Synthesis of C-linked carbo-β2-amino acids and β2-peptides: Design of new motifs for left-handed 12/10- and 10/12-mixed helices

Article abstract of DOI:10.1039/c2ob26615f

C-linked carbo-β²-amino acids (β²-Caa), a new class of β-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new β-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cβ- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-β²-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl₃), CD, and molecular dynamics simulations. The β²-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on β³-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the β²- and β³-peptides.

Full text of DOI:10.1039/c2ob26615f

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PAPER
Synthesis of C-linked carbo-β2-amino acids and β2-peptides: design of new
motifs for left-handed 12/10- and 10/12-mixed helices†
Gangavaram V. M. Sharma,*a Nelli Yella Reddy,‡a Rapolu Ravi,‡b Bommagani Sreenivas,a Gattu Sridhar,a
Deepak Chatterjee,b Ajit C. Kunwar*b and Hans-Jörg Hofmann*c
Received 15th August 2012, Accepted 2nd October 2012
DOI: 10.1039/c2ob26615f
C-linked carbo-β2-amino acids (β2-Caa), a new class of β-amino acid with a carbohydrate side chain
having D-xylo configuration, were prepared from D-glucose. The main idea behind the design of the new
β-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cβ- to the
Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating
(R)- and (S)-β2-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl3), CD,
and molecular dynamics simulations. The β2-peptides of the present study form left-handed 12/10- and
10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and
result in a new motif. These results differ from earlier findings on β3-peptides of the same design,
containing a carbohydrate side chain with D-xylo configuration, which form exclusively right-handed 12/
10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain
chirality as an important factor for the observed definite left- or right-handedness of the helices in the
β2- and β3-peptides.
helix,10,11 was first observed in β-peptides having a backbone
Introduction
with alternating β2- and β3-residues. Similarly, a hairpin turn12
was observed in peptides containing β2-amino acids. Seebach
The research activity on β-peptides has grown immensely since
the appearance of the first reports by Gellman et al.1 and
Seebach et al.,2 leading to the emergence of the area of fold-
amers.3 Meanwhile, a large number of ω-amino acids and their
oligomers have been designed to realize a wide variety of sec-
ondary structures.3,4 In the β-peptide domain, oligomers of
β3-amino acids have been extensively studied,3 whereas β2-peptides
derived from β2-amino acids5 have received less attention.6 Inter-
estingly, a few β2-amino acids form partial structures of natural
products7 and are known to exhibit diverse biological activities.
The natural occurrence of β2-hAla8a as an exclusive component
of rare natural peptides was established in 1951.8b,c Some
unusual motifs9 have been generated in β2-peptides. One of the
most interesting and unique helical structures, the 12/10-mixed
et al.6a reported that homochiral β2-peptides with proteinogenic
side chains adopt a right-handed 14-helix, in contrast to the left-
handed 14-helix observed in β3-peptides.2 Seebach’s group have
also used β2-amino acids in mixed peptides to design 9- and
10-membered (mr) H-bonded turns.13 Kaur et al.14 synthesized
β2-peptides using 2,3-diamino propionic acid, whose CD spectra
suggested the presence of solvent-dependent secondary struc-
tures, while the hydrazino peptides reported by Seebach and
Lelais15 did not show secondary structures. Recently, Mikata
et al.16a reported the synthesis of and conformational studies on
β2-peptides from C-glycosyl β2-amino acids.16b
Earlier we reported11b right-handed 12/10- (10/12-) helices in
peptides from alternating (R)- and (S)-C-linked carbo β3-amino
acids (β3-Caa; epimeric at Cβ),17 while, both the right- and left-
handed 12/10- (10/12-) helices18 were observed in peptides from
(R)- and (S)-β-Caas alternating with β-hGly (β-homo glycine),
respectively. The backbone flexibility available in β-hGly was
found to be responsible for the ‘switch’ in the handedness,18 dic-
tated by the epimeric β3-Caas. Furthermore, the mass spectral
studies19 on the β3-dipeptides, derived from epimeric β3-Caas,
revealed a steric strain at the Cβ-carbon due to the presence of
the carbohydrate side chain. The results described above, in par-
ticular the switch in handedness of the 14-helix2 in homo-oligo-
meric β2-peptides compared to β3-peptides,6a prompted us to
aOrganic and Biomolecular Chemistry Division, CSIR-Indian Institute
of Chemical Technology, Hyderabad-500 007, India
bCentre for Nuclear Magnetic Resonance, CSIR-Indian Institute of
Chemical Technology, Hyderabad-500 007, India.
E-mail: esmvee@iict.res.in, kunwar@iict.res.in,
hofmann@uni-leipzig.de
cInstitute of Biochemistry, Faculty of Biosciences, Pharmacy, and
Psychology, University of Leipzig, Brüderstraße 34, D-04103 Leipzig,
Germany
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26615f
‡These authors contributed equally to this work.
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DIPEA, and DMAP (cat.) in CH2Cl2 at room temperature to give
a separable diastereomeric mixture of 1 (40%) and 2 (23%). The
esters 1 and 2 on exposure to CF3COOH afforded the TFA salts
17 and 18, respectively.
To ascertain the absolute stereochemistry of the new stereo-
centre at C-5 in 1 and 2, a cyclic ether was envisaged and syn-
thesized from the known diol 19.24 Accordingly, 19 on reaction
with dihydropyran in CH2Cl2 and PTSA (cat.) at room temper-
ature gave THP ether 20 in 70% yield (Scheme 2). The carbinol
20 on Swern oxidation afforded the corresponding ketone 21,
which on Wittig olefination furnished 22 (56%). Reaction of 22
with PTSA in aq. MeOH at room temperature gave alcohol 23
(71%), which under Mitsunobu23 conditions furnished 24 in
77% yield.
Treatment of 24 with hydrazine hydrate in MeOH at room
temperature followed by reaction with (Boc)2O at room temper-
ature gave 25 in 79% yield. Hydroboration of 25 with BH3·DMS
in dry THF at room temperature afforded the alcohols 26 (25%)
and 27 (10%) as a separable diastereomeric mixture. The benzyl
ether 26 was subjected to hydrogenolysis with Pd/C (10%) in
EtOAc under a hydrogen atmosphere to give diol 28 in 92%
yield. Reaction of diol 28 with p-TsCl and Et3N in CH2Cl2 at
room temperature furnished the corresponding tosylate, which
underwent concomitant cyclization to afford the cyclic ether 29
in 80% yield. The structure and the stereochemistry at the C-5
stereocentre were unambiguously assigned by NMR studies.
The conformational analysis of 29 (Fig. 3) showed strong
indicative nOes between C3H and –CH2NHBoc reflecting the
presence of C3H and –CH2NHBoc at the same face, clearly
giving evidence for the stereochemical assignment at the C-5
stereocentre.
Subsequently, the diol 28 was converted into the dimethyl
ether using Ag2O and MeI to afford 30 (42%), [α]2D5 = −107
(c 0.14, CHCl3). To further ascertain the absolute stereo-
chemistry of the new β2-Caas 1 and 2, they were converted into
the dimethyl ethers 30 and 33, respectively. Accordingly, 1 and
2 were independently treated with LiAlH4 to afford the corres-
ponding alcohols 31 (87%) and 32 (79%). Alkylation of the
alcohols 31 and 32 with MeI in the presence of Ag2O in DMF
gave the respective ethers 30 (56%), [α]2D5 = −109.2 (c 1.49,
CHCl3) and 33 (47%), [α]D25 = −40.4 (c 0.5, CHCl3). From the
spectral data and optical rotation values, it can be concluded that
30, derived from 28 and 1, is the same and thus has the same
stereochemistry at C-5.
Fig. 1 Structures of β2-amino acids 1 and 2.
investigate the folding propensities in β2-peptides with alternat-
ing chirality. We propose a new set of β-amino acids, which are
C-linked carbo-β2-amino acids (β2-Caa), with a carbohydrate
side chain at the Cα-carbon. The envisaged relief in the strain at
the Cβ-carbon is expected to influence the conformational fea-
tures of the β2-peptides. The β2-Caas 1 and 2 (Fig. 1), which are
epimeric at the Cα-stereocenter, were synthesized to investigate
new motifs.
The predictions of theoretical studies,20,21 that the ‘unlike’
β2-peptides favor the formation of 10/12-mixed helices,
prompted us to explore experimentally the design of ‘alternating
chirality’ in β2-peptides from 1 and 2 for the first time. We report
the synthesis of β2-peptides 3 and 4 with an (S)-β2-Caa at the
N-terminus and 5 and 6 with an (R)-β2-Caa at the N-terminus
(Fig. 2) and their conformational studies employing NMR, CD,
MD, and quantum chemical methods.
Results and discussion
1. Synthesis of β2-amino acids 1 and 2
The main strategy in the synthesis22 of the β2-Caas 1 and 2 from
D-glucose would be the conversion of the C-6 hydroxy group
into the amino group, while the requisite carboxylic group is
introduced at the C-5 stereocentre. Accordingly, the known diol
7 was treated with TrCl and Et3N in CH2Cl2 at room temperature
to afford the trityl ether 8 in 89% yield (Scheme 1). The alcohol
8 was oxidized under Swern reaction conditions to give ketone 9
(98%), which on Wittig olefination with (methyl)triphenylphos-
phonium iodide and t-BuOK in THF at 0 °C gave 10 in 51%
yield. Subsequently, the trityl group in 10 was deprotected using
CF3COOH in CH2Cl2 at room temperature to afford allylic
alcohol 11 in 79% yield. The alcohol 11 under Mitsunobu23 con-
ditions was treated with phthalimide, Ph3P, and DIAD in dry
THF to furnish 12 in 71% yield (along with DIAD byproduct as
an impurity). Compound 12 was treated with hydrazine hydrate
in MeOH to give the amine, which on subsequent treatment with
(Boc)2O at room temperature afforded 13 in 79% yield.
