Design and study of peptides containing 1:1 left- and right-handed helical patterns from aminopyrancarboxylic acids

Article abstract of DOI:10.1002/ejoc.201402123

(R,R)- and (S,S)-aminopyrancarboxylic acids (APyCs) were used in the design of mixed β- and α/β-peptides in alternation with β-hGly and L-Ala/D-Ala, respectively. The enantiomeric β- and α/β- tetrapeptides were then coupled in a 1:1 fashion to give octapeptides with 12/10- and 9/11-mixed helices, respectively. The structure of the helices, with their characteristic hydrogen-bonding patterns and an additional stabilizing interaction between peptide-bond NH groups and pyran-ring oxygen atoms, was investigated by NMR spectroscopy, molecular dynamics, and quantum chemical studies. The presence of equal parts of left- and right-handed helices was confirmed by the cancellation of their respective CD patterns. Despite the disruption of the hydrogen bonding in the transition region, the helical conformation was maintained in the octamers. This study demonstrates the possibility of accommodating helices of opposite handedness within the same peptide. Copyright

Full text of DOI:10.1002/ejoc.201402123

FULL PAPER
DOI: 10.1002/ejoc.201402123
Design and Study of Peptides Containing 1:1 Left- and Right-Handed Helical
Patterns from Aminopyrancarboxylic Acids
Gangavaram V. M. Sharma,*[a] Hajari Ravindranath,[a][‡] Akkala Bhaskar,[a][‡]
Shaik Jeelani Basha,[b][‡] Potti Reddy Gari Gurava Reddy,[b][‡‡] Katukuri Sirisha,[b]
Akella V. S. Sarma,*[b] and Hans-Jörg Hofmann*[c]
Keywords: Amino acids / Peptides / Peptidomimetics / Foldamers / Helical structures
(R,R)- and (S,S)-aminopyrancarboxylic acids (APyCs) were
used in the design of mixed β- and α/β-peptides in alter- ies. The presence of equal parts of left- and right-handed
nation with β-hGly and -Ala/ -Ala, respectively. The enan- helices was confirmed by the cancellation of their respective
troscopy, molecular dynamics, and quantum chemical stud-
L
D
tiomeric β- and α/β-tetrapeptides were then coupled in a 1:1
fashion to give octapeptides with 12/10- and 9/11-mixed hel-
ices, respectively. The structure of the helices, with their
characteristic hydrogen-bonding patterns and an additional
stabilizing interaction between peptide-bond NH groups and
pyran-ring oxygen atoms, was investigated by NMR spec-
CD patterns. Despite the disruption of the hydrogen bonding
in the transition region, the helical conformation was main-
tained in the octamers. This study demonstrates the possibil-
ity of accommodating helices of opposite handedness within
the same peptide.
β-peptides. Gellman et al. and Seebach et al. presented left-
handed 14-helices in β-peptides built from conformationally
constrained oligomers of trans-2-aminocyclohexanecarbox-
ylic acid (ACHC) and homologated α-amino acids, respecti-
vely.[3d,4a,6] Gellman et al.[7] also reported a 12-helix in
trans-2-amino cyclopentanecarboxylic acid (ACPC)-derived
peptides, and Fülöp et al.[8] obtained 12-helices in β-pept-
ides composed of amino-6,6-dimethylbicyclo[3.1.1]heptane-
3-carboxylic acids (ABHC). Sharma et al. reported right-
and left-handed 12/10-mixed helices in β-peptides con-
sisting of alternating (R)-β-Caa/(S)-β-Caa and β-hGly (β-
homo-glycine) constituents, and 9/11-mixed helical struc-
tures occurred in α/β-peptides with alternating l-Ala and
(S)-β-Caa residues.[3g,3h,4d,4f] Considering the role played by
additional interactions in the formation and stabilization of
secondary structures in foldamers,[9] our group presented
(S,S)-aminopyrancarboxylic acid [(S,S)-APyC] as a new
monomer in a previous paper.[10] Peptides with alternating
(S,S)-APyC and d-Ala residues were reported to form a
right-handed 9/11-mixed helix, in which the pyran oxygen
of APyC induced a 5-mr (five-membered ring) hydrogen
bond,[9k,11] which provided additional stability for the 9/11-
Introduction
Oligomers containing unnatural amino acids with strong
and predictable folding propensities stabilized by non-cova-
lent interactions, referred to as foldamers,[1] enable the mim-
icry of large protein recognition surfaces. In the past dec-
ade, much attention has been focused on the design of
oligomers with homogeneous and heterogeneous back-
bones, due to their well-defined folding propensities.[2] Sev-
eral groups have explored unnatural peptide foldamers
composed of β-amino acids[1b,2g,3] or a combination of
α- and β-amino acids to mimic natural peptides and prote-
ins.[1e,1f,2e,2f,3d,3g,4] Based on the helix-coil theory, longer se-
quences and several intramolecular hydrogen bonds are re-
quired to overcome the energetically demanding nucleation
parameters.[5] Despite this theory, β-peptides with very
small numbers of residues show helical conformations,
which suggests that the nucleation parameter is different for
[a] Organic and Biomolecular Chemistry Division, CSIR – Indian
Institute of Chemical Technology,
Hyderabad 500007, India
E-mail: esmvee@iict.res.in
www.iictindia.org
[b] Centre for NMR & SC, CSIR – Indian Institute of Chemical helix.[10]
Technology,
Left-handed secondary structures are rare in nature,[12]
Hyderabad 500007, India
E-mail: ssav@iict.res.in
but they are observed in peptides and proteins at active
sites. However, both left- and right-handed helical struc-
tures are formed in foldamers.[13] A literature survey[14] re-
vealed that different designs could result in a switch of the
helical pattern from one handedness to the other. In earlier
studies, we reported “hybrid helices” in peptides containing
more than one type of secondary structure with high com-
[c] Institute of Biochemistry, Faculty of Biosciences, University of
Leipzig,
Brüderstrasse 34, 04103 Leipzig, Germany
E-mail: hofmann@uni-leipzig.de
[‡] These authors contributed equally.
[‡‡] Deceased
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402123.
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FULL PAPER
Figure 1. Structures of peptides 1–6, ent-1, ent-2, (S,S)-APyC (7), and (R,R)-APyC (ent-7).
patibility.[15] The design and synthesis of peptides contain- ides 1, 2, ent-1, ent-2, and 3, and α/β-peptides 4–6, as well
ing both left- and right-handed helical patterns have also as the conformational analysis (NMR, MD, CD, ab initio
been reported.[16] Recently, Martinek et al.[17] presented MO theory) of peptides 1–6, ent-1, and ent-2 (Figure 1) are
their observations of the switch in helicity in β-peptides described below.
with β-H18/20 mixed helices. In the work presented here,
enantiomeric (R,R)- and (S,S)-aminopyrancarboxylic acids
Results and Discussion
(APyCs) were used to synthesize β- and α/β-peptides to-
gether with alternating β-hGly and l-Ala/d-Ala constitu-
ents, respectively. The left- and right-handed structures in
the peptides were examined by NMR spectroscopy, sup-
Synthesis of β- and α/β-Peptides
Mixed β-peptides 1, 2, ent-1, and ent-2 were synthesised
ported by molecular dynamics (MD), circular dichroism from (S,S)- and (R,R)-aminopyrancarboxylic acids (APyC)
(CD), and quantum chemical studies.[18] The β- and α/β- 7 and ent-7 with alternating use of β-hGly. Peptide 3 was
tetrapeptides of opposite handedness were coupled in a 1:1 prepared from tetrapeptides 1 and ent-1. Similarly, α/β-
fashion. The resulting octapeptides also show the presence peptides 4 and 5 were prepared by alternating ent-7 with l-
of both types of handedness. The details of the syntheses Ala. Octapeptide 6 was prepared from two tetrapeptides.
of (R,R)-aminopyrancarboxylic acid [(R,R)-APyC], β-pept- The required building block (R,R)-APyC ent-7 was synthe-
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Design and Study of Peptides
Scheme 1. Reagents and conditions: a) i) BnNH2, dry MgSO4, diethyl ether, 0 °C to room temp., 2 h; ii) vinyl bromide, Mg, THF, 1,2-
dibromoethane, 0 °C to r.t., 8 h; b) Et3N, (Boc)2O (Boc = tert-butoxycarbonyl), CH2Cl2, 0 °C–r.t., 6 h; c) PTSA (p-toluenesulfonic acid),
MeOH/H2O (9:1), 0 °C–r.t., 6 h; d) TBSCl (tert-butyldimethylsilyl chloride), imidazole, CH2Cl2, 0 °C–r.t., 4 h; e) allyl bromide, NaH,
DMF, 0 °C–r.t., 4 h; f) Grubbs I catalyst, toluene, reflux, 8 h; g) Li, liq. NH3, THF, –78 °C, 1 h; h) Pd-C (10%), H2, MeOH, r.t., 6 h;
i) TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxy], BAIB [bis(acetoxy)iodobenzene], CH2Cl2/H2O (3:1), 0 °C to r.t., 4 h; j) CH2N2, diethyl
ether, 0 °C–r.t., 1 h.
sised from (S)-glyceraldehyde derivative 8 by a procedure was converted into salt 19b by reaction with CF3COOH in
already reported for 7,[10] according to Scheme 1 (for de- CH2Cl2. Acid 19a, on coupling (EDCI, HOBt, and
tails, see Exp. Section).
DIPEA) with salt 19b in CH2Cl2, gave tetrapeptide 1 in
42% yield. Treatment of peptide 1 with LiOH gave acid 1a,
while 1 was converted into salt 1b by reaction with
CF3COOH in CH2Cl2. Coupling (EDCI, HOBt, and
Synthesis and Conformational Analysis of β-Peptides
Peptides 1, 2, ent-1, ent-2, and 3, were prepared from DIPEA) of acid 1a with salt 19b in CH2Cl2 gave hexapept-
β-hGly-OMe·HCl 18, 7a, and ent-7 by standard peptide- ide 2 (38%).
coupling methods[19] using EDCI [1-ethyl-3-(3-dimethyl-
Base (LiOH) hydrolysis of ester ent-7 gave acid 17,
aminopropyl)carbodiimide], HOBt (hydroxybenzotriazole), which, on coupling (EDCI, HOBt, and DIPEA) with 18 in
and DIPEA (diisopropylethylamine) in solution phase CH2Cl2, gave dipeptide ent-19 (66%). Ester ent-19, on base
(Scheme 2). Base hydrolysis of ester 7 with LiOH gave acid (LiOH) hydrolysis, gave acid ent-19a, while CF3COOH-me-
7a, which, on coupling with 18 in the presence of EDCI, diated reaction of 19 in CH2Cl2 gave salt ent-19b.
HOBt, and DIPEA in CH2Cl2, gave dipeptide 19 (89%).
Coupling of acid ent-19a (EDCI, HOBt, and DIPEA)
Hydrolysis of ester 19 with LiOH gave acid 19a, while 19 and salt ent-19b in CH2Cl2 gave ent-1 in 46% yield. Peptide
Scheme 2. Synthesis of peptides 1, 2, ent-1, ent-2, and 3. Reagents and conditions: (a) HOBt (1.2 equiv.), EDCI (1.2 equiv.), DIPEA
(2 equiv.), dry CH2Cl2, 0 °C–r.t., 8 h; (b) LiOH, THF/MeOH/H2O (3:1:1), 0 °C–r.t., 1 h; (c) CF3CO2 H, dry CH2Cl2, 2 h.
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ent-1 gave acid ent-1a upon base hydrolysis with LiOH, The ROESY spectrum of peptide 2 showing various charac-
while ent-1 was converted into salt ent-1b by treatment with teristic correlations, and the characteristic hydrogen-bond-
CF3COOH in CH2Cl2. Acid ent-1a, on coupling (EDCI, ing pattern are shown in Figure 2.
HOBt, and DIPEA) with salt ent-19b in CH2Cl2, gave ent-
2 in 46% yield. Similarly, the coupling of acid 1a with salt
ent-1b in the presence of EDCI, HOBt, and DIPEA in
CH2Cl2 gave octapeptide 3 in 75% yield.
