Solvent-Directed Switch of a Left-Handed 10/12-Helix into a Right-Handed 12/10-Helix in Mixed β-Peptides

Article abstract of DOI:10.1021/acs.joc.6b02856

Present study describes the synthesis and conformational analysis of β-peptides from C-linked carbo-β-amino acids [β-Caa_(l)] with a d-lyxo furanoside side chain and β-hGly in 1:1 alternation. NMR and CD investigations on peptides with an (S)-β-Caa_(l) monomer at the N-terminus revealed a right-handed 10/12-mixed helix. An unprecedented solvent-directed "switch" both in helical pattern and handedness was observed when the sequence begins with a β-hGly residue instead of a (S)-β-Caa_(l) constituent. NMR studies on these peptides in chloroform indicated a left-handed 10/12-helix, while the CD spectrum in methanol inferred a right-handed secondary structure. The NMR data for these peptides in CD₃OH showed the presence of a right-handed 12/10-helix. NMR investigations in acetonitrile indicated the coexistence of both helix types. Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the two helix types, which may explain the possibility of solvent influence. Examples for a solvent-directed switch of both the H-bonding pattern and the handedness of foldamer helices are rare so far. A comparable solvent effect was not found in the corresponding peptides with (R)-β-Caa_(l) residues, where right-handed 12/10-helices are predominating.

Full text of DOI:10.1021/acs.joc.6b02856

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Article
Solvent-directed switch of a left-handed 10/12-helix
into a right-handed 12/10-helix in mixed beta-peptides
Prashanth Thodupunuri, Sirisha Katukuri, Kallaganti V. S. Ramakrishna, Gangavaram
V. M. Sharma, Ajit C. Kunwar, Akella V. S. Sarma, and Hans-Jörg Hofmann
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02856 • Publication Date (Web): 17 Jan 2017
Downloaded from http://pubs.acs.org on January 20, 2017
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Solventꢀdirected switch of a leftꢀhanded 10/12ꢀhelix into a rightꢀ
handed 12/10ꢀhelix in mixed βꢀpeptides
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Prashanth Thodupunuri,a Sirisha Katukuri,b Kallaganti V. S. Ramakrishna,b Gangavaram V.
M. Sharma,a* Ajit C. Kunwar,b Akella V. S. Sarma,b* Hans-Jörg Hofmannc*
a. Organic and Biomolecular Chemistry Division, CSIRꢀIndian
Institute of Chemical Technology, Hyderabad 500 007, India
b. Nuclear Magnetic Resonance & Structural Chemistry Division,
CSIRꢀIndian Institute of Chemical Technology, Hyderabad 500 007,
India
c. Institute of Biochemistry, Faculty of Biosciences, University of
Leipzig, Brüderstrasse 34, Dꢀ04103, Leipzig, Germany
Email: esmvee@iict.res.in; ssav@iict.res.in; hofmann@uniꢀleipzig.de
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Abstract:
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Present study describes the synthesis and conformational analysis of βꢀpeptides from Cꢀlinked
carboꢀβꢀamino acids [βꢀCaa(l)] with a Dꢀlyxo furanoside side chain and βꢀhGly in 1:1 alternation.
NMR and CD investigations on peptides with an (S)ꢀβꢀCaa(l) monomer at the Nꢀterminus
revealed a rightꢀhanded 10/12ꢀmixed helix. An unprecedented solventꢀdirected ‘switch’ both in
helical pattern and handedness was observed when the sequence begins with a βꢀhGly residue
instead of a (S)ꢀβꢀCaa(l) constituent. NMR studies on these peptides in chloroform indicated a
leftꢀhanded 10/12ꢀhelix, while the CD spectrum in methanol inferred a rightꢀhanded secondary
structure. The NMR data for these peptides in CD3OH showed the presence of a rightꢀhanded
12/10ꢀhelix. NMR investigations in acetonitrile indicated the coꢀexistence of both helix types.
Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the two
helix types, which may explain the possibility of solvent influence. Examples for a solventꢀ
directed switch of both the Hꢀbonding pattern and the handedness of foldamer helices are rare so
far. A comparable solvent effect was not found in the corresponding peptides with (R)ꢀβꢀCaa(l)
residues, where rightꢀhanded 12/10ꢀhelices are predominating.
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Introduction
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The structural features of peptides and proteins are the basis for their function.1 However,
the application of native peptides for pharmacological and pharmaceutical purposes is often
limited by their insufficient stability towards proteases and unfavorable transport properties.2 To
circumvent such problems in peptide design, researchers initiated the use of unnatural amino
acids instead of the native αꢀamino acids. Among the great number of possibilities of amino acid
modification, homologous amino acids, in particular βꢀamino acids, have attracted special
attention, at first to substitute single αꢀamino acid constituents of peptides, later as constituents
of peptides completely composed of homologous amino acids.3 It was a rather surprising and
unexpected result to find characteristic secondary structure elements, as for instance helices, in
such homologous peptide sequences. The first helices in short sequences of βꢀamino acids (βꢀ
peptides) were reported in 19964 and stimulated the field of ‘foldamers’.5 This field, over a
period of time, expanded beyond peptides with homogeneous backbones6 to peptides with
heterogeneous backbones composed of different homologous amino acids, as for instance
alternating αꢀ and βꢀamino acids.7 Extensive quantum chemical studies were carried out on
numerous foldamer classes by several groups.8 They were complemented by comprehensive
molecular dynamics simulations on the conformational and dynamic features of foldamer
secondary structures.8 Experimental and theoretical studies indicate a wide variety of competing
secondary structures within the same foldamer class, in particular various helix alternatives with
different hydrogen bonding patterns and handedness, but also sheet and turn structures. As
known from native αꢀpeptides, several internal and external factors, like steric bulk of the
monomers,9 special side chain interactions,10 stereochemical patterning,11 temperature,12 and
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solvent polarity as well as concentration,13 control the secondary structure formation and decide
about the final peptide structure.
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The first examples of βꢀpeptide helices showed hydrogenꢀbonded rings of the same size
with all hydrogen bonds pointing in backward or, alternatively, in forward direction along the
sequence. A novel helix type was presented by Seebach et al.14 in sequences of alternating β2ꢀ
and β3ꢀamino acids. In these peptides, helices with alternating 12ꢀ and 10ꢀmembered (12ꢀmr and
10ꢀmr) hydrogenꢀbonded rings and an alternating change of the hydrogen bond direction appear,
which were called ‘mixed’ helices. Since these helices can either begin with a 10ꢀ or a 12ꢀ
membered hydrogenꢀbonded ring, two alternative mixed helices are possible, which differ in
stability according to theoretical calculations.8c,f,h,i,j The competition between these two structures
may be influenced by the aboveꢀmentioned structural and external factors.
