11/9-Mixed helices in the α/β-peptides derived from alternating α- and β-amino acids with proteinogenic side chains

Article abstract of DOI:10.1021/jo0612980

α/β-Hybrid peptides are prepared from amino acids with proteinogenic side chains on the basis of the concept of "alternating chirality", involving D-Phe and a β³-h Val. Through the extensive NMR, CD, and MD studies, robust left-handed 11/9-mixe

Full text of DOI:10.1021/jo0612980

11/9-Mixed Helices in the r/â-Peptides Derived from Alternating
r- and â-Amino Acids with Proteinogenic Side Chains*
Gannoju Srinivasulu,† Singarapu Kiran Kumar,† Gangavaram V. M. Sharma,*,‡ and
Ajit C. Kunwar*,†
Centre for Nuclear Magnetic Resonance and D-211, DiscoVery Laboratory,
Organic Chemistry DiVision III, Indian Institute of Chemical Technology, Hyderabad 500 007, India
esmVee@iict.res.in; kunwar@iict.res.in
ReceiVed June 23, 2006
R/â-Hybrid peptides are prepared from amino acids with proteinogenic side chains on the basis of the
concept of “alternating chirality”, involving D-Phe and a â3-hVal. Through the extensive NMR, CD, and
MD studies, robust left-handed 11/9-mixed helices were identified in these peptides in CDCl3 solutions,
wherein the 11/9-mixed helix was observed even in a small peptide with three residues.
Introduction
peptides has generated a lot of research interest, creating an
imminent need to find new secondary structural elements for
such exploration in this class of peptides. It has been suggested
that preorganization4 of â-residues is essential for the helix
stability, whereas the â-branched side chains in R-residues lower
the propensity of helix formation. In â-peptides, enhanced helical
stability has been very well documented for â-residues with
branched side chains.8 On the basis of the very robust design
of mixed helices from Caa’s with “alternating chirality”9 as well
as of the above considerations on the helical stability, in the
present communication, we report the synthesis of peptides 1-5
(Figure 1) and the identification of new robust left-handed 11/
Foldamers1 derived from alternating R- and â-amino acid
residues adopt a variety of helical structures.2-5 Such hybrid
peptides containing constrained cyclic â-amino acids, reported
by Gellman et al. and Reiser et al., display 11-, 13-, and 14/
15-helices,2-4 whereas those derived from acyclic C-linked
carbo-â3-amino acid [(S)-Caa] reported5 by us have shown
robust 9/11- (11/9)-mixed helices. The demonstration of anti-
microbial activity6 and protein surface recognition7 by R/â-
* IICT Communication No. 051005.
† Centre for Nuclear Magnetic Resonance.
‡ D-211, Discovery Laboratory.
(1) (a) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071-13072. (b) Gellman,
S. H. Acc. Chem. Res. 1998, 31, 173-180.
(6) Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc.
2004, 126, 6848-6849.
(7) Sadowsky, J. D.; Schmitt, M. A.; Lee, H.-S.; Naoki, U.; Wang, S.;
Tomita, Y.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127, 11966-11968.
(8) (a) Raguse, T. L.; Lai, J. R.; Gellman, S. H. HelV. Chim. Acta 2002,
85, 4154-4164. (b) Hart, S. A.; Bahadoor, A. B. F.; Mathews, E. E.; Qiu,
X. J.; Schepartz, A. J. Am. Chem. Soc. 2003, 125, 4022-4023.
(9) (a) Sharma, G. V. M.; Ravinder Reddy, K.; Radha Krishna, P.; Ravi
Sankar, A.; Narsimulu, K.; Kiran Kumar, S.; Jayaprakash, P.; Jagannadh,
B.; Kunwar, A. C. J. Am. Chem. Soc. 2003, 125, 13670-13671. (b) Sharma,
G. V. M.; Ravinder Reddy, K.; Radha Krishna, P.; Ravi Sankar, A.;
Jayaprakash, P.; Jagannadh, B.; Kunwar, A. C. Angew. Chem. 2004, 116,
4051-4055; Angew. Chem., Int. Ed. 2004, 43, 3961-3965.
(2) Hayen, A.; Schmitt, M. A.; Nagassa, N.; Thomson, K. A.; Gellman,
S. H. Angew. Chem. 2004, 116, 511-516; Angew. Chem., Int. Ed. 2004,
43, 505-510.
(3) De Pol, S.; Zorn, C.; Klein, C. D.; Zerbe, O.; Reiser, O. Angew.
Chem. 2004, 116, 517-520; Angew. Chem., Int. Ed. 2004, 43, 511-514.
(4) Schmitt, M. A.; Choi, S. H.; Guzei, A. I.; Gellman, S. H. J. Am.
Chem. Soc. 2005, 127, 13130-13131.
(5) Sharma, G. V. M.; Nagendar, P.; Jayaprakash, P.; Radha Krishna,
P.; Ramakrishna, K. V. S.; Kunwar, A. C. Angew. Chem. 2005, 117, 6028-
6032; Angew. Chem., Int. Ed. 2005, 44, 5878-5882.
