Pseudoprolines as removable turn inducers: Tools for the cyclization of small peptides

Article abstract of DOI:10.1021/jo0484732

The cyclization of small peptides which do not incorporate turn inducers is often difficult. We have developed a method involving the use of removable turn inducers, in the form of pseudoprolines, for the cyclization of difficult peptide sequences. The ps

Full text of DOI:10.1021/jo0484732

Pseudoprolines as Removable Turn Inducers: Tools for the
Cyclization of Small Peptides
Danielle Skropeta, Katrina A. Jolliffe,* and Peter Turner
School of Chemistry, The University of Sydney, 2006 NSW, Australia
jolliffe@chem.usyd.edu.au
Received August 31, 2004
The cyclization of small peptides which do not incorporate turn inducers is often difficult. We have
developed a method involving the use of removable turn inducers, in the form of pseudoprolines,
for the cyclization of difficult peptide sequences. The pseudoprolines induce a cisoid amide bond in
the peptide backbone which facilitates cyclization. They are then readily removed to yield a cyclic
peptide that does not contain any turn inducers.
Introduction
incorporate turn inducers is a more challenging problem.
Several methods have recently been developed for the
head-to-tail cyclization of small peptides. However, no
single method has been found to be generally applicable,
and many methods result in only a moderate increase in
the yield of the required cyclic product or have only been
employed for the synthesis of peptides that already
contain some type of turn-inducer.3,7-10 We now report
an approach to the cyclization of small peptides involving
the incorporation of turn inducing side chain protecting
groups that can be removed once peptide cyclization is
complete to give cyclic peptide products that do not
contain turn-inducers.
Pseudoprolines (Ψpro’s), derived from serine, threo-
nine, and cysteine residues upon cyclocondensation with
an aldehyde or ketone, were recently introduced as
solubilization aids for solid-phase peptide synthesis,11,12
and dipeptides containing these modified residues are
commercially available.13 The introduction of these modi-
fied residues into a peptide sequence has been found to
induce a predominantly cisoid conformation about the
amide bond immediately preceding the modified amino
acid, resulting in the adoption of a type VI â-turn
structure.12 We anticipated that this would enhance both
the rate and yield of cyclization of peptides containing
these modified amino acid residues.10 Once cyclization
was complete, treatment with acid would result in
Cyclic peptides are currently attracting the attention
of chemists and biochemists alike. Naturally occurring
cyclic peptides exhibit a wide range of biological activities,
and cyclic peptides generally exhibit improved biological
properties when compared to their linear counterparts.
Cyclization may also be employed to constrain a bioactive
peptide in its active conformation, thereby increasing its
potency and/or specificity. As such, the development of
efficient and convenient methods for the synthesis of
cyclic peptides is of great interest.1-3
While it is now commonplace to synthesize linear
peptides in high yield and purity, the head-to-tail cy-
clization of small linear peptides (three to eight amino
acids) remains difficult, often resulting in epimerization
of the C-terminal amino acid and/or the formation of
linear and cyclic oligomers.4,5 This commonly observed
difficulty in cyclization has been attributed to the prefer-
ence of amide bonds to adopt an s-transoid conformation,
as a result of their strong π-character. This results in an
extended and somewhat rigid linear peptide structure
that is highly favored in certain peptide sequences. These
compounds can be virtually impossible to cyclize; for
example, the cyclization of all-L peptides is notoriously
difficult.5
Cyclization of a linear peptide is dependent on a
number of factors including the peptide sequence. The
incorporation of turn-inducing elements (e.g., Gly, Pro,
N-alkylated, or D-amino acids) into a peptide sequence
has been shown to enhance cyclization yields.4-8 How-
ever, the synthesis of cyclic peptides which do not
(8) El Haddadi, M.; Cavelier, F.; Vives, E.; Azmani, A.; Verducci,
J.; Martinez, J. J. Peptide Sci. 2000, 6, 560.
(9) (a) Tam, J. P.; Lu, Y.-A.; Yu, Q. J. Am. Chem. Soc. 1999, 121,
4316. (b) Shao, Y.; Lu, W.; Kent, S. B. Tetrahedron Lett. 1998, 39, 3911.
(c) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363. (d)
Meutermans, W. D. F.; Golding, S. W.; Bourne, G. T.; Miranda, L. P.;
Dooley, M. J.; Alewood, P. F.; Smythe, M. L. J. Am. Chem. Soc. 1999,
121, 9790
(1) Li, P.; Roller, P. P.; Xu, J. Curr. Org. Chem., 2002, 6, 411.
