Selection of amino acids and the biomimetic synthesis of amido bond in the presence of β-CD

Article abstract of DOI:10.1080/00397911.2013.850093

A new method was developed to construct a special amido bond in the presence of β-cyclodextrin. This process is similar to peptide synthesis in organisms. NMR experiments were performed to investigate the possible mechanism. This work has potential application in biomimetic peptide synthesis.

Full text of DOI:10.1080/00397911.2013.850093

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Selection of Amino Acids and the
Biomimetic Synthesis of Amido Bond in
the Presence of β-CD
Jie Su a , Fan Su a , MingFang Ma a , Shangyang Li a , Pengyao Xing a
& Aiyou Hao a
a School of Chemistry and Chemical Engineering, Shandong
University , Jinan , China
Accepted author version posted online: 08 Nov 2013.Published
online: 19 Mar 2014.
To cite this article: Jie Su , Fan Su , MingFang Ma , Shangyang Li , Pengyao Xing & Aiyou Hao (2014)
Selection of Amino Acids and the Biomimetic Synthesis of Amido Bond in the Presence of β-CD,
Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic
Chemistry, 44:8, 1111-1121, DOI: 10.1080/00397911.2013.850093
To link to this article: http://dx.doi.org/10.1080/00397911.2013.850093
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Synthetic Communications®, 44: 1111–1121, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397911.2013.850093
SELECTION OF AMINO ACIDS AND THE BIOMIMETIC
SYNTHESIS OF AMIDO BOND IN THE PRESENCE
OF β-CD
Jie Su, Fan Su, MingFang Ma, Shangyang Li, Pengyao Xing,
and Aiyou Hao
School of Chemistry and Chemical Engineering, Shandong University,
Jinan, China.
GRAPHICAL ABSTRACT
Abstract A new method was developed to construct a special amido bond in the presence of
β-cyclodextrin. This process is similar to peptide synthesis in organisms. NMR experiments
were performed to investigate the possible mechanism. This work has potential application
in biomimetic peptide synthesis.
[Supplementary materials are available for this article. Go to the publisher’s online edi-
tion of Synthetic Communications® for the following free supplemental resource(s): Full
experimental and spectral details.]
Keywords Amino acids; cyclodextrin; tRNA
INTRODUCTION
Peptides, which are short polymers of amino acid monomers linked by peptide
bonds, have recently gained prominence in molecular biology and drug synthesis for
several reasons. First, peptides are involved in many diseases such as human immunode-
ficiency virus-1 (HIV-1) and Alzheimer’s.[1] Second, polypeptide drugs play important
roles in various treatments of disease.[2] Third, peptide antibodies allow molecular cell
biologists to design antibodies for a specific assay of altered forms of proteins. Because
they are amenable to specific immunolocalization of highly similar species, peptide
Received May 28, 2013.
Address correspondence to Aiyou Hao, School of Chemistry and Chemical Engineering, Shandong
University, Jinan 250100, China. E-mail: haoay@sdu.edu.cn
1111

1112
J. SU ET AL.
antibodies can be considered as subcellular probes of cell expression and posttrans-
lational modification.[3] As the building blocks of various biomolecules ranging from
small peptides to large proteins, amino acids, including different kinds and sequences,
are the key factor of peptide properties. To get sequence-specific peptides, solid-phase
peptide synthesis (SPPS) and fermentation from recombinant organisms are the cur-
rent primary methods.[4] Despite many improvements in the area of SPPS the general
concept is still valid: The synthesis proceeds in contrast to peptide biosynthesis from
the C-terminus to the N-terminus and the specificity is ensured by the reaction of a
selected activated amino acid building block with an N-terminally unprotected solid
supported peptide of choice.[5] For the amino acids such as L-lysine or L-ornithine, the
two amino groups almost have the same chemical activity under normal conditions.
Complicated steps, extra reagents, and long times are necessary in the synthesis of
mono-Cbz-Cl-protected L-lysine or L-ornithine.[6] The key point of the mono-Cbz-
protected L-lysine or L-ornithine is the selected activated amino group, which is similar
to peptide synthesis in human.
The key element in peptide formation is the amino acid activation domain, which
selects substrate amino acids and forms adenylates.7 In organisms, sequence-specific
peptides can be synthesized in the presence of free amino acids. Transfer RNA (tRNA),
which carries special amino acids to the protein synthetic machinery of a cell (ribo-
some) as directed by a three-nucleotide sequence in messenger RNA (mRNA), is a nec-
essary component in the synthesis of peptides (Fig. 1).[8] tRNA can selectively transfer
amino acids from free amino acids in organisms. Mimicing the ability of tRNA is not
only important in peptide synthesis but also significant in the origin of life.
Benzyl chloroformate (Cbz-Cl), which can react with an amino group to form
an amido bond and be easily removed by catalytic hydrogenation, is widely used in the
protection of amino acids and synthesis of peptides.[9] Without additional agents, the
selectivity of Cbz-Cl is poor.[10] In our previous work, we found that β-cyclodextrin
(β-CD) (Fig. 2) can catalyze the reaction between Cbz-Cl and special amino groups of
L-lysine.[11] The different orientations of L-lysine in the cavity of β-CD make the amino
groups have different chemical activities under special conditions. High chemoselectivity
Figure 1. Position of tRNA in peptide synthesis. (Figure is provided in color online.)