Based on theoretical predictions, we have reported the synthesis of βꢀpeptides15
composed of the ‘epimeric’ (R)ꢀ and (S)ꢀCꢀlinked carbo βꢀamino acids [(R)ꢀ and (S)ꢀβꢀCaa(x)]16
in 1:1 alternate order. These amino acids bear a Dꢀxylo furanoside side chain. In these peptides,
the two aboveꢀmentioned mixed helix alternatives were found for the first time as rightꢀhanded
12/10ꢀ and 10/12ꢀhelices, indicating a ‘switch’ in helical conformation caused by the ‘epimeric’
nature of the monomers. In the meantime, diverse amino acids in the design of mixed βꢀ
peptides17 and further motifs for a design of 12/10ꢀ and 10/12ꢀhelices were suggested by us18 and
other authors.19
In this context, Kessler et al.20 obtained a 12/10ꢀhelix in βꢀpeptides alternately composed
of furanoid sugar amino acid (fSAA) and the simplest βꢀamino acid βꢀhGly (11), which is
achiral. In comparable studies, we combined the above mentioned (R)ꢀ and (S)ꢀβꢀCaa(x) residues
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bearing a Dꢀxylo furanoside side chain with βꢀhGly in 1:1 alternation. In sequences of (R)ꢀβꢀ
Caa(x) and βꢀhGly, we obtained a rightꢀhanded 12/10ꢀhelix if the sequence begins with an (R)ꢀβꢀ
Caa(x) constituent, whereas a rightꢀhanded 10/12ꢀhelix resulted in peptides with βꢀhGly at the Nꢀ
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terminus. In peptide sequences of the corresponding (S)ꢀβꢀCaa(x) amino acid and βꢀhGly,21
a
switch of handedness occurred and leftꢀhanded 12/10ꢀhelices resulted with an (S)ꢀβꢀCaa(x) at the
Nꢀterminus, but leftꢀhanded 10/12ꢀhelices in sequences beginning with βꢀhGly. This was the first
report on the formation of leftꢀhanded 10/12ꢀhelices and the results clearly showed the impact of
the side chain on helix handedness. It is interesting that stable 12/10ꢀhelix structures were
obtained in oligomers consisting only of βꢀhGly constituents by capping with a chiral amino acid
or a short helical peptide.21
The above findings stimulated the present study to investigate the helix formation in
mixed βꢀpeptides 1ꢀ8 (Figure 1), prepared from (S)ꢀ and (R)ꢀβꢀCaa(l)s 9 and 10 with a Dꢀlyxo
furanoside side chain and βꢀhGly 11 in 1:1 alternation and to compare the results with the former
data reported for the corresponding peptides with Dꢀxylo furanoside side chain.21 The synthesis
of peptides composed of βꢀCaa(l)s with a Dꢀlyxo furanoside side chain was already reported by
us22 in the design of HelixꢀTurnꢀHelix (HTH) motifs, using βꢀhGly residues in the turn region.
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H
N
H
H
N
H
N
O
N
OCH3
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O
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O
O
OCH3
OCH3
n
n = 1; n = 2
1
2
H
H
H
N
H
O
N
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N
OCH3
O
O
O
O
O
O
O
O
O
O
O
O
OCH3
OCH3
n
3 n = 1; 4 n = 2
H
N
H
H
N
H
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OCH3
O
O
O
O
O
O
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O
O
O
O
OCH3
OCH3
n
5 n = 1; 6 n = 2
H
H
N
H
N
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O
N
OCH3
O
O
O
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O
O
O
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O
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OCH3
OCH3
OCH3
n
7 n = 1; 8 n = 2
O
O
O
O
HN
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HN
O
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O
OCH3
H3CO
H3CO
H2N
OH
11
O
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10
O
NHBoc
O
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NHBoc
H3CO
O
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H3CO
O
O
H3CO
O
13
H3CO
12
Figure 1. Structures of the peptides 1ꢀ8 and their βꢀamino acid constituents 9 and 10 with a Dꢀ
lyxo furanoside side chain and βꢀhGly 11 (For comparison with 9 and 10, the βꢀamino acids 12
and 13 with a Dꢀxylo furanoside side chain are given).
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Results and Discussion
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Synthesis of peptides 1ꢀ8: Peptides 1ꢀ8 were prepared by standard peptide coupling methods,23
using EDCI, HOBT and DIPEA in solution phase. The synthesis of peptides 1 and 2 was
achieved from Bocꢀ(S)ꢀβꢀCaa(l)ꢀOMe 9 and 11. Accordingly, acid 9a (prepared from 9) on
reaction with the salt 11a (prepared from 11) in the presence of EDCI, HOBt, and DIPEA in
CH2Cl2 afforded the dipeptide 14 in 67% yield (Scheme 1). Treatment of peptide 14 with base
(LiOH) furnished the acid 14a, while reaction of 14 with CF3COOH in CH2Cl2 gave the salt 14b.
Acid 14a on coupling as above with the salt 14b in CH2Cl2 afforded the tetrapeptide 1 in 55%
yield. Ester 1 on base hydrolysis with LiOH furnished the acid 15, which on further coupling
with the salt 14b gave the hexapeptide 2 in 49% yield.
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Likewise, for the synthesis of peptides 3 and 4, ester 9 was treated with CF3COOH to
furnish salt 9b. Coupling of acid 11b with salt 9b in the presence of EDCI, HOBt, and DIPEA in
CH2Cl2 gave the dipeptide 16 in 57% yield (Scheme 2). Base (aq. LiOH) hydrolysis of 16
afforded 16a, while 16 on reaction with CF3COOH in CH2Cl2 furnished 16b. Coupling (EDCI,
HOBt, and DIPEA) of acid 16a with salt 16b in CH2Cl2 provided the tetrapeptide 3 (48%). Base
hydrolysis of 3 with LiOH furnished the acid 17, which on further coupling with the salt 16b
gave the hexapeptide 4 in 45% yield.
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Peptides 5 and 6 were synthesized from Bocꢀ(R)ꢀβꢀCaa(l)ꢀOH 10a and 11 (Scheme 3).
Accordingly, acid 10a (prepared from 10) on coupling (EDCI, HOBt, and DIPEA) with the salt
11a in the presence of in CH2Cl2 afforded the dipeptide 18 in 65% yield. Peptide 18 on base
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hydrolysis with aq. LiOH furnished 18a, while treatment of 18 with CF3COOH in CH2Cl2 gave
the salt 18b. Coupling (EDCI, HOBt, and DIPEA) of acid 18a with the salt 18b in CH2Cl2
provided the tetrapeptide 5 in 52% yield. Ester 5 on base hydrolysis with LiOH afforded the acid
19, which on further reaction with the salt 18b furnished hexapeptide 6 in 44 % yield.
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Similarly, for the synthesis of peptides 7 and 8, ester 10 was treated with CF3COOH to
give salt 10b. Acid 11b on coupling with 10b in the presence of EDCI, HOBt, and DIPEA in
CH2Cl2 afforded the dipeptide 20 in 57% yield (Scheme 4). Peptide 20 on base hydrolysis with
LiOH gave 20a, while reaction of 20 with CF3COOH in CH2Cl2 afforded 20b. Coupling (EDCI,
HOBt, and DIPEA) of acid 20a with salt 20b in CH2Cl2 furnished the tetrapeptide 7 in 43% yield
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(Scheme 3). Base hydrolysis of 7 with LiOH gave the acid 21, which on further coupling with
the salt 20b provided the hexapeptide 8 in 41% yield.