Hydroboration of 13 with BH3·DMS in dry THF at room
temperature furnished the alcohol 14 (Scheme 1) as an insepar-
able diastereomeric mixture (7 : 3). The terminal alcohol 14 on
oxidation under Swern reaction conditions furnished aldehyde
15 (96%), which on further oxidation with NaClO2 and H2O2 in
tert-BuOH : H2O (7 : 3) gave acid 16 (85%) as an inseparable
diastereomeric mixture. To overcome the racemisation problem,
the acid was converted into the benzyl ester with Bn–Br,
2. Synthesis of peptides
The synthesis of the peptides 3–6 is outlined in Scheme 3.
Accordingly, Boc-(S)-β2-Caa-OBn 2 (Scheme 3) on hydrogen-
olysis with 10% Pd/C in EtOAc gave acid 34 in 92% yield,
which on coupling with 17 in the presence of EDCI, HOBt, and
NMM in CH2Cl2 afforded the dipeptide 35 in 69% yield. Hydro-
genolysis (10% Pd/C in EtOAc) of 35 gave acid 36, while 35 on
exposure to CF3COOH in CH2Cl2 was converted into the salt
37. Condensation of the acid 36 with the salt 37 in the presence
of EDCI, HOBt, and NMM in CH2Cl2 furnished the tetrapeptide
3 in 54% yield. Hydrogenolysis of peptide 3 afforded the
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Fig. 2 Structures of β2-peptides 3 to 6 (arrows indicate the H-bonding pattern deduced from structural studies).
corresponding acid 38, which on coupling (EDCI, HOBt and
NMM) with 37 in CH2Cl2 gave the hexapeptide 4 in 32% yield.
Likewise, coupling (EDCI, HOBt and NMM) of acid 39
(obtained from 1) with salt 17 in CH2Cl2 furnished the dipeptide
40 in 61% yield. Ester 40 on hydrogenolysis gave the corres-
ponding acid 41, which on coupling with the salt 42 (obtained
from 40 by exposure to CF3COOH) gave tetrapeptide 5 in 55%
yield. Hydrogenolysis and coupling of the corresponding acid 43
with 42 in the presence of EDCI, HOBt and NMM in CH2Cl2
afforded the hexapeptide 6 in 36% yield.
N-terminus showed wide dispersion in the chemical shifts (δ) of
amide as well as CβH protons. The NH-3 and NH-4 resonances
appeared at low field (δ >7 ppm), indicating their participation in
hydrogen bonding. The solvent titration studies26 showed that all
amide protons, except NH-2, display modest changes of their
chemical shifts (ΔδNH <0.66 ppm) upon addition of up to 33%
v/v DMSO-d6 in CDCl3 solution, suggesting their inaccessibility
to DMSO-d6, and confirming their involvement in intramolecu-
lar H-bonding. For the residues in the middle of the sequence,
3
the coupling constants JNH–CβH ∼9.0 Hz and ∼4.0 Hz imply
that the torsion angles C(O)–N–Cβ–Cα (ϕ) are constrained with
(R)-β2-Caa and (S)-β2-Caa having values of ∼120° and ∼−120°,
respectively (Fig. 4).
3. Conformational analysis
3
Additionally, JCαH–CβH couplings of about ∼4.0 Hz for (R)-
The 1H NMR studies25 on peptides 3–6 were performed
in ∼5 mM solutions in CDCl3 on a 600 MHz spectrometer.
The spectrum of peptide 3 with an (S)-β2-Caa residue at the
β2-Caa imply that the torsion angles N–Cβ–Cα–C(O) (θ) are
∼−60°, whereas, the 3JCαH–CβH values of ∼10.0 Hz and ∼3.0 Hz
for (S)-β2-Caa correspond to θ values of ∼−60° or ∼180°. The
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Scheme 1 Reagents and conditions: (a) Et3N, TrCl, CH2Cl2, 0 °C–rt, 8 h; (b) (COCl)2, dry DMSO, Et3N, CH2Cl2, −78 °C, 3 h; (c) Ph3P + CH3I,
t-BuOK, dry THF, 0 °C, 17 h; (d) CF3COOH, CH2Cl2, 0 °C–rt, 2 h; (e) phthalimide, DIAD, Ph3P, dry THF, 0 °C–rt, 6 h; (f) NH2NH2·H2O, MeOH,
rt, 5 h; (g) (Boc)2O, MeOH, 0 °C–rt, 6 h; (h) BH3·DMS, 30% H2O2, NaOH, dry THF, 10 h; (i) NaClO2, H2O2, t-BuOH : H2O 10 h; ( j) DIPEA,
BnBr, DMAP, CH2Cl2, rt, 8 h.
sequential nOe correlation (Fig. 5) CβH(pro-S)(2)/NH(3) and
intra-residue nOe correlations NH(1)/CαH(1) and NH(3)/CαH(3)
along with almost extremum values for JCαH–CβH (discussed
NH(5)/NH(6) support the preponderance of structures having
NH(1)–CO(2), NH(3)–CO(4), NH(5)–CO(6), NH(4)–CO(1), and
NH(6)–CO(3) hydrogen bonds. The above data show the pres-
ence of a left-handed mixed helix with a 10/12/10/12/10-hydro-
gen bonding pattern.
3
above) imply a predominant conformation with θ ∼ −60° and
Cβ–Cα–C(O)–N(ψ) of ∼ 120° (Fig. 4) for the second and the
third residue. For the two terminal residues, the lack of the extre-
mum values of the couplings between backbone protons
suggests averaging over several conformers. In addition to these
conclusions on the backbone dihedral angles, the nOes CβH(pro-S)(2)/
NH(4) and CβH(pro-S)(2)/C4H(4) provide support for a well
defined structure with H-bonding between NH(4) and CO(1),
corresponding to a 12-mr pseudo ring, while the nOe corre-
lations NH(1)/NH(2) and NH(3)/NH(4) imply that NH(1) and
NH(3) are H-bonded with CO(2) and CO(4), respectively, to
form 10-mr pseudo rings.
This study establishes β2-peptides with alternating chirality as
a new motif for the realization of 10/12-left-handed mixed
helices. Interestingly, left-handed helices are very rare in natural
proteins, as reflected in the protein data bank (PDB).27,28 An
occasional incidence of left-handed helices has been noticed at
ligand binding sites and other functional sites, which confirms
their significance in protein functions.29
For further investigations on the impact of alternating chirality,
the peptides 5 and 6 were synthesized having an (R)-β2-Caa
residue at the N-terminus. The 1H NMR spectrum of 5 displayed
two sets of resonance peaks in the ratio of 73 : 27. Based on the
presence of exchange peaks in the ROESY spectrum, the exist-
ence of two isomers in 5 is implied, unlike in the case of the cor-
responding β3-peptides.11b Only the isomer with the larger
population was investigated in detail, since it was not possible to
deduce specific structural information for the minor one. The
δ >7.19 ppm and ΔδNH <0.45 ppm for NH-2 and NH-3 imply
Thus, peptide 3 showed a propensity for a left-handed 10/12-
mixed helix (10/12/10…hydrogen bond pattern), unlike the
observed right-handed 10/12-helix in β3-peptides.11b A similar
switch in the handedness of the 14-helix in the homo-oligomeric
β3- and β2-peptides was earlier reported by Seebach et al.2,6a
The 1H NMR spectrum of 4 showed that several amide
protons have δ >7 ppm (except NH-1 and NH-2) and ΔδNH
<0.53 (except NH-2) in solvent titration studies, suggesting their
involvement in H-bonding.26 Like 3, for all central residues
3
their participation in H-bonding.25 The JNH–CβH values of ∼7.5
Hz and ∼5.0 Hz reflect contributions from several conformers,
though the predominance of structures with ϕ ∼ 120° can be
3
(excluding the residues at the termini), JNH–CβH >9.5 Hz and
<3.0 Hz imply ϕ ∼120° and ∼−120° for (R)-β2-Caa and (S)-β2-
inferred. The JCαH–CβH values of ∼5.0 Hz and ∼5.0 Hz for (R)-
3
3
Caa, respectively. Similarly, JCαH–CβH <4.0 Hz and >10.0 Hz
β2-Caa and ∼8.5 Hz and ∼4.0 Hz for (S)-β2-Caa along with the
strong nOes NH(2)/CβH(pro-S)(1), NH(4)/CβH(pro-S)(3), NH(2)/
CαH(2), and NH(4)/CαH(4) indicate the presence of a substan-
tial population of conformers with θ ∼−60° and ψ ∼ 120°. The
nOe correlations CβH(pro-S)(1)/NH(3), CβH(pro-S)(1)/C4H(3), and
NH(2)/NH(3) along with the above information suggest that a
significant population of structures has a left-handed mixed helix
with a 12/10-hydrogen bonding pattern.
suggest the preponderance of a single rotamer about Cα–Cβ.
