Application of C-Terminal 7-Azabicyclo[2.2.1]heptane to Stabilize β-Strand-like Extended Conformation of a Neighboring α-Amino Acid

Article abstract of DOI:10.1021/acs.joc.8b01756

β-Strands are formed by extended linear peptide chains that are usually paired to form β-sheet structure through interstrand hydrogen bonding. Linking a structured organic molecule with α-amino acid(s) can enforce or stabilize β-strand-like extended struc

Full text of DOI:10.1021/acs.joc.8b01756

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Article
Application of C-terminal 7-azabicyclo[2.2.1]heptane to stabilize #
-strand-like extended conformation of a neighboring #-amino acid
Luhan Zhai, Siyuan Wang, Masayuki Nara, Koh Takeuchi,
Ichio Shimada, Yuko Otani, and Tomohiko Ohwada
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01756 • Publication Date (Web): 04 Oct 2018
Downloaded from http://pubs.acs.org on October 5, 2018
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Application of C-terminal 7-azabicyclo[2.2.1]heptane to stabilize β−strand-like
extended conformation of a neighboring α-amino acid
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Luhan Zhai, Siyuan Wang, Masayuki Nara, Koh Takeuchi,
3,4
1,*
1,*
Ichio Shimada, Yuko Otani, Tomohiko Ohwada
1
Laboratory of Organic and Medicinal Chemistry, Graduate School of
Pharmaceutical Sciences, The University of Tokyo,
7
-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
2
Department of Chemistry, College of Liberal Arts and Sciences,
Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan
Molecular Profiling Research Center for Drug Discovery, National Institute of
3
Advanced Industrial Science and Technology (AIST), Aomi, Koto-ku, Tokyo 135-0064,
Japan
4
Laboratory of Physical Chemistry, Graduate School of Pharmaceutical Sciences,
The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
E-mail: otani@mol.f.u-tokyo.ac.jp, ohwada@mol.f.u-tokyo.ac.jp
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Graphical Abstract
β-sheet structure
!-strand like
extended structure
O
H N
Me
H N
O
O
O
R
R
NH
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
2
3
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9
0
N H
R
N H
O
O
R
H
N
R
HN
O
R1
H
H N
R
H N
O
O
O
H
R
R
N
(ABh)
N H
!-strand enhancer R1
N H
O
/stabilizer
R
cis-amide
R
O
Without inter-strand/intra-residual
hydrogen bonding
H N
H N
O
Abstract
β-Strand is formed by extended linear peptide chains that are usually paired to form
β-sheet structure through inter-strand hydrogen bonding. Linking a structured organic
molecule with α-amino acid(s) can enforce or stabilize β-strand-like extended
structures of the jointed amino acids. Spectroscopic and simulation studies indicated
that the presence of a C-terminal 7-azabicyclo[2.2.1]heptane amine (Abh) favors a
β−strand-like extended conformation of the adjacent α-amino acid on the N side. The
bridgehead substitution of the Abh unit biases the amide cis-trans equilibrium of the
adjacent α-amino acid residue to cis conformation. The proximity, specified by the
presence of bond paths (such as H-H bond path) between the bridgehead proton of
Abh and the α-proton of the α-amino acid provides a driving force favoring the
extended conformation, which is independent of solvents. These results provide a
basis for de novo design of β-strand-mimicking extended peptides by using β-strand
enforcer/stabilizer even in the absence of the inter-strand hydrogen bonding.
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Introduction
About 30% of protein secondary structure consists of β-strand/β-sheet structure,
which is formed by extended linear peptide chains that are usually paired to form
antiparallel, parallel or barrel β-sheet structure through inter-strand hydrogen
1
1
1
1
1
1
1
1
1
1
2
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0
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9
0
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9
0
1
2
3
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7
8
9
0
1
-5
bonding. Distinctive phi (φ) and psi (ψ) dihedral torsion angles distinguish β-
strands/β-sheets (e.g. antiparallel, φ=-139°, ψ=-135°; parallel, φ=-119°, ψ=113°)
1
from α-helix (-58°, -47°) and β-turn (e.g. type I -60°, -30°) structures. Amino acids
that favor strand formation in proteins are branched residues such as Val, Ile, and Thr,
1
as well as Tyr, Cys, Trp, and Phe. There have been two strategies of molecular
design to mimic β-strand structures and hydrogen-bonding β-sheet structures. One is
the use of non-peptidic scaffolds to attain shape similarities to β-strand-like extended
6
-20
structures,
and the other is utilizing amino acids and amino acid surrogates to
stabilize inter-strand hydrogen-bonding β-sheet structures, which includes
21
macrocyclization (backbone or side chain to side chain) and replacement of amino
2
2-28
acids.
A major difference between these two strategies is the absence or presence
of the ability to form inter-strand hydrogen bonding. Many examples of the former
approach have been reported, using various non-amino acid units such as
6
,7
8-10
11-16
17
dibenzofurans, oligoureas,
metallopeptides,
indolin-3-ones, imidazolidin-2-
1
8
19
20
ones, epiindolines, and oligothienylpyridines. These mimics do not contain inter-
strand hydrogen bonding sites. Therefore their general applicability to mimic protein-
protein interaction via β-sheets formation is unclear. On the other hand, there are
only a few examples of the latter approach, such as the use of stapling peptides
21
(macrocyclization), and inter-strand or intramolecular hydrogen bond-stabilizing
2
2
scaffolds such as 1,2-dihydro-3(6H)-pyridinone, the unnatural amino acid Hao (5-
2
3-25
26
27
hydrazino-2-methoxybenzoic acid),
aminopyrazole, N-difluoromethyl-triazole,
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and 4,4,4-trifluorothreonine. In these examples, formation of intra- and/or inter-
molecular hydrogen bonding stabilized paired β-sheet structures. Some important
applications of these β-sheet peptide mimics include HIV-1 protease inhibitors and
0
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2
1
amyloid aggregation inhibitors.
(
a)
R
H
N
O
R
H
N
O
R
H
N
Without inter-strand/intra-residual
hydrogen bonding
N
H
N
H
N
H
O
R
O
R
O
H
N
R
O
H
N
R
O
H
N
H
R
O
H
R
O
H
N
N
H
N
H
N
N
O
R
O
R
O
N
H
N
H
β-strand enforcer β-strand-like extended
O
R
O
R
O
/
stabilizer
structure
β-sheet structure
(b)
R
O
N
(c)
O
O
H
O
H
CH3
N
N
H
R2
H
N
O
R
H C
3
O
N
H
2
O
H
O
2
R2
N
R
H
R
H
H
N
R1
N
1
4
H
R1
1
4
2
O
O
cis
trans
cis
trans
Figure 1. Inter-residue interactions to enforce β-strand extended structure by
control of amide isomerization. (a) β-Strand enforcer/stabilizer (b) Amide
equilibrium of homooligomers of bicyclic β-proline peptide surrogates. (c) C-
Terminal 7-azabicyclo[2.2.1]heptane-linked α-amino acid. Heteropeptide dimers and
bicyclic amides were used in this study.
