Synthesis and human neurotensin receptor binding activities of neurotensin(8-13) analogues containing position 8 α-azido-N-alkylated derivatives of ornithine, lysine, and homolysine

Article abstract of DOI:10.1021/jm9903444

A series of neurotensin(8-13) (NT) analogues were synthesized through intermediates in which the N-terminal Arg(8) was replaced by various ω- bromo-2(S)-azido residues spanning 3-5 methylene units in side-chain length. Subsequent nucleophilic substitution

Full text of DOI:10.1021/jm9903444

4914
J . Med. Chem. 1999, 42, 4914-4918
Brief Articles
Syn th esis a n d Hu m a n Neu r oten sin Recep tor Bin d in g Activities of
Neu r oten sin (8-13) An a logu es Con ta in in g P osition 8 r-Azid o-N-a lk yla ted
Der iva tives of Or n ith in e, Lysin e, a n d Hom olysin e
J oseph T. Lundquist, IV and Thomas A. Dix*
Department of Pharmaceutical Sciences, Medical University of South Carolina, 280 Calhoun Street,
P.O. Box 250140, Charleston, South Carolina 29425-2303
Received J uly 6, 1999
A series of neurotensin(8-13) (NT) analogues were synthesized through intermediates in which
the N-terminal Arg(8) was replaced by various ω-bromo-2(S)-azido residues spanning 3-5
methylene units in side-chain length. Subsequent nucleophilic substitution of the ω-bromo
groups with ammonia, methylamine, dimethylamine, or trimethylamine provided peptides
containing N-terminal 2(S)-azido residues containing primary through quaternary N-alkylated
side chains corresponding in length to ornithine, Lys, and homolysine. The synthetic procedure
appears applicable for incorporation of a wide variety of amine-containing nonnatural amino
acids into peptides. The particular modifications were intended to enhance physiochemical
properties of NT(8-13) responsible for human NT 1 receptor (hNTR) binding, overall
lipophilicity, and stability that may influence the potency of the peptides in vivo. When the
peptides were tested for hNTR binding, the affinities in each series followed the order primary
> secondary > tertiary > quaternary amine with the homolysine side-chain length being
favored. All analogues possess binding affinities between acetyl-NT(8-13) and NT(8-13)
indicating that the sterically less bulky R-azido may be inherently preferable to the acetyl
group for N-terminal protection. This study extends the strategy of modifying amine-containing
drugs through alkylations to peptide modification. The set of alkylated side chains also offers
a new method of steric selection between receptor subtypes and could be used to modify the
properties and biological effects of any peptide that contains cationic residues.
In tr od u ction
The C-terminal binding fragment of neurotensin (NT),
NT(8-13) (Arg(8)-Arg(9)-Pro(10)-Tyr(11)-Ile(12)-Leu(13)),
is the minimal structural element able to bind and
trigger biological effects at human NT receptors
(hNTRs).1 Since the peptide possesses an atypical neu-
roleptic profile in the brain,2 there is substantial interest
in developing bioavailable NT(8-13) derivatives as
alternatives for the treatment of schizophrenia. Al-
F igu r e 1. Structural comparison of NT(8-13) analogues.
