Synthesis of a β-turn mimetic suitable for incorporation in the peptide hormone LHRH

Article abstract of DOI:10.1016/j.tet.2005.03.061

LHRH is a decapeptide hormone which plays a central role in neuroendocrinology. Conformational studies have suggested that LHRH may adopt a β-turn involving residues 5-8 when bound to its receptor. A β-turn mimetic with side chains corresponding to those of a Tyr-Gly-Leu-Orn tetrapeptide has therefore been synthesized for incorporation at positions 5-8 in LHRH. In the turn mimetic, residues i and i+1 are connected by a ψ[CH₂O] isostere instead of an amide bond, while a covalent ethylene bridge replaces the hydrogen bond which is often found between residues i and i+3 in β-turns. The turn mimetic was assembled from three types of building blocks: an azido aldehyde, an Fmoc protected amino acid and a protected dipeptide amine.

Full text of DOI:10.1016/j.tet.2005.03.061

Tetrahedron 61 (2005) 4901–4909
Synthesis of a b-turn mimetic suitable for incorporation in the
peptide hormone LHRH
*
ZhongQing Yuan and Jan Kihlberg
˚
˚
Organic Chemistry, Department of Chemistry, Umea University, SE-901 87 Umea, Sweden
Received 13 January 2005; revised 28 February 2005; accepted 18 March 2005
Available online 19 April 2005
Abstract—LHRH is a decapeptide hormone which plays a central role in neuroendocrinology. Conformational studies have suggested that
LHRH may adopt a b-turn involving residues 5–8 when bound to its receptor. A b-turn mimetic with side chains corresponding to those of a
Tyr-Gly-Leu-Orn tetrapeptide has therefore been synthesized for incorporation at positions 5–8 in LHRH. In the turn mimetic, residues i and
iC1 are connected by a j[CH2O] isostere instead of an amide bond, while a covalent ethylene bridge replaces the hydrogen bond which is
often found between residues i and iC3 in b-turns. The turn mimetic was assembled from three types of building blocks: an azido aldehyde,
an Fmoc protected amino acid and a protected dipeptide amine.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
elements found in peptides and proteins.15,16 In addition, b-
turns have often been indicated as recognition elements
involved in intermolecular interactions that regulate many
important functions in organisms.17,18 Therefore, b-turns
have received a great deal of attention and a large number of
b-turn mimetics have been reported. In order to achieve
high-affinity and selective binding to a targeted receptor, the
conformation of the mimetic should accurately resemble
that of the peptide backbone in the turn and the compound
must also mimic side chains which are essential for
receptor-binding.19,10 The existence of a b-turn involving
residues Tyr5-Gly-Leu-Arg8 in LHRH has been suggested
by conformational energy calculations20 and various
physiochemical methods.21,22 Furthermore, replacement of
the Tyr5-Arg8 segment by a0sterically hindered b-lactam,23
which is a model of a type II b-turn, resulted in an analogue
that was more potent than LHRH both in vitro and in vivo.
In addition, a highly potent and orally active nonpeptide
antagonist at the human LHRH receptor based on a type II
b-turn model has been developed.24 Therefore, introduction
of a b-turn mimetic having correct side chain functionalities
will provide invaluable insight into the structural features
essential for binding of LHRH to its receptor. As a first step
towards this goal we now report an approach which provides
access to a conformationally restricted b-turn mimetic for
incorporation in place of residues 5–8 in LHRH.
Luteinizing hormone-releasing hormone (LHRH), pGlu-
His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, plays a key
role in the mammalian reproductive system.1 The decapep-
tide hormone is secreted in pulses from the hypothalamus
and stimulates the anterior pituitary gland to release the
gonadotropic hormones, luteinizing hormone (LH) and
follicle stimulating hormone (FSH). After the sequence of
LHRH was determined in 1971,1–4 intensive studies
directed towards obtaining knowledge of structure–activity
relationships as well as receptor binding have been
performed.5,6 Such studies are of interest in development
of therapeutic agents for treatment of a variety of hormone
dependent disorders, such as breast and prostate cancer.7,8
The use of peptides, such as LHRH, as drugs is often limited
due to rapid degradation by proteolytic enzymes, low
bioavailability and rapid secretion. Design of peptido-
mimetics which resemble the secondary structure of bio-
logically active peptides is one way to circumvent these
problems.9–11 Such, peptidomimetics are valuable not only
for design of new pharmaceuticals, but also serve to help in
elucidating the biologically active conformations of pep-
tides, and provide additional structure–activity relationship
data.12–14
b-Turns are formed by four consecutive amino acid residues
and constitute one of the most common secondary structure
2. Results and discussion
Keywords: Peptidomimetic; b-Turn; LHRH; Peptide hormone.
*
Our group recently reported results from a project aimed at
design and synthesis of a novel type of b-turn mimetics, and
Corresponding author. Tel.: C46 90 7866890; fax: C46 90 138885;
e-mail: jan.kihlberg@chem.umu.se
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.061

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Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
exemplified the approach by incorporation of a mimetic into
Leu-enkephalin.25,26 In these mimetics the amide bond
between residue i and iC1 of the b-turn is replaced by a
methylene ether isostere, while the i/iC3 hydrogen bond
which stabilizes the turn has been exchanged for a covalent
ethylene bridge, thereby forming a 10-membered b-turn
mimetic (cf. 1, Fig. 1). We now describe the synthesis of
b-turn mimetic 2 which has side-chains corresponding to
those of a Tyr-Gly-Leu-Orn tetrapeptide (Fig. 2). Mimetic 2
is intended for incorporation at positions 5–8 in LHRH and
in order to simplify the synthesis ornithine was chosen in
place of arginine, which is found at position 8 in LHRH.
This choice was made since it is possible to convert the side
chain of ornithine to that of arginine on solid phase after
incorporation of the mimetic in LHRH.27 Mimetic 2 was
designed to allow assembly from three building blocks;
azido aldehyde 3, dipeptide amine 4 and Fmoc-Leu-OH 5.
Building block 3 corresponds to Tyr and Gly at positions i
and iC1 of the turn and the azido group serves as a
precursor of the amino group of residue i. N-Alkylated
amino acid derivatives, such as residue iC3 in mimetic 1
(Fig. 1), are more susceptible to epimerization during
activation and coupling than the ordinary amino acids.28,29
Therefore, it was decided to avoid this potential problem by
including Pro9 of LHRH when preparing the turn mimetic
and dipeptide amine 4 was chosen instead of an ornithine
building block. The tert-butyldiphenyl silyl protected
hydroxyl group in 4 corresponds to the carboxyl group of
the proline residue.
The key building block, azido aldehyde 3, was prepared
from the commercially available Cbz-Tyr(Bzl)-OH
(Schemes 1 and 2). This was transformed into a mixed
anhydride by additionof isobutyl chloroformateand N-methyl
Scheme 1. (a) iBuOCOCl, NMM, MeONHMe$HCl, CH2Cl2, K15 8C/rt,
2 h, 92%. (b) Allylmagnesium bromide, THF, K78 8C, 1.5 h, 94%. (c) K-
Selectride, THF, K78 8C 19 h. (d) and (e) THF:MeOH:aq KOH (7.5 N)
(4:2:1), rt, 5 h, flash column chromatography, 72% of 10 over steps (c)–(e).