As a hybrid strategy of the above two methods in terms of shape and hydrogen-
bonding ability (Figure 1(a)), linking a structured organic molecule with α-amino
acid(s) can enforce or stabilize β-strand-like extended structures of the jointed amino
acids even in the absence of inter-strand hydrogen bond formation. Examination of
tertiary amides of the bicyclic amine 7-azabicyclo[2.2.1]heptane (7-
azabicyclo[2.2.1]heptane, Abh) has shown that this bicyclic system induces nitrogen
2
9
pyramidalization. Notably, the derivative bearing a 2-carboxylic acid functionality
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can be regarded as a conformation-constrained β-proline surrogate (see Figure 1b).
Furthermore, in a bridgehead-substituted β-proline analogue (7-azabicyclo[2.2.1]-
heptane-2-carboxylic acid) with an alkoxymethyl substituent installed at the C4-
1
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0
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9
0
bridgehead position, the bridgehead substituent completely biases the amide cis-trans
3
0
equilibrium to the cis side, irrespective of the solvent (Figure 1b). Homooligomers
of a bridgehead-substituted bicyclic β-proline analogue were found to generate
ordered structures such as helix, and spectroscopic analysis showed that only two
units of 7-azabicyclo[2.2.1]heptane-2-carboxylic acid are sufficient to form a pre-
3
0
1
organized structure. The detection of H-NOE between the C -bridgehead-proton
1
and the α-H proton of the N-side adjacent bicycle support the cis amide structure and
2
the proximity of the bicyclic units (see Figure 1b). These observations led us to
consider that this 7-azabicyclo[2.2.1]heptane unit would affect the conformation of
the adjacent α-amino acid on the N-terminal side in a similar manner to that of the
homo-oligomer of 7-azabicyclo[2.2.1]heptane-2-carboxylic acid through the
interaction of the C -bridgehead hydrogen atom (Figure 1c).
1
In this work, we synthesized various kinds of hetero-peptides (Ac-X-Abh) and
showed that they have complete cis-amide preference independently of the N-terminal
1
α-amino acid and solvent (Figure 1c). Furthermore, H NMR and Raman
spectroscopic studies showed how the main-chain freedom of the neighboring α-
amino acid is reduced by incorporation of the C-terminal-bicyclic amine (Abh),
driving the neighboring α-amino acid to take a β-strand-like extended secondary
structure. It has been shown that the dihedral angle φ (conformational preference) of
α-amino acid dipeptides, i.e., Ac-X-NHMe (X = an amino acid) is similar to that of
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31,32
short peptides.
Therefore, it is expected that the conformational effect of C-
terminal Abh on the N-terminal side can be estimated by comparing conformational
preferences of Ac-X-NHMe and Ac-X-Abh.
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Results and Discussion
Synthesis and optimization of coupling reagents
First of all, coupling conditions for various protected α-amino acids and Abh
were optimized (Scheme 1). It was expected that Abh would have low reactivity due
to the presence of the bridgehead substituent as well as the secondary amine.ꢀFifteen
amino acids with either N-protected polar or non-protected non-polar side chains were
connected to the nitrogen atom of Abh (1a-1o) (Table 1).
ꢀ
In the cases of amino acids with relatively small and hydrophobic side chains,
conventional coupling conditions (i.e., DIC/HOBT (condition A)) were effective
Table 1). However, in the cases of polar amino acids with protecting groups on both
(
the side chain and the N-terminal side, the yield was low or the product was not
formed, probably due to the steric bulkiness of the protected α-amino acid and the
low nucleophilic reactivity of the bicyclic amine (Abh). In the case of Fmoc-Trp,
conventional coupling conditions (A) gave the product (Fmoc-Trp-Abh(OBn)) (1i) in
30% yield (Scheme 1). HTBU and COMU (condition B) also failed to give a good
[33]
yield. Finally, we tried 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (Scheme 1),
which is a derivative of 2,4,6-trichloro-1,3,5-triazine (TCT) and causes less
racemization (see Supporting Information), with N-methyl morpholine (NMM) as a
base. The yield of Fmoc-Trp-Abh(OBn) was increased to 79%. We found that CDMT
(
condition C) can give good to moderate yields with amino acids for which other
coupling reagents are unsuccessful (Table 1). The HPLC chromatograms of these
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products were shown in Supporting Information.
Protected
α-amino
acid
O
Protected
α-amino
acid
NH
NHFmoc
Boc
N
H
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
BnO
BnO
BnO
°C-rt, 30 min
N
TFA
(
Fmoc-Trp-OH)
0
Fmoc-Trp-Abh(OBn) (1i)
a) DIC, HOBT, DIPEA, DMF, 12h, rt : yield 30% (two steps)
b) HBTU, DIPEA, DMF, 12h, rt : yield 0% (two steps)
o
c) CDMT, NMM, CH Cl , 12h, 0 C-rt : yield 79% (two steps)
2
2
O
N
NC
-PF
Cl
N
O
Cl
N
6
N
N
N
N
O
-PF6
N
N
N
N
O
N+
N+
Cl
Cl MeO
OMe
N
O
TCT
CDMT
HBTU
COMU
Scheme 1. Synthesis of C-terminal Abh analogue of α-amino acid
Table 1 Synthesis of C-terminal Abh analogue of α-amino acid
Compound
number
coupling
method
A
Yield(%)
Amino acid (AA)
Product
1a
Boc-Gly
Boc-Gly-Abh(OBn)
85
89
88
70
57
77
75
38
43
44
54
72
30
79
14
16
60
trace
C
A
C
C
A
A
B
C
A
A
C
A
C
A
B
C
A
1
b
Boc-Ala
Boc-Val
Boc-Ile
Boc-Ala-Abh(OBn)
Boc-Val-Abh(OBn)
Boc-Ile-Abh(OBn)
Cbz-Leu-Abh(OBn)
Boc-Phe-Abh(OBn)
1c
1
d
1
e
Cbz-Leu
Boc-Phe
1f
1g
Boc-β-Pro
Boc-β-Pro -Abh(OBn)
1
h
Boc-(Trt)-Asn
Boc-(Trt)-Asn -Abh(OBn)
1i
Fmoc-Trp
Fmoc-Trp-Abh(OBn)
1
j
Boc-(OBzl)-Tyr
Boc-(OBzl)-Tyr-Abh(OBn)
1
k
Boc-(Trt)-His
Boc-(Trt)-His-Abh(OBn)
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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C
A
B
C
C
C
C
31
trace
trace
69
1
l
Boc-(OBzl)-Asp
Boc-(OBzl)-Asp-Abh(OBn)
1
m
Boc-(Cbz)-Lys
Boc-(t-Bu)-Ser
Boc-(Cbz)-Lys -Abh(OBn)
Boc-(t-Bu)-Ser -Abh(OBn)
54
0
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9
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1
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9
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9
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1
2
3
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5
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7
8
9
0
1n
28
1
o
Boc-(NO
2
)-Arg
Boc-(NO
2
)-Arg -Abh(OBn)
trace
Coupling conditions: A: AA-OH (1.5 eq.), DIC or EDCI (2 eq.), HOBt (2 eq.),
iPr EtN (3.0 eq.), DMF, rt, 12 h. B: AA-OH (1.5 eq.), COMU (2 eq.), collidine (3.0
2
eq.), DCM, rt, 12 h. C: AA-OH (1.5 eq.), CDMT (2 eq.), NMM (3 eq.), DMAP (0.1
eq.), DCM, rt, 12 h.
