Synthesis of backbone modified cyclic peptides bearing dipicolylamino sidearms

Article abstract of DOI:10.1016/j.tet.2010.11.100

Three analogues of the Lissoclinum class of cyclic peptides, bearing dipicolylamino functionalised side chains, have been synthesised using a stepwise approach followed by macrocyclisation. Attempts to incorporate dipicolylamino functionalised side chains prior to peptide synthesis resulted in epimerisation, but this was overcome by functionalising the ornithine side chains with dipicolylamino groups after the macrocyclisation reaction.

Full text of DOI:10.1016/j.tet.2010.11.100

Tetrahedron 67 (2011) 1019e1029
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of backbone modified cyclic peptides bearing dipicolylamino sidearms
*
Stephen J. Butler, Katrina A. Jolliffe , Wee Yu Gladys Lee, Matthew J. McDonough, Aaron J. Reynolds
School of Chemistry, The University of Sydney, 2006 NSW, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 August 2010
Received in revised form 12 November 2010
Accepted 30 November 2010
Available online 7 December 2010
Three analogues of the Lissoclinum class of cyclic peptides, bearing dipicolylamino functionalised side
chains, have been synthesised using a stepwise approach followed by macrocyclisation. Attempts to
incorporate dipicolylamino functionalised side chains prior to peptide synthesis resulted in epimerisa-
tion, but this was overcome by functionalising the ornithine side chains with dipicolylamino groups after
the macrocyclisation reaction.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic peptide
Oxazole
Macrocycle
1. Introduction
adjacent amino acids in a peptide sequence. The presence of the
modified amino acids influences the three dimensional structures
Macrocyclic molecular scaffolds that present multiple functional
groups in a preorganised fashion, e.g., calixarenes, resorcinarenes,
crown and aza-crown ethers and porphyrins are widely used in
molecular recognition research.1,2 While such scaffolds are often
prepared by a cyclooligomerisation reaction, it is generally difficult
to access unsymmetrical or highly functionalised scaffolds using
this approach. An alternative approach to the preparation of mac-
rocyclic scaffolds is to cyclise a linear precursor that has been
prepared in a stepwise manner. This allows the sequential in-
troduction of individual subunits bearing different functional
groups and can be used to prepare both symmetrical and un-
symmetrical macrocycles. Cyclic peptides are ideal candidates for
synthesis via this approach. The large number of amino acid de-
rivatives available for synthesis allows ready incorporation of
a wide variety of functionalised pendant arms into these molecules
and the stepwise nature of standard peptide synthesis protocols
means that the sequence of functional groups in the macrocycle can
be systematically varied.
In recent years, numerous oxazole and thiazole-containing cy-
clic peptides have been isolated from ascidians, cyanobacteria and
other sources. In particular, the Lissoclinum family of cyclic peptides
contains cyclic hexa-, hepta- and octa-peptides characterised by
the presence of oxazoline/oxazole/thiazoline/thiazole heterocycles
alternating with proteinogenic amino acid residues.3 The azole
heterocycles present in these natural products are derived from the
condensation of cysteine, serine and threonine side chains with the
and bioactivity of these natural products and provides added ri-
gidity to the macrocycles. In addition, the alternating hydrogen
bond donor and acceptor sites that line the interior of the mole-
cules form a network of bifurcated hydrogen bonds that further
rigidifies the macrocycle. This, together with the observation that in
all-syn substituted compounds the side chains are presented on the
same face of the macrocycle, has led to the recent use of analogues
of these natural products as molecular scaffolds for the de-
velopment of molecular receptors, chiral ligands, artificial proteins
and combinatorial libraries.4e6
Our interest in the recognition of large, biologically interesting
anions [e.g., pyrophosphate (P2O74ꢀ, PPi) and adenosine tri-
phosphate (ATP)] led us to explore the use of this class of cyclic
peptide scaffold for the development of anion receptors.5 The cyclic
hexapeptide and octapeptide scaffolds have diameters of approxi-
6
ꢀ
mately 6.6 and 9 A, respectively, allowing binding sites to be po-
sitioned at suitable distances to complex these large anions. We
report here the use of a stepwise synthetic approach to one such
cyclic octapeptide 1,5 with pendant dipicolylamino (Dpa) side
chains suitable for complexation of Zn(II) ions to in turn bind
phosphate oxoanions, together with the related cyclic hexapeptides
2 and 3 (Fig. 1). Compounds 1 and 2 were designed to investigate
the effect that changing the size of the scaffold, and hence the
distance between the two Dpa side chains, would have on the
ability of these compounds to bind pyrophosphate ions, while
scaffold 3, which bears only a single Dpa side chain, was prepared
as a reference compound. The ornithine side chains were chosen to
provide some flexibility in the binding sites, thereby allowing ‘in-
duced fit’ of a target anion.
* Corresponding author. Tel.: þ61 2 9351 2297; fax: þ61 2 9351 3329; e-mail
address: kate.jolliffe@sydney.edu.au (K.A. Jolliffe).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.11.100

1020
S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
Fig. 1. Structures of compounds 1e3.
2. Results and discussion
At this point, careful inspection of the 1H and 13C NMR spectra of
14 suggested that either a mixture of diastereoisomers or rotamers
was present (no evidence for the presence of diastereomers or
rotamers was observed for bisoxazole 11, whose 1H and 13C spectra
contained only a single set of signals). To confirm which was the
case, the C- and N-termini of 14 were deprotected under the
There are a number of possible approaches to the synthesis of
1e3, the most attractive of which involves the coupling of oxazole-
containing amino acid building blocks with appropriately func-
tionalised side chains. We chose to prepare 1ꢀ3 using a stepwise
approach followed by macrocyclisation, rather than via a cyclo-
dimerisation approach in the case of 1,7 as this would provide
a route to allow us to synthesise similar scaffolds with different
sizes and arrangements of functional groups. In our initial approach
we chose to functionalise the amino acid side chains with the re-
quired Dpa groups prior to oxazole formation. This provided us
with two target oxazole building blocks; the known alanine-
derived oxazole 48 and an oxazole bearing a Dpa pendant sidearm,
5 (Scheme 1). We envisaged that coupling of the suitably protected
oxazoles 4 and 5, followed by macrocyclisation would provide the
required anion receptors. Our initial synthetic target was therefore
the Dpa-substituted oxazole 5. We first prepared Dpa-substituted
Boc-ornithine 6a via our recently reported reductive amination
method.9 This was subsequently coupled to serine methyl ester 7
using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride (EDC)/hydroxybenzotriazole (HOBt) as the coupling re-
agents to give the dipeptide 8a in 80% yield. Treatment of 8a with
diethylaminosulfur trifluoride (DAST), followed by oxidation of the
intermediate oxazoline with 1,8-diazabicyclol[5.4.0]undec-7-ene
(DBU) and bromotrichloromethane,10 gave the oxazole 5 in 68%
yield. The alanine-derived oxazole 4 was prepared from Boc-ala-
nine 6b and 7 using the same procedure. Treatment of oxazole 4
with 4 M HCl in dioxane resulted in cleavage of the Boc group to
give the amine 9 as the hydrochloride salt. Hydrolysis of the methyl
ester group of 5 was achieved upon treatment with lithium
hydroxide to yield the carboxylic acid 10. Acid 10 and amine 9 were
then coupled to give the bisoxazole 11 using EDC/HOBt as the
coupling reagents (Scheme 2).
O
R
Cl
H3N
OH
OMe
OH
BocHN
O
6a: R = (CH2)3N[(CH2)Py]2
6b: R = Me
7
6c: R = (CH2)3NHCbz
EDC, HOBt, NMM, CH2Cl2, DMF
or
HBTU, HOBt, DIPEA, CH2Cl2, DMF
R
O
H
N
BocHN
OMe
O
OH
8a: R = (CH2)3N[(CH2)Py]2 (80%)
8b: R = Me (80%)
8c: R = (CH2)3NHCbz (87%)
i) DAST or Deoxofluor, CH2Cl2, -78 0 °C
ii) DBU, BrCCl3, CH2Cl2, 0 °C
R
O
BocHN
For the synthesis of 1, we envisaged that two bisoxazole units
could be coupled to provide the required tetraoxazole linear pre-
cursor. Hence, N-deprotection of 11 under identical conditions to
those described above gave the amine 12 as the hydrochloride salt.
Alternatively, treatment of 11 with lithium hydroxide resulted in C-
deprotection to give the carboxylic acid 13. These two bisoxazole
fragments (12 and 13) were then coupled under standard condi-
tions to yield the protected linear tetraoxazole 14 (Scheme 3).
N
CO2Me
5: R = (CH2)3N[(CH2)Py]2 (68%)
4: R = Me (62%)
16: R = (CH2)3NHCbz (69%)
Scheme 1. Synthesis of the oxazole building blocks.

S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
1021
R1
N
R1
N
LiOH or NaOH,
H2O, MeOH
R2
R2
HN
O
O
O
N
N
R
O
O
LiOH
NHBoc
BocHN
CO2H
BocHN
N
N
MeOH, H2O
13: R = (CH2)3N[(CH2)Py]2 (84%)
20: R = (CH2)3NHCbz (quant.)
CO2Me
CO2H
HN
O
O
5: R1 = R2 = CH2Py
10: R1 = R2 = CH2Py (96%)
17: R1 = H, R2 = Cbz (quant.)
EDC,
16: R1 = H, R2 = Cbz
N
N
O
R
HOBt,
NMM,
DMF
NHBoc
CO2Me
11: R = (CH2)3N[(CH2)Py]2 (76%)
18: R = (CH2)3NHCbz (61%)
O
BocHN
O
4M HCl
dioxane
Cl H3N
N
HN
O
O
N
O
CO2Me
N
X
N
R
CO2Me
9 (97%)
4
4M HCl,
dioxane
NH3
CO2Me
or
12: R = (CH2)3N[(CH2)Py]2, X = Cl- (99%)
19: R = (CH2)3NHCbz, X = CF3CO2- (quant.)
TFA, CH2Cl2
Scheme 2. Synthesis of bisoxazoles.
determined that a mixture of diastereomeric peptides, including
the desired isomer 1 had been obtained. Re-examination of each
step of the synthetic route enabled us to ascertain that substantial
epimerisation was occurring during the synthesis of the oxazole 5
13 + 12
EDC, HOBt, NMM,
CH2Cl2, DMF, 24 h
19 + 20
R
BocHN
CO2Me
EDC, HOBt,
O
N
NMM, MeCN, 24 h
O
N
R
O
N
O
HN
BocHN
CO2Me
NH
O
O
N
N
N
H
N
O
O
O
N
O
O
HN
R
NH
14: R = (CH2)3N[(CH2)Py]2 (68%)
N
H
N
O
i) LiOH, MeOH, H2O , 18 h
O
O
ii) 4 M HCl, dioxane, 4 h
R
21: R = (CH2)3NHCbz (24%)
iii) FDPP, i-Pr2EtN, DMF, 24 h
O
R
O
i) NaOH, MeOH, H2O, 4 h
ii) CF3CO2H, CH2Cl2, 2.25 h
iii) FDPP, i-Pr2EtN, DMF, 23 h
O
N
H
N
N
O
NH
HN
O
O
N
O
O
N
N
O
H
H
N
N
N
RHN
O
O
R
O
NH
HN
15: R = (CH2)3N[(CH2)Py]2
O
NHR
(50% over 3 steps)
N
N
H
N
O
Scheme 3. Synthesis of 15 as a mixture of diastereoisomers.