Figure 1. Design of a b-turn mimetic. In this mimetic, a methylene ether
isostere replaces the amide bond between resides i and iC1 in the turn and a
covalent ethylene bridge connects residues i and iC3, which are often
hydrogen bonded in b-turns.
Scheme 2. (a) EtOH/KOH (aq, 1 M) (1:1), reflux, 6 h, 84%. (b) TfN3,
DMAP, CuSO4, CH2Cl2, rt, 2 h, 85%. (c) BrCH2CO2tBu, Bu4NHSO4,
benzene:aq NaOH (50%) (1:1), rt, 1 h. (d) OsO4, NMO, THF/acetone:H2O
(1:1:1), rt, 2.5 h, 65% over two steps. (e) Pb(OAc)4, Na2CO3, benzene,
0 8C/rt, 88%.
Figure 2. A retrosynthetic analysis shows that the target mimetic 2 may be
assembled from three building blocks; azido aldehyde 3, dipeptide amine 4
and Fmoc-Leu-OH (5).

Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
4903
morpholine, followed by reaction with N,O-dimethylhy-
droxylamine to give Weinreb amide 6 (92%). Grignard
reaction30 of 6 with allylmagnesium bromide furnished
ketone 7 (94%) which was reduced using K-selectride to
give a mixture of 8, 9, and 10. Anti and syn amino alcohols 8
and 9 were difficult to separate from each other by flash
column chromatography, whereas anti-oxazolidinone 10
was readily separated from 8 and 9. After removal of 10, the
mixture of 8 and 9 was converted to a mixture of syn and
anti-oxazolidinones by treatment with aqueous KOH in a
mixture of THF and methanol. Purification by chromato-
graphy then gave 10 in 72% overall yield from 7. The
stereochemistry of anti-oxazolidinone 10 was established
by comparing the NOESY spectrum of 10 with that of the
corresponding syn-isomer. The spectrum of 10 did not show
any cross-peak for H-4 and H-5 in the oxazolidinone ring,
whereas the syn-isomer showed a strong cross-peak between
these two hydrogen atoms.
Scheme 3. (a) tBuPh2SiCl, imidazole, CH2Cl2, rt, 3 h, 96%. (b) Fmoc-
Orn(Aloc)-OH, HATU, DIEA, DMF, 0 8C/rt, 2 h, 82%. (c) Morpholine,
rt, 3 h, 94%.
was used as coupling reagent under conditions found to be
optimal for acylation of a secondary amine in a synthetic
route leading to a g-turn mimetic,36 a very low yield of 18
was obtained as a mixture of diastereomers. Conversion of 5
into the corresponding acid chloride and coupling with 17
also led to a diastereomeric mixture of 18, most likely due to
epimerization of the stereogenic center of 5. Instead 18 was
obtained in enantiomerically pure form by use of HATU as
coupling reagent in the presence of diisopropylethylamine.
In contrast to what can be expected,37 attempts to cleave the
tert-butyl ester of 18 without removing the tert-butyldi-
phenylsilyl protecting group failed. The tert-butyldiphenyl-
silyl protecting group was found to be cleaved much faster
than the tert-butyl ester in formic acid, but fortunately the
resulting hydroxyl group was simultaneously protected as a
formate so that 19 was formed. The carboxyl group of 19
was then activated as a pentafluorophenyl ester which was
added to DBU in hot dioxane under high dilution conditions.
This gave a mixture of 20 and 21 in a one-pot procedure38
involving Fmoc deprotection followed by closure of the ten-
membered ring and partial removal of the formate.
Compound 20 was easily hydrolyzed to give 21, which
thereby was then obtained in 51% yield from 19 over the
three steps. Finally Jones oxidation of 21 furnished the
b-turn mimetic 2 which is ready for incorporation in LHRH
using solid-phase synthesis (Scheme 4).
Oxazolidinone 10 was hydrolyzed under alkaline conditions
to furnish amino alcohol 11 (Scheme 2). Azido transfer was
then performed with freshly prepared triflyl azide in the
presence of catalytic amount of CuSO4 to give azido alcohol
12.31 This transformation served the dual purposes of
introducing an inert amino protective group as well as
reducing the steric bulk in the vicinity of the hydroxyl group
of 12. Compound 12 decomposed on standing but could be
purified by flash column chromatography if performed
rapidly. Due to its instability, 12 was immediately alkylated
with tert-butyl bromoacetate using phase-transfer cataly-
sis,32 thus introducing the iC1 residue of the turn mimetic.
Compound 13 was even more labile than 12 and crude 13
was therefore oxidized immediately to diol 14 by osmium
tetraoxide catalysed dihydroxylation (65% from 12).
Cleavage of diol 14 with lead tetraacetate furnished the
stable azido aldehyde 3 in 88% yield, thereby providing one
of the building blocks required for assembly of the target
b-turn mimetic.
Synthesis of a building block corresponding to 4 was first
attempted for an Arg-Pro sequence. However, due to
purification problems related to the guanidino group of
arginine this approach had to be abandoned. Since ornithine
can be converted to arginine late in synthetic routes,27 an
Orn-Pro dipeptide was chosen instead of Arg-Pro. A
diketopiperazine was formed instead of the desired amine
on cleavage of the Fmoc protecting group of Fmoc-
Orn(Aloc)-Pro-OMe with morpholine. Therefore, prolinol
was chosen as starting material and protected with tert-
butyldiphenylsilyl chloride to give 15 in high yield
(Scheme 3). Coupling of 15 with Fmoc-Orn(Aloc)-OH
was then achieved by using HATU33,34 as coupling reagent
in the presence of diisopropylethylamine in DMF. Finally
cleavage of the Fmoc group of 16 furnished 4, that is, the
other key building block for synthesis of the b-turn mimetic.
3. Conclusions
A b-turn mimetic with side-chains corresponding to a Tyr-
Gly-Leu-Orn tetrapeptide, suitable for incorporation at
positions 5–8 of the hormone LHRH, has been synthesized.
The design of the mimetic is similar to a Tyr-Gly-Gly-Phe
mimetic previously incorporated in Leu-enkephalin by
us.25,26 Thus, residues i and i C1 are connected by a
j(CH2O( isostere instead of an amide bond, while a
covalent ethylene bridge replaces the hydrogen bond that
is often found between the carbonyl group of residue i and
the amino group of residue iC3 in b-turns. In addition, we
have now shown that it is possible to incorporate chiral
amino acids at position iC2 of this type of b-turn mimetics,
that is, the strategy is not limited to glycine which was found
at the iC2 position in the Leu-enkephalin mimetic. After
evaluation of several different procedures it was found that
the iC2 leucine could be coupled without racemization to a
sterically hindered secondary amine using HATU as
Assembly of the b-turn mimetic began with reductive
amination35 of 3 with 4 using sodium triacetoxyborohydride
as reducing agent to give 17 in almost quantitative yield.
Introduction of Fmoc-Leu-OH 5 at position iC2 in the
mimetic turned out to be very difficult, most likely due to
steric hindrance. Therefore, large efforts were made to find
suitable conditions for acylation of 17 with 5. When DIC

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Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
calcium hydride and sodium-benzophenone, respectively.