Cis-amide preference of α-amino acid-Abh derivatives
1
The amide equilibrium of α-amino acid-Abh derivatives was studied by H-NOESY
spectroscopy. As shown in Figure 2, NOE can be detected between the bridgehead
proton (H
1 2
) and the α proton (H ) of the side chain of the α-amino acid, which is
consistent with cis-amide structure. The NMR results indicated that all the dimers
(
Table 1) take the cis-amide structure, and trans-amide structure was not observed.
In the case of alanine, the α-carbon has a methyl group, making it one of the
simplest amino acids. N-Boc-alanine was coupled with the Abh to form a hetero-
dimer (1b). The observation of NOE between the bridgehead proton (H
proton (Figure 2 (a)) is consistent with cis-amide structure.
1
) and methyl
Furthermore, deprotected H-Ala-Abh(OMe) (H-1b(OMe)) also takes cis-amide
structure in D O (Figure 2 (b)). The NOE signal between the bridgehead proton H
and α proton (H ) of Ala was detected even in water. This is consistent with cis-amide
structure, and demonstrates stability in water.
2
1
2
Phenylalanine can be regarded as an amino acid in which a hydrogen of the methyl
group of Ala is substituted with a phenyl group. The NOE signal between the
bridgehead proton H
1
2
and the α proton (H ) of Phe was detected in the NOESY
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spectra of Boc-Phe-Abh(OBn) 1f in CD
3
OD, suggesting cis-amide structure (Figure 2
(c)). The cis-amide preference is also shown in other dimers (see Supporting
Information).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
NH2
CH3
HN
HN
O
O
O
O
CH3
H
2
H
H2
N
N
H
N
1
1
H
Me
1
O
BnO
BnO
Boc-Phe-Abh(OBn)
Boc-Ala-Abh(OBn)
1b)
H-Ala-Abh(OMe)
(1b(OMe)-H)
(1f)
(
(b)
(c)
(a)
H2
H2
H1
H1
NOESY (1b, CDCl , 25 °C) NOESY (1b(OMe), D O, 25 °C) NOESY (1f, CD OD, 25 °C)
3
2
3
Figure 2. Various N-terminal of α-amino acids. NOESY spectra of (a) Boc-Ala-
Abh(OBn) (1b), (b) H-Ala-Abh(OMe) (H-1b(OMe)) and (c) Boc-Phe-Abh(OBn)
(1f).
Effect of the bridgehead group on amide isomerization
2 2
Various kinds of bridgehead substituents of different sizes, CH OBn, CH OMe,
2 2
CH OH, CO Me, and CN, were chosen to investigate the effect of bridgehead
substituents on the amide isomerization (Figure 3). Boc-alanine was coupled with the
bicyclic amines.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
HN
O
O
HN
O
HN
H
O
O
O
O
CH3
CH3
CH3
N
N
H
N
H
1
H
1
1
H CO
3
HO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Boc-Ala-Abh(OMe)
1b(OMe))
Boc-Ala-Abh(OH)
(
(1b(OH))
O
O
HN
O
HN
O
O
O
CH3
CH3
O
N
N
NC
H
H
1
1
H CO
3
Boc-Ala-Abh(CN)
(1b(CN))
Boc-Ala-Abh(CO Me)
2
(
1b(CO Me))
2
Figure 3. Effect of bridgehead substitution
In Boc-Ala-Abh(OMe) 1b(OMe), the bridgehead group was changed from Bn to
smaller Me, but cis-amide conformation was retained, as indicated by the observation
of NOE between the bridgehead proton H1 and the methyl group (Figure 3). The DFT
calculation at 298K showed that the cis structure is 2.4 kcal/mol more stable than the
trans one. The difference in calculated Gibbs free energy is about 2.9 kcal/mol,
suggesting that the cis amide contribution is more than 98%.
2 2
In the cases of Boc-Ala-Abh(CO Me) 1b(CO Me) and Boc-Ala-Abh(CN) 1b(CN),
2
which have an sp carbon and an sp carbon on the bridgehead, respectively (Figure 3),
1 2 3
NOE was observed between the bridgehead proton H and the α-proton H in CD OD.
This is consistent with cis-amide structure. We consider that the cis-amide
conformation is preferred due to the rigid ester group and the linear nitrile group at
the bridgehead position.
In the case of a hydroxy group at the bridgehead position in Boc-Ala-Abh(OH)
1 2
1b(OH), NOE between the bridgehead proton H and α proton H was detected in
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CDCl
3
(Figure 3). This is consistent with cis-amide structure. Temperature coefficient
studies also supported the existence of intramolecular hydrogen bonding between the
amide carbonyl and the hydroxy group in Boc-Ala-Abh(OH) 1b(OH) (Figure 3);
small temperature coefficients for the OH resonance (-3.47 ppb/K) were obtained in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4-36
DMSO-d
6
.
This small value is consistent with intramolecular hydrogen bonding,
which may contribute synergistically to stabilization of the cis-amide structure.
NMR study of the conformation of α-amino acid linked to C-terminal Abh
3
α
N
The temperature dependence of the J(H , H ) coupling constants of amide protons
37-39
of Ac-α-amino acid-NHMe provides a good index of conformational stability.
3
α
N
The following relationship between the J(H , H ) coupling constant and the dihedral
4
0
angle φ of the respective amino acid residue was used in this study:
2
J(φ) = A cos (φ-60) +B cos(φ-60) + C
A = 6.51, B = -1.76 and C = 1.60
1
To obtain residue-level details of the peptide conformations, we performed H NMR
spectroscopy to study the conformational preferences of three kinds of α-amino acids
(Ala, Val, and Ile). Amino acid dipeptides are in equilibrium mainly between α-helix
(α), β-strand (β), and polyproline II (PII) conformations. These three conformations
3
are well populated for most amino acids: β (φ = -120°, J =9.6±0.3Hz), PII (φ = -75°,
3
3
41
3
J = 5.8± 0.4Hz), and α
R
(φ = -60°, J =3.8±0.5Hz). Thus, the J -value of β is
41
R
considerably larger than those of PII and α (right-handed α-helix). Also, it is
reported that alanine dipeptide (Ac-Ala-NHMe) 3b takes predominantly a PII
conformation while valine dipeptide (Ac-Val-NHMe) 3c and isoleucine dipeptide
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1
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2
2
2
2
2
2
2
3
3
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5
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5
5
6
31,42
(
Ac-Ile-NHMe) 3d favor β conformations.