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Conformational analysis
The NMR spectra of peptides 1ꢀ8 were recorded as 3ꢀ5 mM solutions in CDCl3.24 The
proton NMR spectra of peptides 1 and 2 with (S)ꢀβꢀCaa(l) at the Nꢀterminus showed a good
dispersion in the amide region. The appearance of NH(1) signals at δ > 5.68 ppm for peptide 1
and δ > 6.14 ppm for peptide 2 as well as the presence of a weak nOe correlation NH(1)/NH(2)
suggested the participation of NH(1) in 10ꢀmr hydrogen bonding. Solvent titration studies25 on
peptides 1 and 2 with DMSOꢀd6 indicated that, except NH(2), all amide protons, participate in Hꢀ
bonding.
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For peptide 1, the longꢀrange backbone nOe interactions CβH(2)/NH(4), CβH(2)/CαH(pro-
R)(4) and the weak NH(1)/NH(2) and NH(3)/NH(4) nOe correlations provided ample evidence
for a rightꢀhanded 10/12ꢀhelical structure, while, the CD profile did not show distinct structural
features. However, the hexamer 2 exhibited a definite structure in CDCl3.24 The presence of
CβH(2)/NH(4), CβH(2)/CαH(pro-R)(4), CβH(4)/NH(6), and CβH(4)/CαH(pro-R)(6) nOe correlations
in the ROESY spectrum (Figure 2) suggests the existence of 12ꢀmr hydrogenꢀbonded rings,
involving Hꢀbonds from NH(4) to CO(1) and NH(6) to CO(3).
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CβH(4)/CαH(pro-R)(6)
CβH(2)/CαH(pro-R)(4)
CβH(4)/NH(6)
NH(5)/NH(6)
NH(3)/NH(4)
CβH(2)/NH(4)
Figure 2. ROESY spectrum of peptide 2 depicting characteristic nOe correlations.
In addition, the weak NH(3)/NH(4) and NH(5)/NH(6) nOe correlations indicate the
presence of 10ꢀmr rings with Hꢀbonds between NH(3) and CO(4) as well as NH(5) and CO(6).
All the nOe correlations are characteristic for a rightꢀhanded 10/12ꢀhelical structure in 2. For all
3
the βꢀCaa residues, a large JCβH/C4H coupling constant implies the preponderance of a structure
with χ1 ≈ 180o.
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MD simulations on peptide 2 on the basis of the available NMR data revealed the
presence of a rightꢀhanded 10/12ꢀhelix with heavy atom and backbone rmsd values of 0.67 Å and
0.41 Å, respectively (Figure 3). Although the NH(1)/NH(2) correlation is very weak according to
the ROESY spectrum, the MD minimized structures display a distance of 2.5 Å between NH(1)
and NH(2), indicating a 10ꢀmembered hydrogenꢀbonded cycle between NH(1) and CO(2). The
average backbone dihedral angles for peptide 2 are listed in Table 1. In agreement with the NMR
data, the rightꢀhanded 10/12ꢀhelix of the peptide hexamer 2 is most stable among the four mixed
helix alternatives with 10ꢀ and 12ꢀmr hydrogenꢀbonded rings in a systematic quantum chemical
conformational analysis at the B3LYP/6ꢀ31G* level of density functional theory (DFT) (Table 2;
backbone torsion angles in Table 1, for details of the quantum chemical conformational search,
see Experimental section).
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Table 1. Average backbone dihedral anglesa in the tetraꢀ and hexapeptides 1, 2, 3, and 4 from
the MD simulations on the basis of the NMR data in CDCl3 and from quantum chemical studies
at the B3LYP/6ꢀ31G* level of density functional theory
Helix typec
Peptideb
βꢀCaa(l)
βꢀhGly
θ
62
θ
64
65
ϕ
86
88
92.0
98
100
100.4
ψ
ꢀ84
ꢀ100
ϕ
ꢀ102
ꢀ105
ψ
84
85
91.2
95
97
1d
rhꢀ10/12
rhꢀ10/12
rhꢀ10/12
lhꢀ10/12
lhꢀ10/12
lhꢀ10/12
2d
2e
3d
4d
4e
62
61.3 ꢀ99.9 ꢀ98.6
ꢀ67
ꢀ66
61.3
ꢀ65
ꢀ60
ꢀ76
ꢀ83
ꢀ83
ꢀ80
ꢀ59.4 ꢀ85.0 ꢀ89.7 ꢀ67.1
105.4
aIn degrees. See Figure 1; A = βꢀhGly, S = (S)ꢀβꢀCaa(l). rh: rightꢀhanded, lh: leftꢀhanded.
dNMR study in CDCl3. eDFT//B3LYP/6ꢀ31G* level; averaged over residues 2 and 4 and 3 and
5, respectively.
b
c
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51
52
53
54
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60
Figure 3. Stereoview of a su
p
erimposition of 20 minimum energy structurees for peptide hexamer
2 from MD simulations based on NMR data in CDCl3 (hydrogen atoms removed for clarity).
Support for this structure comes also from CD studies in methannol. The CD profile of
peptide 2 shows a characteristic signature21 with a positive molar ellipticity at 202 nm that
corresponds to a rightꢀhanded 10/12ꢀhelical structure.24
Table 2. Relative energies i
peptides 2, 4, 6 and 8 consist
and βꢀhGly in 1:1 alternate ordder at the B3LYP/6ꢀ31G* level of density fu
n
kcal/mol of the mixed 10/12ꢀ and 12/10ꢀh
e
e
lix alternatives of the
iing of (S)ꢀ and (R)ꢀβꢀCaa(l)s bearing a Dꢀlyx
oo
furanoside side chain
nn
ctional theory
Peptidea
rhꢀ10/
1
2b)
lhꢀ10/12b
rhꢀ12/10b
lhꢀ12/10b
0.0e
ꢀd
9.8
ꢀd
8.6
2
4
6
0.0g
0.0c
0.3
0.4
10.9
ꢀd
7.6
0.0f
ꢀd
10.3
8.9
8
b
aSee Figure 1. rh = rightꢀhanded; lh = leftꢀhanded. cET = ꢀ3784.424222 aa.u. dNot possible from
steric reasons. eET = ꢀ3784.435426 a.u. fET = ꢀ3784.430419 a.u. gET = ꢀ37
4.413737 a.u.
8
Different from this result, a leftꢀhanded 12/10ꢀhelix was found in the earlier studies on
the corresponding peptides
w
ith (S)ꢀβꢀCaa(x) bearing a Dꢀxylo furanoside side chain.21 Initial
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studies on the monomeric (S)ꢀ and (R)ꢀβꢀCaa(l)
9
and 10 (Figure 1) with a Dꢀlyxo furanoside side
8
9
chain showed a similarly puckered conformation for the side chain with the C4 atom in the ring
plane. In contrast, in the monomeric (S)ꢀ and (R)ꢀβꢀCaa(x)s 12 and 13 with a Dꢀxylo furanoside
side chain (Figure 1), the C4 atom is out of plane. The different arrangement of the C4 atom is
10
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also confirmed for the peptides
1 and 2 with Dꢀlyxo furanoside side chain and the corresponding
peptides with xylose side chain,21 which might be the origin of the differences in helix formation
in these two peptide classes.