˚
DMF was distilled and then dried over 3 A molecular sieves.
TLC was performed on Silica Gel 60 F254 (Merck) with
detection by UV light and charring with phosphomolybdic
acid in EtOH. Flash column chromatography (eluents given
˚
in brackets) was performed on silica gel (Matrex, 60 A, 35–
70 mm, Grace Amicon).
1H NMR spectra for compounds 2–21 were recorded at
400 MHz whereas 13C NMR spectra were obtained at
100 MHz for solution in CDCl3 [residual CHCl3 (dH
7.26 ppm) or CDCl3 (dC 77.0 ppm) as internal standard] or
in MeOH-d4 [residual CD2HOD (dH 3.31 ppm) or CD3OD
(dC 49.2 ppm) as internal standard] at 298 K. Elemental
analysis was performed by Mikro Kemi AB, Uppsala,
Sweden. Compounds lacking elemental analysis were
characterized by high resolution MS and were O95%
1
pure according to H NMR spectroscopy. Ions for positive
fast atom bombardment mass spectra were produced by a
beam of Xenon atoms (6 keV) from a matrix of glycerol and
thioglycerol.
4.1.1. (S)-N-Methoxyl-N-methyl-2-benzyloxycarbonyl-
amino-3-(4-benzyloxyphenyl)-propanoic amide (6). Iso-
butyl chloroformate (0.845 mL, 6.49 mmol) was added
dropwise to a solution of Cbz-Tyr(Bzl)-OH (2.63 g,
6.49 mmol) and N-methyl morpholine (NMM, 1.57 mL,
14.3 mmol) in CH2Cl2 at K15 8C. After being stirred for
15 min, N,O-dimethylhydroxylamine$HCl (0.633 g,
6.49 mmol) was added. After a further 30 min the cooling
bath was removed and the reaction mixture was stirred at
room temperature for additional 1.5 h. The reaction mixture
was poured into H2O (20 mL) and extracted with CH2Cl2
(3!15 mL). The combined organic layers were washed
sequentially with 0.2 M aq HCl, satd aq NaHCO3 and brine,
then dried with MgSO4, filtered and concentrated. Flash
column chromatography (heptane/ethyl acetate, 3:1/1:1)
of the residue gave 6 as a colourless oil (2.69 g, 92%):
[a]2D0ZC9.3 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) d
7.30–7.45 (10H, m, Ph), 7.09 (2H, d, JZ8.5 Hz, Phtyr), 6.90
(2H, d, JZ8.5 Hz, Phtyr), 5.52 (1H, d, JZ8.6 Hz, NH),
5.04–5.13 (5H, m, CH2Ph, CH2Ph and CHNH), 3.68 (3H, s,
OCH3), 3.18 (3H, s, NCH3), 3.05 (1H, ABX type dd, JZ5.8,
13.6 Hz, Tyr b-H), 2.88 (1H, ABX type dd, JZ7.2, 13.6 Hz,
Tyr b-H); 13C NMR (100 MHz, CDCl3) d 171.9, 157.7,
155.7, 136.9, 136.3, 130.4, 128.5, 128.4, 128.2, 128.0,
127.9, 127.8, 127.4, 114.7, 69.8, 66.6, 61.4, 52.1, 37.7, 32.0;
IR (neat) 3298, 1714, 1657 cmK1; HRMS (FAB) calcd for
C26H29N2O5 449.2079 (MCHC), found 449.2057; Anal.
Calcd C, 69.6; H, 6.3; N, 6.2. Found C, 69.7; H, 6.3; N, 6.1.
Scheme 4. (a) NaBH(OAc)3, DCE, rt, 45 min, 95%. (b) Fmoc-Leu-OH,
HATU, DIEA, DMF, 0 8C/rt, 24 h, 52% (70% based on recovered 17).
(c) HCOOH, rt, 19 h, 65%. (d) PfpOH, DIC, EtOAc, 0 8C, 1.5 h, then DBU,
dioxane, 100 8C, 4 h. (e) aq LiOH (1 M), THF/MeOH:H2O (3:1:1), rt, 3 h,
51% for steps (d) and (e). (f) Jones oxidation, 3.5 h, 70%.
coupling reagent in the presence of diisopropylethylamine.
In addition, the synthetic strategy was revealed to be
compatible with amino acids having protected functional-
ities in their side-chains both at positions i and iC4, such as
those of tyrosine and ornithine in the present mimetic.
4.1.2. (S)-2-Benzyloxycarbonylamino-1-(4-benzyloxy-
phenyl)-hex-5-en-3-one (7). Allylmagnesium bromide
(1 M solutiom in diethyl ether, 8.35 mL, 8.35 mmol) was
added dropwise to a solution of Weinreb amide 6 (1.50 g,
3.34 mmol) in THF (32 mL) at K78 8C. After being stirred
for 1.5 h, the reaction was quenched with satd aq NH4Cl
(20 mL) and the mixture was extracted with CH2Cl2 (3!
20 mL). The combined organic layers were then washed
with brine, dried over MgSO4, filtered and concentrated.
Flash chromatography (toluene/ethanol, 20:1/10:1) of the
residue gave 7 as a white amorphous solid (1.35 g, 94%):
[a]2D0ZC9.6 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) d
4. Experimental
4.1. General data
All reactions were carried out under a nitrogen atmosphere
with dry solvents under anhydrous conditions, unless
otherwise stated. CH2Cl2 and THF were distilled from

Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
4905
7.32–7.43 (10H, m, Ph), 7.02 (2H, d, JZ8.2 Hz, Phtyr), 6.89
(2H, d, JZ8.2 Hz, Phtyr), 5.85 (1H, m, CH]CH2), 5.34
(1H, d, JZ7.7 Hz, NH), 5.02–5.20 (6H, m, CH2Ph, CH2Ph
and CH]CH2), 4.63 (1H, m, CHN), 3.19 (1H, ABX type
dd, JZ7.2, 17.3 Hz, CH2CH]CH2), 3.13 (1H, ABX type dd,
JZ7.2, 17.3 Hz, CH2CH]CH2), 3.03 (1H, ABX type dd,
JZ6.8, 14.2 Hz, Tyr b-H), 2.96 (1H, ABX type dd, JZ6.2,
14.2 Hz, Tyr b-H); 13C NMR (100 MHz, CDCl3) d 206.6,
157.9, 155.6, 136.9, 136.2, 130.2, 129.5, 128.5, 128.4,
128.1, 128.0, 127.9, 127.8, 127.4, 119.4, 115.1, 70.0, 66.9,
60.1, 45.5, 36.8; IR (neat) 3309, 1716, 1689 cmK1; HRMS
(FAB) calcd for C27H28NO4 430.1979 (MCHC), found
430.2024; Anal. Calcd C, 75.5; H, 6.3; N, 3.3. Found C,
75.5; H, 6.3; N, 3.3.