Conformational analysis of mixed β-peptides 1, 2, ent-1,
ent-2, and 3 (Figure 1) was carried out by NMR spec-
troscopy (in CDCl3), CD, MD, and quantum chemical
studies. Peptide 1 showed a very well-resolved amide region
in the proton NMR spectrum,[18] suggesting the existence
of a well-defined structure. The appearance of NH(2) and
NH(3) at low field (δ Ͼ 7.7 ppm) along with a small change
in their chemical shift values during solvent titration stud-
ies[18] provide sufficient support for their participation in
intramolecular hydrogen bonding. In addition, NH(4), res-
onating at δ = 6.9 ppm, showed a small difference in chemi-
cal shift during titration with [D6]DMSO, giving support
for its involvement in non-covalent interactions.[18]
For (S,S)-APyC residue 1 in 1, the coupling constant
3JNH,CβH Ն 9.2 Hz shows that the NH and CβH protons
are antiperiplanar to each other, corresponding to a dihe-
3
dral angle φ[C(O)–N–Cβ–Cα] ≈ –120°. A large JCαH,CβH
≈
9.6 Hz supports a value of –60° for the dihedral angle θ,
which is further supported by the presence of strong nOe
correlation between the NH and CαH protons. For the β-
hGly residues, the presence of a small (Յ4.8 Hz) and a large
3
(Ն7.9 Hz) value for JNH,βH is consistent with φ ≈ –120°,
whereas the 3JCαH,CβH values (Ͻ1.9 and Ͼ12.4 Hz) are con-
sistent with θ ≈ –60°. The prochiral protons of Cα and Cβ
of β-hGly were assigned based on coupling constants and
nOe correlations. CβH(pro-S) is assigned as the proton with
a large coupling and medium nOe with the NH proton,
whereas CβH(pro-R) shows a small coupling and strong nOe
with the NH proton. Similarly, CαH(pro-S) has a large cou-
pling constant with CβH(pro-R) and a strong nOe correlation
with the succeeding NH proton. NH(4) appeared as a trip-
let because of fraying at the terminus.
The nOe correlations CβH(1)/NH(3) and CαH(pro-R)(2)/
NH(3) support a 12-membered (mr) hydrogen bond be-
tween NH(3)···O=C(Boc), whereas the nOe correlation
NH(2)/NH(3) is consistent with a 10-mr hydrogen bond be-
tween NH(2)···O=C(3). From the coupling and nOe infor-
mation, the presence of a 12/10-helix was unambiguously
established in this peptide. This helix was found to be fur-
ther stabilized by additional electrostatic interactions be-
tween NH(2)···OC(1) and between NH(4)···OC(3), which is
supported by the nOe correlations CβH(1)/NH(2), CβH(3)/
NH(4), CεH(1)/NH(2), and CεH(3)/NH(4).
Figure 2. A) ROESY expansion of peptide 2 showing characteristic
nOe correlations. B) Ball-and-stick model of peptide 2 showing
characteristic nOes represented by double arrows. C) Chemical
structure showing various hydrogen bonds and electrostatic interac-
tions (red-colored arrows).
The right-handed helical structure in peptides 1 and 2
was confirmed by their CD profiles. The CD spectra of
peptides 1 and 2 in methanol (0.2 mm) showed a maximum
at 204 nm, which supports the presence of a right-handed
12/10-helix with molar ellipticities of ca. 10000 and ca.
15000 degcm2 dmol–1 (Figure 3). For the restrained molecu-
lar dynamics studies, the constraints were derived from the
intensities of the nOe cross-peaks in the ROESY spectra
using the two-spin approximation.
Having defined the helical pattern (12/10-helix) and the
presence of electrostatic interactions between peptide-bond
NH groups and the pyran ring-oxygen atom in peptide 1,
the study was extended to higher homologue 2. The spectral
analysis of 2 showed all the characteristic nOe correlations
and hydrogen-bonding information required for a propa-
gated 12/10-mixed helix. Furthermore, the 12/10-helix was
additionally stabilized by 5-mr electrostatic interactions. Figure 3. CD profiles of peptides 1–3, ent-1, and ent-2.
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Design and Study of Peptides
The study was then extended to peptides ent-1 and ent- for the β(6) and β(8) monomers, since they have one small
2, prepared from the enantiomeric β-amino acid [i.e., (R,R)- (Յ4.8 Hz) and one large (Ն7.9 Hz) coupling constant
3
APyC ent-7]. Peptides ent-1 and ent-2 are enantiomeric with 3JNH,CβH, and values of Ͻ2.2 or Ͼ12.4 Hz for JCαH,CβH
.
1 and 2. The 1H NMR spectrum[18] of peptide ent-1 in
The nOe correlations CβH(1)/NH(3), CαH(pro-R)(2)/
CDCl3 showed the features of a stable secondary structure. NH(3), and NH(2)/NH(3) along with CβH(1)/NH(2),
The amide protons NH(2) and NH(3) resonate at δ Ͼ CβH(3)/NH(4), and CεH(1)/NH(2) correspond to a right-
7.7 ppm, which indicates their participation in hydrogen handed 12/10-helix, while CβH(5)/NH(7), CαH(pro-S)(5)/
bonding. This was further confirmed by their small chemi- NH(6), NH(6)/NH(7), CβH(5)/NH(6), CβH(7)/NH(8),
cal shift change (Ͻ 0.25 ppm) during solvent titration[18] CεH(5)/NH(6), and CεH(7)/NH(8) support a left-handed
with [D6]DMSO. Furthermore, NH(4) resonated very close 12/10-helix (Figure 4). These helical structures are further
to δ = 7 ppm and showed a small change in chemical shift stabilized by electrostatic interactions between the amide
(Δδ Ͻ 0.34 ppm) during solvent titration, confirming its protons of the β-hGly residues and the oxygen atoms of the
participation in non-covalent interactions.[18] A higher Δδ pyran rings in the preceding residues. Figure 3 shows the
value of 1.94 ppm for NH(1) indicates its exposure to the compensation of the positive and negative profiles of the
solvent.
left-handed and right-handed 12/10-helices in the CD spec-
The nOe correlations, coupling constants, and changes in trum of peptide 3. A stereoview of 20 minimum-energy
chemical shift values are similar to those observed for pept- structures of peptide 3 resulting from molecular dynamics
ides 1 and 2, but the signs of the dihedral angles are oppo- can be seen in Figure 5, demonstrating the presence of the
site. These details indicate a switch of handedness for pept- right- and left-handed 12/10-helices.[18]
ides ent-1 and ent-2. Support for this switch was also ob-
tained from the CD spectra, which showed profiles opposite
to those of peptides 1 and 2. Thus, a left-handed 12/10-
mixed helix is formed by ent-1 and ent-2, according to
NMR, CD, and MD studies.[18] Such a left-handed 12/10-
helix has earlier been observed in mixed peptides made
from (S)-β-Caa and β-hGly in a 1:1 alternating order.[3g]
This study presents the first reported well-designed motif
for a left-handed 12/10-helix. Since left-handed conforma-
tions in natural proteins[12c] are implicated in biological ac-
tivity, this design is important in the foldamer field.
Having unequivocally analyzed the presence of right-
handed 12/10-helices in peptides 1 and 2, as well as left-
handed 12/10-helices in ent-1 and ent-2, we went on to
study peptide 3, whose design connects the two helices of
opposite handedness. It is very interesting to investigate the
structure of peptide 3, because this gives information on the
compatibility of two enantiomeric helical structures within
a single peptide.
1
The H NMR spectrum[18] of 3 showed a well-dispersed
amide region, indicating the existence of a well-defined
structure. Amide protons NH(2)–NH(3) and NH(5)–NH(7)
resonated at low field (δ Ͼ 7.8 ppm), while NH(4) and
NH(8) resonated at δ Ͼ 6.8 ppm. All the amide protons
except NH(1) showed a small change in their chemical shift
values during solvent titration, which indicates that they are
involved in non-covalent interactions.[18]
3
For the APyC residue, the large JNH,CβH Ն 9.2 Hz
shows that the NH and CβH protons are antiperiplanar to
each other, corresponding to a dihedral angle φ[C(O)–N–
Cβ–Cα] ≈ |120°|. A large value for the coupling constant
3JCαH,CβH ≈ 9.6 Hz supports a magnitude of |60°| for the
dihedral angle |θ|, which is further supported by the pres-
Figure 4. A) ROESY expansion of peptide 3 showing characteristic
nOe correlations. B) Ball-and-stick model of peptide 3 showing
characteristic nOes represented by double arrows. C) Chemical
structure showing various hydrogen bonds and electrostatic interac-
tions (red-colored arrows).
The conformational structure of peptide 3 was also con-
ence of strong nOe correlations between the NH and CαH firmed by quantum chemical calculations at the B3LYP/6-
protons. The dihedral angles of the β(1) and β(3) constitu- 31G* level of ab initio MO theory. Most striking is the
ents have opposite signs to those of β(5) and β(7), because smooth transition between the two helical parts of opposite
of the connection of two enantiomeric peptides. A similar handedness, such that an essentially linear helical overall
situation is found with β-hGly, i.e., φ ≈ 120° and θ ≈ 60° for shape of the peptide is maintained. A detailed inspection of
the β(2) and β(4) constituents, and φ ≈ –120° and θ ≈ –60° the structure shows that the NH group of residue 4, which
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Earlier, we reported[10] a right-handed 9/11-helix in pept-
ides containing trans-(S,S)-APyC. Hence, we proposed to
synthesize α/β-peptides 4 and 5, and octapeptide 6 from 4
and the known[10] tetrapeptide prepared from 7 (Scheme 3).
Accordingly, acid 17, upon coupling with 20 in the presence
of EDCI, HOBt, and DIPEA in CH2Cl2 gave dipeptide 21
(90%). Hydrolysis of ester 21 with LiOH gave acid 21a,
while reaction of 21 with CF3COOH in CH2Cl2 gave salt
21b. Coupling (EDCI, HOBt, and DIPEA) of acid 21a with
salt 21b in CH2Cl2 gave tetrapeptide 4 (47%). Hydrolysis of
peptide 4 with LiOH gave acid 4a, while treatment of 4
with CF3COOH in CH2Cl2 gave salt 4b. Coupling (EDCI,
HOBt, and DIPEA) of acid 4a with salt 21b in CH2Cl2
provided hexapeptide 5 (38%). Similarly, coupling of
known acid ent-4a[10] with 4b in the presence of EDCI,
HOBt, and DIPEA in CH2Cl2 gave octapeptide 6 in 75%
yield.
Figure 5. Stereoview of 20 superimposed minimum energy struc-
tures of peptide 3 (hydrogens were removed after calculations for
clarity).
comes from peptide 1, and the CO group of residue 5,
which is the first residue of peptide ent-1, are no longer
involved in the hydrogen-bonding network, as they would
be if the handedness had not been changed. The approxi-
mately linear helical overall structure of the peptide results
from two 12-mr hydrogen bonds between the helices of op-
posite handedness arising from the CO group of residue 2
(first helix) and the NH group of residue 5 (second helix),
and the CO group of residue 4 (first helix) and the NH
group of residue 7 (second helix), respectively. The forma-
tion of a 12-mr hydrogen bond between NH(5) with CO(2),
which stabilizes the hinge region, is well documented by the
NMR spectroscopic data, which indicates an nOe corre-
lation between CβH(3)/NH(5) (Figure 4). The NH group of
residue 4 keeps its stabilizing electrostatic interaction with
the pyran oxygen atom of the preceding residue. According
to the quantum chemical calculations, the corresponding
NH···O distance is 2.18 Å. The backbone torsion angles
calculated for peptide 3 are given in the Supporting Infor-
mation.
Based on our earlier report,[10] we anticipated that the
peptides 4 and 5 would show left-handed 9/11-helices. The
proton NMR spectrum of peptide 4 was very well dis-
persed, and in the solvent titration study of amide protons,
except for NH(1), showed small chemical shift changes,[18]
indicating a well-defined secondary structure with the par-
ticipation of the amide protons in hydrogen bonding. The
nOe correlations and information on dihedral angles ob-
3
tained from J coupling constants indicate that the amide
protons NH(2) and NH(3) formed 9-mr and 11-mr hydro-
gen bonds with the CO(3) and Boc CO groups, respectively.