O
O
standard conditions described above, followed by macrocyclisation
of the resulting peptide upon treatment with pentafluorophenyl
diphenylphosphinate (FDPP)11 to give the cyclic peptide 15 in 50%
yield (Scheme 3). The 1H NMR spectra of 15 in either CD3OD or
DMSO-d6 contained broad signals and notably, when DMSO-d6 was
used as the solvent, multiple signals attributable to the amide
protons were observed rather than the expected two signals.
Multiple sets of signals were also observed in the 13C NMR spec-
trum of 15. These could not be attributed to conformers and it was
22: R = Cbz (55%)
i) HBr, AcOH, 0.5 h
ii) Amberlite IRA-400 resin
23: R = H (quant.)
2-pyridinecarboxaldehyde,
NaBH(OAc)3, CH2Cl2, 18 h
1 (54%)
Scheme 4. Synthesis of dipicolylamino functionalised cyclic peptide 1.

1022
S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
from dipeptide 8a. This was surprising since our synthesis of 4 from
8b under identical conditions proceeded without any detectable
subjected to reductive amination with 2-pyridinecarboxaldehyde in
the presence of sodium triacetoxyborohydride to install the Dpa li-
gands, yielding 1. The 1Hand13C NMR spectra ofboth22and1 exhibit
the expected number of signals for compounds with C2 symmetry,
confirming that only a single diastereoisomer was obtained from the
macrocyclisation.
epimerisation of the a-carbon (as examined by the preparation of
Mosher’s amides12 and comparison of the optical rotation with
reported values8), which is in agreement with results reported in
the literature for similar compounds.10 However, it is known that
the
a
-carbon of dipeptide derived oxazolines is prone to epimer-
With the synthesis of 1 as a single diastereomer confirmed, we
proceeded to prepare compounds 2 and 3 using a similar approach.
Thus, acid 10 was coupled to bisoxazole 19 under standard condi-
tions to provide linear trioxazole 24, while bisoxazole 20 was
coupled to oxazole 9 to provide linear trioxazole 25 (Scheme 5).
Both 24 and 25 were subject to N- and C-deprotection using the
same procedures as described above for the deprotection of tet-
raoxazole 21, and macrocyclisation of the resulting free peptides
gave cyclic peptides 26 and 27 in 64% and 76% yields, respectively.
Removal of the Cbz groups from 26 was achieved by using HBr in
acetic acid, followed by neutralisation to give amine 28, while
compound 27 was subjected to hydrogenolysis in the presence of
Pd/C to give the corresponding free amine 29 (Pd catalysed
hydrogenolysis was sluggish when more than one Cbz-protected
amine was present). Both 28 and 29 were then subject to reductive
amination with 2-pyridinecarboxaldehyde to give the cyclic triox-
azoles 2 and 3 as single diastereoisomers, respectively. Compounds
1e3 were found to be stable when stored for up to 2 years at ꢀ15 ꢁC
and were not observed to undergo epimerisation during this time,
or when stood in CDCl3 solution at rt for up to 7 days.
isation under both acidic and basic catalysis13 and has been ob-
served to occur during oxidation of an oxazoline to the
corresponding oxazole.14 We postulate that the epimerisation oc-
curring in 5 is a result of facile abstraction of the acidic proton at the
stereocentre of the oxazoline by the tethered dipicolylamino group,
which is six atoms distant from this proton. All attempts to opti-
mise the synthesis of
unsuccessful.
5 to minimise epimerisation were
The loss of stereochemical integrity of the oxazole 5 required us to
revise our synthetic route. We did this by delaying functionalisation
of the ornithine side chains until after the cyclic peptide was formed.
Hence, Boc-Orn(Cbz)-OH 6c was coupled with serine methyl ester 7
and the resulting dipeptide 8c was converted to the corresponding
oxazole 16 usingthetwo-stepproceduredescribedabove, employing
the more stable Deoxofluor instead of DAST (Scheme 1). As expec-
ted,15 no detectable epimerisation occurred during the synthesis of
16, (as examined by the preparation of Mosher’s amides12) further
suggesting that it is the basic nitrogen atoms in 5 that are responsible
for the epimerisation observed for this compound. Hydrolysis of the
methyl ester of 16 under standard conditions gave the free acid 17
(Scheme 2), which was coupled with amine 9 to give the tetrapeptide
18 in good yield. Selective removal of the Boc group was achieved
upon treatment of 18 with trifluoroacetic acid to give the amine 19.16
Alternatively, treatment of 18 with sodium hydroxide resulted in C-
deprotection to give the acid 20, which was coupled with 19 to give
the octapeptide 21 (Scheme 4). Sequential C- and N-deprotection of
21 was followed by macrocyclisation upon treatment with FDPP to
give the cyclic peptide 22 in 58% yield over three steps. Cbz depro-
tection occurred upon treatment of 22 with HBr in acetic acid
and, following neutralisation, the corresponding diamine 23 was
3. Conclusions
In summary, cyclic peptide scaffolds 1e3 bearing dipicolylamino
side chains were synthesised using a stepwise approach to couple
the oxazole units, followed by a macrocyclisation step. Installation
of the dipicolylamino group prior to oxazole formation was found
to result in epimerisation of the carbon a- to the azole, presumably
through deprotonation during oxidation of the oxazoline to the
oxazole 5. Given that the similar oxazoles, 4 and 16, were prepared
under identical conditions with minimal epimerisation and that
NHR
NHCbz
O
CbzHN
O
i) NaOH, MeOH, H2O, 4 h
N
H
HN
O
ii) CF3CO2H, CH2Cl2, 2 h
2-pyridinecarboxaldehyde,
NaBH(OAc)3, CH2Cl2, 24 h
O
O
O
N
N
iii) FDPP, i-Pr2EtN, DMF, 65 h
EDC, HOBt
2
N
N
10
19
(40%)
NMM, MeCN, 24 h
NH
HN
O
NHBoc
O
N
H
N
O
24 (19%)
O
N
O
OMe
O
NHR
26: R = Cbz (64%)
28: R = H (quant.)
i) HBr, AcOH, 0.5 h
ii) Amberlite IRA-400 resin
O
CbzHN
O
i) NaOH, MeOH, H2O, 6 h
ii) CF3CO2H, CH2Cl2, 18 h
N
H
HN
O
O
N
O
N
O
2-pyridinecarboxaldehyde,
N
iii) HATU, collidine, DMF, 24 h
NaBH(OAc)3, CH2Cl2, 24 h
(38%)
EDC, HOBt
N
3
20
9
NMM, MeCN, 24 h
NH
HN
O
NHBoc
25 (79%)
O
N
N
O
H
O
N
O
OMe
O
NHR
H2, Pd/C (10%), EtOH
27: R = Cbz (76%)
29: R = H (quant.)
Scheme 5. Synthesis of dipicolylamino functionalised cyclic peptides 2 and 3.

S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
1023
numerous other chiral oxazoles have been prepared by this
method,10 we postulate that epimerisation of 5 is facilitated by the
tethered dipicolylamino group. This loss of stereochemical integrity
was overcome by altering the synthetic route such that the dipi-
colylamino groups were installed in the final step of the synthetic
sequence. The compounds prepared in this manner were obtained
as single stereoisomers, and appear stable under a range of con-
ditions. The bis-zinc(II) complexes of 1 and 2 are under evaluation
for their ability to bind selectively to large phosphate oxoanions
under physiological conditions; some of the results of these studies
have already been published,5 the remainder will be published
elsewhere.
OMe 7 (0.589 g, 3.79 mmol) were dissolved in anhydrous
dichloromethane/DMF (3:1 v/v, 40 mL). EDC (0.799 g, 4.17 mmol),
HOBt (0.638 g, 4.17 mmol) and then N-methylmorpholine
(1.25 mL, 11.4 mmol) were added, and the resulting mixture was
stirred at rt for 24 h. The mixture was partitioned between satd aq
NaHCO3 solution (100 mL) and ethyl acetate (100 mL). The organic
phase was isolated and the aqueous fraction was further extracted
with ethyl acetate (3ꢂ100 mL). The combined organic fractions
were washed first with water (100 mL) and then brine (100 mL),
dried (MgSO4) and concentrated under reduced pressure to give
a yellow oil. Subjection of the crude material to flash chroma-
tography (silica gel; CHCl3/MeOH/aq ammonia, 190:9:1 v/v/v)
gave the desired Dpa-functionalized dipeptide 8a (1.56 g, 80%) as
20
4. Experimental
a pale yellow solid. Mp 54e57 ꢁC; [
a
]
þ12.1 (c 1.0, CHCl3); 1H
D
NMR (200 MHz, CDCl3):
d
8.55 (d, J¼4.9 Hz, 2H), 8.10 (br s, 1H),
4.1. General methods
7.65 (t, J¼7.6 Hz, 2H), 7.41 (d, J¼7.7 Hz, 2H), 7.19 (t, J¼7.0 Hz, 2H),
5.95 (br d, 1H), 4.60 (m, 1H), 3.99 (m, 3H), 3.77 (s, 4H), 3.75 (s, 3H),
2.55 (s, 2H), 1.76e1.25 (m, 4H), 1.39 (s, 9H), OꢀH signal not ob-
Melting points were obtained using a Stanford Research Sys-
tems Optimelt melting point apparatus and are uncorrected. Op-
tical rotations were obtained using a PerkineElmer model 341
polarimeter at 589 nm and 20 ꢁC, using the indicated spectroscopic
grade solvent. 1H nuclear magnetic resonance spectra were recor-
ded using a Bruker Avance DPX 400 spectrometer at a frequency of
400.13 MHz, a Bruker Avance DPX 300 spectrometer at a frequency
of 300.13 MHz, or a Bruker Avance DPX 200 spectrometer at a fre-
quency of 200.13 MHz, and are reported in parts per million (ppm)
relative to the residual isotopomer with one less deuterium then
the quoted perdeuterated solvent. The data is reported as chemical
served; 13C NMR (75.5 MHz, CDCl3):
d 173.1, 171.1, 159.3, 156.0,
149.1, 136.9, 123.8, 122.4, 79.8, 62.5, 60.1, 55.1, 54.4, 53.8, 52.6, 30.1,
28.6, 22.5; IR nmax (NaCl)/cmꢀ1 3296, 2952, 1660, 1519, 1434, 1168,
1049, 734; MS (ESI) m/z 538 [(MþNa)þ, 35%], 516 (100), 438 (16),
416 (26); HRMS (ESI) calcd for C26H37N5O6Na (MþNa)þ 538.2636,
found 538.2634.
4.1.2. Boc-Orn(Dpa)-Oxz(Ser)-OMe (5). Under an atmosphere of
nitrogen, (diethylamino)sulfur trifluoride (1.61 mL, 12.1 mmol) was
added dropwise to a solution of dipeptide 8a (1.56 g, 3.03 mmol) in
anhydrous dichloromethane (50 mL) at ꢀ78 ꢁC. Stirring of the
resulting mixture was continued at ꢀ78 ꢁC for 1 h and then at rt for
a further 24 h. After cooling to 0 ꢁC, the reaction mixture was
quenched by the addition of anhydrous K2CO3 (2.09 g, 15.2 mmol)
in one portion. The reaction mixture was poured into satd aq
NaHCO3 solution (50 mL) and the biphasic mixture was extracted
with ethyl acetate (3ꢂ50 mL). The combined organic fractions were
dried (Na2SO4) and the solvent was removed under reduced pres-
sure to give the corresponding oxazoline as a brown oil.