(2) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243.
(3) For recent reviews, see: (a) Lambert, J. N.; Mitchell, J. P.;
Roberts, K. D. Perkin Trans. 1 2001, 471. (b) Davies, J. S. J. Pept. Sci.
2003, 9, 471.
(10) Sager, C.; Mutter, M.; Dumy, P. Tetrahedron Lett. 1999, 40,
7987. Ru¨ckle, T.; de Lavallaz, P.; Keller, M.; Dumy, P.; Mutter, M.
Tetrahedron 1999, 55, 11281.
(4) Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345.
(11) Wo¨hr, T.; Wahl, F.; Hefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.;
Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218.
(5) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber,
H.; Carpino, L. A.; Bienert, M. J. Org. Chem. 1996, 61, 8831.
(6) Titlestad, K. Acta Chem. Scand. 1997, B31, 641.
(7) Cavelier-Frontin, F.; Achmad, S.; Verducci, J.; Jacquier, R.; Pe`pe,
G. THEOCHEM 1993, 286, 125.
(12) Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wo¨hr, T.;
Mutter, M. J. Am. Chem. Soc. 1997, 119, 918.
(13) Novabiochem and Bachem both supply a range of Fmoc-
protected pseudoproline containing dipeptides.
10.1021/jo0484732 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/12/2004
8804
J. Org. Chem. 2004, 69, 8804-8809

Pseudoprolines as Removable Turn Inducers
imidazole gave the TBS-protected dipeptide 9 (Scheme
1). Alternatively, the pseudoproline-containing dipeptide
12 was prepared upon treatment of 8 with 2-methox-
ypropene in the presence of (()-10-camphorsulfonic acid.
Hydrolysis of the methyl esters 9 and 12 was readily
achieved upon treatment with LiOH in aqueous methanol
to give the carboxylic acids 10 and 13, respectively, in
good yield. Hydrogenolysis of 9 gave the free amine 11,
but when 12 was treated with hydrogen in the presence
of a palladium on charcoal catalyst, only the diketopip-
erazine 15 was isolated.
FIGURE 1. Previous attempts to synthesize 1.14
SCHEME 1
removal of the pseudoprolines to yield cyclic peptides
devoid of turn inducers.
Our initial interest in this area stemmed from the
desire to prepare derivatives of the hexapeptide cyclo-
(Val-Thr)3 (1) for molecular recognition studies. Wipf and
Miller have previously reported that the TBS side-chain-
protected linear peptide H-Val-Thr(TBS)-Val-Thr(TBS)-
Val-Thr(TBS)-OH (2a) does not cyclize under a wide
range of attempted conditions (Figure 1).14 We therefore
investigated the effect that the incorporation of pseudopro-
line residues had upon the head-to-tail cyclization yield
of this hexapeptide sequence.
Results and Discussion
A modular approach to the synthesis of hexapeptides
2a-7a was employed, in which fragment coupling of
appropriately protected dipeptides 10, 11, and 13 (Scheme
1), using standard solution-phase techniques, gave ready
access to the differentially protected hexapeptides 2b-
7b. Thus, dipeptide 8 was prepared according to litera-
ture procedures.15 Treatment of 8 with TBSCl and
The hexapeptides 2b-7b were prepared by coupling
of these dipeptide fragments using standard solution-
phase techniques. While no epimerization was observed
(15) (a) Wipf, P.; Miller, C. P.; Grant, C. G. Tetrahedron 2000, 56,
9143. (b) Wo¨hr, T.; Mutter, M. Tetrahedron Lett. 1995, 36, 3847. (c)
Kurokawa, N.; Ohfune, Y. Tetrahedron 1993, 49, 6195.
(14) Wipf, P.; Miller, C. P. J. Am. Chem. Soc. 1992, 114, 10975.
J. Org. Chem, Vol. 69, No. 25, 2004 8805

Skropeta et al.
