Solid phase synthesis of novel α/β-tetrapeptides, electrospray ionization mass spectrometric evaluation of their metal cation complexation behavior, and conformational analysis using density functional theory (DFT)

Article abstract of DOI:10.1002/poc.1328

Thirty-four novel α/β-tetrapeptides (1-34) have been prepared employing solid-phase and in-parallel synthetic protocols. α/β- Tetrapeptides 1-34 were prepared by a combination of three α-amino acid residues (alanine (Ala), phenylalanine (Phe), and isoleucine (Ile)) with one β-amino acid residue (β³-homophenylglycine). The corresponding complexes of several selected α/β-tetrapeptides with alkali, alkaline earth, and transition metals, [tP + M⁺], were evaluated using ion electrospray-ionization mass spectrometry (ESI-MS). According to the results from analysis of mixtures, we can conclude that the position of the β-amino acid is determinant in the affinity toward different metal cations. Computational modeling (DFT, B3LYP 6-311++G) provided useful information regarding the most likely coordination sites of the metal ions on the receptor α/β-tetrapeptide 12, HO₂C-α- Phe-α-Phe-α-Ile-β³-hPhg-NH₂, as well as the conformational changes induced by the metal upon [tP + M⁺] complex formation. Copyright

Full text of DOI:10.1002/poc.1328

Research Article
Received: 10 December 2007,
Accepted: 21 December 2007,
Published online in Wiley InterScience: 2008
(www.interscience.wiley.com) DOI 10.1002/poc.1328
Solid phase synthesis of novel
a/b-tetrapeptides, electrospray ionization mass
spectrometric evaluation of their metal cation
complexation behavior, and conformational
analysis using density functional theory (DFT)
a
a
b
b
*
˜
´
Yamir Bandala , Judit Avina , Tania Gonzalez , Ignacio A. Rivero
and Eusebio Juaristi
a**
Thirty-four novel a/b-tetrapeptides (1–34) have been prepared employing solid-phase and in-parallel synthetic
protocols. a/b-Tetrapeptides 1–34 were prepared by a combination of three a-amino acid residues (alanine (Ala),
phenylalanine (Phe), and isoleucine (Ile)) with one b-amino acid residue (b3-homophenylglycine). The corresponding
complexes of several selected a/b-tetrapeptides with alkali, alkaline earth, and transition metals, [tP R MR], were
evaluated using ion electrospray-ionization mass spectrometry (ESI-MS). According to the results from analysis of
mixtures, we can conclude that the position of the b-amino acid is determinant in the affinity toward different metal
cations. Computational modeling (DFT, B3LYP 6-311RRG) provided useful information regarding the most likely
coordination sites of the metal ions on the receptor a/b-tetrapeptide 12, HO2C-a-Phe-a-Phe-a-Ile-b3-hPhg-NH2, as well
as the conformational changes induced by the metal upon [tP þ Mþ] complex formation. Copyright ß 2008 John Wiley
& Sons, Ltd.
Keywords: a/b-tetrapeptides; solid phase synthesis; coordination; metal ions; electrospray-ionization mass spectrometry
the interaction between Zn2þ and the His residues. In a particular
INTRODUCTION
example with b-peptides, Seebach and coworkers[15] discovered
that the addition of Zn2þ salts stabilizes the secondary structure
of b-peptides containing b3-hCys and b3-hHis residues. Further-
more, peptides are able to transport metals through cellular
walls;[16] hence, evaluation of peptidic affinity toward metal ions
is fundamental for the understanding of catalytic processes by
proteins.
As a consequence of the fundamental importance of peptides
and proteins in physiological events, the interest in peptide
synthesis as well as in the structural characterization of peptides
has increased exponentially in recent years. Although the
vast majority of natural peptides and proteins are constituted
by a-amino acids, the pioneering and systematic studies of
Seebach[1] and Gellman[2] have shown that the presence of
b-amino acids instead of a-amino acids drastically modifies the
activity and increases the hydrolytic stability of several bioactive
natural peptides. Furthermore, it has been demonstrated that
inclusion of b-amino acids in natural peptides leads in some cases
to an increase in inhibitory ability of platelet aggregation,[3] or to
increased affinity toward opioid receptors.[4,5] It is then very
important to gain knowledge on the nature of the interactions
that allow supramolecular recognition between the peptide’s
secondary structure and potential substrates, since such under-
standing paves the way to potential developments of pharma-
cologically promising peptidomimetics.[6,7]
Electrospray ionization-mass spectrometry (ESI-MS) has proved
to be a versatile method for the analysis of supramolecular
complexes formed in solution and transported into the gas phase
´ ´
Centro de Graduados e Investigacion, Instituto Tecnologico de Tijuana,
*
´
Apartado Postal 1166, 22000 Tijuana, BC, Mexico.
