Design and synthesis of trans-3-aminopyran-2-carboxylic acid (APyC) and α/β-peptides with 9/11-helix

Article abstract of DOI:10.1039/c1ob06279d

A new β-amino acid, trans-3-aminopyran-2-carboxylic acid (APyC), was designed and synthesized from (R)-glyceraldehyde derivative and used in the synthesis of α/β-peptides in a 1:1 alternating pattern with d-Ala. The presence of oxygen atom at the Cβ²

Full text of DOI:10.1039/c1ob06279d

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PAPER
Design and synthesis of trans-3-aminopyran-2-carboxylic acid (APyC) and
a/b-peptides with 9/11-helix†‡
Gangavaram V. M. Sharma,*a Kodeti Srinivas Reddy,a Shaik Jeelani Basha,b Kondreddi Ravinder Reddya and
Akella V. S. Sarma*b
Received 28th July 2011, Accepted 1st September 2011
DOI: 10.1039/c1ob06279d
A new b-amino acid, trans-3-aminopyran-2-carboxylic acid (APyC), was designed and synthesized
from (R)-glyceraldehyde derivative and used in the synthesis of a/b-peptides in a 1 : 1 alternating
2
pattern with D-Ala. The presence of oxygen atom at the Cb -position in APyC was envisaged to provide
opportunity for additional interaction. These hybrid peptides have shown the presence of 9/11-helix
through extensive NMR and MD studies. The amide protons of D-Ala, in addition to participating in
9-mr H-bonding with CO of succeeding b-residue, were also involved in additional electrostatic
interaction with pyran ring oxygen of preceding b-residue, which facilitated further stabilization to the
9/11-mixed helix. The study thus results in a new ‘motif’ for a 9/11-helix, and the first example from a
cyclic b-amino acid.
amino acid. Gellman et al.8 have published several studies on
Introduction
a/b-peptides and arrived at different structural and biological
features. The theoretical studies,9 from the initial years of the
work in this area, complemented the experimental results. Further,
the studies on a/b-peptides by Hofmann et al.10 predicted 9/11-
helix (i→i+1/i←i+3) and 11/9-helix (i←i+3/i→i+1) as the most
stable conformations.
Designing and engineering of bio-molecules such as peptides and
proteins,1 either to understand their functions or to define new
functions, has become an important area of research towards bio-
medical and materials applications. The initial studies of Gellman
et al. and Seebach et al. on b-peptides, resulting in new 12- and
14-helical patterns,2 led to the emergence of a frontier field of
‘foldamers’.3 Further quest of the investigators in this active field
resulted in a wealth of new peptide classes with heterogeneous
backbones having more than one type of monomer residues. These
efforts led to the synthesis of a/b-, a/g-, a/d-, a/e-, b/g- and
other classes of hybrid peptides4,5 with a variety of unidirectional
and mixed helical patterns. More recently, ‘hybrid helices’ resulted
from tethering together different helix types as shown by Gellman
et al.6 and Sharma et al.7
In the case of heterogeneous peptides, extensive studies have
been carried out on a/b-peptides. The first results by Gellman
et al.4a and Reiser et al.4b have shown ‘split’ 11, 14/15-helices and
13-helix respectively. Later designs by Sharma et al.4c with L-Ala
and (S)-b-Caa (C-linked carbo b-amino acid) led to the emergence
of another new 9/11(11/9)-mixed helix, while Chandrasekhar
et al.4d reported a split pattern in a/b-peptides derived from sugar
In the earlier study on a/b-peptides, Gellman et al.4a,11 noticed
that the ACPC-containing peptides showed non-sequential nOes
giving strong evidence for a folded conformation, while, the
peptides made from ACHC showed no helical pattern. In view
of the above observations, it was felt worthwhile to explore
possibilities of additional interactions which may stabilize folded
structures in six membered side chains like ACHC. The studies
by Grierson et al.12 on the oligomers of phenylisoserine, based on
the literature findings13 on taxol side chain, revealed stabilization
of C6 stranded folds, by additional 5-mr H-bonding14 between
the N–H ◊ ◊ ◊ O (hydroxyl). We have thus proposed to introduce
2
an oxygen atom at b -position in ACHC, replacing the ‘–CH2–’
group, to result in the design of a new pyran based b-amino acid,
trans-3-aminopyran-2-carboxylic acid (APyC). A literature survey
on peptides from sugar amino acids (SAA),15a–f though revealed
no such H-bonding pattern, a recent report by Gervay-Hague
et al.15g on sialic acid derived a/d-hybrid peptides has shown such
an interaction of the pyranose ring oxygen of Neu2en with L-Gluc
HN, which appears to be unique to sialic acid moieties. Based on
the above assumptions, the present study describes the design and
synthesis of new b-amino acid, APyC 1. Conversion of 1 into a
series of a/b-peptides 3–10 (Fig. 1 and 2) with 1 : 1 alternation of
D-Ala 2, and the conformational analysis of the above peptides by
extensive NMR spectroscopy supported by Molecular Dynamics
(MD) and Infrared (IR) studies.
aOrganic Chemistry Division III, CSIR-Indian Institute of Chemical Tech-
nology, Hyderabad, 500607, India. E-mail: esmvee@iict.res.in
bNMR Group, CSIR-Indian Institute of Chemical Technology, Hyderabad,
500607, India. E-mail: ssav@iict.res.in
† This article is part of an Organic & Biomolecular Chemistry web theme
issue on Foldamer Chemistry.
‡ Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06279d
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Fig. 1 Structures of the peptides 3–7 (arrows show hydrogen bonding).
Treatment of 17 with diazomethane (CH2N2) generated in situ at
0 ◦C, for 1 h afforded ester 1 in 79% yield (over 3 steps).
Results and discussions
1. Synthesis of amino acid 1
The new pyran b-amino acid (1) was synthesized from the known
compound 11a, which was derived from (R)-2,3-O-isopropylidene-
D-glyceraldehyde 1116 (Scheme 1). Accordingly, reaction of 11a
with allyl bromide (NaH, DMF) afforded the allyl ether 12
(79%). RCM reaction on diene 12 in toluene at reflux for
8 h in the presence of Grubb’s catalyst I17 (5 mol%) afforded
13 (80%).
Debenzylation of 13 under Birch reaction conditions (Li, Liq.
NH3) at -78 ◦C in THF for 1 h afforded the amide 14 (68%),
which on hydrogenation with 10% Pd–C in MeOH under hydrogen
atmosphere gave 15 (93%). Sequential oxidation of alcohol 15
under Swern reaction conditions using (COCl)2, DMSO and Et3N
in CH2Cl2, followed by further oxidation of aldehyde 16 using
NaClO2 and 30% H2O2 in t-BuOH–H2O furnished the acid 17.
2. Synthesis of peptides 3–10
The peptides 3–10 (Fig. 1 and 2) were prepared by alternating use
of 1 and D-Ala 2, under standard peptide coupling18 conditions
with EDCI, HOBt and DIPEA in CH2Cl2. Accordingly, APyC
monomer 1 on hydrolysis with LiOH in THF : MeOH : H2O
(3 : 1 : 1) furnished the acid 17, while, 1 on exposure to CF3COOH
in CH2Cl2 gave the salt 17a (Scheme 2). Peptide coupling of acid
17 in the presence of EDCI, HOBt and DIPEA in CH2Cl2 with
the HCl salt of D-Ala-OMe (18b) gave the dipeptide 3 (93%). Base
(LiOH) hydrolysis of ester 3 afforded the acid 19a, while, reaction
of 3 with CF3COOH in CH2Cl2 furnished the salt 19b. Acid 19a
on coupling (EDCI, HOBt and DIPEA) with salt 17a in CH2Cl2
gave the tripeptide 20 (75%). Hydrolysis of tripeptide 20 with base
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Scheme 1 Synthesis of trans APyC monomer 1.