First, the conformational stability of
Ac-Ala-Abh(OBn) 2b was compared with that of alanine dipeptide (Ac-Ala-NHMe,
1
3
b). Then, H-NMR spectra of Abh compounds with the same bicyclic skeleton but
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
different α-amino-acid side chain bulkiness (valine and iso-leucine) were measured in
3
α
N
organic solvent, and the values of J(H , H ) coupling constant were determined as a
function of temperature. Here, the coupling constant reflects the φ-angle of the α-
amino acid residue in the α-amino acid-Abh system (see Figure 4).
The conformation of alanine dipeptide in various solvents has been widely
43
3
α
N
studied. The values of J(H , H ) of the alanine dipeptide 3b at 25°C in DMSO-d
6
and in water were 7.68 Hz and 6.03 Hz, respectively (Table 2), which are comparable
4
3
3
to the reported values (7.60 Hz, and 6.02 Hz, respectively). The values of the J-
coupling constant of Ac-Ala-Abh(OBn) 2b in DMSO-d and in water were 7.97 Hz
6
and 6.89 Hz, respectively, being larger than those of Ac-Ala-NHMe 3b; this suggests
that Ac-Ala-Abh(OBn) 2b has a higher population of β conformation in both solvents.
3
α
N
The temperature dependency of J(H , H ) of Ac-Ala-Abh(OBn) 2b was compared
with that of alanine dipeptide (Ac-Ala-NHMe, 3b) in DMSO-d (Figure 4(a)). In the
6
case of Ac-Ala-Abh(OBn) 2b, the change of the coupling constants with temperature
is small over a nearly 40-degree temperature range, whereas in the case of the alanine
dipeptide 3b, the change of the coupling constants with temperature is large over a
2
0-degree temperature range. The strong temperature dependency of the alanine
dipeptide 3b suggests that its conformation is very flexible. On the other hand, Ac-
Ala-Abh(OBn) 2b exhibits greater conformational rigidity and stability (Figure 4(a)).
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(a)8.2ꢀ
8.1ꢀ
O
H
N
ϕ
H C
CH3
8ꢀ
3
N
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
O
7.9ꢀ
7.8ꢀ
7.7ꢀ
CH3
3
b
H
7.6ꢀ
H3C
CH3
O
O
N
7
.5ꢀ
.4ꢀ
N
H
H
7
BnO
7.3ꢀ
2
b
7.2ꢀ
15ꢀ
25ꢀ
35ꢀ
45ꢀ
55ꢀ
T (°C)
T/°C
(b)
9.1ꢀ
O
H
H
H C
N
CH3
3
N
H
ϕ
O
9ꢀ
3
c
O
8.9ꢀ
8.8ꢀ
8.7ꢀ
H
N
O
N
ϕ
H
Bn
O
2
c
-
30ꢀ
-25ꢀ
-20ꢀ
-15ꢀ
T/ Cꢀꢀ
-10ꢀ
-5ꢀ
o
T (°C)
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(c)
9.1ꢀ
O
H
H
N
H C
CH3
3
N
H
ϕ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
9ꢀ
3d
O
H
O
N
8.9ꢀ
N
H
Bn
O
2d
8.8ꢀ
-
10ꢀ
-5ꢀ
0ꢀ
5ꢀ
10ꢀ
15ꢀ
20ꢀ
o
T/ Cꢀꢀ
T (°C)
H C
H2
N
3
O
H
O
N
H
N
O
H1
CH3
(
d)
O
4
b
NH1ꢀ
8ꢀ
7
7
7
7
.95ꢀ
.9ꢀ
.85ꢀ
.8ꢀ
.75ꢀ
.7ꢀ
.65ꢀ
7
7
7
ϕ1
7
.6ꢀ
.55ꢀ
.5ꢀ
7
7
15ꢀ
20ꢀ
25ꢀ
30ꢀ
35ꢀ
40ꢀ
T (°C)
ꢀ
o
T/ Cꢀ
NH2ꢀ
8ꢀ
7.95ꢀ
7.9ꢀ
7
.85ꢀ
.8ꢀ
.75ꢀ
.7ꢀ
ϕ27.65ꢀ
.6ꢀ
.55ꢀ
.5ꢀ
7
7
7
7
7
7
15ꢀ
20ꢀ
25ꢀ
30ꢀ
35ꢀ
40ꢀ
ꢀ
T (°C)
T/ Cꢀ
o
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3
Figure 4 (a-c) Comparison of the temperature dependency of J-coupling
constants of α-amino acid moiety with different C-terminal groups (d)
temperature-dependency of two dihedral angles of tripeptide Ac-Ala-Ala-Abh 4b.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Table 2 Values of J-coupling constants at 25°C.
Water (298 K)
DMSO-d (298 K)
6
a
Ac-Ala-Abh(OBn) 2b
Ac-Ala-NHMe 3b
6.89 Hz
7.97 Hz
7.68 Hz
b
6.03 Hz
Water (294 K)
3
CDCl (291 K)
c
Ac-Ile-Abh(OBn) 2d
Ac-Ile-NHMe 3d
7.87 Hz
8.99 Hz
8.84 Hz
d
7.20 Hz
a
c
b
1
0% CD
3
OD+90% H
2
O, pH=2-3. 10% D
2
O+90% H O, pH=2-3.
2
d
1
4% CD
3
OD+86% H
2
O, pH=2-3. 10% D
2
O+90% H O, pH=2-3.
2
To confirm the reproducibility of the restrictiveness of the bicyclic scaffold, the
conformations of Ac-Val-Abh(OBn) 2c and Ac-Ile-Abh(OBn) 2d were studied
(
Figure 4(b)(c)). Compared with alanine, valine has a bulkier side chain. Variable-
temperature NMR spectra of Ac-Val-Abh(OBn) 2c and valine dipeptide (Ac-Val-
3
NHMe, 3c) in CDCl
3
were measured and the values of the J coupling constants were
3
plotted (Figure 4(b)(c)). In the case of Ac-Val-Abh(OBn) 2c, the change of the J
coupling constants upon change in temperature is small over a 25-degree temperature
range, while in the case of the valine dipeptide (Ac-Val-NNMe, 3c), the
corresponding change is large in the same temperature range. The strong temperature
dependency of the valine dipeptide 3c suggests that it is conformationally flexible,
whereas Ac-Val-Abh(OBn) 2c shows conformational rigidity and stability (Figure
4
(b)).