The study is further extended to peptides
3 and 4 with βꢀhGly at the Nꢀterminus. It is
found that both peptides revealed downꢀfield shifts of amide protons with good dispersion in
their proton NMR spectra in CDCl3.24 Solvent titration studies25 with DMSOꢀd6 showed that
several amide protons participate in hydrogen bonding, indicated by small chemical shift
changes. The solvent titration and nOe correlation data suggest a leftꢀhanded 10/12ꢀhelix
structure for tetrapeptide
detail. The distinctive nOe correlations CβH(2)/NH(4) and CβH(4)/NH(6) shown in the ROESY
spectrum of peptide (Figure 4) confirm the existence of 12ꢀmr hydrogenꢀbonded rings between
3. To understand the unusual behavior, hexapeptide 4 was studied in
4
CO(1)⋅⋅⋅NH(4) and CO(3)⋅⋅⋅NH(6). Further, the correlations NH(3)/NH(4) and NH(5)/NH(6)
reveal the presence of 10ꢀmr hydrogen bonding between NH(3) and CO(4) and NH(5) and
CO(6), respectively. For the three βꢀCaa residues, a large 3JCβH/C4H coupling constant implies the
preponderance of a structure with χ1 ≈ 180o. The crucial nOe correlations, CβH(2)/CαH(pro-S)(4)
and CβH(4)/CαH(pro-S)(6), which determine the handedness of the observed helix, confirm a leftꢀ
handed helical structure. In addition, the MD simulations on the basis of the NMR information
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also suggest a leftꢀhanded 10/12ꢀhelix in CDCl3. The average backbone dihedral angles from the
analysis of the NMR data and the quantum chemical calculations are given in the Tables 1 and 3.
10
11
12
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60
CβH(4)/CαH(pro-S)(6)
CβH(4)/NH(6)
CβH(2)/CαH(pro-S)(4)
CβH(2)/NH(4)
Figure 4. ROESY spectrum of peptide 4 in CDCl3 showing the characteristic correlations for a
leftꢀhanded 10/12ꢀ helix.
The CD profile of peptide 4 in methanol shows a maximum at 202 nm (Figure 5), which
corresponds unequivocally to a rightꢀhanded helical structure. This is in contrast to the leftꢀ
handed 10/12ꢀhelix structure found by NMR in the solvent CDCl3. Obviously, a change of the
solvent induces a change of handedness.
Figure 5. CD spectrum of peptide 4 in methanol showing a positive molar ellipticity.
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9
These unusual findings prompted us to undertake an NMR study on peptide 4 in
methanol (CD3OH), a polar protic solvent, to understand the contradictory CD results in the two
solvents. The nOe correlations CβH(1)/CαH(pro-R)(3) and CβH(3)/CαH(pro-R)(5) from the ROESY
spectrum of 4 in CD3OH (Figure 6) clearly support the rightꢀhanded structure in agreement with
the CD profile. In addition, the correlations CβH(1)/NH(3), CβH(3)/NH(5), NH(2)/NH(3), and
NH(4)/NH(5) support a rightꢀhanded 12/10ꢀhelix, resembling structures observed earlier.21 The
12/10ꢀhelix remains stable in the MD simulations. Its average backbone torsion angles are given
in Table 3.24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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50
51
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57
58
59
60
Table 3. Average backbone dihedral anglesa of the rightꢀhanded 12/10ꢀ and the leftꢀhanded
10/12ꢀhelices of peptide 4 from MD simulations on the basis of the NMR data in CD3OH and
CD3CN solvents in comparison with quantum chemical data at the B3LYP/6ꢀ31G* level of
density functional theory
Helix typec
Peptideb
βꢀCaa(l)
βꢀhGly
θ
61
55
θ
60
60
ϕ
106
90
91.8
108
ψ
ꢀ100
ꢀ86
ϕ
ꢀ106
ꢀ107
ψ
81
92
88.8
97
4d
4e
4f
4e
rhꢀ12/10
rhꢀ12/10
rhꢀ12/10
lhꢀ10/12
62.8 ꢀ102.7 ꢀ100.7 59.6
ꢀ65 ꢀ86 ꢀ93 ꢀ63
aIn degrees. See Figure 1; A = βꢀhGly, S = (
S
)ꢀβ
ꢀCaa(l). crh: rightꢀhanded, lh: leftꢀhanded.
b
e
f
dNMR study in CD3OH. NMR study in CD3CN. DFT//B3LYP/6ꢀ31G* level; averaged over
residues 2 and 4 and 3 and 5, respectively.
The theoretical conformational analysis on peptide 4 predicts the leftꢀhanded 10/12ꢀ and
the rightꢀhanded 12/10ꢀhelices as approximately equivalent in energy (Table 2), suggesting that
external factors like solvents may easily influence the structure equilibrium in favor of the one or
the other helical alternative.
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CβH(1)/
CαH(pro-R)(3)
CβH(1)/NH(3)
CβH(3)/CαH(pro-R)(5)
CβH(3)/NH(5)
Figure 6. ROESY spectrum of peptide 4 in CD3OH with the crucial nOe correlations labeled.
The observation of a change of the hydrogen bonding pattern and a ‘switch’ in
handedness from a leftꢀhanded 10/12ꢀhelix to a rightꢀhanded 12/10ꢀhelix by the change of the
solvent from CDCl3 to CD3OH is rather interesting and very unusual. Some relevance for the
present work have the results by Fülöp and coworkers,26 who found a switch from an 18ꢀhelix
into a 12ꢀhelix in βꢀpeptides in the solvent methanol after dilution. Recently, a switch from an
Mꢀ to a Pꢀhelix for polyꢀ(quinoxalineꢀ2,3ꢀdiyl) copolymers was reported by a change from the
solvent benzene to trifluorobenzene.27
The results inspired us to investigate the behavior of peptide 4 in a solvent with
properties different from the other two solvents. Therefore, the NMR study was repeated in the
polar aprotic solvent acetonitrile (CD3CN). Interestingly, the ROESY spectrum of peptide 4 in
CD3CN displays information corresponding to the presence of both mixed helices (see
Supporting information). Thus, the nOe correlations CβH(2)/NH(4), CβH(4)/NH(6),
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CβH(2)/CαH(pro-S)(4), CβH(4)/CαH(pro-S)(6), NH(3)/NH(4), and NH(5)/NH(6) correspond to a
leftꢀhanded 10/12ꢀhelix, whereas, the nOe correlations CβH(1)/NH(3), CβH(3)/NH(5),
CβH(1)/CαH(pro-R)(3), CβH(3)/CαH(pro-R)(5), NH(2)/NH(3), and NH(4)/NH(5) unequivocally
indicate the existence of the rightꢀhanded 12/10ꢀhelix alternative.
10
11
12
13
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The data sets from the analysis of the NMR spectrum in CD3CN were the basis for MD
simulations, which support the presence of both helices in this solvent. The overlays of the
minimum energy structures of the leftꢀhanded 10/12ꢀ and the rightꢀhanded 12/10ꢀhelices are
displayed in Figure 7A and 7B along with various hydrogen bond interactions in different
solvents (Figure 7C). The average backbone dihedral angles resulting from the MD simulations
based on the NMR data for acetonitrile are listed in Table 3 together with those estimated for
methanol and the quantum chemical data.
A
B
Rightꢀhanded 12/10ꢀhelix in CD3OH
Leftꢀhanded 10/12ꢀhelix in CDCl3
C
10
10
O
12
12
12
OMe
H
H
H
H
H
H
O
N
N
N
N
R =
N
N
OMe
O
O
R
R
O
O
O
R
O
O
O
O
12
10
10
10
Both helices are present in CD3CN
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5
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7
8
9
Figure 7. Stereoview of a superimposition of the 20 minimum energy structures of peptide 4: A)
rightꢀhanded 12/10ꢀ and B) leftꢀhanded 10/12ꢀhelices from the MD simulations on the basis of
the available NMR data for CD3CN (hydrogen atoms removed for clarity); C) Hꢀbonding
interactions of all the helices in different solvents.