HOBt and DIPEA in CH2Cl2 afforded the tripeptide 24 (83%).
Base (LiOH) hydrolysis of peptide 24 gave the acid 24a, which
on further coupling with the salt 19b under standard peptide
coupling conditions furnished the pentapeptide 9 (67%). Acid 21a
on coupling with 21b in the presence of EDCI, HOBt and DIPEA
in CH2Cl2 gave the tetrapeptide 8 (75%). Similarly, base hydrolysis
of 8 gave the acid 25a, which on coupling with the salt 21b in
the presence of EDCI, HOBt and DIPEA in CH2Cl2 afforded the
hexapeptide 10 (65%).
3. Conformational analysis of peptides 3–10
The peptides 3–10 (Fig. 1 and 2), having alternating 1 and
2 residues, were prepared under standard peptide coupling18
19
conditions with EDCI, HOBt and DIPEA in CH2Cl2 and the
1H NMR studies for all the peptides were undertaken as 3–5 mM
solution in CDCl3.19
1
The H NMR spectrum19 of 3 showed low field chemical shift
(d) > 7 ppm for NH(2), which along with small value of Dd
(0.56) in solvent titration study20 confirmed its participation in
3
possible interaction. JCaH-CbH > 9 Hz corresponds to a trans
orientation of CaH-CbH, implying a value of (q) ª 60◦ for dihedral
angle N-C(b)-C(a)-CO. A strong nOe correlation NH(1)/CaH(1)
further supports this value. The nOe correlation NH(1)/Cg¢H,
Fig. 2 Structures of the peptides 8–10 (arrows show hydrogen bonding).
3
CbH(1)/CdH(1) together with large value of JCg¢H-CbH and four
4
bond ‘w’ coupling confirmed C1 chair conformation of pyran
(LiOH) gave the acid 20a. Likewise, acid 18a on coupling with
the salt 17a under standard peptide coupling conditions furnished
the dipeptide 21 (86%). Hydrolysis of dipeptide 21 with LiOH
gave the acid 21a, while, reaction of 21 with CF3COOH in CH2Cl2
afforded the salt 21b. Peptide coupling of acid 19a with the salt 19b
in the presence of EDCI, HOBt and DIPEA in CH2Cl2 afforded
the tetrapeptide 4 (80%). Base (LiOH) hydrolysis of peptide 4 gave
the acid 22a, which on further coupling with 19b in the presence
of EDCI, HOBt and DIPEA afforded the hexapeptide 6 (64%).
Similarly, peptide coupling of acid 20a with the salt 21b in the
presence of EDCI, HOBt and DIPEA in CH2Cl2 afforded the
pentapeptide 5 (75%).
ring as shown in the Fig. 3. Additionally, the presence of weak nOe
correlation between CdH(1)/NH(2) and CeH(1)/NH(2) suggests
the proximity of NH of D-Ala and ring oxygen in the pyran
of APyC, providing evidence for a weak interaction between
NH(2) and O(Cb(1)). In order to understand such conformational
constraints in more detail, the study was extended to higher
homologues (Fig. 1 and 2).
The proton NMR spectrum19 of tetrapeptide 4 in CDCl3
showed a substantial dispersion in the amide region, indicating
the presence of a secondary structure. The d > 7 ppm for NH(2)–
NH(4) along with small change in the chemical shift values in
solvent titration studies20 confirmed their participation in H-
bonding. For b-amino acid residues b(1) and b(3), the value of
3JNH-CbH > 8.9 Hz was consistent with a fb (C(O)-N-Cb-Ca) ª
Peptide 5 on hydrolysis of with LiOH gave the acid 23a, which
on further coupling with the salt 21b under standard peptide
coupling conditions afforded the heptapeptide 7 (43%). Likewise,
acid 21a on coupling with the salt 18b in the presence of EDCI,
-1◦20◦ and large JCaH-CbH value of ~10.0 Hz corresponds to q ª
3
60 . For Ala(2), 3JNH-CaH value of 9.3 Hz correspond to a fa ª 120
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Scheme 2 Synthesis of peptides 3–10.
The nOe correlations CbH(1)/NH(3), CaH(2)/NH(3) in 4
support an 11-membered H-bond between Boc CO and NH(3).
Further, the nOe correlation NH(2)/NH(3) supports the presence
of a 9-mr H-bonding between NH(2) and CO(3). The above
characteristic nOes confirm the presence of a 9/11-helix21 in
peptide 4. Similar to the observation made for peptide 3, the
MD structures of 4 revealed the presence of proximity for NH(2)
and NH(4) to the pyran ring oxygen15g of preceding b(1) and
˚
b(3) residues. The distance of 2.4 A provides an evidence for
weak electrostatic interactions in peptide 4.22 This observation
was further supported by weak nOe correlations CdH(1)/NH(2),
CeH(1)/NH(2), CdH(3)/NH(4), CeH(3)/NH(4).
A systematic propagation of H-bonding pattern was observed in
peptides 5 and 6. All the nOe correlations and coupling constants
defined the presence of 9/11-helix, with additional stabilization
from the weak electrostatic interactions between the amide protons
a(i) and pyran oxygen of b(i-1). The characteristic nOes have been
labeled in the ROESY spectrum of peptide 6 (Fig. 4).
1H NMR spectrum19 of peptide 7 showed a very well resolved
amide region. Their low field d and small Dd values in solvent titra-
tion studies,20 imply that many of the amide protons participated
in H-bonding. Large values of 3JNH-CbH > 9 Hz and 3JCbH-CaH > 9 Hz
are consistent with f ª -120◦ and q ª 60◦ for b-amino acid residues
b(1), b(3) and b(5). For the second and the fourth D-Ala residues,
Fig.
3
Representation of electrostatic interaction of (D-Ala)-
NH—O-(pyran) in peptide 3.
3
whereas for Ala (4), JNH-CaH value of 7.2 Hz is not very distinct
and may reflect fraying in the termini.
3
large JNH-CaH > 9 Hz are consistent with fa (C(O)-N-Ca-C(O))
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Fig. 4 ROESY spectrum of peptide 6.
ª 120◦. The characteristic nOe correlation CbH(i)/NH(i+2) was
observed for the first and third b-residues, while, due to the spectral
overlap, several characteristic nOe correlations were ambiguous,
though NH(i)/NH(i+1) (i = 2 and 4) were distinctly observed.