E-mail: irivero@tectijuana.mx
´
** Departamento de Qu´ımica, Centro de Investigacion y de Estudios Avanzados
del Instituto Politecnico Nacional, Apartado Postal 14-740, 07000 Mexico, DF,
´
´
´
Mexico.
E-mail: juaristi@relaq.mx
In this regard, the binding of metal ions to certain functional
groups in the peptide chain can induce the formation of stable
helix or turn conformations.[8–13] For example, Searle and
coworkers[14] synthesized one b-peptide with two and three
histidine (His) fragments close to the N- and C-terminal and
studied the effect of the addition of Zn2þ ions, finding a hairpin
stabilizing effect in the peptide’s secondary structure as a result of
˜
a
Y. Bandala, J. Avina, E. Juaristi
Departamento de Qu´ımica, Centro de Investigacio´n y de Estudios Avanzados
´
del Instituto Politecnico Nacional, Apartado Postal 14-740, 07000 Mexico, DF,
´
´
Mexico
´
T. Gonzalez, A. Rivero
Centro de Graduados e Investigacion, Instituto Tecnologico de Tijuana,
Apartado Postal 1166, 22000 Tijuana, BC, Me´xico
b
´
´
J. Phys. Org. Chem. 2008, 20 349–358
Copyright ß 2008 John Wiley & Sons, Ltd.

Y. BANDALA ET AL.
for detection. In particular, electrospray ionization is sufficiently
gentle that non-covalent complexes can be transferred from the
solution to the gas phase without disruption of the binding
interactions of the complexes.[17–30] Furthermore, based upon
the intensities of complexes in the resulting mass spectra, it is
possible to estimate the relative binding affinities of different
hosts toward different guests.[21–27] This general method
provides the basis for the examination of the metal ion
binding selectivities and stoichiometries of several novel a/
b-tetrapeptides reported in the present study.
Methods
(S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropionic
acid, (S)-b3-hPhg[31–37]
A round-bottom flask equipped with magnetic stirrer was
charged with tert-butyl (S)-3-amine-3-phenylpropionate (2.18 g,
9.9 mmol) in 3 ml of CH2Cl2 at 08C and was added 4 ml of TFA,
and stirred for 16 h at that temperature. After that, the tem-
perature was increased to room temperature and the solvent
was totally removed under vacuum. The lightly brown solid
[(S)-3-amino-3-phenylpropionic acid] was dissolved in a THF/
H2O (92 ml 1:1 v/v) mixture and cooled to 08C, then, 3.3 g
(9.77 mmol) of FmocSuc and 8.21 g (97.74 mmol) of NaHCO3 was
added. The reaction was stirred for 3 h at room temperature and
then the solvent was removed under vacuum. The aqueous
phase was washed with diethyl ether and acidified with HCl 1N
before the extraction with CH2Cl2; the organic phases were dried
under anhydrous Na2SO4 filtered and evaporated under vacuum.
The product was purified by recrystallization from ethyl acetate
obtaining a white solid (73% yield after two steps, 2.8 g,
7.23 mmol); mp ¼ 185–1878C (lit.[42] mp ¼ 1848C). [a]D ¼ ꢀ22.8
(c ¼ 1.1, DMF). Lit.[42] [a]D ¼ ꢀ22.2 (c ¼ 1, DMF).
EXPERIMENTAL
Instrumentation and materials
Wang resin, reagents, solvents, and amino acids employed in this
work were high-purity reagents (>99.5%) and were purchased
from commercial suppliers. Glassware, needles, and magnetic
bars were dried in an oven at 1508C for 24 h before their use.
Anhydrous solvents were obtained by conventional methods.
The purification of organic products was achieved by means of
flash chromatography on 230–400 mesh silica gel, followed by
recrystallization or Kugelrohr distillation. Melting points were
measured on an Electrothermal apparatus and are uncorrected.
Infrared (IR) spectra were recorded on a Perkin Elmer FT-IR 1600
spectrometer. 1H and 13C-NMR spectra were recorded on a JEOL
GSX-270 at 270 MHz, JEOL GSX-400 at 400 MHz, and Bruker
WM300 at 300 MHz Spectrometers in CDCl3 or DMSO-d6 with TMS
as internal standard. The optical rotations were determinated in a
Perkin-Elmer polarimeter model 241 at l ¼ 589 nm and 25–288C
of temperature. The b-amino acid (S)-3-amino-3-phenylpropionic
acid [(S)-b3-homophenylglycine, (S)-b3-hPhg] was prepared
according to the described procedure[31,32] via the highly
diastereoselective conjugate addition of N-benzyl-a-phenylethy-
lamine to t-butylcinnamate.[33,34] The syntheses of a/b-
tetrapeptides were performed on a Vantage aapptec Automation
reactor from Advanced Chem. Tech. Inc. or under microwave
irradiation using a CEM Discover SPS or a CEM Liberty reactor and
were obtained with 90% of purity (determined by HPLC analysis
with a column C18 and 0.1 or 0.05% TFA in H2O—CH3CN with
gradient). Mass spectra were recorded on an Agilent 1100 series
LC/MSD Trap SL spectrometer under the following conditions:
normal scanning mode, 50–2200 m/z range, 13 000 m/z/seg
speed, 14.0 psi nebulizer pressure, drying gas (nitrogen), flow
of 7.0 L/min, 3258C temperature, normal optimization of data
with a ꢀ4500.0 to ꢀ1500.0 V gradient and positive polarity and
the samples were analyzed by direct insertion. Relative
abundances were determined in quintuplicate measurements.
