Synthesis and conformational analysis of new troponyl aromatic amino acid

Article abstract of DOI:10.1016/j.tet.2014.08.014

Synthetic peptides are in huge demand in expansion of potential peptide mimics, which may have improved or comparable function as natural one. With these concerns, phenyl bearing aromatic amino acids and peptides has extensively explored, because phenyl r

Full text of DOI:10.1016/j.tet.2014.08.014

Accepted Manuscript
Synthesis and Conformational Analysis of New Troponyl Aromatic Amino Acid
Chenikkayala Balachandra, Nagendra K. Sharma
PII:
S0040-4020(14)01177-6
10.1016/j.tet.2014.08.014
TET 25914
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 May 2014
Revised Date: 22 July 2014
Accepted Date: 8 August 2014
Please cite this article as: Balachandra C, Sharma NK, Synthesis and Conformational Analysis of New
Troponyl Aromatic Amino Acid, Tetrahedron (2014), doi: 10.1016/j.tet.2014.08.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and Conformational Analysis of
Leave this area blank for abstract info.
New Troponyl Aromatic Amino Acid
Chenikkayala Balachandra and Nagendra K. Sharma*
School of chemical Sciences, National Institute of Science Education and Research (NISER) IOP Campus,
Sachivalaya Marg, Bhubaneswar-751005, Odisha, India

ACCEPTED MANUSCRIPT
2
Tetrahedron
Tetrahedron
journal homepage: www.elsevier.com
Synthesis and Conformational Analysis of New Troponyl Aromatic Amino Acid
Chenikkayala Balachandra and Nagendra K. Sharma∗
School of chemical Sciences, National Institute of Science Education and Research (NISER) IOP Campus, Sachivalaya Marg, Sainik School (P.O) Bhubaneswar-
7
51005, Odisha, India
ABSTRACT
ARTICLE INFO
Article history:
Received
Received in revised form
Accepted
Synthetic peptides are in huge demand in expansion of potential peptide mimics, which may
have improved or comparable function as natural one. With these concerns, phenyl bearing
aromatic amino acids and peptides has extensively explored, because phenyl residue has high
probability in forming stable secondary structure, owing to the presence of an extra stabilizing
factor as π-π non-covalent interactions. Apart from phenyl bearing benzenoid aromatic amino
acids, a few non-benzenoid aromatic derivatives such as tropolone and related compounds are
also occurred in nature, but troponyl containing amino acids and peptides are very poorly
understood. Tropolonyl derivatives also contain carbonyl functional group, which may play an
important role to provide stable conformation in peptide. Herein we report the synthesis, and
Available online
Keywords:
Keyword_1 Tropolone
conformational analysis of rationally designed new unnatural δ-amino acid, troponyl aminoethyl
Keyword_2 Aaminotropone
Keyword_3 Conformational analysis
Keyword_4 Aminoethylglycine (aeg)
Keyword_5 δ-amino acid, hybrid Peptide
glycine (Tr-aeg), which contains troponyl residue as side chain in flexible aminoethylglycine
(aeg) amino acid backbone. We also demonstrate the role of troponyl carbonyl of Tr-aeg residue
in hydrogen bonding with adjacent amide NH of their hybrid di/tri-peptides with NMR methods
and DFT calculations. In future, Tr-aeg amino acid would be a potential building block in
development of promisable peptide mimics.
2
009 Elsevier Ltd. All rights reserved.
—
——
∗
Corresponding author. Tel.: +91-674-230-4130; fax: +91-674-230-2436; e-mail: nagendrar@niser.ac.in

ACCEPTED MANUSCRIPT
Introduction
Conformational analysis of a molecule defines the type of
structural organization, which explain the functional behavior of
that molecule. In case of macromolecules, the conformation of
in development of synthetic aromatic amino acids and their
peptides. In nature, non-benzenoid aromatic compounds such as
tropolone (1) and related compounds are also available (Figure
1
19,20
building blocks has crucial role in controlling the overall
1).
Tropolone related bioactive natural products as β-
2
21 22
functionality of molecules. For an instance, the function of
thuzaplicinol (hinokitiol), and α-manicil, are studied in details.
Other tropolonyl derivatives including α-hydroxy tropolone and
2-aminotropone analogues are also considered as target drugs
molecules. In addition, tropolone derivatives contain metal
chelating properties especially with Zn and Cu metal ion.
protein and enzyme relay on favorable conformation of their
building block such as amino acid residue. The side chain
functionality of that amino acid residue having stable
conformation is also responsible in modulation of non-covalent
2
3
2
4-26
3
,4
interactions for being functional respective protein/enzymes.
Very recently, new electronic properties such as proton transfer
Due to variable side chain of amino acids residue, various non-
covalent interactions are present in functional protein and
enzymes. Among them, hydrogen bonding, one of the important
non-covalent interactions, plays an important role to acquire the
within tropolone (1) molecule7-30have been observed via
2
intramolecular hydrogen bonding.
Owing to many remarkable
features, the tropolone and related compounds would be
employed in synthetic peptides, to examine the conformational
stability of their peptides. To expand the synthetic peptides
4
,5
well defined structural organization in protein/enzymes. To
improve the structural and functional properties, many synthetic
peptide mimics have been synthesized from natural and unnatural
repertoire, we rationally designed an
aminoethylglycine (Tr-aeg), from
aminoethylglycine (aeg) -amino acid backbone derivative
δ
-amino acid, troponyl
tropolone and
6
amino acids and nicely explored. Some of these peptide mimics
δ
have shown exceptional functional properties and then
considered as therapeutics drug candidate. For ideal peptide
(Figure 1). In this report, we describe the synthesis of rationally
designed new Tr-aeg amino acid where troponyl motif linked
covalently at aeg backbone. We also demonstrate the role of
troponyl carbonyl group of Tr-aeg, in hydrogen bonding with
amide N-H of peptide containing Tr-aeg residue by NMR
techniques. So far, there is no such report, which describes the
role of troponyl carbonyl group in conformational stability of
peptide via hydrogen bonding with adjacent amide N-H.
7
mimic, the presence of following secondary structural elements-
helices, turns and sheets are required in target peptide to adopt
5
,8
protein-like conformations and folding pattern. To meet these
requirements, many unnatural synthetic peptides from non-
natural amino acid, including backbone expanded amino acids
8
-10
11
12,13
such as β-,
γ-, and δ-amino acid
were synthesized and
studied extensively to find the interesting structurally folding
behaviors. Some of these synthetic peptides have shown
promiseable folding behavior with significant secondary
structural elements. It is also learnt that the substituent of
synthetic amino acids (β-, γ- and δ-amino acid) analogues also
play an important role in acquiring secondary structure by
restricting their allowed conformational space. For an instance, β-
alanine peptide reportedly forms random structures in solution
Results and discussions
The synthesis of newly designed
δ-amino acid, Tr-aeg, was
planned from commercially available materials: tropolone (1) and
ethylenediamine (3) (Scheme 1). Herein, the hydroxyl functional
group of tropolone (1) was derivatized into 2-tosyloxy tropolonyl
intermediate (2) after treatment with tosylchloride by following
1
4
phase, while sheet like packing in its solid state. However, the
substituted ring constrained β-amino acid analogues, containing
cycloalkane ring, also facilitate the formation of helical type of
31
literature procedure. And ethylenediamine (3) was modified to
N-Boc-aminoethylglycinate (aeg) (4) backbone by using known
1
5
secondary structure formation in solid state. Interestingly, the
synthetic peptides, even with four appropriate residues,
reportedly form stable secondary structures and inhibit protein-
32
synthetic procedure. Subsequently, the troponylation reactions
performed at N-atom of aeg backbone (4) by refluxing with
reactive tropolonyl tosylate intermediates (2), under mild basic
1
0,16
protein interactions and other biological events.
Some of the
synthetic peptides have also shown high stability towards enzyme
degradation at cellular level. Recently, many new synthetic
aromatic amino acids have been synthesized by positioning of
chain propagating groups, amino and carboxyl group, on or with
aromatic frameworks to generate conformationally constrained β-
Scheme 1. Synthesis of Tr-aeg amino acid monomer
17,18
turn type of peptide foldamers.
The structural analysis of
phenyl bearing aromatic, benzenoid, peptides has revealed that π-
π non-covalent interactions provide extra stabilities in formation
of their secondary structure. So far many benzenoid aromatic
compounds are known, but a very few of them have been utilized
Figure 1. Chemical structure of tropolone (1) and troponyl amino acid
(Tr-aeg) and Tr-aeg peptide

