An improved solid-phase methodology for the synthesis of putative hexa- and heptapeptide intermediates in vancomycin biosynthesis

Article abstract of DOI:10.1039/b418908f

The biosynthesis of the vancomycin aglycone involves three oxidative phenol coupling reactions, each catalyzed by a discrete cytochrome P450-like enzyme. Studies on the mechanism and specificity of the enzyme (called OxyB) catalyzing the first coupling, require access to suitable linear peptide precursors, each conjugated as a thioester to a peptide carrier domain of the vancomycin non-ribosomal peptide synthetase. An efficient route to representative free linear peptides is described here. The method makes use of Alloc-chemistry during solid-phase assembly of the peptide backbone, but importantly and in contrast to earlier efforts, largely avoids the use of amino acid side chain protecting groups. In this way, the target linear peptides can be released directly from the solid support under very mild conditions. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b418908f

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
An improved solid-phase methodology for the synthesis of putative
hexa- and heptapeptide intermediates in vancomycin biosynthesis†
Dong Bo Li and John A. Robinson*
Organic Chemistry Institute, University of Zurich, Winterthurerstrasse 190, 8057-Zurich,
Switzerland. E-mail: robinson@oci.unizh.ch; Fax: (+41) 1-635-6833
Received 17th December 2004, Accepted 27th January 2005
First published as an Advance Article on the web 23rd February 2005
The biosynthesis of the vancomycin aglycone involves three oxidative phenol coupling reactions, each catalyzed by a
discrete cytochrome P450-like enzyme. Studies on the mechanism and specificity of the enzyme (called OxyB)
catalyzing the first coupling, require access to suitable linear peptide precursors, each conjugated as a thioester to a
peptide carrier domain of the vancomycin non-ribosomal peptide synthetase. An efficient route to representative free
linear peptides is described here. The method makes use of Alloc-chemistry during solid-phase assembly of the
peptide backbone, but importantly and in contrast to earlier efforts, largely avoids the use of amino acid side chain
protecting groups. In this way, the target linear peptides can be released directly from the solid support under very
mild conditions.
No such obstacle exists with the P450 proteins implicated in
Introduction
OPCRs during glycopeptide antibiotic biosynthesis, where the
The biosynthesis of vancomycin and related glycopeptide an-
relevant genes have now been cloned and sequenced from several
tibiotics is presently attracting attention.1,2 Amongst the many
glycopeptide producers,9–17 and two of these proteins (OxyB and
interesting enzymic transformations involved in the assembly
OxyC from the vancomycin producer) have been crystallized
of these complex natural products (Fig. 1) are several oxidative
and their 3D structures determined.17,18
phenol coupling reactions (OPCRs). These reactions lead to
Functions have been assigned by gene inactivation experi-
cross-linking of aromatic amino acid side chains, which together
ments to the three P450 proteins catalyzing OPCRs during
constrain the antibiotic aglycone into a conformation that
balhimycin biosynthesis (balhimycin shares the same aglycone
is ideal for binding to its biological target, an N-acyl-D-
with vancomycin).19–21 The first OPCR, catalyzed by OxyB (the
Ala-D-Ala intermediate of peptidoglycan assembly in Gram-
Oxy lettering corresponds to the order of the three contiguous
genes in the gene cluster, viz. oxyA-oxyB-oxyC), occurs between
positive bacteria.3,4 The importance of OPCRs in natural
product biosynthesis, for example in alkaloid biosynthesis, is
rings C and D, the second catalyzed by OxyA occurs between
well appreciated. Recently, it has become clear that a family of
rings D and E, and the final coupling catalyzed by OxyC takes
cytochrome P450 proteins have evolved in various organisms
place between rings A and B (Fig. 1). Recently, it was shown
to catalyze specific OPCRs. However, the P450s of plant origin
that OxyB, cloned from the vancomycin producer, catalyzes the
studied so far, for example those acting in the biosynthesis of
conversion of 1 to 2 shown in Fig. 2. The linear hexapeptide
1, must be attached through its C-terminus to a holo-peptide
carrier domain (PCD) derived from the vancomycin non-
benzylisoquinoline alkaloids, are associated with microsomes,5–8
which has no doubt hindered structural and mechanistic studies.
ribosomal peptide synthetase (NRPS) in order to function as
a substrate for OxyB.22 The corresponding peptide with a free
C-terminus was not turned-over by OxyB. It is currently not
clear, however, whether the enzyme can also catalyze an OPCR,
and perhaps more efficiently, on a heptapeptide–PCD conjugate,
such as 3a or 3b (Fig. 3). The influence on the rate of the OxyB
Fig. 1 Structure of vancomycin and related glycopeptides.