AMINO ACID SELECTED PROTECTION
1113
Scheme 1. L-ornithine reacts with Cbz-Cl in the presence of β-CD.
(>99%) and yield (90%) are acquired in short times (10min). L-Ornithine has a struc-
ture similar to L-lysine and is necessary in the ornithine cycle which converts toxic
ammonia to less toxic water-soluble urea. Based on previous study, we envisioned
that β-CD can form inclusions with L-ornithine that may affect the chemical activ-
ity of amino groups. The results confirmed our proposal, as only ε-amino groups
reacted with Cbz-Clin the presence of β-CD (Scheme 1, selectivity >99% yield 90%).
These observations inspired us to investigate the selectivity of β-CD to form inclu-
sions with amino acids in the construction of amido bonds, which is similar to the
ability of tRNA in peptide synthesis in organisms. Chain and aromatic amino acids
were applied to investigate the selectivity separately.
Cyclodextrins (CDs) are cyclic oligomers of a-1, 4-linked D-glucopyranose.[12]
The hydrophobic and hydrophilic features are what make the CDs extremely attrac-
tive components of mimic enzyme systems. In this way CDs provide a chemically
and sterically specific reaction field, to provide by multiple molecular recognition
means of different binding forces as required for an effective enzymelmodel.[13] The
CDs and their derivative are wieldy used in the recognition of peptides and targeted
delivery of DNA.[14] Among the CDs, β-CD is widely used for its water solubility,
low cost, commercial availability, biocompatibility, and easy functionalization.[15]
β-CD can form inclusion complexes with numerous guest molecules. The different
inclusion stability constants allow β-CD to have potential applications in molecular
recognition.
We studied a wide range of easily available and human essential amino acids for
testing selectivity. The result was interesting. In the presence of chain amino acids,
both the L-lysine and L-ornithine showed good selectivity. The selectivity was still
good even when two chain amino acids existed (Table 1). This is similar to tRNA,
which transfers special amino acids in the course of peptide synthesis. In the pres-
ence of an aromatic amino acid, the selectivity decreased. In theory, the cavity size of
Figure 2. Sketch of the proton positions in a CD molecule.

1114
J. SU ET AL.
Table 1. Chemoselectivity between L-lysine/L-ornithine and other amino acids in the presence of β-CD
Entry
n
R1
R2
Yield (a)a %
Yield (b)
Yield (c)
1
2
4
3
HOCH2
HOCH2
(CH3)2CHCH2
(CH3)2CHCH2
80
85
ND
ND
ND
ND
aIsolated yield. ND, not determined.
β-CD is fit for a benzene ring,[16] but L-ornithine still showed good selectivity when
L-tryptophane was applied in the system. In the same condition the selectivity of
L-lysine was poor (Table 2, entries 3, 8).
We propose that the β-CD plays two distinct roles in the reactions. The first
is selectivity. The amino groups of L-lysine and L-ornithine almost have the same
Table 2. Chemoselectivity between L-lysine/L-ornithine and amino acid in the
presence of β-CD
Entry
n
R1
Yield (a)a %
Yield (b)a %
1
2
4
4
HOCH2
(CH3)2CHCH2
90
90
ND
ND
90
3
4
10
4
5
4
4
ND
40
ND
60
6
7
3
3
HOCH2
(CH3)2CHCH2
90
90
ND
ND
8
3
80
ND
9
3
3
ND
50
ND
50
10
aIsolated yield. ND, not determined.

