β- and γ-Di- and tripeptides as potential substrates for the oligopeptide transporter hPepT1

Article abstract of DOI:10.1021/jm070148u

The hPepT1-mediated transport properties of a series of 11 synthesized β- and γ-peptides have been studied in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but only two β-dipeptides act as substrates a

Full text of DOI:10.1021/jm070148u

5238
J. Med. Chem. 2007, 50, 5238-5242
Brief Articles
â- and γ-Di- and Tripeptides as Potential Substrates for the Oligopeptide Transporter hPepT1
Ina Hubatsch,*,† Per I. Arvidsson,§ Dieter Seebach,‡ Kristina Luthman,# and Per Artursson†
Department of Pharmacy, Uppsala UniVersity, Box 580, SE-751 23 Uppsala, Sweden, Department of Biochemistry and Organic Chemistry,
Uppsala UniVersity, Box 576, SE-751 23 Uppsala, Sweden, Laboratorium fu¨r Organische Chemie der Eidgeno¨ssischen Technischen
Hochschule, ETH Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland, Department of ChemistrysMedicinal Chemistry,
Go¨teborg UniVersity, SE-412 96 Go¨teborg, Sweden
ReceiVed February 8, 2007
The hPepT1-mediated transport properties of a series of 11 synthesized â- and γ-peptides have been studied
in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but
only two â-dipeptides act as substrates and are transported across the cell monolayers. These two are less-
efficient substrates than R-peptides. Larger derivatives than â-dipeptides do not act as hPepT1 substrates,
but instead, they appear to be substrates for P-glycoprotein efflux.
Introduction
that several of the compounds interact with the peptide
transporter and that two compounds act as substrates and are
actually transported across Caco-2 cell monolayers. However,
larger derivatives than â-dipeptides, for example, γ-tripeptides,
appear to be substrates for P-glycoprotein efflux.
Peptides composed of homologated amino acids, that is, â-
and γ-peptides, are of increasing significance as novel pepti-
domimetic agents.1 Although, intrinsically more flexible than
R-peptides, â- and γ-peptides have been shown to form stable
secondary structure elements, such as helices and turns,2 more
readily than R-peptides. In fact, only two â- or γ-amino acid
residues are needed for the preparation of turn fragments that
may act as somatostatin mimicking peptidomimetics with good
binding affinities to the sst-receptors.2c,3 Somewhat longer
â-peptides, typically containing less than 10 residues, have also
been shown to possess antibacterial,4 antiproliferative, and
hemolytic activities;4a,b examples of â-peptides capable of
disrupting protein-protein interactions5 are also known. Peptides
composed of â- and γ-amino acids are completely resistant
toward proteolytic and metabolic degradation,6 which has
fortified the interest of such molecules as peptidomimetic agents
for medicinal and biological purposes.
Intrigued by the structural properties of â- and γ-peptides,
we became interested in their potential interaction with the
oligopeptide transport protein hPepT1 found mainly in the
enterocytes in the human intestine. It has been shown that
hPepT1 is involved in the transport of most di-and tripeptides
as well as peptide-based drugs such as â-lactam antibiotics, ACE
and renin inhibitors, and various prodrug derivatives.7 The
structure-transport relationships for hPepT1 have been studied,8
as well as the use of dipeptidomimetics as drug transport
vehicles.9 â-Amino acids and â-dipeptides have also been
investigated for their hPepT1 mediated transport, and it was
shown that the introduction of â-amino acids in the N- or
C-terminal positions of a dipeptide usually decreased the affinity,
but was compatible with binding and transport.8e,10
Results and Discussion
Selection of â- and γ-Di- and Tripeptides. The structures
of the â- and γ-di- and tripeptides 1-11 used in this study are
shown in Figure 1. Based on the amino acids at hand, we aimed
at making the selection of compounds as diverse as possible.
Among the tripeptides investigated were both mono- and
disubstitited aliphatic γ-peptides, that is, 1 and 2, â-peptides
composed of all-â3-amino acids (3-5), and of all-â2-amino acids
(6). Two â-dipeptides with a lysine side chain either in the
2-position (7) or in the 1-position (8) were tested, as well as
the regioisomerically mixed â2/â3-peptides 9 and 10. Also
included was a â3-dipeptide with a thioamide bond, that is, 11.