10.1021/jo0612980 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/27/2006
J. Org. Chem. 2006, 71, 8395-8400
8395

Srinivasulu et al.
FIGURE 2. Hydrogen-bonding pattern in (A) 11/9- and (B) 9/11-
mixed helices in R/â-hybrid peptides.
â-amide proton is involved in an 11-membered (11-mr) H-bond
is referred to as a 9/11-helix (Figure 2). On the other hand,
when the R-amide proton makes an 11-mr pseudoring and the
â-amide proton participates in a 9-mr ring, it is called an 11/
9-helix (Figure 2). However, in our earlier studies,5 the mixed
9/11- and 11/9-helices have been differentiated depending upon
whether the first H-bonded pseudoring was 9-mr or 11-mr,
respectively. On the basis of the new convention,10 these mixed
helices correspond to the family of the 11/9-helix.5
Results and Discussions
1. Synthesis of Peptides 1-5. The R/â-peptides 1-5
(Scheme 1) have been synthesized in the solution phase from
Boc-â3-hVal-OMe 6 and Boc-D-Phe-OH 7 by the standard
peptide coupling method (EDCI/HOBt). First, 6 was converted
into the corresponding amine salt 8 on exposure to CF3COOH
in CH2Cl2 for 3 h and then coupled with acid 7 in the presence
of EDCI/HOBt and Et3N in CH2Cl2 for 12 h to afford the
dipeptide 9. The ester group in 9 was saponified with LiOH in
THF/MeOH/H2O (2:1:1) to give acid 10 (99%), which on
coupling with H-D-Phe-OMe 11 under the above conditions gave
tripeptide 1 (61.3%). Unmasking of Boc protection in 1 with
CF3COOH and coupling of the resulting salt 12 with acid 10
afforded pentapeptide 2 (93.6%). Similarly, treatment of 2 with
CF3COOH gave the amine salt 13, which on further coupling
with acid 10 furnished the heptapeptide 3 (38.8%).
Likewise, the peptides 4 and 5 (Scheme 1), having â3-hVal
at the N-terminus, were prepared adopting the above procedure
using the amine salts 12 and 13. Accordingly, 6 was saponified
to acid 14 and coupled independently with 12 and 13 to afford
the tetrapeptide 4 and hexapeptide 5, respectively.
FIGURE 1. Structures of peptides 1-5; H-bonds (solid lines) and
diagnostic nOe’s (curved lines) in 1-4.
9-mixed helices in the peptides containing alternating R-D-Phe
and â3-hVal, from incisive NMR, CD, and molecular dynamics
(MD) studies.
2. Conformational Analysis of Peptides 1-5. The 1H NMR
spectrum11 of peptide 1 in CDCl3 shows the signatures of a
stable secondary structure. Though none of the amide protons
has a chemical shift (δNH) >7 ppm, a small change of 0.54
ppm in the NH(2) chemical shift (∆δNH), during the solvent
titration studies,12 confirms its participation in H-bonding. For
Hofmann et al.10 have carried out a detailed theoretical study
of R/â-peptides with a dimeric repetitive pattern. They have
shown that the most favored structure in apolar solvents is a
mixed helix with a nine-membered H-bond in the forward
direction and an 11-membered H-bond in the backward direction
of the peptide chain. They further distinguish between the two
possibilities of such helices. A structure where an R-amide
proton participates in a nine-membered (9-mr) H-bond and a
3
3
the â-hVal residue, JNH-CâH ) 9.8 Hz and JCRH-CâH < 5 Hz
are consistent with the values of dihedral angles φ (C(O)-N-
Câ-CR) ∼ 100° and θ (N-Câ-CR-C(O)) ∼ ( 60°,
(11) See Supporting Information.
(12) Solvent titration studies were carried out by sequentially adding up
to 33% of DMSO-d6 to CDCl3 solutions of the peptides.
(10) Baldauf, C.; Gunther, R.; Hofmann, H. Biopolymers (Pept. Sci.)
2006, 84, 408-413.
8396 J. Org. Chem., Vol. 71, No. 22, 2006

11/9-Mixed Helices in the R/â-Peptides
FIGURE 3. Characteristic nOe’s in the ROESY spectrum of 2: (1) CRH(1)/NH(3), (2) CâH(2)/NH(3), (3) CRH(3)/NH(5), (4) CâH(4)/NH(5), (5)
NH(2)/NH(3), (6) NH(4)/NH(5).