AMINO ACID SELECTED PROTECTION
1115
Scheme 2. Chemoselectivity between L-lysine and L-ornithine in the presence of β-CD. Conversion deter-
mined from 1H NMR spectrum of crude reaction mixture.
chemical activities under normal conditions, but mono-Cbz-protected L-lysine and
L-ornithine can be observed in the presence of β-CD. The second is catalysis. The
reaction between Cbz-Cl and amino groups are slow in the absence of β-CD.[17] The
catalysis could play a major factor in Table 2, entry 3; the selectivity could play a
major factor in Table 2, entry 8. This could be the reason that the selectivities in
entries 3 and 8 are quite different.From the experimental results, it was proposed that
the selectivity of L-ornithine is stronger than L-lysine. To confirm this, the reaction
with L-ornithine and L-lysine were performed simultaneously and the main product
of L-ornithine was observed (Scheme 2).
NMR spectroscopy is one of the most important tools to obtain inclusion
modes and geometries. It is very useful in providing evidence because the proton
environment in both host and guest will be reflected by chemical shifts. From the
chemical shifts, we can conclude the orientations in the inclusions.1H NMR experi-
ments were used to investigate the inclusion between β-CD and L-lysine with differ-
ent molar ratios.
The inclusion of a guest molecule in a CD generally provokes a change in the
environment of only the protons inside the cavity.[18] From the spectra (Fig. 3), the
chemical shifts of protons d and e significantly changed in the β-CD lysine solution
(1:1molar ratio). Compared to protons d and e, little chemical shift of protons a, b,
and c were observed in the spectra (Tables 3 and 4). These observations unambiguously
Figure 3. 1H NMR (400-MHz) spectra in D2O at 298K of β-CD (10−3mol·L−1) upon addition of L-lysine.
Chemical shifts (δ) are reported in ppm and referenced to the residual solvent peak as an internal standard
(δ=4.7000ppm for 1H NMR in D2O). (Figure is provided in color online.)

1116
J. SU ET AL.
Table 3. δ of L-lysine protons in L-lysine/β-CD complex (1:1) and alone
δ (ppm) of
L-lysine protons
Alone
In complex (1:1)
∆δ
a
b
c
d
e
2.9452
1.6451
1.3929
1.8258
3.6753
2.9451
1.6450
1.3928
1.8200
3.6651
−0.0001
−0.0001
−0.0001
−0.0058
−0.0102
Table 4. δ of β-CD protons in L-lysine/β-CD complex (1:1) and alone
δ (ppm) of β-CD
Alone
In complex (1:1)
∆δ
H1
H2
H3
H4
H5
H6
4.9892
3.5684
3.8857
3.5032
3.7426
3.7975
4.9893
3.5685
3.8911
3.5033
3.7497
3.7976
−0.0001
−0.0001
−0.0054
−0.0001
−0.0071
−0.0001
proved that the protons d and e of L-lysine are inside the cavity of β-CD, which allows
only one amino group to react with Cbz-Cl. This is also in accorded with the observed
experimental results. The similar results were also observed in the β-CDL-ornithine1H
NMR spectrums (Fig. 4). The chemical shifts of L-ornithine protons next to α-amino
groups significantly changed. Little chemical shifts of other L-ornithine protons were
observed (Table 5 and 6).
Figure 4. 1H NMR (400 MHz) spectra in D2O at 298K of β-CD (10−3 mol·L−1) upon addition of
L-ornithine. Chemical shifts (δ) are reported in ppm and referenced to the residual solvent peak as an inter-
nal standard (δ=4.7000 ppm for 1H NMR in D2O). (Figure is provided in color online.)

AMINO ACID SELECTED PROTECTION
1117
Table 5. δ of L-ornithine protons in L-ornithine/β-CD complex (1:1) and alone
δ (ppm) of
L-ornithine protons
Alone
In complex (1:1)
∆δ
a
b
c
2.9800
1.7395
1.8557
3.7067
2.9799
1.7394
1.8556
3.7037
−0.0001
−0.0001
−0.0001
−0.0030
d
Table 6. δ of β-CD protons in L-ornithine/β-CD complex (1:1) and alone
δ (ppm) of β-CD
Alone
In complex (1:1)
∆δ
H1
H2
H3
H4
H5
H6
4.9892
3.5684
3.8857
3.5032
3.7426
3.7975
4.9893
3.5685
3.8921
3.5033
3.7506
3.7976
−0.0001
−0.0001
−0.0064
−0.0001
−0.0080
−0.0001
Figure 5. NOESY 2D spectra of β-CD/L-lysine/L-tryptophane (1:1:1 mol/mol/mol) in D2O at room tem-
perature. (Figure is provided in color online.)