For computational modeling, molecular mechanics conforma-
tional distribution searches were performed using MMFF force
fields method[38] using PC Spartan Pro software (Wavefunction
Inc.[39]). The equilibrium geometries of the lowest energy
conformers were optimized using semiempirical PM3 calcula-
tions and the lowest energy conformer was then subjected to
higher level methods in ab initio calculations using the
Hartree–Fock Model, initially at the 3-21G level and subsequently
at the 6-31G level of theory using Gaussian 03W software
(Gaussian Inc.[41]). Finally, the lowest energy conformers were
subjected to DFT B3LYP 6-311þþG//B3LYP 6-31G calculation. To
ensure the validity of the final structures, all the conformers
obtained were subject of a frequency analysis.
IR (KBr) C ¼O carbamate 1704 cmꢀ1 (lit.[42] 1705 cmꢀ1).
1H NMR (DMSO-d6, 270 MHz) d 2.61–2.78 (m, 2H), 3.44–3.56 (b,
1H), 4.17–4.33 (m, 3H), 4.92–5.00 (m, 1H), 7.16-7.99 (m, 13H), 1.27
(b, 1H).
13C NMR (DMSO-d6, 68 MHz) d 41.1, 46.7, 51.6, 65.3, 120.1, 125.1,
126.3, 127.0, 127.6, 128.3, 140.7, 142.9, 143.7, 143.9, 155.3, 171.7.
Coupling of the first amino acid to the Wang resin
In a round-bottom flask was placed the resin and suspended in
9:1 v/v of CH2Cl2/DMF (approximately 15 ml of solution per gram
of resin) and stirred for 30 min. Separately, 3–5 equivalents of the
N-Fmoc protected b3-amino acid were dissolved in the minimum
amount of DMF. One equivalent of HOBt was then added and the
resulting mixture was stirred before addition of the solution
containing the resin. The reaction mixture was stirred for 10 min
before the addition of 0.1 equiv. of DMAP dissolved in the
minimum amount of DMF as well as 3–5 equiv. of DIC. The
reaction mixture was stirred for 24 h at ambient temperature and
then the resin was separated by means of a glass-sintered filter
and washed three times with DMF, three times with CH2Cl2, three
times with MeOH, and three times with Et2O. The complete
procedure was repeated and the modified resin was treated for
30 min with 2 equiv. of acetic anhydride and 2 equiv. of pyridine
in order to protect the unreacted sites. The amino acid-
containing resin was separated by means of a glass-sintered
filter and washed three times with DMF, three times with CH2Cl2,
three times with MeOH, and three times with Et2O and dried
under vacuum. A small amount of the resin was treated with a
20% solution of piperidine in DMF in order to liberate the amino
acid, whose presence was confirmed by means of the ninhydrin
test.[43] The degree of substitution was determined according to
the procedure of Hamper et al.[44]
Deprotection in the Vantage reactor
A 3-ml solid-phase reactor was charged with the Wang-
AA1-NH-Fmoc (50 mg, 0.03 mmol) product, which was suspended
in a solution of DCM and NMP (9:1 v/v, 1 ml). The resulting
suspension was stirred for 3 min at 600 rpm. The Fmoc group
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METAL CATION COMPLEXATION BY NOVEL ꢀ/ꢁ-TETRAPEPTIDES
bound to the amino acid was removed with 20% of piperidine in
NMP (2 ml, stirring for 40 min) and the amino acid was filtered,
washed with MeOH (2 ꢁ 2 ml) and CH2Cl2 (2 ꢁ 2 ml), and dried
under vacuum.
during 90 min. The resin was recovered by filtration and the
liberated product was collected in a 10 ml vials and treated with
cold anhydrous ether to precipitate the product, then allowed to
stand for 12 h at 0–48C. The supernatant liquid was decanted and
centrifuged and the product dried under vacuum.
Deprotection by means of microwave irradiation
Liberation of HO2C-a/b-tetrapeptide-NH2 by means of
microwave irradiation
In a solid-phase reactor was placed the Wang-AA1-NH-Fmoc resin
and treated with 10 ml of DMF for 15 min before treatment with a
solution of 20% piperidine in DMF. The resulting mixture was
irradiated at 50 W and at 758C during 12 min. The modified resin
was washed with CH2Cl2 and dried under vacuum.