In addition, NH(2) and NH(4) are involved in weak electro-
static interactions with the oxygen atom of the pyran ring
of APyC. Solvent titration and nOe studies on peptide 5
confirmed that the NHs of Ala(2) and Ala(4) participate in
9-mr hydrogen bonding with the CO group of APyC(3) and
APyC(5), respectively, whereas the NH groups of APyC(3)
and APyC(5) participate in 11-mr hydrogen bonding with
the Boc CO and CO(2) groups. The amide protons of
Ala(2), Ala(4), and Ala(6) showed weak interactions with
the oxygen of the pyran rings of APyC(1), APyC(3), and
APyC(5), respectively, which may provide further stabiliza-
tion of the 9/11-helical structure. The CD spectra of 4 and
5 showed minima at 203 and 206 nm, respectively, indicat-
ing the presence of a left-handed 9/11-helical pattern, which
was further confirmed by molecular dynamics simula-
Synthesis and Conformational Analysis of α/β-Hybrid
Peptides 4–6
After the synthesis and analysis of peptides 1, 2, ent-1,
ent-2, and 3 and the identification of the occurrence of left-
and right-handedness in the mixed β-peptides, the study
was extended to α/β-peptides with right- and left-handed 9/
11-helices.
Scheme 3. Synthesis of peptides 4–6. Reagents and conditions: (a) HOBt (1.2 equiv.), EDCI (1.2 equiv.), DIPEA (2 equiv.), dry CH2Cl2,
0 °C–r.t., 8 h; (b) LiOH, THF/MeOH/H2O (3:1:1), 0 °C–r.t., 1 h; (c) CF3CO2 H, dry CH2Cl2, 2 h.
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Design and Study of Peptides
Figure 6. ROESY spectrum of peptide 6.
tions.[18] Peptide 5 was also studied in CD3OH, a polar sol-
vent, and the majority of the nOe correlations that are in-
dicative of a 9/11-helix, and which were observed in CDCl3,
were also observed here. The weak intensitites of the cross-
peaks imply a diminishing of the ordered structure.
To understand the compatibility between two different
helix types with opposite handedness, peptide 6 was
studied. As was observed for peptide 3, the amide protons
of 6 resonated at low field (δ Ͼ 7 ppm), and showed small
changes in their chemical shifts in solvent titration,[18] ex-
cept for NH(1) and NH(4). The nOe correlations CβH(1)/
NH(3) and NH(2)/NH(3), along with CβH(1)/NH(2) and
CβH(3)/NH(4), correspond to a right-handed 9/11-helix,
while CβH(5)/NH(7), NH(6)/NH(7), CβH(5)/NH(6), and
CβH(7)/NH(8) are consistent with a left-handed 9/11-helix.
In addition, it was observed that the amide protons of Ala
showed electrostatic interactions with the pyran oxygen
atoms of the preceding residues, as indicated by the charac-
teristic nOe correlations CεH(APyC)/NH(α) (Figure 6).
This also concerns NH(4), which does not take part in the
helical hydrogen-bonding system.
Figure 7. Stereoview of 20 superimposed minimum-energy struc-
tures of peptide 6 (hydrogens were removed after calculations for
clarity).
As observed for peptide 3, the smooth transition of han-
dedness in the hinge region between the two helical parts is
documented by the presence of a characteristic nOe be-
tween CβH(3)/NH(5), indicating the formation of a stabiliz-
ing 11-mr hydrogen bond between NH(5) and CO(2). This
finding is also supported by the absence of an nOe between
NH(4)/CαH(5). All data confirm the presence of right- and
left-handed 9/11-helices in peptide 6, as can also be seen in
the stereoview of a set of superimposed minimum-energy
structures in Figure 7, resulting from molecular dynamics.
The CD profile of peptide 6 is similar to that of peptide
3, reflecting the compensation of the positive and negative
profiles of the connected mixed helices (Figure 8).
Figure 8. CD profile of peptide 6.
tween the peptide bond NH groups and pyran ring oxygen
atoms of the preceding residue, was also confirmed by MO
calculations at the B3LYP/6-31G* level of ab initio MO
theory. The backbone torsion angles from these calculations
are given in the Supporting information.
Conclusions
The discussion on the structural features of the helical
In this paper, we report the synthesis of (R,R)-APyC, the
pattern of peptide 3 given above, in particular in the transi- enantiomer of the already known (S,S)-APyC. Both (R,R)-
tion range between the two 12/10-helices of opposite hand- and (S,S)-APyC were used together with β-hGly to synthe-
edness, can be completely transferred to peptide 6 with its size mixed β-peptides in a 1:1 alternating order to form
mixed 9/11-helices. The structure of peptide 6 with its char- right- and left-handed 12/10-helices. Coupling of the enan-
acteristic helical folds, and the electrostatic interactions be- tiomeric tetrapeptides 1 and ent-1 gave peptide 3 showing
Eur. J. Org. Chem. 2014, 4295–4308
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4301

G. V. M. Sharma, A. V. S. Sarma, H.-J. Hofmann et al.
FULL PAPER
(R,R)-trans-3-Aminopyran-2-carboxylic Acid [(R,R)-APyC] (ent-7):
Reaction of 8[20] with benzylamine in diethyl ether, followed by
treatment of the imine with vinylmagnesium bromide in THF at
0 °C to room temperature for 8 h, gave 9[21] in 71% yield
(Scheme 1). Treatment of amine 9 with (Boc)2O and Et3N in
CH2Cl2 at 0 °C to room temperature for 6 h gave 10 in 86% yield.
Acid (PTSA)-mediated hydrolysis of 10 in MeOH/H2O (9:1) at 0 °C
to room temperature for 6 h gave diol 11 in 93% yield. Selective
protection of 11 by reaction with TBSCl and imidazole in CH2Cl2
at 0 °C to room temperature gave TBS ether 12 (84%), which, on
both right- and left-handed 12/10 hydrogen-bonding pat-
terns. The extension of this concept to α/β-peptides com-
posed of (R,R)-APyC/l-Ala and (S,S)-APyC/d-Ala led to
enantiomeric 9/11-helical peptides. Peptide 6, with equal
portions of these oligomers, revealed the presence of the
left- and right-handed helices also in this foldamer class. In
both cases, the linear helical overall structure is essentially
maintained, despite the change of handedness and the inter-
ruption of the hydrogen bonding network in the transition
region between the helical peptide parts. These results dem- reaction with allyl bromide in the presence of NaH in DMF at 0 °C
to room temperature for 4 h, gave allyl ether 13 in 59% yield. Ring-
closing metathesis of bis-olefin 13 in the presence of Grubbs I cata-
lyst[22] (5 mol-%) gave 14 in 89% yield. Subjection of pyrene 14 to
Birch reaction conditions using Li and liquid NH3 at –78 °C in
THF for 1 h gave amide 15 (65%), which, upon hydrogenation with
Pd-C (10%) in MeOH under a hydrogen atmosphere, gave alcohol
16 in 91% yield. Sequential oxidation of alcohol 16 with TEMPO
and BAIB in aq. CH2Cl2 gave acid 17 (59%), which, upon treat-
ment with diazomethane, generated in situ, at 0 °C for 1 h, gave
ester ent-7 in 55% yield.
onstrate the design of peptides with more than one type of
handedness, where the screwing sense alternates, depending
on the order of the connected enantiomeric oligomers. This
may pave the way for new types of peptide oligomers.
Experimental Section
General Remarks: NMR spectra were recorded with Bruker
AVANCE III-700, Bruker AVANCE II-600, Bruker AVANCE III-
500, Varian INOVA-500, and Bruker AVANCE-300 spectrometers
at 278–303 K in chloroform, using tetramethylsilane as an internal
reference. Chemical shifts were measured from 1D NMR spectra,
and coupling constants were measured using resolution-enhanced
1D spectra. Chemical shift correlations were done using two-di-
mensional NMR experiments, such as Total Correlation Spec-
troscopy (TOCSY) and rotating-frame Overhauser effect spec-
troscopy (ROESY) in phase-sensitive mode with mixing times of
80 ms and 250–350 ms, respectively. The spectra were acquired
from 2ϫ 192 or 2ϫ 256 experiments containing 8–16 transients
with a relaxation delay of 1.5–2 s. The 2D spectra were processed
with a Gaussian apodization in both dimensions. Solvent titration
studies were done using sequential additions of [D6]DMSO (33%
v/v) (up to 300 μL) to solutions of peptides in chloroform (600 μL).
(R)-N-Benzyl-1-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl]prop-2-en-1-
amine (9): Benzylamine (14.9 mL, 137.0 mmol) was slowly added
to a stirred and cooled (0 °C) suspension of 8 (18.4 g, 138.4 mmol)
and dry MgSO4 (5 g) in diethyl ether (100 mL), and the reaction
mixture was stirred at room temperature for 2 h. A portion (50 mL)
of this solution of chiral imine in diethyl ether was added dropwise
over 30 min to a stirred solution of vinylmagnesium bromide [pre-
pared from Mg (17.0 g, 709.5 mmol) and vinyl bromide (51 mL,
709 mmol) in THF (50 mL)] at 0 °C under a nitrogen atmosphere.
The reaction mixture was stirred at room temperature for 8 h, then
it was quenched with aq. NH4Cl (70 mL) and extracted with
EtOAc (3ϫ 100 mL). The combined organic layers were washed
with water (2 ϫ 100 mL) and brine (2 ϫ 100 mL), and dried
(Na2SO4). The solvent was evaporated, and the residue was puri-
fied by column chromatography (60–120 mesh silica gel, 5% ethyl
acetate in petroleum ether) to give 9 (10 g, 71%) as a colorless
CD Spectra were acquired with a JASCO-810 spectrometer at room
temperature in methanol, using a 2 mm path length CD cell. Spec-
tra were recorded with an average of 2 scans (100 ms time constant,
2 nm bandwidth), background-corrected and smoothed over 2–5
data points. The scans were carried out from 250 to 193 nM, at
200 μm concentration. The molar ellipticities (θ) were normalized,
and the data are presented as degcm2 dmol–1 per residue. The pept-
ides with N-terminus B-residues showed higher molar ellipticities
of 70000 degcm2 dmol–1.
liquid. [α]D = –9.5 (c = 0.7, CHCl ). IR (KBr): ν = 3446, 3069,
˜
3
3027, 2984, 2930, 1665, 1455, 1375, 1255, 1214, 1155, 1061, 997,
1
924, 756, 701, 509 cm–1. H NMR (300 MHz, CDCl3, 293 K): δ =
7.26–7.15 (m, 5 H, ArH), 5.70–5.58 (m, 1 H, olefinic), 5.27–5.17
(m, 2 H, olefinic), 4.08 (m, 1 H, OCH), 3.97–3.84 (m, 3 H, PhCH2,
OCH), 3.62–3.58 (m, 1 H, OCH), 3.16 (qt, J = 4.53, 3.39 Hz, 1 H,
NCH), 1.5 (br. s, 1 H, NH), 1.37 (s, 3 H, CH3), 1.31 (s, 3 H, CH3)
ppm. 13C NMR (75 MHz, CDCl3, 293 K): δ = 25.0, 26.2, 50.6,
61.9, 66.0, 78.0, 109.0, 118.7, 126.8, 128.0 (2 C), 128.2 (2 C), 136.1,
140.2 ppm. HRMS (ESI): calcd. for C15H22NO2 [M + H]+
248.1650; found 248.1640.
Model building and molecular dynamics simulations on 1–6, ent-
1, and ent-2 were carried out using the Insight II (97.0)/Discover
program[18] with a Silicon Graphics O2 workstation. The CVFF
force field with default parameters was used throughout the simula-
tions. Minimizations were first done with steepest descent, followed
by conjugate gradient method for a maximum of 1000 iterations
each or until the RMS deviation reached 0.001 kcal/mol, whichever
was earlier. The molecules were initially equilibrated for 1 ps, and
then subjected to a simulated annealing protocol. Starting from
300 K, they were heated to 1500 K in four steps, increasing the
temperature by 300 K and simulating for 2.5 ps at each step, and
then subsequently cooled back to 300 K by decreasing the tempera-
ture in four steps, and again simulating for 2.5 ps. The structure
was saved and the above process was repeated 100 times. The 100
structures thus generated were energy minimized using the protocol
described above, and 20 of the best structures were superimposed.