ACCEPTED MANUSCRIPT
4
Tetrahedron
troponyl ring. Due to ring puckering, non-planarity character in
seven member closed ring compounds have also been noticed
3
3,34
previously.
Our structural analysis results also reveal the
orientation of troponyl carbonyl group toward the C’-end of Tr-
aeg amino acid derivative (5), not to the N’-end. After
conformation analysis of derivative 5, the role of its troponyl
carbonyl group in peptide folding was planned to examine. That
is why, a few hybrid peptides, combination of synthetic Tr-aeg
and natural α-amino acid, planned to synthesize. The ester group
of Tr-aeg derivative (5), therefore, was hydrolyzed into
carboxylic acid functionalized Tr-aeg derivative (6) by treatment
with aq. NaOH (2.0M) followed by neutralization with HCl
(
scheme 1). This carboxylic acid functionalized derivative 6 was
Figure 2. ORTEP diagram of δ-aminoacid monomer 5
coupled with amino group of methyl ester derivative of following
α-amino acids- L-phenylalanine (Phe), L-proline (Pro), and L-
conditions, in Et N. After completion this reaction, mixture was
3
isoleucine, under peptide coupling reagents, EDC/Et N (Scheme
3
purified by column chromatographic method and then
2). Thus hybrid dipeptides 7/8/9 were achieved from 6 and
respective methyl ester derivative of Phe/Pro/Ilu with moderate
yield ca. 45%. For control studies, without containing troponyl
residue, dipeptide 10 (Boc-NH-Gly-Phe-COOMe) from α-amino
acid derivatives: N-Boc-glycine acid and phenylalanine (Phe)
ester amine. With silica gel column purification method, purified
1
13
characterized by NMR ( H/ C) and HRMS (ESI-Tof) analysis.†
These analytical data suggest the formation of desired compound
as Tr-aeg protected amino acid derivative, BocNH-Tr-aeg-
COOEt, (5). The characterization data (NMR and HRMS spectra)
of newly synthesized compound 5 are provided separately as
Supplementary Information (SI). Moreover, the single crystal X-
ray analysis was performed to crystalline solid form of Tr-aeg
derivative 5. Finally, X-ray studies confirm the chemical
structure of 5 and its ORTEP diagram is depicted in Figure 2,
whiles its crystal packing pattern is provided in SI. X-ray data of
1
13
hybrid peptides (7/8/9/10) were characterized by H-/ C-
NMR/HRMS (ESI-Tof) techniques. The characterization data
(NMR and HRMS spectra) are provided in SI. Furthermore, the
hybrid tri-peptide (11) was also prepared from Tr-aeg derivative
(6) and dipeptide (10) and then characterized by similar
techniques. The characterization data of tri-peptide 11 are
provided in SI. After successful synthesis and characterizing of
hybrid di-/tri-peptides, we planned to examine the role of
troponyl carbonyl group in hydrogen bonding with adjacent
amide N-H or carbamate N-H within peptides (7/8/9/11). Thus,
the conformational studies of hybrid peptides were essential. In
this report, we used NMR methods to find most favorable
conformation and orientation of troponyl residue within peptide.
So that, 2D-NMR (COSY, NOESY, and HSQC) spectra of
synthesized hybrid peptides (7/8/9/11) were recorded, which are
5
as “.cif” file, are submitted to the Cambridge structural
database (CSD) with CCDC No. 975954. Our structural studies
demonstrate that troponyl ring of Tr-aeg derivative (5) is non-
planar, possible owing to ring puckering. Tropnyl carbonyl
substituent C(2)-O(3) of 5, therefore, is not in same plane of
Scheme 2. Synthesis of hybrid Peptides
1
1
provided in SI. In case of hybrid peptide 7, the H- H COSY
spectrum analysis assigns chemical shift ( ) of all protons, while
H- C-HSQC spectrum analysis furnishes chemical shift (δ) of
all CH/CH /CH type of carbons in peptide 7. After proton and
δ
1
13
2
3
1
1
Carbon analysis, H- H NOESY 2D NMR spectrum is used to
extract non vicinal coupling protons partner in peptide 7. The
NOE analysis results show a spatial interaction of troponyl ring
C(7)-H proton (t7H), phenyl (ortho) protons or both with
adjacent amide N-H of phenylalanine residue in dipeptide 7.
Amazingly, no NOE interaction observes for t7H proton with
Table1. Chemical shift value of amide NH and troponyl C=O
Entry Compound Carbamate
N-H
Amide
N-H
(ppm)
Troponyl
Carbonyl
(C=O) group
(ppm)
(
ppm)
1
2
3
4
5
6
5
7
8
9
10
11
5.6
5.5
5.8
5.6
5.1
5.5
no
7.7
no
181.7
182.5
181.7
183.2
no tr-aeg
182.7
7.9
6.6
*
7.9
*
Amide (i+1)th residue