This information along with the nOes NH(3)/CβH(pro-S)(2),
NH(5)/CβH(pro-S)(4), NH(1)/CαH(1), NH(3)/CαH(3), and NH
(5)/CαH(5) indicate that the angles θ and ψ for these residues are
∼−60° and ∼ 120°, respectively. Additionally, the nOe corre-
lations CβH(pro-S)(2)/NH(4), CβH(pro-S)(2)/C4H(4), CβH(pro-S)(4)/
NH(6), CβH(pro-S)(4)/C4H(6), NH(1)/NH(2), NH(3)/NH(4), and
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Scheme 2 Reagents and conditions: (a) 3,4-dihydropyran, PTSA, CH2Cl2, 0 °C–rt, 3 h; (b) (COCl)2, dry DMSO, Et3N, CH2Cl2, −78 °C, 3 h;
(c) Ph3P + CH3I, t-BuOK, dry THF, 0 °C, 17 h; (d) PTSA, aq. MeOH, rt, 2 h; (e) phthalimide, DIAD, Ph3P, dry THF, 0 °C–rt, 6 h; (f) NH2NH2·H2O,
MeOH, rt, 5 h; (g) MeOH, (Boc)2O, 0 °C–rt, 6 h; (h) BH3·DMS, 30% H2O2, NaOH, dry THF, 12 h; (i) H2, 10% Pd/C, EtOAc, rt, 12 h; ( j) p-TsCl,
Et3N, DMAP, CH2Cl2, 0 °C–rt, 12 h; (k) Ag2O, DMF, Mel, rt, 12 h; (l) LiAlH4, dry THF, 0.5 h, 0 °C.
CβH(pro-S)(3)/C4H(5), NH(2)/NH(3), and NH(4)/NH(5) provide
sufficient evidence for the presence of a left-handed mixed helix
with a 12/10/12/10 H-bonding pattern.
The couplings and nOes involving the side chains in peptides
3–6 have been used to obtain information on the structure of the
furanoside sugar ring as well as on the dihedral angle Cβ–Cα–
3
C4–O (χ1). For all residues, values of JCαH–C4H ≥9.0 imply χ1
∼180° for the (S)-residues and ∼60° for the (R)-residues
Fig. 3 Characteristic nOes (shown by double arrow) observed in 29.
(Fig. 7). This was further confirmed by the intra-residue nOe cor-
relations CβH(pro-S)/C4H and CβH(pro-R)/C4H in all (S)-β-Caa
residues and NH/C4H in all (R)-β2-Caa residues. The couplings
3
3
3JC1H–C2H ∼4 Hz, JC2H–C3H ∼0 Hz, and JC3H–C4H ∼3.0 Hz for
Peptide 6 showed a very wide dispersion in chemical shifts of
amide proton (3.30 ppm) and CβH (1.58 ppm), while partici-
pation of several amide protons in H-bonding25 was indicated by
the sugar rings correspond to a sugar pucker of T2.11b Strong
nOes Me(pro-S)/C1H, Me(pro-S)/C2H as well as a weak Me(pro-R)
C4H nOe correlation support an envelope conformation of the
isopropylidine ring.
3
/
3
δ >7 ppm and ΔδNH <0.45 ppm.26 The values of JNH–CβH ∼9
Hz and ∼4 Hz imply that ϕ is ∼ 120°. Also 3JCαH–CβH couplings
of about ∼4.0 Hz for the (R)-β2-Caa-residues and ∼9.5 Hz and
∼4.0 Hz for the (S)-β2-Caa-residues along with the nOes (Fig. 6)
NH(2)/CβH(pro-S)(1), NH(4)/CβH(pro-S)(3), NH(6)/CβH(pro-S)(5),
For the restrained molecular dynamics (MD) studies25
(Fig. 8), the constraints were derived from the volume integrals
obtained from the ROESY spectra using ISPA (Isolated Spin-
Pair Approximation).30 Fig. 8A and 8B show the superimposi-
tion of the 10 lowest energy structures of 3 and 4, respectively.
NH(2)/CαH(2),
NH(4)/CαH(4),
and
NH(6)/CαH(6),
CβH(pro-S)(1)/NH(3), CβH(pro-S)(1)/C4H(3), CβH(pro-S)(3)/NH(5),
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Scheme 3 Reagents and conditions: (a) H2, 10% Pd/C, EtOAc, rt, 2 h; (b) HOBt (1.2 equiv.), EDCI (1.2 equiv.), NMM (4 equiv.), dry CH2Cl2,
0 °C–rt; (c) CF3COOH, dry CH2Cl2, 0 °C–rt, 2 h.
Fig. 4 Schematic Newman projections showing the orientation of the
substituents in a left-handed 10/12-helix for the ideal values of torsional
angles ϕ, θ, and ψ.20,21 (A) depicts the view along the backbone atoms
in (R)-β2-Caa and (B) that in (S)-β2-Caa.
The MD structures depict the features already alluded to with
backbone and heavy atom RMSDs of 0.58 Å and 1.21 Å for 3
and 0.85 Å and 1.64 Å for 4, respectively. Similar structural
details were found for 5 and 6 from the MD analysis.25 In order
to highlight the contributions of the peptidic backbone atoms,
Fig. 5 Characteristic nOes of peptide 3 are shown in 3D model: (1)
while taking RMSD values, the tert-butyl group in Boc and the
CβH(pro-S)(2)/C4H(4); (2) CβH(pro-S)(2)/NH(4); (3) CβH(pro-S)(2)/NH(3);
benzyl group in the –OBn moieties were replaced by hydrogen
(4) NH(3)/NH(4); (5) NH(3)/CαH(3); (6) NH(1)/NH(2); (7) NH(1)/CαH
atoms for the peptides 3–6. The NMR data suggest that the
(1). P = Boc; P′ = OBn.
fraying of the termini is more conspicuous in the β2-peptides
compared to β3-peptides, suggesting that the robustness of the
helices in the β2-peptides is compromised. The MD data confirm
these observations with somewhat larger RMSD deviations for
also in agreement with the reduced robustness of the 14-helix in
the heavy atoms and the backbone atoms.11b This observation is
β2-peptides compared to the β3-peptides.2,6a
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Fig. 9 CD spectra of peptides 3–6 in MeOH.
corresponding to the signatures of a left-handed 10/12-helix in
these β-peptides. Interestingly, like the results from NMR in
CDCl3 solution, the helices in β2-peptides appear less robust, as
reflected from the smaller molecular ellipticities observed for the
peptides 3–6, compared to those obtained for β3-peptides.11b
Peptide 5, however, showed a very small molecular ellipticity
and a loss of structure, as was exhibited by the two populations
from the NMR study.
Fig. 6 ROESY spectrum of peptide 6 with the characteristic nOe cor-
relations supporting a 12/10 helix (dotted lines represent H-bonds).
4. Theoretical studies
The most striking result of the present work on β2-peptides with
alternating chirality design employing (R)- and (S)-β2-amino
acids, which are epimeric at the amine stereocentre, is the occur-
rence of exclusively left-handed mixed 12/10- and 10/12-helical
conformations, independent of the order of the chiral amino acid
constituents. The left-handed 10/12/10-hydrogen bonding
pattern is favored in sequences with an (S)-β2-Caa at the N-
terminus, while the left-handed 12/10/12-hydrogen bonding
pattern appears in sequences with an (R)-β2-Caa at the N-termi-
nus. A similarly surprising result was already observed in
β3-peptides of the same class, where right-handed mixed helices
are exclusively formed.10b,17
Fig. 7 Newman projections showing the orientations of the substitu-
ents for the predominant conformers around the Cα–C4 (χ1) bond,
appending the sugar side chains to the peptide backbone. (A) (S)-β2-Caa
and (B) (R)-β2-Caa.
According to general ideas on the side chain influence on the
structure of mixed helices,20a,c,21a,b a switch in handedness can
be expected with a change in the chirality of the amino acids.
Referring to the β-peptide design of 12/10- and 10/12-mixed
helices, four helix possibilities can formally be expected, viz.
left-handed or right-handed helices with 12/10- or, alternatively,
10/12-hydrogen bonded pseudo rings. Moreover, it can be
assumed that, if a left-handed 12/10-helix is preferred in β2-pep-
tides having a (R,S,R…)-amino acid sequence, the corresponding
right-handed helix with the same 12/10-hydrogen bonded ring
order is the preferred one in β2-peptides with a β2-(S,R,S…)-
amino acid sequence. Likewise, the corresponding conclusions
are valid for 10/12-ring sequences. This is confirmed by the stab-
ilities of all mixed 12/10- and 10/12-helices of blocked β2- and
β3-hexapeptides consisting of mono-substituted β2- and β3-
amino acid constituents with achiral methyl side chains, obtained
at the HF/6-31G* level of ab initio MO theory. These data are
given in Table 5 of the ESI† (see also ref. 20c). In Table 5
(ESI†), both the cases with alternating chirality, which is the
subject of this manuscript, and also the cases of non-alternating
chirality were considered for completeness. The study indicates
Fig. 8 Stereoview of the MD structures of peptide 3 (A) and peptide
4 (B); sugars are replaced by methyl groups after the calculations for
clarity.