All â- and γ-peptides were synthesized in solution by standard
EDC/HOBta-mediated coupling of the protected homoamino
acids (N-Boc- or N-Cbz-; O-Bn), as described in the Supporting
Information.
hPepT1-Affinity Studies. A first assessment of the ability
of the compounds to bind to the human peptide transporter
hPepT1 was performed by testing whether the compounds were
able to inhibit the uptake of the PepT1-substrate glycylsarcosine
(GlySar). Due to the limited amount available of the test
compounds, only one inhibitor concentration was used (4 mM).
All the compounds except 1 and 3 showed significant inhibition
of the [3H]GlySar uptake but at a lower level than the positive
control GlySar (Figure 2).
In the present study, we report the hPepT1-mediated transport
properties of a series of â- and γ-peptides. The results show
a Abbreviations: Papp, apparent permeability coefficient; P-gp, perme-
ability-affecting glycoprotein; HEPES, 4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid; MES, 2-morpholinoethanesulfonic acid; Bn, benzyl;
Boc, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl; 2-Cl-Cbz, 2-chloroben-
zyloxycarbonyl; EDC, N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide
hydrochloride; ESI, electrospray ionization; HOBt, 1-hydroxy-1H-benzo-
triazole hydrate; TFA, trifluoroacetic acid. Three-letter amino-acid ab-
breviations are used for homologated amino acid derivatives: âhXaa (ân,
homoamino acid)1 and γhhXaa (γm,n, doubly homologated amino acid),
where n indicates the position of the proteinogenic side chain.
* To whom correspondence should be addressed. Telephone: +46-18-
471 4628. Fax: +46-18-471 4223. E-mail: ina.hubatsch@farmaci.uu.se.
† Department of Pharmacy, Uppsala University.
§ Department of Biochemistry and Organic Chemistry, Uppsala Univer-
sity.
‡ ETH Ho¨nggerberg.
# Go¨teborg University.
10.1021/jm070148u CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/21/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5239
Table 1. Apparent Permeability Coefficients (Papp) for the
Transepithelial Transport of 1-11 Across Caco-2 Monolayers (24 ( 1
days after Seeding)a
Papp × 10-8 cm s-1 ( S.D.
apical-to-basolateral
apical-to-
basolateral
in the presence of
5 mM GlySar
basolateral-to-
apical
cmpd
1
2
3
4
5
6
7
8
9
3.4 ( 0.4
3.3 ( 0.2
4.4 ( 0.3
3.7 ( 0.7
4.9 ( 0.1
2.4 ( 0.1
3.7 ( 0.5b
7.2 ( 0.1
9.5 ( 0.6
61.3 ( 3.3
75.4 ( 6.6
2.6 ( 0.1
2.8 ( 0.3
4.3 ( 0.6
3.1 ( 0.2
3.9 ( 0.7
2.3 ( 0.3
n.d.c
4.5 ( 0.2
5.5( 0.6
29.1 ( 1.8
39.3 ( 3.0
9.4 ( 1.1 (n ) 2)
9.7 ( 1.0
n.d.
7.1 ( 1.4
6.1 ( 0.9
7.1 ( 1.0
7.8 ( 0.5
7.5 ( 1.1
8.4 ( 0.9
12.9 ( 1.5d
12.4 ( 0.4
10
11
a Measurements done in buffered HBSS, apical pH 6.0, basolateral pH
7.4 (n ) 3-4). See Supporting Information for experimental conditions.
b Because some time-dependent samples (45, 90, and 120 min) were below
the detection limit, the apical-to-basolateral value is based on measurements
after one single time point (90 min). c n.d. ) not determined. d Evaluation
45-90 min.
also acted as substrates. These studies were done both in the
apical-to-basolateral (absorptive) and in the basolateral-to-apical
direction to determine if there is a vectorial transport. The
transport in the absorptive direction was also studied in the
presence of the competitive inhibitor GlySar (33-fold excess).