For the restrained molecular dynamics (MD) studies,19 the
constraints were derived from the intensities of the nOe cross peaks
in the ROESY spectra using two spin approximation. Fig. 5 shows
20 superimposed minimum energy structures of peptides 4 and 6,
in which the RMSD of the backbone and heavy atoms are 0.35
˚
˚
and 0.53 A for 4 and 0.40 and 0.65 A for 6 respectively. The average
backbone dihedral angles are derived by excluding the first and
last residues. For 4, f, q and y values for APyC are -92 2◦, 65
2◦, 74 3◦ respectively, whereas, f = 140 3◦ and y = -75 2◦ for
D-Ala. Similarly, corresponding values for 6 are -95 2◦, 66 1◦,
77 3◦, 136 3◦ and -75 2◦. The f, q and y in a/b-peptides for
9/11-helix predicted theoretically by Hofmann et al.10 are ~-90◦,
~60◦, ~90◦ respectively for b-residue, and f ~ 130◦ and y ~ -60◦
for a-residue.
The above peptides consistently showed absorption maxima
in the NH- stretching region around 3310–3330, 3400 and 3440
cm-1 (Fig. 6). The small NH stretch at 3440 cm-1 corresponds
to non-hydrogen bonded NH. The major band at 3310–3330
cm-1 corresponds to the conventional H-bonding and increases
with length of the peptide chain. Another small NH stretch at
3400 cm-1 may be attributed to weak interaction between NH
of a(i) and pyran oxygen of b(i-1), which is in accordance with
the observation made by MD studies.19 From the IR studies it
was concluded that the folding of these peptides is dominated
by 9/11-H-bonding and further stabilized by weak electrostatic
interaction.22 The distance between the amide proton and pyran
Fig. 5 MD structures of peptides: (A) 4 and (B) 6 (stereoview of 20
superimposed minimum energy structures; hydrogens are removed after
the calculations for clarity).
˚
oxygen of preceding residue was measured as 2.4 A and an angle
of 102◦.15g Though the distance is higher than for a normal H-
bond, similar observation in the form of bifurcated H-bonds were
reported by Gellman et al.14c Further, they compared the relative
stabilities of these H-bonds using IR data. The 5-mr H-bonding is
characterized by N–H ◊ ◊ ◊ O angle of 101◦ and H ◊ ◊ ◊ O distance of
˚
2.34 A. In another study, inter (5-mr)/intra (6-mr) bifurcated H-
bonding interactions were reported in a homo b-peptide.13 The
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mixed helix, the amide proton of a(i) showed a weak electrostatic
2
interaction with the oxygen atom present in the Cb -position of
the b(i-1), which may strengthen the 9/11-folds realized. The
present design generated a new motif for 9/11-helix, for the first
time from a cyclic b-amino acid (APyC). The participation of
oxygen substituent in weak electrostatic interaction in APyC,
opens up new fronts for the use of such monomers in different
designs. Further studies on the use of new b-amino acid are in
progress.
Experimental
NMR spectra (1D and 2D experiments) for peptides 3–10 were
obtained at 500 and 600 MHz (1H), and at 75 MHz, 100 MHz, and
150 MHz (13C). Chemical shifts are reported in d scale with respect
to internal TMS reference. IR spectra were recorded with FT-IR
spectrometer. Melting points were determined in open capillaries
and were not corrected.
Fig. 6 IR spectra of peptides 4–7.
Restrained molecular dynamics (MD) studies were carried
out using the INSIGHT-II Discover13 module employing an
SGI workstation. The constraints were derived from the volume
integrals obtained from the ROESY spectra using a two-spin
˚
5-mr H-bond shows a mean distance O ◊ ◊ ◊ HN ~ 2.26 A and
mean angle O ◊ ◊ ◊ H–N ~ 109◦, whereas, the 6-mr H-bond shows a
˚
mean distance NH ◊ ◊ ◊ O ~ 2.35 A and mean angle formed by N–
H ◊ ◊ ◊ O C ~ 118◦, which spans the length of the peptide. Though
the 5-mr and 6-mr H-bonding14b is commonly observed in natural
products,14a their overall contribution to the conformations in the
oligomers is small.
˚
approximation and a reference distance of 1.8 A for the geminal
protons. The upper and lower bound of the distance constraints
have been obtained by enhancing and reducing the derived
distance by 10%.
Based on the above results, the study was extended to peptides
8–10 (Fig. 2) with an a-residue at the N-terminus. As was observed
in peptides 4–7, these peptides also showed a 9/11-mixed helical
pattern with a-residue participating in 9-mr H-bonding and
b-residue in 11-mr H-bonding. Though several amide protons
showed their involvement in H-bonding as deduced from dNH and
the solvent titration studies,20 the first and second amide protons
do not seem to participate in H-bonding. The amide protons of
terminal a-residues show significantly large value of Dd (~ 1 ppm)
Boc-(S,S)-APyC-D-Ala -OMe (3)
To a solution of ester 1 (0.6 g, 2.31 mmol) in THF : MeOH : H2O
(3 : 1 : 1), LiOH (0.13 g, 5.41 mmol) was added at 0 ◦C and stirred
at room temperature for 2 h. The reaction mixture was adjusted to
pH 2–3 with aq. 1 N HCl and extracted with EtOAc (2 ¥ 15 mL).
The organic layer was dried (Na2SO4) and evaporated to give acid
17.
3
in solvent titration studies. The value of JNH-CaH > 9.0 Hz for
D-Ala(3) was consistent with a f ª 120◦. Large values of 3JNH-CbH
A mixture of acid 17 (0.6 g, 2.44 mmol), HOBt (0.39 g, 2.93
mmol) and EDCI (0.56 g, 2.93 mmol) in CH2Cl2 (25 mL) was
stirred at 0 ◦C for 15 min and treated with salt 18b (0.39 g,
2.93 mmol) under N2 atmosphere and continued stirring at room
temperature for 8 h. The reaction mixture was quenched at 0 ◦C
with sat. NH4Cl (15 mL) solution. After 10 min, reaction mixture
was diluted with CHCl3 (50 mL), washed with 1 N HCl (25 mL),
water (25 mL), aq. sat. NaHCO3 (15 mL) and brine (25 mL). The
organic layers were dried (Na2SO4), evaporated and the residue
was purified by column chromatography (60–120 mesh Silica gel,
50% ethyl acetate in pet. ether) to afford 3 (0.72 g, 90%) as a white
solid; m.p. 155 ◦C; [a]D = +45.8 (c 0.5, CHCl3); IR (CHCl3): 3410,
3009, 2981, 2863, 1741, 1710, 1673, 1510, 1502, 1452, 1367, 1343,
1307 cm-1;1H NMR (600 MHz, CDCl3, 298 K): d 7.03 (d, 1H,
J = 7.5 Hz, NH-2), 5.38 (br, 1H, NH-1), 4.58 (p, 1H, J = 7.5 Hz,
CaH-2), 4.01 (dddd, 1H, J = 1.4, 3.0, 4.3, 11.3 Hz, CeH-1), 3.76
(s, 3H, COOCH3), 3.63 (d, 1H, J = 9.1 Hz, CaH-1), 3.51 (m, 1H,
CbH-1), 3.48 (dt, 1H, J = 2.7, 11.3 Hz, Ce¢H-1), 2.42 (m, 1H,
CgH-1), 1.72 (m, 1H, CdH-1), 1.67 (m, 1H, Cd¢H-1), 1.42 (m, 1H,
Cg¢H-1), 1.42 (d, 1H, J = 7.5 Hz, CH3-2) 1.42 (s, 9H, Boc);13C
NMR (CDCl3, 100 MHz): d 173.0, 169.4, 155.5, 79.3, 78.9, 67.6,
52.5, 50.1, 47.5, 30.1, 28.3 (3C), 24.2, 18.3; HRMS (ESI): m/z
calcd for C15H26N2O6Na: 353.1688 [M+Na]+; found: 353.1686.