though NT(8-13) possesses high receptor binding af-
comparison to NT(8-13), which is known to enhance
finity, it is rapidly degraded by peptidase action and
the potential for blood-brain barrier access.5 It is,
does not cross the blood-brain barrier to a sufficient
however, 3-25 times less potent as a NT agonist than
degree to permit peripheral administration. A successful
strategy to increase biological half-lives of peptides has
been to produce analogues with N-terminal alterations
to prevent inactivation of the peptide through ami-
NT(8-13) depending on the type of assay in which the
peptides are evaluated.6
In this work, preparation and initial evaluation of a
series of NT(8-13) analogues featuring novel modifica-
nopeptidase-catalyzed removal of the N-terminal resi-
tions of the N-terminal Arg(8) residue are reported. The
dues. For example, in NT derivatives, substitution of
modifications were designed to enhance the physio-
the Arg(8)-Arg(9) amide with a reduced peptide bond
chemical parameters of the peptide to maintain receptor
binding while increasing overall lipophilicity that ulti-
mately may translate into higher in vivo activity. The
general structure of these peptides is provided in Figure
has enhanced stability in the NT(8-13) analogue
[Lys(8)ψ[CH2NH]Lys(9)]NT(8-13) while maintaining
comparable agonist activity.3 In comparison, the N-R-
methylated analogue NT-1 (Figure 1) is stable and
1. All peptides feature substitution of an R-azido group
reportedly crosses the blood-brain barrier.4 This likely
for the N-terminal R-amino group. This modification
was intended to hinder aminopeptidase action while
results from the peptide’s enhanced lipophilicity in
enhancing overall lipophilicity and thus potential for
* To whom correspondence should be addressed. Phone: 843-953-
6614. Fax: 843-953-6615. E-mail: dixta@musc.edu.
blood-brain barrier access.5 This free amino group is
10.1021/jm9903444 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/21/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4915
Sch em e 1
not essential for receptor binding since it is present in
an amide bond in the parent peptide. In addition, the
peptides feature substitution of the Arg(8) side chain
with alkylated derivatives of L-Orn, L-Lys, and L-
homolysine (L-Hlys). Peptides in which L-Orn and L-Lys
were substituted for Arg(8) in NT(8-13) have been
shown to maintain high receptor binding affinity.7 These
modifications were designed to further increase the
peptides’ overall lipophilicity while exploring electro-
static and steric requirements for receptor binding at
this site. Of considerable interest is that, although
proteins are known to be methylated posttranslationally
in vivo8 and the properties of a number of nonpeptidic
drugs are improved through amine alkylation,9 this
strategy has not been applied to any degree in peptide
design. The relative binding of all new compounds to
hNTR 1 (the “high-affinity” NT receptor) demonstrates
the validity of the structural changes, which indicates
that the strategies utilized may be applicable to the
modification of any peptide of potential therapeutic
interest that contains important cationic residues.
Resu lts
Ch em istr y. The specific structures of the NT(8-13)
analogues prepared for this work, peptides 1-12, are
shown in Scheme 1. It was thought that these nonnatu-
ral cationic side chains could be prepared routinely
using standard solid-phase peptide synthetic proce-
dures, with condensation of the appropriately protected
alkylated amino acid with resin-bound NT(9-13). Ac-
cordingly, the ω-bromo-2(S)-azido acids 13-15, synthe-
sized previously in this laboratory,10 were readily
converted to N-ω-alkylated analogues of Orn, Lys, and
Hlys, respectively, and N-R-Fmoc derivatized in prepa-
ration for solid-phase peptide synthesis. It was found,
however, that solubility incompatibilities and stability
problems due to the inability to protect the side chains
of the N-R-Fmoc tertiary and quaternary alkylated
amino acids hindered facile peptide bond condensation.
A simpler and more efficient route (Scheme 1) evolved
from incorporation of 13-15 directly into the N-terminal
position of NT(9-13) through DCC-HOBt-mediated
condensation to produce intermediates 16-18 (yields ca.
50%). These intermediates then were treated with 100
equiv of the requisite amine nucleophile in aqueous
ethanol and stirred overnight at ambient temperature
and pressure to produce the alkylation patterns in
1-12. After solvent removal, purification by RP-HPLC
resulted in product yields of 80-90% from 16-18. At
this point the azido group could be reduced to the
R-amino functionality if desired, but for the current
work, it was retained as a more lipophilic group
expected to be aminopeptidase-resistant. Notably, the
choice of nucleophilic substitution of the ω-bromo-2(S)-
azido acids 13-15 enables unequivocal definition of the
alkylation state of the resulting amine, in contrast to
the classical treatment of amines with alkyl halides
which can lead to polyalkylations.11 Overall, this paral-
lel method for the synthesis of 1-12 was efficient in that
side-chain protections were not necessary and only three
brominated intermediates (16-18) needed to be syn-
thesized and purified to produce the desired products
(and potentially many others) by nucleophilic substitu-
tions.