SCHEME 2
be the trans form, although this could not be confirmed
by chemical shift or NOE measurements. The cis/trans
ratios in CDCl3 were determined to be 85:15 and 80:20
for 12 and 13, respectively, which is in accordance with
the ratios observed by Keller et al. for similar Fmoc-
protected dipeptides.17 These ratios did not change
significantly with variations in either the solvent (CDCl3,
toluene-d8, DMSO-d6, CD3CN, or CD3OD) or temperature
1
(300-370 K). In contrast, the H and 13C NMR spectra
of the TBS-protected dipeptide 9 exhibit only a single set
of resonances, and the observed 1H-15N 1J coupling
constant (93 Hz) indicates that 9 is present as a single
conformer with trans geometry.18
While the 1H and 13C NMR spectra for the tetrapeptide
1
20 contain only a single set of resonances, the H and
13C NMR spectra of the tetrapeptide 16 contain two sets
of resonances in an 85:15 ratio (CDCl3 and DMSO-d6).
In this case, 2D NMR NOESY and ROESY experiments
indicate that the major conformer is that in which the
amide bond preceding the pseudoproline residue is cisoid
with the other amide bonds adopting a transoid config-
uration. The minor conformer is likely to be that in which
all amide bonds are trans. The 1H and 13C NMR spectra
of tetrapeptide 18 (CD3CN) indicate that this peptide
exists as a mixture of 4 conformers in a 12:6:1:1 ratio
which interconvert slowly on the NMR time scale. While
overlapping of the signals from different rotamers makes
it difficult to observe the characteristic NOE cross-peaks
for the cis rotamers, it can be assumed by analogy with
the other peptides discussed here that the major con-
former is that in which both pseudoproline residues are
preceded by cis amide bonds.
in couplings of the Ψpro-containing acid 13, some epimer-
ization (up to 30%) was observed during couplings of the
TBS-protected acid 10, as expected for this hindered
fragment coupling. Diastereoisomers resulting from
epimerization were readily separated by column chro-
matography. Notably, peptides containing Ψpro residues
were easier to handle in terms of both solubility and
stability than the corresponding TBS-protected deriva-
tives, indicating that the Ψpro’s are useful serine, threo-
nine, and cysteine protecting groups for the preparation
of difficult peptide sequences by solution-phase peptide
synthesis as well as for solid-phase synthesis.
Since the dipeptide 14 (Scheme 1) was not available
for formation of hexapeptides containing a C-terminal
pseudoproline moiety, it was necessary to adopt an
alternative route to the synthesis of hexapeptides 6b and
7b (Scheme 2). In this case, the tetrapeptide 16 was
treated with TBAF to give the corresponding tetrapeptide
17 with a free threonine side chain. Subsequent treat-
ment with 2-methoxypropene in a “post-insertion” reac-
tion gave the required tetrapeptide 18 in 82% yield.
Deprotection of the N-terminus to give amine 19, followed
by coupling with the carboxylic acids 10 and 13, respec-
tively, gave the hexapeptides 6b and 7b in good yield over
the two steps. Hexapeptides 2b-5b were similarly
prepared by coupling both acids 10 and 13 with both
amines 21 and 22. Sequential deprotection of the C- and
N-terminii of the hexapeptides 2b-7b using the standard
conditions described above gave the free peptides 2a-
7a, which were immediately subjected to cyclization
experiments.
Linear Peptide Conformation. Where possible, the
cis/trans ratios of the amide bonds in the linear peptides
were determined using NMR spectroscopic techniques.
In the case of dipeptides 12 and 13, a major and minor
set of resonances are clearly observed in both the 1H and
13C NMR spectra. The major conformer was determined
to be that with a cisoid amide bond on the basis of the
typical NOE cross-peaks observed by 2D NMR ROESY
1
The H and 13C NMR spectra of the protected linear
hexapeptides 3b-7b are either broad (particularly for
spectra obtained in CDCl3) or exhibit overlapping reso-
nances for at least two confomers for each peptide
(DMSO-d6, CD3CN). However, the 1H NMR spectrum of
hexapeptide 2b exhibits a single set of relatively well
resolved signals in DMSO-d6, CD3CN, and CDCl3, sug-
gesting that this peptide adopts a single conformation
in solution. The absence of NOE cross-peaks between the
R protons of adjacent Val and Thr residues suggests that
this conformer has all-trans amide bonds, but this could
not be confirmed. Interestingly, 1H and 13C NMR spectra
of the N- and C-deprotected hexapeptide 7a show a highly
predominant set of resonances in DMSO-d6, CD3CN, and
and NOESY experiments (i.e., RHi-1 - RHi and RHi-1
-
âHi cross-peaks).16 The minor conformer was assumed to
(17) Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G.
S.; Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714.
(18) Bystrov, V. F. Prog. Nucl. Magn. Reson. Spectrosc. 1976, 10,
41.
(16) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; John Wiley
& Sons: New York, 1986; p 117.