ACCEPTED MANUSCRIPT
carbamate N-H in dipeptide 7. In addition, the NOE interactions
of troponyl ring C(3)-H proton (t3H) with aminoethyl protons
met /met (γH/δH,) are noticeably observed. These 2D-NMR
analysis support the troponyl ring conformation in peptide 7,
where troponyl carbonyl group pointing to the C’-end of peptide
8/11. Their results show that the troponyl residue protons (t3H)
of peptides (8/11) have similar NOE interaction with aminoethyl
protons γH/δH, which also support similar kind of troponyl
carbonyl group conformation as of di-peptide 7. After
conformational analysis, the role of troponyl carbonyl group of
synthesized hybrid peptides in hydrogen bonding examined with
adjacent amide NH by following NMR titration method.
According to this method, the chemical shift (proton resonance)
1
2
(
7), not to the N’-end. Hence, tropnyl ring of peptide 7 possibly
has similar conformation as of Tr-aeg derivative (5). Similar sets
of 2D-NMR analysis completed within other hybrid peptides
Figure 3. DMSO-d6 titration profile of dipeptide (7)
8
9
8.0
11
B
6.0
A
7.8
7.6
7.4
7.2
7.0
6.8
6.6
9
5
7
5.8
7
1
0
5.6
1
1
5.4
1
0 (controle)
5.2
5.0
0
10
20
30
40
0
10
20
30
40
Volume of DMSO-d6 (µl)
Volume of DMSO-d6
(µl)
Figure. 4 NMR titration profile: (A) Boc-NH at N’- end of 5/7/8/9/10/11; (B) Adjacent amide NH at C’-end of 7/9/11/10

ACCEPTED MANUSCRIPT
6
Tetrahedron
of intramolecular hydrogen bonded proton (amide N-H) in
CDCl reportedly exhibit slight downfield/upfield shift
deshielded/shielded region) or unchanged with sequential
), which is strong hydrogen bond
NMR peak of hybrid peptide 7/9/11, which is marginally higher
than that of 8 (Tr-aeg-Pro) and 5 (Tr-aeg). These significant
3
1
3
(
changes in C-NMR peak of hybrid peptide 7/9/11 also
substantially suggest the involvement of troponyl carbonyl
(C=O) in hydrogen bonding, possibly with adjacent amide N-H
(n+1). In further search of troponyl carbonyl group (C=O)
participation in hydrogen bonding with amide adjacent N-H,
addition 5of (DMSO-d
6
3
acceptor. Whereas the chemical shift of exposed or non-
hydrogen bonded amide N-H protons exhibit significant
downfield shift (desheilded region) with sequential addition of
1
3
DMSO-d
6
to the same NMR sample, because of hydrogen
once again solvent dependent C-NMR spectra of hybrid peptide
11 were recorded in following solvent system: aprotic polar
solvents CDCl (100%), DMSO-d (100%) and mixture of both
bonding with strong hydrogen bond acceptor solvent DMSO-d6.
We also performed similar NMR titration experiments with
synthesized peptides, before that the chemical shift (δ) of amide
NH, The carbamate NH, and troponyl carbonyl (C=O) of di/tri
peptides (7-11) were extracted from their respective spectrum
and given in table 1. Thereafter, a series of proton spectra of
hybrid/control peptides (7-11) were recorded with volume wise
3
6
solvent CDCl
3
:DMSO-d
6
(3:1, 1:1). All these spectra are provide
1
3
in SI, while extended regions (δ 70-200 ppm) of C-NMR
spectra of peptide 11 is given in Figure 5, where the chemical
shift all carbonyl groups of that peptide are appeared. The
chemical shift of troponyl carbon peak (ca ~180 ppm) of hybrid
sequential addition of DMSO-d
6
(see SI). For a representation,
peptide 11 is more desheilded in CDCl
which also indicates the formation of intramolecular hydrogen
bonding by troponyl carbonyl is better in CDCl in comparison to
DMSO-d . However the chemical shift of troponyl carbonyl
carbon NMR peak in mixture of solvent CDCl :DMSO-d (3:1
and 1:1) are almost same as in DMSO-d . This would be possible
3 6
, in respect to DMSO-d ,
1
herein only extended regions (5-8 ppm) of sequential H-NMR
spectra of hybrid di-peptide 7 are depicted (Figure 3). Further in,
the NMR titration profiles as plot of chemical shift (ppm) vs
3
6
DMSO-d
6
volume (ꢀL) are generated for both carbamate NH
3
6
(
Figure 4A)/ amide NH (Figure 4B) of hybrid peptides 7 and
6
other peptides (8-11). The titration profile of hybrid peptide 7
indicates the significant downfield shift (deshielding effect) in
due to disturbance of intramolecular hydrogen bonding, between
troponyl carbonyl and amide N-H, in CDCl3 with DMSO-d6.
cabamate N-H proton peak (
little downfield shift (deshielding effect) in amide N-H peak (
.8-7.74, Figure 4B). These NMR titration results for peptide 7
advocate that only amide N-H is involved in intramolecular
hydrogen bonding even though one carbamate N-H is available.
In case of control peptide 10, NMR titration profiles for both
carbamate (Figure 4A) and amide-NH (Figure 4B) show rapid
downfield shift (deshielding effect) with sequential addition of
δ
5.5-5.67, Figure 4A), while very
Because DMSO-d
acceptor solvent and form strong intermolecular hydrogen
is also known as strong hydrogen bond
6
δ
36
7
bonding with exposed amide N-H. However the chemical shift
1
3
of C-NMR peak (∼ δ 126.0 ppm) for Boc carbonyl group remain
same in both CDCl and DMSO-d solvent (Figure 5). Hence the
3
6
1
3
results of C-NMR studies further support the involvement of
troponyl carbonyl group in hydrogen bonding with amide N-H
3
aprotic polar solvent as CDCl .
DMSO-d , such as δ +0.47 ppm (aprox) downfield shift of amide
6
6
N-H with addition of 40 ꢀL of DSO-d (see SI, Table S13). This
titration results clearly show that no intramolecular hydrogen
bonding is formed in non-hybrid dipeptide (10). The NMR
titration experimental results performed with other hybrid
6 3
peptides 8/9/11 with addition of DMSO-D in CDCl NMR
sample. The NMR titration profiles for amide/carbamate N-H of
other hybrid peptides 8/9/11 are provided in Figure 4. The
titration profiles for carbamte N-H of 8/9/11 show a similar
shielding effect trend as of hybrid peptide 7 (Figure 4A). The
titration profiles for amide N-H of peptides 9/11 exhibit
desshielding effect (downfield shift) tendency in amide NH of
9/11 as similar to dipeptide 7 (Figure 4B). Structurally hybrid
peptide (8) does not contain amide N-H so there is no amide NH
titration profile observed. Overall the comaparative NMR
titration studies of hybrid di/tri-peptide (7/9/11) with control
peptide 10 are strongly suggested that only adjacent amide N-
H’s, rather than carbamate NH, of 7/9/11 are only involved in
intramolecular hydrogen bonding. This intramolecular hydrogen
bonding participation by amide N-H of hybrid peptide (7/9/11)
may take place with troponyl carbonyl group of Tr-aeg residue of
peptide (7/9/11), even though the presence of their carbamate
carbonyl group. In further the involvement of troponyl carbonyl
group of hybrid peptide were investigated in hydrogen bonding
1
3
Figure 5. C-NMR of tripeptide 11 (Expanded region) in DMSO-d6 and
CDCl
3
To end with, a DFT calculation was performed with one of
hybrid dipeptide 9 to acquire geometrically optimized structure in
gas phase by using with B97 TURBOMOLE software.
resultant, the optimized structure of 9 is depicted in Fig. 6.,
which also indicates the conformation of troponyl carbonyl group
as pointing to the C’-end of peptide 9, like monomer 5 and other
peptides. The theoretical studies realted file as “.pdb” for peptide
37,38
As
1
3
with adjacent amide NH. We, therefore, carefully studied the C-
NMR spectra of hybrid peptide (7/8/9/11) and Tr-aeg derivative
9
is provided separately (see SI). In addition, the theoretically
5, especially to troponyl carbonyl carbon peak (SI). The chemical
optimized structure of 9, show following strong non-covalent
hydrogen bond interaction: C=O----H-πC (2.41A); πC----H-
C(2.47Å); C=O----H-C (2.05Å); and C=O----H-N (2.08Å). These
interactions further support the formation of hydrogen bonding
shift values of troponyl carbonyl carbon of Tr-aeg containg
compounds 5/7/8/9/11 are given in Table 1. Herein the
deshielding effects are observed in troponyl carobonyl carbon