† Electronic supplementary information (ESI) available: experimen-
tal details of the synthesis of the amino acid building blocks. See
http://www.rsc.org/suppdata/ob/b4/b418908f/
Fig. 2 OxyB transforms peptide 1 linked to a peptide carrier domain
(PCD) into 2.22
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9
1 2 3 3
©

View Article Online
Fig. 3 The preferred biosynthetic route to vancomycin aglycone is presently not clear (i.e. the steps indicated by dotted arrows).1,2 The first OPCR
catalyzed by OxyB may occur preferentially on 3a/b and/or 4a/b.
reaction of the b-hydroxy groups and m-chlorine substituents in
the m-chloro-3-hydroxytyrosine residues (compare 4a/4b and
3a/b with 1) also remain to be defined.
More detailed studies of the substrate specificity of OxyB, as
well as of its mechanism of action, now hinge upon the avail-
ability of suitable linear peptide–PCD conjugates. A method
has been established for linking peptides N-methylated at the
N-terminus to PCDs (e.g. 1).22,23 As long as the N-terminus is N-
methylated, the peptide C-terminus can be efficiently activated
to a thioester for coupling to the PCD; the N-methylamino N-
terminus does not react rapidly with the thioester under the
conditions used. More challenging, however, is the synthesis
of the required linear peptides, because they contain amino
acids, which due to their ease of epimerization and sensitivity to
acids and bases, are incompatible with the two current standard
methods of solid-phase peptide assembly (Boc- and Fmoc-
chemistry). For this reason, we introduced earlier a method for
solid-phase peptide assembly of such peptides, under neutral
conditions, based on (allyloxy)carbonyl (Alloc) protection of
the a-amino groups. Benzyl ether protection was still used
for the phenolic groups, and methyltrityl (Mtt) for the side
chain of Asn.24,25 Although this synthetic method provided
small amounts of heptapeptides 5 and 6 (Fig. 4), it was
Fig. 4 Peptides 5–11.
problematic, because a final side chain deprotection with strong
acid is still required. This deprotection step can lead to rapid
groups that require removal with acid. This required the
degradation, poor reproducibility, and a difficult and inefficient
optimization of methods for activating and coupling amino acids
HPLC purification of the end product. Also, the required
to the growing chain on the resin, whilst avoiding acylation of the
protected amino acids are not commercially available and
phenolic groups and dehydration of the asparagine side chain.
demand considerable effort to produce in multi-gram amounts.
For the synthesis of hexapeptides 9 and 10, the building
blocks 12, 13, 15, 18 and 19 were prepared (Scheme 1). Thus,
In this work, we describe a new, more efficient, epimerization-
free approach to the synthesis of a representative collection of
D-4-hydroxyphenylglycine was converted into 12 in high yield
hexa- and heptapeptides (7–11), which should facilitate studies
and without detectable racemization upon reaction with Alloc-
of these interesting glycopeptide OPCRs. The key improvements
O-(N-hydroxysuccinimide) (Alloc-OSu). This reaction did not
proceed smoothly using Alloc-Cl. Alloc-OSu was also used
to produce the crystalline pentafluorophenyl ester (OPfp) 13
from L-asparagine, using DCC for activation. D-Tyrosine was
converted in four steps via 14 into the protected and activated
derivative 15. Finally, both N-Alloc-D-Leu-OPfp (18) and N-
Alloc-N-methyl-D-Leu-OPfp (19) were prepared from the cor-
responding commercially available Boc-protected amino acids
are methods that largely eliminate the need for amino acid side
chain protecting groups during peptide assembly. This in turn
allows cleavage of the desired end product directly from the
solid support under very mild conditions. No further functional
group manipulations are then needed. Relevant methods for
the stereoselective synthesis of suitably protected amino acid
building blocks are also reviewed.
(Scheme 1).
Results and discussion
The assembly of 9 and 10 (Scheme 2) was performed on
In planning the syntheses of peptides 7–11, key considerations
were the use of the Alloc group as a temporary N-a-amino
protecting group, and the avoidance of side chain protecting
2-chlorotrityl chloride resin, following loading of Fmoc-Tyr-
OH and treatment with piperidine to remove the Fmoc-group.
The first coupling between 20 and 12 was performed with
1 2 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9

View Article Online
that PhSiH3 could not efficiently remove the N-Alloc group
in the so formed tetrapeptide. Further chain elongation to
pentapeptide 24 was achieved smoothly by coupling 15 and
subsequent Pd(PPh3)4–Bu3SnH treatment to remove the two
Alloc groups. Finally, the last coupling of 18 or 19 onto resin
bound pentapeptide 24 followed again by Alloc removal, gave
25 and 25a, respectively. The final products (9 and 10) could
then be released from the resin with 0.6% TFA in CH2Cl2, and
purified by HPLC in up to 52% overall yield. A full assignment
of the 1H NMR spectra of 9 and 10 was possible from an
analysis of 2D NMR (TOCSY, COSY and ROESY) spectra.