Assa ys. After synthesis, the structures of NT(8-13)
analogues 1-12, NT(8-13), acetyl-NT(8-13), [Hlys(8)]-,
[Lys(8)]-, and [Orn(8)]NT(8-13), NT(9-13), and bro-
moazido intermediates 16-18 were characterized by
mass spectrometry. All compounds exhibited correct
molecular weights (Table 1) and sequential breakdown
patterns (data not shown). The peptides were assayed
by RP-HPLC to assess purity and demonstrate that the
P ep tid e Ch a r a cter iza tion s a n d h NTR Bin d in g

4916 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ta ble 1. Binding Affinity Comparison and Analytical Data
Brief Articles
philic “space saver” to be converted into the desired
cationic side chain, and the R-azido group can be
maintained as an unreactive/uncharged form of the
R-amino group or reduced to the R-amino group if
desired. Although not performed for the current set of
experiments, further couplings to produce peptides
containing internal alkylated amino acids appears
straightforward, although tertiary and quaternary
amines may lead to the solubility problems noted in
Results. Therefore, this synthetic strategy potentially
is applicable to the modification of any peptide contain-
ing cationic residues in which alteration to improve its
physiochemical characteristics is desired. The parallel
nature of the strategy enables a wide variety of novel
peptides to be produced rapidly or in sets to undergo
biological evaluation with the intent of sequentially
optimizing the alkylation pattern of a given residue in
an efficient fashion.
Substitution of the R-azido for the R-amino group has
significance toward the pharmacokinetic properties of
NT(8-13) analogues 1-12, and peptides in general, in
which it is incorporated. Since aminopeptidase action
is a major determinant of biological half-life for peptides,
R-azido substitutions are expected to increase the pep-
tides’ in vivo stabilities. This is under evaluation in our
laboratory at this time. A second effect of the R-azido
group is to increase the lipophilicity of the peptides by
removal of a cationic charge. Traditionally, these effects
have been accomplished in peptide modifications by
acetylation of the N-terminus. While both changes will
inhibit aminopeptidase action, the R-azido group also
is more lipophilic than the acetyl group since analogues
1-12 have RP-HPLC retention times 1-2 min longer
than acetyl-NT(8-13). This increase in lipophilicity for
the azido analogues can be attributed to removal of the
hydrogen-bonding ability in the acetyl amide. Most
significantly, comparison of the relative binding of
NT(8-13) analogues 1-12 to acetyl-NT(8-13) demon-
strates that the R-azido is a better choice than acety-
lation due almost certainly to the latter group’s greater
steric bulk. As with acetyl-NT(8-13), some overall loss
in binding affinity in the R-azido analogues, in com-
parison to the [Hlys(8)]-, [Lys(8)]-, and [Orn(8)]NT(8-
13) standards, was observed. This indicates that a minor
unfavorable steric effect is associated with the R-azido
group. This effect of substituting the R-azido for R-amino
group will have variable importance in different pep-
tide-receptor interactions, depending on the nature of
the active site binding motifs that are involved.
relative
affinitya
MW
peptide
tRc (min) foundd (calcd)
NT(8-13)
100b ( 12.7
15 ( 3.7
22.1
23.2
21.6
25.1
25.4
25.6
25.6
21.7
24.5
24.9
25.1
25.1
21.6
24.1
24.3
24.4
24.6
22.4
36.2
34.4
32.9
817.0 (817.0)
859.2 (859.0)
803.0 (803.0)
828.8 (829.0)
843.0 (843.0)
856.8 (857.1)
872.0 (872.1)
789.0 (789.0)
815.0 (815.0)
829.0 (829.0)
843.0 (843.0)
858.0 (858.1)
775.0 (775.0)
800.8 (801.0)
814.8 (815.0)
829.0 (829.0)
844.0 (844.1)
660.8 (660.8)
893.3 (892.9)
879.3 (878.9)
865.3 (864.9)
acetyl-NT(8-13)
[Hlys(8)]NT(8-13)
1
2
3
106 ( 10.2
74 ( 3.4
54 ( 10.7
35 ( 6.4
4
34 ( 13.8
64 ( 7.3
[Lys(8)]NT(8-13)
5
6
7
57 ( 4.2
55 ( 15.5
39 ( 0.6
8
32 ( 6.4
[Orn(8)]NT(8-13)
9
10
11
63 ( 18.9
41 ( 13.6
29 ( 8.7
24 ( 9.5
22 ( 1.5
0.4 ( 0.084
0.6 ( 0.078
2.2 ( 0.25
2.1 ( 0.18
12
NT(9-13)
16
17
18
a
Binding data were obtained by assaying the ability of an
analogue to compete with [125I-Tyr(3)]NT (0.15 nM) for human NT
(Leu194) receptors as described in the Experimental Section. Assays
were performed in triplicate and are the geometric means reported
relative to NT(8-13) with (SE given as a percentage of the
b
geometric mean. The measured Kd which corresponds to 100%
relative affinity for NT(8-13) was 5.4 nM. This compares well to
the literature value of Cusack et al.7 (12 nM). c Analytical RP-
HPLC retention times were obtained using system 1 in the
d
Experimental Section. A mass spectrum for each analogue was
obtained.