SCHEME 1. Synthesis of Peptides 1-5
respectively. The unequivocal assignments for CRH(pro-S) and
CRH(pro-R), based on strong NH(3)/CRH(pro-S)(2) and more
intense CRH(pro-S)(2)/NH(2) compared to CRH(pro-R)(2)/NH(2)
nOe’s, support a value of θ of about -60°, an important
signature for a left-handed helix. From the conformationally
diagnostic nOe’s, CRH(1)/NH(3), CâH(2)/NH(3), and NH(2)/
NH(3), nucleation of an 11/9-helix is evident. 3JNH-CRH values
of 7.5 and 8.5 Hz for D-Phe(1) and D-Phe(3), respectively, are
not very distinctive and reflect the influence of fraying in the
termini. However, in light of the expected value of C(O)-N-
CR-C(O) (φ) ∼ 70° for the R-residues in 11/9-helices,5 the
couplings might even correspond to a predominant single
conformation. The data presented above, along with the CD
spectrum (discussed in latter sections), show a significant
propensity of an unprecedented left-handed 11/9-helix in 1. To
have more definitive evidence for the underlying structures in
this family, peptides 2 and 3 were investigated.
tion of the lowest-energy conformers, which showed values of
3JNH-CRH ) 6.1 and 5.6 Hz for D-Phe(1) and D-Phe(3),
respectively. CRH(pro-S) and CRH(pro-R) protons were assigned
as discussed for 1, and a value of about -60° for θ was deduced.
In the ROESY spectrum (Figure 3), the distinctive nOe’s for
an 11/9-helix, CRH(1)/NH(3) and CRH(3)/NH(5), consistent
with an 11-mr H-bond between Boc CO-NH(3) and NH(5)-
CO(2) and NH(2)/NH(3) and NH(4)/NH(5) supporting 9-mr
H-bonds between NH(2)-CO(3) and NH(4)-CO(5) were
observed. The NMR data provide compelling evidence for a
left-handed 11/9/11/9-H-bonded pattern for 2.
In a detailed study of 2 in CD3OD and CD3OH, weak
signatures of the 11/9-helix are noticed. 3JNH-CRH and 3JNH-CâH
are similar to those in CDCl3 solution. The disappearance time
of the NH(2) and NH(4) resonances is about 8 h (Figure 4),
suggesting some of the molecular population is involved in
H-bonding. The theoretical calculations of Hofmann et al.10 also
showed that the mixed helices are more stable in apolar solvents.
Despite the poor solubility in CDCl3, propagation of an 11/
1
For 2, the H NMR spectrum showing four amide protons,
except NH(1), at δ > 7 ppm and the solvent titration studies
(∆δNH < 0.31 ppm)12 support their participation in H-bonding.
For â-hVal(2) and â-hVal(4), 3JCRH-CâH < 4.7 Hz and 3JNH-CâH
) 10.0 Hz are in conformity with θ ∼ (60° and φ ∼ 100°,
respectively. In light of our deductions of φ ∼ 70°, for the R-
residues in the 11/9-helix,5 we tentatively believe that, for D-Phe-
1
9-helix in peptide 3 was unmistakably noticed. The H NMR
spectrum showed NH(2)-NH(7) at δ > 7 ppm as well as a
very small ∆δNH of <0.26 ppm in the solvent titration studies12
confirming their involvement in H-bonding. Line broadening
precluded determination of some of the 3JNH-CRH values at 303
K; however, studies at 243 K showed 3JNH-CRH ) 6.7, 5.8, and
6.4 Hz for D-Phe(1), D-Phe(3), and D-Phe(5), respectively,
corresponding to a value of φ ∼ 70°. Similarly, for â-hVal
3
(1) and D-Phe(3), the values of JNH-CRH ) 7.0 and 6.7 Hz,
respectively, are arising due to a predominant population with
|φ| ∼ 70°. This observation was also supported by the low-
temperature studies at 263 K, resulting in an enhanced popula-
3
3
residues, JNH-CâH > 9.8 Hz and JCRH-CâH < 5.0 Hz, along
J. Org. Chem, Vol. 71, No. 22, 2006 8397

Srinivasulu et al.
FIGURE 4. H/D exchange of amide protons for 2 in CD3OD (500 MHz) at 303 K.
with the unequivocal assignment for CRH(pro-S) and CRH(pro-R)
protons, imply that φ ∼ 100° and θ ∼ -60°. The nOe
correlations, CRH(1)/NH(3), CRH(3)/NH(5), CRH(5)/NH(7),
NH(2)/NH(3), NH(4)/NH(5), and NH(6)/NH(7), in addition to
the information on H-bonding and the dihedral angles, confirmed
11-mr H-bonds between NH(3)-Boc CO, NH(5)-CO(2), and
NH(7)-CO(4) and 9-mr H-bonds between NH(2)-CO(3), NH-
(4)-CO(5), and NH(6)-CO(7). These observations amply
support a left-handed 11/9-helix with an 11/9/11/9/11/9-H-
bonded configuration in 3.
Like our earlier studies on R/â-peptides with â-Caa,5 4 and
5 with the â-hVal residue at the N-terminus are also expected
to exist as 11/9-helices. The H NMR spectrum of 4 showed
FIGURE 5. CD spectra of peptides (a) 1, (b) 2, and (c) 4 in CH3OH.
1
to the broad peak at about 225 nm in an 11/9-helix.5 The
negative intensities of the peaks very clearly indicate the change
in the handedness in these helices compared to those reported
earlier.