A flask containing the resin-a/b-tetrapeptide material was
suspended in TFA-triisopropylsilane-H2O (95:2.5:2.5) and irra-
diated for 18 min at 20 W, 388C. The resin was separated by
filtration and the filtrate was concentrated under vacuum before
addition of diethyl ether to induce precipitation of the peptide
(12 h at 0–48C). The supernatant liquid was decanted and
centrifuged and the product dried under vacuum.
Identification of the 34 synthesized a/b-tetrapeptides was
performed by ESI mass spectrometry and the purity was
determinated by HPLC analysis.
Amino acid coupling reactions in the Vantage reactor
Three-milliliter solid-phase reactors were charged with the amino
acid-containing resin Wang-AA1-NH2 (50 mg, 0.03 mmol) and
a CH2Cl2-NMP (9:1 v/v, 1 ml) solution. The resulting mixture was
stirred for 3 min at 600 rpm before addition of the corresponding
Fmoc-AA-OH (0.036 mmol) amino acid and HOBt (0.036 mmol) in
NMP (0.75 ml). The reaction mixture was stirred for 2 min before
the addition of DMAP (0.003 mmol) and DIC (0.036 mmol) in NMP
(0.1 ml) and the resulting mixture was stirred for 3 h at ambient
temperature. The modified resin was filtered and washed with
NMP (1 ꢁ 2 ml), MeOH (1 ꢁ 2 ml) and CH2Cl2 (1 ꢁ 2 ml).
Sample preparation for affinity studies
Two saline solutions with mixtures of LiCl-NaCl-KCl-CuCl2-ZnCl2
and LiCl-MgCl2-CaCl2-CuCl2-ZnCl2, and one of the a/
b-tetrapeptide in methanol (1.5 ꢁ 10ꢀ5 M) were prepared.
Mixtures (1:1) of the peptide with each of the saline solutions
were prepared and then let stand for 30 min. The resulting
solutions were analyzed by direct insertion in the mass
spectrometer with electrospray ionization, using a syringe pump
(Kd Scientific, model 100), at 240 ml/h.[45]
Amino acid coupling by means of microwave irradiation
(on a 0.1 mmol scale)
In a solid-phase reactor was placed Wang-AA1-NH2 resin and
10 ml of DMF and the resulting mixture was treated with 5 equiv.
of the Fmoc-AA-OH protected amino acid and 5 equiv. of HBTU.
The coupling reaction was realized at 40 W and 758C for 20 min,
and the modified resin was washed with CH2Cl2. The addition of
the subsequent amino acids was performed using the same
coupling and deprotection procedures in order to obtain the
desired a/b-tetrapeptide chain.
RESULTS AND DISCUSSION
Solid-phase in-parallel synthesis of novel
a/b-tetrapeptides 1–34
Initially, the key b-amino acid (S)-3-amino-3-phenylpropionic acid
[(S)-b3-homophenylglycine, (S)-b3-hPhg] was prepared according
to the described procedure[31,32] via the highly diastereoselective
conjugate addition of N-benzyl-a-phenylethylamine to t-butylci-
nnamate[33,34] (Scheme 1).
b-Amino acid (S)-b3-hPhg was N-protected using 9-fluorenyl-
methoxycarbonyl succinimide (FmocSuc)[35–37] to give derivative
(S)-Fmoc-b3-hPhg in 73% yield (Scheme 1). The synthesis of the
The addition of the subsequent amino acids was performed
using the same coupling and deprotection procedures in order to
obtain the desired a/b-tetrapeptide chain.
Liberation of HO2C-a/b-tetrapeptide-NH2 in the Vantage reactor
Liberation of the modified Wang resin was achieved by treatment
with 2 ml of a TFA-triisopropylsilane-H2O (95:2.5:2.5) mixture
Scheme 1. Synthesis of enantiopure (S)-Fmoc-b3-hPhg
J. Phys. Org. Chem. 2008, 20 349–358
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Y. BANDALA ET AL.
Scheme 2. General scheme for amino acid coupling
desired a/b-tetrapeptides was achieved by means of the Wang
resin,[46–47] which was selected because the liberation of the
peptides is readily carried out under mild conditions, allowing for
smooth liberation in 30 min. The incorporation of b-amino acid
(S)-b3-hPhg to the Wang resin was accomplished by means of a
standard protocol employing 1-hydroxybenzotriazole (HOBt),
4-dimethylaminopyridine (DMAP), and diisopropylcarbodiimide
(DIC) in N-methypyrrolidone (NMP) or N,N-dimethylformamide
(DMF).[48] The deprotection was performed with piperidine at
20% in NMP or DMF. This coupling–deprotection method was
also used to incorporate the other three a-amino acids Ala, Phe,
and Ile, until achievement of the desired amino acid sequence in
the final a/b-tetrapeptide (Scheme 2).