CD spectroscopy, unlike for natural proteins and peptides,31
has been less extensively used for the understanding of folding
patterns of β- and α/β-peptides,32 which can be attributed to the
scarce availability of high resolution structural information.32,33
The CD studies (Fig. 9) on peptides 3–6 have been undertaken
in 0.2 mM solution in methanol, since chloroform absorptions
interfere with the requisite CD absorptions.34 The CD spectra
very clearly depict a negative maximum at about 203 nM,
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Table 1 Relative energiesa of all right-handed and left-handed 12/10-
and 10/12-mixed helices of blocked β2- and β3-hexapeptides with
alternating chirality consisting of mono-methyl-substituted β-amino
acids obtained at the HF/6-31G* level of ab initio MO theoryb
Table 2 Relative energiesa of the right-handed and left-handed 12/10-
and 10/12-mixed helices obtained for the β2- and β3-peptides with
alternating chirality consisting of β2- and β3-Caas at the HF/6-31G*
level of ab initio MO theoryb
Helixb
Relative energy
Helixc
Relative energy
β2-peptides
β2-Caa peptides
β(2R,2S)rh10/12
β(2R,2S)lh12/10
β(2S,2R)lh10/12
β(2S,2R)rh12/10
β3-Caa-peptides
β(3R,3S)rh12/10
β(3R,3S)lh10/12
β(3S,3R)rh10/12
β(3S,3R)lh12/10
β(2R,2S)rh10/12 ≡ β(2S,2R)lh10/12
β(2S,2R)rh10/12 ≡ β(2R,2S)lh10/12
β(2S,2R)rh12/10 ≡ β(2R,2S)lh12/10
β(2R,2S)rh12/10 ≡ β(2S,2R)lh12/10
β3-peptides
β(3R,3S)rh10/12 ≡ β(3S,3R)lh10/12
β(3S,3R)rh10/12 ≡ β(3R,3S)lh10/12
β(3S,3R)rh12/10 ≡ β(3R,3S)lh12/10
β(3R,3S)rh12/10 ≡ β(3S,3R)lh12/10
42.2
1.3
28.8
41.0
47.7
0.0d
0.0c
137.9
1.3
93.9
0.0d
96.6
21.7
112.8
0.0e
72.2
a In kJ mol−1
.
b The first substitution and configuration symbols in the
a In kJ mol−1 b The β2-peptide structures correspond to peptide 6, in the
.
parentheses correspond to the first mono-substituted amino acid
β3-peptides only the side chains are shifted to Cβ; for details of the
calculations, see ESI.† c The first substitution and configuration symbols
in the parentheses correspond to the first mono-substituted amino acid
constituent in the sequence, the second symbols to the second one; rh =
right-handed, lh = left-handed. d Total energy ET = −5590.195546 a.u.
e Total energy ET = −5590.200755 a.u.
constituent in the sequence, the second symbols to the second
constituent; rh = right-handed, lh = left-handed. c Total energy ET
−1956.369187 a.u. d Total energy ET = −1956.378003 a.u.
=
that all formally possible mixed 12/10- and 10/12-helices can be
localized as minimum conformations for the blocked model
hexapeptides. The theoretical data are impressively in agreement
with the experimental data reported by Seebach and co-work-
ers,10d where the formation of right-handed 10/12-helices is
particularly favored in alternate sequences beginning with an
(S)-β2-amino acid constituent followed by an (S)-β3-amino acid
constituent, while right-handed 12/10-helices are favored in
sequences beginning with an (S)-β3-amino acid residue followed
by an (S)-β2-amino acid residue.
for details of the calculations, see ESI†). In the case of a β3-(R,S,
R…)-amino acid sequence, the right-handed 12/10-hydrogen-
bonded ring sequence is favored, whereas, a right-handed 10/12-
ring order is preferred in the β3-(S,R,S...)-amino acid sequence.
Likewise, the theoretical and experimental data are in good
agreement for the (S,R,S…)-amino acid sequences of β2-peptides,
which favor the formation of left-handed mixed 10/12-helices. In
the case of the β2-(R,S,R…)-amino acid sequences, two confor-
mers were indicated in the NMR experiments, wherein the left-
handed 12/10-helix was the most stable conformer, while the
structure of the other could not be analyzed. However, according
to the theoretical data (Table 2), a right-handed 10/12-helix is
slightly favored over the experimentally determined left-handed
12/10-helix. As long as the nature of the second conformer is
unknown, a final evaluation of the theoretical data is difficult.
In comparison to the model peptides with achiral methyl side
chains of Table 1, the space-filling properties of the chiral side
chains in the β2- and β3-Caas exclude the existence of most
mixed helix types. These findings further reiterate the role of the
chiral side chains in defining the handedness of the observed
helices in β2- and β3-peptides.
Since the focus of the present work involves the concept of
alternating chirality, we selected only the data for these cases
from Table 5 (ESI†) and present them in Table 1. As expected,
right-handed mixed helices of β2- and β3-peptides of the same
hydrogen bonding type (10/12- or 12/10-) with an (S,R,S…)-
sequence are equivalent to the left-handed mixed helices of the
same hydrogen bonding type in the corresponding (R,S,R…)-
sequences and vice versa. Moreover, the stability of the right-
handed 10/12-helix in β2-peptides with an (S,R,S…)-sequence of
β2-amino acids is comparable with that of the left-handed 12/10-
helices with the same chirality sequence. The same conclusions
can be drawn for β2-peptides with an (R,S,R…)-sequence of β2-
amino acids and for the corresponding situations in β3-peptides.
In order to explain the preference for the same handedness in
β2- (left-handed) and β3-peptides (right-handed) independent of
the order of the alternate chiral β-Caas, we have to look for
another explanation. Unlike the model hexapeptides with achiral
methyl side chains in Table 1, the epimeric β2- and β3-Caa con-
stituents in our study with their chiral carbohydrate side chains,
are diastereomeric. Obviously, the preference for the same
handedness independent of the order of the chiral constituents in
the peptides from alternating (S)- and (R)-β-Caas is governed by
the different steric situation arising from the chiral carbohydrate
side chains.
5. Conclusions
In this study, β2-Caas with carbohydrate side chain at the Cα
stereocentre have been prepared for the first time, inspired by our
observations on the mass spectral studies on β3-peptides derived
from β3-Caas. The study provided experimental proof of the
theoretical predictions on the concept of unlike β2-peptides
resulting in 12/10- or 10/12-mixed helices. The design of pep-
tides with alternating chirality, employing the epimeric (R)- and
(S)-β2-Caas resulted in a new motif for the generation of left-
handed 12/10- or 10/12-mixed helices. The preference for left-
handedness in the helices of β2-peptides, independent of the
order of the alternate epimeric (R)- and (S)-β2-Caas in the
sequence, suggests a determining influence of the side chain
A systematic theoretical conformational analysis for β3-pep-
tides with the carbohydrate (D-xylo configuration) side chains,
absolutely confirms the preference for right-handedness both in
β3-(R,S,R…)- and β3-(S,R,S…)-amino acid sequences (Table 2,
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chirality. Obviously, the same reasons determine the formation
of exclusively right-handed 12/10- and 10/12-mixed helices in
the corresponding β3-peptides. Furthermore, this study shows
that the shift of the carbohydrate side chain from the Cβ to the
Cα positions, reduces the steric strain at Cβ, and may support the
switch in the handedness. Interestingly, the relief in steric strain
seems to decrease the helix stability in the β2-peptides compared
to the β3-peptides. The suggested new motifs for a predictable
rational design of left-handed secondary structures could stimu-
late further studies towards skeletal and conformational diversity
in foldamers.
initiation of the dynamics the molecular model was built using
the values of the dihedral angles deduced from the coupling con-
stants as well as the nOes. As a first step, the steric contacts were
removed by a mild minimization with the constraints. Sub-
sequently energy minimizations were performed using
steepest descent methods, followed by conjugate gradient
methods for a maximum of 5000 iterations each or RMS deviation
of 0.001 kcal mol−1, whichever was achieved earlier. The 100
structures generated so were energy minimized with the above
protocol and ten of the best possible structures were superimposed
for display.
The HRMS spectra were recorded on Q-TOF mass
spectrometer.
The quantum chemical conformational analysis of the pep-
tides was performed at the HF/6-31G* level of ab initio MO
theory. The details are described in the ESI.†
Experimental section
For peptides 3–6, NMR spectra (1D and 2D experiments) were
acquired at 300 MHz, 400 MHz, 500 MHz, and 600 MHz (1H),
and at 75 MHz, 100 MHz, and 150 MHz (13C). TMS was used
as an internal reference for both 1H and 13C spectra and the
chemical shifts are reported in the δ scale. The low field amide
proton chemical shifts (>7 ppm) were used as an indicator of
their involvement in H-bonding. The presence of H-bonds was
further validated by solvent titration studies by sequentially
adding up to 300 μL of DMSO-d6 in 600 μL of CDCl3 solution
of peptides. The NMR spectra were obtained by using the stan-
dard programs provided by the manufacturer of the instruments.
The 2-D spectra were run in phase sensitive mode by using the
States-TPPI procedure,35 while the TOCSY experiments were
run with a spin locking field of about 10 KHz and a mixing time
of 80 ms, the ROESY experiments were usually performed with
mixing times of 300 ms using a continuous spin-locking field of
about 3 KHz. The 2D data were processed with Gaussian apodi-
zation in both dimensions. The distance constraints for the MD
calculation were obtained from the volume integrals of the cross
peaks in the ROESY spectra using two spin approximation and a
reference distance of 1.8 Å for the geminal protons at Cβ.
tert-Butyl N-2-[(3aR,5R,6S,6aR)-6-methoxy-2,2-dimethyl-
perhydrofuro[2,3-d][1,3]-dioxol-5-yl]allylcarbamate (13)
To a stirred solution of alcohol 11 (4.0 g, 17.39 mmol), Ph3P
(6.83 g, 26.0 mmol) and phthalimide (3.78 g, 26.08 mmol) in
dry THF (80 mL), DIAD (5.16 mL, 26.08 mmol) was added
dropwise at 0 °C and the reaction stirred at room temperature for
6 h. Solvent was evaporated and the residue purified by column
chromatography (silica gel 60–120 mesh, 25% EtOAc in
petroleum ether) to furnish 2-2-[(3aR,5R,6S,6aR)-6-methoxy-
2,2-dimethylperhydrofuro[2,3-d][1,3]dioxol-5-yl]allyl-1,3-isoin-
dolinedione (12; 4.45 g, 71%) as a pale yellow syrup (along with
DIAD byproduct as impurity).