10
11
12
13
14
15
16
17
18
19
20
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57
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60
These results clearly demonstrate the role of solventꢀsolute interactions for structure
formation. The switch of the handedness in peptide 4 going from solvent CDCl3 to solvent
CD3OH is a nice example for the importance of such interactions. Phenomena like this become
especially visible when the competing structures are of comparable stability.
Based on the results presented above, it seemed to be worthwhile to continue the study
with the corresponding peptides composed of (
spectral analysis of the peptides 5ꢀ8 suggests a rightꢀhanded 12/10ꢀhelix for the peptides 5 and 6
with ( )ꢀ ꢀCaa(l) at the Nꢀterminus, and a rightꢀhanded 10/12ꢀhelix for the peptides 7 and 8 with
ꢀhGly at the Nꢀterminus. Again, molecular dynamics simulations on the basis of the NMR
R)ꢀβꢀCaa(l) and βꢀhꢀGly constituents. A detailed
R
β
β
data24 confirmed these structures. The average backbone dihedral angles for the preferred mixed
helix conformers of the peptides 5, 6 and 7, 8 resulting from the MD simulations are given in
Table 4.
The rightꢀhanded 12/10ꢀhelix of the peptide hexamer 6, indicated by NMR spectroscopy,
is only by 0.4 kcal/mol more unstable than the leftꢀhanded 10/12ꢀhelix alternative, which was
predicted as the most stable among the four mixed helix alternatives with 10ꢀ and 12ꢀmr
hydrogenꢀbonded rings according to a systematic quantum chemical conformational analysis at
the B3LYP/6ꢀ31G* level of density functional theory (DFT) (Table 2). Despite the relatively
small energy difference between both helix alternatives, a comparable solvent effect as found for
the (S
)ꢀβꢀCaa(l) peptides was not observed. In agreement with the NMR data, the quantum
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5
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7
8
Table 4. Average backbone dihedral anglesa in the tetraꢀ and hexapeptides 5, 6 and 7, 8 from
the MD simulations on the basis of the NMR data in CDCl3 and from quantum chemical studies
at the B3LYP/6ꢀ31G* level of density functional theory
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Helix typec
Peptideb
βꢀCaa(l)
θ
βꢀhGly
θ
65
67
64.3
75
ϕ
ꢀ101
ꢀ87
ꢀ94.9
ꢀ106
ꢀ108
ꢀ99.1
ψ
82
96
93.3
86
85
ϕ
91
110
85.8
97
ψ
ꢀ103
ꢀ81
ꢀ93.7
ꢀ110
ꢀ106
ꢀ104.0
5d
6d
6e
7d
8d
8e
65
70
54.5
65
67
rh-12/10
rhꢀ12/10
rhꢀ12/10
rhꢀ10/12
rhꢀ10/12
rhꢀ10/12
87
86.8
62
63.5
63.5
89.6
aIn degrees. See Figure 1; A = βꢀhGly, R = (
R
)ꢀβꢀCaa(l). crh: rightꢀhanded, lh: leftꢀhanded.
b
e
dNMR study in CDCl3. DFT//B3LYP/6ꢀ31G* level; averaged over residues 2 and 4; 3 and 5,
respectively.
chemical conformational analysis provides the rightꢀhanded mixed 10/12ꢀhelix as most stable
helix conformer for the peptide hexamer 8. The average backbone torsion angles for the peptides
6 and 8 arising from the quantum chemical geometry optimizations are compared with those
from the MD simulations in Table 4.
The structure data for peptides 5, 6 and 7, 8 are similar to those observed for the
corresponding peptides with (R)ꢀβ
ꢀCaa(x) constituents bearing a Dꢀxylo furanoside side chain.21
Interestingly, the molar ellipticities in the CD profiles of the peptides with Dꢀlyxo furanoside side
chains are higher than found for the corresponding peptides with a Dꢀxylo furanoside side chain,
which hints to more stable secondary structures in the peptides with the Dꢀlyxo furanoside side
chains.
Conclusions
The present study describes the synthesis and conformational analysis of a new series of
peptides 1ꢀ8 composed of βꢀhGly in 1:1 alternation with (
S
)ꢀ or (
R
)ꢀβꢀCaa bearing a Dꢀlyxo
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furanoside side chain. The peptides with an (
S
)ꢀβꢀCaa(l) monomer at the Nꢀterminus reveal the
8
9
presence of a rightꢀhanded 10/12ꢀhelix, as supported by a theoretical study. Especially interesting
10
11
12
13
14
15
16
17
18
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
are the results for the peptides with a βꢀhGly residue at the Nꢀterminus in alternation with (S)ꢀβꢀ
Caa(l) constituents. The NMR studies show a leftꢀhanded 10/12ꢀhelix in the solvent CDCl3,
while, CD spectrum of this peptide in solvent MeOH indicates the opposite handedness. Indeed,
the NMR studies in the more polar solvent CD3OH suggest the presence of a rightꢀhanded 12/10ꢀ
helix. This is an unprecedented ‘switch’ between two helical structures with different hydrogen
bonding pattern and different handedness, which is reported in 12/10ꢀhelices for the first time.
Moreover, the structural features of both helix types were observed in the polar aprotic solvent
acetonitrile. The unusual solvent effect can well be understood by looking at the approximately
energetic equivalence of both helices according to quantum chemical calculations. To the best of
our knowledge, no other examples with such remarkable effects of an excessive change of helix
type and handedness by a mere solvent change were found until now.
The peptides composed of (R)ꢀβꢀCaa(l) constituents in alternation with βꢀhGly revealed
results, that are in agreement with data of former investigations on the corresponding peptides
with (R)ꢀβꢀCaa(x) residues bearing a Dꢀxylo furanoside side chain.
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The data of this study demonstrate several possibilities to influence helix type and
handedness, as for instance by a different configuration of the amino acid constituents and the
variation of their side chains and even by the nature of the solvent. The study shows the wide
variety of possibilities to realize special types of secondary structures. Thus, experimental and
theoretical methods may efficiently contribute to a rational peptide and protein design in a
concerted action.
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Experimental Section:
8
9
All the NMR spectra (1D and 2D experiments) were obtained on 300 MHz, 400 MHz,
500 MHz, 600 MHz and 700 MHz spectrometers at 278ꢀ303 K, sample concentrations were 3ꢀ5
mM in CDCl3, CD3OH, and CD3CN solvents, using tetramethylsilane (TMS) as internal
standard. The chemical shifts are shown in scales. All chemical shifts were measured from 1D
10
11
12
13
14
15
16
17
18
19
20
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23
24
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27
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
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60
°
δ
NMR spectra and the coupling constants were measured with resolutionꢀenhanced 1D spectrum.