Compared with Val, Ile has a bulkier side chain. The corresponding variable-
temperature NMR spectra of Ac-Ile-Abh(OBn) 2d and the Ile dipeptide (Ac-Ile-
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
6
NHMe, 3d) measured in CDCl
3
showed little change in the case of Ac-Ile-Abh(OBn)
2
d over a 25-degree temperature range, whereas the Ile dipeptide 3d showed a
marked change in the same temperature range (Figure 4(c)). The strong temperature
dependency of the Ile dipeptide 3d suggests that its conformation is flexible, whereas
Ac-Ile-Abh(OBn) 2d shows conformational rigidity and stability (Figure 4(c)). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
α
N
values of J(H , H ) of the Ile dipeptide 3d in CDCl and in water were 8.84 Hz and
3
3
7
.20 Hz, respectively (Table 2). The values of the J-coupling constant of Ac-Ile-
Abh(OBn) 2d in CDCl and in water were 8.99 Hz and 7.87 Hz respectively (Table 2),
3
being larger than those of the Ile dipeptide 3d; this result suggests that Ac-Ile-
Abh(OBn) 2d has a higher population of β-strand conformation than 3d in both
solvents.
To know how far the restriction can be transferred, we elongated the main chain and
studied the conformation of Ac-Ala-Ala-Abh(OBn) 4b. Variable-temperature NMR
3
spectra were measured in DMSO-d
6
and the values of the J coupling constant of the
two NHs were plotted against temperature (Figure 4(d)). For both NHs (NH1 and
NH2), the change was rather flat over a 20-degree temperature range, suggesting that
the whole molecule is conformationally restricted. Thus, the conformational
restriction arising from the C-terminal bicyclic moiety appears to suppress rotation for
at least two α-amino acid residues from the N-terminal (Figure 4(d)).
Raman spectroscopy
Raman spectroscopy is a powerful tool to detect the distribution of main-chain
31, 42
conformations of peptides.
Here, we examined the skeletal vibrations (810-950
-
1
cm ) in the Raman spectrum of the alanine dipeptide (Ac-Ala-NHMe, 3b). These
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vibrations consist of one main-chain Cα-C stretching vibration, and two skeletal Cα-N
3
1
stretching and N-Cα-C bending modes. We measured the Raman spectrum of the
alanine dipeptide (Ac-Ala-NHMe, 3b) and Ac-Ala-Abh(OBn) 2b (Figure 5) and
-
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
observed three characteristic bands in the region between 810 and 950 cm . In the
case of the alanine dipeptide 3b, the band frequencies for PII, α
R
and β are well
−
1
−1
−1
separated and were assigned as 863 cm , 915 cm , and 849 cm , respectively
31
(
Table 3), in accordance with reported data. In the case of Ac-Ala-Abh(OBn) 2b,
−
1
the band frequencies for PII and β are well separated, and were assigned as 879 cm ,
−
1
8
R
54 cm , respectively, while α is lost (Figure 5b)). The conformational populations
can be calculated from the band areas (Table 3). In the case of alanine dipeptide 3b,
the predominant conformation is PII (calculated population: 63%), whereas the
R
population of α conformation is 23%, and the population of β conformation is 14%.
In the case of Ac-Ala-Abh(OBn) 2b, the predominant conformation is β (66%), and
the population of PII conformation is 34%. Thus, the Abh scaffold alters the
predominant conformation from PII to β conformation in the Ala moiety.
(a)
(b)
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0
1
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9
0
1
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9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Comparison of Raman skeletal vibrations of C-NHMe and C-Abh
amino acids (a) Raman spectrum of alanine dipeptide 3b (b) Raman spectrum of Ac-
Ala-Abh(OBn) 2b. Color: black=observed spectrum, green= deconvolution spectrum
R
of α , blue= deconvolution spectrum of PII, red= deconvolution spectrum of β.
Table 3. Raman band frequencies of skeletal vibrations.
β
αR
P
II
a
−1
−1
−1
Alanine dipeptide 3b
849 cm (14%)
915 cm (23%)
863 cm (63%)
a
−1
−1
Ac-Ala-Abh(OBn) 2b
854 cm (66%)
ND
879 cm (34%)
a
The sample was measured in hexane dry film. ND= not detected.
Metadynamics simulation of the conformations of α-amino acid moiety bound to
Abh
We carried out metadynamics simulation in explicit DMSO (in the cases of Ala
derivatives) and in chloroform (in the cases of Val and Ile derivatives) to obtain the
44
potential energy landscape of the conformations of the peptides (Figure 6). The
solvent corresponds to the NMR experiments, but water environment provided similar
results. Metadynamics simulations at 298K in DMSO suggested that Ac-Ala-
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Abh(OBn) 2b favors mainly β and PPII conformations (Figure 6(a)), while the
alanine dipeptide 3b exhibits β, PPII, and α-helix conformations, and its low potential
energy region is much broader than that of 2b.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
3
3
3
4
4
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5
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5
5
5
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6
0
1
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5
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7
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9
0
1
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9
0
1
2
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9
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1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
Ac-Ala-Abh (OBn) 2bꢀ
Ac-Ala-NHMe 3b
O
DMSO 298K
DMSO 298K
O
N
H
O
H
N
N
H C
CH3
3
N
H
O
ϕ
φ
CH3
O
βꢀ
PPIIꢀ
βꢀ PPIIꢀ
ϕ
ϕ
αꢀ
αꢀ
φ
φ
(kcal/mol)ꢀ
(b)
Ac-Val-Abh (OBn) 2cꢀ
Ac-Val-NHMe 3c
CHCl 298K
φ
O
CHCl 298K
3
3
O
H
O
N
H
H C
N
CH3
3
N
N
H
ϕ
O
φ
O
PPIIꢀ
βꢀ
PPIIꢀ
βꢀ
ϕ
ϕ
αꢀ
φ
φ
(kcal/mol)ꢀ
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3
4
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5
5
5
5
6
(
c)
Ac-Ile-Abh (OBn) 2dꢀ
CHCl 298K
Ac-Ile-NHMe 3d
CHCl 298K
3
3
O
O
φ
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
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5
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7
8
9
0
O
H C
N
CH3
3
N
H
N
H
ϕ
N
φ
O
O
βꢀ
βꢀ
PPIIꢀ
PPIIꢀ
ϕ
ϕ
(
kcal/mol)ꢀ
φ
φ
(d)
O
φ1
H2
N
O
N
H
1
φ2
N
O
4bꢀ
DMSO 298K
O
NH1:
NH2:
βꢀ
PPIIꢀ
PPIIꢀ
βꢀ
ϕ1
ϕ2
φ1
φ2 (kcal/mol)ꢀ
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(
e)
Ac-Ala-Abh(OBn) ꢀ2 bꢀ
Ac-Ala-Abh(H)ꢀ2b(H)ꢀ
φ
O
φ
O
O
O
N
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
N
N
H
O
ϕ
ϕ
βꢀ
PPIIꢀ
βꢀ PPIIꢀ
φꢀ
φꢀ
(kcal/mol)ꢀ
Figure 6. Energy landscapes of metadynamics simulations. (a) Ac-Ala-Abh(OBn)
b and alanine dipeptide 3b in DMSO, (b) Ac-Val-Abh(OBn) 2c and valine
dipeptide 3c in chloroform and (c) Ac-Ile-Abh(OBn) 2d and isoleucine dipeptide
d in chloroform. (d) Ac-Ala-Ala-Abh(OBn) 4b and Ac-Ala-Ala-NHMe
Supporting Information) (e) alanine derivatives bearing bridgehead-
2
3
(
substituted and unsubstituted Abh
Since Ac-Ala-Abh(OBn) 2b shows markedly fewer accessible minimum
conformations, it is more restricted in terms of the conformation of the Ala moiety
(Figure 6(a)). The low potential energy region of Ac-Val-Abh(OBn) 2c and Ac-Ile-
Abh(OBn) 2d is mainly localized in the β conformation region (Figure 6 (b)) and is
narrower than those of the valine (3c) and isoleucine (3d) dipeptides, indicating a
stronger restriction of the conformations of the Val and Ile moieties (Figure 6(b) and
(c)).