azido-containing peptides were more lipophilic than the
N-terminal amino standards. As indicated in Table 1,
all R-azido-N-alkylated peptides exhibited longer reten-
tion times and thus increased lipophilicity; the un-
charged 8-position ω-bromo compounds 16-18 are
drastically more lipophilic. Within the 8-position N-
alkylated series, greater lipophilicity was exhibited with
extent of alkylation, as expected.
Table 1 also provides relative hNTR receptor binding
affinity of all peptides. The two standards, NT(8-13)
and acetyl-NT(8-13), exhibited comparable binding to
those reported previously in the literature,12 with acety-
lation decreasing the relative binding affinity of NT(8-
13) by almost 1 order of magnitude. While only
[Hlys(8)]NT(8-13) was comparable to NT(8-13) in
absolute binding efficiency, all of the other peptide
analogues were well within 1 order of magnitude (22-
74%) as efficient. Severe loss of binding efficiency was
demonstrated with removal of the cationic 8-position
residue in NT(9-13), while the 8-position ω-bromo-R-
azido-containing peptides also exhibited poor binding.
Among NT(8-13) analogues, [Lys(8)ψ[CH2NH]Lys(9)]-
NT(8-13) (Figure 1) and the N-R-methylated compound
NT-1 (Figure 1) are most closely related to 1-12 in that
all analogues were designed for enhanced stability
through inhibition of aminopeptidase degradation. The
current analogues may have the most potential for use
in vivo, based on the following considerations. These
compounds are more lipophilic than [Lys(8)ψ[CH2NH]-
Lys(9)]NT(8-13) and NT-1 as evidenced by the follow-
ing comparison. Each analogue demonstrated an in-
crease of about 3 min in RP-HPLC retention time (Table
1) compared to the standards NT(8-13) and [Orn(8)]-,
[Lys(8)]-, and [Hlys(8)]NT(8-13), which is primarily
attributable to loss of the N-terminal charge. Each
methyl or methylene addition within this series adds
approximately 15 s to retention time, with the homol-
ysine group of five methylene units being the most
Discu ssion
The NT(8-13) derivatives of interest, peptides 1-12
(Scheme 1), were prepared by an extension of a proce-
dure for the synthesis of N-alkylated amino acids that
was developed recently in our laboratory.10 Incorpora-
tion of the appropriate ω-bromo-2(S)-azido acid into a
nascent peptide chain at the site in which substitution
of an N-alkylated side-chain amino acid is desired leads
to the ability to substitute virtually any alkylated
nucleophile of interest and the potential for preparation
of a wide variety of novel N-terminal residue-modified
peptides in a parallel fashion. In the current synthetic
strategy, the ω-bromo group is used as an inert lipo-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4917
lipophilic. Under the conditions in which the RP-HPLC
runs were performed, a 1-min retardation of elution
time corresponded to approximately a 10-fold difference
in octanol/water partitioning coefficient (data not shown);
hence the range in relative lipophilicities within the set
of analogues 1-12 is fairly broad. This could be a
distinct advantage for the current set of compounds as
there may be an optimal degree of lipophilicity that
allows for sufficient barrier access in the absence of
undesired sequestering in other lipophilic sites of the
blood and various tissues.5 Most importantly, all new
analogues exhibited comparable binding affinities to
NT(8-13) and [Lys(8)ψ[CH2NH]Lys(9)]NT(8-13) (which
does not cross the blood-brain barrier) and significantly
better than NT-1 (which does). The abilities of analogues
1-12 to cross the blood-brain barrier will be evaluated
shortly.