8806 J. Org. Chem., Vol. 69, No. 25, 2004

Pseudoprolines as Removable Turn Inducers
MeOD-d4, consistent with the presence of a major con-
former in solution (a minor set of resonances [<5% as
estimated by integration] is also detected). The major
conformer was assigned as that in which the three amide
bonds preceding the pseudoproline residues are cisoid,
using 2D NOESY experiments as described above for the
dipeptides. An NOE cross-peak between the R protons
of the N-terminal and C-terminal amino acid residues of
this peptide was also observed, indicating that the
terminii of the peptide must be in close proximity.
Cyclization Experiments. Cyclization studies were
initially attempted using a variety of coupling reagents,
solvents and concentrations. However, pentafluorophenyl
diphenylphosphinate (FDPP) was found to give consis-
tently higher yields of cyclic peptide products than the
other coupling reagents employed (e.g., HATU, DEPBT)
and as such this reagent was used for comparative
cyclization experiments. In accordance with previous
experiments,14 peptide 2a gave no identifiable cyclic
products under our optimal cyclization conditions. How-
ever, the incorporation of a single Ψpro residue in the
center of the linear peptide backbone (hexapeptides 3a
and 4a) gave trace amounts (<5%) of a cyclic monomer
at high dilution (0.005 mM). Subsequent characterization
of the cyclic products obtained from these experiments
and comparison to an authentic sample (see below)
indicated that, in both cases, cyclization proceeded only
after epimerisation of the C-terminal amino acid had
occurred.
Treatment of the partially deprotected cyclic peptides
25 and 27 and the fully deprotected 1 with excess TBS-
Cl under standard conditions provided authentic samples
of the cyclic peptides (26, 28, and 23, respectively) that
would have been obtained if cyclization of hexapeptides
3a-5a occurred without epimerization. Comparison of
these samples (NMR, TLC, RD) with the products ob-
tained after peptide cyclization should have enabled us
to determine if the expected products were obtained.
However, the TBS-protected cyclic peptides exist as a
mixture of conformers in solution, and the number and
ratio of conformers present was found to be very sensitive
to the solvent and the presence of impurities, making
comparison by NMR difficult. Therefore, to confirm the
structures of the cyclized products, they were treated
with a 4 M solution of HCl in dioxane to give the fully
deprotected cyclic peptides which were then compared
to 1. While 1 exhibits C3 symmetry in solution (see
below), the products obtained after cyclization of 3a-5a,
followed by global deprotection, all gave identical 1H
NMR spectra in which six signals are clearly observed
for the six amide protons present in the now unsym-
metrical epi-1. The product obtained after global depro-
tection of 28 was identical in all respects to 1, confirming
that the cyclization of 6a which bears a pseudoproline
residue at the C-terminus proceeds without epimeriza-
tion.
Increasing the number of Ψpro residues present in the
peptide backbone led to increased yields of cyclic peptide
products. Cyclization of hexapeptide 5a gave a 40% yield
of a single cyclic peptide, but comparison to an authentic
sample indicated that only peptide in which the C-
terminal residue had epimerized underwent cyclization
to yield epi-28. Since we had observed during the
synthesis of the linear peptides that peptides with
C-terminal Ψpro residues were less likely to epimerize
than the corresponding TBS-protected derivatives, we
attempted cyclization of the linear peptides 6a and 7a,
both of which contain a C-terminal pseudoproline residue.
Upon treatment with FDPP, a 0.005 M solution of
hexapeptide 6a gave the corresponding cyclic peptide 28
in 10% yield. In this case, none of the epimeric product
was observed. In the case of hexapeptide 7a, which
contains three Ψpro residues, high yields (84-99%) of
the corresponding cyclic hexapeptide 24 were consistently
obtained, even when the concentration of linear precursor
was increased to 0.01 M and no product corresponding
to C-terminal epimerisation was observed. The high yield
obtained from this cyclization experiment suggests that
linear peptide 7a must be preorganized for cyclization.
This is consistent with the end-to-end NOE cross-peak
observed for the linear peptide.
Removal of the Ψpro “protecting” group from the cyclic
hexapeptide products was achieved upon treatment with
4 M HCl in dioxane or, alternatively, with trifluoroacetic
acid. The deprotection is somewhat slow; nevertheless,
it proceeds in high yield to give the corresponding cyclic
peptide 1, which does not contain any turn inducers.