For better visualization, the protons have been removed in the fig-
ures.
tert-Butyl Benzyl(R)-1-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl]allyl-
carbamate (10): Et3N (16.9 mL, 121.4 mmol) was added to a stirred
solution of 9 (10.1 g, 40.8 mmol) in CH2Cl2 (50 mL) at 0 °C, and
the mixture was stirred for 15 min. (Boc)2O (10.91 mL, 49.0 mmol)
was added, and the reaction mixture was stirred at room tempera-
ture for 6 h. It was washed with water (2ϫ 100 mL) and brine
(100 mL), and dried (Na2SO4). The solvent was evaporated, and
the residue was purified by column chromatography (60–120 mesh
silica gel, 2% ethyl acetate in petroleum ether) to give 10 (12.28 g,
86%) as a colorless liquid. [α]D = +3.60 (c = 1.0, CHCl3). IR (KBr):
ν = 3458, 2981, 2838, 1728, 1612, 1513, 1373, 1247, 1175, 1032,
˜
1
833, 771 cm–1. H NMR (300 MHz, CDCl3, 293 K): δ = 7.29–7.12
(m, 5 H, ArH), 5.92 (m, 1 H, olefinic), 5.21 (d, J = 9.8 Hz, 2 H,
olefinic), 4.58–4.02 (m, 4 H, 2 OCH, PhCH2), 3.78–3.36 (m, 2 H,
4302
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4295–4308

Design and Study of Peptides
NCH, OCH), 1.62–1.09 (m, 15 H, acetonide, Boc) ppm. 13C NMR
80.8, 80.0, 72.2, 63.0, 60.8, 50.7, 28.3 (3 C), 25.9 (3 C), 18.24, –5.49,
(75 MHz, CDCl3, 293 K): δ = 155.5, 139.3, 132.8, 128.3, 128.1, –5.41 ppm. HRMS (ESI): calcd. for C26H44NO4Si [M + H]+
127.1, 126.7, 126.9, 118.9, 109.5, 80.2, 76.4, 66.9, 60.54, 48.8, 28.2 462.3039; found 462.3029.
(3 C), 26.4, 25.2 ppm. HRMS (ESI): calcd. for C20H29NO4 [M +
Na]+ 370.1994; found 370.1993.
tert-Butyl Benzyl[(2R,3R)-2-(tert-butyldimethylsilyloxy)methyl]-
[3,6-dihydro-2H-pyran-3-yl]carbamate (14): A solution of 13 (7.5 g,
16.2 mmol) in freshly distilled degassed anhydrous toluene
(140 mL) was treated with Grubbs I catalyst (0.14 g, 5 mol-%), and
the mixture was heated at reflux for 8 h. Most of the solvent was
then distilled off, and the concentrated solution was left to stir at
room temperature for 8 h with air bubbling, in order to decompose
the catalyst. The mixture was evaporated to dryness, and the resi-
due was purified by column chromatography (60–120 mesh silica
gel, 2.5% ethyl acetate in petroleum ether) to give 14 (6.3 g, 89%)
as a pale brown liquid. [α]D = –56.7 (c = 0.25, CHCl3). IR (KBr):
tert-Butyl Benzyl[(2R,3R)-1,2-dihydroxypent-4-en-3-yl]carbamate
(11): PTSA (cat.) was added to a stirred solution of 10 (12.2 g,
35.15 mmol) in MeOH/H2O (60 mL, 9:1), and the mixture was
stirred at room temperature for 6 h. The reaction was quenched
with solid NaHCO3 (5 g), and the mixture was extracted with
EtOAc (2ϫ 200 mL). The organic phase was washed with water
(2ϫ 100 mL) and brine (100 mL), and dried (Na2SO4). The solvent
was evaporated, and the residue was purified by column
chromatography (60–120 mesh silica gel, 25% ethyl acetate in pe-
troleum ether) to give 11 (10.1 g, 93%) as a colorless liquid. [α]D
=
ν = 3449, 2929, 2857, 1694, 1452, 1400, 1365, 1251, 1163, 1116,
˜
+50.4 (c = 0.4, CHCl ). IR (KBr): ν = 3411, 2975, 2930, 1668,
˜
839, 773, 698 cm–1. 1H NMR (300 MHz, CDCl3): δ = 7.43–7.32
(m, 5 H, Ar-H), 6.05–5.52 (m, 2 H, olefinic), 4.93–4.15 (m, 5 H,
PhCH2, NCH, 2 OCH), 3.9–3.6 (m, 3 H, 3 OCH), 1.68–1.36 (m, 9
H, Boc), 1.04 (s, 9 H, tBu), 0.19 (s, 6 H, 2 CH3) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.8, 139.6, 128.3, 128.1, 127.4, 126.9,
126.6, 126.5, 126.1, 80.3, 80.1, 76.4, 64.6, 64.0, 51.1, 28.1 (3 C),
26.0 (3 C) 18.4, –5.3 (2 C) ppm. HRMS (ESI): calcd. for
C24H39NO4SiNa [M + Na]+ 456.2546; found 456.2556.
3
1456, 1407, 1364, 1249, 1165, 1075, 880, 700 cm–1 1H NMR
.
(300 MHz, CDCl3): δ = 7.29 (m, 5 H, ArH), 6.07–5.9 (m, 1 H,
olefinic), 5.25–5.14 (m, 2 H, olefinic), 4.48–4.28 (m, 2 H, PhCH2),
4.05–3.69 (m, 2 H, OCH2), 3.4 (br. s, 2 H, NH, OCH) 2.7 (br. s, 1
H, OH), 1.8 (br. s, 1 H, OH), 1.4 (s, 9 H, Boc) ppm. 13C NMR
(75 MHz, CDCl3): δ = 156.6, 138.3, 132.7, 128.3 (3 C), 127.2 (2 C),
119.8, 81.7, 72.5, 63.1, 61.6, 50.2, 28.2 (3 C) ppm. HRMS (ESI):
calcd. for C17H25NO4Na [M + Na]+ 330.1681; found 330.1687.
tert-Butyl [(2R,3R)-2-[(tert-Butyldimethylsilyloxy)methyl]-3,6-di-
hydro-2H-pyran-3-yl]carbamate (15): Lithium metal (0.43 g,
72.7 mmol) was added to a solution of liq. NH3 (50 mL) in dry
THF (20 mL) at –78 °C under a nitrogen atmosphere, and the mix-
ture was stirred for 1 h at the same temperature. A solution of 14
(6.25 g, 14.43 mmol) in THF (30 mL) was added slowly, and the
mixture was stirred for an additional 1 h. Aqueous NH4Cl (50 mL)
was then added dropwise, and the mixture was extracted with
EtOAc (2ϫ 50 mL). The organic layers were washed with brine
(25 mL), and dried (Na2SO4). The solvent was evaporated, and the
residue was purified by column chromatography (60–120 mesh sil-
ica gel, 16% ethyl acetate in petroleum ether) to give 15 (3.1 g,
65%) as a colorless liquid. [α]D = –13.57 (c = 0.14, CHCl3). IR
tert-Butyl Benzyl[(3R,4R)-5-(tert-butyldimethylsilyloxy)-4-
hydroxypent-1-en-3-yl]carbonate (12): Imidazole (6.6 g, 97.7 mmol)
was added to a stirred and cooled (0 °C) solution of 11 (10 g,
32.5 mmol) in CH2Cl2 (50 mL), and the mixture was stirred for
15 min. TBSCl (5.3 g, 35.8 mmol) was added portionwise, and the
mixture was stirred at room temperature for 4 h. The reaction mix-
ture was washed with water (2ϫ 100 mL) and brine (100 mL), and
dried (Na2SO4). The solvent was evaporated, and the residue was
purified by column chromatography (60–120 mesh silica gel, 5%
ethyl acetate in petroleum ether) to give 12 (11.1 g, 84%) as a color-
less liquid. [α]D = +40.7 (c = 0.4, CHCl ). IR (KBr): ν = 3423,
˜
3
2933, 2861, 1678, 1463, 1416, 1362, 1251, 1165, 1112, 841, 774,
1
700 cm–1. H NMR (300 MHz, CDCl3, 293 K): δ = 7.34–7.14 (m,
(KBr): ν = 3391, 2928, 1701, 1517, 1380, 1252, 1161, 1086, 816,
˜
5 H, ArH), 6.19–5.82 (m, 1 H, olefinic), 5.23–4.94 (m, 2 H, olefin),
4.8–3.19 (m, 2 H, NCH, PhCH), 4.17–3.25 (m, 5 H, 3 OCH, PhCH,
OH), 1.46 (s, 9 H, Boc), 0.9–0.84 (m, 9 H, tBu), 0.09–0.01 (s, 6 H,
2 CH3) ppm. 13C NMR (75 MHz, CDCl3, 293 K): δ = 156.7, 138.3,
131.5, 128.2 (3 C), 127.4, 127.1, 118.3, 80.7, 74.1, 63.4, 63.9, 52.9,
28.2 (3 C), 25.9 (3 C), 18.0, –5.5, –5.6 ppm. HRMS (ESI): calcd.
for C23H39NO4SiNa [M + Na]+ 444.2489; found 444.2498.
753, 703 cm–1. 1H NMR (300 MHz, CDCl3): δ = 6.0–5.77 (m, 2 H,
olefinic), 4.6 (br. s, 1 H, NH), 4.32–4.02 (m, 3 H, 2 OCH, NCH),
3.92–3.74 (m, 3 H, 3 OCH), 1.56 (s, 9 H, Boc), 1.03 (s, 9 H, tBu),
0.19 (s, 6 H, 2 CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 155.3,
128.1, 126.5, 79.2, 79.5, 64.6, 64.1, 45.8, 28.3 (3 C), 25.9 (3 C), 18.4,
–5.3, –5.2 ppm. HRMS (ESI): calcd. for C17H34NO4Si [M + H]+
344.2293; found 344.2289.
tert-Butyl [(3R,4R)-4-(Allyloxy)-5-(tert-butyldimethylsilyloxy)pent-
1-en-3-yl](benzyl)carbamate (13): NaH (1.97 g, 82.3 mmol) and
then allyl bromide (3.03 mL, 32.8 mmol) were added to a stirred
solution of alcohol 12 (11 g, 27.4 mmol) in dry DMF (55 mL) at
0 °C, and the mixture was stirred at room temperature for 4 h. The
reaction was quenched with aq. NH4Cl (30 mL) solution, and the
mixture was extracted with EtOAc (2ϫ 50 mL). The combined or-
ganic layers were washed with brine (50 mL) and dried (Na2SO4),
and the solvents were evaporated. The residue was purified by col-
umn chromatography (60–120 mesh silica gel, 2% ethyl acetate in
petroleum ether) to give 13 (7.5 g, 59%) as a colorless liquid. [α]D
tert-Butyl [(2R,3R)-Tetrahydro-2-(hydroxymethyl)-2H-pyran-3-yl]-
carbamate (16): Compound 15 (3.1 g, 9.0 mmol) was dissolved in
MeOH (15 mL), and Pd-C (10%; cat.) was added. The mixture was
stirred at room temperature for 6 h under a hydrogen atmosphere.
The reaction mixture was filtered through a Celite pad, eluting with
ethyl acetate (3ϫ 30 mL). The solvents were evaporated, and the
residue was purified by column chromatography (60–120 mesh sil-
ica gel, 25% ethyl acetate in petroleum ether) to give 16 (1.6 g,
91%) as a white solid, m.p. 75 °C. [α]D = +0.36 (c = 0.5, CHCl3).
