (see Sup-
porting Information). Dde-Lys(Ns)-OAll was then coupled with
5 to give 6b. Finally allyl deprotection of 6b with Pd(PPh3)4/
pyrrolidine afforded 1b in good yield (87%).
The polyamine-NT was synthesized on preloaded Wang resin
using PyBop reagent. Amino acid coupling was performed on
an automated peptide synthesizer. The final addition of 1b was
done manually. Common Dde deprotection with hydrazine could
not be used due to the Ns nitro groups. An alternate deprotection
method27 (NH2OH·HCl/imidazole in NMP/CH2Cl2) was applied
successfully, followed by N-terminal acetylation. Ns deprotec-
tion on the resin using (p-methoxythiophenol/K2CO3 in CH3CN/

1516 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Letters
DMSO28) was accomplished in 2 h. The peptide was cleaved
from the resin with reagent K and purified by HPLC (overall
yield 12%).
Supporting Information Available: Experimental details for
synthetic procedures, full characterization of all compounds, and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
Polyamine-NT was first tested for its ability to bind and
activate the NT receptors, as described previously.29 This
analogue exhibited subnanomolar affinity (Ki 0.25 nM) as well
as low nanomolar agonist potency (EC50 1.4 nM) for the human
NTS1 (Figure 2A,B). These values were similar to those for
the full-length, unmodified NT (Ki 0.5 nM and EC50 1.1 nM),29
suggesting that the polyamine moiety did not affect the
interactions between the polyamine-NT and the NTS1. This
finding is in agreement with other reports (e.g. demotensin
analogues30) and our recent study on glycosylated NT analogues,
e.g., the presence of N-terminal extensions in NT(8-13),
including addition of various glycoamino acids, did not change
the affinity of the analogues toward NTS1.29 The subnanomolar
affinity of polyamine-NT is comparable with another BBB-
permeable NT analogue, such as NT67L,9 and with some of
Arg8 substituted NT(8-13) analogues.10,31 Two other previously
described analgesic NT analogues, NT69L and JMV2012,
exhibited Kd of 1.55 nM and 150 nM, respectively, for human
NTS1.11,12
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analogue displayed analgesic properties. Although the antinoci-
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NT analogue and NTS2, to address these pharmacological
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polyamine amino acids should be explored more frequently
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There are multiple mechanisms by which modified peptides can
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are more likely to be transported by AME.32 Other applications
for polyamine amino acids are to explore them as substitutes
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mers assembled via SPPS.
Acknowledgment. This work was supported in part by
grants from the NIH (R21 NS059669), the Epilepsy Therapy
Project, the Epilepsy Research Foundation, and the University
of Utah Startup Funds. G.B. also acknowledges a financial
support from the NIH program project GM 48677. We would
like to thank Dr. J. Hinshaw and C. R. Robertson for a critical
reading of the manuscript and helpful suggestions. G.B. and
H.S.W. are scientific cofounders of NeuroAdjuvants, Inc.
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