The proton resonance assignments were carried out by using twoꢀdimensional total correlation
spectroscopy (TOCSY) and rotating frame Overhauser effect spectroscopy (ROESY)
experiments in phase sensitive mode. The TOCSY experiments were performed with a mixing
time of 0.08 s, whereas the ROESY experiments, which provide the spatial proximity of the
protons, were performed with mixing times of 0.2ꢀ0.4 s. TOCSY and ROESY spectra were
acquired with 4096 complex data points in the t2 domain and 192 or 256 increments in the t1
domain containing 16ꢀ24 scans with relaxation delay of 1.5 s. Information on the Hꢀbonding in
CDCl3 was obtained from solvent titration studies at 298º K by sequentially adding up to 300 ꢁL
of DMSOꢀd6 in 600 ꢁL of CDCl3 solution of the peptides. The twoꢀdimensional data were
processed with shifted sine bell or Gaussian apodization in both dimensions. The acquired free
induction decays (FID) were zeroꢀfilled during processing to give a final matrix of 2048×2048.
The CD spectra of peptides in MeOH (0.2 mM) were recorded in quartz cells (2 mm path length)
at roomꢀtemperature within a scan range of 190ꢀ260 nm and a scanning speed of 20 nm/min.. All
spectra represent one scan, each of 100 ms time constant and are backgroundꢀcorrected and
smoothened over 2ꢀ5 data points using binomial method.
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Model building and restrained molecular dynamics simulations28 on peptides 1ꢀ8 were
carried out employing the Insight II (97.0) / Discover program.29 The CVFF force field was used
throughout the simulations with default parameters using a distanceꢀdependent dielectric
9
10
11
12
13
14
15
16
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constant with
ε = 4.8 for chloroform. The distance restraints were obtained from the volume
integrals in the ROESY spectra30 using a twoꢀspin approximation and a reference distance of 1.8
Å for geminal protons. The dihedral angles were estimated from threeꢀbond protonꢀproton Jꢀ
couplings, but were not used in the calculations. The complete sets of nOe distance constraints
considered for the structure calculations are given in the Supporting information. The initial
structures were minimized by steepest descent algorithm at first and followed by conjugate
gradient method for a maximum of 1000 iterations each or an RMS deviation of 0.001 kcal/mol,
whichever was earlier. The energyꢀminimized structures were the starting point for the MD
simulations. A number of interatomic distance restraints at a corresponding temperature were
used for the MD runs. The molecules were subjected to a simulated annealing protocol after an
initial equilibration for 50 ps. Starting from an initial temperature of 300 K, they were heated to
1500 K in four steps with an increment of 300 K and simulated for 2.5 ps at each step and
subsequently cooled back to 300K in four steps by decreasing the temperature by 300 K in each
step, again simulating for 2.5 ps at each step. The structure was saved and the above process
repeated 100 times, saving one structure each time. The 100 structures generated were energyꢀ
minimized again. From these 100 energyꢀminimized structures, the twenty lowest energy
structures were superimposed for display. For a better visualization, some atoms/atomic groups
have been removed from the figures.
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The quantum chemical calculations were performed on the hexamers 2, 4, 6, and 8 at the
B3LYP/6ꢀ31G* level of density functional theory, employing the Gaussian03 program
package.31 Four alternative mixed helix types with 10ꢀ and 12ꢀmr hydrogenꢀbonded cycles can
formally be expected: 10/12ꢀleftꢀ and rightꢀhanded and 12/10ꢀleftꢀ and rightꢀhanded helices,
respectively. Some of the 10/12ꢀ and 12/10ꢀhelix arrangements can a priori be excluded from
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steric reasons (Table 2). The starting values for the backbone torsion angles
ϕ, θ, and ψ in the
geometry optimizations came from former theoretical studies,8c,h,i those for the side chain
orientations (χ1) from the present NMR studies. The side chain torsion angles χ1 were
systematically varied in steps of 60°. In short oligomers like hexamers, the 10/12ꢀhelices could
be advantaged over the 12/10ꢀhelix alternatives since one hydrogen bond more can be formed
along the sequence. Deviations from this rule in favor of 12/10ꢀhelices may be caused by steric
effects or special side chainꢀbackbone interactions. It should be kept in mind that if one of the
four possibilities is realized for a special peptide, the helix with the same hydrogen bonding
pattern, but opposite handedness must be preferred in the peptide with the opposite configuration
of the amino acid constituents, because it represents the mirror image of the original peptide.
Since the side chains of the peptides 1ꢀ8 are chiral themselves and their chirality does not change
with that of the constituent, the last condition is only approximately fulfilled and the two
peptides represent only approximate mirror images of more or less different stability.
Bocꢀ(S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀOMe (14):
A cooled solution (0 oC) of 9 (0.75 g, 2.0 mmol) in THF:MeOH:H2O (3:1:1) (1 mL) was
treated with LiOH (0.06 g, 2.4 mmol) and stirred at room temperature. After 1 h, pH was
adjusted to 2ꢀ3 with 1 N HCl solution at 0 oC and extracted with ethyl acetate (2 x 50 mL). The
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organic layer was dried (Na2SO4) and evaporated to give 9a (0.69 g, 95%) as a white solid,
which was used as such for further reaction.
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To a stirred suspension of βꢀhGly 11 (0.4 g, 4.4 mmol) in MeOH (5 mL), dry HCl gas
was bubbled at room temperature until the solid was dissolved. MeOH was evaporated and the
residual salt 11a was dried under high vacuum and used as such for the next reaction.
A solution of acid 9a (0.53 g, 1.46 mmol), HOBt (0.29 g, 2.19 mmol), EDCI (0.42 g, 2.19
mmol) in CH2Cl2 (5 mL) was stirred at 0 °C under N2 atmosphere for 15 min, treated with the
amine salt 11a and DIPEA (0.37 mL, 4.38 mmol) and stirred at room temperature for 8 h. The
reaction mixture was quenched with aq. satd. NH4Cl solution (10 mL). After 10 min, it was
diluted with CHCl3 (2 x 10 mL) and washed with water (10 mL), NaHCO3 solution (10 mL), and
brine (10 mL). The organic layers were dried (Na2SO4) and evaporated. Purification of the
residue by column chromatography (60ꢀ120 mesh silica gel, 60% Ethyl acetate in pet. ether)
20
afforded 14 (0.48 g, 67%) as a colorless syrup; [
α]
= +90.0 (
c
0.05, CHCl3); IR (KBr): ν
D
1
3746, 2954, 2923, 2851, 2310, 1709, 1692, 1646, 1525, 1219, 1054, 772 cmꢀ1; H NMR (500
MHz, 298 K, CDCl3):
1H, = 3.8 Hz, C3Hꢀ1), 4.53 (d, 1H,
3H, COOMe), 3.52 (m, 2H, CβHꢀ2, Cβ Hꢀ2), 3.30 (s, 3H, OMe), 2.62 (s, 2H, CαHꢀ1, CαHꢀ2),
δ
6.40 (b, 1H, NHꢀ2), 5.22 (b, 1H, NHꢀ1), 4.89 (s, 1H, C1Hꢀ1), 4.68 (d,
J
J
= 5.5 Hz, CβHꢀ1), 4.15 (m, 2H, C4Hꢀ1, C2Hꢀ1), 3.70 (s,
'
2.54 (t, 2H,
J = 6.0 Hz, Cα Hꢀ1, Cα Hꢀ2), 1.45 (s, 3H, Ac), 1.44 (s, 9H, Boc), 1.28 (s, 3H, Ac);
' '
13C NMR (125 MHz, 298 K, CDCl3):
δ
172.7, 170.6, 155.7, 112.5, 106.5, 85.0, 79.5 (2C), 79.3,
54.4, 51.7, 48.0, 38.4, 34.7, 33.7, 28.3 (3C), 25.9, 24.6; HRMS (ESI+): m/z calculated for
C20H34N2O9Na(M++Na) 469.2156, found 469.2122.