5.05 (2H, s, CH2Ph), 3.48 (1H, m, CHOH), 2.88 (2H, m,
CHNH2 and Tyr b-H), 2.47 (1H, ABX type dd, JZ
8.9,12.9 Hz, Tyr b-H), 2.33 (2H, m, CH2CH]CH2), 2.06
(3H, br s, OH and NH2); 13C NMR (100 MHz, CDCl3) d
157.5, 137.0, 134.9, 131.1, 130.2, 128.6, 127.9, 127.5,
117.5, 115.0, 72.3, 70.0, 56.0, 39.9, 39.2; IR (neat) 3359,
3298, 1610, 1581, 1510 cmK1; HRMS (FAB) calcd for
C19H24NO2 298.1779 (MCHC), found 298.1798.
4.1.5. (2S,3S)-2-Azido-1-(4-benzyloxy-phenyl)-hex-5-en-
3-ol (12). A solution of freshly prepared triflyl azide31
(14.8 mmol) in CH2Cl2 (40 mL) was added dropwise to a
solution of 11 (1.05 g, 3.53 mmol), DMAP (216 mg,
1.77 mmol) and CuSO4 (28.2 mg, 0.177 mmol) in CH2Cl2
(10 mL) at room temperature. After being stirred for 2 h, the
reaction mixture was washed with 10% citric acid (2!),
satd aq NaHCO3 (2!) and brine. The combined organic
layers were dried over Na2SO4, filtered and concentrated.
Flash column chromatography (heptane/ethyl acetate,
10:1/3:1) of the residue gave azido alcohol 12 as white
amorphous solid (0.966 g, 85%): 1H NMR (400 MHz,
CDCl3) d 7.31–7.45 (5H, m, Ph), 7.18 (2H, d, JZ8.6 Hz,
Phtyr), 6.95 (2H, d, JZ8.6 Hz, Phtyr), 5.79 (1H, m,
CH]CH2), 5.17 (2H, m, CH]CH2), 5.06 (2H, s,
CH2Ph), 3.62 (1H, m, CHOH), 3.44 (1H, ddd, JZ3.3, 6.4,
8.2 Hz, CHN3), 2.99 (1H, ABX type dd, JZ6.4, 13.8 Hz,
Tyr b-H), 2.91 (1H, ABX type dd, JZ6.4, 13.8 Hz, Tyr
b-H), 2.36 (2H, m, CH2CH]CH2), 1.82 (1H, d, JZ5.8 Hz,
OH); 13C NMR (100 MHz, CDCl3) d 157.8, 137.0, 133.7,
130.3, 129.7, 128.6, 128.5, 127.9, 127.5, 127.5, 127.4,
118.7, 71.3, 70.1, 67.1, 39.1, 36.3; IR (neat) 3365, 3031,
2106, 1610, 1510 cmK1; HRMS (FAB) calcd for
C19H21N3O2 323.1679 (MC), found 323.1631.
4.1.3. (4S,5S)-5-Allyl-4-(4-benzyloxybenzyl)-oxazolidin-
2-one (10). K-Selectride (1 M solution in THF, 18.0 mL,
18.0 mmol) was added dropwise to a solution of allyl ketone
7 (5.15 g, 12.0 mmol) in THF (82 mL) at K78 8C. After
being stirred for 19 h, the reaction was quenched with H2O
(20 mL) and acidified with 10% citric acid to adjust the pH
to w4. The reaction mixture was extracted with CH2Cl2
(3!30 mL). The combined organic layers were then
washed with brine, dried over MgSO4, filtered and con-
centrated. Flash column chromatography (heptane/ethyl
acetate, 10:1/1:1.5) of the residue gave a mixture of 8 and
9 (4.93 g) and pure anti-oxazolidinone 10 (0.47 g). Then the
mixture of 8 and 9 was stirred in a mixture of THF, MeOH
and aq 7.5 M KOH (4:2:1, 140 mL) at room temperature for
5 h and poured into H2O (130 mL). The phases were
separated and the aqueous phase was extracted with EtOAc
(4!50 mL). The combined organic phases were washed
with brine and dried over MgSO4. Flash column chromato-
graphy (heptane/ethyl acetate 10:1/1:1.5) of the residue
gave anti-oxazolidinone 10 as a white amorphous solid
(2.38 g in total, 72% for 7): [a]2D0ZK63.2 (c 0.8, CHCl3);
1H NMR (400 MHz, CDCl3) d 7.32–7.44 (5H, m, Ph), 7.08
(2H, d, JZ8.3 Hz, Phtyr), 6.94 (2H, d, JZ8.3 Hz, Phtyr),
5.92 (1H, s, NH), 5.68 (1H, m, CH]CH2), 5.11 (2H, m,
CH]CH2), 5.05 (2H, s, CH2Ph), 4.32 (1H, q, JZ5.7, 5.7,
5.7 Hz, CHO), 3.68 (1H, q, JZ6.3, 6.3, 6.3 Hz, CHN), 2.78
(2H, m, Tyr b-H), 2.34 (2H, m, CH2CH]CH2); 13C NMR
(100 MHz, CDCl3) d 158.9, 158.0, 137.0, 131.5, 130.3,
128.7, 128.3, 128.1, 127.6, 119.4, 115.5, 80.7, 70.1, 58.2,
40.7, 38.7; IR (neat) 3269, 1749, 1687, 1512 cmK1; HRMS
(FAB) calcd for C20H22NO3 324.1679 (MCHC), found
324.1609; Anal. Calcd C, 74.3; H, 6.6; N, 4.3. Found C,
74.5; H, 6.6; N, 4.3.
4.1.6. a-[(4S,5S)-5-Azido-6-(4-benzyloxyphenyl)-1,2-
dihydroxy-hex-4-oxy]-acetic acid tert-butyl ester (14).
tert-Butyl bromoacetate (0.72 mL, 4.89 mmol) was added
dropwise to a vigorously stirred mixture of azido alcohol 12
(0.878 g, 2.71 mmol) and tetrabutyl ammonium hydrogen
sulphate (257 mg) in benzene–aq 50% NaOH (1:1, 30 mL)
at room temperature. After being stirred for 1.5 h, the
mixture was separated and the aqueous phase was extracted
with CH2Cl2 (3!20 mL). The combined organic layers
were dried over Na2SO4 and concentrated to give crude
O-alkylated azido alcohol 13. Compound 13 was dissolved
in a mixture of THF, acetone and water (1:1:1, 30 mL).
OsO4 (69 mg, 0.21 mmol) and NMO (0.586 g, 4.34 mmol)
were added sequentially to the solution. After being stirred
for 2.5 h the solution was acidified with 0.2 M aq HCl to
adjust the pH to w2 and then extracted with EtOAc (4!