Typical HPLC chromatograms of crude 10 from the resin, and
after HPLC purification, are shown in Fig. 5. This method
has afforded 100 mg quantities of 10, which were sufficient to
establish methods for coupling 10 to a PCD (Fig. 2) and for
assays of the conjugate with OxyB, as reported elsewhere.22
Our next target was the assembly of hexapeptide 11, which
contains (2S,3R)-m-chloro-b-hydroxytyrosine (Cht) (Fig. 4),
using the same protocol developed from the synthesis of 9
and 10. The required building block 29 was prepared following
the procedure of Evans and Weber (Scheme 3).26 Following
the Sn(OTf)2-mediated aldol reaction between isothiocyanate
26 and aldehyde 27, the aldol adduct was treated with magne-
sium methoxide in MeOH to afford the corresponding methyl
ester 28. This was subjected to Alloc-protection and further
transformation into 29. The final hydrolysis step was marred by
an accompanying oxazolidinone (29a) formation, which could
nevertheless be recycled to 29, but lowered the overall yield.
Alloc-Cht(Allyl)-OH (29) was then loaded onto 2-chlorotrityl
chloride resin and the Alloc- and allyl-groups were removed
to afford 20a, which was ready for assembly of hexapeptide
11 (Scheme 2). HPLC-MS analysis of resin at each stage of
the assembly revealed no evidence of epimerization or major
side reactions. After cleaving 25b from the resin, analytical
Scheme 1 Reagents. a) Alloc-OSu, NaHCO3, acetone–water (1 : 1), rt;
b) DCC, Pfp-OH, dioxane, 0 ◦C–rt; c) AcCl, MeOH, reflux, 99%; d)
Alloc-Cl, NaHCO3, dioxane–water (1 : 1), rt; e) LiOH, THF–water (1 :
1), 0 ◦C; f) TFA–CH2Cl2 (1 : 1) containing 5% TIS.
DIC–HOBt activation in DMF, and subsequent Alloc-removal
was with Pd(PPh3)4–PhSiH3 for 3 h. HPLC-MS analysis of
material cleaved (0.6% TFA–CH2Cl2) from a small portion of
the resin showed clean formation of the desired resin-bound
dipeptide 21. Elaboration to the tripeptide 22 in the same
way also proceeded cleanly (>95%). Further extension to the
tetrapeptide 23 was performed with 13 and subsequent Alloc
removal with Pd(PPh3)4–Bu3SnH. It was necessary to change
the allyl scavenger, since HPLC-MS analysis at this stage showed
Scheme 2 Reagents. a) 12 (4 eq.), DIC (4 eq.) and HOBt (8 eq.), DMF, overnight; b) Ph(PPh3)4 (1 eq.), PhSiH3 (60 eq.), CH2Cl2, 3 h; c) 13 (5 eq.),
HOBt (10 eq.), DMF, overnight; d) Ph(PPh3)4 (1 eq.), Bu3SnH (60 eq.), CH2Cl2, 3 h; e) 15 (5 eq.), HOBt (10 eq.), DMF, overnight; f) Ph(PPh3)4
(2 eq.), Bu3SnH (120 eq.), CH2Cl2, 3 h; g) for 25, 18 (5 eq.), HOBt (10 eq.), DMF, overnight; for 25a and 25b, 19 (5 eq.), HOBt (10 eq.), DMF,
overnight; h) 0.6% TFA in CH2Cl2.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9
1 2 3 5

View Article Online
2-chlorotrityl chloride resin and then treated with Pd(PPh3)4–
PhSiH3 to afford resin 32. (S)-Alloc-Tyr-OH 34 was conveniently
prepared by a three-step sequence starting from L-Tyr-OH
(Scheme 4).
Scheme
4 Reagents. a) LiOH, THF–H2O (3 : 1), 0
◦C; b)
TFA–thioanisole (3 : 1), rt, 81% over two steps; c) 2-chlorotrityl chloride
resin, NMM, DMF–CH2Cl2 (10 : 1), overnight; d) Ph(PPh3)4, PhSiH3,
CH2Cl2, 3 h; e) AcCl, MeOH, reflux, 97%; f) Alloc-OSu, NaHCO3,
acetone–water (1 : 1), rt, 100%; g) LiOH, THF–water (1 : 1), 0 ◦C, 87%.