A stirred solution of oxazoline (1.47 g, 2.96 mmol) in anhydrous
dichloromethane (40 mL) at 0 ꢁC under an atmosphere of nitrogen
was treated dropwise with bromotrichloromethane (0.583 mL,
5.92 mmol), followed by 1,8-diazabicyclo[5.4.0]undec-7-ene
(0.885 mL, 5.92 mmol). The reaction mixture was warmed to rt and
stirred for 24 h prior to quenching with satd aq NaHCO3 solution
(40 mL) and extracted with ethyl acetate (3ꢂ40 mL). The combined
organic fractions were dried (Na2SO4) and the solvent was removed
under reduced pressure. The crude material thus obtained was
subjected to flash chromatography (silica gel; CHCl3/MeOH/aq
ammonia, 190:9:1 v/v/v) to afford the desired Dpa-functionalized
shift (
d
), multiplicity (br¼broad, s¼singlet, d¼doublet, t¼triplet,
q¼quartet, dd¼doublet of doublets, ddd¼doublet of doublet of
doublets, m¼multiplet), coupling constant (J Hz) and relative in-
tegral. 13C nuclear magnetic resonance spectra were recorded on
a
Bruker Avance DPX 400 spectrometer at a frequency of
100.61 MHz, or a Bruker Avance DPX 300 spectrometer at a fre-
quency of 75.47 MHz and are reported in parts per million (ppm)
relative to the internal perdeuterated solvent resonance, unless
otherwise stated. Low resolution mass spectra were recorded on
a Thermo Finnigan LCQ Deca Ion Trap mass spectrometer using
electrospray ionisation (ESI). High resolution mass spectra were
recorded on a Bruker BioApex Fourier Transform Ion Cyclotron
Resonance mass spectrometer (FT-ICR) with an analytical ESI
source, operating at 4.7 T. Analytical thin layer chromatography
(TLC) was performed using pre-coated silica gel plates (Merck
Kieselgel 60 F254). Preparative flash chromatography was carried
out using Merck Kieselgel Silica Gel 60 (particle size
0.040e0.065 mm) with the indicated ratio of solvents (volume/
volume), which were mixed as specified. Liquid chromatography
mass spectrometry (LCMS) was performed on a Thermo Separation
Products: spectra System using a P400 Pump and a UV6000LP
photodiode array detector. Separation was achieved using a Phe-
oxazole 5 (1.01 g, 68%) as a yellow oil. [
NMR (200 MHz, CDCl3):
a
]
20 ꢀ4.24 (c 0.7, CHCl3); 1H
D
d
8.55 (d, J¼4.8 Hz, 2H), 8.13 (s, 1H), 7.63
nomenex Jupiter column (5
mm, 150ꢂ2.1 mm ID), which was cou-
(dt, J¼7.6 and 1.7 Hz, 2H), 7.45 (d, J¼7.7 Hz, 2H), 7.14 (t, J¼6.5 Hz,
2H), 5.43 (d, J¼7.2 Hz, 1H), 4.86 (d, J¼5.6 Hz, 1H), 3.90 (s, 3H), 3.77
(s, 4H), 2.56 (t, J¼6.8 Hz, 2H), 1.87e1.21 (m, 4H), 1.41 (s, 9H); 13C
pled to a Thermoquest Finnigan LCQ Deca mass spectrometer (ESI).
Flow rate was maintained at 0.2 mL minꢀ1 over a linear gradient
from 0% to 100% solvent B (solvent A: 100:0.1 v/v Milli-Q water/
formic acid, solvent B: 100:0.1 v/v acetonitrile/formic acid) over
30 min. Reactions were performed under an inert atmosphere of
anhydrous nitrogen unless otherwise stated. Dichloromethane,
methanol and acetonitrile were distilled from calcium hydride.
Tetrahydrofuran (THF) was dried over sodium wire. HPLC grade
N,N-dimethylformamide was obtained from LabScan and used
without further purification. Boc-Orn(Dpa)-OH was prepared
according to a previously reported procedure.9 All other reagents
were commercially available and were used as supplied.
NMR (75.5 MHz, CDCl3):
d 165.9, 161.9, 159.4, 155.5, 149.4, 144.2,
136.9, 133.6, 123.6,122.5, 80.5, 60.6, 54.0, 52.6, 49.3, 32.3, 28.7, 23.2;
IR nmax (NaCl)/cmꢀ1 3300, 2950, 1737, 1699, 1589, 1434, 1169, 999,
760; MS (ESI) m/z 518 [(MþNa)þ, 70%], 496 (100), 418 (27), 396
(87); HRMS (ESI) calcd for C26H34N5O5Na (MþH)þ 518.2379, found
518.2373.
4.1.3. Boc-Ala-Ser-OMe17 (8b). To a solution of the amino acid Boc-
Ala-OH 6b (6.00 g, 31.7 mmol) and HCl$NH2-Ser-OMe 7 (4.93 g,
31.7 mmol) in anhydrous dichloromethane/DMF (3:1 v/v, 100 mL)
under an atmosphere of nitrogen were added EDC (6.68 g,
34.9 mmol), HOBt (5.34 g, 34.9 mmol) and N-methylmorpholine
(10.5 mL, 95.1 mmol). The reaction mixture was stirred at rt for 4 h
4.1.1. Boc-Orn(Dpa)-Ser-OMe (8a). Under an atmosphere of nitro-
gen, Boc-Orn(Dpa)-OH 6a (1.57 g, 3.79 mmol) and HCl$NH2-Ser-

1024
S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
after which time it was partitioned between hydrochloric acid
(0.50 M, 100 mL) and ethyl acetate (100 mL). The aqueous phase
was isolated and extracted with ethyl acetate (3ꢂ100 mL). The
combined organic fractions were washed first with water
(2ꢂ100 mL) and then brine (100 mL), dried (MgSO4) and concen-
trated under reduced pressure to give a colourless oil, which was
purified by flash chromatography (silica gel; CHCl3/MeOH, 9:1 v/v)
481 (Mþ, 21%), 504 (100), 404 (24); HRMS (ESI) calcd for
C25H31N5O5Na (MþNa)þ 504.2218, found 504.2223.
4.1.7. Boc-Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)-OMe (11). Under an at-
mosphere of nitrogen, carboxylic acid 10 (0.463 g, 0.962 mmol) and
the hydrochloride salt 9 (0.198 g, 0.962 mmol) were dissolved in
anhydrous dichloromethane/DMF (3:1 v/v, 20 mL). EDC (0.203 g,
1.06 mmol), HOBt (0.162 g, 1.06 mmol) and N-methylmorpholine
(0.317 mL, 2.89 mmol) were added and the resulting solution was
stirred at rt for 7 h. The mixture was partitioned between hydro-
chloric acid (0.50 M, 50 mL) and ethyl acetate (50 mL), the aqueous
phase was isolated and further extracted with ethyl acetate
(3ꢂ50 mL). The combined organic fractions were washed first with
water (2ꢂ50 mL) and then brine (50 mL), dried (MgSO4) and con-
centrated under reduced pressure. The crude material thus
obtained was subjected to flash chromatography (silica gel; CHCl3/
MeOH/aq ammonia, 93:6:1 v/v/v) to give the desired bisoxazole 11
to afford the desired dipeptide 8b (7.36 g, 80%) as a colourless oil.
20
[
a]
þ11.2 (c 1.0, CHCl3); 1H NMR (200 MHz, CDCl3)
d 6.98 (d,
D
J¼4.9 Hz, 1H), 5.11 (d, J¼7.0 Hz, 1H), 4.64 (m, 1H), 4.14 (m, 1H), 3.95
(m, 2H), 3.79 (s, 3H), 1.43 (s, 9H), 1.40 (d, J¼7.0 Hz, 3H), OH not
observed. Compound 8b had spectroscopic data identical to that
reported in the literature.17
4.1.4. Boc-Ala-Oxz(Ser)-OMe8 (4). (Diethylamino)sulfur trifluoride
(1.93 mL,14.5 mmol) was added dropwise to a solution of 8b (2.11 g,
7.27 mmol) in anhydrous dichloromethane (40 mL) at ꢀ78 ꢁC under
an atmosphere of nitrogen. The resulting mixture was stirred at
ꢀ78 ꢁC for 3 h after which time it was quenched by the addition of
anhydrous K2CO3 (2.51 g, 18.2 mmol). After warming to rt the
mixture was poured into satd aq NaHCO3 solution (40 mL) and the
biphasic mixture was extracted with dichloromethane (3ꢂ40 mL).
The combined organic fractions were dried (Na2SO4) and the sol-
vent was removed under reduced pressure to give the corre-
sponding oxazoline as a yellow oil.
(0.465 g, 76%) as a yellow solid. Mp 59e60 ꢁC; found: C, 57.2; H, 6.3;
20
N, 14.2% (C32H39N7O7$2H2O requires C, 57.4; H, 6.5; N, 14.6%); [a]
D
ꢀ11.5 (c 0.8, CHCl3); 1H NMR (200 MHz, CDCl3):
8.52 (d, J¼4.3 Hz,
d
2H), 8.19 (s, 1H), 8.11 (s, 1H), 7.62 (t, J¼7.5 Hz, 2H), 7.45 (d, J¼8.2 Hz,
2H), 7.16 (t, J¼5.4 Hz, 2H), 5.48 (br d, 2H), 4.81 (br s, 2H), 3.91 (s,
3H), 3.78 (s, 4H), 2.57 (t, J¼6.6 Hz, 2H), 1.84e1.25 (m, 7H), 1.44 (s,
9H); 13C NMR (75.5 MHz, CDCl3):
d 165.5, 161.8, 160.3, 159.7, 149.4,
144.5, 141.8, 136.8, 135.8, 133.7, 123.5, 122.4, 80.5, 60.7, 53.9, 52.6,
49.3, 43.2, 32.3, 29.7, 28.7, 23.1; IR nmax (NaCl)/cmꢀ1 3402, 2929,
1708, 1670, 1593, 1506, 1112, 730; MS (ESI) m/z 634 [(MþH)þ, 49%],
828 (21), 656 (100), 556 (47), 534 (32); HRMS (ESI) calcd for
C32H39N7O7Na (MþNa)þ 656.2801, found 656.2809.
A stirred solution of oxazoline (1.74 g, 6.39 mmol) in anhydrous
dichloromethane(35 mL)at0 ꢁCunderanatmosphereofnitrogenwas
treated dropwise with bromotrichloromethane (1.26 mL,12.8 mmol),
followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (1.91 mL,12.8 mmol).