15 mL). The combined organic layers were washed with
brine, dried with Na2SO4 and concentrated. Flash column
chromatography (heptane/ethyl acetate, 5:1/1:3) of the
residue gave azido diol 14 as a colourless oil (0.832 g,
4.1.4. (2S,3S)-2-Amino-1-(4-benzyloxyphenyl)-hex-5-en-
3-ol (11). Anti-oxazolidinone 10 (1.54 g, 4.76 mmol) was
refluxed in a mixture of EtOH (35 mL) and aq KOH (1 M,
35 mL) for 6 h, after which the solvent was evaporated and
the residue was partitioned between H2O (15 mL) and
CH2Cl2 (15 mL). The aqueous phase was extracted with
CH2Cl2 (4!30 mL). The combined organic layers were
then washed with brine, dried over MgSO4, filtered and
concentrated. Flash column chromatography (toluene/ethanol,
10:1/1:2) of the residue furnished amino alcohol 11 as a
white amorphous solid (1.18 g, 84%): [a]2D0ZK9.9 (c 1.1,
1
65%): H NMR (400 MHz, CDCl3) d for a 1:1 diastereo-
meric mixture 7.30–7.45 (5H, m, Ph), 7.15 (2H, d, JZ
8.5 Hz, Phtyr), 6.94 (2H, d, JZ8.5 Hz, Phtyr), 5.06 (2H, s,
CH2Ph), 4.17–4.28 (1.5H, m, CH2COOtBu), 4.15 (0.5H, m,
CHO), 4.05 (0.5H, d, JZ16.3 Hz, CH2COOtBu), 3.95
(0.5H, m, CHO), 3.76 (0.5H, m, CHO), 3.64 (2.5H, m,
CHN3, CHO and CH2OH), 3.54 (1H m, CH2OH), 2.92–3.02
(1H, m, Tyr b-H), 2.58–2.67 (1H, m, Tyr b-H), 1.59–1.83
(2H, m, CH2CHO), 1.52 (4.5H, s, tBu), 1.49 (4.5H, s, tBu);
1
CHCl3); H NMR (400 MHz, CDCl3) d 7.33–7.45 (5H, m,
Ph), 7.12 (2H, d, JZ8.4 Hz, Phtyr), 6.93 (2H, d, JZ8.4 Hz,
Phtyr), 5.90 (1H, m, CH]CH2), 5.14 (2H, m, CH]CH2),

4906
Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
13C NMR (100 MHz, CDCl3) d 171.1, 170.2, 157.8, 157.8,
137.0, 137.0, 130.2, 130.0, 129.9, 128.6, 127.9, 127.5,
115.1, 115.1, 82.9, 82.8, 78.4, 71.0, 70.1, 68.2, 67.7, 66.8,
66.7, 66.7, 66.0, 35.5, 35.0, 34.3, 33.8, 28.1; IR (neat) 3408,
2106, 1732, 1612, 1512 cmK1; HRMS (FAB) calcd for
C25H33N3O6Na 494.2198 (MCNaC), found 494.2191.
(2 mL) was added to the solution which was stirred for 3 h
before being diluted with water (100 mL) and extracted with
CH2Cl2 (5!10 mL). The combined organic layers were
washed with aq 0.2 M HCl, H2O, satd aq NaHCO3 and
brine, dried with Na2SO4, filtered and concentrated. Flash
column chromatography (toluene/ethanol, 20:1/10:1) of
the residue gave 16 as a white amorphous solid (0.502 g,
82%): [a]2D0ZK35.7 (c 0.8, CHCl3); H NMR (400 MHz,
1
4.1.7. (1S,2S)-2-Azido-3-(4-benzyloxyphenyl)-1-(2-oxo-
ethyl)-propoxy]-acetic acid tert-butyl ester (3). Lead
tetraacetate (0.237 g, 0.534 mmol) was added to a mixture
of azido diol 14 (0.168 g, 0.356 mmol) and Na2CO3
(94.3 mg, 0.890 mmol) in benzene (10 mL) at 0 8C. After
being stirred for 10 min, the cooling bath was removed and
stirring was continued for 1 h. Then the reaction was
quenched with 10 drops of ethylene glycol and stirring was
continued for 5 min. Et2O and satd aq NaHCO3 were added
and the phases were separated. The aqueous phase was
extracted with Et2O (4!10 mL), the combined organic
phases were washed with brine, dried with Na2SO4, filtered
and concentrated. Flash column chromatography (heptane/
ethyl acetate, 5:1/3:1) of the residue gave azido aldehyde
3 as a colourless oil (0.138 g, 88%): [a]2D0ZK22.5 (c 0.1,
CHCl3); 1H NMR (400 MHz, CDCl3) d 9.84 (1H, s, CHO),
7.31–7.45 (5H, m, Ph), 7.19 (2H, d, JZ8.3 Hz, Phtyr), 6.95
(2H, d, JZ8.3 Hz, Phtyr), 5.06 (2H, s, CH2Ph), 4.11 (2H, s,
CH2COOtBu), 4.02 (1H, m, CHN3CHO), 3.60 (1H, dt, JZ
4.6, 9.2 Hz, CHN3), 3.06 (1H, ABX type dd, JZ4.6,
14.0 Hz, Tyr b-H), 2.80–2.95 (3H, m, Tyr b-H and
CH2CHO), 1.49 (9H, s, tBu); 13C NMR (100 MHz,
CDCl3) d 200.1, 169.2, 157.7, 136.9, 130.2, 129.6, 128.6,
128.5, 127.9, 127.5, 115.0, 81.9, 67.0, 68.9, 65.8, 45.7, 35.3,
28.1; IR (neat) 2107, 1743, 1722 cmK1; HRMS (FAB) calcd
for C24H28N3O5 (MCHC) 438.2021, found 438.2030.
CDCl3, rotamer ratio 4:1) d (major rotamer) 7.76 (2H, d, JZ
7.4 Hz, Ph), 7.62 (6H, m, Ph), 7.29–7.43 (10H, m, Ph), 5.90
(1H, m, CH]CH2), 5.75 (1H, d, JZ8.5 Hz, Orn a-NH),
5.28 (1H, d, JZ17.1 Hz, CH]CH2), 5.19 (1H, d, JZ
17.1 Hz, CH]CH2), 4.75 (1H, br s, Orn d-NH), 4.53 (3H,
m, CH2CH]CH2 and Orn a-H), 4.37 (2H, m, Fmoc-CH2),
4.26 (1H, m, Pro a-H), 4.21 (1H, t, JZ6.9 Hz, Fmoc-CH),
3.85 (1H, m, CH2OSi), 3.72 (1H, m, CH2OSi), 3.64 (1H, m,
Pro d-H), 3.48 (1H, m, Pro d-H), 3.15 (2H, m, Orn d-H),
2.08 (2H, m, Pro b-H and Pro g-H), 1.93 (2H, m, Pro b-H
and Pro g-H), 1.73 (1H, m, Orn b-H), 1.56 (3H, m, Orn b-H
and Orn g-H), 1.06 (9H, s, tBu); 13C NMR (100 MHz,
CDCl3) d 170.4, 156.2, 143.9, 143.8, 141.3, 135.5, 135.5,
133.4, 133.3, 132.9, 129.7, 127.7, 127.7, 127.7, 127.0,
125.2, 125.1, 119.9, 119.9, 117.5, 66.9, 65.4, 63.3, 58.6,
52.1, 47.6, 47.2, 40.7, 30.7, 26.9, 26.8, 25.5, 24.5, 19.2; IR
(neat) 3307, 3278, 3970, 1716, 1633, 1525, 1448 cmK1
;
HRMS (FAB) calcd for C45H54N3O6Si 760.3779 (MCHC),
found 760.3768; Anal. Calcd C, 71.1; H, 7.0; N, 5.5. Found
C, 70.8; H, 7.0; N, 5.5.