The reactions were carried out in an instrument for
combinatorial chemistry by using a solid-phase reactor or under
microwave irradiation using a manual or automatic peptide
synthesizer, so that a/b-tetrapeptides 1–7, 8–19, and 33, and
20–26 were obtained from commercially available resin-
supported Ala, Phe, and Ile, respectively. Finally, a/b-tetrap-
eptides 27–32 and 34 were prepared in parallel from resin bound
(S)-b3-hPhg. Thus, the library design involved three a-amino acids
(Ala, Phe, and Ile) and one b-amino acid (b3-hPhg) to give a total
number of possible combinations equal to 256. We synthesized
the 34 linear a/b-tetrapeptides shown in Table 1, which includes
the corresponding molecular ions obtained by mass spectro-
metry.
As discussed in the introduction, ESI-MS was deemed as the
most convenient analytical tool to determine the potential
formation of metal ion-a/b-tetrapeptide complexes [tP ꢂ Mþ] in
solution. The method by which binding selectivities are
determined via ESI-MS relies upon comparison of signal
intensities obtained upon spraying solutions containing a single
host molecule and multiple metal ions.[49] A competitive
equilibrium is established in solution, thus leading to a
distribution of a/b-tetrapeptide-metal complexes that reflects
the relative binding constants of the a/b-tetrapeptide-metal
complexes or the selectivity of the peptide host.[23] Upon
electrospraying the solutions, the complexes generated in
solution are transported to the gas phase.[50]
Nevertheless, comparison of the ion intensities for the [tP ꢂ Mþ]
complexes in the mass spectra requires consideration of the
relative ionization efficiencies, which are estimated from the
analysis of the relative response attained by ionization of
individual solutions containing a single a/b-tetrapeptide and a
single metal ion.[50] Thus, the relative spray efficiencies of
different a/b-tetrapeptide-metal complexes were estimated
based on spraying solutions containing a single peptide and a
single metal and comparing the observed intensities for the
different complexes.
In particular, tP 12 (HO2C-a-Phe-a-Phe-a-Ile-b3-hPhg-NH2) in a
2.6 ꢁ 10ꢀ5 M methanolic solution was treated with individual salt
solutions of LiCl, NaCl, KCl, MgCl2, CaCl2, CuCl2 y ZnCl2 at three
different concentrations in MeOH to afford 1:1 mixtures of a/b-tP
and salt. The results from ESI-MS are shown in Fig. 1, which
includes the R2 and m parameters for each salt.
Analysis of mass spectra
A lineal correlation between the ratio of a/b-tetrapeptide and
metal ion in a range of 0.5 to 1.0 equiv. of salt is clearly found.
More important, the substantial difference in ion abundances for
individual complexes confirm rather significant differences in
spray efficiencies. This observation confirms the need for
calibration of measured abundances in competition experiments
(as described below) in order to estimate binding affinities.
Because the slopes for each individual metal ion in Fig. 1 are also
different, we used the relative abundances obtained at a 1:1 a/
Binding properties of alkali/alkaline earth and transition metal
ions to several selected a/b-tetrapeptides (a/b-tP 12, 18, and 31)
were evaluated via ESI-MS methods. These a/b-tetrapeptides
were selected as representative of peptides presenting
the b-amino acid residue in different positions of the a/
b-tetrapeptide. Salts in the form of chlorides for the following
metal ions were used: Liþ, Naþ, Kþ (alkali metals), Mg2þ, Ca2þ
(alkaline earth metals), and Cu2þ, Zn2þ (transition metals).