The results are shown in Table 1. The apparent permeability
coefficient (Papp) values measured at the different conditions
show that the permeability for most of the â- and γ-peptides is
considerably lower than for R-peptides (for example, Val-Val11,
Papp ) 1.9 × 10-7 cm s-1 or the metabolically stable GlySar,
Papp ) 1.1 ( 0.1 × 10-5 cm s-1, own data). The Papp values
indicate active, PepT1-mediated transport only for the smaller
â-dipeptides 10 and 11. These compounds show significantly
higher transport in the apical-to-basolateral direction compared
to the reversed direction and the apical-to-basolateral transport
is significantly reduced when GlySar is added at the pH value
when PepT1 is activated (pH 6). Again, a comparison between
the isomeric 9 and 10 shows that 10 interacts more favorably
with PepT1, indicating that a substituent in the â2-position seems
to induce a more favorable conformation for proper PepT1
interaction than in the â3-position.
Figure 1. Structure of the synthesized and tested â- and γ-di- and
tripeptides.
Figure 2. Relative [3H]GlySar uptake into Caco-2 cell monolayers in
the presence or absence of the 4 mM competitor (1-10, GlySar); error
bars represent SD (n ) 3); *significant difference against the control
(-) at p < 0.05; **significant at p < 0.005.
However, the size of the whole compound is also of
importance, it cannot be too large, as, for example, 2, 5, or 7.
It has previously been shown that an N-terminal valine residue
enhancestheaffinityandtransportbyPepT1ofbothpeptidomimetics9c
and of drugs such as valacyclovir12 and valganciclovir.13
However, for the γ- and â2-derivatives 1 and 6, it is likely that
the size prevented an efficient binding to PepT1, whereas the
smaller â3-dithiopeptide 11 fits considerably better and can even
be translocated across the Caco-2 cell monolayer by PepT1.
Thioamides have previously been shown to be good substrates
for PepT1, studies on AlaΨ[CS-N]Pro showed that the trans-
amide conformer of the thioamide is preferentially transported
by PepT1.14
Surprisingly, compounds 2 and 10 were found to be the most
potent inhibitors, though the size of 2 would be expected to
prevent an efficient interaction with the hPepT1 binding site.
Compounds 4-9 were of equal activity. A comparison between
the activities of 9 and 10 is of special interest as these
compounds are positional isomers; in 9 the methyl group is
positioned R to the N-terminal amino group but in 10 it is in
the â-position. As shown in Figure 2, there is a significant
difference in the inhibitory properties between the isomers, with
compound 10 being most active. Thus, the results show that
only small changes in substrate structures can have a large
impact on binding to PepT1.
To investigate if the difference in PepT1 transport properties
of the tested dipeptide derivatives 9-11 depended on their
conformational properties, the compounds were subjected to
conformational analysis using molecular mechanics calculations
(Amber force field as implemented in the MacroModel program,
version 7.1). The results showed that 10 and 11 had similar
conformational preferences, whereas the low-energy conforma-
Transepithelial Transport Studies. As most of the com-
pounds were found to interact with PepT1, transepithelial
transport studies were carried out to determine whether they

5240 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Brief Articles
mL/min, with UV detection at 220 nm, and then lyophilized. The
purified peptides were then analyzed by analytical RP-HPLC on a
Merck Lichrospher 100 column (RP-18, 100 × 4.6 mm) with the
gradient given for each peptide and by analytical HPLC on a
Thermo HyPurity Aquastar column (250 × 2.1 mm) using another
gradient system (see Supporting Information).
Mass Spectrometry. Mass spectrometrical analyses of the
synthesized peptides were performed on a Finnigan MAT TSQ 700
(ESI) and reported as m/z (% of basis peak).
Synthetic Procedures. Peptide Synthesis. In short, all peptides
were synthesized in solution by standard EDC/HOBt-mediated
coupling of the protected homoamino acids (N-Boc- or N-Cbz-;
O-Bn). Detailed descriptions for the preparation of protected â-
and γ-peptides can be found in references 2a, 17, and 18 and in
the Supporting Information.