> 8.9 Hz and JCbH-CaH > 9.0 Hz are consistent with f ª -120◦
3
and q ª 60◦ for the second and the fourth b residues. Similar to
peptides 4–7, the peptides 8–10 exhibited the characteristics of a
9/11-mixed helix. Further proof of structural confirmation was
obtained from the MD studies.19
NMR studies on peptide 9 were carried out in CD3OH to
understand the stability of the secondary structure in a polar
solvent. The presence of characteristic nOes such as Cb(2)/NH(4),
Ce(4)/NH(5), NH(1)/NH(2) and NH(3)/NH(4) with low inten-
sities support small population of 9/11-helical structures. The
exchange studies in CD3OD do not seem to suggest very robust
folds in the pentapeptide 9.19 These observations are consistent
with the conclusions derived from theoretical studies by Hofmann
et al.10
4. Conclusion
In summary, the peptides prepared with 1 : 1 alternating APyC, the
new b-amino acid (trans-3-aminopyran-2-carboxylic acid) and D-
Ala, have shown the participation of the amide proton of a-residue
in a 9-mr H-bonding and that of b-residue in 11-mr H-bonding,
giving rise to a right-handed 9/11-mixed helix. In addition to the
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Boc-(S,S)-APyC-D-Ala-(S,S)-APyC-OMe (20)
chromatography (60-120 mesh Silica gel, 2.5% methanol in CHCl3)
afforded 4 (0.2 g, 80%) as a white solid; m.p. 250 ◦C; [a]D = 157.3
(c 0.5, CHCl3); IR (CHCl3): 3432, 3404, 3331, 3006, 2934, 2860,
A solution of ester 3 (0.5 g, 1.51 mmol) in THF: MeOH: H2O
(3 : 1 : 1) was treated with LiOH (0.09 g, 3.78 mmol) at 0 ◦C
and continued stirring at room temperature for 2 h. Work up
as described for 17 gave the acid 19a.
1
1741, 1672, 1513, 1452, 1371, 1310, 1231, 1163, 1090 cm-1; H
NMR (600 MHz, CDCl3, 278 K): d 7.60 (d, 1H, J = 8.9 Hz, NH-
3), 7.22 (d, 1H, J = 9.3 Hz, NH-2), 7.02 (d, 1H, J = 7.2 Hz, NH-4),
4.75 (d, 1H, J = 9.8 Hz, NH-1), 4.63 (dq, 1H, J = 9.3, 7.0 Hz,
CaH-2), 4.51 (d, 1H, J = 7.2 Hz, CaH-4), 4.04 (m, 1H, CeH-3),
4.03 (m, 1H, CeH-1), 3.96 (dq, 1H, J = 3.9, 9.6 Hz, CbH-3), 3.84
(dq, 1H, J = 4.1, 9.8 Hz, CbH-1), 3.79 (d, 1H, J = 9.6 Hz, CaH-3),
3.77 (s, 3H, COOCH3), 3.47 (d, 1H, J = 9.8 Hz, CaH-1), 3.44
(dt, 1H, J = 2.4, 11.9 Hz, Ce¢H-3), 3.38 (dt, 1H, J = 2.4, 12.0 Hz,
Ce¢H-1), 2.13 (m, 1H, CgH-3), 2.11 (m, 1H, CgH-1), 1.82 (m, 1H,
CdH-1), 1.80 (m, 1H, CdH-3), 1.69 (m, 1H, Cd¢H-1), 1.68 (m, 1H,
Cd¢H-3), 1.64 (d, 1H, J = 3.9, 12.6 Hz, Cg¢H-3) 1.43 (s, 9H, Boc),
1.42 (d, 1H, J = 7.2 Hz, CH3-4), 1.39 (dq, 1H, J = 3.8, 12.5 Hz,
Cg¢H-1), 1.30 (d, 1H, J = 7.0 Hz, CH3-2); 13C NMR (CDCl3, 150
MHz): d 173.1, 171.5, 169.4, 169.3, 155.7, 82.4, 80.0, 79.9, 67.8,
67.4, 52.4, 49.7, 48.6, 47.8, 47.0, 30.8, 30.0, 28.2 (3C), 25.2, 25.1,
18.1, 16.9; HRMS (ESI): m/z calcd for C24H40N4O9Na: 551.2692
[M+Na]+; found: 551.2702.
A mixture of acid 19a (0.45 g, 1.42 mmol), HOBt (0.23 g, 1.70
mmol), EDCI (0.32 g, 1.70 mmol) in CH2Cl2 (15 mL) was stirred
at 0 ◦C for 15 min and treated with the salt 17a (0.36 g, 1.42
mmol) under N2 atmosphere for 8 h. Workup as described for 3
and purification by column chromatography (60–120 mesh silica
gel, 1.5% methanol in CHCl3) afforded 20 (0.49 g, 75%) as a white
solid; m.p. 230 ◦C; [a]D = +32.5 (c 0.25, CHCl3); IR (CHCl3): 3400,
3325, 3008, 2930, 2858, 1735, 1676, 1514, 1238, 1167, 1090, 1053
1
cm-1; H NMR (CDCl3, 298 K, 600 MHz): d 7.38 (d, 1H, J =
9.1 Hz, NH-3), 6.63 (d, 1H, J = 9.6 Hz, NH-2), 4.75 (d, 1H, J =
9.6 Hz, NH-1), 4.63 (dq, 1H, J = 9.6, 7.0 Hz, CaH-2), 4.11 (dq,
1H, J = 3.9, 9.6 Hz, CbH-3), 4.06 (m, 2H, CeH-1, CeH-3), 3.94
(d, 1H, J = 9.7 Hz, CaH-3), 3.77 (s, 3H, COOCH3), 3.70 (m, 1H,
CbH-1), 3.47 (d, 1H, J = 9.6 Hz, CaH-1), 3.43 (dt, 1H, J = 2.4,
11.9 Hz, Ce¢H-3), 3.41 (dt, 1H, J = 2.4, 12.0 Hz, Ce¢H-1), 2.14
(m, 1H, CgH-1), 2.08 (m, 1H, CgH-3), 1.82 (m, 1H, CdH-3), 1.81
(m, 1H, CdH-1), 1.74 (m, 1H, Cd¢H-1), 1.73 (m, 1H, Cd¢H-3), 1.68
(m, 1H, Cg¢H-3), 1.43 (m, 1H, Cg¢H-1), 1.43 (s, 9H, Boc), 1.30 (d,
3H, J = 7.0 Hz, CH3-2);13C NMR (CDCl3, 150 MHz): d 171.5,
170.2, 169.9, 155.8, 82.1, 80.2, 80.1, 67.5 (2C), 52.6, 50.1, 47.6,
46.7, 30.7, 29.3, 28.2 (3C), 25.4, 24.8, 16.9; HRMS (ESI): m/z
calcd for C21H35N5O8Na: 480.2473 [M+Na]+; found: 480.2488.