NH(3) and NH(4) at δ > 7 ppm, and solvent titration studies
confirmed their participation in H-bonding.12 Further, the
characteristic long-range nOe’s, CRH(2)/NH(4), NH(1)/NH(2),
and NH(3)/NH(4), as well as the couplings, providing informa-
tion on some of the backbone dihedral angles, and the CD
spectrum yield strong evidence for the presence of a unique
left-handed helix with a 9/11/9-H-bonded pattern. For peptide
5, poor solubility did not permit determination of detailed
structural information. However, CD spectra (discussed below)
showed characteristic signatures of a left-handed mixed 11/9-
helix.
The MD calculations for 1-4 were carried out using the
quantitative restraints obtained from the ROESY spectra using
the volume integrals and a two-spin approximation. Figure 6
depicts a superposition of the 25 lowest-energy structures for
2. For the sake of clarity, the side chains have been replaced
with a methyl group after the MD calculations. Maximum
violations for the NMR-derived constraints for 2 and 3 are 0.18
and 0.20 Å, respectively. Average pairwise backbone and heavy
atom root-mean-square deviations are 0.19 and 0.41 Å for 2
and 0.17 and 0.42 Å for 3, respectively. The average values of
backbone dihedral angles were obtained by excluding the first
and the last residues. In 2 the φ, θ, and Câ-CR-C(O)-N (ψ)
for the â-residues are -93 ( 8°, -58 ( 1°, and 85 ( 1°,
respectively, whereas for the R-residue, φ ) 72 ( 3° and CR-
C(O)-N-CR (ψ) ) -141 ( 2°. The corresponding values for
3 are -94 ( 4°, -60 ( 2°, 86 ( 2°, 76 ( 2°, and -142 ( 5°,
respectively.11 These values are very similar to those reported
for mixed 11/9-helices in R/â-peptides derived from a C-linked
carbo-â3-amino acid.5
For the â-hVal residue, especially in 2 and 3, 3JCâH-CγH was
found to be ∼10 Hz, suggesting the preponderance of a single
conformer with CâH and CγH being antiperiplanar to each
other. Further support for this comes from the nOe’s between
CγH of â-hVal(i) and CRH(pro-S) of â-hVal(i+2) in 2 and 3.
On the other hand, it was intriguing that only the D-Phe residue
at the C-terminus shows 3JCRH-CâH(pro-R) > 10 Hz and 3JCRH-CâH
(pro-
< 5 Hz indicating a large population of a single conformer
S)
about the CR-Câ bond, which is more conspicuous as the length
of the peptide increases. It was observed that the most populated
conformer, with ø1(N-CR-Câ-Cγ) ∼ 180°, had populations
of about 80% for 2 and of 90% for 3.13
The CD studies for 1, 2, and 4 were undertaken in 50 µM
MeOH solutions (Figure 5), whereas for 3 and 5, because of
poor solubility, the spectra were obtained in saturated solutions.11
The spectra show two very distinct minima at 196 and 220 nm,
with no isodichroic point. The stronger peak at about 198 nm
resembles those observed in 10/12-9,14 as well as in 11/9-mixed
helices5 at about 200 nm, whereas the weaker peak corresponds
Conclusions
We have found that alternating sequences of â3-hVal and
D-Phe provide stable and robust mixed 11/9-helices in peptides
(14) (a) Seebach, D.; Gademann, K.; Schreiber, J. V.; Mathews, J. L.;
Hintermann, T.; Jaun, B.; Ober, L.; Hommel, U.; Widmer, H. HelV. Chim.
Acta 1997, 80, 2033-2037. (b) Seebach, D.; Abele, S.; Gademann, K.;
Guichard, G.; Hintermann, T.; Jaun, B.; Mathews, J. L.; Schreiber, J. V.;
Ober, L.; Hommel, U.; Widmer, H. HelV. Chim. Acta 1998, 81, 932-982.
(13) Demarco, A.; Llinas, M.; Wuthrich, K.; Biopolymers 1978, 17, 617-
636.
8398 J. Org. Chem., Vol. 71, No. 22, 2006

11/9-Mixed Helices in the R/â-Peptides
FIGURE 6. Superimposed MD structures for 2: (A) stereoview, (B) top view from the C-terminal end.
temperature. Solvent was evaporated, and the residue was diluted
1-5. Despite the markedly inherent lower propensity for helix
formation in proteinogenic amino acids,4,15 even peptides with
as few as only three residues display the signatures of a helix.
The hindrance in the studies due to the poor solubility of these
hydrophobic peptides, during both synthesis and NMR studies,
may be alleviated by incorporating amino acids with charged
side chains, which may additionally endow this class of
molecules with other desirable properties. Controlled folding
of biomolecules into well-defined compact structures allows
precise orientation and placement of the functional groups to
enable them to carry out desired functions. Extending the
conformational space for the family of mixed helices by using
R- and â-amino acids with proteinogenic side chains opens up
novel options and enhances the variety of secondary structural
elements available for bioevaluation and obtaining higher-order
assemblies in synthetic peptides and proteins.
with H2O (10 mL) and washed with ether (2 × 10 mL). The aq
layer was neutralized with citric acid at 0 °C and extracted with
ether (2 × 10 mL). The organic layer was dried (Na2SO4) and
evaporated to give Boc-D-Phe-â-hVal-OH (10), which was used
as such for a further coupling reaction.