The reaction mixture was warmed to rt and stirred for 24 h prior to
quenching with satd aq NaHCO3 solution (40 mL) and extracted with
ethyl acetate (3ꢂ40 mL). The combined organic fractions were dried
(Na2SO4) and the solvent was removed under reduced pressure. The
crude material thus obtained was subjected to flash chromatography
(silica gel; EtOAc/hexane, 2:3 v/v) to afford the desired oxazole 4
4.1.8. HCl.H2N-Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)-OMe (12). The bis-
oxazole 11 (0.465 g, 0.734 mmol) was dissolved in a solution of
hydrochloric acid in dioxane (4.0 M, 1.92 mL) and stirred under an
atmosphere of nitrogen for 4 h. The reaction mixture was concen-
trated under reduced pressure to give the desired hydrochloride
salt 12 (0.414 g, 99%) as a colourless foam, which was used without
(1.20g, 62%)asawhite solid.Mp 108ꢀ110 ꢁC;[
a]
D
20 ꢀ53.2(c0.5, CHCl3)
{lit.8b
[a]
20 ꢀ50.0 (c 0.5, CHCl3)}; 1H NMR (200 MHz, CDCl3)
d
8.18 (s,
further purification. 1H NMR (200 MHz, CD3OD):
d
8.83 (d, J¼5.9 Hz,
D
1H), 5.30(br s,1H), 5.02(d, J¼8.2 Hz,1H), 3.91 (s, 3H),1.54 (d, J¼5.9 Hz,
3H),1.43 (s, 9H). Compound 4 had spectroscopic data identical to that
reported in the literature.8
2H), 8.53 (d, J¼2.1 Hz, 2H), 8.44 (t, J¼7.2 Hz, 2H), 7.99 (d, J¼7.5 Hz,
2H), 7.90 (t, J¼6.8 Hz, 2H), 5.41 (m, 1H), 4.68 (m, 1H), 4.38 (s, 2H),
3.86 (s, 3H), 3.66 (s, 4H), 2.86 (t, J¼7.6 Hz, 2H), 1.71e1.30 (m, 7H),
one NeH signal not observed; MS (ESI) m/z 534 (Mþ, 100%), 556
(18), 535 (40).
4.1.5. HCl$H2N-Ala-Oxz(Ser)-OMe(9). Oxazole 4 (0.289 g,1.07 mmol)
was dissolved in a solution of hydrochloric acid in dioxane (4.0 M,
2.80 mL) and stirred under an atmosphere of nitrogen for 4 h. The
reaction mixture was concentrated under reduced pressure to give
the desired hydrochloride salt 9 (0.214 g, 97%) as a colourless foam,
which was used without further purification. 1H NMR (200 MHz,
4.1.9. Boc-Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)-OH (13). To a solution of
the bisoxazole 11 (0.169 g, 0.266 mmol) in methanol (3 mL) was
added a solution of LiOH (19 mg, 0.798 mmol) in water (1 mL) and
the resulting mixture was stirred at rt for 18 h. The mixture was
neutralized (pH 7) and the organic solvent was removed under
reduced pressure. The residue thus obtained was partitioned be-
tween water (30 mL) and CHCl3/iPrOH (3:1 v/v, 30 mL). The organic
phase was isolated and the aqueous phase was further extracted
with CHCl3/iPrOH (3:1 v/v, 3ꢂ30 mL). The combined organic frac-
tions were dried (Na2SO4) and concentrated under reduced pres-
sure to afford the carboxylic acid 13 (0.140 g, 84%), which was used
CDCl3):
d 9.41 (d, 3H), 8.25 (s, 1H), 4.96 (d, 1H), 3.89 (s, 3H), 1.93 (d,
J¼6.6 Hz, 3H); MS (ESI) m/z 171 (Mþ, 100%), 363 (18), 341 (86).
4.1.6. Boc-Orn(Dpa)-Oxz(Ser)-OH (10). To a solution of the oxazole
5 (0.459 g, 0.926 mmol) in methanol (9 mL) was added a solution of
LiOH (66 mg, 2.8 mmol) in water (3 mL) and the resulting mixture
was stirred at rt for 18 h. The mixture was neutralized (pH 7) and
the organic solvent was removed under reduced pressure. The
residue thus obtained was partitioned between water (30 mL) and
CHCl3/iPrOH (3:1 v/v, 30 mL), and the isolated aqueous phase was
further extracted with CHCl3/iPrOH (3:1 v/v, 3ꢂ30 mL). The com-
bined organic fractions were dried (Na2SO4) and concentrated
without further purification. 1H NMR (200 MHz, DMSO-d6):
d 8.76
(br d, J¼8.0 Hz, 1H), 8.68 (s, 1H), 8.57 (s, 1H), 8.47 (d, J¼4.0 Hz, 2H),
7.75 (t, J¼7.5 Hz, 2H), 7.51 (t, J¼9.3 Hz, 2H), 7.24 (t, J¼5.8 Hz, 2H),
5.26 (m, 1H), 4.35 (d, J¼3.5 Hz, 6H), 3.78 (t, J¼5.9 Hz, 2H), 1.90e1.22
(m, 7H), 1.36 (s, 9H), OeH signal not observed; MS (ESI) m/z 620
[(MþH)þ, 100%], 642 (25), 520 (34); HRMS (ESI) calcd for
C31H38N7O7 (MþH)þ 620.2831, found 620.2791.
under reduced pressure to afford the carboxylic acid 10 (0.428 g,
20
96%), which was used without further purification; [
a]
ꢀ58.0 (c
D
1.0, CHCl3); 1H NMR (200 MHz, DMSO-d6):
d
8.46 (d, J¼4.8 Hz, 2H),
7.86 (s, 1H), 7.74 (t, J¼6.1 Hz, 2H), 7.48 (d, J¼7.9 Hz, 2H), 7.23 (t,
J¼6.1 Hz, 2H), 4.67 (br s, 1H), 4.33 (br s, 1H), 3.69 (s, 4H), 2.42 (m,
2H), 1.90e1.23 (m, 4H), 1.35 (s, 9H), OꢀH signal not observed; IR
nmax (NaCl)/cmꢀ1 3296, 2932, 1616, 1435, 1217, 754; MS (ESI) m/z
4.1.10. Boc-[Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)]2-OMe (14). Under an
atmosphere of nitrogen, compound 13 (0.140 g, 0.226 mmol) and
compound 12 (0.129 g, 0.226 mmol) were dissolved in anhydrous
dichloromethane/DMF (3:1 v/v, 2.4 mL). EDC (48 mg, 0.249 mmol),

S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
1025
HOBt (38 mg, 0.249 mmol) and then N-methylmorpholine (75
m
L,
132.4, 130.1,# 129.9, 124.9, 123.8, 61.2, 54.9, 44.4, 43.9,# 43.8,# 43.5,#
40.2, 33.0,# 32.5,# 32.0,# 31.8,# 31.6, 24.9,# 24.3, 24.2,# 24.0,# 23.7,#
19.4,# 19.2,# 19.1, 18.7,# 18.4,# (#minor diastereomer); (numerous
signals obscured or overlapping); MS (ESI) m/z 1003 [(MþH)þ,
100%], 1026 (26); HRMS (ESI) calcd for C52H55N14O8 (MþH)þ
1003.4329, found 1003.4322.
0.680 mmol) were added, and the resulting mixture was stirred at
rt for 24 h. The mixture was partitioned between satd aq NaHCO3
solution (10 mL) and ethyl acetate (10 mL). The organic phase was
isolated and the aqueous fraction was further extracted with ethyl
acetate (3ꢂ10 mL). The combined organic fractions were washed
sequentially with water (10 mL) and brine (10 mL), then dried
(MgSO4) and concentrated under reduced pressure to give a yellow
oil. Subjection of the crude material to flash chromatography (silica
4.1.13. Boc-Orn(Cbz)-Ser-OMe (8c). Under an atmosphere of nitro-
gen, Boc-Orn(Cbz)-OH 6c (10.0 g, 27.3 mmol) and the hydrochloride
salt of Ser-OMe 7 (4.25 g, 27.3 mmol) were dissolved in anhydrous
dichloromethane/DMF (1:4 v/v, 110 mL). The solution was stirred
and cooled to 0 ꢁC and then HBTU (12.4 g, 32.8 mmol), HOBt (4.43 g,
gel; CHCl3/MeOH, 9:1 v/v) afforded the tetraoxazole 14 (0.174 g,
20
68%) as a white solid. Mp 68e71 ꢁC; [
a]
ꢀ12.9 (c 0.7, CHCl3); 1H
D
NMR (200 MHz, CDCl3): d 8.52 (s, 4H), 8.13 (m, 3H), 8.11 (s, 1H), 7.63
€
(t, J¼6.7 Hz, 4H), 7.44 (d, J¼5.7 Hz, 4H), 7.19 (t, J¼7.0 Hz, 4H), 5.44
(m, 4H), 4.79 (br s, 2H), 3.90 (s, 3H), 3.78 (m, 8H), 2.58 (s, 4H), 2.35
(s, 2H), 1.79e1.27 (m, 14H), 1.45 (s, 9H); 13C NMR (75.5 MHz, CDCl3):
32.8 mmol) and Hunig’s base (14.3 mL, 81.9 mmol) were added. The
resulting mixture was slowly warmed to rt and stirred for 18 h. The
mixture was concentrated under reduced pressure to half of the
initial volume and then partitioned between water (200 mL) and
CHCl3/iPrOH (3:1 v/v, 200 mL). The organic phase was extracted
with water (3ꢂ200 mL) and the combined aqueous fractions were
back-extracted with CHCl3/iPrOH (3:1 v/v, 2ꢂ300 mL). The organic
fractions were combined and washed with satd aq (NH4)2CO3 so-
lution (400 mL), half-strength brine solution (400 mL) and then
dried (MgSO4). The solvent was removed under reduced pressure to
give a yellow oil, which was purified by flash chromatography
(silica gel: CHCl3/MeOH, 95:5 v/v) to afford the desired dipeptide 8c
d
165.5e162.5, 161.4, 160.0, 149.4, 144.5, 142.0, 136.8, 135.5, 133.2,
123.1, 122.0, 80.1, 60.3, 53.9, 52.1, 48.9, 43.2, 31.5, 28.2, 23.3, 19.6;
MS (ESI) m/z 1135 [(MþH)þ, 100%], 1157 (25), 1069 (98), 968 (35),
770 (51), 619 (34); HRMS (ESI) calcd for C58H67N14O11 (MþH)þ
1135.5116, found 1135.5068.
4.1.11. HCl$H2N-[Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)]2-OH (30). To
a
solution of the tetraoxazole 14 (0.151 g, 0.133 mmol) in methanol
(1.5 mL) was added a solution of LiOH (9.5 mg, 0.399 mmol) in water
(0.5 mL) and the resulting mixture was stirred at rt for 18 h. The
mixture was neutralized (pH 7) and the organic solvent was re-
moved under reduced pressure. The residue thus obtained was
partitioned between water (15 mL) and CHCl3/iPrOH (3:1 v/v,
15 mL). The organic phase was isolated and the aqueous phase was
further extracted with CHCl3/iPrOH (3:1 v/v, 3ꢂ15 mL). The com-
bined organic fractions were dried (Na2SO4) and concentrated un-
der reduced pressure to afford the desired carboxylic acid. This
material was immediately dissolved in a solution of hydrochloric
acid in dioxane (4.0 M, 0.322 mL) and stirred under an atmosphere
of nitrogen for 4 h. The reaction mixture was concentrated under
reduced pressure to give the desired hydrochloride salt 30 (0.128 g,
99%) as a colourless foam, which was used without further purifi-
(11.1 g, 87%) as a colourless solid. IR nmax (NaCl)/cmꢀ1 3319, 2972,
20
2881, 2359; mp 67e68 ꢁC; [
a
]
ꢀ2.09 (c 1.1, MeOH); 1H NMR
D
(300 MHz, CDCl3):
d
7.33 (m, 5H), 5.39 (d, J¼8.1 Hz, 1H), 5.15 (t,
J¼6.6 Hz, 1H), 5.08 (m, 2H), 4.64 (m, 1H), 4.27 (t, J¼7.3 Hz, 1H), 3.93
(m, 2H), 3.74 (s, 3H), 3.42 (br s, 1H), 3.34 (m, 1H), 3.16 (m, 1H), 1.86
(m, 1H), 1.62 (m, 3H), 1.42 (s, 9H), OeH signal not observed; 13C
NMR (75.5 MHz, CDCl3):
d 172.7, 171.0, 157.2, 156.1, 136.6, 128.6,
128.2, 80.4, 66.9, 62.8, 54.9, 53.7, 52.7, 40.1, 29.9, 28.4, 26.0, one
signal obscured or overlapping; HRMS (ESI) calcd for C22H33N3O8Na
(MþNa)þ 490.2160, found 490.2157.