ACCEPTED MANUSCRIPT
th
between Troponyl carbonyl and adjacent, (i+1) amide NH. Then
Synthetic procedure of Tr-aeg monomer (5): A solution of 2-
tosyloxy tropone (2) (1.2g, 4.34 mmol) in ethanol (25 mL) was
refluxed in presence of Ethyl (2-N-Boc-aminoethyl) glycinate (4)
(3.2 g, 13.04 mmol) for 48 hours. After completion of reaction,
which was monitored by thin layer chromatography (TLC),
reaction mixture was cooled down to room temperature and then
concentrated to dryness under vacuum. The dried solid reaction
residue was re-dissolved in DCM (50 mL) and washed with
water (50ML) ,at least three times, followed by brine solution (10
mL) using separating flask. The DCM layer was kept over
the formation of 8-membered ring hydrogen bond motif is
reasonably possible, which may provide the interesting helical
structure in longer peptide. The theoretical folding pattern of
hybrid peptide 9 also predicted from backbone dihedral angles ф
phi) and ψ (psi) of peptide 9. Since peptide backbone dihedral as
ф and ψ are being used to generate Ramachandran plot, ф vs ψ,
which predict the secondary structure of peptide and protein.
With geometrically optimized structure of hybrid peptide 9, the
backbone dihedral angles as ф = -76.0 , θ1 = 170.3 ; θ2= 89.3 ;
(
39
o
o
o
o
o
θ3=81.9 ; ψ=92.7 are calculated and then generate
a
2 4
Na SO for 30 min and then concentrated to dryness under
Ramachandran plot with help of software GNUPLOT 4.7 (SI).
vacuum. The concentrated reaction mixture was subject for
purification by column chromatographic methods. Then The
major component of reaction residue was purified with solvent
system ethylacetate:hexane (1:3) and then characterized as
o
o
The coordinate (-76.0 , 92.7 ) of ramachandran plot for peptide 9
indicates the formation of β-sheet or β-turns type of secondary
structure, which is really provide encouraging information about
development of conformationally defined useful peptide mimics.
1
13
desired product 5 (1.2 g, 80%) by H/ C-NMR and mass
1
spectrometric method. H NMR (400 MHz, CDCl3) δ (ppm) 7.12
–
5
3
6.96 (m, 2H), 6.92 (d, J = 11.8 Hz, 1H), 6.75–6.55 (m, 2H),
.60 (s, 1H), 4.20 (s and q, 4H), 3.59 (t, J = 6.1 Hz, 2H), 3.45 –
1
3
.31 (m, 2H), 1.40 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H). C-NMR
): δ (ppm) 181.69, 170.41, 157.93, 156.21,
(
101 MHz, CDCl
3
1
35.62, 133.92, 133.78, 125.10, 115.79, 79.37, 61.15, 53.57,
+
52.37, 37.57, 28.33, 14.11. HRMS (ESI-TOF) m/z: [M+H]
26 2 5
calcd. for C18H N O 351.1914, found 351.1921.
Acknowledgment
CB thank to UGC & NISER for research fellowship. This studied
was supported by Grant-in Aid from NISER, Department of
Atomic Energy (DAE)-India. We also thank Dr. Himanshu.
Biswal for DFT calculation.
Figure 6. Energy minimized structure of di-peptide 9 from DFT
calculations
Supplementary Information
Electronic Supplementary Information (ESI) available: [All
1
13
NMR ( H, C, 2D NMR and solvent dependent NMR) and mass
spectra of all new compounds: monomer (5 & 6) and peptide (7-
Conclusions
11) are provided]. See DOI: 10.1039/c000000x/
In summary, we have successfully synthesized new unnatural δ-
amino acid (Tr-aeg) containing troponyl residue and then have
been used in synthesis of hybrid di-tri-peptides with α-amino
acid. With experimental and theoretical studies, we have also
shown the role of troponyl carbonyl group in intramolecular
hydrogen bonding with adjacent amide NH. As resultant, the
formation of 8-membered ring type of intramolecular hydrogen
bond motif and β-sheet or β-
References
1) C. Branden; J. Tooze Introduction to Protein Structure; Garland:
NewYork, 1991.
2) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter,
P. Molecular Biology of the Cell. 4th edition, New York: Garland
Science; 2002.
(
(
turns type of secondary structure
are observed. Our future works
(3) Kotha, S. Acc Chem Res 2003, 36, 342.
are in progress to synthesize and
study the role of troponyl
(4) Padmanabhan, S.; Marqusee, S.; Ridgeway, T.; Laue, T. M.;
Baldwin, R. L. Nature 1990, 344, 268.
carbonyl
group
in
(
5) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem
Rev 2011, 111, 657.
6) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
Chem Rev 2001, 101, 3893.
homologous/heterologous long
peptides containing Tr-aeg unit
in development of potential peptide mimics.
(
(
(
7) Hicks, R. P.; Russell, A. L. Methods Mol Biol 2012, 794, 135.
8) Seebach, D.; Matthews, J. L. Chem. Commun. (Camb) 1997, 21,
2
015.
(
Experimental Procedure
9) Seebach, D.; Hook, D. F.; Glattli, A. Biopolymers 2006, 84, 23.
10) Seebach, D.; Gardiner, J. 2008, 41, 1366.
(11) Basuroy, K.; Dinesh, B.; Reddy, M. B. M.; Chandrappa, S.;
(
Synthesis of 2-tosyloxy tropone (2). From tropolone by following
literature procedure.
1
5
Raghothama, S.; Shamala, N.; Balaram, P. Org Lett 2013, 15. 4866.
(12) Peng, Y.; Gong, T.; Cui, Y. Chem Commun (Camb) 2013, 49,
Synthesis of aminoethylglycine (aeg) backbone (4). From
tropolone by following literature procedure.
1
9
8
253.