Intriguingly, deprotection of 24 can be carried out in a
stepwise manner to give cyclic peptides 25 and 27
containing one or two Ψpro residues, respectively, or the
fully deprotected cyclo(Val-Thr)3, 1.
Conformational Studies of the Cyclic Peptides.
1H NMR spectra of 24 indicate that it adopts a C3-
symmetric structure which does not vary with changes
in solvent (CD3CN, CDCl3, CD3OD), consistent with the
presence of a single conformer in solution. NOE cross-
peaks observed between the R protons of the ThrΨMe,Mepro
residues and both the R and â protons of the Val residues
J. Org. Chem, Vol. 69, No. 25, 2004 8807

Skropeta et al.
in the peptide, incorporation of a single Ψpro residue in
a linear precursor is sufficient to substantially improve
the head-to-tail cyclization yield. We have now found that
multiple pseudoproline residues can be used to enable
the cyclization of peptides that do not contain natural
turn inducers. Additionally, we have found that the
cyclization of peptides in which the C-terminal amino
acid is protected as a Ψpro derivative proceeds without
epimerization of this center. This greatly expands the
range of linear precursors available for peptide cyclization
(currently proline and glycine are the only preferred
C-terminal residues). Deprotection of the Ψpro groups
can be carried out with concurrent cis-trans isomeriza-
tion of the preceding amide bonds to give cyclic peptides
devoid of turn inducers. We are currently investigating
modifications of this methodology that will permit the
synthesis of a wider range of cyclic peptides, including
those that lack serine, threonine, or cysteine residues.
Experimental Section
General procedures are available in the Supporting Infor-
mation.
FIGURE 2. ORTEP depiction of 24 with 50% displacement
ellipsoids. Water molecules and hydrogen atoms have been
omitted for clarity.
General Method for N-Deprotection. Catalytic Pd/C (10
mol %) was added to a solution of the Z-protected peptide in
MeOH (20 mL/1 g peptide) in a single-necked, round-bottom
flask fitted with a three-way tap attached to a balloon of H2(g).
The flask was evacuated (water aspirator) and then filled with
H2(g), and the process was repeated five times. The reaction
mixture was then stirred under a H2(g) atmosphere for 5 h,
after which the contents of the flask were filtered through a
pad of Celite, the Celite was washed several times (CHCl3/
MeOH/NH3(aq) 90:9:1), the organic fractions were combined,
and the solvent was removed by rotary evaporation with
toluene to give the desired free amine in high yield. The free
amine was used immediately in subsequent steps, generally
without requiring further purification.
General Method for C-Deprotection. A solution of LiOH
(3 equiv) in water (5 mL/1 g substrate) was added dropwise to
a solution of methyl ester protected peptide (1 equiv) in MeOH
(15 mL/1 g peptide) and the reaction mixture stirred at rt until
the reaction was complete as judged by TLC (typically 6 h).
The reaction mixture was then poured into a 10% aqueous
solution of citric acid and extracted several times with EtOAc.
The combined organic fractions were washed with brine and
dried over Na2SO4, the solvent was evaporated, and the crude
compound was recrystallized to give the desired free carboxylic
acid.
General Method for Pseudoproline Formation. 2-Meth-
oxypropene (2.5 equiv) was added to a cooled solution of the
threonine-containing peptide (1 equiv) in CH2Cl2 (10 mL/1 g
peptide at 0 °C), followed by the addition of a catalytic amount
of camphorsulfonic acid (10 mol %). Upon addition of the latter,
the solution immediately became red. The reaction mixture
was stirred at 0 °C for 5 h, diluted with a further 10 mL of
CH2Cl2, and washed with satd NaHCO3(aq). The organic
fraction was dried over MgSO4, filtered, and concentrated and
the residue purified by flash chromatography to give the
desired pseudoproline-protected peptide as a colorless solid.
General Method for Peptide Coupling. Under anhy-
drous conditions, NMM (1.1 equiv) was added to a solution of
the Z-protected peptide carboxylic acid (1 equiv), EDC (1.1
equiv), and HOBt (1.1 equiv) in CH2Cl2 (20 mL/1 g substrate)
and the mixture stirred at 0 °C for 10 min. A precooled solution
of the methyl ester protected free amine peptide (1.1 equiv)
and NMM (1.1 equiv) in DMF (8 mL/1 g substrate) was then
added and the reaction mixture allowed to warm to rt and
stirred overnight. The reaction mixture was then poured into
a 10% aqueous solution of citric acid and extracted several
times with EtOAc. The combined organic fractions were
washed with 10% citric acid (aq), NaHCO3(aq), water, and then
indicated that the amide bonds preceding the Ψpro
residues adopt a cisoid configuration.16 This was con-
firmed by a single-crystal X-ray diffraction structure
determination for cyclic hexapeptide 24 (Figure 2), which
clearly illustrates the alternating arrangement of cis and
trans amide bonds in the cyclic peptide.