IR (KBr): ν = 3328, 2936, 2861, 1684, 1531, 1455, 1367, 1311, 1249,
˜
= +36.0 (c = 1.2, CHCl ). IR (KBr): ν = 3430, 2931, 2858, 1693,
1171, 1081, 1039, 982, 856, 756 cm–1. 1H NMR (300 MHz, CDCl3):
˜
3
1456, 1407, 1365, 1252, 1165, 928, 837, 777, 701 cm–1. 1H NMR
δ = 4.43 (d, J = 8.4 Hz, 1 H, NH), 4.13–3.92 (m, 1 H, OCH), 3.60–
(300 MHz, CDCl3): δ = 7.26–6.56 (m, 5 H, ArH), 6.09–5.49 (m, 2 3.46 (m, 3 H, 3 OCH), 3.46–3.20 (m, 2 H, NCH, OCH), 2.08 (d, J
H, olefinic), 5.37–4.76 (m, 4 H, olefin), 4.67–4.98 (m, 2 H, PhCH2), = 9.0 Hz, 1 H, OH), 1.87–1.59 (m, 4 H, 2 CH2), 1.43 (s, 9 H, Boc)
4.22–3.23 (m, 6 H, 5 OCH, NCH), 1.55–1.11 (s, 9 H, Boc), 0.86 (s,
ppm. 13C NMR (75 MHz, CDCl3): δ = 156.4, 82.6, 80.3, 67.7, 62.2,
9 H, tBu), 0.06 (s, 6 H, 2 CH3) ppm. 13C NMR (75 MHz, CDCl3): 46.1, 30.2, 28.1 (3 C), 25.5 ppm. HRMS (ESI): calcd. for
δ = 155.2, 139.3, 135.1, 133.8, 128 (3 C), 127.2, 126.8, 118.3, 116.7,
C11H21NO4Na [M + Na]+ 254.1368; found 254.1362.
Eur. J. Org. Chem. 2014, 4295–4308
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4303

G. V. M. Sharma, A. V. S. Sarma, H.-J. Hofmann et al.
(2R,3R)-3-(tert-Butoxycarbonylamino)tetrahydro-2H-pyran-2- (m, 2 H, CδH-1, CδЈH-1), 1.37 (s, 9 H, Boc) ppm. 13C NMR
FULL PAPER
carboxylic Acid (17): Compound 16 (1.9 g, 12.3 mmol) was dis-
solved in CH2Cl2/H2O (3:1; 4 mL), and the solution was cooled to
0 °C. TEMPO (0.38 g, 2.46 mmol) and BAIB (7.9 g, 24.6 mmol)
were added, and the mixture was stirred for 4 h. The reaction was
quenched with aq. Na2S2O3 (10 mL), and the mixture was ex-
tracted with CH2Cl2 (2ϫ 100 mL). The combined organic layers
were washed with water (2ϫ 25 mL) and brine (25 mL), and dried
(Na2SO4). The solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography (60–120 mesh
silica gel, 70% ethyl acetate in petroleum ether) to give 17 (1.2 g,
(75 MHz, CDCl3): δ = 172.9, 170.0, 155.6, 79.6, 79.3, 67.7, 51.8,
50.4, 34.1, 33.5, 30.6, 28.2 (3 C), 24.6 ppm. HRMS (ESI): calcd.
for C15H26N2O6Na [M + Na]+ 353.1683; found 353.1681.
Boc-(S,S)-APyC-β-hGly-(S,S)-APyC-β-hGly-OMe (1): LiOH
(0.02 g, 1.13 mmol) was added to a solution of ester 19 (0.15 g,
0.45 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C, and the mixture
was stirred at room temperature for 2 h. Work-up as described for
7a gave 19a, which was used as such for further reaction.
A solution of acid 19a (0.14 g, 0.45 mmol), HOBt (0.07 g,
0.55 mmol), and EDCI (0.10 g, 0.55 mmol) in CH2Cl2 (2 mL) was
stirred at 0 °C under a nitrogen atmosphere for 15 min. Then salt
19b [prepared from 19 (0.15 g, 0.45 mmol) and CF3COOH
(0.1 mL) in dry CH2Cl2 (1 mL) at 0 °C] and DIPEA (0.11 mL,
0.68 mmol) were added sequentially, and the mixture was stirred
for 8 h. Work-up as described for 19, and purification of the residue
by column chromatography (60–120 mesh silica gel, 1.5% methanol
in CHCl3), gave 1 (0.09 g, 42%) as a white solid, m.p. 188–190 °C.
59%), m.p. 110–112 °C. [α]D = –3.0 (c = 0.5, CHCl ). IR (KBr): ν
˜
3
= 3374, 2931, 2860, 1700, 1522, 1368, 1309, 1250, 1168, 1089, 1043,
960, 756, 610 cm–1. 1H NMR (500 MHz, CDCl3): δ = 4.81 (s, 1 H,
NH), 3.95 (d, J = 11.4 Hz, 1 H, OCH), 3.76–3.69 (m, 2 H, OCH2),
3.43 (m, 1 H, NCH), 2.05 (m, 1 H, CH), 1.66 (m, 2 H, 2 CH), 1.47
(m, 1 H, CH), 1.36 (s, 9 H, Boc) ppm. 13C NMR (75 MHz, CDCl3):
δ = 172.5, 155.9, 78.9, 77.8, 66.8, 29.4, 28.2 (3 C), 23.8 (2 C) ppm.
HRMS (ESI): calcd. for C11H19NO5Na [M + Na]+ 268.1155; found
268.1153.
[α]D = +200 (c = 0.77, CHCl ). IR (KBr): ν = 3295, 3094, 2962,
˜
3
2854, 1743, 1743, 1687, 1661, 1556, 1442, 1369, 1311, 1248, 1172,
1089, 956, 684 cm–1. 1H NMR (600 MHz, CDCl3, 278 K): δ = 8.42
(dd, J = 4.8, 7.9 Hz, 1 H, NH-2), 7.70 (d, J = 9.2 Hz, 1 H, NH-3),
6.95 (t, J = 6.2 Hz, 1 H, NH-4), 4.79 (d, J = 9.9 Hz, 1 H, NH-1),
4.04 (m, 1 H, CεH-3), 4.02 (m, 1 H, CεH-1), 3.96 (m, 1 H, CβH-
3), 3.87 (dddd, J = 3.1, 4.7, 7.7, 12.6 Hz, 1 H, CβH-2), 3.75 (m, 1
H, CβH-1), 3.72 (s, 3 H, COOMe), 3.63 (dddd, J = 5.1, 6.2, 6.6,
13.3 Hz, 1 H, CβH-4), 3.48 (d, J = 9.5 Hz, 1 H, CαH-3), 3.46 (d,
J = 9.8 Hz, 1 H, CαH-1), 3.43 (m, 1 H, CβЈH-4), 3.43 (m, 1 H,
CεЈH-3), 3.35 (dt, J = 2.1, 11.9 Hz, 1 H, CεЈH-1), 3.08 (ddt, J =
1.9, 4.7, 12.6 Hz, 1 H, CβЈH-2), 2.65 (ddd, J = 5.2, 7.7, 17.4 Hz, 1
H, CαЈH-4), 2.55 (ddd, J = 5.1, 6.2, 17.4 Hz, 1 H, CαЈH-4), 2.25
(dt, J = 3.1, 12.4 Hz, 1 H, CαH-2), 2.13 (ddd, J = 1.9, 4.7, 12.4 Hz,
1 H, CαЈH-2), 2.10 (m, 1 H, CγH-1), 2.00 (m, 1 H, CγH-3), 1.77
(m, 1 H, CδH-3), 1.73 (m, 1 H, CδH-1), 1.69 (m, 1 H, CδЈH-3),
1.69 (m, 1 H, CδЈH-1), 1.59 (dq, J = 3.7, 12.4 Hz, 1 H, CγЈH-3),
1.43 (s, 9 H, Boc), 1.39 (dq, J = 3.9, 12.5 Hz, 1 H, CγЈH-1) ppm.
13C NMR (150 MHz, CDCl3): δ = 173.3, 172.1, 169.9, 169.6, 155.7,
83.9, 81.4, 80.1, 67.4, 67.3, 51.8, 49.7, 47.8, 37.4, 36.7, 34.2, 33.3,
31.3, 29.7, 28.3 (3 C), 25.5, 25.1 ppm. HRMS (ESI): calcd. for
C24H40N4O9Na [M + Na]+ 551.2687; found 551.2682.
tert-Butyl [(2R,3R)-2-(Methoxycarbonyl)-tetrahydro-2H-pyran-3-yl-
]carbamate (ent-7): A solution of 17 (1.2 g, 48.9 mmol) in diethyl
ether (15 mL) at 0 °C was treated with ethereal diazomethane [pre-
pared from N-nitrosomethyl urea (3.8 g) and KOH (50%; 40 mL)],
and the mixture was stirred at 0 °C for 1 h. The solvent was evapo-
rated, and the residue was purified by column chromatography (60–
120 mesh silica gel, 20% ethyl acetate in petroleum ether) to give
ent-7 (0.7 g, 55%) as a white solid, m.p. 96–98 °C. [α]D = –25.1 (c
= 0.5, CHCl ). IR (KBr): ν = 3373, 2981, 2940, 1752, 1681, 1519,
˜
3
1440, 1369, 1246, 1171, 1115, 1088, 1038, 1012, 949, 865, 748,
702 cm–1. 1H NMR (600 MHz, CDCl3, 293 K): δ = 4.60 (br. s, 1
H, NH), 4.01 (dt, J = 11.7, 4.5 Hz, 1 H, CεH), 3.79–3.75 (m, 5 H,
CβH, CαH, COOMe), 3.46 (m, 1 H, CεЈH), 2.1–1.98 (m, 1 H,
CγH), 1.77–1.66 (m, 2 H, CδH, CδЈH), 1.55–1.35 (m, 10 H, CγЈH,
Boc) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.0, 154.9, 80.1 (2
C), 66.8, 52.5, 48.2, 29.3, 28.3 (3 C), 23.9 ppm. HRMS (ESI): calcd.
for C12H21NO5Na [M + Na]+ 282.1317; found 282.1325.
Boc-(S,S)-APyC-β-hGly-OMe (19): LiOH (0.10 g, 4.4 mmol) was
added to a solution of 7 (0.46 g, 1.77 mmol) in THF/MeOH/H2O
(3:1:1), at 0 °C, and the mixture was stirred for 2 h. The reaction
was quenched with HCl (1 n; 3 mL) at 0 °C, and the mixture was
extracted with EtOAc (2ϫ 50 mL). The organic layers were dried
(Na2SO4) and evaporated to give acid 7a as a semi-solid, which was
used as such for further reaction.
(S,S)-APyC-β-hGly-(S,S)-APyC-β-hGly-(S,S)-APyC-β-hGly-OMe
(2): LiOH (0.01 g, 0.46 mmol) was added to a solution of ester 1
(0.11 g, 0.18 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C, and the
mixture was stirred at room temperature for 2 h. Work-up as de-
scribed for 7a gave 1a, which was used as such for further reaction.
A solution of acid 7a (0.4 g, 1.63 mmol), HOBt (0.26 g,
1.95 mmol), and EDCI (0.37 g, 1.95 mmol) in CH2Cl2 (25 mL) was A solution of acid 1a (0.09 g, 0.15 mmol), HOBt (0.02 g,
stirred at 0 °C for 15 min under a nitrogen atmosphere, and then 0.18 mmol), and EDCI (0.03 g, 0.18 mmol) in CH2Cl2 (10 mL) was
salt 18 (0.27 g, 1.95 mmol) was added. The mixture was stirred at stirred at 0 °C for 15 min. Then 19b [prepared from 19 (0.05 g,
room temperature for 8 h. The reaction mixture was quenched with
aq. NH4Cl (10 mL) at 0 °C, and diluted with CHCl3 (10 mL). The
mixture was washed with HCl (1 n; 10 mL), water (10 mL), and
brine (10 mL). The organic layers were dried (Na2SO4), the solvents
0.154 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (2 mL)] and
DIPEA (0.04 mL, 0.23 mmol) were added, and the mixture was
stirred under a nitrogen atmosphere for 8 h. Work-up as described
for 19, and purification of the residue by column chromatography
were evaporated, and the residue was purified by column (60–120 mesh silica gel, 3.0% methanol in CHCl3), gave 2 (0.04 g,
chromatography (60–120 mesh silica gel, 50% ethyl acetate in pe-
38%) as a white solid, m.p. 212–216 °C. [α]D = +370 (c = 0.12,
CHCl ). IR (KBr): ν = 3298, 3092, 2940, 2855, 1732, 1887, 1653,
troleum ether) to give 19 (0.70 g, 89%) as a white solid, m.p. 104–
˜
3
106 °C. [α]D = +160 (c = 0.16, CHCl ). IR (KBr): ν = 3340, 3311, 1553, 1443, 1365, 1310, 1177, 1090, 1054, 651 cm–1
.