Catalytic Hydrogenation of Peptides (GP 1). The Cbz-, 2-Cl-
Cbz-, or -OBn-protected peptide was dissolved in MeOH (0.1 M),
and Pd/C (10%; 0.1 equiv) was added after careful degassing and
saturation of the solvent with argon. The reaction flask was
evacuated and flushed with H2 several times, and the reaction
mixture was stirred under an atmosphere of H2 (1 bar) for 16 h.
Subsequent filtration through Celite and concentration under
reduced pressure afforded the desired product.
tion of 9 was slightly different, especially in the N-terminal
region (data not shown). These differences might prevent the
proper interaction of 9 with hPepT1. It was, however, observed
that the low-energy conformations of all three compounds were
quite similar, and the analysis could not clearly illustrate which
small modifications of a structure are needed for a compound
to act as substrate for a transporter.
Compounds 2 and 7 showed a considerably higher transport
rate in the basolateral-to-apical direction than in the apical-to-
basolateral direction in the pH gradient system (which is needed
for PepT1-activity) and were therefore studied further at pH
7.4 to try to find the cause of this apparent efflux. For 7, the
Papp values measured at pH 7.4 did not show any significant
difference between apical-to-basolateral and basolateral-to-apical
transport measured over a time interval of 180 min and neither
did the P-gp inhibitor GF12091815 influence the transport [the
values were Papp ) 4.4 ( 1.7 × 10-8 cm s-1 (apical-to-
basolateral), 5.1 ( 0.4 × 10-8 cm s-1 (basolateral-to-apical),
and, in the presence of 2 µM GF120918, Papp ) 2.9 ( 1.3 ×
10-8 cm s-1 (apical-to-basolateral) and 4.9 ( 0.7 × 10-8 cm
s-1 (basolateral-to-apical)]. From these results, we conclude
that 7 is not a substrate for an efflux transporter. Furthermore,
Papp values measured in the absorptive direction, either with
or without a pH gradient, are comparable, confirming that
PepT1 should not be involved in the transepithelial transport
of 7.
On the other hand, the Papp values measured in the secretive
direction for the γ-tripeptide 2 differ in absence (11.5 ( 1 ×
10-8 cm s-1) and presence (5.3 ( 0.9 × 10-8 cm s-1) of the
P-gp inhibitor, indicating that 2 is a potential P-gp substrate;
the absorptive transport of 2 was not significantly changed at
pH 7.4 (Papp ) 2.1 ( 1 × 10-8 cm s-1) compared to the pH
gradient, but seems slightly higher in presence of GF120918
(Papp ) 5.3 ( 2.4 × 10-8 cm s-1), an observation that would
strengthen the hypothesis of 2 being a substrate for P-gp. In
line with these observations, Gao et al. have reported that three
Ala-Phe hydroxyethylamine bioisostere-containing peptidomi-
metics are substrates for P-gp.16
Boc-Deprotection of Amino Acids or Peptides (GP 2). The
Boc-protected peptide was dissolved in CH2Cl2 (1 M) at rt under
argon. After cooling to 0 °C, the same amount of TFA was added,
and the resulting homogeneous reaction mixture was stirred for 2
h at rt. The solvent was removed under reduced pressure, and the
resulting oily residue was stripped three times with diethyl ether.
Finally, the residue was dried at high vacuum (0.01-0.1 Torr)
overnight.
H2N-(S)-â3hLys-(S)-â3hPhe-OH (8). Boc-â3hLys(2-Cl-Cbz)-â3-
hPhe-OBn (100 mg; 0.14 mmol)6 was hydrogenated as described
in GP 1. The Bn-deprotected peptide was directly treated with TFA
(GP 2) to yield crude 8. Purification by preparative HPLC (Method
A), followed by lyophilization, gave â-dipeptide H2N-â3hLys-â3-
hPhe-OH as a di-TFA salt (15 mg; 19% yield). Analytical HPLC
(5% A for 15 min; 5 f 50% A over 15 min; C18): tR 23.7 min,
purity >98%. ESI-MS (positive mode): 322.2 (100, [M + H]+).