While analogues 1-12 all possess subnanomolar
binding affinities to hNTR 1 (Table 1), the relative
binding affinities reveal important information about
the structure of the peptide-receptor interaction. The
order of affinity for position 8 amino groups was primary
> secondary > tertiary > quaternary, irrespective of
side-chain length. Therefore it is evident that addition
of alkyl functionalities decreases the interaction at the
receptor by either steric or electrostatic means since all
of the subgroups follow the same order. With respect to
side-chain length, the homolysine side chain provides
binding similar to that of the native guanidino group,
indicating that it places the localized charge in a similar
location to that of the resonating guanidino group of
native Arg(8). In general, increasing the overall chain
lengths at this position led to overall moderate increases
in binding efficiencies across the homologous series,
which indicates that the binding site for this residue in
the hNTR is fairly elastic. The binding affinities of the
brominated analogues 16-18 also were evaluated;
removal of charge in these analogues results in a 98-
99% loss in binding affinity. This demonstrates that
alkylation of the cationic side chains results in only
minimal losses in binding efficiency through adverse
steric interactions as long as the cationic nature of the
residue is maintained. The minor losses in binding
efficiencies with side-chain alkylations parallel the effect
of the R-azido group, which exhibits only a 25-35% loss
in affinity at the hNTR when substituted for the
R-amino group. Thus, while these analogues may exhibit
minor decreases in overall potency compared to the
standards, this may be tolerable with the gains in
lipophilic character (and potential for blood-brain bar-
rier access) and overall stability when the relative in
vivo potencies are evaluated.
proteins13,14 or alkylate with one to three methyl sub-
stituents in others.15 The role of alkylations on peptide
activity is not fully understood, but it appears that these
posttranslational modifications serve to balance overall
peptide activity or serve as markers for degradative
processes. The current modification strategy thus can
be considered biomimetic.
Con clu sion
In summary, two types of modifications intended to
enhance peptide stability and potential for increased
barrier accesssconversion of the N-R-amino group to an
N-R-azido moiety and alkylation of cationic side chainss
were explored in the context of NT(8-13) binding to
hNTR 1. Both modifications led to more lipophilic
analogues with comparable binding efficiencies to the
parent peptide. The parallel mode of synthesis allows
for quick optimization of sterics and electrostatics at
cationic positions and hence appears to be applicable
to peptide design in general. Relative stability and
blood-brain barrier access of analogues 1-12 in com-
parison to NT(8-13) standards and other active NT
analogues are under investigation, with results to be
reported in due time.
Exp er im en ta l Section
St a r t in g Ma t er ia ls. p-Benzyloxybenzyl alcohol, resin-
bound L-N-R-Fmoc-leucine, L-N-R-Fmoc-isoleucine, L-N-R-Fmoc-
(t-Bu)-tyrosine, L-N-R-Fmoc-proline, L-N-R-Fmoc-(Pmc)-argi-
nine, L-N-R-Fmoc-(t-Boc)-ornithine, and L-N-R-Fmoc-(t-Boc)-
lysine were purchased from Bachem. L-N-R-Fmoc-(t-Boc)-
homolysine was prepared previously in this laboratory.16
Acetyl-NT(8-13) was purchased from Bachem. Ammonia
(29.6% in H2O) was purchased from Mallinckrodt. Methy-
lamine (40% in H2O), dimethylamine (40% in H2O), and
trimethylamine (25-27% in H2O) were obtained from Aldrich.
Other reagents and solvents were analytical reagent grade.
P ep tid e Syn th esis. The leader portion of the peptide was
synthesized in bulk using p-benzyloxybenzyl alcohol solid-
phase methodology on a manual shaker17 and stored in fully
protected form for use in small-scale couplings. Final resin
loading of the pentapeptide was calculated and confirmed by
quantitative ninhydrin test18 of the position 9 Fmoc-depro-
tected R-amino groups. Aliquots of the resin-bound pentamer
(12.5 µmol) were weighed for couplings in
a small-scale
synthesizer19 following general washing protocol. Standard
peptides NT(8-13) and [Orn(8)]-, [Lys(8)]-, and [Hlys(8)]NT(8-
13) were prepared by activating 50 µmol of the requisite
diprotected amino acids with 50 µmol each of HOBt and DCC.