1H NMR
(600 MHz, CDCl3, 288 K): δ = 8.67 (dd, J = 4.5, 8.4 Hz, 1 H, NH-
1H NMR (500 MHz, 4), 8.62 (dd, J = 4.8,7.9 Hz, 1 H, NH-2), 8.28 (d, J = 9.3 Hz, 1 H,
˜
3
3093, 2954, 2855, 1733, 1680, 1656, 1558, 1523, 1446, 1388, 1313,
1250, 1173, 1091, 1030, 958, 620 cm–1
.
CDCl3, 288 K): δ = 6.91 (s, 1 H, NH-2), 5.30 (s, 1 H, NH-1), 3.96
(d, J = 10.38 Hz, 1 H, CαH-1), 3.69 (s, 3 H, COOCH3), 3.56–3.46
NH-5), 8.12 (d, J = 9.6 Hz, 1 H, NH-3), 6.97 (t, J = 6.3 Hz, 1 H,
NH-6), 4.78 (d, J = 10.3 Hz, 1 H, NH-1), 4.07 (m, 1 H, CεH-1),
(m, 5 H, CβH-1, CεH-1, CεЈH-1, CβH-2, CβЈH-2), 2.54 (m, 2 H, 4.05 (m, 2 H, CεH-3, CεH-5), 4.04 (m, 1 H, CβH-3), 3.98 (m, 1
CαH-2, CαЈH-2), 2.42–2.32 (m, 2 H, CγH-1, CγЈH-1), 1.73–1.59 H, CβH-5), 3.93 (m, 1 H, CβH-4), 3.89 (m, 1 H, CβH-2), 3.78 (m,
4304
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4295–4308

Design and Study of Peptides
1 H, CβH-1), 3.72 (s, 3 H, COOMe), 3.67 (m, 1 H, CβH-6), 3.50 H, CβH-1), 3.71 (s, 3 H, COOMe) 3.62 (dddd, J = 5.1, 6.2, 6.6,
(d, J = 9.9 Hz, 1 H, CαH-3), 3.47 (d, J = 9.9 Hz, 1 H, CαH-1),
3.44 (d, J = 9.7 Hz, 1 H, CαH-5), 3.43 (m, 1 H, CβЈH-6), 3.40 (m,
13.3 Hz, 1 H, CβH-4), 3.48 (d, J = 9.4 Hz, 1 H, CαH-3), 3.46 (d,
J = 9.7 Hz, 1 H, CαH-1), 3.43 (m, 2 H, CεЈH-3, CβЈH-4), 3.35 (dt,
1 H, CεЈH-3), 3.37 (m, 1 H, CεЈH-5), 3.36 (m, 1 H, CεЈH-1), 3.04 J = 2.1, 11.9 Hz, 1 H, CεЈH-1), 3.08 (ddt, J = 1.9, 4.7, 12.6 Hz, 1
(m, 1 H, CβЈH-4), 2.99 (m, 1 H, βЈH-2), 2.70 (ddd, J = 4.8, 8.1, H, CβЈH-2), 2.65 (ddd, J = 5.2, 7.7, 17.4 Hz, 1 H, CαH-4), 2.56
17.3 Hz, 1 H, CαH-6), 2.57 (ddd, J = 4.8, 6.3, 17.4 Hz, 1 H, CαЈH- (ddd, J = 5.1, 6.2, 17.4 Hz, 1 H, CαЈH-4), 2.25 (dt, J = 3.1, 12.4 Hz,
6), 2.33 (m, 2 H, CαH-2, CαH-4), 2.15 (m, 1 H, CαЈH-2), 2.12 (m,
1 H, CαH-2), 2.13 (ddd, J = 1.9, 4.7, 12.4 Hz, 1 H, CαЈH-2), 2.10
1 H, CγH-1), 2.08 (m, 1 H, CαЈH-4), 2.06 (m, 1 H, CγH-5), 2.04 (m, 1 H, CγH-1), 2.0 (m, 1 H, CγH-3), 1.77 (m, 1 H, CδH-3), 1.73
(m, 1 H, CγH-3), 1.81 (m, 1 H, CδH-1), 1.77 (m, 1 H, CδH-3), (m, 1 H, CδH-1), 1.70 (m, 1 H, CδЈH-3), 1.68 (m, 1 H, CδЈH-1),
1.73 (m, 2 H, CδH-5, CγЈH-5), 1.70 (m, 1 H, CδЈH-1), 1.68 (m, 2
1.59 (dq, J = 3.7, 12.4 Hz, 1 H, CγЈH-3), 1.42 (s, 9 H, Boc), 1.39
H, CδЈH-5, CδЈH-3), 1.54 (m, 1 H, CγЈH-3), 1.43 (m, 9 H, Boc), (dq, J = 4.1, 12.5 Hz, 1 H, CγЈH-1) ppm. 13C NMR (150 MHz,
1.42 (m, 1 H, CγЈH-1) ppm. 13C NMR (150 MHz, CDCl3): δ =
173.5, 173.0, 172.5, 170.1, 170.0, 169.7, 155.9, 83.5, 83.2, 82.0, 80.3,
67.6, 67.4, 67.3, 51.9, 50.0, 48.5, 47.8, 37.5, 37.0, 36.8, 34.3, 33.4,
31.4, 29.9, 29.6, 28.4, 25.7, 25.5, 25.2 ppm. HRMS (ESI): calcd. for
C33H54N6O12Na [M + Na]+ 749.3691; found 749.3698.
CDCl3): δ = 173.1, 172.0, 169.9, 169.6, 155.7, 83.2, 81.4, 80.0, 67.4,
67.3, 51.7, 49.9, 47.9, 37.4, 36.7, 34.2, 33.4, 31.3, 29.8, 28.7 (3 C),
25.5, 25.1 ppm. HRMS (ESI): calcd. for C24H40N4O9Na [M +
Na]+ 551.2692; found 551.2685.
(R,R)-APyC-β-hGly-(R,R)-APyC-β-hGly-(R,R)-APyC-β-hGly-
OMe (ent-2): LiOH (0.01 g, 0.45 mmol) was added to a solution of
ester ent-1 (0.1 g, 0.18 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C,
and the mixture was stirred at room temperature for 2 h. Work-up
as described for 7a gave ent-1a, which was used as such for further
reaction.
Boc-(R,R)-APyC-β-hGly-OMe (ent-19): LiOH (0.10 g, 4.3 mmol)
was added to a solution of ent-7 (0.45 g, 1.7 mmol) in THF/MeOH/
H2O (3:1:1) at 0 °C, and the mixture was stirred at room tempera-
ture for 2 h. Work-up as described for 7a gave 17, which was used
as such for further reaction.
A solution of acid 17 (0.4 g, 1.7 mmol), HOBt (0.31 g, 2.08 mmol),
and EDCI (0.39 g, 2.08 mmol) in CH2Cl2 (25 mL) was stirred at
0 °C for 15 min, and then salt 18 (0.39 g, 2.93 mmol) and DIPEA
(0.45 mL, 2.6 mmol) were added under a nitrogen atmosphere. The
mixture was stirred at room temperature for 8 h. Work-up as de-
scribed for 19, and purification of the residue by column
chromatography (60–120 mesh silica gel, 50% ethyl acetate in pe-
troleum ether), gave ent-19 (0.38 g, 66%) as a white solid, m.p. 103–
A solution of acid ent-1a (0.09 g, 0.18 mmol), HOBt (0.03 g,
0.22 mmol), and EDCI (0.04 g, 0.22 mmol) in CH2Cl2 (10 mL) was
stirred at 0 °C for 15 min. Then ent-19b [prepared from ent-19
(0.06 g, 0.18 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (2 mL)]
and DIPEA (0.04 mL, 0.27 mmol) were added, and the mixture
was stirred under a nitrogen atmosphere for 8 h. Work-up as de-
scribed for 19, and purification of the residue by column
chromatography (60–120 mesh silica gel, 3.0 % methanol in
CHCl3), gave ent-2 (0.06 g, 46%) as a white solid, m.p. 212–216 °C.
105 °C. [α]D = –190 (c = 0.03, CHCl ). IR (KBr): ν = 3385, 3108,
˜
3
2926, 2855, 1739, 1700, 1656, 1570, 1522, 1448, 1374, 1309, 1252,
1169, 1120, 1088, 1029, 957, 900, 870, 689, 614 cm–1. 1H NMR
(500 MHz, CDCl3, 288 K): δ = 6.90 (s, 1 H, NH-2), 5.32 (s, 1 H,
NH-1), 3.91 (d, J = 10.8 Hz, 1 H, CαH-1), 3.65 (s, 3 H, COOCH3),
3.51–3.33 (m, 5 H, CβH-1, CεH-1, CεЈH-1, CβH-2, CβЈH-2), 2.50
(m, 2 H, CαH-2, CαЈH-2), 2.3 (d, J = 10 Hz, 2 H, CγH-1, CγЈH-
1), 1.72–1.52 (m, 2 H, CδH-1, CδЈH-1), 1.37 (s, 9 H, Boc) ppm.
13C NMR (75 MHz, CDCl3): δ = 172.8, 169.8, 155.5, 79.6, 79.2,
67.6, 51.7, 50.3, 34.1, 33.4, 30.5, 28.2 (3 C), 24.5 ppm. HRMS
(ESI): calcd. for C15H26N2O6Na [M + Na]+ 353.1688; found
353.1687.
[α]D = –380.6 (c = 0.31, CHCl ). IR (KBr): ν = 3295, 3091, 2927,
˜
3
2853, 1741, 1688, 1660, 1551, 1442, 1369, 1310, 1245, 1173, 1090,
1
1060, 1034, 956, 872, 653, 583 cm–1. H NMR (600 MHz, CDCl3,
288 K): δ = 8.64 (dd, J = 8.3, 4.4 Hz, 1 H, NH-4), 8.60 (dd, J =
4.7, 7.8 Hz, 1 H, NH-2), 8.26 (d, J = 9.3 Hz, 1 H, NH-5), 8.09 (d,
J = 9.7 Hz, 1 H, NH-3), 6.96 (t, J = 6.3 Hz, 1 H, NH-6), 4.77 (d,
J = 10.4 Hz, 1 H, NH-1), 4.06 (m, J = 9.4 Hz, 1 H, CεH-1), 4.04
(m, 3 H, CεH-3, CεH-5, CβH-3), 3.99 (m, 1 H, CβH-5), 3.93 (m,
1 H, CβH-4), 3.90 (m, 1 H, CβH-2), 3.76 (m, 1 H, CβH-1), 3.72
(s, 3 H, COOMe), 3.66 (ddt, J = 13.7, 4.9, 6.3 Hz, 1 H, CβH-6),
3.50 (d, J = 9.7 Hz, 1 H, CαH-3), 3.47 (d, J = 9.9 Hz, 1 H, CαH-
1), 3.44 (d, J = 9.6 Hz, 1 H, CαH-5), 3.42 (m, 1 H, CβЈH-6), 3.41
(m, 1 H, CεЈH-3), 3.37 (m, 1 H, CεЈH-5), 3.35 (m, 1 H, CεЈH-1),
3.04 (ddt, J = 1.8, 4.6, 13.2 Hz, 1 H, CβЈH-4), 3.0 (ddt, J = 1.8,
4.7, 12.8 Hz, 1 H, βH-2), 2.69 (ddd, J = 4.9, 8.1, 17.3 Hz, 1 H,
CαH-6), 2.57 (ddd, J = 4.7,6.3, 17.3 Hz, 1 H, CαЈH-6), 2.33 (dt, J
= 6.1, 12.1 Hz, 1 H, CαH-2), 2.33 (dt, J = 6.5, 12.2 Hz, 1 H, CαH-
4), 2.14 (m, 1 H, CαЈH-2), 2.11 (m, 1 H, CγH-1), 2.07 (m, 1 H,
CαЈH-4), 2.06 (m, 1 H, CγH-5), 2.03 (m, 1 H, CγH-3), 1.81 (m, 1
H, CδH-1), 1.77 (m, 1 H, CδH-5), 1.74 (m, 2 H, CδH-3, CδЈH-3),
1.70 (m, 1 H, CδЈH-1), 1.68 (m, 1 H, CδЈH-5), 1.67 (m, 1 H, CγЈH-
3), 1.54 (dq, 1 H, CγЈH-5), 1.42 (s, 9 H, Boc), 1.41 (dq, J = 3.7,
12.8 Hz, 1 H, CγЈH-1) ppm. 13C NMR (150 MHz, CDCl3): δ =
173.2, 172.7, 171.3, 169.9, 169.5, 169.4, 155.8, 82.7, 81.7, 80.0, 79.6,
67.6, 67.4 (2 C), 52.4, 50.0, 48.7, 48.4, 47.7, 47.2, 46.6, 30.6, 29.5,
29.3, 28.1 (3 C), 25.4, 25.2, 25.0, 18.1, 17.1, 17.0 ppm. HRMS
(ESI): calcd. for C33H54N6O12Na [M + Na]+ 749.3687; found
749.3687.