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J. Phys. Org. Chem. 2008, 20 349–358

METAL CATION COMPLEXATION BY NOVEL ꢀ/ꢁ-TETRAPEPTIDES
Table 1. Molecular ions of the 34 a/b-tetrapeptides synthe-
sized in this work
Table 2. Relative ESI efficiencies for a/b-tetrapeptides 12, 18,
and 31 with alkali metals
HO2C-a/b-tetrapeptide-NH2
[MtP þ H]þ
a/b-Tetrapeptide
1, a-Ala-a-Phe-a-Ala-b3-hPhg
2, a-Ala-a-Phe-a-Ile-b3-hPhg
3, a-Ala-a-Phe-b3-hPhg-a-Ile
4, a-Ala-a-Ile-a-Phe-b3-hPhg
5, a-Ala-a-Ile-b3-hPhg-a-Phe
6, a-Ala-b3-hPhg-a-Phe-a-Ile
7, a-Ala-b3-hPhg-a-Ile-a-Phe
8, a-Phe-a-Ala-a-Phe-b3-hPhg
9, a-Phe-a-Ala-a-Ile-b3-hPhg
10, a-Phe-a-Ala-b3-hPhg-a-Ile
11, a-Phe-a-Phe-a-Ala-b3-hPhg
12, a-Phe-a-Phe-a-Ile-b3-hPhg
13, a-Phe-a-Ile-a-Ala-b3-hPhg
14, a-Phe-a-Ile-a-Ile-b3-hPhg
15, a-Phe-a-Ile-b3-hPhg-a-Ala
16, a-Phe-b3-hPhg-a-Ala-a-Phe
17, a-Phe-b3-hPhg-a-Ala-a-Ile
18, a-Phe-b3-hPhg-a-Phe-a-Ile
19, a-Phe-b3-hPhg-a-Ile-a-Ala
20, a-Ile-a-Ala-a-Phe-b3-hPhg
21, a-Ile-a-Ala-b3-hPhg-a-Phe
22, a-Ile-a-Phe-a-Ala-b3-hPhg
23, a-Ile-a-Phe-b3-hPhg-a-Ala
24, a-Ile-b3-hPhg-a-Ala-a-Phe
25, a-Ile-b3-hPhg-a-Phe-a-Ala
26, a-Ile-b3-hPhg-a-Phe-a-Phe
27, b3-hPhg-a-Ala-a-Phe-a-Ile
28, b3-hPhg-a-Ala-a-Ile-a-Phe
29, b3-hPhg-a-Phe-a-Ala-a-Ile
30, b3-hPhg-a-Phe-a-Ile-a-Ala
31, b3-hPhg-a-Ile-a-Ala-a-Phe
32, b3-hPhg-a-Ile-a-Phe-a-Ala
33, a-Phe-a-Ile-b3hPhg-a-Phe
34, b3-hPhg-a-Phe-a-Phea-Ile
455
497
497
497
497
497
497
531
497
497
531
573
497
539
497
531
497
573
497
497
497
497
497
497
497
573
497
497
497
497
497
497
573
573
Adduct
12
18
31
[M þ Li]þ
[M þ Na]þ
[M þ K]þ
1.88
13.01
4.13
1.00
11.77
2.36
1.00
15.85
4.13
Table 3. Relative ESI efficiencies for a/b-tetrapeptides 12, 18,
and 31 with alkaline earth metals
a/b-Tetrapeptide
Adduct
12
18
31
[M–H þ Mg]þ
[M–H þ Ca]þ
[M–H þ Cu]þ
[M–H þ Zn]þ
4.25
3.19
1.30
1.00
6.78
1.06
6.37
2.37
7.16
1.33
1.03
1.22
Table 4. Relative abundance (estimated affinities, %, multi-
plied by ESI efficiencies) for a/b-tetrapeptides 12, 18, and 31
with alkali metals
a/b-Tetrapeptide
Adduct
12
5.6
1157.9
78.5
18
31
[M þ Li]þ
[M þ Na]þ
[M þ K]þ
5
211.9
33
24
317
66
Table 5. Relative abundance (estimated affinities, %, multi-
plied by ESI efficiencies) for a/b-tetrapeptides 12, 18, and 31
with alkaline earth metals
b-tP to Mþ ratio equal to calculate the normalization factor that
was subsequently used before interpretation of data recorded
when a/b-tetrapeptide 12 was exposed to mixtures of the salts of
interest.
a/b-Tetrapeptide
Accordingly, the relative ESI efficiencies for a/b-tetrapeptide 12
complexes among the series Naþ, Mg2þ, Kþ, Ca2þ, Liþ, Cu2þ
,
Adduct
12
18
31
and Zn2þ are 13.0, 4.25, 4.1, 3.2, 1.9, 1.3, and 1.0, respectively.
Similarly, the relative ESI efficiencies for metal complexes of a/
b-tetrapeptides 18 and 31 were estimated and the results are
collected in Tables 2 and 3.
Calibration of the experimentally obtained abundances
recorded from competitive experiments where a particular a/
b-tetrapeptide is exposed to mixtures of metal ions dissolved in
methanol (a/b-tetrapeptides 1–34 are insoluble in water) affords
the data collected in Tables 4 and 5.
In the case of a/b-tetrapeptides 12 and 18, where the b-amino
acid is located at the amino group terminus or in the inner
section, it is observed that the relative affinity among the alkali
metal mixture is Naþ > Kþ > Liþ, whereas the relative abun-
[M–H þ Mg]þ
[M–H þ Ca]þ
[M–H þ Cu]þ
[M–H þ Zn]þ
357
47.8
3.9
3
67.8
6.4
114.7
18.9
608.6
95.7
568.6
420.9
dances of adducts produced in the presence of divalent alkaline
earth metals were Mg2þ > Ca2þ. On the other hand, for transition
metals Cu2þ and Zn2þ a/b-tetrapeptide 12 showed similar
affinity whereas the trend for tetrapeptide 18 was Cu2þ > Zn2þ
.