Boc-(S,S)-APyC-[D-Ala-(S,S)-APyC]2-D-Ala-OMe (6)
To a solution of ester 4 (0.06 g, 0.11 mmol) in THF: MeOH:
H2O (3 : 1 : 1), LiOH (0.005 g, 0.22 mmol) was added at 0 ◦C
and continued stirring at room temperature for 2 h. Work up as
described for 17 gave the acid 22a.
A mixture of acid 22a (0.05 g, 0.09 mmol), HOBt (0.01 g, 0.1
mmol) and EDCI (0.02 g, 0.1 mmol) in CH2Cl2 (5 mL) was stirred
at 0 ◦C for 15 min and treated with 19b [prepared from 3 (0.03
g, 0.09 mmol) and CF3COOH (0.1 mL) in CH2Cl2 (1 mL)] and
DIPEA (0.1 mL, 0.18 mmol) under nitrogen atmosphere at room
temperature for 8 h. Workup as described for 3 and purification by
column chromatography (60–120 mesh Silica gel, 3.5% methanol
in CHCl3) gave 6 (0.05 g, 64%) as a white solid; m.p. 280 ◦C; [a]D =
+262.8 (c 0.5, CHCl3); IR (CHCl3): 3428, 3402, 3317, 3005, 2932,
2858, 1740, 1686, 1672, 1666, 1514, 1451, 1372, 1311, 1237, 1212,
1161, 1091 cm-1; 1H NMR (600 MHz, CDCl3, 278 K): d 7.69 (d,
1H, J = 9.8 Hz, NH-3), 7.47 (d, 1H, J = 8.3 Hz, NH-5), 7.29 (d, 1H,
J = 9.4 Hz, NH-2), 7.15 (d, 1H, J = 7.3 Hz, NH-6), 7.11 (d, 1H, J =
9.1 Hz, NH-4), 4.79 (d, 1H, J = 9.9 Hz, NH-1), 4.65 (dq, 1H, J =
9.4, 7.0 Hz, CaH-2), 4.58 (dq, 1H, J = 9.1, 7.0 Hz, CaH-4), 4.54
(p, 1H, J = 7.3 Hz, CaH-6), 4.14 (dq, 1H, J = 4.0, 9.8 Hz, CbH-3),
4.08 (m, 1H, CeH-1), 4.03 (m, 1H, CeH-3), 4.01 (m, 1H, CeH-5),
3.98 (m, 1H, CbH-5), 3.94 (d, 1H, J = 9.7 Hz, CaH-5), 3.78 (s,
3H, COOCH3), 3.75 (m, 1H, CbH-1), 3.58 (d, 1H, J = 9.8 Hz,
CaH-3), 3.49 (d, 1H, J = 9.7 Hz, CaH-1), 3.48 (m, 1H, Ce¢H-5),
3.44 (m, 1H, Ce¢H-3), 3.39 (m, 1H, Ce¢H-1), 2.10 (m, 2H, CgH-5,
CgH-1), 2.03 (m, 1H, CgH-3), 1.86 (m, 1H, CdH-1), 1.79 (m, 1H,
CdH-3), 1.77 (m, 1H, CdH-5), 1.76 (m, 1H, Cg¢H-3), 1.75 (m, 1H,
Cg¢H-5), 1.72 (m, 1H, Cd¢H-1), 1.69 (m, 1H, Cd¢H-3), 1.67 (m,
1H, Cd¢H-5), 1.47 (s, 9H, Boc), 1.43 (d, 3H, J = 7.3 Hz, CH3-6),
1.43 (m, 1H, Cg¢H-1), 1.33 (d, 3H, J = 7.1 Hz, CH3-4), 1.26 (d, 3H,
J = 7.0 Hz, CH3-2); 13C NMR (CDCl3, 150 MHz): d 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
(2C), 52.4, 50.0, 48.7, 48.4, 47.7, 47.2, 46.6, 30.6, 29.5, 29.3, 28.1
(3C), 25.4, 25.2, 25.0, 18.1, 17.1, 17.0; HRMS (ESI): m/z calcd for
C33H54N6O12Na: 749.3697 [M+Na]+; found: 749.3724.
Boc-D-Ala-(S,S)-APyC-OMe (21)
A solution of acid 18a (0.13 g, 0.67 mmol), HOBt (0.11 g, 0.80
mmol) and EDCI (0.16 g, 0.80 mmol) in CH2Cl2 (15 mL) was
stirred at 0 ◦C for 15 min and treated with the salt 17a [prepared
from 1 (0.18 g, 0.67 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (0.5
mL)] and DIPEA (0.3 mL, 1.34 mmol) under nitrogen atmosphere
at room temperature for 8 h. Workup as described for 3 and
purification by column chromatography (60–120 mesh Silica gel,
60% ethyl acetate in pet. ether) gave 21 (0.19 g, 86%) as a white
solid; m.p. 143 ◦C; [a]D = +107.6 (c 0.25, CHCl3); IR (CHCl3):
3431, 3013, 2980, 2932, 2860, 1690, 1494, 1448, 1369, 1286, 1231,
1165, 1122, 1094 cm-1;1H NMR (600 MHz, CDCl3, 278 K): d 6.54
(d, 1H, J = 8.6 Hz, NH-2), 4.92 (d, 1H, J = 7.3 Hz, NH-1), 4.17
(p, 1H, J = 7.3 Hz, CaH-2), 4.12 (m, 1H, CbH-2), 4.02 (m, 2H,
CeH-2), 3.82 (d, 1H, J = 8.1 Hz, CaH-2), 3.75 (s, 3H, COOCH3),
3.51 (m, 1H, Ce¢H-2), 2.03 (m, 1H, CgH-2), 1.74 (m, 2H, CdH-2,
Cd¢H-2), 1.56 (m, 1H, Cg¢H-2), 1.46 (s, 9H, Boc), 1.34 (d, 3H,
J = 7.2 Hz, CH3-1);13C NMR (CDCl3, 150 MHz): d 172.0, 169.8,
155.7, 80.4, 79.2, 66.7, 53.4, 52.5, 50.1, 46.7, 28.6 (3C), 23.5, 17.6;
HRMS (ESI): m/z calcd for C15H26N2O6Na: 353.1688 [M+Na]+;
found: 353.1701.