Boc-(R,R)-APyC-β-hGly-(R,R)-APyC-β-hGly-OMe (ent-1): LiOH
(0.02 g, 0.98 mmol) was added to a solution of ester ent-19 (0.13 g,
0.39 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C, and the mixture
was stirred at room temperature for 2 h. Work-up as described for
7a gave ent-19a, which was used as such for further reaction.
A solution of acid ent-19a (0.12 g, 0.39 mmol), HOBt (0.07 g,
0.47 mmol), and EDCI (0.09 g, 0.47 mmol) in CH2Cl2 (2 mL) was
stirred at 0 °C under a nitrogen atmosphere for 15 min. Salt ent-
19b [prepared from ent-19 (0.12 g, 0.39 mmol) and CF3COOH
(0.1 mL) in dry CH2Cl2 (1 mL) at 0 °C] and DIPEA (0.1 mL,
0.5 mmol) were then added sequentially, and the mixture was
stirred for 8 h. Work-up as described for 19, and purification of the
residue by column chromatography (60–120 mesh silica gel, 1.5%
methanol in CHCl3), gave ent-1 (0.1 g, 46%) as a white solid, m.p.
188–190 °C. [α]D = –228.1 (c = 0.235, CHCl ). IR (KBr): ν = 3290,
˜
3
3093, 2927, 2855, 1742, 1662, 1556, 1446, 1369, 1310, 1246, 1172,
1090, 957, 683 cm–1. 1H NMR (600 MHz, CDCl3, 278 K): δ = 8.40
(dd, J = 4.7, 7.7 Hz, 1 H, NH-2), 7.68 (d, J = 9.2 Hz, 1 H, NH-3),
(S,S)-APyC-β-hGly-(S,S)-APyC-β-hGly-(R,R)-APyC-β-hGly-
6.94 (t, J = 6.3 Hz, 1 H, NH-4), 4.80 (d, J = 10.1 Hz, 1 H, NH-1), (R,R)-APyC-β-hGly-OMe (3): LiOH (0.01 g, 0.47 mmol) was
4.03 (m, 1 H, CεH-3), 4.02 (m, 1 H, CεH-1), 3.96 (m, 1 H, CβH-
3), 3.86 (dddd, J = 3.1, 4.7, 7.7, 12.6 Hz, 1 H, CβH-2), 3.75 (m, 1
added to a solution of ester 1 (0.1 g, 0.18 mmol) in THF/MeOH/
H2O (3:1:1) at 0 °C, and the mixture was stirred at room tempera-
Eur. J. Org. Chem. 2014, 4295–4308
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4305

G. V. M. Sharma, A. V. S. Sarma, H.-J. Hofmann et al.
FULL PAPER
ture for 2 h. Work-up as described for 7a gave 1a, which was used
as such for further reaction.
H, CβH-1), 3.56–3.44 (m, 2 H, CεH-1, CεЈH-1), 2.40 (dd, J = 1.8,
9.82 Hz, 1 H, CγH-1), 1.61 (m, 3 H, CγЈH-1, CδH-1, CδЈH-1),
1.47–1.37 (m, 12 H, Boc, CH3-2) ppm. 13C NMR (75 MHz,
CDCl3): δ = 172.9, 169.4, 155.5, 79.2, 78.9, 67.5, 52.4, 50.0, 47.5,
30.1, 28.3 (3 C), 24.1, 18.3 ppm. HRMS (ESI): calcd. for
C15H26N2O6Na [M + Na]+ 353.1688; found 353.1680.
A solution of acid 1a (0.07 g, 0.13 mmol), HOBt (0.025 g,
0.163 mmol), and EDCI (0.03 g, 0.16 mmol) in CH2Cl2 (15 mL)
was stirred at 0 °C for 15 min. Salt ent-1b [prepared from ent-1
(0.08 g, 0.16 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (2 mL)]
and DIPEA (0.34 mL, 0.20 mmol) were added, and the mixture
was stirred under a nitrogen atmosphere for 8 h. Work-up as de-
scribed for 19, and purification of the residue by column
chromatography (60–120 mesh silica gel, 4.5 % methanol in
CHCl3), gave 3 (0.49 g, 75%) as a white solid, m.p. 248–250 °C.
Boc-(R,R)-APyC-L-Ala-(R,R)-APyC-L-Ala-OMe (4): LiOH
(0.02 g, 1.13 mmol) was added to a solution of ester 21 (0.15 g,
0.45 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C, and the mixture
was stirred at room temperature for 2 h. Work-up as described for
7a gave 21a, which was used as such for further reaction.
[α]D = +4.5 (c = 0.22, CHCl ). IR (KBr): ν = 3305, 2926, 2855,
˜
3
A solution of acid 21a (0.06 g, 0.31 mmol), HOBt (0.07 g,
0.55 mmol), and EDCI (0.10 g, 0.55 mmol) in CH2Cl2 (2 mL) was
stirred at 0 °C under a nitrogen atmosphere for 15 min. Then salt
21b [prepared from 21 (0.15 g, 0.45 mmol) and CF3COOH
(0.1 mL) in dry CH2Cl2 (1 mL) at 0 °C] and DIPEA (0.11 mL,
0.68 mmol) were added sequentially, and the mixture was stirred
for 8 h. Work-up as described for 19, and purification of the residue
by column chromatography (60–120 mesh silica gel, 1.5% methanol
in CHCl3), gave 4 (0.13 g, 47%) as a white solid, m.p. 225–228 °C.
1659, 1540, 1446, 1374, 1304, 1249, 1175, 1089, 760, 577 cm–1. H
1
NMR (600 MHz, CDCl3, 278 K): δ = 8.55 (dd, J = 4.2, 8.1 Hz, 1
H, NH-6), 8.38 (dd, J = 4.7, 7.4 Hz, 1 H, NH-2), 7.99 (d, J =
9.4 Hz, 1 H, NH-3), 7.88 (d, J = 9.2 Hz, 1 H, NH-7), 7.81 (d, J =
19.8 Hz, 1 H, NH-5), 6.98 (t, J = 6.1 Hz, 1 H, NH-8), 6.89 (t, J =
6.3 Hz, 1 H, NH-4), 4.83 (d, J = 10.2 Hz, 1 H, NH-1), 4.09 (m, 1
H, CβH -5), 4.06 (m, 1 H, CεH-7), 4.04 (m, 1 H, CεH-1), 4.03 (m,
1 H, CεH-3), 4.01 (m, 1 H, CεH-5), 4.00 (m, 1 H, CβH-7), 3.88
(m, 2 H, CβH-2, CβH-3), 3.87 (m, 1 H, CβH-6), 3.78 (m, 1 H,
CβH-4), 3.72 (s, 3 H, COOMe), 3.71 (m, 1 H, CβH-1), 3.64 (m, 1
H, CβH-8), 3.59 (d, J = 9.8 Hz, 1 H, CαH-5), 3.51 (d, J = 9.8 Hz,
1 H, CαH-7), 3.48 (m, 1 H, CεЈH-1), 3.48 (d, J = 9.8 Hz, 1 H,
CαH-1), 3.48 (d, J = 9.8 Hz, 1 H, CαH-3), 3.44 (m, 2 H, CεЈH-3,
CεЈH-7), 3.43 (m, 1 H, CβЈH-8), 3.38 (m, 1 H, CεЈH-5), 3.24 (m,
1 H, CβЈH-4), 3.07 (m, 2 H, CβЈH-2, CβЈH-6), 2.67 (ddd, J = 4.9,
7.9, 17.4 Hz, 1 H, CαH-8), 2.56 (ddd, J = 4.9, 6.2, 17.4 Hz, 1 H,
CαЈH-8), 2.48 (ddd, J = 3.7, 10.9, 13.9 Hz, 1 H, CαH-4), 2.31 (dt,
J = 3.2, 12.4 Hz, 1 H, CαH-2), 2.25 (ddd, J = 3.2, 5.0, 13.9 Hz, 1
H, CαЈH-4), 2.21 (dt, J = 3.3, 12.3 Hz, 1 H, CαH-6), 2.18 (m, 1 H,
CαЈH-2), 2.12 (ddd, J = 2.2, 4.5, 12.3 Hz, 1 H, CαЈH-6), 2.10 (m,
1 H, CγH-1), 2.07 (m, 1 H, CγH-7), 2.03 (m, 2 H, CγH-3, CγH-
5), 1.79 (m, 1 H, CδH-7), 1.76 (m, 1 H, CδH-3), 1.75 (m, 1 H,
CδH-1), 1.74 (m, 1 H, CδH-5), 1.71 (m, 1 H, CδЈH-1), 1.70 (m, 1
H, CδЈH-7), 1.69 (m, 1 H, δЈH-3), 1.68 (m, 1 H, CγЈH-3), 1.67 (m,
1 H, CδЈH-5), 1.65 (m, 1 H, CγЈH-5), 1.64 (m, 1 H, CγЈH-7), 1.43
(s, 9 H, Boc), 1.41 (m, 1 H, CγЈH-1) ppm. 13C NMR (150 MHz,
CDCl3, 278 K): δ = 173.6, 172.5, 172.4, 172.1, 170.2, 170.1, 169.8,
169.7, 156.0, 82.9, 82.4, 81.9, 81.5, 80.1, 67.5 (2 C), 67.4, 67.2, 51.7,
50.0, 48.6, 48.5, 48.0, 37.2, 36.7, 36.6, 36.2, 36.0, 34.4, 33.5, 31.9,
29.7 (2 C), 29.6, 29.6, 28.4 (3 C), 25.5, 25.4, 25.2, 24.9 ppm. HRMS
(ESI): calcd. for C42H68N8O15Na [M + Na]+ 947.4701; found
947.4683.
[α]D = –256.1 (c = 0.12, CHCl ). IR (KBr): ν = 3300, 3086, 2931,
˜
3
2854, 1734, 1689, 1664, 1543, 1453, 1368, 1312, 1250, 1165, 1093,
1054, 964, 662 cm–1. 1H NMR (600 MHz, CDCl3, 278 K): δ = 7.50
(d, J = 8.3 Hz, 1 H, NH-3), 7.15 (d, J = 9.1 Hz, 1 H, NH-2), 6.98
(d, J = 7.2 Hz, 1 H, NH-4), 4.76 (d, J = 9.2 Hz, 1 H, NH-1), 4.60
(dq, J = 9.1, 7.0 Hz, 1 H, CαH-2), 4.51 (q, J = 7.2 Hz, 1 H, CαH-
4), 4.03 (m, 1 H, CεH-3), 4.01 (m, 1 H, CεH-1), 3.94 (m, 1 H,
CβH-3), 3.83 (m, 1 H, CβH-1), 3.78 (d, J = 10 Hz, 1 H, CαH-3),
3.76 (s, 3 H, COOCH3), 3.47 (d, J = 9.6 Hz, 1 H, CαH-1), 3.43 (dt,
J = 2.4, 11.9 Hz, 1 H, CεЈH-3), 3.37 (dt, J = 2.3, 12.0 Hz, 1 H,
CεЈH-1), 2.14 (m, 1 H, CγH-3), 2.11 (m, 1 H, CγH-1), 1.82 (m, 1
H, CδH-1), 1.79 (m, 1 H, CδH-3), 1.67 (m, 1 H, CδЈH-1), 1.66 (m,
1 H, CδЈH-3), 1.62 (dq, J = 3.9, 12.6 Hz, 1 H, CγЈH-3), 1.42 (s, 9 H,
Boc), 1.42 (d, J = 7.2 Hz, 3 H, CH3-4), 1.39 (dq, J = 3.8, 12.5 Hz, 1
H, CγЈH-1), 1.30 (d, J = 7.0 Hz, 3 H, CH3-2) ppm. 13C NMR
(75 MHz, CDCl3): δ = 173.1, 171.5, 169.2, 169.4, 155.7, 82.3, 79.8,
79.9, 67.4, 67.7, 52.4, 49.7, 47.0, 47.7, 48.5, 30.7, 28.22 (3 C), 30.0,
31.5, 25.0, 16.9, 18.0 ppm. HRMS (ESI): calcd. for C24H40N4O9Na
[M + Na]+ 551.2692; found 551.2685.