Bocꢀ(S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀ(
S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀOMe (1):
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o
A cooled solution (0 C) of 14 (0.17 g, 0.38 mmol) in THF:MeOH:H2O (3:1:1) (1 mL)
was treated with LiOH (0.01 g, 0.46 mmol) and stirred at room temperature. Work up as
described for 9a gave 14a (0.15 g, 90%) as a colorless solid, which was used as such for further
reaction.
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To a solution of 14a (0.15 g, 0.34 mmol), HOBt (0.07 g, 0.52 mmol), and EDCI (0.1 g,
0.52 mmol) in CH2Cl2 (5 mL), amine salt 14b [prepared from 14 (0.18 g, 0.4 mmol) and
CF3COOH (0.2 mL) in CH2Cl2 (1 mL)] and DIPEA (0.08 g, 0.67 mmol) were added and stirred
at room temperature for 8 h. Work up as described for 14 and purification of the residue by
column chromatography (60ꢀ120 mesh silica gel, 2.6% CH3OH in CHCl3) afforded 1 (0.15 g,
20
55%) as a colorless solid; mp 136ꢀ138
°C; [
α]
D = +50.0 (
c
0.05, CHCl3); IR (KBr):
ν
3284,
2983, 2931, 1739, 1692, 1650, 1535, 1439, 1369, 1289, 1271, 1106, 1079, 1019, 963, 857, 771,
1
666 cmꢀ1; H NMR (500 MHz, 278 K, CDCl3):
δ
7.67 (b, 1H, NHꢀ2), 7.62 (d, 1H,
= 9.8 Hz, NHꢀ1), 4.94 (s, 1H, C1Hꢀ3), 4.93 (s, 1H,
=3.8, 5.9 Hz, C3Hꢀ3), 4.68 (dd, 1H, = 3.9, 5.9 Hz, C3Hꢀ1), 4.60 (m, 1H,
= 5.9 Hz, C2Hꢀ1, C2Hꢀ3), 4.44 (m, 1H, CβHꢀ1), 4.06 (dd, 1H, = 3.8, 9.5
Hz, C4Hꢀ3), 3.99 (m, 1H, CβHꢀ2), 3.91 (dd, 1H, = 3.9, 7.3 Hz, C4Hꢀ1), 3.76 (m, 1H, CβHꢀ4),
J = 9.5 Hz,
NHꢀ3), 7.00 (b, 1H, NHꢀ4), 5.68 (d, 1H,
C1Hꢀ1), 4.74 (dd, 1H,
CβHꢀ3), 4.54 (d, 2H,
J
J
J
J
J
J
3.71 (s, 3H, COOMe), 3.32 (s, 3H, OMe), 3.30 (s, 3H, OMe), 3.12 (m, 1H, Cβ'Hꢀ2), 2.77 (dd,
,
1H,
J
= 3.9, 13.7 Hz, CαHꢀ1), 2.73 (dddd, 1H,
J
= 3.8, 3.8, 8.2, 16.5 Hz, Cβ Hꢀ4), 2.54 (m, 1H,
CαHꢀ3), 2.50 (m, 1H, CαHꢀ4, Cα'Hꢀ4), 2.43 (m, 1H, Cα Hꢀ1), 2.41 (m, 1H, Cα Hꢀ3), 2.28 (m, 2H,
'
'
CαHꢀ2, Cα Hꢀ2), 1.64 (s, 6H, Ac), 1.48 (s, 6H, Ac), 1.29 (s, 9H, Boc); 13C NMR (125 MHz, 298
'
K, CDCl3): δ 173.3, 171.7, 171.1, 170.7, 156.0, 112.7, 112.5, 106.8, 106.5, 85.1, 85.0, 79.9, 79.8,
79.6, 79.5, 54.6, 54.4, 51.8, 48.2, 47.4, 39.7, 38.4, 37.2, 36.2, 35.1, 33.7, 28.4 (2C), 26.0, 25.9,
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24.8, 24.6; HRMS (ESI+): m/z calculated for C34H56N4O15Na(M++Na) 783.3634, found
783.3593.
Bocꢀ(S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀ(
S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀ(S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀOMe (2):
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A cooled solution (0 oC) of 1 (0.05 g, 0.07 mmol) in THF:MeOH:H2O (3:1:1) (1 mL) was
treated with LiOH (0.002 g, 0.1 mmol) and stirred at room temperature. Work up as described
for 9a gave 15 (0.05 g, 95%) as a colorless solid, which was used as such for further reaction.
To a solution of 15 (0.05 g, 0.07 mmol), HOBt (0.01 g, 0.10 mmol) and EDCI (0.02 g,
0.10 mmol) in CH2Cl2 (5 mL), amine salt 14b [prepared from 14 (0.04 g, 0.07 mmol) and
CF3COOH (0.1 mL) in CH2Cl2 (0.5 mL)] and DIPEA (0.02 g, 0.13 mmol) were added and stirred
at room temperature for 8 h. Work up as described for 14 and purification of the residue by
column chromatography (60ꢀ120 mesh silica gel, 2.8% CH3OH in CHCl3) afforded 2 (0.04 g,
20
49%) as a colorless solid; mp 157ꢀ158
°C; [
α]
D = +94.0 (
c
0.05, CHCl3); IR (KBr):
ν
3286,
3019, 2924, 2853, 1692, 1651, 1541, 1441, 1373, 1213, 1105, 1080, 1022, 964, 746, 667 cmꢀ1;
1H NMR (500 MHz, 298 K, CDCl3):
8.36 (dd, 1H, J = 3.5, 9.4 Hz, NHꢀ4), 8.24 (d, 1H, J = 7.8
δ
Hz, NHꢀ3), 7.85 (b, 1H, NHꢀ6), 7.56 (d, 1H,
J
= 8.2 Hz, NHꢀ5), 6.94 (b, 1H, NHꢀ2), 6.14 (d, 1H,
= 7.8 Hz, NHꢀ1), 4.91 (s, 1H, C1Hꢀ1), 4.87 (s, 2H, C1Hꢀ3, C1Hꢀ5), 4.80 (dd, 1H, = 3.5, 5.9 Hz
= 3.8, 5.9 Hz, C3Hꢀ1), 4.59 (m, 1H, CβHꢀ3), 4.52 (d, 3H, = 5.9 Hz,
J
J
C3Hꢀ3), 4.74 (dd, 1H,
J
J
C2Hꢀ1, C2Hꢀ3, C2Hꢀ5), 4.50 (m, 1H, CβHꢀ5), 4.40 (m, 1H, CβHꢀ1), 4.14 (m, 1H, CβHꢀ2), 4.06
(dd, 1H, = 3.2, 9.5 Hz, C4Hꢀ5), 4.05 (dd, 1H, = 3.2, 9.5 Hz, C4Hꢀ5), 4.04 (m, 1H, CβHꢀ4),
4.03 (dd, 1H, = 3.8, 9.6 Hz, C4Hꢀ1), 4.01 (dd, 1H, = 3.5, 9.6 Hz, C4Hꢀ3), 3.92 (m, 1H, CβHꢀ
J
J
J
J
6), 3.63 (s, 3H, COOMe), 3.33 (s, 3H, OMe), 3.32 (s, 3H, OMe), 3.30 (s, 3H, OMe), 3.23 (m,