Metadynamics simulation of the two alanine moieties of Ac-Ala-Ala-Abh(OBn)
4
b (Figure 6(e)) also showed the restriction as compared with Ac-Ala-Ala-NHMe 5b
(see Supporting Figure). This is consistent with the weak temperature dependency of
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2
2
2
2
2
3
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4
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4
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5
5
5
5
5
6
3
α
N
the J(H , H ) values of the two Ala NH protons of Ac-Ala-Ala-Abh(OBn) 4b
(
Figure 4(d)).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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9
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0
1
2
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4
5
6
7
8
9
0
As described in the previous section, the NMR study indicated that the bridgehead
proton is close to the α-proton of the α-amino acid, due to the characteristic upward-
projecting direction of the bridgehead proton, and we postulated that this proximity
would influence the main chain rotation of the adjacent α-amino acid. To test this idea,
we compared the simulated potential energy landscapes of Ac-Ala-Abh(OBn) 2b and
Ac-Ala-Abh(H) 2b(H) (Figure 6(e)). The energy maps were similar, suggesting
similar conformational preferences. This implies that some interaction between the
bridgehead proton of the Abh and the α-proton and the side chain of the α-amino acid
both contribute to the restriction of the main chain rotation of the linked α-amino acid
residue.
Interaction of the C-terminal Abh unit with the adjacent α-amino acid in β-
strand conformation
Proximity interaction of the Abh unit with the β-strand conformers of adjacent α-
amino acids (2b, 2c and 2d) can be validated in terms of molecular graphs of quantum
45
theory of atoms-in-molecules (QTAIM) analysis. Figure 7 showed the presence of
through-space weak accumulation of electron density (bond path, red line in Figure 7)
between the Abh unit and the β-strand conformers of adjacent Ala, Val and Ile. In all
cases ((a) Ala 2b, (b) Val 2c and (d) Ile 2d) a bond path between the carbonyl oxygen
atom and the methylene H atom (in the case of 2b and 2d) or the C atom (in the case
of 2c) of the bridgehead substituent, and several bond paths between the methylene H
atom(s) in the amino acid side chain and the Abh bridgehead H atom and/or the Abh
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bicyclic ethano-bridge H atom can be found (Figure 7 (a)(c)(d)). In the case of Ala
(2b, Figure 7(a)), the intraresidual carbonyl O---amide N bond path was detected
within the Ala moiety. This kind of interaction may be relevant to the putative weak
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
46
intraresidual carbonyl O---amide H(N) C5 hydrogen bonding, particularly important
4
7
in β-sheet formation. While there have been arguments of the presence and the
4
8-49
interpretation of H—H bond path,
the present H—H interactions, detected in
Figure 7, are at least consistent with the proximity of these relevant atoms and also
indicative of interactions in the C-terminal Abh derivatives. In the non-Abh derivative,
the NHMe C-terminal case (3b), no such weak interactions were detected in the Ala
derivative (Figure 7(b) (Val and Ile derivative cases, see also Supporting Figures).
Therefore these results indicated that such intramolecular weak atomic link arising
from the Abh unit can contribute to enhancement of taking β-strand-like extended
structures of the adjacent α-amino acid residue.
(a) Ac-Ala-Abh(OBn) 2b
CH3
density
CO
1
.00
H
N
N
O
0.750
0.500
H
C
H
H
0
.250
0
.00
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2
2
2
2
2
2
3
3
3
3
3
3
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3
3
3
4
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4
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4
4
4
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5
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5
5
5
5
5
6
(
b) Ac-Ala-NHMe 3b
O
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
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8
9
0
C
N
N
H
C
CH3
CH3
O
(c) Ac-Val-Abh(OBn) 2c
H
O
N
H
C
H
H
C
H
(d) Ac-Ile-Abh(OBn) 2d
CH3
H
CO
N
O
C
H
H
H
H
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Figure 7. Molecular Graphs obtained in QTAIM, indicating proximity (red
colored bond paths) of the C-terminal Abh unit in the β-strand conformation of
α-amino acids (a, c, d). The β-strand conformer of NHMe C-terminal Ala
derivative (b) is a reference compound.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Conclusion
In summary, introduction of the C-terminal bicyclic scaffold (Abh) restricted the
1
overall conformation of the adjacent α-amino acid residue(s). The combination of H-
NMR and Raman spectroscopies and metadynamics simulations suggest that the
present bicyclic amine (Abh) can work as an effective scaffold to constrain or enforce
local peptide conformation. Both of the bridgehead groups, even the hydrogen atom
(H-H bond path), have proximity interactions with the α-amino acid side chain and
furthermore influence the adjacent α-amino acid to favor extended β-strand-like
conformation. These results provide a new perspective for enforcing/stabilizing
extended β-strand-like conformation through the proximity interactions and also
provide a basis for de novo design of β-strand-mimicking peptides and small proteins
by using β-strand enforcer/stabilizer even in the absence of the inter-strand hydrogen
bonding.
Experimental Section
General methods
Open column chromatography was carried out on silica gel (silica gel 60N (100-210
µm), Kanto Chemicals, Japan). All the NMR experiments were recorded on a Bruker
1
13
Avance 400 NMR spectrometer. H-NMR and C-NMR chemical shifts (δ) were
calibrated with the solvent peak and are shown in ppm. Coupling constants are given
in Hz. Mass spectra were recorded on a Bruker micrOTOF-05. All the melting points
were measured with a Yanaco Micro Melting Point Apparatus without corrected. The
combustion analyses were carried out in the microanalytical laboratory of this faculty.
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5
6
HPLC data were obtained using a Hitachi instrument with Senshu Pak Pegasil Silica
SP-100 (250 mm x 20 mm) for a normal phase column, and Mightysil RP-18 GP
Aqua (250 mm x 10 mm) for a reverse phase column. Flow rate: 5.0 ml/min. The
HPLC chromatograms of these products were shown in Supporting Information.