Boc-(R,R)-APyC-L-Ala-(R,R)-APyC-L-Ala-(R,R)-APyC-L-Ala-
OMe (5): LiOH (0.01 g, 0.46 mmol) was added to a solution of
ester 4 (0.12 g, 0.18 mmol) in THF/MeOH/H2O (3:1:1) at 0 °C, and
the mixture was stirred at room temperature for 2 h. Work-up as
described for 7a gave 4a, which was used as such for further reac-
tion.
Boc-(R,R)-APyC-L-Ala-OMe (21): LiOH (0.106 g, 4.4 mmol) was
added to a solution of ent-7 (0.46 g, 1.77 mmol) in THF/MeOH/
H2O (3:1:1) at 0 °C, and the mixture was stirred at room tempera-
ture for 2 h. Work-up as described for 7a gave 17, which was used
as such for the next reaction.
A solution of acid 4a (0.09 g, 0.15 mmol), HOBt (0.03 g,
0.18 mmol), and EDCI (0.03 g, 0.18 mmol) in CH2Cl2 (2 mL) was
stirred at 0 °C under a nitrogen atmosphere for 15 min. Salt 21b
[prepared from 21 (0.05 g, 0.15 mmol) and CF3COOH (0.1 mL) in
dry CH2Cl2 (1 mL) at 0 °C] and DIPEA (0.04 mL, 0.23 mmol) were
A solution of acid 17 (0.4 g, 1.63 mmol), HOBt (0.37 g,
1.95 mmol), and EDCI (0.26 g, 1.95 mmol) in CH2Cl2 (25 mL) was added sequentially, and the mixture was stirred for 8 h. Work-up
stirred at 0 °C for 15 min. Salt 20 (0.27 g, 1.95 mmol) was added,
and the mixture was stirred under a nitrogen atmosphere at room
temperature for 8 h. Work-up as described for 19, and purification
of the residue by column chromatography (60–120 mesh silica gel,
50% ethyl acetate in petroleum ether), gave 21 (0.72 g, 90%) as a
white solid, m.p. 145–147 °C. [α]D = –40.55 (c = 0.12, CHCl3). IR
as described for 19, and purification of the residue by column
chromatography (60–120 mesh silica gel, 2.5 % methanol in
CHCl3), gave 5 (0.05 g, 38%) as a white solid, m.p. 296–298 °C.
[α]D = –266.5 (c = 0.16, CHCl ). IR (KBr): ν = 3299, 3088, 2965,
˜
3
2931, 2854, 1728, 1690, 1657, 1550, 1451, 1370, 1309, 1290, 1247,
1165, 1094, 1052, 964, 670 cm–1 1H NMR (600 MHz, CDCl3,
.
(KBr): ν = 3358, 3259, 3084, 2945, 2853, 1757, 1698, 1658, 1659, 288 K): δ = 7.63 (d, J = 9.8 Hz, 1 H, NH-3), 7.43 (d, J = 8.4 Hz,
˜
1527, 1365, 1310, 1250, 1167, 1092, 1039, 973, 909, 856, 774, 1 H, NH-5), 7.24 (d, J = 9.4 Hz, 1 H, NH-2), 7.10 (d, J = 7.4 Hz,
616 cm–1. 1H NMR (300 MHz, CDCl3, 288 K): δ = 7.01 (d, 1 H, J
1 H, NH-6), 7.07 (d, J = 9.1 Hz, 1 H, NH-4), 4.79 (d, J = 9.9 Hz,
= 7.17 Hz, NH-2), 5.35 (s, 1 H, NH-1), 4.58 (m, 1 H, CαH-2), 4.00 1 H, NH-1), 4.64 (dq, J = 9.4, 7.0 Hz, 1 H, CαH-2), 4.57 (dq, J =
(d, J = 11.3 Hz, 1 H, CαH-1), 3.76 (s, 3 H, COOCH3), 3.62 (m, 1 9.1, 7.0 Hz, 1 H, CαH-4), 4.54 (p, J = 7.4 Hz, 1 H, CαH-6), 4.15
4306
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4295–4308

Design and Study of Peptides
(dq, J = 4.0, 9.8 Hz, 1 H, CβH-3), 4.07 (m, 1 H, CεH-1), 4.02 (m,
1 H, CεH-3), 4.00 (m, 1 H, CεH-5), 3.98 (m, 1 H, CβH-5), 3.93
(d, J = 9.7 Hz, 1 H, CαH-5), 3.78 (s, 3 H, -COOCH3), 3.76 (m, 1
H, CβH-1), 3.58 (d, J = 9.8 Hz, 1 H, CαH-3), 3.48 (d, J = 9.6 Hz,
1 H, CαH-1), 3.46 (m, 1 H, CεЈH-5), 3.43 (m, 1 H, CεЈH-3), 3.37
(m, 1 H, CεЈH-1), 2.10 (m, 1 H, CγH-1), 2.10 (m, 1 H, CγH-5),
2.20 (m, 1 H, CδH-3), 1.85 (m, 1 H, CδH-1), 1.79 (m, 1 H, CδH-
3), 1.77 (m, 1 H, CδH-5), 1.76 (m, 1 H, CγЈH-3), 1.75 (m, 1 H,
CγЈH-5), 1.7 (m, 1 H, CδЈH-1), 1.68 (m, 1 H, CδЈH-3), 1.66 (m, 1
H, CδЈH-5), 1.46 (m, 1 H, CγЈH-1), 1.42 (d, J = 7.4 Hz, 3 H, CH3-
6), 1.47 (s, 9 H, Boc), 1.33 (d, J = 7.0 Hz, 3 H, CH3-4), 1.25 (d, J
= 7.0 Hz, 3 H, CH3-2) ppm. 13C NMR (150 MHz, CDCl3): δ =
173.1, 172.6, 171.3 169.9, 169.5, 169.3, 155.8, 82.6, 81.7, 79.9, 79.6,
67.6, 67.4 (2 C), 52.3, 50.5, 48.7, 48.4, 47.7, 47.2, 46.7, 30.6, 29.6,
29.3, 28.1 (3 C), 25.4, 25.2, 25.0, 18.1, 17.1, 16.9 ppm. HRMS
(ESI): calcd. for C33H54N6O12Na [M + Na]+ 749.3697; found
749.3667.
Acknowledgments
The authors are grateful for financial support from the Council of
Scientific and Industrial Research (CSIR), New Delhi (CSC-0406/
CSC-0108). H. R. and A. B. are grateful to the University Grants
Commission (UGC), New Delhi. S. J. B. and P. G. R. are grateful
to CSIR for financial support in the form of fellowships.
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Boc-(S,S)-APyC-
L-Ala-(S,S)-APyC-L-Ala-(R,R)-APyC-L-Ala-
(R,R)-APyC-
L
-Ala-OMe (6): A solution of acid ent-4a[10] (0.04 g,
0.87 mmol), HOBt (0.02 g, 0.10 mmol), and EDCI (0.02 g,
0.105 mmol) in CH2Cl2 (2 mL) was stirred at 0 °C under a nitrogen
atmosphere for 15 min. Then salt 4b [prepared from 4 (0.05 g,
0.10 mmol) and CF3COOH (0.1 mL) in dry CH2Cl2 (1 mL) at 0 °C]
and DIPEA (0.17 mL, 0.13 mmol) were added sequentially, and the
mixture was stirred for 8 h. Work-up as described for 19, and puri-
fication of the residue by column chromatography (60–120 mesh
silica gel, 3.0% methanol in CHCl3), gave 6 (0.15 g, 75%) as a white
solid, m.p. 321–324 °C. [α]D = +1.78 (c = 0.14, CHCl3). IR
(CHCl ): ν = 3298, 2923, 2853, 1652, 1539, 1453, 1371, 1346, 1311,
˜
3
1219, 1162, 1091, 965, 772 cm–1 1H NMR (500 MHz, CDCl3,
.
288 K): δ = 7.73 (d, J = 9.8 Hz, 1 H, NH-2), 7.62 (d, J = 10 Hz, 1
H, NH-3), 7.51 (d, J = 8.5 Hz, 1 H, NH-7), 7.37 (d, J = 9.5 Hz, 1
H, NH-5), 7.11 (d, J = 7.5 Hz, 1 H, NH-8), 7.0 (d, J = 9.0 Hz, 1
H, NH-7), 6.45 (d, J = 9.8 Hz, 1 H, NH-4), 4.86 (d, J = 10 Hz, 1
H, NH-1), 4.70 (dq, J = 9.8, 7.0 Hz, 1 H, CαH-2), 4.66 (dq, J =
9.8, 7.0 Hz, 1 H, CαH-4), 4.55 (dq, J = 9.0, 7.0 Hz, 1 H, CαH-6),
4.54 (dq, J = 7.5, 7.0 Hz, 1 H, CαH-8), 4.08 (m, 2 H, CεH-1, CβH-
5), 4.04 (m, 1 H, CεH-7), 4.00 (m, 2 H, CεH-3, CεH-5), 3.98 (m,
2 H, CαH-7, CβH-7), 3.97 (m, 1 H, CαH-3), 3.83 (d, J = 9.8 Hz,
1 H, CαH-5), 3.76 (s, 3 H, -COOCH3), 3.73 (m, 1 H, CβH-1), 3.67
(d, J = 9.8 Hz, 1 H, CαH-3), 3.52 (d, J = 9.5 Hz, 1 H, CαH-1),
3.45 (m, 1 H, CεЈH-3), 3.43 (m, 1 H, CεЈH-7), 3.39 (m, 1 H, CεЈH-
5), 3.37 (m, 1 H, CεЈH-1), 2.10 (m, 1 H, CγH-1), 2.09 (m, 1 H,
CγH-7), 2.0 (m, 1 H, CγH-3), 1.99 (m, 1 H, CγH-5), 2.0 (m, 1 H,
CδH-5), 1.86 (m, 1 H, CδH-3), 1.85 (m, 1 H, CδH-7), 1.83 (m, 1
H, CδH-1), 1.8 (m, 1 H, CγЈH-3), 1.79 (m, 2 H, CδЈH-7, CδЈH-5),
1.75 (m, 1 H, CδЈH-3), 1.69 (m, 1 H, CδЈH-5), 1.68 (m, 1 H, CδЈH-
1), 1.67 (m, 1 H, CγЈH-7), 1.43 (s, 9 H, Boc), 1.42 (m, 1 H, CγЈH-
1), 1.42 (m, J = 7.0 Hz, 3 H, CH3-8), 1.37 (d, J = 7 Hz, 6 H, CH3-4,
CH3-6), 1.33 (s, J = 7 Hz, 3 H, CH3-2) ppm. 13C NMR (150 MHz,
CDCl3): δ = 173.3, 173.1, 171.9, 171.4, 170.2, 170, 169.9, 169.4,
155.7, 81.8, 81.1, 80.4, 79.7 (2 C), 67.8, 67.5, 67.4, 67.2, 52.4, 49.8,
49.1, 48.7, 48.5, 47.7, 47.4, 47.0, 46.4, 30.7, 29.8, 29.7, 29.4, 29.2,
28.2 (3 C), 25.4, 25.3, 25.1, 18.2, 17.5, 17.0, 16.7 ppm. HRMS
(ESI): calcd. for C42H68N8O15Na [M + Na]+ 947.4701; found
947.4683.
Supporting Information (see footnote on the first page of this arti-
cle): NMR (1H, 13C, 2D) spectra, solvent titration studies, mini-
mum energy structures and distant constraints used in the MD
calculations for all the compounds of interest are available.
Supporting Information (see footnote on the first page of this arti-
cle): General; solvent titration studies; NMR data; molecular dy-
namics; CD spectra; quantum chemical calculations.
Eur. J. Org. Chem. 2014, 4295–4308
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4307

G. V. M. Sharma, A. V. S. Sarma, H.-J. Hofmann et al.
FULL PAPER
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Published Online: June 3, 2014
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