ACCEPTED MANUSCRIPT
8
Tetrahedron
(
13) Schramm, P.; Sharma, G. V. M.; Hofmann, H.-J.
Biopolymers 2010, 94, 279.
14) Narita, M.; Doi, M.; Kudo, K.; Terauchi, Y. Bull. Chem. Soc.
Jpn 1986, 59, 3553.
15) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071.
(
(
(16) Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399.
(17) Ramesh, V. V. E.; Roy, A.; Vijayadas, K. N.; Kendhale, A. M.;
Prabhakaran, P.; Gonnade, R.; Puranik, V. G.; Sanjayan, G. J. Org.
Biomol. Chem. 2011, 9, 367.
(18) Hecht, S. a. H., I. Foldamer: structure, properties and
applications, Eds. Wiley-VCH, Weinheim, 2007.
(
(
19) Bentley, R.; Thiessen, C. P. Nature 1959, 184(Suppl 8), 552.
20) Dewar, M. J. Nature 1950, 166, 790.
(
21) Byeon, S. E.; Lee, Y. G.; Kim, J. C.; Han, J. G.; Lee, H. Y.; Cho,
J. Y. Planta Med 2008, 74, 828.
22) Koufaki, M.; Theodorou, E.; Alexi, X.; Nikoloudaki, F.; Alexis,
M. N. Eur J Med Chem 2010, 45, 1107.
23) Sanz, P.; Mo, O.; Yanez, M.; Elguero, J. Chem. Eur. J. 2008,
4, 4225.
24) Fullagar, J. L.; Garner, A. L.; Struss, A. K.; Day, J. A.; Martin,
D. P.; Yu, J.; Cai, X.; Janda, K. D.; Cohen, S. M. Chem. Commun.
Camb.) 2013, 49, 3197.
25) Nishinaga, T.; Aono, T.; Isomura, E.; Watanabe, S.; Miyake, Y.;
(
(
1
(
(
(
Miyazaki, A.; Enoki, T.; Miyasaka, H.; Otani, H.; Iyoda, M. Dalton
Trans 2010, 39, 2293.
(26) Miyake, Y.; Watanabe, S.; Aono, S.; Nishinaga, T.; Miyazaki,
A.; Enoki, T.; Miyasaka, H.; Otani, H.; Iyoda, M. Chem Commun
(
Camb) 2008, 46, 6167.
27) Wojcik, M. J.; Boda, L.; Boczar, M. J Chem Phys 2009, 130,
64306.
(
1
(
(
(
(
28) Jose, D.; Datta, A. Angew Chem Int Ed Engl 2012, 51, 9389.
29) Fernandez-Ramos, A. Angew Chem Int Ed Engl 2013, 52, 8204.
30) Datta, A. Angew Chem Int Ed Engl 2013, 52, 8206.
31) Dochnahl, M.; Lohnwitz, K.; Pissarek, J. W.; Biyikal, M.;
Schulz, S. R.; Schon, S.; Meyer, N.; Roesky, P. W.; Blechert, S. Chem.
Eur. J. 2007, 13, 6654.
(32) Sharma, N. K.; Ganesh, K. N. Chem. Commun. (Camb) 2005,
3
4, 4330.
(
(
33) Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354.
34) Boessenkool, I. K.; Boyens, J. C. A. J. Cryst. Mol. Struct. 1980,
1
1
0, 11.
(35) Rai, R.; Raghothama, S.; Balaram, P. J. Am. Chem. Soc. 2006,
28, 2675.
(
36) Urry, D. W.; Mitchell, L. W.; Onishi, T. Proc Natl Acad Sci U S
A 1974, 71, 3265.
37) Ahlrichs, R.; Bar, M.; Haser, M.; Horn, H.; Kolmel, C. Chem.
Phys. Lett. 1989, 162, 165.
(
(38) Furche, F.; Ahlrichs, R. J. Chem. Phys. 2002, 117, 7433.
(39) Kolaskar, A. S.; Sawant, S. Int. J. Pept. Protein. Res. 1996, 47,
1
10.