The solid-phase assembly of heptapeptide 7, illustrated in
Scheme 5, was straightforward until hexapeptide 35. However,
after the final coupling of OPfp ester 19 and subsequent
Alloc-deprotection and cleavage from the resin, one main
byproduct was observed by HPLC/HPLC-MS, in addition to
the desired heptapeptide. MS and NMR analysis indicated that
the byproduct lacked the N-methylleucine moiety, although the
heptapeptide appeared to be stereochemically homogeneous.
This indicated that no epimerization(s) had occurred during
the assembly, but that a significant byproduct was formed due
to inefficient coupling in the final step. In order to overcome
this problem, we explored the use of a dipeptide building block
in the last phase of the assembly. Thus, Alloc-N(Me)-D-Leu-D-
Tyr(OAllyl)-OPfp 41 was prepared (Scheme 6) and used in the
assembly of heptapeptide 8. For the synthesis of 41, the known27
tyrosine derivative 39 was treated with TFA and then coupled to
Alloc-N(Me)-D-(Leu)-OH. Subsequent hydrolysis and coupling
to Pfp-OH provided 41, which was used directly for peptide
assembly without further purification. Starting from Dhpg-resin
32, pentapeptide 37 (Scheme 5) was assembled using the same
protocol as for 35. Thereafter, coupling of the dipeptide OPfp
ester 41 afforded resin-bound heptapeptide 38 as a single main
component. HPLC-MS analysis revealed no epimerization at
this dipeptide coupling step. Finally, after cleavage from the
resin and HPLC purification, heptapeptide 8 was obtained in
Fig. 5 HPLC chromatograms showing: A, the crude product (gradient
from 5–40% MeCN–H2O + 0.15% TFA over 15 min) from the
synthesis of 10; and B, after HPLC purification (gradient from 5–35%
MeCN–H2O + 0.15% TFA over 15 min).
1
11% overall yield and was characterized by MS and H 1D
and 2D NMR. The use of 41 as a viable building block for
heptapeptide assembly was thus established.
Scheme 3 Reagents. a) Sn(OTf)2, N-ethylpiperidine, THF, −78 ◦C,
81%; b) MeMgBr, MeOH, 0 ◦C, 87%; c) Alloc-Cl, Et3N, DMAP,
CH2Cl2, rt; d) HCO2H–35% H2O2, 0 ◦C, 76% over two steps; e) LiOH,
dioxane–water (10 : 1), rt (29, 19%; 29a, 58%).
In summary, a judicious choice of protecting groups and
coupling strategies has provided efficient access to a range of
hexa- and heptapeptides that are of interest in ongoing studies
of vancomycin biosynthesis. The coupling conditions were
optimized to allow an essentially epimerization-free assembly
of these peptides on a solid support. Also, the avoidance as far
as possible of side-chain protecting groups allowed the cleavage
of the end products from the resin under very mild conditions.
The methodology presented here should be readily applicable to
the synthesis of other related peptides, as potential precursors
of PCD-conjugates to explore the specificity of the OPCRs
catalyzed by OxyB.
HPLC-MS of the crude product showed only one main peak
with the correct mass. The overall yield of hexapeptide 11 after
HPLC purification was ca. 13% and its structure was confirmed
by MS and NMR spectroscopy. These results demonstrate the
compatibility of a free unprotected b-hydroxy group in residue-
6 with the method of peptide assembly and cleavage from the
resin.
As a next step, we show that this method is also amenable
to the efficient synthesis of heptapeptides 7 and 8. The
building blocks 31 and 34 required for heptapeptide 7 were
synthesized as shown in Scheme 4. Preparation of the (S)-
3,5-dihydroxyphenylglycine derivative 31 proceeded via oxazo-
lidinone 30, reported in earlier work.24 After removal of the
chiral auxiliary and deprotection with TFA–thioanisole, Alloc-
Dhpg-OH 31 was obtained in 81% yield. This was loaded onto
Experimental
For solid-phase synthesis, DMF was redistilled under reduced
pressure from ninhydrin, CH2Cl2 was redistilled under N2
1
from CaH2 and HPLC-grade MeOH was used. For H NMR
1 2 3 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9

View Article Online
Scheme 5 Reagents. a) for coupling 34 and 12 (each 4 eq.), DIC–HOBt (4 and 8 eq.), DMF, overnight; for deprotection Ph(PPh3)4 (1 eq.) and PhSiH3
(60 eq.), CH2Cl2, 3 h; b) for coupling 13 and 15 (each 5 eq.), HOBt (10 eq.), DMF, overnight; for deprotection of 13, Ph(PPh3)4 (1 eq.), Bu3SnH (60
eq.), CH2Cl2, 3 h; of 15, Ph(PPh3)4 (2 eq.), Bu3SnH (120 eq.), CH2Cl2, 3 h; c) 19 (5 eq.), HOBt (10 eq.), DMF, overnight; d) Ph(PPh3)4(1 eq.), Bu3SnH
(60 eq.), CH2Cl2, 3 h; e) 0.6% TFA in CH2Cl2; f) for coupling: 29 and 12 (each 4 eq.), DIC–HOBt (4 and 8 eq.), DMF, overnight; for deprotection of
29, Ph(PPh3)4 (2 eq.), PhSiH3 (120 eq.), CH2Cl2, 3 h; of 12, Ph(PPh3)4 (1 eq.) and PhSiH3 (60 eq.), CH2Cl2, 3 h; g) for coupling 13 (5 eq.), HOBt (10
eq.), DMF, overnight; for deprotection Ph(PPh3)4 (1 eq.), Bu3SnH (60 eq.), CH2Cl2, 3 h; h) 41 (5 eq.), HOBt (10 eq.), DMF, overnight; i) Ph(PPh3)4
(2 eq.), Bu3SnH (120 eq.), CH2Cl2, 3 h.