A solution of the above crude 12 (4.41 g, 12.32 mmol) and
hydrazine hydrate (1.20 mL, 24.64 mmol) in MeOH (40 mL)
was stirred at room temperature for 5 h. The reaction mixture
was filtered and solvent evaporated. The above free amine dis-
solved in MeOH (50 mL) was treated with (Boc)2O (4.24 mL,
18.48 mmol) at 0 °C and stirred at room temperature for 6 h.
Solvent was evaporated and the residue purified by column
chromatography (silica gel 60–120 mesh, 14% EtOAc in pet-
roleum ether) to furnish 13 (3.19 g, 79%) as a pale yellow syrup;
[α]2D5 = −69.4 (c 1.1, CHCl3); IR (neat): 3331, 2979, 1705,
The CD spectra were run at room temperature (∼298 K) on
0.2 mM peptide in MeOH using rectangular fused quartz cells of
0.2 cm path length with scan range of 190–260 nm and scanning
speed of 50 nm min−1. The spectra were smoothened using bino-
mial method. The values are expressed in terms of [θ], the total
molar ellipticity (deg cm2 dmol−1 res−1).
1395, 1260, 1125, 1052 cm−1 1H NMR (CDCl3, 300 MHz):
;
δ 5.88 (d, 1H, J = 3.7 Hz, C1H), 5.24 (s, 1H, olefinic), 5.14 (s,
1H, olefinic), 4.67–4.63 (m, 2H, NH, C4H), 4.53 (d, 1H, J =
3.7 Hz, C2H), 3.87 (dd, 1H, J = 7.5, 15.1 Hz, CHN) 3.77 (d, 1H,
J = 3.2 Hz, C3H), 3.66 (dd, 1H, J = 7.5, 15.1 Hz, CHN), 3.39 (s,
3H, OMe), 1.47 (s, 9H, Boc), 1.43 (s, 6H, 2 × CH3); 13C NMR
(CDCl3, 75 MHz): δ 155.9, 140.7, 113.6, 111.49, 104.4, 84.9,
81.4, 92.0, 80.7, 79.2, 57.7, 43.1, 28.3, 28.1, 26.6, 26.1; ESIMS
(m/z, %): 352 (M+ + Na, 100); HRMS (ESI): m/z calculated for
C16H27NO6 (M+ + Na) 352.1736, found 352.1742.
The structural analysis of different conformations, including
molecular modeling calculations and energy minimization, were
carried out with the help of the Insight-II(97.0)/Discover
program. The CVFF force field with default parameters was
employed for all the calculations using a distance dependent
dielectric constant with ε = 4.7 (dielectric constant of CDCl3),
which allows the inclusion of the solvent implicitly. A large
number of restraints were used quantitatively in the restraint MD
calculations. The upper and lower bound of the distance con-
straints have been obtained by diminishing and enhancing the
derived distance by 10%. These constraints with a force constant
of 15 kcal mol−1 Å−2 were applied in the form of a flat-bottom
potential. No restraints for dihedral angles and hydrogen bonds
were included in the structure determination. The complete set of
distance constraints used for peptides 3–6 have been presented in
tabular form in the ESI.† These restraints were used throughout
the MD simulations as well as the minimization. For the
tert-Butyl N-2-[(3aR,5R,6S,6aR)-6-methoxy-2,2-
dimethylperhydrofuro [2,3-d][1,3]-dioxol-5-yl]-3-
hydroxypropylcarbamate (14)
To a cooled (0 °C) solution of 13 (4.6 g, 13.98 mmol) in dry
THF (30 mL) under N2 atmosphere, BH3·DMS (1.32 mL,
13.98 mmol) was added dropwise and the reaction stirred at
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Benzyl (2R)-2-[(3aR,5R,6S,6aR)-6-methoxy-2,2-
dimethylperhydrofuro[2,3-d][1,3]dioxol-5-yl]-3-[(tert-
butoxycarbonyl)amino]propanoate (1) and benzyl (2S)-2-
[(3aR,5R,6S,6aR)-6-methoxy-2,2-dimethylperhydrofuro[2,3-d]-
[1,3]dioxol-5-yl]-3-[(tert-butoxycarbonyl)amino]propanoate (2)
room temperature for 10 h. Subsequently, the above cold (0 °C)
reaction mixture was treated with 2N aq. NaOH solution
(13.8 mL, 3 mL g−1) followed by 30% H2O2 (4.6 mL, 1 mL
g−1). It was warmed to room temperature and stirred for 3 h.
Water (20 mL) was added and the mixture extracted with EtOAc
(3 × 40 mL). Combined organic layers were washed with brine
(20 mL), dried (Na2SO4) and evaporated. The residue was
purified by column chromatography (silica gel 60–120 mesh,
25% EtOAc in petroleum ether) to furnish 14 as a 7 : 3 diastereo-
meric mixture (2.93 g, 60%) as a pale yellow syrup; IR (neat):
To a stirred solution of 16 (2.50 g, 6.92 mmol) and DIPEA
(2.41 mL, 13.84 mmol) in CH2Cl2 (35 mL), BnBr (0.82 mL,
6.92 mmol) and DMAP (cat.) were added at 0 °C and the reac-
tion stirred at room temperature for 8 h. The reaction mixture
was washed with water (20 mL) and brine (20 mL). Solvent was
evaporated and the residue purified by column chromatography.
First eluted (silica gel, 60–120 mesh, 12% EtOAc in petroleum
ether) was 1 (1.25 g, 40%) as a pale yellow syrup; [α]2D5 = +6.07
(c 0.73, CHCl3); IR (neat): 3432, 2928, 1713, 1675, 1249, 1152,
3415, 2980, 2934, 1690, 1518, 1370, 1167, 1079, 1021 cm−1
;
1H NMR (CDCl3, 300 MHz): δ 5.84–5.80 (m, 1H, C1H), 4.92
(m, 1H, NH), 4.54–4.51 (m, 1H, C2H), 4.08 (dd, 0.3H, J = 3.0,
9.8 Hz, C4H), 3.97 (dd, 0.7H, J = 3.0, 9.8 Hz, C4H), 3.78–3.71
(m, 0.6H, C6H), 3.66 (d, 0.3H, J = 3.0 Hz, C2H), 3.63 (d, 0.7H,
J = 3.0 Hz, C2′H), 3.55–3.07 (m, 3.4H, C6H, CHN), 3.43 (s,
0.9H, OMe), 3.41 (s, 2.1H, OMe′), 2.07–1.97 (m, 1H, C5H),
1.50 (s, 3H, CH3), 1.47 (s, 9H, Boc), 1.32 (s, 3H, CH3); 13C
NMR (CDCl3, 125 MHz): δ 158.0, 111.3, 104.9, 104.3, 86.1,
83.5, 81.8, 81.2, 80.0, 78.8, 59.6, 57.5, 57.4, 40.8, 38.3, 28.4,
28.3, 26.6, 26.5, 26.2, 26.1; ESIMS (m/z, %): 370 (M+ + Na,
100); HRMS (ESI): m/z calculated for C16H29NO7 (M+ + Na)
370.1841, found 370.1837.
1
1041 cm−1; H NMR (CDCl3, 300 MHz): δ 7.43–7.31 (m, 5H,
Ar-H), 5.86 (d, 1H, J = 3.7 Hz, C1H), 5.21 (d, 1H, J = 12.2 Hz,
benzylic CH), 5.08 (d, 1H, J = 12.2 Hz, benzylic CH), 4.98 (t,
1H, J = 5.2 Hz, NH), 4.51 (d, 1H, J = 3.7 Hz, C2H), 4.36 (dd,
1H, J = 3.2, 9.8 Hz, C4H), 3.71 (d, 1H, J = 3.2 Hz, C3H),
3.64–3.54 (m, 2H, CβH), 3.14 (s, 3H, OMe), 3.01–2.92 (m, 1H,
CαH), 1.5 (s, 3H, CH3), 1.42 (s, 9H, Boc), 1.31 (s, 3H, CH3);
13C NMR (CDCl3, 75 MHz): δ 171.6, 155.6, 135.7, 128.2,
126.8, 111.5, 105.8, 104.7, 86.0, 84.1, 81.3, 79.2, 66.5, 57.4,
45.0, 40.7, 28.2, 26.7, 26.1; HRMS (ESI): m/z calculated for
C23H33NO8Na (M+ + Na) 474.2103, found 474.2127.