4.1.14. Boc-Orn(Cbz)-Oxz(Ser)-OMe (16). Under an atmosphere of
nitrogen, Deoxo-FluorÒ (5.90 mL, 32.0 mmol) was added dropwise
to a stirred solution of the dipeptide 8c (12.5 g, 26.7 mmol) in an-
hydrous dichloromethane (270 mL) at ꢀ20 ꢁC (EtOH/ice/CO2 chips).
The reaction mixture was stirred at ꢀ20 ꢁC for 1 h, then slowly
warmed to rt and stirred for a further 6 h. Analysis by TLC at this
time revealed complete conversion of starting material. The solu-
tion was quenched with satd aq (NH4)2CO3 solution (250 mL) at
ꢀ20 ꢁC, and then warmed to rt and extracted with chloroform
(3ꢂ250 mL). The combined organic fractions were washed with
half-strength brine solution (400 mL), dried (MgSO4) and concen-
trated under reduced pressure to afford the intermediate oxazoline
as a yellow oil.
cation. 1H NMR (200 MHz, CD3OD):
d
8.87 (d, J¼5.1 Hz, 4H),
8.54e8.25 (m, 8H), 8.15e7.99 (m, 8H), 4.36 (d, J¼4.9 Hz, 4H), 3.69
(s, 8H), 2.84 (m, 4H), 1.74e1.31 (m, 14H), NeH and OeH signals not
observed; MS (ESI) m/z 1022 [(MþH)þ, 73%], 1044 (24), 511 (100).
4.1.12. Cyclo[Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)]2 (15). To
a stirred
solution of the hydrochloride salt 30 (0.140 g, 0.132 mmol) in an-
hydrous DMF (26 mL) under an atmosphere of nitrogen was added
pentafluorophenyl diphenylphosphinate (70 mg, 0.198 mmol) and
€
Hunig’s base (69 mL, 0.396 mmol). The reaction mixture was stirred
at rt for 24 h after which time the solvent was removed under re-
duced pressure to give a crude orange coloured oil, which was
partitioned between water (10 mL) and CHCl3/iPrOH (3:1 v/v,
20 mL). The organic phase was isolated and the aqueous phase was
further extracted with CHCl3/iPrOH (3:1 v/v, 2ꢂ20 mL). The com-
bined organic fractions were washed sequentially with 2 M aq
NaOH solution (30 mL), water (30 mL), and brine (30 mL), then
dried (MgSO4) and concentrated under reduced pressure to give an
orange oil. Purification by flash chromatography (silica gel; CHCl3/
MeOH/aq ammonia, 90:9:1 v/v/v) afforded the cyclic peptide 15
Under a nitrogen atmosphere, the crude oxazoline (12.0 g,
26.7 mmol) was dissolved in anhydrous dichloromethane (270 mL)
and cooled to 0 ꢁC. The solution was then treated with bromotri-
chloromethane (5.27 mL, 53.4 mmol) and 1,8-diazabicyclo[5.4.0]
undec-7-ene (8.00 mL, 53.4 mmol) and the resulting mixture was
stirred at rt for 4 h. The mixture was quenched by the addition of
hydrochloric acid (0.30 M, 400 mL) and extracted with CHCl3/iPrOH
(3:1 v/v, 3ꢂ300 mL). The combined organic fractions were washed
with half-strength brine solution (600 mL), dried (MgSO4) and
concentrated under reduced pressure to give a brown oil. Purifica-
tion of the crude material by flash chromatography (silica gel;
eluting first with hexane/EtOAc,1:1 v/v, then hexane/EtOAc, 2:3 v/v)
afforded the desired oxazole 16 (8.63 g, 69% over two steps) as
(66 mg, 50%) as a yellow solid and an inseparable mixture of di-
20
astereoisomers. [
CD3OD):
a]
ꢀ36.0 (c 0.7, MeOH); 1H NMR (200 MHz,
D
d
8.40 (br m, 8H), 7.74 (br m, 4H), 7.59 (br m, 4H), 7.26 (br
m, 4H), 5.36 (br m, 4H), 3.77 (br m, 8H), 2.58 (br m, 4H), 1.69e1.29
(br m, 14H), NeH signals not observed; 13C NMR (75.47 MHz,
a colourless solid. Mp 83e84 ꢁC; [
(300 MHz, CD3OD):
a
]
20 ꢀ9.40 (c 1.0, MeOH); 1H NMR
D
MeOD)
d
166.3,# 166.0,# 165.9,# 165.8,# 165.6, 165.5, 164.8,# 162.4,#
d
8.46 (s, 1H), 7.27e7.34 (m, 5H), 7.02 (t,
162.2,# 162.0,# 161.9, 161.8,# 161.7,# 161.6,# 160.6, 160.5,# 149.4,
149.3,# 144.2,# 144.1,# 143.9,# 143.8,# 143.7,# 143.6,# 143.5, 138.6,
138.5,# 137.2,# 137.0,# 136.9, 136.8,# 136.7,# 133.6,# 132.7,# 132.6,#
J¼6.6 Hz, 1H), 5.05 (s, 2H), 4.79 (m, 1H), 3.87 (s, 3H), 3.15 (m, 2H),
1.75e2.02 (m, 2H), 1.56 (m, 2H), 1.43 (s, 9H), one NeH signal not
observed; 13C NMR (75.5 MHz, CD3OD):
d 167.2, 163.0, 159.0, 157.7,

1026
S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
146.0,138.4,133.9,129.4,128.9,128.8, 67.4, 52.5, 50.0, 41.3, 41.2, 31.4,
28.7, 27.2; MS (ESI) m/z 470 [(MþNa)þ, 100%], 414 (28); HRMS (ESI)
calcd for C22H29N3O7Na (MþNa)þ 470.1898, found 470.1908.
with CHCl3/iPrOH (3:1 v/v, 3ꢂ25 mL). The combined organic frac-
tions were washed with brine (50 mL), dried (MgSO4) and con-
centrated under reduced pressure to give the desired carboxylic
acid 20 (342 mg, 100%) as a colourless foam. 1H NMR (400 MHz,
4.1.15. Boc-Orn(Cbz)-Oxz(Ser)-OH (17). To a stirred solution of the
oxazole 16 (5.88 g, 13.1 mmol) in methanol (167 mL) and water
(55 mL) was added NaOH (1.58 g, 39.3 mmol). Stirring was con-
tinued at rt for 2 h after which time analysis by TLC revealed
complete conversion of the starting material. The reaction mixture
was acidified (ca. pH 5) with 2 M HCl and extracted with CHCl3/i-
PrOH (3:1 v/v, 3ꢂ100 mL). The combined organic fractions were
dried (MgSO4) and concentrated under reduced pressure to give the
desired carboxylic acid 17 (5.70 g, 100%) as a colourless foam. 1H
CD3OD): d 8.42 (s, 1H), 8.33 (s, 1H), 7.33e7.27 (m, 5H), 5.40e5.34
(m, 1H), 5.05 (s, 2H), 4.79 (br m, 1H), 3.17e3.14 (m, 2H), 1.99e1.81
(m, 2H), 1.66 (d, J¼6.9 Hz, 3H), 1.61e1.48 (m, 2H), 1.43 (s, 9H), NꢀH
and OꢀH signals not observed; 13C NMR (100.6 MHz, CD3OD):
d
165.2, 164.8, 162.6, 161.0, 157.5, 156.3, 144.6, 141.9, 137.0, 135.4,
133.5, 128.0, 127.5, 127.4, 79.4, 66.0, 43.0, 39.8, 30.0, 27.3, 25.8, 23.9,
17.3; MS (ESI) m/z 594 [(MþNa)þ, 100%]; HRMS (ESI) calcd for
C27H33N5O9Na (MþNa)þ 594.2171, found 594.2188.
NMR (400 MHz, CD3OD):
d
8.41 (s, 1H), 7.34e7.27 (m, 4H), 5.06 (s,
4.1.19. Boc-[Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)]2-OMe (21). To a stir-
red solution of the bisoxazole carboxylic acid 20 (663 mg,
1.16 mmol) and the bisoxazole amine TFA salt 19 (1.16 mmol) in
anhydrous acetonitrile (30 mL) was added HOBt (196 mg,
1.28 mmol), EDC (245 mg, 1.28 mmol) and N-methylmorpholine
2H), 4.80 (br m, 1H), 3.14 (t, J¼6.7 Hz, 2H), 1.99e1.82 (m, 2H),
1.63e1.48 (m, 4H), 1.43 (s, 9H), NꢀH and OꢀH signals not observed;
13C NMR (100.6 MHz, CD3OD):
d 165.6, 162.6, 157.5, 156.3, 144.4,
137.0, 133.2, 128.0, 127.5, 127.4, 79.4, 66.3, 39.8, 30.0, 27.3, 25.8,
23.9; MS (ESI) m/z 456 [(MþNa)þ, 100%], 889 [(2MþNa)þ, 55%];
HRMS (ESI) calcd for C21H27N3O7Na (MþNa)þ 456.1742, found
456.1737.
(383
mL, 3.48 mmol). The reaction mixture was stirred at rt for
23.5 h after which time the solvent was removed under reduced
pressure to give an orange coloured oil, which was partitioned
between satd aq NaHCO3 solution (25 mL) and CHCl3/iPrOH (3:1 v/
v, 25 mL). The organic phase was isolated and the aqueous phase
was further extracted with CHCl3/iPrOH (3:1 v/v, 2ꢂ25 mL). The
combined organic fractions were washed first with water (25 mL)
and then brine (30 mL), dried (MgSO4) and concentrated under
reduced pressure to give an orange coloured oil, which was purified
by flash chromatography on silica gel to afford the desired tet-
4.1.16. Boc-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-OMe (18). To a stirred
solution of the carboxylic acid 17 (3.53g, 6.04 mmol) and the hy-
drochloride salt 9 (1.37 g, 6.61 mmol) in anhydrous acetonitrile/
DMF (7:1 v/v, 125 mL) was added HOBt (1.11 g, 7.27 mmol), EDC
(1.39 g, 7.27 mmol) and N-methylmorpholine (3.30 mL, 29.7 mmol).