ACCEPTED MANUSCRIPT
Synthesis and Conformational Analysis of New Troponyl Aromatic
Amino Acid
Chenikkayala Balachandra and Nagendra K. Sharma*
School of chemical Sciences, National Institute of Science Education and Research (NISER)
IOP Campus, Sachivalaya Marg, Sainik School (P.O)
Bhubaneswar-751005, Odisha, India
Phone: +91-674-2304130, Fax: +91-674-2302436
Email: nagendra@niser.ac.in
Contents
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
. General:.....................................................................................................................................................3
. Experimental procedure:...........................................................................................................................3
1
. H-NMR spectrum of 2: ...........................................................................................................................6
1
. H-NMR spectrum of monomer 5:............................................................................................................7
1
3
. C-NMR spectrum of monomer 5:...........................................................................................................8
. Mass spectrum of dipeptide 5: ..................................................................................................................9
1
. H-NMR spectrum of dipeptide 7: ..........................................................................................................10
1
3
. C-NMR spectrum of dipeptide 7:.........................................................................................................11
1
. Deuterium exchange H-NMR experiment of dipeptide 7:.....................................................................12
0. Mass spectrum of dipeptide 7: ..............................................................................................................13
1
1. H-NMR spectrum of dipeptide 8:........................................................................................................14
1
3
2. C-NMR spectrum of dipeptide 8:.......................................................................................................15
3. Mass spectrum of dipeptide 8: ..............................................................................................................16
1
4. H-NMR spectrum of dipeptide 9:........................................................................................................17
1
3
5. C-NMR spectrum of dipeptide 9:.......................................................................................................18
6. Mass spectrum of dipeptide 9: ..............................................................................................................19
1
7. H-NMR spectrum of dipeptide 10: ......................................................................................................20
S1

ACCEPTED MANUSCRIPT
1
3
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
8. C-NMR spectrum of dipeptide 10:.....................................................................................................21
9. Mass spectrum of dipeptide 10: ............................................................................................................22
1
0. H-NMR spectrum of tripeptide 11:......................................................................................................23
1
3
1. C-NMR spectrum of tripeptide 11:.....................................................................................................24
2. Mass spectrum of tripeptide 11:............................................................................................................25
3. 1H-1H COSY and NOESY 2D NMR spectrum of monomer 5............................................................26
1
1
4. H- H COSY 2D NMR spectrum of dipeptide 7: .................................................................................27
1
1
5. H- H NOESY 2D NMR spectrum of dipeptide 7:...............................................................................28
6. HSQC 2D NMR spectrum of dipeptide 7:............................................................................................29
1
1
7. H- H COSY 2D NMR spectrum of dipeptide 8: ..................................................................................32
1
1
8. H- H NOESY 2D NMR spectrum of dipeptide 8:...............................................................................33
9. HSQC 2D-NMR spectrum of Dipeptide 8:...........................................................................................34
1
1
0. H- H COSY 2D-NMR spectrum of tripeptide 11:...............................................................................35
1
1
1. H- H NOESY 2D NMR spectrum of tripeptide 11: ............................................................................36
2. HSQC 2D NMR sptectrum of tripeptide 11: ........................................................................................37
3. NMR titration studies of 5/7/8/9/10/11:................................................................................................39
1
4. H-NMR titration results of monomer (5) and peptides (7/8/9/10/11) with DMSO-d : .......................44
6
5. Proposed Hydrogen bonding in peptides ..............................................................................................44
1
3
6. C-NMR of tripeptide (11) in CDCl and DMSO-d6 solvent pairs:....................................................45
3
Expanded region of above given spectra: ...................................................................................................45
1
3
1
3
3
3
4
7. C-NMR and H-NMR of monomer (5) and peptide (7/8/9/10/11):....................................................46
8. Crystal data of monomer 5:..................................................................................................................47
9. DFT calculated density map .................................................................................................................49
0. Theoretical Ramachandran Plot............................................................................................................49
S2

ACCEPTED MANUSCRIPT
1
. General:
Materials and instrumentation: All required materials were obtained from commercial suppliers
and used without further purification. Dry dichloromethane was freshly prepared by distilling
over KOH and Calcium hydride sequentially. Reactions were monitored by thin layer
chromatography, visualized by UV and Ninhydrin. Column chromatography was performed in
1
13
1
00-200 mesh silica. NMR spectra were recorded on Bruker AV-400 ( H: 400 MHz, C: 100.6
1
13
MHz). H and C{1H} NMR chemical shifts were recorded in ppm downfield from tetramethyl
silane. Splitting patterns are abbreviated as: s, Singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quartet; dq, doublet of quartet; m, multiplet. Mass spectra were obtained from Bruker
micrOTOF-Q II Spectrometer.
2
. Experimental procedure:
. Syntheses of Ethyl-(2-N-Boc-aminoethyl)troponyl)glycinate (5): To a solution of 2-
1
tosyloxy tropone (2) (1.2g, 4.34 mmol) in ethanol (25 mL) was added Ethyl (2-N-Boc-
aminoethyl) glycinate (4) (3.2 g, 13.04 mmol) and refluxed. Reaction was monitored using thin
layer chromatography (TLC) and found the completion of reaction after two days. After cooling
to room temperature, reaction mixture was concentrated under vacuum on rota vapour. The
reaction residue was redissolved in DCM (50 mL) and washed with water (50mL) three times
and then with brine solution (10 mL) by using separating flask. The organic layer was kept over
Na SO for 30 min and then concentrated to dryness under vacuum. The concentrated residue
2
4
was subjected for purification on silica gel by column chromatographic methods. The major
component of reaction residue was purified with solvent mixture Ethylacetate:Hexane (1:3) and
1
13
characterized as desired product (1.2 g, 80%) by H/ C-NMR and Mass spectrometric method.
1
H NMR (400 MHz, CDCl ) δ (ppm) 7.12 – 6.96 (m, 2H), 6.92 (d, J = 11.8 Hz, 1H), 6.75–6.55
3
(m, 2H), 5.60 (s, 1H), 4.20 (s and q, 4H), 3.59 (t, J = 6.1 Hz, 2H), 3.45-3.31 (m, 2H), 1.40 (s,
1
3
1
1
0H), 1.26 (t, J = 7.1 Hz, 3H). C-NMR (101 MHz, CDCl ): δ (ppm) 181.69, 170.41, 157.93,
3
56.21, 135.62, 133.92, 133.78, 125.10, 115.79, 79.37, 61.15, 53.57, 52.37, 37.57, 28.33, 14.11.
+
HRMS (ESI-TOF) m/z: [M+H] calcd. for C H N O 351.1914, found 351.1922. Summary of
1
8
26
2
5
X-ray data of monomer 5 is deposited to the Cambridge Structural Database (CSD) and their
deposition number CCDC 975954. From X-ray analysis revealed the unit cell parameters: a
1
1.1125(4) b 15.3001(7) c 12.2394(5) P21/c and molecular formula C H N O of monomer 5.
18 26 2 5
S3