1118
J. SU ET AL.
A cross peak in nuclear Overhause effect spectroscopy (NOESY) or rotat-
ing frame NOESY (RNOESY) spectra occurs if two hydrogen atoms are within
~5 Å.[19] In L-tryptophane β-CD water solution, a cross peak between aromatic pro-
tons and inner proton of β-CD (H3, H5) occurred, which proved that the inclusion
was observed in the two-dimensional (2D) NMR spectrum.[20] To further investigate
inclusion selectivity, a 2D NMR experiment of β-CD, L-tryptophane, and L-lysine
water solution was performed. No cross peaks between the inner proton of β-CD (H3,
H5) and the aromatic proton of L-tryptophane were observed. Cross peaks between
the inner proton of β-CD and proton e were observed in the spectrum, which proved
the inclusion between β-CD and L-lysine (Fig. 5). It seems that L-lysine expelled
the benzene ring of L-tryptophane from the cavity of β-CD. Similar results can be
observed from the 2D NMR spectrum of β-CD, L-tryptophane, and L-ornithine
water solution (Fig. 6).No cross peaks between the inner proton of β-CD (H3, H5)
and the aromatic proton of L-tryptophane were observed. Cross peaks between the
Figure 6. NOESY 2D spectra of β-CD/L-ornithine/L-tryptophane (1:1:1 mol/mol/mol) in D2O at room
temperature. (Figure is provided in color online.)

AMINO ACID SELECTED PROTECTION
1119
inner proton of β-CD and proton e were observed in the spectrum, which proved
the inclusion between β-CD and L-ornithine. These observations were in accord
with the experiment results. A computational study of host–guest inclusion com-
plexes among β-CD, 20 natural L-amino acids, and some selected pentapeptides also
proved that there were different stability constants in different inclusions.[21] Liu and
coworkers also proved the molecular recognition abilities and enantioselectivity for
different amino acid guests.[22]
EXPERIMENTAL
1H NMR spectra were measured on a Bruker AM-400 spectrometer at room
temperature with D2O as the solvent. 1H-1H NOESY 2D experiments were recorded
using an Inova-600 (600-MHz) spectrometer at ambient temperature. A mixing
time of 0.200s, a relaxation delay time of 1.000s, and an acquisition time of 0.228s
were used. All plus sequences were set according to the manufacturer’s standards.
Tetramethylsilane (TMS) was used as internal standard. β-CD and derivatives were
purchased from ShangdongZiyuan Chemical Reagent Co., China. Other reagents
were all commercially available from Country Medicine Reagent Co. Ltd, Shanghai,
China. Liquid chromatography was recorded on an Agilent 1200 series. The separa-
tion was performed using mobile phase consisting of MeOH, H2O (50: 50 v/v), with
0.1% triethylamine (TEA). The column used was a Waters C18 column (250×4.6mm
internal diameter with 5-µm particle size). The flow rate was 1.0ml/min. Mass spec-
trawere recorded on an Agilent 6510 Q-TOF, equipped with an electrospray ioniza-
tion source (ESI). The capillary temperature was 350 °C, dryinggas is 12L/min, and
the spray voltage was 4.0kV for all applications.
β-Cyclodextrin (0.1mmol) was dissolved in 0.1 M carbonate buffer (pH=8)
(10mL) at room temperature, the L-lysine (1mmol) and other amino acids (1mmol)
were added, and the reaction mixture was stirred for 5 min. Cbz-Cl (1mmol) was
added and stirring was continued at room temperature until the reaction was com-
plete. The reaction mixture was extracted with ethyl acetate (2×5ml). The solvent was
removed under vacuum and the products were obtained.
CONCLUSIONS
The inclusion complexes between β-CD and amino acids were studied. Special
amino acids and activation domain were achieved in the construction of an amido
1
bond, which is similar to the ability of tRNA in peptide synthesis. The H NMR
experiments in D2O confirmed the inclusion and gave information on the position
of guests in the CD cavity. The NOESY experiments gave information of inclu-
sion complexes in mixtures. These studies showed that special amino acids can
selectively form inclusion with β-CD. These properties have potential applications
in mimic peptide synthesis. By accurate modification of CD, special selectivity can
be accessed.

1120
J. SU ET AL.
ACKNOWLEDGMENT
Thanks go to Edward C. Mignot, Shandong University, for linguistic advice.
The authors declare no competing financial interests.
SUPPORTING INFORMATION
Full experimental detail, 1H NMR spectra, and HPLC traces can be found via
the “Supplementary Content” section of this article’s Web page.
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