Table 3 1H NMR assignment of 11 (d6 DMSO, 300 K, 500 MHz)
Residue
OH
—
NH
—
a
b
Others
N(Me)Leu1
3.56 1.50
1.39 (c), 1.98 (NMe),
0.85/0.80(d)
D-Tyr2
Asn3
Hpg4
Hpg5
Cht6
9.17 8.79 4.75 2.97, 2.58 7.05 (2,6), 6.63(3,5)
8.43 4.68 2.47, 2.33 7.30 (NH), 6.92(NH)
—
Scheme 6 Reagents. a) TFA–thioanisole (3 : 1), rt; b) 17, EDC–HOBt,
DIEA, 0 ◦C–rt, DMF; c) LiOH, THF–H2O (1 : 1), 80% yield over three
steps; d) DCC, Pfp-OH, dioxane, 0 ◦C–rt.
9.32 8.04 5.53
9.28 8.69 5.58
9.95 8.14 4.33 4.97
—
—
7.17 (2,6), 6.67(3,5)
6.98 (2,6), 6.61(3,5)
7.25(2), 6.87 (6),
6.74(5)
assignments of the hexa- and heptapeptides, see Tables 1–5. A
description of the synthesis of each of the amino acid building
blocks is given in the electronic supplementary information†.
Table 4 1H NMR assignment of 7 (d6 DMSO, 300 K, 600 MHz)
Residue
OH NH
a
b
Others
Table 1 1H NMR assignment of 9 (d6 DMSO, 300 K, 600 MHz)
N(Me)Leu1
—
—
3.60 1.52
1.41 (c), 1.97 (NMe),
0.85/0.80 (d)
D-Tyr2
Asn3
9.17 8.81 4.77 2.97, 2.59
8.45 4.68 2.43, 2.33
7.05 (2,6), 6.64 (3,5)
7.30 (NH), 6.93 (NH)
7.19 (2,6), 6.68 (3,5)
6.98 (2,6), 6.62 (3,5)
6.78 (2,6), 6.49 (3,5)
6.28 (2,6), 6.18 (4)
Residue OH NH
a
b
Others
—
Leu1
—
—
3.71 1.55
1.55 (c), 0.86/0.85(d)
Hpg4
9.33 8.05 5.52
9.31 8.69 5.58
9.08 8.08 4.53 2.81, 2.59
9.33 8.69 5.06
—
—
D-Tyr2
Asn3
9.18 8.59 4.53 2.89, 2.66 7.04 (2,6), 6.64 (3,5)
8.33 4.64 2.41, 2.28 7.30 (NH), 6.92(NH)
Hpg5
L-Tyr6
Dhpg7
—
Hpg4
Hpg5
L-Tyr6
9.34 8.01 5.52
9.37 8.75 5.42
9.15 8.25 4.25 2.78, 2.66 6.71 (2,6), 6.51 (3,5)
—
—
7.18 (2,6), 6.67 (3,5)
7.08 (2,6), 6.65 (3,5)
—
L-Tyr-resin (20)
Fmoc-Tyr-OH (564 mg, 1.4 mmol) and NMM (400 lL,
3.6 mmol) in CH2Cl2 and DMF (17 ml, 10 : 1) were added
to 2-chlorotrityl chloride resin (817 mg, 1.4 mmol g−1) and the
mixture was agitated overnight. MeOH (5 mL) was added and
the mixture was agitated for 10 min. The resin was filtered and
washed with DMF (4 × 25 mL), MeOH (4 × 25 mL), CH2Cl2
(4 × 25 mL) and DMF (4 × 25 mL). The resin was treated with
a 20% solution of piperidine in DMF (15 mL) with agitation
for 2 h. The resultant resin was filtered and washed with DMF
(4 × 25 mL), CH2Cl2 (4 × 25 mL) and DMF (4 × 25 mL). The
loading of L-Tyr was ca. 0.25 mmol g−1.