The cyclic peptides 25-28 containing either one or two
pseudoproline residues exhibit multiple conformations in
solution, depending on the solvent, temperature, and
sample concentration. While the presence of multiple
conformers complicates analysis of the structures, the
presence of cisoid amide bonds preceding the pseudo-
proline residues in some of the conformers is clear from
the presence of the expected RHi-1-RHi NOE cross-peaks
between the Vali-1 and Thri[ΨMe,Mepro] R-protons. In
contrast, the 1H and 13C NMR spectra of 1, in both
methanol-d4 and dimethyl-d6 sulfoxide at 22 °C, contain
a single set of well-resolved valine and threonine signals.
However, since the coupling constants and chemical
shifts vary between solvents, it is likely that the observed
C3 symmetry is a result of rapid equilibration between
different conformers, which precludes the determination
of the geometry about the amide bonds.19 Based on the
presence of multiple cis-trans conformers for the more
restricted cyclic peptides (25-28), together with the
absence of any NOE cross-peaks attributable to a cisoid
amide bond geometry, it can be concluded that the amide
bonds of 1 are free to adopt a transoid configuration.
Conclusions
The above results indicate that Ψpro derivatives can
act as removable turn inducers to induce linear peptide
conformations amenable to cyclization. The overall yields
of cyclic peptides are still somewhat dependent on peptide
sequence, but can be significantly improved if more than
one turn inducer is incorporated into the peptide back-
bone. Previous work by Mutter and co-workers10 indicates
that when other turn inducers (e.g., proline) are present
(19) Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512.
8808 J. Org. Chem., Vol. 69, No. 25, 2004

Pseudoprolines as Removable Turn Inducers
brine, dried over Na2SO4, and filtered, the solvent was
evaporated, and the crude compound was purified by flash
chromatography to give the desired coupled peptide.
were washed with brine and dried over Na2SO4, the solvent
was evaporated, and the residue was purified by flash chro-
matography (silica gel: 95:5 f 80:20 EtOAc/MeOH) to give
the di-deprotected hexapeptide 25 (81 mg, 47% yield) as a
colorless solid: mp 118-120 °C; [R]D ) -34 (c 1.6, MeOH); 1H
NMR (CDCl3/200 MHz) δ 0.91-1.06 (m, 18H), 1.21 (d, J 6.3,
3H), 1.29 (d, J 6.4, 3H), 1.43 (d, J 6.4, 3H), 1.58 (s, 3H), 1.68
(s, 3H), 1.93 (m, 1H), 2.13 (m, 2H), 3.40-3.50 (m, 3H), 3.60
(d, J 6.1, 1H), 3.86 (app t, J 5.8, 1H), 3.93-4.46 (m, 6H), 6.86
(br s, 1H), 6.95 (d, J 9.2, 1H, NH), 7.68 (d, J 7.3, 1H), 8.15 (d,
J 6.0, 1H), 8.47 (d, J 8.8, 1H) [minor conformer (<10%) also
observed]; ESI-MS (positive mode) m/z ) 641 ([MH]+, 30), 663
([MNa]+, 52), 1303 ([2M + Na]+, 100); HR-MS m/z ) 663.3665
[MNa]+, 663.3687 calcd for C30H52N6O9Na.
Cyclo[-Val-Thr-Val-Thr-Val-Thr-] (1) (Method 1). The
pseudoproline-derivatized hexapeptide 24 (183 mg, 0.25 mmol)
was treated with 4 M HCl in dioxane (1 mL) at 0 °C, allowed
to warm to rt, and then stirred at rt for 3 d. The reaction
mixture was diluted with water (15 mL) and extracted several
times with EtOAc. The combined organic fractions were
washed with brine, dried over Na2SO4, and filtered, the solvent
was evaporated, and the residue was purified by flash chro-
matography [silica gel: 80:18:2 CHCl3-MeOH-NH3(aq)] to
give two fractions A (Rf 0.6) and B (Rf 0.5).