ACCEPTED MANUSCRIPT
2
. Syntheses of dipeptide (7): A solution of N-(2-amioethyl)troponyl)glycine (6) (200 mg, 0.62
o
mmol) in anhydrous dichloromethane (10 mL) was cooled to 0 C, and then EDC-HCl (142 mg,
o
0
.744 mmol) was added, stirred for 5 min at 0 C. Then L-Phenylalanine methyl ester
hydrochloride (160 mg, 0.744 mmol) and Triethylamine (0.26 mL, 1.86 mmol) was added
together. This reaction mixture was continued to stirrer at room temperature (rt) for overnight.
After completion of reaction, the reaction mixture was concentrated to dryness under reduced
pressure. Concentrated reaction residue was re-dissolved in DCM (30 mL) and then washed with
water thrice (3*30mL) followed by saturated sodium bicarbonate (20 mL) by following
extraction method. The washed organic layers were combined together and concentrated under
reduced pressure and then loaded on silicagel column for purification by EtOAc/Hexane to
obtain desired product (7). The major component was isolated with EtOAc/Hexane (30:70) and
1
13
characterized as desired product (120 mg, 40%) by H/ C-NMR and Mass spectrometric
1
method. H NMR (400 MHz, CDCl ) δ (ppm) 7.76 (d, J = 7.9 Hz, 1H), 7.24 – 7.06 (m, 7H), 7.05
3
–
3
3
1
1
6.92 (m, 2H), 6.72 (dd, J = 21.4, 13.0 Hz, 2H), 5.59 (s, 1H), 4.78 (dd, J = 14.0, 6.7 Hz, 1H),
.93 (d, J = 16.7 Hz, 1H), 3.88 – 3.75 (m, 1H), 3.72 – 3.60 (m, 3H), 3.55 – 3.37 (m, 1H), 3.36 –
1
3
.12 (m, 4H), 3.05 – 2.93 (m, 1H), 1.38 (s, 9H). C NMR (101 MHz, CDCl ): δ (ppm) 182.52,
3
71.78, 169.39, 157.22, 156.02, 135.87, 134.34, 133.79, 129.02, 128.91, 128.27, 126.75, 126.38,
18.09, 79.02, 55.60, 53.25, 52.12, 51.18, 37.65, 37.46, 28.19. HRMS (ESI-TOF) m/z: [M+H]+
calcd for C H N O 484.2442, found 484.2486.
2
6
33
3
6
3
. Syntheses of dipeptide (8): Similarly the dipeptide 8 was synthesized from Tr-aeg (6) and
1
L-prolime methyl ester . (150 mg, 41%). H NMR (400 MHz, CDCl ) δ 7.15 – 6.98 (m, 3H), 6.92
3
(
dd, J = 11.7, 5.5 Hz, 1H), 6.78 (t, J = 11.6 Hz, 1H), 6.70 – 6.58 (m, 1H), 5.91 – 5.77 (m, 1H),
.72 (d, J = 17.2 Hz, 1H), 4.49 (dd, J = 8.4, 4.0 Hz, 1H), 4.20 (t, J = 13.9 Hz, 1H), 3.90 – 3.81
m, 1H), 3.79 (s, 1H), 3.74 (dd, J = 11.2, 5.9 Hz, 1H), 3.69 (s, 3H), 3.64 (dd, J = 12.7, 5.5 Hz,
H), 3.55 (dt, J = 17.1, 8.8 Hz, 2H), 3.40 (d, J = 5.7 Hz, 3H), 2.35 – 2.27 (m, 1H), 2.27 – 2.15
4
(
1
1
3
(
m, 2H), 2.13 – 1.94 (m, 4H), 1.44 (d, J = 19.3 Hz, 9H). C NMR (101 MHz, CDCl ): δ (ppm)
3
1
5
81.71, 172.49, 167.89, 157.49, 156.37, 135.66, 134.02, 133.72, 124.99, 116.67, 79.07, 58.92,
3.37, 52.49, 52.26, 46.21, 37.87, 28.83, 28.42, 24.89, ( cis and trans isomers are existing around
S4