Table 2 1H NMR assignment of 10 (d6 DMSO, 300 K, 600 MHz)
Residue
OH
—
NH
—
a
b
Others
N(Me)Leu1
3.59 1.50
1.40 (c), 1.96 (NMe),
0.84/0.79(d)
7.04 (2,6), 6.63 (3,5)
7.30 (NH), 6.92(NH)
7.18 (2,6), 6.67 (3,5)
7.08 (2,6), 6.65 (3,5)
6.72 (2,6), 6.51 (3,5)
D-Tyr2
Asn3
9.17 8.81 4.77 2.97, 2.61
8.45 4.68 2.48, 2.33
—
Hpg4
Hpg5
L-Tyr6
9.33 8.07 5.53
9.35 8.75 5.43
—
—
9.15 8.26 4.26 2.78, 2.61
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9
1 2 3 7

View Article Online
Table 5 1H NMR assignment of 8 (d6 DMSO, 300 K, 600 MHz)
and CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in the dark,
a solution of Pd(PPh3)4 (2 eq.) and Bu3SnH (120 eq.) in CH2Cl2
(15 mL) was added to the resin and the mixture was agitated for
3 h. The resultant resin 24 was filtered, washed with DMF (4 ×
25 mL) and CH2Cl2 (4 × 25 mL), and divided into two equal
parts.
Residue
OH NH
a
b
Others
N(Me)Leu1
—
—
3.58 1.50
1.40 (c), 1.96 (NMe),
0.84/0.78 (d)
D-Tyr2
Asn3
Hpg4
Hpg5
Cht6
9.17 8.80 4.75 2.96, 2.55 7.04 (2,6), 6.62 (3,5)
8.43 4.65 2.45, 2.31 7.29 (NH), 6.91 (NH)
—
9.31 8.03 5.47
9.34 8.67 5.46
9.88 7.78 4.56 4.84
—
—
7.16 (2,6), 6.65 (3,5)
7.00 (2,6), 6.65 (3,5)
7.16 (2), 6.70 (6),
Hexapeptide 9
Step 5. A solution of 18 (5 eq.) and HOBt (10 eq.) in DMF
(20 mL) was added to 24 and the mixture was agitated overnight.
The resultant resin was filtered and washed with DMF (4 ×
25 mL) and CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in
the dark, a solution of Pd(PPh3)4 (1 eq.) and Bu3SnH (60 eq.)
in CH2Cl2 (15 mL) was added to the resin and the mixture was
agitated for 3 h. The resultant resin 25 was filtered and washed
with DMF (4 × 25 mL), CH2Cl2 (4 × 25 mL) and DMF (4 ×
25 mL). A 0.6% solution of TFA in CH2Cl2 (20 mL) was added
to 25 and the mixture was agitated for 5 min, and then filtered.
After repeating this cleavage procedure four times, the resultant
resin was washed with MeOH (4 × 20 mL) and the resultant
filtrate was collected, concentrated under reduced pressure to
give a yellow oil, and product was purified by HPLC (preparative
C18 column, gradient from 5–35% MeCN–H2O + 0.15% TFA)
to afford 9 (≥95% purity by HPLC) as a white powder (16 mg,
ca. 22% overall yield). MS(ESI): 870.5 [M + H+].
6.65(5), 5.57 (b-OH)
6.27 (2,6), 6.16 (4)
Dhpg7
9.32 8.50 5.11
—
Cht-resin (20a)
To Alloc-Cht(Allyl)-OH 29 (240 mg, 0.68 mmol) and NMM
(187 lL, 1.7 mmol) in CH2Cl2 and DMF (17 mL, 10 : 1) was
added 2-chlorotrityl chloride resin (600 mg, 0.95 mmol g−1) and
the mixture was agitated overnight. MeOH (5 mL) was added
and the mixture was agitated for 10 min. The resin was then
filtered and washed with DMF (4 × 25 mL), MeOH (4 × 25 mL)
and CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in the
dark, a solution of Pd(PPh3)4 (416 mg, 0.36 mmol) and PhSiH3
(2.7 mL, 22 mmol) in CH2Cl2 (15 mL) was added to the resin
(600 mg) and the mixture was agitated for 3 h. The resin was
then filtered and washed with DMF (4 × 25 mL), CH2Cl2 (4 ×
25 mL) and DMF (4 × 25 mL). The loading of Cht was ca.