As described for the synthesis of 9, a mixture of 10 (0.38 g,
1.02 mmol), HOBt (0.16 g, 1.22 mmol), and EDCI (0.23 g, 1.22
mmol) in CH2Cl2 (8 mL) was stirred at 0 °C for 30 min and treated
with H-D-Phe-OMe‚HCl (11; 0.28 g, 1.3 mmol) and Et3N (0.4 mL,
3.9 mmol) under a nitrogen atmosphere for 12 h. Workup and
purification of the residue by column chromatography (silica gel,
25% ethyl acetate, and pet ether) afforded 1 (0.35 g, 61.3%) as a
white solid: mp 178-180 °C; IR (KBr) 3283 (m), 2954 (m), 1744
(m), 1688 (m), 1656 (m), 1648 (m), 1641 (m), 1539 (m), 1455
1
(w), 1366 (w), 1219 (m), 1170 (w), 771 (s), 698 (w); H NMR
(500 MHz, CDCl3) δ 7.42-7.15 (m, 10H, Ar-H), 7.0 (d, J ) 8.5
Hz, 1H, NH-3), 6.86 (d, J ) 9.8 Hz, 1H, NH-2), 5.08 (d, J ) 7.5
Hz, 1H, NH-1), 4.84 (ddd, J ) 5.0, 9.6, 8.5 Hz, 1H, CRH-3), 4.26
(q, J ) 7.5 Hz, 1H, CRH-1), 3.75 (s, 3H, OMe), 3.20 (dd, J ) 5.0,
14.2 Hz, 1H, CâH(pro-S)-3), 3.08 (dd, J ) 7.5, 13.6 Hz, 1H,
CâH(pro-S)-1), 3.01 (dd, J ) 9.6, 14.2 Hz, 1H, CâH(pro-R)-3), 2.96
(dd, J ) 7.5, 13.6 Hz, 1H, CâH(pro-R)-1), 2.38 (dd, J ) 4.5, 14.0
Experimental Section
Boc-D-Phe-â-hVal-OMe (9). A solution of 6 (0.88 g, 3.6 mmol)
and TFA (5 mL) in CH2Cl2 (5 mL) was stirred for 2 h at room
temperature. The solvent was evaporated under reduced pressure
and resulting H-â-hVal-OMe‚CF3COOH (8) was dried under high
vacuum and used without further purification.
Hz, 1H, CRH(pro-R)-1), 2.30 (dd, J ) 4.5, 14.0 Hz, 1H, CRH(pro-S)
-
1), 1.58 (m, 1H, CγH-2), 1.39 (s, 9H, Boc), 0.87 (d, J ) 6.6 Hz,
3H, δCH3), 0.62 (d, J ) 6.6 Hz, 3H, δ′CH3); 13C NMR (75 MHz,
CDCl3) δ 173.6, 171.3, 170.8, 155.9, 136.7, 129.3, 129.0, 128.6,
128.5, 126.9, 80.2, 56.7, 53.6, 52.7, 52.4, 38.6, 38.1, 37.2, 30.4,
29.7, 28.2, 19.7, 19.2; FAB-MS 545 ([M + Li]+).
A solution of Boc-D-Phe-OH 7 (0.95 g, 3.6 mmol), HOBt (0.58
g, 4.3 mmol), and EDCI (0.8 g, 4.3 mmol) in CH2Cl2 (10 mL) at
0 °C was stirred under a N2 atmosphere for 30 min and treated
sequentially with the above TFA salt 8 and Et3N (1.5 mL, 10.8
mmol) and stirred for 12 h. Solvent was evaporated, and the residue
was dissolved in CHCl3 (10 mL). The organic layer was washed
with aq NH4Cl solution (2 × 10 mL), NaHCO3 solution (2 × 10
mL), and brine (10 mL), dried (Na2SO4), and evaporated. The
residue obtained was purified by column chromatography (silica
gel, 10% ethyl acetate, and pet ether) to afford dipeptide 9 (1.12 g,
79.8%) as a white solid: mp 122-125 °C; IR (KBr) 3477 (w),
3340 (m), 3306 (m), 2952 (m), 1732 (m), 1687 (m), 1653 (s), 1519
(s), 1441 (m), 1368 (m), 1290 (m), 1241 (m), 1165 (m), 1046 (w),
Boc-D-Phe-â-hVal-D-Phe-â-hVal-D-Phe-OMe (2): A solution
of 1 (0.18 g, 0.33 mmol) and TFA (5 mL) in CH2Cl2 (5 mL) was
stirred for 2 h at room temperature. The solvent was evaporated
under reduced pressure, and the resulting H-D-Phe-â-hVal-D-Phe-
OMe‚CF3COOH (13) was dried under high vacuum and used
without further purification.