Reactions were monitored with the Kaiser test20 and recoupled
as necessary. The N-terminal Fmoc groups were removed with
20% piperidine in DMF prior to cleavage from the resin. The
halogenated peptide intermediates 16-18 were prepared in a
similar fashion using the required ω-bromo-2(S)-azido acids
13-15. Cleavage from the resin was effected with 300 µL of
H2O:TFA (20:80) (scavengers were not employed due to the
lability of the halogen groups) for 3 h. The acidic solution was
collected in a test tube and the resin was washed twice with
the same solution. To complete final deprotection of the Arg(9)
Pmc group, solutions were stirred vigorously for an additional
3 h in the stoppered test tubes. The crude peptides were
concentrated and purified by preparative RP-HPLC.
Previous to this work, utilization of N-alkyl groups
has proven essential in optimizing selectivity, potency,
lipophilicity/pharmacokinetics, and stability of a number
of drug entities; this work extends the strategy to
peptide-based compounds. Interestingly, this strategy
is one employed in nature as methylation is a standard
component of posttranslational modifications of pro-
teins.8 Cellular enzymes use S-adenosyl-L-methionine
as the methyl donor in O-esterification of aspartyl
residues, N-methylation of the imidazolidine side chain
of histidine, and N-methylation of the ꢀ-N of lysine. The
enzyme responsible for alkylation of lysyl residues,
protein methylase III, is unique in that it can effectively
add three methyl groups on a single side chain in some
Nu cleop h ilic Su bstitu tion . Analogues 16-18 were dis-
solved in 0.5 mL of EtOH. The required amine nucleophiles,
ammonia, methylamine, dimethylamine, or trimethylamine
(100 equiv in H2O), were added and the mixtures were stirred
overnight in stoppered test tubes. The reaction mixtures were
then concentrated prior to purification by RP-HPLC, giving
1-12.
P u r ifica tion . Preparative RP-HPLC was performed on a
Waters dual pump system in combination with a Vydac (10 ×
250 mm, 300 Å, 5 µm) semipreparative column. The mobile
phase consisted of 0.1% trifluoroacetic acid in water (solvent

4918 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Brief Articles
A) and 0.084% trifluoroacetic acid in acetonitrile (solvent B).
Purification of intermediates 16-18 was performed at a flow
rate of 3 mL/min. Linear gradients of 15% to 70% B over 55
min were used and the effluent was detected by UV absorbance
at 254 nm. All standard peptides and final products 1-12 were
purified using a linear gradient of 2% to 50% B over 55 min.
Ch a r a cter iza tion of NT(8-13) An a logu es. Data for
peptide characterization are provided in Table 1. All peptides
were analyzed in two separate analytical HPLC systems to
assess purity and lipophilic character. A mass spectrum for
each analogue was obtained using electrospray conditions since
this method was mild enough to detect the intact azido group.
1. An a lytica l HP LC. System 1: RP-HPLC analysis was
performed on a Waters dual pump HPLC system equipped
with a Bakerbond (C18, 4.6 mm × 250 mm) column. The
solvent system consisted of 0.1% TFA in water (solvent A) and
0.1% TFA in 83% acetonitrile (solvent B). Samples (ap-
proximately 10-30 µg) were eluted with a linear gradient from
5% to 50% B over 30 min at a flow rate of 1 mL/min and
detected by UV absorbance at 220 nm.
System 2: RP-HPLC analysis was performed using an ABI
chromatograph (model 130A, Applied Biosystems) equipped
with an Aquapore 300 column (C8, 2.1 × 30 mm). The solvent
system consisted of 0.1% TFA in water (solvent A) and 0.1%
TFA in 80% acetonitrile (solvent B). Approximately 1-2 µg of
peptide were injected automatically and separated at a flow
rate of 100 µL/min. Usually 60-min gradients from 5% to 50%
B were employed and UV absorbance of the effluent was
recorded at 230 nm. All peptides were of greater than 95%
purity in both systems.
Ack n ow led gm en t. J .T.L. is an American Founda-
tion for Pharmaceutical Education predoctoral scholar.
We thank Prof. Kevin L. Schey (MUSC Mass Spectrom-
etry Facility) for electrospray mass spectrum data and
analysis. The MUSC NMR Facility was used in support
of this work.
1
Su p p or tin g In for m a tion Ava ila ble: 400-MHz H NMR
spectra for one analogue of the new class of peptides, the
methylated peptide 2. This material is available free of charge
via the Internet at http://pubs.acs.org.