In the case of a/b-tetrapeptide 31, where the b-amino acid is
located at the carboxylic group terminus the relative affinity
J. Phys. Org. Chem. 2008, 20 349–358
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Y. BANDALA ET AL.
Figure 1. Ion abundances of complexes formed from a/b-tetrapeptide 12 in the absence of competition with other metals. Range of values from 0.5 to 1
equivalent of salt. m, slope.
among the alkali metal mixture is again Naþ > Kþ > Liþ.
Interestingly, among divalent metal ions a very large affinity
toward Cu2þ and Zn2þ is observed.
Computational analysis
In addition to methodologies such as X-ray diffraction crystal-
lography and NMR spectroscopy that are commonly used to
determine the structural characteristics of peptides and proteins,
molecular modeling using computational methods is becoming
increasingly reliable to predict their lower-energy conformations
and behavior.[51,52]
Figure 2. Conformation of lowest energy obtained for a/b-tetrapeptide
Conformational analysis in the absence of metal ions
12 (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code: gray for carbon,
white for hydrogen, red for oxygen, and blue for nitrogen.
In this computational study, a/b-tetrapeptide 12 (HO2C-a-Phe-a-
Phe-a-Ile-b3-hPhg-NH2) was selected as model system in view of
the well-defined behavior exhibited in the ESI-MS affinity
measurements (as described above). Three initial conformations,
chosen after consideration of the structural behavior already
reported in representative peptides,[53–56] were used as starting
structures in the iterative energy-minimization process. These
starting structures were then subjected to a conformational
analysis where the conformers were distributed according to
their relative energy, using the PC Spartan Pro (Wavefunction Inc.)
program. This conformational analysis is based in MMFF
molecular mechanics MonteCarlo methods that have proved
quite efficient in similar conformational searches.[40]
The structure of nearly forty conformers of low energy
obtained from this process were then optimized by ab initio
Hartree–Fock methods at the 3-21G level, and then the three
lowest energy structures obtained at this level were re-optimized
at the DFT B3LYP 6-31G level of theory and then realized a
single point calculation at B3LYP 6-311þþG level by means of
the Gaussian 03 (Gaussian Inc.) software package.[41] Figure 2
depicts the conformation of lowest energy calculated for a/
b-tetrapeptide 12. That this is a true minimum was confirmed by
frequency analysis.
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METAL CATION COMPLEXATION BY NOVEL ꢀ/ꢁ-TETRAPEPTIDES
Figure 3. Electronic potential surface calculated for a/b-tP 12. The
relative size of the green contours correlates with the relative concentra-
tion of electron density. The hydrogen atoms were removed for clarity.
Figure 5. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Naþ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code:
gray for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
purple for sodium.
Figure 4. Most likely metal coordination sites on a/b-tP 12.
Figure 6. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Kþ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code: gray
for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
purple for potassium.
It can be appreciated in Fig. 2 that the lowest energy
conformation obtained for a/b-tP 12 corresponds to a linear
conformation, where steric repulsion among substituents is
minimum. On the other hand, the arrangement adopted by the
b-amino acid residue b3-hPhg (far right in Fig. 2) allows for
hydrogen-bond formation between the terminal amino group
and the carbonyl group in the same amino acid. Finally, the
lowest energy conformation of a/b-tP 12 depicted in Fig. 2 allows
for p–p interaction between the aromatic groups of the
phenylalanine segments.
Conformational analysis in the presence of metal ions
The potential coordination sites in a/b-tetrapeptide 12 (the
terminal amino and carboxylic groups, internal amide segments
and aromatic phenyl substituents[57]) are highlighted in Fig. 3,
they were derived from the electronic potential surface
calculation using for that purpose the PC Spartan Pro software.
According to the information provided by Fig. 3, one may
anticipate that the most likely binding sites in a/b-tetrapeptide
12 are the carbonyl groups on the peptidic segments, as well as
the terminal amino and carboxylic groups (Fig. 4).
Consideration of the potential coordinating sites depicted in
Fig. 4 suggested[57–63] the use of nine starting structures for the
complexes generated from a/b-tP 12 and each one of the seven
metal ions studied in this work. The nine starting structures for
each cation were then optimized following the same protocol
described for the optimization of a/b-tetrapeptide 12 (as
described above). The most stable complexes a/b-tP 12 ꢂ Mþ
presented in Figs 5–11 were obtained in this fashion.
Figure 7. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Liþ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code: gray
for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
purple for lithium.
Significant conformational differences are appreciated among
the complexes of a/b-tP 12 and the three alkali metals studied.