Synthesis
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis scheme before compound 6 is shown in the supporting information.ꢀ
Boc
N
COOMe
HO
6
ꢀ
To a solution of 5 (6.2 g, 19.8 mmol) in MeOH (40 ml) was added DBU (5.9 ml 39.7
mmol). The reaction mixture was stirred for 20 h at room temperature. After solvent
was evaporated, AcOEt was added to the residue and the organic phase was washed
with saturated aqueous solution of NH
4
Cl. The aqueous layer was combined and
SO and the
extracted with CHCl . The organic layers were combined, dried over Na
3
2
4
solvent was evaporated. Column chromatography (hexane: AcOEt= 3:7) gave
compound 6 (4.7 g, 88%) as colorless oil.
1
H-NMR (400 MHz, CDCl
3
): 4.223 (1H, d, J=5.2 Hz), 3.953-3.915 (1H, m), 3.805
(3H, s), 2.183-2.133 (1H, m), 2.128-2.094 (1H, m), 2.044 (3H, s), 1.994-1.902(1H, m),
1
.676-1.507 (1H, m), 1.428 (9H, s).
Boc
N
COOMe
MeS
O
S
7
ꢀ
2
To a suspension of NaH in THF (80 ml) under argon were added CS (6.8 mL, 113.0
mmol) and 6 (3.1 g, 11.3 mmol) in THF (20ml) at 0 °C. The reaction mixture was
stirred for 2 h at 40-50 °C and then cooled to room temperature. MeI (3.5 ml, 56.5
mmol) was added at room temperature and the reaction mixture was stirred at 30-
4
0 °C for 2 h. The reaction was quenched by addition of saturated aqueous solution of
NH Cl (30 ml), extracted with AcOEt, and the organic layer was dried over Na SO
4
2
4
and the solvent was evaporated. Column chromatography (hexane: AcOEt= 10:1-20:3)
gave compound 7 (4.0 g, 97%) as a yellow oil.
1
3
H-NMR (400 MHz, CDCl ): 5.442-5.417 (1H, m), 4.538 (1H, brd, J = 5.6 Hz), 3.805
(3H, s), 2.543 (3H, s), 2.381-2.175 (2H, m), 2.124-2.105 (1H, m), 2.019-1.932 (1H,
1
3
m), 1.691-1.521 (1H, m), 1.509-1.487 (1H, m), 1.425 (9H, s). C-NMR (100 MHz,
CDCl ): 215.5, 170.3, 155.7, 85.1, 81.3, 67.9, 63.2, 52.5, 41.7, 32.8, 28.2, 24.1, 19.4.
3
+
+
+
5 2
HRMS (ESI , [M+Na] ): Calcd. for C15H23NNaO S , 384.0910. Found: 384.0906.
Boc
N
COOMe
8
To a solution of 7 (4.0 g, 10.9 mmol) in toluene (150 ml) under argon were added
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Tris(trimethylsilyl)silane (TTMSS, 5.03 ml, 16.4 mmol) and AIBN (179 mg, 1.1
mmol) at room temperature. The mixture was heated at reflux for 30min and the
solvent was evaporated. Column chromatography (hexane: AcOEt= 10:1) gave
compound 8 (2.6 g, 92%) as a colorless oil.
1
3
H-NMR (400 MHz, CDCl ): 4.327-4.303 (1H, m), 3.799 (3H, s), 2.217-2.134 (2H,
m), 1.976-1.883 (2H, m), 1.777-1.714 (2H, m), 1.523-1.461 (2H, m), 1.416 (9H, s).
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
C-NMR (100 MHz, CDCl ): 171.8, 156.6, 80.8, 68.8, 59.8, 52.2, 33.5, 29.4, 28.2.
+
+
+
4
HRMS (ESI , [M+Na] ): Calcd. for C13H21NNaO , 278.1363. Found: 278.1366.
Boc
N
OH
9
To a solution of 8 (2.7 g, 10.7 mmol) in EtOH/THF (3:2, 50 ml) were added CaCl
2.4 g, 21.4 mmol) and NaBH (1.6 g, 42.8 mmol) at 0 °C. The reaction mixture was
stirred for 30 min at 0 °C and then stirred for 3 h at room temperature. The reaction
was quenched by adding cold saturated aqueous solution of NH Cl (30 ml). The water
phase was extracted with AcOEt, dried over Na SO and the solvent was evaporated.
2
(
4
4
2
4
Column chromatography (hexane: AcOEt= 3:1) gave compound 9 (2.3 g, 94%) as a
colorless oil.
1
H-NMR (400 MHz, CDCl
3
): 4.894 (1H, brs), 4.231-4.208 (1H, m), 3.882 (2H, brd, J
t
=
6.8 Hz), 1.875-1.705 (4H, m), 1.478-1.319 (13H, m, at 1.428 ppm ( Bu group, 9H,
1
3
3
s)). C-NMR (100 MHz, CDCl ): 155.3, 80.2, 69.2, 62.1, 58.5, 31.9, 29.4, 28.5.
HRMS (ESI , [M+Na] ): Calcd. for C12H21NNaO , 250.1414. Found: 250.1423.
3
+
+
+
Boc
N
OBn
1
0
To a solution of NaH (420 mg, 10.5 mmol) in THF (20 ml), 9 (796 mg, 3.5 mmol) in
THF (10 ml) was added dropwise at 0°C under argon. The solution was stirred for
30min at 0 °C. Then BnBr (0.83 ml, 7 mmol) was added dropwise. The solution was
warmed to room temperature and stirred at 50°C overnight. The reaction was
quenched by adding water (20 ml), extracted with Et
dried over Na SO and evaporated the solvent. Column chromatography (hexane:
EtOAc = 10:1) gave compound 10 (949 mg, 85%) as a colorless oil.
2
O, and the organic layer was
2
4
1
H-NMR (400 MHz, CDCl
m), 4.068 (2H, s), 1.787-1.425 (8H, m), 1.425 (9H, s). C-NMR (100 MHz, CDCl
155.6, 139.0, 128.4, 127.7, 127.5, 79.6, 73.5, 72.7, 67.3, 58.9, 33.9, 29.0, 28.5.
3
): 7.345-7.244 (5H, m), 4.612 (2H, s), 4.279-4.260 (1H,
1
3
3
):
+
+
+
3
HRMS (ESI , [M+Na] ): Calcd. for C19H27NNaO , 340.1883. Found: 340.1884.
Boc
N
OMe
11
To a solution of NaH (117.6 mg, 2.94 mmol) in THF (10 ml), 9 (223 mg, 0.98 mmol)
in THF (4 ml) was added dropwise at 0°C under argon. The solution was stirred for
3
0 min at 0°C. Then MeI (0.122 ml, 1.96 mmol) was added dropwise. The solution
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was warmed to room temperature and stirred for 3 h. The reaction was quenched by
adding water (20 ml) and extracted with Et
Na SO and the solvent was evaporated. Column chromatography (hexane: EtOAc =
:1) gave compound 11 (194 mg, 82%) as a colorless oil.