4.1.10. Nd-(Allyloxycarbonyl)-L-ornithyl-O-(tert-butyl-
diphenylsilyl)-L-prolinol (4). Compound 16 (3.35 g,
4.41 mmol) was stirred in morpholine (24 mL) at room
temperature for 2 h. Then the precipitate was filtered off and
the filtrate was co-evaporated with toluene. Flash column
chromatography (toluene/ethanol, 10:1/1:1) of the residue
gave 4 as a colourless oil (2.22 g, 94%): [a]2D0ZK20.8
4.1.8. O-(tert-Butyldiphenylsilyl)-L-prolinol (15). tert-
Butyldiphenylsilyl chloride (4.92 mL, 21.0 mmol) was
added to a solution of L-prolinol (0.987 mL, 10.0 mmol)
and imidazole (1.50 g, 22.0 mmol) in CH2Cl2 (100 mL) at
0 8C. After being stirred for 3 h the resulting precipitate was
filtered off and the reaction was quenched by addition of
satd aq NH4Cl. The aqueous phase was extracted with
CH2Cl2 (2!20 mL), the combined organic phases were
dried with Na2SO4, filtered and concentrated. Flash colum
chromatography (toluene/ethanol, 10:1/3:1) of the residue
gave 15 as a colourless oil (3.26 g, 96%): [a]2D0ZK5.9
(c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.67 (4H, m,
Ph), 7.35–7.44 (6H, m, Ph), 3.65 (1H, ABX type dd, JZ4.9,
10.0 Hz, CH2OSi), 3.59 (1H, ABX type dd, JZ6.1, 10.0 Hz,
CH2OSi), 3.23 (1H, m, Pro a-H), 2.97 (1H, m, Pro d-H),
2.85 (1H, m, Pro d-H), 2.13 (1H, br s, NH), 1.69–1.82 (3H,
m, Pro b-H and Pro g-H), 1.48 (1H, m, Pro b-H), 1.06 (9H,
s, tBu); 13C NMR (100 MHz, CDCl3) d 135.6, 135.6, 133.7,
129.6, 127.7, 127.7, 66.5, 59.9, 46.5, 27.5, 26.9, 25.4, 19.3;
IR (neat) 2856, 1111 cmK1; HRMS (FAB) calcd for
C21H30NOSi 340.2079 (MCHC), found 340.2086; Anal.
Calcd C, 74.3; H, 8.6; N, 4.1. Found C, 73.9; H, 8.6; N, 4.1.
1
(c 0.1, CHCl3); H NMR (400 MHz, CDCl3, rotamer ratio
4:1) d (major rotamer) 7.62 (4H, m, Ph), 7.34–7.44 (6H, m,
Ph), 5.89 (2H, m, Orn d-NH and CH]CH2), 5.28 (2H, m,
CH]CH2), 4.53 (2H, m, CH2CH]CH2), 4.24 (1H, m, Pro
a-H), 3.82 (1H, ABX type dd, JZ5.3, 9.8 Hz, CH2OSi),
3.72 (1H, ABX type dd, JZ2.8, 9.8 Hz, CH2OSi), 3.53 (2H,
m, Orn a-H and Pro d-H), 3.41 (1H, m, Pro d-H), 3.14 (2H,
m, Orn d-H), 2.07 (4H, m, Orn a-NH2, Pro b-H and Pro
g-H), 1.81–1.95 (2H, m, Pro b-H and Pro g-H), 1.58–1.71
(3H, m, Orn g-H and Orn b-H), 1.49 (1H, m, Orn b-H), 1.04
(9H, s, tBu); 13C NMR (100 MHz, CDCl3) d 173.9, 156.2,
135.5, 135.4, 133.4, 132.9, 132.7, 129.9, 129.6, 128.2,
127.8, 127.6, 127.6, 117.4, 65.3, 63.4, 58.4, 52.6, 47.1, 40.6,
32.4, 26.8, 26.7, 26.3, 24.4, 19.2; IR (neat) 3298, 3070,
1712, 1631, 1531 cmK1; HRMS (FAB) calcd for
C30H44N3O4Si 538.3079 (MCHC), found 538.3096.
4.1.11. Compound 17. NaBH(OAc)3 (101 mg, 0.478 mmol)
was added to a solution of azido aldehyde 3 (132 mg,
0.30 mmol) and primary amine 4 (258 mg, 0.48 mmol) in
dichloroethane (7.5 mL) at room temperature. After being
stirred for 1 h the reaction was quenched with satd aq
NaHCO3 and extracted with CH2Cl2 (3!10 mL). The
combined organic layers were washed with brine, dried with
Na2SO4, filtered and concentrated. Flash column chromato-
graphy (toluene/ethanol, 20:1/5:1) of the residue gave
secondary amine 17 as a colourless oil (274 mg, 95%):
4.1.9. Nd-(Allyloxycarbonyl)-Na-(fluoren-9-ylmethoxy-
carbonyl)-L-ornithyl-O-(tert-butyldiphenylsilyl)-L-pro-
linol (16). Fmoc-Orn(Aloc)-OH (0.386 g, 0.881 mmol) was
activated for 15 min with HATU (0.305 g, 0.801 mmol) in
the presence of DIEA (0.308 mL, 1.76 mmol) in DMF
(2 mL) at 0 8C. Then 15 (0.275 g, 0.801 mmol) in DMF

Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
4907
[a]2D0ZK31.1 (c 2.5, CHCl3); 1H NMR (400 MHz, CDCl3,
rotamer ratio 4:1) d (major rotamer) 7.62 (4H, m, Ph), 7.30–
7.45 (11H, m, Ph), 7.18 (2H, d, JZ8.6 Hz, PhTyr), 6.93 (2H,
d, JZ8.6 Hz, PhTyr), 5.90 (1H, m, CH]CH2), 5.25–5.32
(2H, m, CH]CH2 and Orn d-NH), 5.18 (1H, m, CH]CH2),
5.05 (2H, s, CH2Ph), 4.54 (2H, dd, JZ5.5, 13.0 Hz,
CH2CH]CH2), 4.29 (1H, m, Pro a-H), 4.12 (1H, d, JZ
16.1 Hz, CH2COOtBu), 4.04 (1H, d, JZ16.1 Hz, CH2-
COOtBu), 3.82 (1H, ABX type dd, JZ5.6, 9.8 Hz,
CH2OSi), 3.73 (1H, dd, JZ2.8, 9.8 Hz, CH2OSi), 3.55
(2H, m, Pro d-H and CHN3CHO), 3.45 (2H, m, Pro d-H and
CHN3), 3.25 (1H, m, Orn a-H), 3.12 (2H, m, Orn d-H), 3.02
(1H, ABX type dd, JZ4.2, 13.7 Hz, Tyr b-H), 2.77 (1H,
ABX type dd, JZ9.3, 14.0 Hz, Tyr b-H), 2.69 (1H, m,
CH2NH), 2.51 (1H, m, CH2NH), 2.08 (2H, m, Pro b-H and
Pro g-H), 1.91 (2H, m, Pro b-H and Pro g-H), 1.81 (2H, m,
CH2CHO), 1.59 (3H, m, Orn b-H and Orn g-H), 1.49 (9H, s,
COOtBu), 1.46 (1H, m, Orn b-H), 1.05 (9H, s, OSiPh2tBu);
13C NMR (100 MHz, CDCl3) d 173.2, 169.3, 157.6, 156.2,
133.4, 133.0, 133.4, 133.3, 133.0, 130.2, 129.7, 128.5,
127.9, 127.8, 127.7, 127.6, 127.4, 117.4, 114.9, 81.6, 80.2,
70.0, 68.4, 65.3, 65.2, 63.3, 59.6, 58.3, 47.2, 44.6, 40.7,
35.4, 31.0, 28.1, 27.9, 26.8, 26.6, 24.4, 21.1 19.2; IR (neat)
d 173.8, 169.3, 168.6, 157.7, 156.2, 156.1, 143.8, 143.7,
141.3, 137.0, 135.7, 135.5, 135.5, 133.4, 133.0, 130.3,
130.1, 129.7, 129.7, 128.5, 127.9, 127.7, 127.5, 127.0,
125.2, 125.1, 120.0, 119.9, 117.4, 115.0, 81.6, 79.3, 70.0,
67.7, 67.0, 65.3, 64.7, 63.2, 58.7, 54.6, 49.9, 47.3, 47.1,
42.3, 40.5, 40.4, 34.9, 31.7, 28.1, 27.2, 27.0, 26.9, 26.2,
24.6, 24.4, 23.5, 21.3, 19.3; IR (neat) 3301, 2108, 1720,
1635, 1511 cmK1; ESMS calcd for C75H94N7O11Si 1297.6
(MCHC), found 1297.4.