The reaction mixture was stirred at rt for 22 h after which time the
solvent was removed under reduced pressure to give an orange
coloured oil, which was partitioned between satd aq NaHCO3 so-
lution (70 mL) and CHCl3/iPrOH (3:1 v/v, 70 mL). The organic phase
was isolated and the aqueous phase was further extracted with
CHCl3/iPrOH (3:1 v/v, 2ꢂ50 mL). The combined organic fractions
were washed first with water (70 mL) and then brine (70 mL), dried
(MgSO4) and concentrated under reduced pressure to give an or-
ange coloured oil, which was purified by flash chromatography on
raoxazole 21 (290 mg, 24%) as a colourless foam. [
a
]
25 ꢀ37.8 (c 0.91,
D
CHCl3); 1H NMR (400 MHz, CD3OD):
d
8.46 (s, 1H), 8.35 (s, 1H), 8.32
(s, 2H), 7.31e7.26 (m, 10H), 5.40e5.30 (m, 3H), 5.04 (s, 4H), 4.79 (br
m, 1H), 3.85 (s, 3H), 3.19e3.14 (m, 4H), 2.13e1.46 (m, 14H), 1.42 (s,
9H), NꢀH signals not observed; 13C NMR (100.6 MHz, CD3OD):
d
163.8, 163.2, 163.0, 162.2, 159.9, 159.7, 159.4(2), 158.9(5), 155.9,
154.7, 143.2, 140.7, 140.6, 140.5, 135.4, 133.9, 133.8, 131.1, 126.5,
126.0, 125.8, 77.8, 64.4, 49.5, 47.1, 41.4, 38.2, 28.4, 27.9, 25.7, 24.3,
15.7(2), 15.6(9), 11 signals obscured or overlapping; MS (ESI) m/z
1061 [(MþNa)þ, 100%], 939 (80); HRMS (ESI) calcd for
C50H58N10O15Na (MþNa)þ 1061.3976, found 1061.3973.
silica gel to afford the desired bisoxazole 18 as a colourless foam
25
(2.18 g, 61%). [
a
]
ꢀ25.0 (c 0.80, CHCl3); 1H NMR (400 MHz,
D
CD3OD):
d
8.47 (s, 1H), 8.33 (s, 1H), 7.33e7.27 (m, 5H), 5.36 (q,
J¼7.2 Hz, 1H), 5.05 (s, 2H), 4.79 (br m, 1H), 3.86 (s, 3H), 3.18e3.14
(m, 2H), 2.01e1.92 (m, 2H), 1.66 (d, J¼7.2 Hz, 3H), 1.63e1.47 (m, 2H),
1.43 (s, 9H), NꢀH signals not observed; 13C NMR (100.6 MHz,
4.1.20. Boc-[Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)]2-OH (31). To a stirred
solution of the tetraoxazole 21 (287 mg, 0.276 mmol) in methanol
(3.5 mL) and water (1.2 mL) was added NaOH (33 mg, 0.828 mmol).
Stirring was continued at rt for 4 h after which time analysis by TLC
revealed complete conversion of the starting material. The solution
was acidified (ca. pH 5) with 2 M HCl before extracting with
CHCl3/iPrOH (3:1 v/v, 3ꢂ25 mL). The combined organic fractions
were washed with brine solution, dried (MgSO4) and concentrated
under reduced pressure to give the desired carboxylic acid 31
(268 mg, 95%) as a colourless foam. 1H NMR (400 MHz, CD3OD):
CD3OD):
d 165.4, 164.8, 161.5, 161.1, 157.5, 156.3, 144.8, 141.9, 137.0,
135.3, 132.7, 128.0, 127.5, 127.4, 79.4, 66.0, 51.9, 43.0, 40.0, 30.0, 27.3,
25.8, 17.3, one signal obscured or overlapping; MS (ESI) m/z 608
[(MþNa)þ, 100%], 552 (42), 530 (35), 486 (48); HRMS (ESI) calcd for
C28H35N5O9Na (MþNa)þ 608.2327, found 608.2331.
4.1.17. CF3CO2H$H2N-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-OMe
(19). Under an atmosphere of nitrogen, trifluoroacetic acid
(10.7 mL, 0.138 mmol) was added to a solution of the bisoxazole 18
(404 mg, 0.691 mmol) in anhydrous dichloromethane (10.8 mL).
The reaction mixture was stirred at rt for 1.5 h after which time the
solvent was removed under reduced pressure to give a crude oil,
which was azeotropically dried with toluene to afford the desired
TFA salt 19 (485 mg, 100%) as a pale yellow foam, which was used
immediately in the subsequent peptide coupling reaction; MS (ESI)
m/z 486 (Mþ, 100%); HRMS (ESI) calcd for C23H28N5O7 (Mþ)
486.1984, found 486.1971.
d
8.41 (s,1H), 8.34 (s,1H), 8.31 (s, 2H), 7.31e7.24 (m,10H), 5.39e5.28
(m, 3H), 5.03 (s, 4H), 4.78 (br m, 1H), 3.18e3.11 (m, 4H), 2.10e1.48
(m, 8H), 1.63 (d, J¼9.2 Hz, 6H), 1.41 (s, 9H), NꢀH and OꢀH signals
not observed; 13C NMR (100.6 MHz, CD3OD):
d 166.6, 166.2, 165.9,
165.1, 163.9, 162.6, 162.4, 162.3, 158.9, 157.7, 146.0, 143.7, 143.5,
143.4, 138.4, 136.9, 136.8, 136.7, 134.8, 80.8, 67.4, 44.3, 41.2, 31.4,
30.8, 28.7, 27.2, 18.7(4), 18.6(8), 14 signals obscured or overlapping;
MS (ESI) m/z 1047 [(MþNa)þ, 100%], 925 (47), 663 (31); HRMS (ESI)
calcd for C49H56N10O15Na (MþNa)þ 1047.3819, found 1047.3803.
4.1.18. Boc-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-OH (20). To a stirred
solution of the bisoxazole 18 (328 mg, 0.561 mmol) in methanol
(7.1 mL) and water (2.4 mL) was added NaOH (67 mg, 1.68 mmol).
Stirring was continued at rt for 1 h 35 min after which time analysis
by TLC revealed complete conversion of the starting material. The
solution was acidified (ca. pH 5) using 2 M HCl before extracting
4.1.21. CF3CO2H$H2N-[Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)]2-OH
(32). Under an atmosphere of nitrogen, a stirred solution of the
carboxylic acid 31 (269 mg, 0.262 mmol) in anhydrous dichloro-
methane (4.0 mL) was treated with trifluoroacetic acid (4.0 mL).
Stirring was continued at rt for 2 h 15 min after which time analysis
by TLC revealed complete conversion of the starting material. The

S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
1027
reaction mixture was concentrated under reduced pressure to give
(d, J¼5.4 Hz, 6H), 1.60 (m, 4H); 13C NMR (75.5 MHz, CD3OD):
d
165.1,
the desired TFA salt 32 (251 mg, 100%) as a colourless foam. 1H NMR
164.7, 161.0, 160.7, 159.6,148.4, 143.0, 142.8,137.6, 135.8, 123.9,122.8,
60.3, 53.9, 46.1, 42.5, 30.8, 23.4, 18.1; MS (ESI) m/z 1026 [(MþNa)þ,
100%]; HRMS (ESI) calcd for C52H54N14O8Na (MþNa)þ 1025.4147,
found 1025.4142.
(300 MHz, CD3OD):
d 8.51 (s, 1H), 8.43 (s, 1H), 8.36 (s, 1H), 8.34 (s,
1H), 7.32e7.28 (m, 10H), 5.41e5.30 (m, 3H), 5.04 (s, 4H), 4.72e4.68
(m,1H), 3.17 (br m, 4H), 2.16e1.97 (m, 4H), 1.68e1.49 (m,10H), NꢀH
and OꢀH signals not observed; 13C NMR (75.5 MHz, CD3OD):
d
167.1, 166.3, 165.6, 164.4, 163.0, 162.7, 162.1, 161.7, 159.3, 146.5,
4.1.24. Boc-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-OMe
(24). To a stirred solution of compound 10 (503 mg, 1.16 mmol) and
compound 19 (696 mg, 1.16 mmol) in acetonitrile (30 mL) was
added HOBt (196 mg, 1.28 mmol), EDC (245 mg, 1.28 mmol) and N-
145.1, 144.1, 143.9, 137.7, 137.3, 135.2, 129.9, 129.44, 129.37, 129.2,
67.9, 44.9, 41.5, 41.2, 31.3, 30.6, 27.7, 27.0, 19.1, 11 signals obscured or
overlapping; MS (ESI) m/z 925 (Mþ, 100%); HRMS (ESI) calcd for
C44H49N10O13 (MþH)þ 925.3476, found 925.3458.
methylmorpholine (383 mL, 3.48 mmol). The reaction mixture was
stirred at rt for 23.5 h after which time the solvent was removed
under reduced pressure to give an orange coloured oil, which was
partitioned between satd aq NaHCO3 solution (25 mL) and
CHCl3/iPrOH (3:1 v/v, 25 mL). The organic phase was isolated and
the aqueous phase was further extracted with CHCl3/iPrOH (3:1 v/v,
2ꢂ25 mL). The combined organic fractions were washed with
water (25 mL) and saturated brine (30 mL), then dried (MgSO4) and
concentrated under reduced pressure to give an orange coloured
oil, which was purified by flash chromatography on silica gel to
afford the desired trisoxazole 24 (234 mg, 19%) as a colourless foam.
4.1.22. Cyclo[Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)]2 (22). To
a stirred
solution of the TFA salt 32 (243.9 mg, 0.235 mmol) in anhydrous
DMF (47 mL) under an atmosphere of nitrogen was added penta-
fluorophenyl diphenylphosphinate (124 mg, 0.353 mmol) and
€
Hunig’s base (123 mL, 0.705 mmol). The reaction mixture was stir-
red at rt for 23 h after which time the solvent was removed under
reduced pressure to give a crude orange coloured oil, which was
partitioned between water (20 mL) and CHCl3/iPrOH (3:1 v/v,
20 mL). The organic phase was isolated and the aqueous phase was
further extracted with CHCl3/iPrOH (20 mL, 3:1 v/v). The combined
organic fractions were washed sequentially with 2 M aq NaOH
solution (30 mL), water (30 mL) and brine (30 mL), dried (MgSO4)
and concentrated under reduced pressure to give an orange oil.
Purification by flash chromatography on silica gel afforded the
desired cyclic peptide 22 (123 mg, 58%) as a colourless foam. Mp
[a
]
25 ꢀ23.0 (c 1.2, CHCl3); 1H NMR (400 MHz, CD3OD):
d 8.45 (s,1H),
D
8.33 (s, 2H), 7.31e7.25 (m, 10H), 5.38e5.30 (m, 2H), 5.03 (s, 4H),
4.78 (br s, 1H), 3.84 (s, 3H), 3.21e3.15 (m, 4H), 2.15e1.83 (m, 4H),
1.64 (d, J¼7.0 Hz, 3H), 1.65e1.55 (m, 8H), 1.42 (s, 9H); 13C NMR
(100.6 MHz, CD3OD):
d 165.4, 164.8, 163.8, 161.5, 161.3, 161.0, 157.5,
156.3, 144.8, 142.2, 137.0, 135.5, 135.3, 132.7, 79.4, 66.0, 51.1, 43.0,
40.0, 30.0, 29.5, 27.3, 25.8, 13.1, 14 signals obscured or overlapping;
MS (ESI) m/z 923 (MþNa, 100%), 801 (79); HRMS (ESI) calcd for
C44H52N8O13Na (MþNa)þ 923.3547, found 923.3531.