Caco-2 Cell Studies. In short, the studies were performed on
Caco-2 cell monolayers (ATCC, passage 94-100) grown on cell
culture inserts maintained at 10% CO2 in an incubator with a
humidified atmosphere at 37 °C. Cells were seeded on polycar-
bonate cell culture inserts (4.4 × 105 cells per cm2, Transwell
system, diameter 12 mm, pore size 0.4 µm, Corning Costar, The
Netherlands) and were allowed to differentiate for 24 ( 1 days in
DMEM supplemented with 10% fetal calf serum, 1% nonessential
amino acids, and PEST.
The transepithelial resistance values of the monolayers were 285
( 45 Ω cm2 (n ) 49 filters) before the experiment and had
decreased in average 30 Ω cm2 after the transport studies.
Briefly, experiments were performed in Hank’s balanced salt
solution (HBSS) and a pH-gradient was applied, which is required
for PepT1 activity (apical side pH 6.0 by addition of 10 mM MES,
basolateral side pH 7.4 by addition of 25 mM HEPES); exceptions
were the experiments performed to evaluate P-gp interaction, which
were done at pH 7.4. For solubility reasons, a final concentration
of 0.5% DMSO was included during the experiments. During
transport experiments, samples were withdrawn at regular time
intervals and replaced by the same volume of pretempered HBSS
+ 0.5% DMSO. The Papp were calculated according to Papp ) dQ/
dt × 1/(A × c0), where dQ/dt is the steady-state flux (µmol/s), A is
the surface area of the filter (cm2), and c0 is the initial concentration
in the donor chamber at each time interval (µM).
Conclusions
The â- and γ-peptides studied here are less-efficient substrates
for hPepT1 than their R-homologues. Larger compounds than
â-dipeptides do not act as substrates for hPepT1, instead they
appear to be substrates for P-glycoprotein efflux. However,
many of the tested derivatives act as inhibitors of Gly-Sar
transport, which show that they do interact with the binding
site of hPepT1. Interestingly, also for this type of derivative
the results show that small modifications in structure result in
loss of transport properties.
Experimental Section
Peptide Synthesis and Purification. HPLC Analysis and
Purification. All HPLC related to peptide purification and analysis
after preparation was done on a Merck LaChrom HPLC system
(L-7150 pump, L-7400 UV detector (variable wavelength monitor)).
RP-HPLC analysis of the crude product was performed on a Merck
Lichrospher 100 column (RP-18, 100 × 4.6 mm) using a linear
gradient (5-100% A in 30 min) of A (acetonitrile with 0.1% TFA)
and B (H2O with 0.1% of TFA) at a flow rate of 1.2 mL/min, with
UV detection at 220 nm. Crude products were separated and purified
by preparative RP-HPLC on a Macherey-Nagel C18 column/
Nucleosil 100-7 C18 (250 × 21 mm) using a gradient of A and B
(Method A: 5 f 30% A over 25 min, 30% isocratic for 3 min, 30
f 50% A over 2 min; Method B: 10 f 40% A over 30 min, 40%
isocratic for 2 min, 40 f 50% A over 2 min) at a flow rate of 20
For affinity studies, cells monolayers grown on the filter supports
were incubated for 5 min, including 1 µCi [3H]GlySar/mL (250
nM) with or without addition of 4 mM of the respective test
compound in the apical donor chamber. Uptake was stopped by
removing the uptake solution, followed by three washes of the filters
in ice-cold HBSS, and the radioactivity of the monolayers was
determined.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5241
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Acknowledgment. We thank Drs. Ju¨rg Schreiber, Meinrad
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protected â- and γ-peptides. Financial support was obtained from
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Supporting Information Available: Detailed experimental
procedures for the Caco-2 cell studies and the synthesis of
compounds 1-9. This material is available free of charge via the
Internet at http://pubs.acs.org.
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5242 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Brief Articles
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strates for the oligopeptide transporter and P-glycoprotein in the
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(17) Seebach, D.; Ciceri, P. E.; Overhand, M.; Jaun, B.; Rigo, D.; Oberer,
L.; Hommel, U.; Amstutz, R. H. Widmer Probing the helical
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Synthesis and solution structures of constitutional isomers, a novel
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