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(1) Carraway, R.; Leeman, S. E. Structural Requirements for the
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(2) Litwin, L. C.; Goldstein, J . M. Effects of Neurotensin on Midbrain
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(3) Lugrin, D.; Vecchini, F.; Doulut, S.; Rodriguez, M.; Martinez,
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(4) Banks, W. A.; Wustrow, D. J .; Cody, W. L.; Davis, M. D.; Kastin,
A. J . Permeability of the Blood-Brain Barrier to the
Neurotensin8-13 Analogue NT1. Brain Res. 1995, 695, 59-63.
(5) Pardridge, W. M. Peptide Drug Delivery to the Brain; Raven
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(6) Akunne, H. C.; Demattos, S. B.; Whetzel, S. Z.; Wustrow, D. J .;
Davis, D. M.; Wise, L. D.; Cody, W. L.; Pugsley, T. A.; Heffner,
T. G. Agonist Properties of a Stable Hexapeptide Analogue of
Neurotensin, NaMeAtg-Lys-Pro-Trp-tLeu-Leu (NT1). Biochem.
Pharmacol. 1995, 49, 1147-1154.
(7) Cusack, B.; McCormick, D. J .; Pang, Y.-P.; Souder, T.; Garcia,
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2. Ma ss Sp ectr oscop y. A mass spectrum for each analogue
was obtained using electrospray conditions on a Finnigan LCQ
instrument. The tandem mass spectrum of the [M + 2H]2+ ion
for each analogue gave correct sequential data.
3. Ra d ioliga n d Bin d in g Assa ys. Gen er a l p r oced u r e:
Binding affinity assays for each NT(8-13) standard and
analogues 1-12 were performed on hNTR 1 (Leu194) produced
in CHO Cells from New England Nuclear (Boston, MA).
Membrane preparations (0.5 mL) were diluted with 7.0 mL of
incubation buffer (0.2% BSA in 50 mM Tris-HCl, pH 7.4) at 4
°C. The radioligand [125I-Tyr(3)]NT diluted in 10 µL of incuba-
tion buffer (amount necessary to produce a concentration of
0.15 nM for competition assays) was introduced to each of nine
Eppendorf tubes. Next, after serial dilution through the
appropriate concentration range, 10 µL of a NT(8-13) ana-
logue was added to each of the nine Eppendorf tubes. These
were cooled to 4 °C at which time 150 µL of diluted membranes
was added and allowed to incubate for 60 min at 4 °C.
Incubation of the samples was terminated by dilution with 50
mM Tris-HCl, pH 7.4, buffer (0.5 mL) followed by rapid
filtration through Whatman GF/A filters (25-mm diameter
presoaked in 0.3% poly(ethylenimine)) using a Millipore 1225
sampling vacuum manifold. Each filter was immediately
washed nine times with 3 mL of ice-cold 50 mM Tris-HCl, pH
7.4, and placed in tubes for gamma counting. Assays were
performed in triplicate and are the geometric means reported
relative to NT(8-13) ( SE. Nonspecific binding was deter-
mined in the presence of 100 nM of each analogue and was
found to be 1-2% of total binding in each assay.
Mod ified p r oced u r e: Assays for brominated peptides 16-
18 were modified slightly to lower the probability of nucleo-
philic substitution of the halogen functionalities by high
concentrations of buffer amines. The incubation buffer used
was 0.02% BSA in 25 mM HEPES, pH 7.4, and the wash buffer
employed was 25 mM HEPES, pH 7.4. NT(9-13) was used in
both buffer systems for standardization. Nonspecific binding
was determined in the presence of 20 µM of each ligand.
Abbr evia tion s: all natural amino acids used are of L-
configuration; BSA, bovine serum albumin; t-Bu, tert-butyl;
t-Boc, tert-butoxycarbonyl; CHO, Chinese hamster ovary; DCC,
1,3-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide;
Fmoc, 9-fluorenylmethoxycarbonyl; HPLC, high-performance
liquid chromatography; hNTR, human neurotensin receptor
(Leu194); HOBt, 1-hydroxybenzotriazole; NT, neurotensin;
Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP, reversed-
phase; Tris-HCl, tri(hydroxymethyl)aminomethane hydrochlo-
ride.
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Quantitative Monitoring of Solid-Phase Peptide Synthesis by the
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J M9903444