116e118 ꢁC; [
a]
25 ꢀ108 (c 0.51, CHCl3); 1H NMR (400 MHz, CD3OD):
D
d
8.37 (s, 2H), 8.36 (s, 2H), 7.24e7.33 (m, 10H), 5.48 (m, 2H), 5.41 (m,
2H), 5.10 (s, 4H), 3.20 (dt, J¼2.9, 6.9 Hz, 4H), 2.13 (m, 2H), 2.03 (m,
2H), 1.66 (d, J¼6.9 Hz, 6H), 1.60 (m, 4H), NeH signals not observed;
13C NMR (100.6 MHz, CD3OD):
d
166.2, 165.5, 162.2, 161.8, 159.0,
4.1.25. CF3CO2H$H2N-Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala-Oxz
(Ser)-OH (33). To a stirred solution of the trisoxazole 24 (210.8 mg,
0.234 mmol) in methanol (3.0 mL) and water (1.0 mL) was added
NaOH (28 mg, 0.702 mmol). Stirring was continued at rt for 4 h after
which time analysis by thin layer chromatography revealed com-
plete conversion of the starting material. The solution was acidified
(ca. pH 5) with 2 M hydrochloric acid before extracting with
CHCl3/iPrOH (3:1 v/v, 3ꢂ25 mL). The combined organic fractions
were washed with saturated brine (25 mL), then dried over anhy-
drous MgSO4 and concentrated under reduced pressure to give the
carboxylic acid (194 mg, 93%) as a colourless foam. The acid was
immediately dissolved in anhydrous dichloromethane (3.3 mL)
under an atmosphere of nitrogen and treated with trifluoroacetic
acid (3.3 mL). Stirring was continued at rt for 2 h 10 min after which
time analysis by thin layer chromatography revealed complete
conversion of the starting material. The reaction mixture was
concentrated under reduced pressure to give the desired TFA salt 33
(208 mg, quant.) as a colourless foam, which was used immediately
144.0, 143.9, 138.4, 136.9, 136.7, 129.4, 128.9, 128.8, 67.4, 47.5, 43.6,
41.1, 31.4, 27.3, 19.1; MS (ESI) m/z 929 [(MþNa)þ, 100%], 824 (58);
HRMS (ESI) calcd for C44H46N10O12Na (MþNa)þ 929.3189, found
929.3184.
4.1.23. Cyclo[Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)]2 (1). Under an at-
mosphere of nitrogen, a solution of hydrogen bromide in acetic acid
(33% v/v, 4.0 mL) was added to the cyclic tetraoxazole 22 (100 mg,
0.110 mmol) and the resulting mixture was stirred at rt for 30 min.
The orange solution that resulted was combined with anhydrous
ether (30 mL) to give a cream coloured precipitate. The precipitate
was centrifuged and the ethereal layer was removed. Subsequent
trituration of the precipitate with anhydrous ether (8ꢂ30 mL) and
removal of the final clear ethereal layer under reduced pressure,
gave the dihydrobromide salt as a cream solid. The free amine was
liberated from the dihydrobromide salt by treatment of the meth-
anolic solution with basic resin (Amberlite lRA-400). Removal of
the resin by filtration, followed by concentration of the filtrate, gave
the crude amine 23.
in the next step. 1H NMR (300 MHz, CD3OD):
d 8.52 (s, 1H), 8.43 (s,
1H), 8.36 (s,1H), 7.31e7.28 (m,10H), 5.40e5.33 (m, 2H), 5.05 (s, 4H),
4.72e4.67 (br m, 1H), 3.19e3.15 (m, 4H), 2.23e2.00 (m, 4H), 1.65 (d,
J¼7.0 Hz, 3H), 1.66e1.57 (m, 4H), NeH and OeH signals not ob-
served; MS (ESI) m/z 787 (Mþ, 100%); HRMS (ESI) calcd for
C38H43N8O11 (MþH)þ 787.3046, found 787.3058.
A suspension of 23 in dichloromethane (10 mL) was treated with
2-pyridinecarboxyaldehyde (150
acetoxyborohydride (510 mg, 2.4 mmol) under an atmosphere of
nitrogen for 4 h. More 2-pyridinecarboxyaldehyde (75 L, 0.78
mL, 1.57 mmol) and sodium tri-
m
mmol) and sodium triacetoxyborohydride (255 mg, 1.2 mmol) were
then added and the mixture was stirred overnight. The mixture was
filtered through a plug of silica (EtOAc) and the solvent was removed
under reduced pressure. The residue thus obtained was purified by
flash chromatography (silica gel; eluting first with EtOAc/toluene/
Et3N, 50:50:1 v/v/v, then MeOH/EtOAc/Et3N, 10:90:1 v/v/v) to afford
the Dpa-functionalized cyclic peptide 1 (66 mg, 54%) as a pale yellow
4.1.26. Cyclo[Orn(Cbz)-Oxz(Ser)-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)]
(26). To a stirred solution of the TFA salt 33 (208 mg, 0.231 mmol) in
anhydrous DMF(46 mL)underan atmosphereof nitrogenwasadded
pentafluorophenyl diphenylphosphinate (122 mg, 0.347 mmol) and
€
Hunig’s base (121 mL, 0.693 mmol). The reaction mixture was stirred
at rt for 65 h after which time the solvent was removed under
reduced pressure to give a crude orange coloured oil, which was
partitioned between water and CHCl3/iPrOH (3:1 v/v, 20 mL). The
organic phase was isolated and the aqueous phase was further
extracted with CHCl3/iPrOH (3:1 v/v, 3ꢂ20 mL). The combined
oil. [
a
]
25 ꢀ21.7 (c 0.65, MeOH); 1H NMR (300 MHz, CD3OD):
d 8.45 (br
D
d, J¼4.4 Hz, 4H), 8.42 (s, 2H), 8.41 (s, 2H), 7.74 (m, 4H), 7.62 (br d,
J¼7.8 Hz), 7.25 (m, 4H), 5.48 (ABquartet, 4H), 5.28 (dd, J¼6.4, 8.7 Hz,
4H), 3.74 (s, 8H), 2.60 (ABquartet, 4H), 2.06 (m, 4H),1.92 (m, 4H),1.67

1028
S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
organic extracts were washed sequentially with 2 M aqueous NaOH
solution (30 mL), water (30 mL) and brine (30 mL), then dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
a dark orange oil. Purification by flash chromatography on silica gel
afforded the desired cyclic peptide 26 (113 mg, 64%) as a colourless
2.23 (m, 2H), 1.43e1.72 (m, 8H), 1.40 (br s, 9H); 13C NMR (75.5 MHz,
CDCl3): 165.7, 164.9, 164.6, 161.7, 160.5, 156.9, 155.6, 144.4, 142.2,
142.0, 136.9, 135.8, 135.7, 128.9, 128.4, 80.7, 67.0, 52.5, 48.6, 43.3,
43.2, 40.8, 31.0, 28.7, 26.6, 19.8, 19.2, three signals obscured or
overlapping; MS (ESI) m/z 741 [(MþNH4)þ, 100%]; HRMS (ESI) calcd
for C34H41N7O11Na (MþNa) þ 746.2764, found 746.2756.
d
foam. [
a]
25 ꢀ41.2 (c 0.5, CHCl3); 1H NMR (300 MHz, CDCl3):
d 8.50 (d,
D
J¼8.3 Hz, 1H), 8.39 (dd, J¼12.2, 9.2 Hz, 2H), 8.19 (s, 2H), 8.17 (s, 1H),
7.30e7.25 (m,10H), 5.26e5.13 (m, 5H), 5.04 (s, 4H), 3.23e3.21 (br m,
4H), 2.17e2.09 (m, 2H), 2.00e1.91 (m, 2H), 1.68 (d, J¼9.0 Hz, 5H),
4.1.29. CF3CO2H$H2N-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-Ala-Oxz(Ser)-
OH (35). To a stirred solution of the trisoxazole 25 (458 mg,
0.632 mmol) in methanol (3.0 mL) and water (1.0 mL) was added
NaOH (76 mg, 1.90 mmol). Stirring was continued at rt for 6 h after
which time analysis by thin layer chromatography revealed com-
plete conversion of the starting material. The solution was acidified
(ca. pH 5) with 2 M HCl before extracting with dichloromethane
(3ꢂ25 mL). The combined organic fractions were washed with
saturated brine (25 mL), dried over anhydrous MgSO4 and con-
centrated under reduced pressure to give the desired carboxylic
acid (445 mg, quant.) as a colourless foam. This material was im-
mediately treated with trifluoroacetic acid (2 mL) in dichloro-
methane (10 mL) overnight. The solution was concentrated under
reduced pressure to give the desired TFA salt 35 (340 mg) as an
orange oil, which was used without further purification. 1H NMR
1.54e1.50 (m, 2H); 13C NMR (75.5 MHz, CDCl3):
d 164.8, 163.9, 163.7,
159.7,159.6, 159.3, 156.7,141.9,136.8,135.5(3),135.4(7), 128.7,128.3,
68.8, 48.2(5), 48.1(5), 44.7, 40.7, 32.3, 25.6, 20.9, 13 signals obscured
or overlapping; MS (ESI) m/z 792 [(MþNa)þ,100%]; HRMS (ESI) calcd
for C38H40N8O10Na (MþNa)þ 791.2760, found 791.2749.
4.1.27. Cyclo[Orn(Dpa)-Oxz(Ser)-Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)]
(2). The cyclic peptide 26 (50 mg, 0.063 mmol) was treated with
hydrogen bromide in acetic acid (33% v/v, 3 mL) for 30 min. The
solution was then slowly added to a stirred solution of anhydrous
ether (20 mL), resulting in the formation of a colourless solid,
presumably the hydrobromide salt. This solid was collected by fil-
tration and then subsequently dissolved in MeOH (20 mL). The free
amine was liberated from the hydrobromide salt by treatment of
the methanolic solution with base resin (Amberlite lRA-400).
Removal of the resin by filtration, followed by concentration of the
filtrate, gave the desired crude amine 34.
(300 MHz, CD3OD):
d 8.49 (s, 1H), 8.42 (s, 1H), 8.34 (s, 1H),
7.29e7.25 (m, 5H), 5.36 (m, 2H), 5.03 (br s, 2H), 4.71 (m, 1H), 3.16
(m, 2H), 2.09 (m, 2H) 1.53e1.65 (m, 8H), NeH and OeH signals not
observed; MS (ESI) m/z 610.1 [(MþH)þ, 100%]; HRMS (ESI) calcd for
C28H32N7O9Na (MþNa)þ 610.2262, found 610.2256.
A suspension of the amine 34 in dichloromethane (5 mL) was
treated with 2-pyridinecarboxyaldehyde (50
sodium triacetoxyborohydride (102 mg, 0.624 mmol) for 4 h. More
2-pyridinecarboxyaldehyde (20 L, 0.780 mmol) and sodium tri-
mL, 0.520 mmol) and
4.1.30. Cyclo[Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-Ala-Oxz(Ser)]
(27). The TFA salt 35 (173 mg, 0.284 mmol) was dissolved in
methanol and treated with weak base resin. Removal of the resin by
filtration, followed by concentration of the filtrate (with co-evap-
oration with toluene), gave the crude amine. The amine was dis-
solved in a solution of DMF and dichloromethane (1:2 v/v, 180 mL),
m
acetoxyborohydride (102 mg, 0.624 mmol) were then added and
the mixture was left to stir overnight. The mixture was filtered
through a plug of silica (EtOAc) and the filtrate concentrated under
reduced pressure. The residue was then purified by flash chroma-
tography (silica gel; eluting first with EtOAc/toluene/Et3N, 50:50:1
v/v/v, then EtOAc/toluene/Et3N, 10:90:1 v/v/v) to give the Dpa-
to which was added collidine (250
mL, 1.87 mmol) and HATU
(237 mg, 0.620 mmol). After stirring for 24 h the solution was
concentrated under reduced pressure. The residue was partitioned
between ethyl acetate (25 mL) and 1 M hydrochloric acid (25 mL).