0.15 mmol g−1 as determined after coupling Fmoc-Gly-OH, by
Fmoc and HPLC analyses.
Hexapeptide 10
Step 5ꢀ. Compound 19 was coupled to 24 and cleavage from
the resin gave product 10, as above, white powder (90 mg, ca.
52% overall yield). MS(ESI): 884.5 [M + H+].
Dhpg-resin (32)
To Alloc-Dhpg-OH 31 (380 mg, 1.42 mmol) and NMM (400 lL,
3.55 mmol) in CH2Cl2 and DMF (17 mL, 10 : 1) was added
2-chlorotrityl chloride resin (1.20 g, 0.95 mmol g−1) and the
mixture was agitated overnight. MeOH (5 mL) was added and
the mixture was agitated for 10 min. The resultant resin was
filtered and washed with DMF (4 × 25 mL), MeOH (4 × 25 mL)
and CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in the
dark, a solution of Pd(PPh3)4 (208 mg, 0.18 mmol) and PhSiH3
(1.3 mL, 11 mmol) in CH2Cl2 (15 mL) was added to the resin
(600 mg) and the mixture was agitated for 3 h. The resulting
resin was filtered and washed with DMF (4 × 25 mL), CH2Cl2
(4 × 25 mL) and DMF (4 × 25 mL). The substitution level
of Dhpg-resin 32 was ca. 0.25 mmol g−1 as determined after
coupling Fmoc-Gly-OH by Fmoc and HPLC analyses.
Hexapeptide 11
MS(MALDI): 934.4 [M + H+].
Heptapeptide 7
MS(ESI): 1049.4 [M + H+].
Heptapeptide 8
MS(ESI): 1099.6 [M + H+].
Abbreviations
Alloc, allyloxycarbonyl (= (prop-2-enyloxy)carbonyl); Boc,
(tert-butoxy)carbonyl; Cht, b-hydroxy-m-chlorotyrosine; Dhpg,
Hexapeptides 9 and 10
D-3,5-dihydroxyphenylglycine;
DMAP,
4-N,N-di-methyl-
Step 1. A solution of 12 (4 eq.), DIC (4 eq.) and HOBt
(8 eq.) in DMF (20 mL) was added to L-Tyr-resin 20 (660 mg,
0.25 mmol g−1) and the mixture was agitated overnight. The
resin was then filtered and washed with DMF (4 × 25 mL) and
CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in the dark,
a solution of Pd(PPh3)4 (1 eq.) and PhSiH3 (60 eq.) in CH2Cl2
(15 mL) was added to the resin and the mixture was agitated for
3 h. The resultant resin 21 was filtered and washed with DMF
(4 × 25 mL), CH2Cl2 (4 × 25 mL) and DMF (4 × 25 mL).
aminopyridine; DCC, dicyclohexyl-carbodiimide; DIC, 1,3-
diisopropylcarbodiimide; DIEA, N,Nꢀ-diisopropylethylamine;
DMF, N,N-dimethylformamide; EDC, 1-(3-(dimethylamino)
propyl)-3-ethylcarbodiimide;
Fmoc,
[(9H-fluorenyl)-
methoxy]carbonyl; HOBt, 1-hydroxybenzotriazole; Hpg, D-4-
hydroxyphenylglycine; NMM, N-methylmorpholine; Pfp-OH,
pentafluorophenol; TFA, trifluoroacetic acid.
Acknowledgements
Step 2. The coupling of 12 was repeated as above to give 22.
The authors thank the European Union and the Swiss National
Science Foundation for financial support.
Step 3. A solution of 13 (5 eq.) and HOBt (10 eq.) in DMF
(20 mL) was added to 22 and the mixture was agitated overnight.
The resin was then filtered and washed with DMF (4 × 25 mL)
and CH2Cl2 (4 × 25 mL). Under Ar-atmosphere and in the dark,
a solution of Pd(PPh3)4 (1 eq.) and Bu3SnH (60 eq.) in CH2Cl2
(15 mL) was added to the resin and the mixture was agitated for
3 h. The resultant resin 23 was filtered and washed with DMF
(4 × 25 mL), CH2Cl2 (4 × 25 mL) and DMF (4 × 25 mL).
Step 4. A solution of 15 (5 eq.) and HOBt (10 eq.) in DMF
(20 mL) was added to 23 and the mixture was agitated overnight.
The resin was then filtered and washed with DMF (4 × 25 mL)
References
1 B. K. Hubbard and C. T. Walsh, Angew. Chem. Int. Ed., 2003, 42,
730–765.