4.1.13. Compound 19. Compound 18 (0.240 mg,
0.185 mmol) was stirred in formic acid (6.0 mL) for 19 h,
after which the solution was poured into H2O (15 mL) and
extracted with EtOAc (5!5 mL). The combined organic
layers were washed with brine, dried with Na2SO4, filtered
and concentrated. Flash column chromatography (toluene/
ethanol, 30:1/10:1) of the residue gave 19 as a white
amorphous solid (124 mg, 65%): [a]2D0ZK53.5 (c 0.2,
1
CHCl3); H NMR (400 MHz, CDCl3, rotamer ratio 4:1) d
(major rotamer) 8.05 (1H, s, HCOO), 7.75 (2H, d, JZ
7.5 Hz, Ph), 7.56 (2H, d, JZ7.5 Hz, Ph), 7.28–7.43 (9H, m,
Ph), 7.15 (2H, d, JZ8.2 Hz, PhTyr), 6.91 (2H, d, JZ8.2 Hz,
PhTyr), 5.88 (1H, m, CH]CH2), 5.54 (1H, d, JZ9.1 Hz,
Leu-NH), 5.12–5.29 (3H, m, CH]CH2 and Orn a-H), 5.01
(2H, s, CH2Ph), 5.00 (1H, m, Orn d-NH), 4.68 (1H, m, Leu
a-H), 4.47–4.58 (2H, m, CH2CH]CH2), 4.16–4.43 (8H, m,
CH2COOH, Fmoc-CHCH2, Pro a-H, and CH2OCHO), 3.80
(1H, m, CH2N), 3.45–3.60 (5H, m, CHO, CHN3, Pro d-H
and CH2N), 3.17 (2H, m, Orn d-H), 3.01 (1H, ABX type dd,
JZ3.2, 13.4 Hz, Tyr b-H), 2.74 (1H, ABX type dd, JZ9.6,
13.4 Hz, Tyr b-H), 1.96–2.06 (3H, m, Pro b-H, Pro g-H and
CH2CHO), 1.82–1.92 (4H, m, Pro b-H, Pro g-H, CH2CHO
and Orn b-H), 1.61–1.76 (3H, m, Orn b-H, Leu b-H), 1.41–
1.51 (2H, m, Orn g-H), 1.33 (1H, m, Leu g-H), 1.01 (6H, d,
JZ5.3 Hz, CH(CH3)2); 13C NMR (100 MHz, CDCl3) d
173.9, 171.1, 169.4, 160.8, 157.8, 156.2, 143.6, 141.2,
136.9, 132.8, 130.2, 129.3, 128.5, 127.9, 127.7, 127.5,
127.0, 125.1, 125.0, 120.0, 119.9, 117.6, 115.1, 79.6, 69.4,
67.9, 67.1, 65.5, 64.9, 62.9, 56.0, 54.1, 49.6, 47.2, 47.0,
42.4, 40.5, 40.2, 35.1, 32.2, 27.3, 26.9, 26.1, 24.6, 24.2,
3325, 3070, 2933, 2860, 2110, 1747, 1722, 1635, 1511 cmK1
;
HRMS (FAB) calcd for C54H73N6O8Si 961.5279 (MCHC),
found 961.5258; Anal. Calcd C, 67.5; H, 7.6; N, 8.7. Found
C, 67.4; H, 7.6; N, 8.7.
4.1.12. Compound 18. Fmoc-Leu-OH (5, 0.192 g,
0.543 mmol) was activated with diisopropylethylamine
(DIEA, 0.19 mL, 1.09 mmol) and HATU (0.188 g,
0.494 mmol) at 0 8C for 30 min in DMF (1.0 mL). Then a
solution of 17 (0.475 g, 0.494 mmol) in DMF (2.0 mL) was
added. After stirring for 24 h the solution was diluted with
CH2Cl2 (30 mL) and then poured into H2O (150 mL) and
the aqueous phase was extracted with CH2Cl2 (5!10 mL).
The combined organic layers were washed with 0.2 M aq
HCl, H2O, satd aq NaHCO3 and brine. The organic layer
was dried with Na2SO4, filtered and concentrated. Flash
column chromatography (toluene/ethanol, 30:1/15:1 and
heptane/ethyl acetate 10:1/2:1) of the residue gave 18 as a
white amorphous solid (333 mg, 52, 70% yield based on the
recovered 17): [a]D20ZK54.2 (c 0.3, CHCl3); 1H NMR
(400 MHz, CDCl3, rotamer ratio 4:1) d (major rotamer) 7.75
(2H, d, JZ7.3 Hz, Ph), 7.60 (6H, m, Ph), 7.29–7.44 (15H,
m, Ph), 7.18 (2H, d, JZ8.1 Hz, PhTyr), 6.90 (2H, d, JZ
8.1 Hz, PhTyr), 5.85 (1H, m, CH]CH2), 5.33 (1H, dZ
9.0 Hz, Leu a-NH), 5.19–5.26 (2H, m, CH]CH2 and Orn
a-H), 5.13 (1H, d, JZ10.0 Hz, CH]CH2), 5.02 (2H, s,
CH2Ph), 4.90 (1H, m, Orn d-NH), 4.67 (1H, m, Leu a-H),
4.48 (2H, m, CH2CH]CH2), 4.31–4.43 (2H, m, Fmoc-
CHCH2), 4.21 (2H, m, Fmoc-CHCH2 and Pro a-H), 4.11
(1H, d, JZ16.3 Hz, CH2COOtBu), 4.02 (1H, d, JZ16.3 Hz,
CH2COOtBu), 3.87 (1H, ABX type dd, JZ5.0, 10.0 Hz,
CH2OSi), 3.72 (1H, ABX type dd, JZ2.8, 10.1 Hz,
CH2OSi), 3.65 (1H, m, CH2N), 3.56 (1H, m, CHN3), 3.49
(2H, m, Pro d-H), 3.43 (2H, m, CH2N and CHO), 3.07–3.17
(3H, m, Orn d-H and Tyr b-H), 2.81 (1H, ABX type dd, JZ
9.6, 14.0 Hz, Tyr b-H), 1.85–2.10 (6H, m, Pro b-H, Pro g-H,
CH2CHO and Orn b-H), 1.58–1.82 (4H, m, Pro g-H, Orn
b-H, Leu b-H, Leu g-H), 1.45 (9H, s, COOtBu), 1.28–1.36
(3H, m, Orn g-H and Leu b-H), 1.05 (9H, s, OSitBu), 0.98
(6H, d, JZ6.3 Hz, Leu g-H); 13C NMR (100 MHz, CDCl3)
23.4, 21.6; IR (neat) 3309, 2107, 1720, 1635, 1511 cmK1
;
HRMS (FAB) calcd for C56H67N7O12Na 1052.4698 (MC
NaC), found 1052.4734; Anal. Calcd C, 65.2; H, 6.6; N, 9.5.