ACCEPTED MANUSCRIPT
secondary amide bond in 1:4, not predicted due to overlapping signals). HRMS (ESI-TOF) m/z:
+
[
M+H] calcd. for C H N O 456.2105, found 456.2123.
2
2
31
3
6
3
. Syntheses of dipeptide (9): Similarly the dipeptide 9 was synthesized from Tr-aeg (6) and L-
1
Isoleucice methyl ester. (45 mg 36%) H NMR (400 MHz, CDCl ) δ in ppm 7.90 (d, J = 8.2 Hz,
3
1
1
H), 7.24 – 7.12 (m, 1H), 7.11 – 7.00 (m, 2H), 6.92 – 6.81 (m, 1H), 6.79 – 6.70 (m, 1H), 5.65 (s,
H), 4.65 – 4.43 (q, 1H), 4.18 – 4.05 (m, 1H), 3.72 (d, J = 5.3 Hz, 1H), 3.69 (s, 3H), 3.63 – 3.50
(
1
m, 1H), 3.47 – 3.24 (m, 4H), 2.32 (dd, J = 17.1, 9.6 Hz, 1H), 2.02 – 1.85 (m, 1H), 1.52 (dd, J =
.0, 6.1 Hz, 1H), 1.47 – 1.34 (m, 9H), .21 – 1.12 (m, 1H), 0.96 – 0.82 (m, 6H). ( cis and trans
isomers are existing around carbamate amide bond in 1:4, not predicted due to overlapping
1
3
signals). C-NMR (101 MHz, CDCl ) δ in ppm 183.22, 172.43, 169.79, 157.71, 156.27, 136.95,
3
1
5
36.41, 136.18, 135.01, 133.83, 132.73, 127.70, 119.33, 113.09, 79.21, 64.92, 56.65, 56.25,
2.06, 51.39, 37.81, 37.28, 28.33, 24.94, 22.64, 15.68, 14.24, 11.53. ( cis and trans isomers are
+
existing around carbamate amide bond in 1:4). HRMS (ESI-TOF) m/z: [M+H] calcd. for
C H N O 472.2418, found 472.2428.
23
35
3
6
4
. Syntheses of dipeptide (10): Similarly the dipeptide 10 was synthesized from N-Boc glycine
1
and L-Phenylalanine methyl ester. H NMR (400 MHz, CDCl ) δ 7.36 – 7.17 (m, 3H), 7.16 –
3
7
3
1
.03 (m, 2H), 6.59 (s, 1H), 5.14 (s, 1H), 4.94 – 4.82 (m, 1H), 3.78 (dd, J = 18.7, 5.5 Hz, 2H),
1
3
.71 (s, 3H), 3.20 – 3.06 (m, 2H), 1.44 (s, 9H). C NMR (101 MHz, CDCl ) δ in ppm 171.70,
3
69.12, 155.91, 135.61, 129.17, 128.56, 127.09, 80.16, 77.00, 53.04, 52.29, 44.10, 37.83, 28.22.
+
HRMS (ESI-TOF) m/z: [M+H] calcd. for C H N O 337.1758, found 337.1723.
1
7
24
2
5
5
. Syntheses of tripeptide (11): Similarly the tripeptide 11 was synthesized from Tr-aeg (6) and NH -Gly-
2
1
Phe-OMe. (138 mg,41%). H NMR (400 MHz, CDCl ) δ 7.91 (s, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.27 –
3
7
4
3
.16 (m, 3H), 7.15 – 7.01 (m, 4H), 6.94 (dd, J = 25.0, 10.4 Hz, 2H), 6.81 – 6.68 (m, 1H), 5.55 (s, 1H),
.81 (dd, J = 13.4, 6.5 Hz, 1H), 4.08 (dd, J = 16.7, 6.2 Hz, 1H), 3.97 – 3.73 (m, 3H), 3.75 – 3.54 (m, 4H),
1
3
.53 – 3.34 (m, 2H), 3.29 (d, J = 5.3 Hz, 2H), 3.09 (qd, J = 13.8, 6.4 Hz, 2H), 1.39 (s, 9H). C NMR (101
MHz, CDCl ) δ in ppm 182.74, 172.02, 170.96, 169.07, 157.65, 156.18, 136.39, 136.00, 135.01, 134.05,
3
129.19, 128.48, 126.97, 118.83, 79.37, 56.85, 53.42, 52.13, 42.90, 37.58, 37.09, 28.30. HRMS (ESI-
+
TOF) m/z: [M+H] calcd. for C H N O 541.2657, found 541.2565.
2
8
36
4
7
S5

ACCEPTED MANUSCRIPT
1
3
. H-NMR spectrum of 2:
1
Figure S1: H-NMR of 2–tosyloxy tropone (2)
S6

ACCEPTED MANUSCRIPT
1
4
. H-NMR spectrum of monomer 5:
1
Figure S2: H-NMR of monomer (Boc-Tr-aeg-OEt).
S7

ACCEPTED MANUSCRIPT
1
3
5
. C-NMR spectrum of monomer 5:
*
1
3
Figure S3: C-NMR of monomer (Boc-Tr-aeg-OEt). (* hexane peaks)
S8

ACCEPTED MANUSCRIPT
6
. Mass spectrum of dipeptide 5:
HRMS (ESI-MS–Tof)
+
(M+H)
+
(M+Na)
S9

ACCEPTED MANUSCRIPT
1
7
. H-NMR spectrum of dipeptide 7:
*
*
1
Figure S4: H-NMR of dipeptide (Boc-Tr-aeg-Phe-OMe). (* hexane peaks)
S10

ACCEPTED MANUSCRIPT
1
3
8
. C-NMR spectrum of dipeptide 7:
*
*
1
3
Figure S5: C-NMR of dipeptide (Boc-Tr-aeg-Phe-OMe). (* hexane peaks)
S11

ACCEPTED MANUSCRIPT
1
9
. Deuterium exchange H-NMR experiment of dipeptide 7:
5
mg of dipeptide was dissolved in 0.5 ml of CDCl , to this three drops of D O was added and recorded NMR after 5
3
2
hrs.
1
Figure S6: H-NMR of dipeptide (Boc-Tr-aeg-Phe-OMe, 7) in CDCl and CDCl +D O.
3
3
2
S12

ACCEPTED MANUSCRIPT
1
0. Mass spectrum of dipeptide 7:
HRMS (ESI-MS–Tof) (7)
+
(M+H)
S13

ACCEPTED MANUSCRIPT
1
1
1. H-NMR spectrum of dipeptide 8:
*
*
1
Figure S7: H-NMR of dipeptide (Boc-Tr-aeg-Pro-OMe). (* hexane peaks)
S14

ACCEPTED MANUSCRIPT
13
1
2. C-NMR spectrum of dipeptide 8:
*
*
1
3
Figure S8: C-NMR of dipeptide (Boc-Tr-aeg-Pro-OMe). (* hexane peaks)
S15

ACCEPTED MANUSCRIPT
1
3. Mass spectrum of dipeptide 8:
HRMS (ESI-MS–Tof)
+
(M+H)
S16

ACCEPTED MANUSCRIPT
1
1
4. H-NMR spectrum of dipeptide 9:
1
Figure S9: H-NMR of dipeptide (Boc-Tr-aeg-Ile-OMe).
S17

ACCEPTED MANUSCRIPT
13
1
5. C-NMR spectrum of dipeptide 9:
*
1
3
Figure S10: C-NMR of dipeptide (Boc-Tr-aeg-Ile-OMe). (* hexane peaks)
S18

ACCEPTED MANUSCRIPT
1
6. Mass spectrum of dipeptide 9:
HRMS (ESI-MS–Tof)
S19

ACCEPTED MANUSCRIPT
1
1
7. H-NMR spectrum of dipeptide 10:
1
Figure S11: H-NMR of dipeptide (Boc-Gly-Phe-OMe).
S20

ACCEPTED MANUSCRIPT
13
1
8. C-NMR spectrum of dipeptide 10:
1
3
Figure S12: C-NMR of dipeptide (Boc-Gly-Phe-OMe).
S21