2-[(3aR,5R,6S,6aR)-6-Methoxy-2,2-dimethylperhydrofuro[2,3-d]-
[1,3]dioxol-5-yl]-3-[(tert-butoxycarbonyl)amino]propanoic acid
(16)
Second eluted (silica gel, 15% EtOAc in petroleum ether) was
2 (0.72 g, 23%) as a colourless syrup; [α]2D5 = −36.4 (c 0.48,
CHCl3); IR (neat): 3435, 2931, 1715, 1370, 1166, 1079 cm−1
;
1H NMR (CDCl3, 300 MHz): δ 7.38–7.30 (m, 5H, Ar-H), 5.87
(d, 1H, J = 3.8 Hz, C1H), 5.26 (d, 1H, J = 12.4 Hz, benzylic
CH), 5.13 (d, 1H, J = 12.4 Hz, benzylic CH), 4.96–4.78 (m, 1H,
NH), 4.56 (d, 1H, J = 3.8 Hz, C2H), 4.37 (dd, 1H, J = 2.6,
9.9 Hz, C4H), 3.79 (d, 1H, J = 2.6 Hz, C3H) 3.56–3.43 (m, 1H,
CβH), 3. 43 (s, 3H, OMe), 3.35–3.26 (m, 1H, CβH), 3.12–3.06
(m, 1H, CαH), 1.49 (s, 3H, CH3), 1.43 (s, 9H, Boc), 1.33 (s, 3H,
CH3); 13C NMR (CDCl3, 75 MHz): δ 172.5, 135.6, 135.7,
128.4, 128.0, 127.8, 111.5, 104.5, 99.8, 83.3, 80.9, 78.6, 66.5,
57.3, 44.8, 38.9, 28.2, 26.5, 26.1; HRMS (ESI): m/z calculated
for C23H33NO8Na (M+ + Na) 474.2103, found 474.2125.
To a stirred solution of oxaloyl chloride (2.04 mL, 23.4 mmol),
in dry CH2Cl2 (25 mL), DMSO (3.32 mL, 46.8 mmol) was
added at −78 °C and the reaction stirred at the same temperature
for 15 min. A solution of alcohol 14 (5.41 g, 15.6 mmol) in
CH2Cl2 (25 mL) was added at −78 °C and the reaction stirred at
the same temperature for 2.5 h. The reaction mixture was treated
with Et3N (13.02 mL, 93.6 mmol) at −78 °C. Work up as
described for 9 afforded 15 (5.21 g, 96%) as a yellow syrup,
which was used as such for the next reaction.
To a stirred solution of 15 (2.80 g, 8.11 mmol) in t-butanol :
water (10 mL; 7 : 3), sodium chlorite (1.10 g, 12.17 mmol) and
H2O2 (4.5 mL, 40.57 mmol, 30% aqueous solution) were added
and the reaction stirred at room temperature for 10 h. The reac-
tion mixture was concentrated and the residue dissolved in ethyl
acetate (20 mL). It was washed with water (5 mL), brine (5 mL),
dried (Na2SO4) and evaporated under reduced pressure. The
residue was purified by column chromatography (Silica gel,
60–120 mesh, 60% EtOAc in petroleum ether) to give 16 as a
diastereomeric mixture (2.50 g, 85%) as a colorless liquid; IR
Boc-[(S)-β2-Caa-(R)-β2-Caa]2-OBn (3)
A solution of 35 (0.37 g, 0.53 mmol) in EtOAc (3 mL) was
treated with 10% Pd/C (0.03 g) and stirred at room temperature
for 6 h. Work up as described for 34 gave 36 (0.3 g, 96%) as a
white solid, which was used as such for further reaction.
A solution of 36 (0.28 g, 0.46 mmol) was treated with HOBt
(0.075 g, 0.55 mmol), EDCI (0.106 g, 0.55 mmol) in CH2Cl2
(10 mL) and stirred at 0 °C under N2 atmosphere for 15 min and
treated with 37 (0.30 g, 0.50 mmol) [obtained from 35 on
exposure to CF3COOH (0.5 mL)] and NMM (0.20 mL,
1.84 mmol) under nitrogen atmosphere for 24 h. Work up as
described for 35 and purification of the residue by column
chromatography (silica gel 60–120 mesh, 1.3% methanol in
CHCl3) gave 3 (0.3 g, 54%) as a white solid; m.p. 109–110 °C;
[α]2D5 = −54.5 (c 0.36, CHCl3); IR (KBr): 3379, 2986, 1714,
1666, 1539, 1378, 1167, 1080, 1012 cm−1; 1H NMR (600 MHz,
CDCl3): δ 7.64 (dd, 1H, J = 5.0, 8.1 Hz, NH-4), 7.42–7.32 (m,
(neat): 3419, 2992, 2925, 1708, 1671, 1412, 1163 cm−1 1H
;
NMR (CDCl3, 300 MHz): δ 5.90 (d, 1H, J = 3.9 Hz, C1H),
4.69–4.66 (m, 1H, C4H), 4.55 (d, 1H, J = 3.7 Hz, C2H), 3.84 (d,
1H, J = 3.5 Hz, C3H), 3.50 (s, 3H, OMe), 3.68–3.37 (m, 2H,
CβH), 3.06–2.91 (m, 1H, CαH), 1.47 (s, 3H, CH3) 1.43 (s, 9H,
Boc), 1.32 (s, 3H, CH3); ESIMS (m/z, %): 384 (M+ + Na, 100);
13C NMR (CDCl3, 125 MHz): δ 177.1, 177.0, 155.9, 155.8,
111.5, 111.4, 104.6, 84.4, 83.6, 81.4, 81.0, 80.2, 79.1, 78.9,
65.5, 65.4, 57.7, 57.5, 45.5, 45.2, 41.3, 39.1, 36.9, 29.6, 28.3,
27.4, 26.7, 26.1; HRMS (ESI): m/z calculated for C16H27NO8
(M+ + Na) 384.1634, found 384.1646.
9200 | Org. Biomol. Chem., 2012, 10, 9191–9203
This journal is © The Royal Society of Chemistry 2012

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5H, Ar-H), 7.08 (dd, 1H, J = 4.0, 8.9 Hz, NH-3), 6.70 (dd, 1H,
J = 4.0, 9.2 Hz, NH-2), 5.90 (bd, 1H, NH-1), 5.88 (d, 1H, J =
3.9 Hz, C1H-2), 5.86 (d, 1H, J = 3.9 Hz, C1H-1), 5.84 (d, 1H,
J = 3.9 Hz, C1H-4), 5.83 (d, 1H, J = 3.9 Hz, C1H-3), 5.18 (d,
1H, J = 12.1 Hz, benzylic CH), 5.05 (d, 1H, J = 12.1 Hz,
benzylic CH), 4.63 (dd, 1H, J = 3.4, 9.3 Hz, C4H-4), 4.56 (d,
1H, J = 3.9 Hz, C2H-2), 4.55 (d, 1H, J = 3.9 Hz, C2H-1), 4.54
(d, 1H, J = 3.9 Hz, C2H-3), 4.49 (d, 1H, J = 3.9 Hz, C2H-4),
4.44 (dd, 1H, J = 3.4, 10.0 Hz, C4H-2), 4.24 (dd, 1H, J = 3.1,
9.9 Hz, C4H-1), 4.24 (dd, 1H, J = 3.1, 9.9 Hz, C4H-3), 3.71 (m,
1H, CβH (pro-R)-4), 4.22 (m, 1H, CβH (pro-R)-2), 3.92 (d, 1H,
J = 3.1 Hz, C3H-1), 3.76 (d, 1H, J = 3.4 Hz, C3H-2), 3.74 (ddd,
1H, J = 3.2, 8.9, 11.7 Hz, CβH (pro-R)-3), 3.71 (d, 1H, J =
3.4 Hz, C3H-4), 3.70 (d, 1H, J = 3.1 Hz, C3H-3), 3.48 (s, 3H,
OMe), 3.44 (s. 3H, OMe), 3.39 (ddd, 1H, J = 3.0, 5.5, 10.8 Hz,
CβH (pro-S)-1), 3.39 (m, 1H, CβH (pro-R)-1), 3.35 (m, 1H,
CβH (pro-S)-4), .48 (s, 3H, OMe), 3.34 (s. 3H, OMe), 3.31 (m,
CβH (pro-S)-2), 3.06 (ddd, 1H, J = 4.0, 9.9, 11.7 Hz, CβH (pro-
S)-3), 3.05 (s, 3H, OMe), 2.96 (ddd, 1H, J = 4.6, 6.3, 9.3 Hz,
CαH-4), 2.87 (td, 1H, J = 4.9, 10.0 Hz, CαH-2), 2.73 (dt, 1H,
J = 3.2, 9.9 Hz, CαH-3), 2.67 (ddd, 1H, J = 3.0, 5.9, 9.9 Hz,
CαH-1), 1.47 (s, 3H, CH3), 1.45 (s, 3H, CH3), 1.45 (s, 3H,
CH3), 1.43 (s, 9H, Boc), 1.33 (s, 3H, CH3), 1.30 (s, 9H, 3 ×
CH3), 1.27 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ 172.9
(2C), 172.5, 171.1, 156.2, 135.8, 128.6 (2C), 128.5 (2C), 128.3,
111.9, 111.7, 111.6, 104.9, 104.7 (2C), 104.6, 84.3, 84.0, 83.8,
83.4, 81.7, 81.2, 81.1, 81.0, 79.3, 78.9, 78.8, 78.4, 77.2, 66.7,
57.8, 57.7, 57.6, 57.3, 45.9, 45.7, 45.6, 45.3, 39.8, 39.3, 38.8,
37.5, 29.7, 28.4 (3C), 26.9, 26.8 (2C), 26.7, 26.3 (2C), 26.2
(2C); HRMS (ESI): m/z calculated for C56H84N4O23 (M+ + Na)
1203.5424, found 1203.5391.