A solution of 10 (0.12 g, 0.32 mmol), HOBt (0.05 g, 0.37 mmol),
and EDCI (0.75 g, 0.37 mmol) in CH2Cl2 (8 mL) was stirred at 0
°C for 30 min and treated with the above H-D-Phe-â-hVal-D-Phe-
OMe‚CF3COOH (12) and Et3N (0.04 mL, 10 mmol) and stirred
for 12 h. Solvent was removed, and the resultant solid was washed
with pet ether and a 1:1 mixture of pet ether and EtOAc to give 2
(0.24 g, 93.6%) as a white solid: mp 206-210 °C; IR (KBr) 3301
(s), 3065 (m), 2961 (s), 1756 (s), 1691 (s), 1653 (s), 1541 (s), 1460
(s), 1383 (s), 1169 (s), 1045 (m), 990 (m), 855 (w), 748 (s), 699
1
1019 (w), 865 (w), 766 (s), 695 (m), 664 (m), 626 (m); H NMR
(400 MHz, CDCl3) δ 7.35-716 (m, Ar-5H), 6.27 (d, J ) 9.5 Hz,
1H), 4.97 (br s, 1H), 4.31 (q, J ) 7.3 Hz, 1H), 4.03 (m, 1H), 3.64
(s, 3H), 3.06 (d, J ) 6.9 Hz, 2H), 2.45 (d, J ) 6.0 Hz, 2H), 1.72
(m, 1H), 1.41 (s, 9H), 0.79 (d, J ) 6.8 Hz, 3H), 0.77 (d, J ) 6.8
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 172.0, 170.7, 155.3, 136.7,
129.2, 128.6, 126.8, 80.1, 56.1, 51.7, 51.4, 38.2, 36.3, 31.2, 28.2,
19.1, 18.4; FAB-MS 393 ([M + 1]+).
Boc-D-Phe-â-hVal-D-Phe-OMe (1): A solution of ester 9 (0.40
g, 1.02 mmol) in a mixture of THF/MeOH:H2O (2:1:1) was treated
with LiOH (0.10 g, 2.5 mmol) at 0 °C and stirred for 3 h at room
1
(s), 493 (m); H NMR (500 MHz, CDCl3) δ 7.42-7.10 (m, 15H,
Ar-H), 7.74 (d, J ) 8.5 Hz, 1H, NH-5), 7.63 (d, J ) 6.72 Hz, 1H,
NH-3), 7.45 (d, J ) 10.0 Hz, 1H, NH-2), 7.09 (d, J ) 10.0 Hz,
1H, NH-4), 5.20 (d, J ) 7.0 Hz, 1H, NH-1), 4.79 (ddd, J ) 10.9,
8.5, 4.0 Hz, 1H, CRH-5), 4.46 (q, J ) 6.7 Hz, 1H, CRH-3), 4.44
(q, J ) 7.0 Hz, 1H, CRH-1), 3.87 (tt, J ) 10.0, 4.5 Hz, 1H, CâH-
1), 3.77 (s, 3H, OCH3), 3.18 (dd, J ) 13.8, 4.0 Hz, 1H, Câ′H-5),
3.12 (dd, J ) 13.4, 7.0 Hz, 1H, Câ′H-1), 3.07 (dd, J ) 12.9, 6.7
(15) Leplae, P. R.; Fisk, J. D.; Porter, E. A.; Weisblum, B.; Gellman, S.
H. J. Am. Chem. Soc. 2002, 124, 6820-6821.
J. Org. Chem, Vol. 71, No. 22, 2006 8399

Srinivasulu et al.
Hz, 1H, Câ′H-3), 3.04 (dd, J ) 12.9, 6.7 Hz, 1H, CâH-3), 2.98
(dd, J ) 13.4, 7.0 Hz, 1H, CâH-1), 2.97 (dd, J ) 13.8, 10.9 Hz,
1H, CâH-5), 2.42 (dd, J ) 13.0, 4.5 Hz, 1H, CRH(pro-R)-2), 2.40
(dd, J ) 13.0, 4.5 Hz, 1H, CRH(pro-S)-2), 2.35 (dd, J ) 12.5, 3.4
30 min and treated with TFA salt 12 (0.03 g, 0.55 mmol) and Et3N
(0.08 mL, 1.65 mmol) and stirred for 12 h under a nitrogen
atmosphere. Solvent was removed, and the resultant solid was
washed with pet ether and a 1:1 mixture of pet ether and EtOAc to
give 3 (0.055 g, 29.6%) as a white solid: mp 250-254 °C; IR
(KBr) 3308 (s), 3064 (m), 2962 (s), 1755 (s), 1691 (s), 1646 (s),
1536 (s), 1454 (m), 1368 (m), 1247 (m), 1173 (s), 1047 (m), 960
Hz, 1H, CRH(pro-R)-4), 2.17 (dd, J ) 12.5, 4.7 Hz, 1H, CRH(pro-S)
-
4), 1.57 (m, 1H, CγH-2), 1.52 (m, 1H, CγH-4), 1.38 (s, 9H, Boc),
0.95 (d, J ) 6.7 Hz, 3H, CδMe-2), 0.93 (d, J ) 6.7 Hz, 3H, CδMe-
4), 0.64 (d, J ) 6.7 Hz, 3H, Cδ′Me-2), 0.51 (d, J ) 6.7 Hz, 3H,
Cδ′Me-4); 13C NMR (75 MHz, CDCl3) δ 174.6, 172.6, 172.3,
171.8, 171.0, 156.2, 137.3, 137.0, 136.6, 129.4, 129.2, 129.1,
128.58, 128.3, 126.9, 126.7, 80.2, 57.4, 56.7, 54.3, 53.7, 53.1, 52.5,
38.6, 38.4, 38.0, 37.1, 36.5, 30.1, 29.7, 28.3, 20.0, 19.8, 19.7, 19.5;
ES+ MS: 801 ([M + 1]+).