2 R. D. Sussmuth and W. Wohlleben, Appl. Microbiol. Biotechnol.,
2004, 63, 344–350.
3 D. H. Williams and B. Bardsley, Angew. Chem. Int. Ed., 1999, 38,
1172–1193.
4 J. C. J. Barna and D. H. Williams, Annu. Rev. Microbiol., 1984, 38,
339–357.
5 R. Stadler and M. H. Zenk, J. Biol. Chem., 1993, 268, 823–831.
1 2 3 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9

View Article Online
6 P. F. X. Kraus and T. M. Kutchan, Proc. Natl. Acad. Sci. U. S. A.,
1995, 92, 2071–2075.
7 R. Gerardy and M. H. Zenk, Phytochemistry, 1993, 32, 79–86.
8 A. Nasreen, M. Rueffer and M. H. Zenk, Tetrahedron Lett., 1996,
45, 8161–8164.
9 A. M. A. van Wageningen, P. N. Kirkpatrick, D. H. Williams, B. R.
Harris, J. K. Kershaw, N. J. Lennard, M. Jones, S. J. M. Jones and
P. J. Solenberg, Chem. Biol., 1998, 5, 155–162.
10 S. Pelzer, R. D. Su¨ssmuth, D. Heckmann, J. Recktenwald, P. Huber,
G. Jung and W. Wohlleben, Antimicrob. Agents Chemother., 1999, 43,
1565–1573.
11 T.-L. Li, F. Huang, S. F. Haydock, T. Mironenko, P. F. Leadlay and
J. B. Spencer, Chem. Biol., 2004, 11, 107–119.
12 M. Sosio, A. Bianchi, E. Bossi and S. Donadio, Antonie van
Leeuwenhoek, 2000, 78, 379–384.
13 M. Sosio, H. Kloosterman, A. Bianchi, d. P. L. Dijkhuizen and S.
Donadio, Microbiology, 2004, 150, 95–102.
14 H.-T. Chiu, B. K. Hubbard, A. N. Shah, J. Eide, R. A. Fredenburg,
C. T. Walsh and C. Khosla, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
8548–8553.
17 K. Zerbe, O. Pylypenko, F. Vitali, W. W. Zhang, S. Rouse, M. Heck,
J. W. Vrijbloed, D. Bischoff, B. Bister, R. D. Su¨ssmuth, S. Pelzer, W.
Wohlleben, J. A. Robinson and I. Schlichting, J. Biol. Chem., 2002,
277, 47476–47485.
18 O. Pylypenko, F. Vitali, K. Zerbe, J. A. Robinson and I. Schlichting,
J. Biol. Chem., 2003, 278, 46727–46733.
19 D. Bischoff, S. Pelzer, B. Bister, G. J. Nicholson, S. Stockert, M.
Schirle, W. Wohlleben, G. Jung and R. D. Su¨ssmuth, Angew. Chem.
Int. Ed., 2001, 40, 4688–4691.
20 D. Bischoff, S. Pelzer, A. Ho¨ltzel, G. J. Nicholson, S. Stockert, W.
Wohlleben, G. Jung and R. D. Su¨ssmuth, Angew. Chem. Int. Ed.,
2001, 40, 1693–1696.
21 R. D. Su¨ssmuth, S. Pelzer, G. Nicholson, T. Walk, W. Wohlleben and
G. Jung, Angew. Chem. Int. Ed., 1999, 38, 1976–1979.
22 K. Zerbe, K. Woithe, D. B. Li, F. Vitali, L. Bigler and J. A. Robinson,
Angew. Chem. Int. Ed., 2004, 43, 6709–6713.
23 F. Vitali, K. Zerbe and J. A. Robinson, Chem. Commun., 2003, 2718–
2719.
24 E. Freund, F. Vitali, A. Linden and J. A. Robinson, Helv. Chim. Acta,
2000, 83, 2572–2579.
15 J. Pootoolal, M. G. Thomas, C. G. Marshall, J. M. Neu, B. K.
Hubbard, C. T. Walsh and G. D. Wright, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 8962–8967.
16 M. Sosio, S. Stinchi, F. Beltrametti, A. Lazzarini and S. Donadio,
Chem. Biol., 2003, 10, 541–549.
25 E. Freund and J. A. Robinson, Chem. Commun., 1999, 2509–2510.
26 D. A. Evans and A. E. Weber, J. Am. Chem. Soc., 1987, 109, 7151–
7157.
27 S. D. Erickson, J. A. Simon and W. C. Still, J. Org. Chem., 1993, 58,
1305–1308.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 3 3 – 1 2 3 9
1 2 3 9