Boc-(S,S)-APyC-D-Ala-(S,S)-APyC-D-Ala-OMe (4)
A mixture of acid 19a (0.15 g, 0.46 mmol), HOBt (0.08 g, 0.56
mmol) and EDCI (0.11 g, 0.56 mmol) in CH2Cl2 (10 mL) was
stirred at 0 ◦C for 15 min and treated with 19b [prepared from 3
(0.16 g, 0.46 mmol) and CF3COOH (0.2 mL) in CH2Cl2 (2 mL)]
and DIPEA (0.16 mL, 0.92 mmol) under N2 atmosphere for
8 h. Workup as described for 3 and purification by column
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Boc-(S,S)-APyC-D-Ala-(S,S)-APyC-D-Ala-(S,S)-APyC-OMe
(5)
4.14 (dq, 1H, J = 4.1, 9.7 Hz, CbH-3), 4.09 (m, 1H, CbH-7), 4.07
(m, 4H, CeH-1, CbH-5, CeH-5, CaH-7), 4.06 (m, 1H, CeH-3,
CeH-7), 3.77 (s, 3H, -COOCH3), 3.74 (dq, 1H, J = 4.1, 9.8 Hz,
CbH-1), 3.68 (d, 1H, J = 9.7 Hz, CaH-5), 3.57 (d, 1H, J = 9.7
Hz, CaH-3), 3.47 (m, 1H, Ce¢H-5), 3.45 (m, 1H, Ce¢H-7), 3.43
(dt, 1H, J = 2.5, 11.7 Hz, Ce¢H-3), 3.38 (dt, 1H, J = 2.4, 11.8 Hz,
Ce¢H-1), 2.11 (m, 1H, CgH-1), 2.04 (m, 1H, CgH-7), 2.01 (m, 1H,
CgH-3), 2.00 (m, 1H, CgH-5), 1.96 (m, 1H, CdH-5), 1.89 (m, 1H,
CdH-7), 1.87 (m, 1H, CdH-1), 1.86 (m, 1H, CdH-3), 1.82 (m, 2H,
Cd¢H-5, Cd¢H-7), 1.81 (m, 1H, Cd¢H-3), 1.74 (m, 1H, Cg¢H-5),
1.72 (m, 1H, Cd¢H-1), 1.71 (m, 1H, Cg¢H-3), 1.68 (m, 1H, Cg¢H-
7), 1.47 (s, 9H, Boc), 1.43 (m, 1H, Cg¢H-1), 1.32 (d, 3H, J = 7.1
Hz, CH3-6), 1.27 (d, 3H, J = 7.0 Hz, CH3-4), 1.25 (d, 3H, J = 7.0
Hz, CH3-2); 13C NMR (CDCl3, 150 MHz): d 173.1, 172.5, 171.4,
170.3, 170.0, 169.9, 169.4, 155.8, 82.7, 82.1, 81.2, 80.0, 79.8, 67.5
(2C), 67.4 (2C), 52.3, 50.1, 49.0 (2C), 47.7, 46.9, 46.6, 46.5, 30.6,
29.1, 28.9 (2C), 28.2 (3C), 25.4 (2C), 25.3, 24.8, 17.2, 17.1, 17.0;
HRMS (ESI): m/z calcd for C39H63N7O14Na: 876.4413 [M+Na]+;
found: 876.4434.
To a solution of ester 20 (0.2 g, 0.17 mmol) in THF: MeOH:
H2O (3 : 1 : 1), LiOH (0.015 g, 0.34 mmol) was added at 0 ◦C
and continued stirring at room temperature for 2 h. Work up as
described for 17 gave the acid 20a.
A mixture of acid 20a (0.1 g, 0.31 mmol), HOBt (0.05 g,
0.37 mmol) and EDCI (0.07 g, 0.37 mmol) in CH2Cl2 (10 mL)
was stirred at 0 ◦C for 15 min and treated with 21b [prepared
from 21 (0.14 g, 0.31 mmol) and CF3COOH (0.2 mL) in CH2Cl2
(2 mL)] and DIPEA (0.11 mL, 0.63 mmol) under N2 atmosphere
for 8 h. Workup as described for 3 and purification by column
chromatography (60–120 mesh Silica gel, 3.0% methanol in
CHCl3) afforded 5 (0.15 g, 75%) as a white solid; m.p. 255 ◦C;
[a]D = +220.1 (c 0.5, CHCl3); IR (CHCl3): 3432, 3400, 3315, 3008,
2942, 2861, 1734, 1665, 1516, 1447, 1370, 1238, 1160, 1089 cm-1;
1H NMR (600 MHz, CDCl3, 288 K): d 7.71 (d, 1H, J = 9.7 Hz,
NH-3), 7.38 (d, 1H, J = 8.7 Hz, NH-5), 7.30 (d, 1H, J = 9.4 Hz,
NH-2), 6.65 (d, 1H, J = 9.6 Hz, NH-4), 4.80 (d, 1H, J = 10.1 Hz,
NH-1), 4.66 (dq, 1H, J = 9.4, 6.9 Hz, CaH-2), 4.62 (dq, 1H, J =
9.6, 7.0 Hz, CaH-4), 4.10 (m, 1H, CbH-5), 4.08 (m, 1H, CeH-1),
4.07 (m, 3H, CbH-3, CeH-3, CeH-5), 3.78 (s, 3H, COOCH3), 3.73
(m, 1H, CbH-1), 3.58 (d, 1H, J = 9.7 Hz, CaH-3), 3.49 (d, 1H,
J = 9.7 Hz, CaH-1), 3.47 (dt, 1H, J = 2.5, 11.9 Hz, Ce¢H-3), 3.45
(dt, 1H, J = 2.4, 11.6 Hz, Ce¢H-5), 3.39 (dt, 1H, J = 2.4, 11.9 Hz,
Ce¢H-1), 2.11 (m, 1H, CgH-1), 2.05 (m, 1H, CgH-3), 2.04 (m, 1H,
CgH-5), 1.88 (m, 1H, CdH-5), 1.84 (m, 2H, CdH-1, Cd¢H-5), 1.82
(m, 2H, CdH-3, Cd¢H-3), 1.75 (m, 1H, Cg¢H-3), 1.72 (m, 1H, CdH-
1), 1.69 (m, 1H, Cg¢H-5), 1.44 (m, 1H, Cg¢H-1), 1.43 (s, 9H, Boc),
1.33 (d, J = 7.0 Hz, 3H, CH3-4), 1.25 (d, 3H, J = 6.9 Hz, CH3-2);
13C NMR (CDCl3, 150 MHz): d 172.9, 171.3, 170.3, 169.8, 169.4,
155.9, 82.7, 81.5, 80.1, 79.9, 67.5, 67.4 (2C), 52.4, 50.1, 49.0, 47.7,
46.9, 46.6, 30.7, 29.2, 28.9, 28.2 (3C), 25.4 (2C), 24.8, 17.0, 16.9;
HRMS (ESI): m/z calcd for C30H49N5O11Na: 678.3326 [M+Na]+;
found: 678.3349.
Boc-D-Ala-(S,S)-APyC-D-Ala-OMe (24)
To a solution of ester 21 (0.5 g, 1.51 mmol) in THF: MeOH: H2O
(3 : 1 : 1) LiOH (0.09 g, 3.78 mmol) was added at 0 ◦C continue
stirring at room temperature for 2 h. Work up as described for 17
gave the acid 21a.