1H, Cβ'Hꢀ6) 3.12 (m, 1H, Cβ'Hꢀ4), 3.04 (m, 1H, Cβ'Hꢀ2), 2.70 (m, 1H, Cα'Hꢀ6), 2.69 (m, 1H,
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CαHꢀ1), 2.54 (m, 1H, CαHꢀ3), 2.52 (m, 1H, Cα'Hꢀ2), 2.50 (m, 1H, Cα'Hꢀ1), 2.47 (m, 1H, Cα'Hꢀ4),
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2.44 (m, 1H, CαHꢀ6), 2.42 (dd, 1H,
J = 4.2, 9.8 Hz, CαHꢀ5), 2.40 (m, 1H, Cα Hꢀ3), 2.38 (dd, 1H,
'
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J
= 5.5, 8.6 Hz, Cα Hꢀ5), 2.27 (m, 1H, CαHꢀ4), 2.16 (m, 1H, Cα Hꢀ2), 1.45 (s, 3H, Ac), 1.44 (s,
'
'
9H, Boc), 1.43 (s, 3H, Ac), 1.41 (s, 3H, Ac), 1.30 (s, 3H, Ac), 1.29 (s, 3H, Ac), 1.28 (s, 3H, Ac);
13C NMR (125 MHz, 298 K, CDCl3):
δ
173.8, 171.5, 171.4 (3C), 170.8, 156.1, 112.5, 112.4,
112.4, 106.8, 106.6, 106.1, 85.0, 84.9, 84.8, 79.7, 79.5, 79.5, 79.4, 79.3, 79.1, 54.3, 54.2, 54.2,
54.1, 52.0, 48.1, 46.9, 46.7, 39.9, 38.6, 38.3, 38.2, 37.8, 36.9, 36.8, 35.6, 34.6, 28.5 (3C), 26.0
(2C), 25.9 (2C), 24.7 (2C), 24.6 (2C); HRMS (ESI+): m/z calculated for
C48H78N6O21Na(M++Na) 1097.5112, found 1097.5044.
BocꢀβꢀhGlyꢀ(S)ꢀβꢀCaa(l)ꢀOMe (16):
To a stirred solution of βꢀhGly 11 (0.31 g, 3.5 mmol) in 4N NaOH (1 mL), (Boc)2O (0.8
mL, 3.5 mmol) was added slowly for 2 h and stirred at room temperature for 18 h. The reaction
mixture was washed with petroleum ether (3 x 2 mL) and washed with aq. sat. NaHCO3 (5 mL).
Both the aq. NaOH and aq. sat. NaHCO3 layers were mixed and acidified with aq. sat. KHSO4
(pH = 2) and extracted with ethyl acetate (2 x 5 mL). The organic layer was dried (Na2SO4) and
evaporated to give 11b (0.3 g, 94%) as a colorless solid, which was used for the next reactions.
A solution of acid 11b (0.30 g, 1.93 mmol) [prepared from 11 (0.31 g, 3.5 mmol) in 4N
NaOH (1 mL) and (Boc)2O (0.8 mL, 3.5 mmol)], HOBt (0.32 g, 2.34 mmol), and EDCI (0.46 g,
2.34 mmol) in CH2Cl2 (5 mL) was stirred at 0 °C under N2 atmosphere for 15 min. Amine salt 9b
[prepared from 9 (0.6 g, 0.16 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (2 mL)] and DIPEA
(0.40 g, 3.16 mmol) were added and stirred at room temperature for 8 h. Work up as described
for 14 and purification of the residue by column chromatography (60ꢀ120 mesh silica gel, 55%
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ethyl acetate in pet. ether) afforded 16 (0.60 g, 57%) as a colorless syrup; [α] D = +84.0 (c 0.05,
8
9
CHCl3); IR (KBr):
ν 3753, 3611, 3355, 2921, 2852, 1741, 1709, 1691, 1549, 1531, 1464, 1377,
10
11
12
13
14
15
16
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1168, 1076, 720, 602 cmꢀ1; 1H NMR (500 MHz, 298 K, CDCl3):
δ
6.15 (d, 1H,
J
= 7.7 Hz, NHꢀ
= 7.3 Hz, C4Hꢀ
J = 3.6, 7.3 Hz, C3Hꢀ1), 3.69 (s, 3H, COOMe),
2), 5.20 (b, 1H, NHꢀ1), 4.89 (s, 1H, C3Hꢀ2), 4.65 (m, 1H, CβHꢀ1), 4.60 (t, 1H,
2), 4.55 (d, 1H, = 5.6 Hz, C2Hꢀ1), 4.16 (dd, 1H,
J
J
3.41 (m, 2H, CβHꢀ1, Cβ Hꢀ1), 3.30 (s, 3H, OMe), 2.78 (m, 2H, CαHꢀ1, CαHꢀ2), 2.38 (m, 2H,
'
13
Cα Hꢀ1, Cα Hꢀ2), 1.63 (s, 3H, Ac), 1.45 (s, 9H, Boc), 1.29 (s, 3H, Ac); C NMR (125 MHz,
'
'
CDCl3, 298 K):
δ 171.9, 171.0, 155.9, 112.6, 106.6, 84.9, 79.4, 79.0, 78.7, 54.6, 51.7, 45.8, 36.6,
36.2, 35.6, 28.3 (3C), 25.9, 24.5; HRMS (ESI+): m/z calculated for C20H34N2O9Na(M++Na)
469.2156, found 469.2121.
BocꢀβꢀhGlyꢀ(
S)ꢀβꢀCaa(l)ꢀβꢀhGlyꢀ(S)ꢀβꢀCaa(l)ꢀOMe (3):
o
A cooled solution (0 C) of 16 (0.16 g, 0.34 mmol) in THF:MeOH:H2O (3:1:1) (1 mL)
was treated with LiOH (0.01 g, 0.52 mmol) and stirred at room temperature. Work up as
described for 9a gave 16a (0.14 g, 90%) as a colorless solid, which was used as such for further
reaction.
A solution of 16a (0.14 g, 0.38 mmol), HOBt (0.06 g, 0.48 mmol), and EDCI (0.09 g,
o
0.48 mmol) in CH2Cl2 (5 mL) was stirred at 0 C under N2 atmosphere for 15 min, treated
sequentially with 16b [prepared from 16 (0.18 g, 0.38 mmol) and CF3COOH (0.4 mL) in CH2Cl2
(1.2 mL) at 0 °C] and DIPEA (0.16 mL, 0.93 mmol) and stirred for 8 h. Work up as described for
14 and purification of the residue by column chromatography (60ꢀ120 mesh silica gel, 2.2%
20
CH3OH in CHCl3) afforded 3 (0.14 g, 48%) as a colorless solid; mp 142ꢀ143
°C; [
α]
D = +90.0
(c
0.05, CHCl3); IR (KBr):
ν
3743, 3353, 3272, 2952, 2842, 1725, 1708, 1691, 1661, 1645, 1449,
56
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60
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