Boc-D-Phe-â-hVal-D-Phe-â-hVal-D-Phe-â-hVal-D-Phe-OMe (3):
A solution of 2 (0.13 g, 0.16 mmol) and TFA (5 mL) in CH2Cl2 (5
mL) was stirred for 2 h at room temperature. The solvent was
evaporated under reduced pressure, and the resulting H-D-Phe-â-
hVal-D-Phe-â-hVal-D-Phe-OMe‚CF3COOH (13) was dried under
high vacuum and used without further purification.
1
(w), 749 (m), 699 (s), 587 (w); H NMR (500 MHz, CDCl3) δ
7.20 (d, J ) 7.9 Hz, 1H, NH-4), 7.07 (d, J ) 9.2 Hz, 1H, NH-3),
6.79 (d, J ) 6.5 Hz, 1H, NH-2), 5.05 (d, J ) 9.2 Hz, 1H, NH-1),
4.75 (ddd, J ) 9.4, 7.9, 5.0 Hz, 1H, CRH-4), 4.51 (q, J ) 6.5 Hz,
1H, CRH-2), 3.80 (m, 1H, CâH-3), 3.74 (s, 3H, OMe), 3.64 (dddd,
J ) 10.0, 7.7, 6.4, 4.4 Hz, 1H, CâH-1), 3.14 (dd, J ) 13.5, 5.0
Hz, 1H, CâH(pro-S)-4), 3.13 (dd, J ) 13.5, 6.5 Hz, 1H, CâH(pro-S)-
2), 2.99 (dd, J ) 13.5, 6.5 Hz, 1H, CâH(pro-R)-2), 2.97 (dd, J )
13.5, 9.4 Hz, 1H, CâH(pro-R)-4), 2.39 (dd, J ) 14.5, 7.7 Hz, 1H,
CRH(pro-R)-1), 2.36 (m, 2H, CRH(pro-R)-3 and CRH(pro-S)-3), 2.33
(dd, J ) 14.5, 4.4 Hz, 1H, CRH(pro-S)-1), 1.67 (m, 1H, CγH-1),
1.59 (m, 1H, CγH-3), 0.88 (d, J ) 6.6 Hz, 3H, CδH-Me-3), 0.87
(d, J ) 6.6 Hz, 6H, CδH-Me-1 and CδH-Me′-1), 0.65 (d, J ) 6.6
Hz, 3H, CδH-Me′-3); 13C NMR (75 MHz, CDCl3) δ 174.0, 171.6,
171.2, 171.0, 157. 0, 136.8, 129.3, 129.1, 128.6, 126.9, 79.5, 55.5,
54.0, 52.6, 52.4, 39.6, 38.8, 37.5, 36.9, 31.9, 30.6, 28.4, 19.7, 19.3,
A solution of 10 (0.05 g, 0.15 mmol), HOBt (0.025 g, 0.18
mmol), and EDCI (0.035 g, 0.18 mmol) in CH2Cl2 (5 mL) was
stirred at room temperature for 30 min and treated with the above
TFA salt 13 and Et3N (0.05 mL, 0.8 mmol) and then stirred for 12
h under a nitrogen atmosphere. Solvent was removed, and the
resultant solid was washed with pet ether and a 1:1 mixture of pet
ether and EtOAc to give 3 (0.06 g, 38.8%) as a white solid: mp >
250 °C; IR (KBr) 3281 (s), 3066 (m), 3031 (m), 2963 (s), 1722
(s), 1644 (s), 1550 (s), 1454 (m), 1368 (m), 1292 (m), 1242 (m),
1172 (m), 1119 (m), 1050 (w), 960 (w), 914 (w), 742 (m), 698 (s),
621 (w); 1H NMR (750 MHz, CDCl3, 5 °C) δ 8.21 (d, J ) 6.4 Hz,
1H, NH-5), 7.89 (d, J ) 10.1 Hz, 1H, NH-4), 7.86 (d, J ) 8.4 Hz,
1H, NH-7), 7.66 (d, J ) 5.8 Hz, 1H, NH-3), 7.65 (d, J ) 9.8 Hz,
1H, NH-2), 7.33 (d, J ) 10.0 Hz, 1H, NH-6), 7.0-7.4 (m, 20H,
Ar-H), 5.14 (d, J ) 6.7 Hz, 1H, NH-1), 4.72 (ddd, J ) 12.0, 8.4,
3.6 Hz, 1H, CRH-1), 4.66 (dq, J ) 7.6, 5.8 Hz, 1H, CRH-3), 4.36
(ddd, J ) 9.0, 6.4, 5.4 Hz, 1H, CRH-5), 3.81 (m, 1H, CâH-2),
3.75 (s, 3H, OMe), 3.71 (m, 1H, CâH-4), 3.68 (m, 1H, CâH-6),
3.11 (dd, J ) 14.0, 3.6 Hz, 1H, CâH(pro-S)-7), 3.08 (dd, J ) 13.7,
6.7 Hz, 1H, CâH(pro-S)-1), 3.05 (m, 2H, CâH-3), 3.04 (dd, J )
13.0, 9.0 Hz, 1H, CâH(pro-S)-5), 2.93 (dd, J ) 13.7, 6.7 Hz, 1H,
CâH(pro-R)-1), 2.92 (dd, J ) 13.0, 6.4 Hz, 1H, CâH(pro-R)-5), 2.87
(dd, J ) 14.0, 12.0 Hz, 1H, CâH(pro-R)-7), 2.39 (dd, J ) 13.0, 4.5
18.4; FAB MS 653 [M + 1]+
.