General Method for TBS Protection. Under anhydrous
conditions, TBS-Cl (2 equiv per hydroxyl) and imidazole (4
equiv per hydroxyl) were added to a solution of cyclic peptide
in DMF (1 mL/100 mg substrate) and the mixture stirred at
rt for 36 h. The mixture was partitioned between EtOAc and
brine, and then the aqueous layer was extracted with EtOAc
(2×). The combined organic phases were dried (Na2SO4), and
then the solvent was removed under reduced pressure. The
residue was purified by flash chromatography to give the
desired TBS-protected products.
Cyclo[-Val-Thr(ΨMe.Mepro)-Val-Thr(ΨMe.Mepro)-Val-Thr-
(ΨMe.Mepro)-] (24). Under anhydrous conditions, 1.5 equiv of
FDPP (154 mg, 0.40 mmol) was added to a solution of the
linear hexapeptide 7a (197 mg, 0.26 mmol) in acetonitrile (54
mL, C ) 0.005 M), followed by the addition of 3 equiv of DIPEA
(0.14 mL, 0.80 mmol). The reaction mixture was then stirred
at rt overnight, after which the solvent was removed by rotary
evaporation and the residue purified by flash chromatography
(silica gel: 1:1 f 2:1 ethyl acetate/hexane) to give the desired
cyclized hexapeptide 24 (174 mg, 91% yield) as a colorless
solid: mp 234-236 °C; [R]D ) -65 (c 1.6, CHCl3). 1H NMR
(CD3CN/200 MHz) δ 0.83 (d, J 6.8, 9H), 0.92 (d, J 6.8, 9H),
1.31 (d, J 6.2, 9H), 1.47 (s, 9H), 1.50 (s, 9H), 2.06 (dseptet, J
5.9, 6.8, 3H), 4.05 (d, J 5.9, 3H), 4.23 (dq, J 5.9, 6.2, 3H), 4.33
(dd, J 5.9, 6.6, 3H), 6.47 (d, J 6.6 Hz, 3H); 13C NMR (CD3CN/
75 MHz) δ 16.8, 17.9(0), 17.9(1), 24.9, 25.8, 30.3, 57.2, 66.9,
75.0, 96.2, 167.3, 168.0; ESI-MS (positive mode) m/z ) 663 (32),
721 ([MH]+, 42), 743 ([MNa]+, 100); HR-ESI-MS m/z )
743.4318 [MNa]+, 743.4314, calcd for C36H60N6O9Na. Anal.
Calcd for C36H60N6O9‚H2O: C, 58.52; H, 8.46; N, 11.37.
Found: C, 58.37; H,8.44; N, 11.24.
Concentration of fraction A gave the di-deprotected hexapep-
tide 25 (50 mg, 33% yield), which was identical in all respects
to the material obtained above.
Concentration of fraction B gave the desired hexapeptide 1
(85 mg, 56% yield) as a colorless solid: mp 188-190 °C; [R]D
1
) -98 (c 0.9, MeOH); H NMR (MeOD/200 MHz) δ 0.90 (d, J
7.0, 9H), 0.93 (d, J 7.0, 9H), 1.13 (d, J 6.4, 9H), 2.30 (dseptet,
J 5.6, 7.0, 3H), 4.02 (d, J 5.6, 3H), 4.07 (d, J 3.7, 3H), 4.20 (dq,
J 3.7, 6.4, 3H), (NH, OH not observed); 13C NMR (MeOD/75
MHz) δ 19.0, 20.3, 21.2, 31.4, 61.7, 62.3, 68.8, 173.3, 174.5;
ESI-MS (positive mode) m/z ) 601 ([MH]+, 7), 623 ([MNa]+,
20), 1223 ([2M + Na]+, 100); HR-MS m/z ) 623.3380 [MNa]+,
623.3381 calculated for C27H48N6O9Na.