1H, CβH (pro-R)-2), 4.61 (d, 1H, J = 3.9 Hz, C2H-2), 4.54 (d,
1H, J = 3.9 Hz, C2H-1), 4.52 (d, 1H, J = 3.9 Hz, C2H-5), 4.50
(d, 1H, J = 3.8 Hz, C2H-6), 4.39 (ddd, 1H, J = 4.5, 10.0,
14.1 Hz, CβH (pro-R)-4), 4.34 (dd, 1H, C4H-5), 4.33 (m, 1H,
CβH (pro-R)-6), 4.28 (dd, 1H, J = 3.1, 10.1 Hz, C4H-3), 4.18
(dd, 1H, J = 3.1, 10.2 Hz, C4H-1), 3.99 (d, 1H, J = 3.1 Hz,
C3H-1), 3.92 (d, 1H, J = 3.3 Hz, C3H-2), 3.82 (m, 1H, CβH
(pro-R)-5), 3.80 (m, 1H, CβH (pro-R)-3), 3.76 (d, 1H, J =
3.3 Hz, C3H-4), 3.69 (d, 1H, J = 3.5 Hz, C3H-6), 3.64 (d, 1H,
J = 3.1 Hz, C3H-3), 3.56 (d, 1H, J = 3.2 Hz, C3H-5), 3.47 (s,
3H, OMe), 3.41 (m, 1H, CβH (pro-R)-4, 3.41 (m, 1H, CβH
(pro-S)-1), 3.40 (s, 3H, OMe), 3.35 (ddd, 1H, J = 2.6, 9.7,
12.1 Hz, CβH (pro-S)-4), 3.34 (s, 3H, OMe), 3.31 (m, 1H, CβH
(pro-S)-2), 3.29 (m, 1H, CβH (pro-S)-6), 3.29 (s, 3H, OMe),
3.22 (s, 3H, OMe), 3.01 (ddd, 1H, J = 4.7, 6.4, 9.0 Hz, CαH-6),
3.00 (s, 3H, OMe), 2.97 (td, 1H, J = 3.2, 9.0 Hz, CαH-2), 2.85
(m, 1H, CαH-4), 2.81 (m, 1H, CαH-5), 2.81 (m, 1H, CβH (pro-
S)-5), 2.62 (ddd, 1H, J = 3.9, 3.9, 10.2 Hz, CαH-1), 2.53 (ddd,
1H, J = 2.6, 10.1, 10.6 Hz, CαH-3), 1.57 (s, 3H, CH3), 1.49 (s,
3H, CH3), 1.48 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.44 (s, 9H,
Boc), 1.40 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.29 (s, 6H, CH3),
1.27 (s, 9H, CH3), 1.25 (s, 3H, CH3); 13C NMR (75 MHz,
CDCl3): δ 173.6, 173.4, 173.1, 173.0, 171.8, 170.4, 156.3,
135.4, 128.8 (2C), 128.5 (2C), 128.2, 112.1, 112.0, 111.6 (2C),
111.5 (2C), 104.8 (2C), 104.7, 104.5, 104.2, 84.3, 84.0, 83.8,
83.7, 83.5, 83.2, 81.8, 81.5, 81.2 (2C), 81.1, 81.0, 79.2, 78.8
(2C), 78.6, 78.3, 77.6, 77.2, 66.8, 57.9, 57.7, 57.6, 57.5, 57.2,
57.1, 47.0, 46.6, 46.3, 45.9, 45.1, 44.9, 39.9, 38.9 (2C), 38.4,
37.2, 36.9, 29.7, 28.4 (3C), 26.9 (2C), 26.8 (2C), 26.7 (2C),
26.4 (2C), 26.3 (2C), 26.2 (2C); HRMS (ESI): m/z calculated for
C78H118N6O33 (M+ + Na) 1689.7632, found 1689.7606.
Boc-[(S)-β2-Caa-(R)-β2-Caa]3-OBn (4)
Boc-[(R)-β2-Caa-(S)-β2-Caa]2-OBn (5)
A solution of 3 (0.26 g, 0.22 mmol) in EtOAc (2 mL) was
treated with 10% Pd/C (0.03 g) and stirred at room temperature
for 6 h. Work up as described for 34 gave 38 (0.22 g, 94%) as a
white solid, which was used as such for further reaction.
A solution of ester 40 (0.31 g, 0.44 mmol) in EtOAc (3 mL) was
treated with 10% Pd/C (0.06 g) and stirred at room temperature
for 6 h. Work up as described for 34 gave 41 (0.26 g, 98%) as a
white solid, which was used as such for further reaction.
A solution of 38 (0.19 g, 0.17 mmol) was treated with HOBt
(0.028 g, 0.20 mmol), EDCI (0.040 g, 0.20 mmol) in CH2Cl2
(5 mL) and stirred at 0 °C under N2 atmosphere for 15 min and
treated with 37 (0.10 g, 0.18 mmol) [obtained from 35 on
exposure to CF3COOH (0.3 mL)] and NMM (0.076 mL,
0.69 mmol) in CH2Cl2 (3 mL) under nitrogen atmosphere for
24 h. Work up as described for 35 and purification of the residue
by column chromatography (silica gel 60–120 mesh, 1.7%
methanol in CHCl3) afforded 4 (0.09 g, 32%) as a white solid;
m.p. 130–131 °C; [α]2D5 = −107.3 (c 0.395, CHCl3); IR (KBr):
A solution of 41 (0.26 g, 0.43 mmol) was treated with HOBt
(0.07 g, 0.52 mmol), EDCI (0.10 g, 0.52 mmol) in CH2Cl2
(5 mL) and stirred at 0 °C under N2 atmosphere for 15 min and
then treated with 42 (0.32 g, 0.47 mmol) [obtained from 40 on
exposure to CF3COOH] and NMM (0.19 mL, 1.72 mmol) in
CH2Cl2 (3 mL) under nitrogen atmosphere for 24 h. Workup as
described for 35 and purification of the residue by column
chromatography (silica gel 60–120, 1.2% methanol in CHCl3)
gave 5 (0.27 g, 55%) as a white solid; m.p. 110–111 °C; [α]D25
−71.0 (c 0.27, CHCl3); IR (KBr): 3379, 2986, 1714, 1666,
1539, 1378, 1167, 1080, 1012 cm−1 1H NMR (600 MHz,
=
3325, 2987, 1715, 1656, 1456, 1380, 1219, 1080 cm−1
;
1H
;
NMR (600 MHz, CDCl3): δ 8.46 (dd, 1H, J = 4.8, 8.3 Hz,
NH-6), 8.31 (dd, 1H, J = 2.8, 10.0 Hz, NH-4), 8.10 (dd, J = 2.8,
9.6 Hz, NH-3), 7.54 (bd, 1H, NH-5), 7.45–7.32 (m, 5H, Ar-H),
6.78 (bd, 1H, NH-2), 5.88 (d, 1H, J = 3.9 Hz, C1H-2), 5.86 (d,
1H, J = 3.9 Hz, C1H-5), 5.84 (d, 1H, J = 3.8 Hz, C1H-6), 5.82
(d, 1H, J = 3.9 Hz, C1H-1), 5.78 (bd, 1H, NH-1), 5.75 (d, 1H,
J = 3.8 Hz, C1H-3), 5.20 (d, 1H, J = 11.9 Hz, benzylic CH),
5.07 (d, 1H, J = 11.9 Hz, benzylic CH), 4.83 (dd, 1H, J = 3.3,
9.9 Hz, C4H-4), 4.70 (dd, 1H, J = 3.3, 9.9 Hz, C4H-2), 4.68 (m,
CDCl3): δ 7.49 (dd, 1H, J = 4.8, 7.5 Hz, NH-3), 7.45–7.29 (m,
5H, Ar-H), 7.19 (dd, 1H, J = 4.8, 7.6 Hz, NH-2), 6.65 (dd, 1H,
J = 4.8, 7.3 Hz, NH-4), 5.89 (d, 1H, J = 3.8 Hz, C1H-4), 5.87
(d, 1H, J = 3.8 Hz, C1H-1), 5.86 (d, 1H, J = 3.8 Hz, C1H-3),
5.83 (d, 1H, J = 3.8 Hz, C1H-2), 5.37 (dd, 1H, J = 5.3, 7.4 Hz,
NH-1), 5.22 (d, 1H, J = 12.5 Hz, benzylic CH), 5.17 (d, 1H, J =
12.5 Hz, benzylic CH), 4.58 (d, 1H, J = 3.8 Hz, C2H-4), 4.54 (d,
1H, J = 3.8 Hz, C2H-1), 4.54 (d, 1H, J = 3.8 Hz, C2H-2), 4.53
(d, 1H, J = 3.8 Hz, C2H-3), 4.48 (dd, 1H, J = 3.3, 9.1 Hz,
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C4H-3), 4.43 (dd, 1H, J = 3.3, 9.7 Hz, C4H-4), 4.40 (dd, 1H, J =
3.3, 10.0 Hz, C4H-1), 4.25 (dd, 1H, J = 3.3, 9.0 Hz, C4H-2),
3.91 (ddd, 1H, J = 5.0, 7.5, 13.5 Hz, CβH (pro-R)-3), 3.79 (ddd,
1H, J = 4.9, 7.4, 13.9 Hz, CβH (pro-R)-1), 3.78 (d, 1H, J =
3.3 Hz, C3H-4), 3.77 (d, 1H, J = 3.3 Hz, C3H-1), 3.74 (d, 1H,
J = 3.3 Hz, C3H-2), 3.70 (d, 1H, J = 3.3 Hz, C3H-3), 3.69 (ddd,
1H, J = 4.0, 7.6, 13.5 Hz, CβH (pro-R)-2), 3.63 (ddd, 1H, J =
4.0, 7.6, 13.5 Hz, CβH (pro-R)-4), 3.45 (ddd, 1H, J = 5.0, 5.3,
13.9 Hz, CβH (pro-S)-1), 3.45 (s. 3H, OMe), 3.40 (s. 3H, OMe),
3.39 (td, 1H, J = 4.8, 13.5 Hz, CβH (pro-S)-3), 3.37 (ddd, 1H,
J = 4.8, 6.5, 13.5 Hz, CβH (pro-S)-4), 3.32 (s, 3H, OMe), 3.31
(s. 3H, OMe), 3.16 (ddd, 1H, J = 4.8, 8.7, 13.5 Hz, CβH (pro-
S)-2), 3.07 (ddd, 1H, J = 4.1, 6.5, 9.7 Hz, CαH-4), 2.82 (ddd,
1H, J = 4.9, 5.0,, 10.0 Hz, CαH-1), 2.74 (ddd, 1H, J = 4.0,. 8.7,
9.0 Hz, CαH-2), 2.72 (ddd, 1H, J = 4.8, 5.0, 9.1 Hz, CαH-3),
1.51 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.45 (s, 3H, CH3), 1.43 (s,
3H, CH3), 1.41 (s, 9H, Boc), 1.31 (s, 3H, CH3), 1.29 (s, 3H,
CH3), 1.27 (s, 3H, CH3), 1.24 (s, 3H, CH3); 13C NMR (75 MHz,
CDCl3): δ 172.6, 172.4, 172.1, 171.4, 155.9, 135.7, 128.5 (2C),
128.1, 128.0 (2C), 111.6 (2C), 111.5, 104.7, 104.6 (2C), 83.9,
83.8, 83.5, 83.4, 81.3, 81.2, 81.1, 81.0, 79.4 (2C), 79.0, 78.9,
77.2, 66.7, 57.7, 57.6 (2C), 57.4, 45.9, 45.6 (2C), 44.7, 40.7,
39.3, 37.8, 37.7 (2C), 28.3 (4C), 26.8 (2C), 26.7 (2C), 26.6
(2C), 26.1 (2C); HRMS (ESI): m/z calculated for C56H84N4O23
(M+ + Na) 1203.5424, found 1203.5390.