A mixture of acid 21a (0.3 g, 0.94 mmol), HOBt (0.15 g, 1.13
mmol) and EDCI (0.21 g, 1.13 mmol) in CH2Cl2 (10 mL) was
stirred at 0 ◦C for 15 min and treated with the salt 18b (0.15 g, 1.13
mmol) under N2 atmosphere for 8 h. Workup as described for 3
and purification by column chromatography (60–120 mesh Silica
gel, 2% methanol in CHCl3) afforded 24 (0.31 g, 83%) as a white
solid; m.p. 140 ◦C; [a]D = +41.46 (c 0.5, CHCl3); IR (KBr): 3317,
3296, 2979, 2849, 2361, 1748, 1666, 1535, 1452, 1369, 1330, 1216,
1164, 1097, 1047, 963, 640 cm-1; 1H NMR (600 MHz, CDCl3, 298
K): d 7.02 (d, 1H, J = 7.5 Hz, NH-2), 6.78 (d, 1H, J = 7.1 Hz,
NH-3), 5.11 (br, 1H, NH-1), 4.52 (p, 1H, J = 7.1 Hz, CaH-3), 4.17
(p, 1H, J = 7.2 Hz, CaH-1), 4.04 (m, 1H, CeH-2), 3.80 (m, 1H,
CbH-2), 3.75 (s, 3H, COOCH3), 3.65 (d, 1H, J = 9.4 Hz, CaH-2),
2.38 (m, 1H, CgH-2), 1.85 (m, 1H, CdH-2), 1.76 (m, 1H, Cd¢H-2),
1.68 (m, 1H, Cg¢H-2), 1.45 (s, 9H, Boc), 1.42 (d, 3H, J = 7.1 Hz,
CH3-1), 1.32 (d, 3H, J = 7.1 Hz, CH3-3); 13C NMR (CDCl3, 150
MHz): 172.9, 172.5, 169.3, 155.5, 79.9, 78.8, 67.8, 52.6, 50.3, 49.1,
47.7, 29.8, 28.3 (3C), 24.3, 18.6, 18.2; HRMS (ESI): m/z calcd for
C18H31N3O7Na: 401.2241 [M+Na]+; found: 401.2258.
Boc-(S,S)-APyC-[D-Ala-(S,S)-APyC]2-D-Ala-(S,S)-APyC-OMe
(7)
To a solution of ester 5 (0.12 g, 0.11 mmol) in THF: MeOH:
H2O (3 : 1 : 1), LiOH (0.016 g, 0.22 mmol) was added at 0 ◦C
and continued stirring at room temperature for 2 h. Work up as
described for 17 gave the acid 23a.
A mixture of acid 23a (0.1 g, 0.31 mmol), HOBt (0.05 g,
0.37 mmol) and EDCI (0.07 g, 0.37 mmol) in CH2Cl2 (10 mL)
was stirred at 0 ◦C for 15 min and treated with 21b [prepared
from 21 (0.20 g, 0.31 mmol) and CF3COOH (0.2 mL) in CH2Cl2
(2 mL)] and DIPEA (0.11 mL, 0.63 mmol) under N2 atmosphere
for 8 h. Workup as described for 3 and purification by column
chromatography (60–120 mesh Silica gel, 4.0% methanol in
CHCl3) afforded 7 (0.12 g, 43%) as a white solid; m.p. 295 ◦C;
[a]D = +358.7 (c 0.25, CHCl3); IR (CHCl3): 3430, 3339, 3309,
3007, 2944, 2862, 1661, 1543, 1447, 1372, 1237, 1154, 1089 cm-1;1H
NMR (600 MHz, CDCl3, 278 K): d 7.64 (d, 1H, J = 9.7 Hz, NH-
3), 7.55 (d, 1H, J = 9.6 Hz, NH-5), 7.35 (d, 1H, J = 8.4 Hz, NH-7),
7.20 (d, 1H, J = 9.2 Hz, NH-2), 7.12 (d, 1H, J = 9.4 Hz, NH-4),
6.60 (d, 1H, J = 9.4 Hz, NH-6), 4.73 (d, 1H, J = 9.8 Hz, NH-1),
4.65 (m, 1H, CaH-2), 4.63 (m, 1H, CaH-4), 4.60 (m, 1H, CaH-6),
Boc-D-Ala-(S,S)-APyC-D-Ala-(S,S)-APyC-D-Ala-OMe (9)
To a solution of ester 24 (0.08 g, 1.51 mmol) in THF: MeOH:
H2O (3 : 1 : 1) LiOH (0.02 g, 3.78 mmol) was added at 0 ◦C
and continued stirring at room temperature for 2 h. Work up
as described for 17 gave the acid 24a.
A solution of acid 24a (0.02 g, 0.06 mmol), HOBt (0.01 g, 0.07
mmol) and EDCI (0.01 g, 0.07 mmol) in CH2Cl2 (10 mL) was
stirred at 0 ◦C for 15 min and treated with 19b [prepared from
3 (0.03 g, 0.06 mmol) and CF3COOH (0.02 mL) in CH2Cl2 (0.5
mL)] and DIPEA (0.1 mL, 0.59 mmol) under nitrogen atmosphere
at room temperature for 8 h. Workup as described for 3 and
purification by column chromatography (60–120 mesh Silica gel,
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3.0% methanol in CHCl3) gave 9 (0.03, 67%) as a white solid; m.p.
265 ◦C; [a]D = +41.0 (c 0.5, CHCl3); IR (CHCl3): 3409, 3331, 3005,
2933, 2861, 2300, 1670, 1525, 1451, 1371, 1242, 1162, 1093, 1670,
1525, 1450, 1371, 1242, 1162, 1093 cm-1;1H NMR (600 MHz,
CDCl3, 278 K): d 7.46 (d, 1H, J = 8.6 Hz, NH-4), 7.19 (d, 1H,
J = 8.9 Hz, NH-3), 7.07 (d, 1H, J = 7.3 Hz, NH-5), 6.63 (d, 1H,
J = 8.9 Hz, NH-2), 5.32 (d, 1H, J = 8.1 Hz, NH-1), 4.52 (d, 1H,
J = 7.3 Hz, CaH-5), 4.50 (dq, 1H, J = 8.9, 7.0 Hz, CaH-3), 4.21
(p, 1H, J = 7.4 Hz, CaH-1), 4.09 (m, 1H, CbH-2), 4.05 (m, 1H,
CeH-2), 4.03 (m, 1H, CeH-4), 3.94 (m, 1H, CbH-4), 3.87 (d, 1H,
J = 9.9 Hz, CaH-4), 3.77 (s, 3H, -COOCH3), 3.48 (d, 1H, J = 9.5
Hz, CaH-2), 3.47 (dt, 1H, J = 2.1, 11.9 Hz, Ce¢H-4), 3.41 (dt,
1H, J = 2.3, 12.1 Hz, Ce¢H-2), 2.09 (m, 1H, CgH-4), 2.08 (m,
1H, CgH-2), 1.83 (m, 1H, CdH-2), 1.79 (m, 1H, CdH-4), 1.72 (m,
2H, Cd¢H-4, Cd¢H-2), 1.68 (m, 1H, Cg¢H-4), 1.51 (dq, 1H, J =
4.0, 12.5 Hz, Cg¢H-2), 1.43 (s, 9H, Boc), 1.43 (d, 3H, J = 7.3 Hz,
CH3-5), 1.33 (d, 3H, J = 7.4 Hz, CH3-1), 1.31 (d, 3H, J = 7.0
Hz, CH3-3); 13C NMR (CDCl3, 150 MHz): d 173.0, 172.9, 171.2,
169.6, 169.4, 155.8, 128.4, 96.0, 81.5, 80.2, 79.5, 67.7, 67.5, 52.4,
50.1, 48.5, 48.2, 47.7, 47.4, 30.2, 29.6, 28.2 (3C), 25.0, 18.1, 17.0;
HRMS (ESI): m/z calcd for C27H45N5O11Na: 622.3064 [M+Na]+;
found: 622.3079.
and continued stirring at room temperature for 2 h. Work up
as described for 17 gave the acid 25a.