For example, with Naþ the resulting complex preserves a linear
conformation with the metal dicoordinated to the amino and
carbonyl groups of the b-amino acid residue, b3-hPhg (Fig. 5). In
the case of Kþ (softer and bigger), there is an additional
coordination with the aromatic ring in the same residue[58–63]
(Fig. 6). By contrast, with Liþ (smaller and harder) a tricoordinated
species is predicted, where the metal ion induces a folded
J. Phys. Org. Chem. 2008, 20 349–358
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Y. BANDALA ET AL.
Figure 8. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Ca2þ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code:
gray for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
green for calcium.
Figure 10. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Cu2þ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code:
gray for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
pink for copper.
Figure 11. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Zn2þ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code:
gray for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
pink for zinc.
Figure 9. Optimized conformation and molecular structure for a/b-tP
12 ꢂ Mg2þ complex (B3LYP 6-311þþG//B3LYP 6-31G level). Colour code:
gray for carbon, white for hydrogen, red for oxygen, blue for nitrogen, and
green for magnesium.
significant folding of the original, ‘lineal’ conformation of the
free peptide (Fig. 7), but the metal affinity studies reported in the
first part of this report indicate that complexation to lithium
cation is not so favorable. That is, coordination to sodium cation
causes little conformational changes in the original peptide, and
the resulting complex seems to be most stable by comparison
with those where metal coordination affects the receptor
peptide’s native conformation. This observation might be
relevant in examination of related substrate–receptor phenom-
ena such as in enzymatic activity where conformational changes
of the native protein structure probably lead to modified
structures in the active adducts.[65]
The complex formed by coordination of the soft Ca2þ ion and
a/b-tP 12 (ionic radius for Ca2þ ¼ 100 pm)[64] is calculated to
present the metal ion associated to the a-Phe-a-Ile-b3-hPhg
segment, apparently via coordination to both carbonyl groups, to
the amino group, and to both aromatic rings.[58–63] This
coordination mode gives rise to a folded conformation of the
conformation via association with the a-Phe-a-Ile-b3-hPhg
segment (Fig. 7). It is apparent then that the ionic radius of
the metal is determinant, where the larger Naþ y Kþ ions (102 y
138 pm, respectively)[64] bind more easily to the terminal b-amino
acid residue inducing only small changes in the peptidic
conformation, whereas the electrostatic stabilization afforded
by the smaller and harder Liþ ion (76 pm radius)[50] upon
coordination to the three carbonyls of the a-Phe-a-Ile-b3-hPhg
segment compensates for the otherwise increased energy of the
folded peptide.
The contrasting binding modes for Naþ and Kþ noticed in Figs
5 and 6 do not modify substantially the original conformation of
a/b-tP 12, since the three a-amino acid residues remain
unaltered. Interestingly, complexation to Liþ does provoke
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J. Phys. Org. Chem. 2008, 20 349–358

METAL CATION COMPLEXATION BY NOVEL ꢀ/ꢁ-TETRAPEPTIDES
peptide where the b-amino acid residue constitutes a ‘side chain’
(Fig. 8).
graduate fellowships to T. G., J. A., and Y. B. The authors are also
grateful to M. Parra-Hake for useful discussions.
By the same token, tight Mg2þ ion (ionic radius
for Mg2þ ¼ 72 pm)[64] affords a pentacoordinated adduct with
a/b-tetrapeptide 12. In particular, Mg2þ binds to the four
available carbonyl groups as well as the amino group on the
b-amino acid residue, b3-hPhg. This coordination gives rise to a
drastically folded conformation (Fig. 9).[66,67]
Transition metals Cu2þ and Zn2þ are predicted to form
complexes with a/b-tP 12 that resemble those with Ca2þ
and Mg2þ. Thus, adduct a/b-tP 12 ꢂ Cu2þ presents partial folding
as a consequence of the coordination of the Cu2þ ion with the
carbonyl groups on the terminal a-Phe and the internal peptidic
carbonyls. Interestingly, the calculated conformation of the a/
b-tP 12 ꢂ Cu2þ complex exhibits significant steric hindrance
among the substituents on the peptide, and the anticipated
repulsive interaction may be responsible for the low affinity of the
complex observed in the experimental ESI-MS study (as
described above).
Finally, the adduct arising from coordination between a/
b-tetrapeptide 12 and transition metal Zn2þ shows a distorted
tetrahedral core with the metal bound to both the carbonyl and
amino groups of the b-amino acid residue in addition to the
carbonyl of the inside phenylalanine residue as well as the
carboxylate group of the terminal phenylalanine residue (Fig. 11).
This molecular structure suggests several unfavorable steric
interactions among the a-Phe-a-Phe segment, and such repulsive
effects may be responsible for the experimentally observed low
abundance of [a/b-tP 12 ꢂ Zn2þ] complex.
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Summary
Thirty-four novel a/b-tetrapeptides containing both a and b
amino acid residues (a/b-tetrapeptides 1–34) have been
prepared by solid-phase synthesis and using in-parallel meth-
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J. Phys. Org. Chem. 2008, 20 349–358
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