2
O. The organic layer was dried over
2
4
4
1
3
H-NMR (400 MHz, CDCl ): 4.265-4.242 (1H, m), 3.970 (2H, s), 3.415 (3H, s),
1
3
1
.798-1.251 (17H, m, at 1.437 ppm (t-Bu group, 9H, s)). C-NMR (100 MHz,
+
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
CDCl ): 155.6, 79.5, 74.9, 67.3, 59.5, 58.8, 33.6, 28.9, 28.5. HRMS (ESI , [M+Na] ):
Calcd. for C13H23NNaO , 264.1570. Found: 264.1568.
3
+
Boc
N
CN
12
To a solution of 9 (220 mg, 1 mmol) in MeCN-H
mol%), NH OAc (308 mg, 4 mmol), and PhI(OAc)
successively added. The suspension was stirred at room temperature overnight. The
reaction was quenched by adding water and the whole was extracted with Et O. The
organic layer was dried over Na SO and the solvent was evaporated. Column
2
O (9:1, 3 ml), TEMPO (7.8 mg, 5
4
2
(708 mg, 2.2 mmol) were
2
2
4
chromatography (hexane: EtOAc = 6:1) gave compound 12 (92 mg, 43%) as a
colorless oil and 9 (110 mg, 50% recovery).
1
3
H-NMR (400 MHz, CDCl ): 4.325-4.302 (1H, m), 2.217-2.133 (2H, m,), 1.956-
13
1
1
C
.854 (4H, m), 1.536-1.472 (2H, m), 1.486 (9H, s). C-NMR (100 MHz, CDCl
3
):
+
+
55.6, 118.8, 82.1, 58.5, 56.1, 36.0, 29.0, 28.2. HRMS (ESI , [M+Na] ): Calcd. for
+
12
H
18
N
2
NaO
2
, 245.1260. Found: 245.1263.
O
O
N
H
O
N
O
ꢀ
ꢀ
Boc-Gly-Abh(OBn) (1a)
2 2
To a solution of 10 (102 mg, 0.32 mmol) in CH Cl (1 mL) was added TFA (1.0 mL)
at 0 °C. The reaction mixture was allowed to warm to room temperature, and stirred
for 20 min. The reaction was then quenched with 2 M aqueous solution of NaOH. The
resulting solution was extracted with EtOAc (3 times). The combined organic layer
was dried over Na
amine, which was used in the next reaction without further purification. To a solution
of crude amine in CH Cl (5 mL) were added Boc-Gly-OH (84 mg, 0.48 mmol),
2 4
SO , and filtered. The solvent was evaporated to afford crude
2
2
CDMT (112 mg, 0.64 mmol), DMAP (5 mg, 0.04 mmol) and NMM (70 µl, 0.64
mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature, and
stirred for 24 h. The reaction mixture was diluted with Et
aqueous solution of HCl and saturated aqueous solution of NaHCO
2
O, washed with 1M
, and the organic
3
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2
3
4
5
6
7
8
9
2 4
layer was dried over Na SO . The solution was concentrated, and the residue was
purified with column chromatography (Hexane/Acetone = 4/1) to afford Boc-Gly-
Abh(OBn) (1a) (107 mg, 89%), as a colorless oil.
1
3
H NMR (CD OD, 400MHz) δ 7.365-7.242 (5H, m), 4.585 (2H, s), 4.376 (1H, s),
1
3
4.161 (2H, m), 3.839 (2H, s), 1.822-1.444 (8H, m), 1.444 (9H, s) C-NMR (100 MHz,
CDCl ): 168.1, 158.3, 139.8, 129.3, 128.9, 128.6, 80.5, 74.4, 72.9, 69.8, 58.6, 43.6,
4.2, 30.1, 28.7. HRMS (ESI ): Calcd. for C21
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
3
H
30
N
2
NaO
4
([M+Na] ): 397.2098.
Found: 397.2099. HPLC (Hexane: EtOAc = 1:1, 265 nm): t
R
21.47min, 98% purity.
O
NHBoc
N
OBn
Boc-Ala-Abh(OBn) (1b)
To a solution of compound 10 (194 mg, 0.61 mmol) in CH
2 ml) at 0°C. The solution was stirred for 40 min at room temperature. The whole
was washed with 10% aqueous solution of Na CO (20 ml), and the aqueous layer
was extracted with EtOAc. The organic layer was dried over Na SO and evaporated.
2 2
Cl (3 ml) was added TFA
(
2
3
2
4
The crude amine was obtained and was used to the next step directly. To a solution of
crude amine in DMF (5 ml), Boc-L-Ala (174 mg, 0.92 mmol), DIC (0.188 ml, 1.22
mmol), HOBT (164.85 mg, 1.22 mmol) and DIPEA (0.168 ml, 1.83 mmol) were
added. The solution was stirred overnight at room temperature. The reaction was
quenched by adding water (10 ml) and then the whole was extracted with CH
The organic phase was washed with 5% aqueous solution of citric acid and saturated
aqueous solution of NaHCO , dried over Na SO and evaporated the solvent. Column
2 2
Cl .
3
2
4
chromatography (hexane: EtOAc = 3:1) gave compound Boc-Ala-Abh(OBn) (1b)
(209 mg, 88%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
): 7.338-7.230 (5H, m), 5.439 (1H, d, J = 7.60 Hz), 4.646-
4
.583 (2H, m), 4.405-4.369 (1H, m), 4.296 (1H, s), 4.214 (2H, s), 1.832-1.423 (8 H,
13
m), 1.423 (9H, s), 1.299 (3H, d, J = 6.8 Hz). C-NMR (100 MHz, CDCl
3
): 169.4,
1
55.3, 139.0, 128.6, 127.8, 127.7, 79.6, 73.7, 72.3, 68.5, 58.3, 48.5, 34.0, 33.3, 30.2,
+
+
29.5, 28.7, 20.3. HRMS (ESI, [M+Na] ): Calcd. for C22
Found: 411.2254.
32 2 4
H N NaO , 411.2254.
O
NHBoc
N
OMe
Boc-Ala-Abh(OMe) (1b(OMe))ꢀ
To a solution of compound 11 (194 mg, 0.82 mmol) in CH
2 ml) at 0 °C. The solution was stirred for 40 min at room temperature. The solution
was washed with 10% Na CO (20 ml), and the aqueous phase was extracted with
EtOAc. The organic layer was dried over Na SO and evaporated. The crude amine
2 2
Cl (3 ml) was added TFA
(
2
3
2
4
was obtained and was used to the next step directly. To a solution of crude amine in
DMF (5 ml), Boc-L-Ala (227 mg, 1.2 mmol), DIC (0.246 ml, 1.6 mmol), HOBT (216
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