The organic phase was isolated and washed sequentially with satd
aq NaHCO3 solution (20 mL), water (20 mL) and brine (20 mL), then
dried (MgSO4) and concentrated under reduced pressure. Purifi-
cation by flash chromatography (silica gel; EtOAc/hexane 1:1 v/v,
functionalized cyclic peptide 2 (34 mg, 40%) as a pale yellow oil.
25
[
a]
ꢀ18.8 (c 0.68, MeOH); 1H NMR (300 MHz, CD3OD):
d 8.47 (s,
D
1H), 8.46 (s, 1H), 8.44 (s, 1H), 8.41 (br d, J¼4.4 Hz, 4H), 7.75 (m, 4H),
7.56 (br d, J¼7.8 Hz, 4H), 7.22 (m, 4H), 5.12e5.23 (m, 3H), 4.83 (br s,
8H), 2.52e2.56 (br m, 4H), 1.82e2.23 (m, 5H), 1.34e1.72 (m, 5H),
1.28e1.49 (d, J¼9.0 Hz, 4H); 13C NMR (75.5 MHz, CD3OD):
d 164.8,
164.1, 164.0, 160.3, 160.2, 159.7, 148.4, 148.3, 142.7, 137.6, 135.2,
123.9, 122.7, 60.3, 54.2, 44.8, 31.8, 22.3, 19.6, 15 signals obscured or
overlapping; MS (ESI) m/z 887 [(MþNa)þ, 100%]; HRMS (ESI) calcd
for C46H49N12O6 (MþH)þ 865.3898, found 865.3893.
then 8:2 v/v) gave the desired cyclic peptide 27 (128 mg, 76%) as
25
a colourless oil. [
CDCl3):
a]
ꢀ28.6 (c 1.09, CHCl3); 1H NMR (300 MHz,
D
d
8.60e8.53, (m, 2H), 8.21 (br d, J¼15 Hz, 1H), 8.23 (s, 1H),
8.21 (s, 2H), 7.32e7.29 (m, 5H), 5.19 (m, 3H), 5.05 (s, 2H), 4.83 (m,
1H), 3.24 (m, 2H), 2.23 (m, 2H), 1.96 (m, 1H), 1.70 (d, J¼6.6 Hz, 6H),
4.1.28. Boc-Orn(Cbz)-Oxz(Ser)-Ala-Oxz(Ser)-Ala-Oxz(Ser)-OMe
(25). To a stirred solution of the compound 20 (1.80 g, 3.15 mmol)
and the compound 9 (1.7 g, 8.22 mmol) in acetonitrile (100 mL) was
added HOBt (1.39 g, 9.10 mmol), EDC (1.69 g, 8.80 mmol) and
N-methylmorpholine (4.12 mL, 37.6 mmol). The reaction mixture
was stirred overnight after which time the solvent was removed
under reduced pressure to give an orange coloured oil, which was
partitioned between satd aq NaHCO3 solution (50 mL) and ethyl
acetate (50 mL). The organic phase was isolated and the aqueous
phase was further extracted with ethyl acetate (3ꢂ50 mL). The
combined organic fractions were washed with water (50 mL) and
saturated brine (50 mL), then dried over anhydrous MgSO4 and
concentrated under reduced pressure to give an orange coloured
oil, which was purified by flash chromatography (silica gel; eluting
first with EtOAc/hexane, 1:1 v/v, then 6:4 v/v) to afford the desired
1.51 (m, 1H); 13C NMR (75.5 MHz, CDCl3):
d 164.9, 164.8, 163.8,
159.9, 159.6, 156.8, 141.9, 135.6, 128.8, 128.4, 67.0, 48.4, 45.0, 32.2,
21.0, 20.9, 10 signals obscured or overlapping; MS (ESI) m/z 592.3
[(MþH)þ, 100%]; HRMS (ESI) calcd for C28H30N7O8 (MþH)þ
592.2156, found 592.2151.
4.1.31. Cyclo[Orn(Dpa)-Oxz(Ser)-Ala-Oxz(Ser)-Ala-Oxz(Ser)] (3). The
cyclic peptide 27 (78 mg, 0.13 mmol) was dissolved in ethanol
(10 mL) and treated with hydrogen in the presence of palladium on
charcoal (20 mg, 10%) overnight. The mixture was subsequently
filtered through Celite and concentrated under reduced pressure to
afford the crude amine 29.
dichloromethane (10 mL) was treated with 2-pyridinecarbox-
aldehyde (50 L, 0.520 mmol) and sodium triacetoxyborohydride
(180 mg, 0.850 mmol) for 4 h. More 2-pyridinecarboxyaldehyde
(50 L, 0.520 mmol) and sodium triacetoxyborohydride (180 mg,
A suspension of the amine in
m
trisoxazole 25 (1.80 g, 79%) as a colourless foam. [
a
]
25 ꢀ31.2 (c 1.0,
m
D
CHCl3); 1H NMR (300 MHz, CDCl3):
d
8.23 (m, 2H), 8.10 (s, 1H), 8.07
0.850 mmol) were then added and the mixture was left to stir
overnight. The mixture was filtered through a plug of silica (EtOAc)
and the filtrate concentrated under reduced pressure. The residue
(s, 1H), 8.04 (s, 1H), 7.29e7.25 (m, 5H), 5.58 (m, 1H), 5.42 (m, 2H),
5.18 (m, 1H), 5.04 (s, 2H), 4.83 (m, 1H), 3.83 (s, 3H), 3.17 (m, 2H),

S.J. Butler et al. / Tetrahedron 67 (2011) 1019e1029
1029
3. Wipf, P. In Alkaloids: Chemical and Biological Perspectives; Pelletier, S. W., Ed.;
Elsevier: Amsterdam, 1998; pp 187e228.
was then purified by flash chromatography (silica gel; MeOH/
CHCl3, 5:95 v/v) to give the Dpa-functionalized cyclic peptide 3
4. (a) Mink, D.; Mecozzi, S.; Rebek, J., Jr. Tetrahedron 1998, 39, 5709; (b) Jolliffe, K.
(32 mg, 38%) as a pale yellow oil. [
a
]
25 ꢀ15.0 (c 0.8, MeOH); 1H NMR
ꢁ
D
A. Supramol. Chem. 2005, 17, 81; (c) Pinter, A.; Haberhauer, G. Eur. J. Org. Chem.
ꢁ
2008, 2375; (d) Pinter, A.; Haberhauer, G.; Hyla-Kryspin, I.; Grimme, S. Chem.
(300 MHz, CD3OD):
d
8.47 (s,1H), 8.45 (s,1H), 8.43 (s,1H), 8.39 (br d,
Commun. 2007, 3711; (e) Haberhauer, G.; Oeser, T.; Romiger, F. Chem. Commun.
2005, 2799; (f) Haberhauer, G.; Oeser, T.; Romiger, F. Chem.dEur. J. 2005, 11,
6718; (g) Ziegler, E.; Haberhauer, G. Eur. J. Org. Chem. 2009, 3432; (h) Singh, Y.;
Stoermer, M. J.; Lucke, A. J.; Glenn, M. P.; Fairlie, D. P. Org. Lett. 2002, 4, 3367; (i)
Singh, Y.; Stoermer, M. J.; Lucke, A. J.; Guthrie, T.; Fairlie, D. P. J. Am. Chem. Soc.
2005, 127, 6563; (j) Ceide, S. C.; Trembleau, L.; Haberhauer, G.; Somogyi, L.; Lu,
J¼4.1 Hz, 2H), 7.75 (m, 2H), 7.58 (br d, J¼7.9 Hz, 2H), 7.25 (m, 2H),
5.19 (m, 3H), 3.74 (s, 4H), 2.55 (m, 2H), 2.23e1.87 (m, 10H), NeH
signals not observed; 13C NMR (75.5 MHz, CD3OD):
d 164.9, 164.8,
164.0, 160.2, 159.7, 148.3, 142.7, 137.8, 137.6, 135.3, 123.9, 122.7, 64.5,
60.5, 54.2, 44.9, 31.6, 22.1, 19.5, 19.4, six signals obscured or over-
lapping; MS (ESI) m/z 662.3 [(MþNa)þ, 100%]; HRMS (ESI) calcd for
C32H34N9O6 (MþH)þ 640.2632, found 640.2627.
ꢁ
X.; Bartfai, T.; Rebek, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16727; (k) Pinter,
ꢁ
A; Haberhauer, G. Synlett 2009, 3082.
5. McDonough, M. J.; Reynolds, A. J.; Lee, W. Y. G.; Jolliffe, K. A. Chem. Commun.
2006, 2971.
6. Lucke, A. J.; Tyndall, J. D. A.; Singh, Y.; Fairlie, D. P. J. Mol. Graph. Model. 2003, 21, 341.
7. Haberhauer, G.; Pinter, A.; Oeser, T.; Rominger, F. Eur. J. Org. Chem. 2007, 1779;
Haberhauer, G.; Rominger, F. Eur. J. Org. Chem. 2003, 3209.
Acknowledgements
8. (a) Meyers, A. I.; Tavares, F. X. J. Org. Chem. 1996, 61, 8207; (b) Freeman, D. J.;
Pattenden, G. Tetrahedron Lett. 1998, 39, 3251.
9. DiVittorio, K. M.; Johnson, J. R.; Johansson, E.; Reynolds, A. J.; Jolliffe, K. A.;
Smith, B. D. Org. Biomol. Chem. 2006, 4, 1966.
We thank the Australian Research Council for financial support
and for the award of a Queen Elizabeth II research fellowship to K.A.J.
10. Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org. Lett. 2000, 2, 1165.
ꢁ
ꢁ
11. Bertram, A.; Blake, A. J.; Gonzalez-Lopez de Turiso, F.; Hannam, J. S.; Jolliffe, K.
A.; Pattenden, G.; Skae, M. Tetrahedron 2003, 59, 6979.
Supplementary data
12. Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org. Chem. 1973, 38, 2143.
13. Yonetani, K.; Hirotsu, Y.; Shiba, T. Bull. Chem. Soc. Jpn. 1975, 48, 3302.
14. Smith, A. B., III; Friestad, G. K.; Barbosa, J.; Bertounesque, E.; Duan, J. J.-W.; Hull,
K. G.; Iwashima, M.; Qiu, Y.; Spoors, P. G.; Salvatore, B. A. J. Am. Chem. Soc. 1999,
121, 10478; Bull, J. A.; Balskus, E. P.; Horan, R. A. J.; Langner, M.; Ley, S. V. Angew.
Chem., Int. Ed. 2006, 45, 6714.
Supplementary data associated with this article can be found
in online version at doi:10.1016/j.tet.2010.11.10. These data include
MOL files and InChIKeys of the most important compounds
described in this article.
15. The analogous threonine derived oxazole has been reported previously and no
detectable epimerisation was reported. See: Black, R. J.; Dungan, V. J.; Li, R. Y. T.;
Young, P. Y.; Jolliffe, K. A. Synlett 2010, 551.
References and notes
16. Use of our standard conditions (4 M HCl/dioxane) resulted in partial Cbz
deprotection.
17. (a) Wang, L. X.; Tang, M.; Suzuki, T.; Kitajima, K.; Inoue, Y.; Inoue, S.; Fan, J. Q.;
Lee, Y. C. J. Am. Chem. Soc. 1997, 119, 11137; (b) Ferreira, P. M. T.; Maia, H. L. S.;
Monterio, L. S.; Sacramento, J. J. Chem. Soc., Perkin Trans. 1 1999, 3697.
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2. Macrocyclic Chemistry: Current Trends and Future Perspectives; Gloe, K., Ed.;
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