A solution of acid 25a (0.04 g, 0.07 mmol), HOBt (0.01 g, 0.08
mmol), and EDCI (0.02 g, 0.08 mmol) in CH2Cl2 (10 mL) was
stirred at 0 ◦C for 15 min and treated with 21b [prepared from
21 (0.02 g, 0.07 mmol) and CF3COOH (0.02 mL) in CH2Cl2 (0.5
mL)] and DIPEA (0.12 mL, 0.14 mmol) in CH2Cl2 under N2
atmosphere for 8 h. Workup as described for 3 and purification by
column chromatography (60–120 mesh Silica gel, 4.0% methanol
in CHCl3) gave 10 (0.03 g, 65%) as a white solid; m.p. 270 ◦C; [a]D =
+142.4 (c 0.5, CHCl3); IR (CHCl3): 3402, 3310, 3008, 2935, 2861,
1665, 1533, 1449, 1373, 1236, 1205, 1158, 1092 cm-1;1H NMR (600
MHz, CDCl3, 278 K): d 7.62 (d, 1H, J = 9.6 Hz, NH-4), 7.37 (d,
1H, J = 8.6 Hz, NH-6), 7.31 (d, 1H, J = 9.1 Hz, NH-3), 6.66 (d, 1H,
J = 9.5 Hz, NH-5), 6.60 (d, 1H, J = 9.6 Hz, NH-2), 5.33 (d, 1H, J =
8.1 Hz, NH-1), 4.60 (dq, 1H, J = 9.6, 7.1 Hz, CaH-5), 4.55 (dq,
1H, J = 9.1, 7.0 Hz, CaH-3), 4.22 (p, 1H, J = 7.2 Hz, CaH-1), 4.07
(m, 1H, CeH-4), 4.06 (m, 6H, CbH-2, CeH-2, CbH-4, CaH-6,
CbH-6, CeH-6), 3.78 (s, 3H, -COOCH3), 3.63 (d, 1H, J = 9.8 Hz,
CaH-4), 3.48 (d, 1H, J = 9.9 Hz, CaH-2), 3.47 (m, 1H, Ce¢H-4),
3.45 (m, 1H, Ce¢H-6), 3.41 (m, 1H, Ce¢H-2), 2.08 (m, 1H, CgH-2),
2.03 (m, 1H, CgH-6), 2.00 (m, 1H, CgH-4), 1.92 (m, 1H, CdH-4),
1.88 (m, 1H, CdH-6), 1.85 (m, 1H, CdH-1), 1.83 (m, 1H, Cd¢H-6),
1.81 (m, 1H, Cd¢H-4), 1.75 (m, 1H, Cg¢H-4), 1.74 (m, 1H, Cd¢H-2),
1.68 (m, 1H, Cg¢H-6), 1.52 (dq, 1H, J = 4.0, 12.5 Hz, Cg¢H-2), 1.47
(s, 9H, Boc), 1.33 (d, 3H, J = 7.1 Hz, CH3-1), 1.33 (d, 3H, J = 7.1
Hz, CH3-5), 1.26 (d, 3H, J = 7.0 Hz, CH3-3); 13C NMR (CDCl3,
150 MHz): d 173.2, 172.4, 171.4, 170.3, 169.9, 169.6, 155.8, 82.0,
81.2, 80.4, 79.8, 67.5 (2C), 67.4, 52.4, 49.0, 48.4, 47.7, 47.0 (2C),
46.9, 30.2, 29.7, 29.0, 28.9 (3C), 28.4, 25.4, 25.1, 17.1 (2C), 17.0;
HRMS (ESI): m/z calcd for C33H54N6O12Na: 749.3697 [M+Na]+;
found: 749.3737.
Boc-D-Ala-(S,S)-APyC-D-Ala-(S,S)-APyC-OMe (8)
A solution of ester 21 (0.06 g, 0.18 mmol) in THF: MeOH:
H2O (3 : 1 : 1) LiOH (0.01 g, 0.45 mmol) was added at 0 ◦C
and continued stirring at room temperature for 2 h. Work up
as described for 17 gave the acid 21a.
A solution of acid 21a (0.05 g, 0.17 mmol), HOBt (0.03 g, 0.20
mmol) and EDCI (0.04 g, 0.20 mmol) in CH2Cl2 (10 mL) was
stirred at 0 ◦C for 15 min and treated with the salt 21b [prepared
from 21 (0.06 g, 0.17 mmol) and CF3COOH (0.1 mL) DIPEA (0.05
mL, 0.34 mmol) in CH2Cl2 under N2 atmosphere for 8 h. Workup
as described for 3 and purification by column chromatography
(60–120 mesh Silica gel, 2.8% methanol in CHCl3) gave 8 (0.07,
75%) as a white solid; m.p. 198 ◦C; [a]D = +103.0 (c 0.5, CHCl3);
IR (CHCl3): 3406, 3334, 3010, 2934, 2860, 1670, 1530, 1448, 1371,
1238, 1226, 1163, 1091 cm-1; 1H NMR (600 MHz, CDCl3, 278 K):
d 7.38 (d, J = 8.5 Hz, 1H, NH-4), 6.71 (d, 1H, J = 8.9 Hz, NH-2),
6.65 (d, 1H, J = 9.1 Hz, NH-3), 5.37 (d, 1H, J = 8.3 Hz, NH-1),
4.52 (dq, 1H, J = 9.1, 7.1 Hz, CaH-3), 4.21 (d, 1H, J = 7.2 Hz,
CaH-1), 4.08 (m, 1H, CeH-2), 4.06 (m, 1H, CaH-4), 4.06 (m, 1H,
CbH-4), 4.02 (m, 1H, CeH-4), 3.99 (m, 1H, CbH-2), 3.78 (s, 3H,
-COOCH3), 3.46 (m, 2H, Ce¢H-2, CaH-2), 3.45 (m, 1H, Ce¢H-4),
2.10 (m, 1H, CgH-2), 2.02 (m, 1H, CgH-4), 1.88 (m, 1H, CdH-4),
1.83 (m, 2H, CdH-2, Cd¢H-4), 1.76 (m, 1H, Cd¢H-2), 1.71 (m, 1H,
Cg¢H-4), 1.52 (m, 1H, Cg¢H-2), 1.43 (s, 9H, Boc), 1.33 (m, 3H,
CH3-1), 1.32 (m, 3H, CH3-2); 13C NMR (CDCl3, 150 MHz): d
173.1, 171.3, 170.4, 169.5, 156.0, 81.3, 80.3, 79.7, 67.6, 67.4, 52.5,
50.0, 48.4, 47.9, 47.3, 30.2, 29.7, 28.3 (3C), 25.2, 24.7, 17.7, 17.0;
HRMS (ESI): m/z calcd for C24H40N4O9Na: 551.2692 [M+Na]+;
found: 551.2682.
Acknowledgements
The authors are thankful to CSIR, New Delhi, for financial
support (S.R.K., S.J.B. for fellowship). The financial support for
the work was obtained from MLP-0010 project.
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19 See supporting information.
20 Solvent titration studies were carried out by sequentially adding up to
33% (v/v) of DMSO-d6 to 600 mL CDCl3 solutions of the peptides.
21 (a) As defined by Hofmann et al.,10 a 9/11-helix is one in which the
amide proton of a-residue participates in 9-mr H-bonding, whereas
that of the b-residue participates in 11-mr H-bonding; (b) In our earlier
studies,4c we have defined, based on the convention of Wu and Wong,9b
a 9/11-helix as one in which the first H-bonding is a 9-membered one.
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