Boc-â-hVal-D-Phe-â-hVal-D-Phe-â-hVal-D-Phe-OMe (5): A
solution of 14 (0.01 g, 0.045 mmol), HOBt (0.008 g, 0.055 mmol),
and EDCI (0.01 g, 0.055 mmol) in CH2Cl2 (5 mL) was stirred at
room temperature for 30 min and treated with TFA salt (13) of 2
(0.03 g, 0.04 mmol) and Et3N (pH adjusted to basic) and then stirred
under a nitrogen atmosphere for 12 h. Solvent was removed, and
the resultant solid was washed with pet ether and a 1:1 mixture of
pet ether and EtOAc to give 5 (0.036 g, 90.7%) as a white solid:
mp > 250 °C; IR (KBr) 3294 (s), 3064 (w), 3031 (w), 2961 (m),
2873 (w), 1745 (m), 1690 (s), 1645 (s), 1538 (s), 1455 (m), 1368
1
(m), 1247 (m), 1134 (s), 748 (m), 699 (m), 638 (m), 616 (m); H
NMR (300 MHz, CDCl3) δ 7.86 (d, J ) 8.3 Hz, 1H), 7.75 (d, J )
6.5 Hz, 1H), 7.61 (d, J ) 9.8 Hz, 1H), 7.41-7.13 (m, 15H, Ar-
H), 5.10 (d, J ) 9.8 Hz, 1H), 4.77 (m, 1H), 4.69 (q, J ) 7.1 Hz,
1H), 4.37 (m, 1H), 3.86 (m, 1H), 3.78 (s, 3H, OMe), 3.68 (m, 2H),
3.30-2.87 (m, 6H), 2.5-2.15 (m, 6H), 1.44 (s, 9H, Boc), 0.96 (d,
J ) 6.2 Hz, 3H), 0.93 (d, J ) 6.2 Hz, 3H), 0.85 (d, J ) 6.6 Hz,
6H), 0.70 (d, J ) 6.2 Hz, 3H), 0.57 (d, J ) 6.2 Hz, 3H); FAB MS
914 [M + 1]+.
Hz, 1H, CRH(pro-R)-2), 2.35 (m, 2H, CRH(pro-R)-4 and CRH(pro-S)
-
2), 2.31 (dd, J ) 12.6, 3.1 Hz, CRH(pro-R)-6), 2.20 (m, 1H,
CRH(pro-S)-4), 2.09 (dd, J ) 12.6, 5.0 Hz, 1H, CRH(pro-S)-6), 1.56
(m, 1H, CγH-6), 1.55 (m, 1H, CγH-2), 1.49 (m, 1H, CγH-4), 1.31
(s, 9H, Boc), 0.94 (d, J ) 6.7 Hz, 1H, Cδ′ Me-6), 0.93 (d, J ) 6.4
Hz, 1H, Cδ′ Me-4), 0.92 (d, J ) 6.5 Hz, 1H, Cδ′ Me-2), 0.61 (d,
J ) 6.7 Hz, 1H, Cδ′ Me-6), 0.60 (d, J ) 6.5 Hz, 1H, Cδ′ Me-2),
0.47 (d, J ) 6.4 Hz, 1H, Cδ′ Me-4); HRMS-MS (C60H81N7O10Na)
1082.5942 (calcd), 1082.5906 (obsd).
Acknowledgment. S.K.K. is thankful to CSIR, New Delhi,
for financial support. A.C.K. is thankful to M. Goerlach and R.
Ramachandran for 750 MHz NMR and to INSA and DFG for
financial support.
Supporting Information Available: NMR details, solvent
titration plots, and distance constraints used in MD calculations.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Boc-â-hVal-D-Phe-â-hVal-D-Phe-OMe (4): A solution of 14
(0.07 g, 0.46 mmol), HOBt (0.07 g, 0.55 mmol), and EDCI (0.1 g,
0.55 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for
JO0612980
8400 J. Org. Chem., Vol. 71, No. 22, 2006