Found C, 64.8; H, 6.6; N, 9.4.
4.1.14. Compound 21. Compound 19 (120 mg, 0.116 mmol)
was activated by treatment with DIC (18.0 mL, 0.116 mmol)
in ethyl acetate (0.8 mL) at 0 8C for 10 min, after which
pentafluorophenol (32.2 mg, 0.175 mmol) was added. After
stirring for 1 h the solvent was evaporated and the residue
was purified by flash column chromatography (heptane/
ethyl acetate, 5:1/1:1) to give the pentafluorophenyl ester
corresponding to 19 as a white amorphous solid (115 mg).
The pentafluorophenyl ester (115 mg) in dioxane (25 mL)
was then added dropwise to a solution of DBU (43.9 mg,
0.288 mmol) in refluxing dioxane (25 mL) during 3 h. After
adding all of the ester, the solution was allowed to reflux for
a further 30 min and was then concentrated. Flash column
chromatography (heptane/ethyl acetate, 2:1 then toluene/
ethanol 25:1/5:1) of the residue gave 20 (39 mg) and 21
(5 mg) as white solids, as well as a mixture of 20 and 21
(7 mg). Then aq LiOH solution (1 M, 67.0 mL, 67.0 mmol)
was added to a solution of 20 (39 mg) and the mixture of 20

4908
Z. Q. Yuan, J. Kihlberg / Tetrahedron 61 (2005) 4901–4909
and 21 (7 mg) in THF, MeOH and H2O (3:1:1, 2.0 mL) at
0 8C. After being stirred for 30 min the cooling bath was
removed and the reaction was stirred for a further 3 h. The
solution was acidified with aq HCl (1 M) (5 drops), then
poured into EtOAc and H2O (4:1, 10 mL). The phases were
separated and the aqueous phase was extracted with EtOAc
(3!4 mL). The combined organic phases were then washed
with brine, dried over Na2SO4, filtered and concentrated.
Flash column chromatography (toluene/EtOH, 25:1/10:1)
of the residue gave 21 as a white amorphous solid (40 mg in
total, 51% from 19): [a]2D0ZK69.7 (c 0.3, CHCl3); 1H NMR
(400 MHz, CDCl3, rotamer ratio 4:1) d (major rotamer)
7.33–7.45 (5H, m, Ph), 7.14 (2H, d, JZ8.3 Hz, PhTyr), 6.95
(2H, d, JZ8.4 Hz, PhTyr), 6.50 (1H, d, JZ8.4 Hz, Leu NH),
5.90 (1H, m, CH]CH2), 5.15–5.35 (3H, m, Orn a-H and
CH]CH2), 5.06 (2H, s, CH2Ph), 4.93 (1H, m, Orn d-NH),
4.71 (1H, m, Leu a-H), 4.54 (2H, m, CH2CH]CH2), 4.41
(1H, d, JZ15.3 Hz, OCH2CON), 3.91 (1H, m, Pro a-H),
3.78 (1H, m, bridge CH2N), 3.70 (1H, m, CH2OH), 3.55
(1H, m, CH2OH and Pro d-H), 3.53 (1H, d, JZ15.4 Hz,
OCH2CON), 3.43 (3H, m, Pro d-H, bridge CH2N and
CHN3), 3.35 (1H, m, CHO), 3.21 (2H, m, Orn d-H), 3.01
(1H, ABX type dd, JZ7.1, 13.8 Hz, Tyr b-H), 2.93 (1H,
ABX type dd, JZ7.5, 13.9 Hz, Tyr b-H), 1.90–2.02 (4H, m,
bridge CH2, Orn b-H, Pro b-H and OH), 1.66–1.85 (5H, m,
Pro g-H, Leu g-H and Leu b-H), 1.52–1.63 (3H, m, Orn
b-H, bridge CH2 and Pro b-H), 1.45 (2H, m, Orn g-H), 1.03
(3H, t, JZ5.7 Hz, Leu d-CH3), 0.98 (3H, t, JZ5.6 Hz, Leu
d-CH3); 13C NMR (100 MHz, CDCl3) d 171.8, 171.2, 170.8,
158.0, 156.3, 136.8, 132.9, 130.3, 128.6, 128.6, 128.0,
127.5, 117.6, 115.2, 80.8, 70.2, 70.1, 66.5, 65.5, 65.0, 62.2,
54.6, 54.3, 47.9, 41.0, 40.5, 38.0, 36.2, 31.8, 28.3, 25.9,
25.9, 25.0, 24.2, 23.0, 21.2; IR (neat) 3334, 2106, 1701,
1679, 1624, 1512 cmK1; HRMS (FAB) calcd for
C40H55N7O8Na 784.3998 (MCNaC), found 784.3990.
g-H), 1.06 (3H, t, JZ6.5 Hz, Leu d-CH3), 1.00 (3H, t, JZ
6.5 Hz, Leu d-CH3); 13C NMR (100 MHz, CD3OD) d 173.9,
172.8, 169.4, 157.9, 157.4, 137.3, 133.1, 130.4, 130.2,
130.1, 129.7, 129.6, 128.2, 127.6, 127.3, 127.3, 116.4,
115.0, 114.9, 114.7, 82.3, 71.3, 70.9, 66.9, 66.2, 62.5, 56.0,
55.5, 48.4, 41.3, 41.2, 39.1, 37.1, 31.7, 30.5, 26.6, 26.5,
25.8, 25.5, 23.3, 21.3; IR (neat) 3325, 2106, 1714, 1624,
1512 cmK1; HRMS (FAB) calcd for C40H54N7O9 776.3979
(MCHC), found 776.3999.
Acknowledgements
This work was funded by grants from the Swedish Research
˚
Council, the Biotechnology Fund at Umea University and
the Goran Gustafsson Foundation for Research in Natural
¨
Sciences and Medicine.
References and notes
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