Cyclo[-Val-Thr-Val-Thr(ΨMe.Mepro)-Val-Thr(ΨMe.Mepro)-]
(27). The pseudoproline-derivatized hexapeptide 24 (149 mg,
0.21 mmol) was treated with 4 M HCl in dioxane (1 mL) and
stirred at 0 °C for 1 h after which the reaction mixture was
diluted with water (15 mL) and extracted several times with
EtOAc. The combined organic fractions were washed with
brine and dried over Na2SO4, the solvent was evaporated, and
the residue was purified by flash chromatography (silica gel:
95:5 EtOAc/MeOH) to give the monounprotected hexapeptide
27 (46 mg, 32% yield) as a colorless solid: mp 148-150 °C;
[R]D ) -140 (c 1.7, CHCl3); 1H NMR (CD3CN/200 MHz) δ 0.84
(m, 15H), 1.05 (d, J 6.3, 3H), 1.10 (d, J 6.9, 3H), 1.33 (d, J 6.3,
3H), 1.40 (d, J 6.1, 3H), 1.43 (s, 6H), 1.52 (s, 3H), 1.54 (s, 3H),
2.12 (dseptet, J 3.7, 6.6, 1H), 2.39 (m, 2H), 3.67 (dd, J 4.9, 7.4,
1H), 3.90 (app. t, J 8.4, 1H), 3.98 (m, 1H), 4.03 (m, 1H), 4.07-
4.17 (m, 2H), 4.17-4.25 (m, 2H), 4.35 (br.d, J 4.8, 1H, OH
(disappears on D2O exchange)), 4.49 (dq, J 3.7, J 6.3, 1H), 6.70
(d, J 4.8, 1H), 7.35 (m, 2H), 7.76 (d, J 8.4, 1H); 13C NMR (CD3-
CN/75 MHz) δ 15.1, 17.5, 17.8 (3), 17.8 (4), 18.1, 18.9, 19.0,
20.0, 22.4, 25.1, 25.2, 26.8, 27.3, 27.7, 32.4, 32.4, 57.6, 58.2,
60.7, 65.4, 65.5, 66.4, 75.1, 76.0, 95.8, 96.6, 167.7, 168.8, 169.1,
169.6, 170.4, 171.9 (1 signal obscured or overlapping); ESI-
MS (positive mode) m/z 681 ([MH]+, 100), 703 ([MNa]+, 37);
HR-MS m/z ) 703.3960 [MNa]+, 703.4001 calcd for C33H56N6O9-
Na.
Cyclo[-Val-Thr(TBS)-Val-Thr(TBS)-Val-Thr(TBS)-] (23).
Treatment of 1 (78 mg, 0.13 mmol) according to the general
method for TBS protection gave the cyclic hexapeptide 23 as
a colorless solid (88 mg, 70%) after flash chromatography
(silica gel: 2:1 EtOAc/hexane): mp 170-172 °C; [R]D ) -18
(c 1.2, CHCl3); 1H NMR (CDCl3/200 MHz) δ 0.00 (s, 9H), 0.01
(s, 9H), 0.78 (s, 27 H), 0.87 (d, J 6.8, 9H), 0.91 (d, J 6.8, 9H),
1.03 (d, J 6.3, 9H), 2.38 (m, 3H), 3.73 (dd, J 6.8, 6.8, 3H), 4.12
(dd, J 4.3, 5.6, 3H), 4.46 (dd, J 4.3, 6.3, 1H), 7.18 (d, J 6.8,
3H), 7.35 (d, J 5.6, 3H); 13C NMR (CDCl3/75 MHz) δ -4.5, -4.3,
18.3, 19.5, 19.9, 20.2, 26.2, 29.2, 60.5, 63.3, 67.2, 170.3, 171.3;
ESI-MS (positive mode) m/z ) 966 ([MNa]+, 100); HR-MS m/z
) 965.5927, 965.5970 calcd for C45H90N6O9Si3Na.
Acknowledgment. We thank the Australian Re-
search Council for financial support and for the award
of a Queen Elizabeth II research fellowship to K.A.J.
Supporting Information Available: Experimental de-
tails and characterization data for compounds 1-13, 15-22,
and 28 and details of single-crystal X-ray diffraction structure
determination for 24. Copies of 1H and 13C spectra for
compounds 1, epi-1, 2b, 7a, 8, 9, 12, 13, 15-18, 23-25, and
27 and NOESY spectra for compounds 7a, 12, 16, and 24.
X-ray crystal structure of 24 (CIF). This material is available
free of charge via the Internet at http://pubs.acs.org.
Cyclo-[-Val-Thr-Val-Thr-Val-Thr(ΨMe.Mepro)-] (25). A
5% (v/v) TFA solution was added dropwise to a solution of the
pseudoproline-derivatized hexapeptide 24 (194 mg, 0.27 mmol)
in CH2Cl2 (10 mL) at 0 °C. The reaction mixture was stirred
at rt for 3 h and then diluted with water (15 mL) and extracted
several times with EtOAc. The combined organic fractions
JO0484732
J. Org. Chem, Vol. 69, No. 25, 2004 8809