C4H-4), 4.37 (ddd, 1H, CβH (pro-R)-3), 4.33 (m, 1H, CβH (pro-
R)-1), 4.26 (dd, 1H, J = 3.2, 9.7 Hz, C4H-2), 4.21 (ddd, 1H, J =
6.9, 8.5, 14.0 Hz, CβH (pro-R)-5), 3.93 (d, J = 3.4 Hz, C3H-1),
3.88 (ddd, 1H, J = 2.7, 10.1, 13.1 Hz, CβH (pro-R)-2), 3.82
(ddd, 1H, J = 3.7, 10.0, 12.1 Hz, CβH (pro-R)-4), 3.79 (d, 1H,
J = 3.4 Hz, C3H-6), 3.77 (d, 1H, J = 3.4 Hz, C3H-5), 3.73 (d,
1H, J = 3.4 Hz, C3H-3), 3.69 (d, 1H, J = 3.2 Hz, C3H-2), 3.59
(m, 1H, CβH (pro-R)-6), 3.57 (d, 1H, J = 3.2 Hz, C3H-4), 3.45
(s, 3H, OMe), 3.41 (m, 1H, CβH (pro-S)-6, 3.40 (m, 1H, CβH
(pro-S)-1), 3.40 (s, 3H, OMe), 3.35 (s, 3H, OMe), 3.30 (m, 1H,
CβH (pro-S)-3), 3.29 (s, 3H, OMe), 3.26 (s, 3H, OMe), 3.23 (s,
3H, OMe), 3.19 (td, 1H, J = 4.1, 14.0, CβH (pro-S)-5), 3.06
(ddd, 1H, J = 4.3, 7.1, 9.8 Hz, CαH-6), 2.94 (td, 1H, J = 3.2,
10.4 Hz, CαH-1), 2.85 (ddd, 1H, J = 4.1, 6.9, 9.4 Hz, CαH-5),
2.85 (m, 1H, CβH (pro-S)-2), 2.79 (ddd, 1H, J = 2.6, 9.7,
12.1 Hz, CβH (pro-S)-4), 2.79 (ddd, 1H, CαH-3), 2.73 (ddd,
1H, J = 3.7, 9.7, 9.8 Hz, CαH-4), 2.64 (ddd, 1H, J = 2.7, 9.7,
10.3 Hz, CαH-2), 1.57 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.47 (s,
6H, CH3), 1.46 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.39 (s, 9H,
Boc), 1.34 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.29 (s, 6H, CH3),
1.27 (s, 6H, CH3); 13C NMR (75 MHz, CDCl3): δ 173.6, 172.4,
172.3 (2C), 171.8, 170.4, 156.3, 135.8, 128.5 (2C), 128.3, 128.1
(2C), 111.9, 111.7, 111.6 (2C), 111.5, 111.4, 104.8, 104.7,
104.6, 104.5 (2C), 104.2, 84.0, 83.9 (2C), 83.4, 83.3 (2C), 81.8,
81.5, 81.1, 81.0, 79.9 (2C), 79.1, 79.0, 78.9, 78.7, 77.9, 77.3,
66.7, 57.9, 57.8, 57.5 (3C), 57.1, 47.0, 46.9 (2C), 45.1, 45.0,
44.9, 40.3, 40.1, 38.8, 37.9, 37.1, 37.0, 29.7, 28.4 (3C), 26.9
(2C), 26.8 (2C), 26.7 (2C), 26.3 (5C), 26.0; HRMS (ESI): m/z
calculated for C78H118N6O33 (M+ + Na) 1689.7632, found
1689.7647.
Boc-[(R)-β2-Caa-(S)-β2-Caa]3-OBn (6)
A solution of 5 (0.23 g, 0.19 mmol) in EtOAc (3 mL) was
treated with 10% Pd/C (0.03 g) and stirred at room temperature
for 6 h. Work up as described for 34 gave 43 (0.2 g, 97%) as a
white solid, which was used as such for further reaction.
Acknowledgements
A solution of 43 (0.18 g, 0.16 mmol) was treated with HOBt
(0.026 g, 0.19 mmol), EDCI (0.038 g, 0.19 mmol) in CH2Cl2
(5 mL) and stirred at 0 °C under N2 atmosphere for 15 min and
then treated with 42 (0.11 g, 0.166 mmol) [obtained from 40 on
exposure to CF3COOH] and NMM (0.072 mL, 0.664 mmol) in
CH2Cl2 (4 mL) under nitrogen atmosphere for 24 h. Work up as
described for 35 and purification of the residue by column
chromatography (silica gel 60–120 mesh, 1.7% methanol in
CHCl3) furnished 6 (0.10 g, 36%) as a white solid; m.p.
127–129 °C; [α]2D5 = −115.2 (c 0.35, CHCl3); IR (KBr): 3325,
NYR, RR, GS, DC, BS are thankful to UGC and CSIR, New
Delhi for financial support in the form of fellowship. H.-J. H. is
obliged to Deutsche Forschungsgemeinschaft for financial
support (Sonderforschungsbereich 610 and project HO/2346/
1–3).
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(600 MHz, CDCl3) δ 8.36 (dd, 1H, J = 2.9, 10.6 Hz, NH-3),
8.16 (dd, 1H, J = 4.1, 8.5 Hz, NH-5), 7.94 (dd, J = 2.5, 10.6 Hz,
NH-2), 7.68 (dd, J = 2.6, 10.0 Hz, NH-3), 7.39–7.30 (m, 5H,
Ar-H), 6.96 (t, 1H, J = 6.2 Hz, NH-6), 5.89 (d, 1H, J = 3.9 Hz,
C1H-6), 5.87 (d, 1H, J = 3.9 Hz, C1H-1), 5.86 (d, 1H, J =
3.9 Hz, C1H-4), 5.86 (d, 1H, J = 3.9 Hz, C1H-5), 5.80 (d, 1H,
J = 3.9 Hz, C1H-3), 5.72 (d, 1H, J = 3.9 Hz, C1H-2), 5.21 (d,
1H, J = 12.3 Hz, benzylic CH), 5.15 (d, 1H, J = 12.3 Hz,
benzylic CH), 5.06 (dd, J = 2.8, 10.0 Hz, NH-1),4.79 (dd, 1H,
J = 3.4, 10.0 Hz, C4H-3), 4.60 (dd, 1H, J = 3.4, 9.4 Hz, C4H-5),
4.60 (d, 1H, J = 3.9 Hz, C2H-1), 4.59 (d, 1H, J = 3.9 Hz,
C2H-6), 4.53 (d, 1H, J = 3.9 Hz, C2H-4), 4.51 (d, 1H, J =
3.9 Hz, C2H-2), 4.51 (dd, 1H, J = 3.4, 10.4 Hz, C4H-1), 4.49 (d,
1H, J = 3.9 Hz, C2H-3), 4.48 (d, 1H, J = 3.9 Hz, C2H-5), 4.41
(dd, 1H, J = 3.4, 9.8 Hz, C4H-6), 4.37 (dd, 1H, J = 3.2, 9.8 Hz,
9202 | Org. Biomol. Chem., 2012, 10, 9191–9203
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