Mixing of peptides and electrophilic traps gives rise to potent, broad-spectrum proteasome inhibitors

Article abstract of DOI:10.1039/b702268a

The synthesis and evaluation of hybrid proteasome inhibitors that contain structural elements of the known inhibitors bortezomib, epoxomicin and peptide vinyl sulfones is described. From the panel of 15 inhibitors some structure activity relationships can be deduced with regard to inhibitory activity in relation to peptide recognition element, inhibitor size and nature of the electrophilic trap. Further, the panel contains one of the most potent peptide-based pan-proteasome inhibitors reported to date. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b702268a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Mixing of peptides and electrophilic traps gives rise to potent, broad-spectrum
proteasome inhibitors
Martijn Verdoes,† Bogdan I. Florea,† Wouter A. van der Linden, Didier Renou, Adrianus M. C. H. van den
Nieuwendijk, Gijs A. van der Marel and Herman S. Overkleeft*
Received 13th February 2007, Accepted 8th March 2007
First published as an Advance Article on the web 23rd March 2007
DOI: 10.1039/b702268a
The synthesis and evaluation of hybrid proteasome inhibitors that contain structural elements of the
known inhibitors bortezomib, epoxomicin and peptide vinyl sulfones is described. From the panel of 15
inhibitors some structure activity relationships can be deduced with regard to inhibitory activity in
relation to peptide recognition element, inhibitor size and nature of the electrophilic trap. Further, the
panel contains one of the most potent peptide-based pan-proteasome inhibitors reported to date.
Introduction
substrates are marked for proteasomal degradation through the
attachment of ubiquitin chains at specific sites. The ubiquitin
modifications allow docking to one of the two 19S caps that,
The development and use of proteasome inhibitors has found
wide attention in recent years, both in fundamental and applied
together with the inner catalytic 20S core, form the mammalian
1
sciences. The proteasome is a multicatalytic proteinase complex
3
26S proteasome. The approval of the peptide boronic acid PS341
that is involved in many biological processes in man. Its primary
function is the processing to oligopeptides of cytosolic and nuclear
proteins, as well as N-linked glycoproteins that are rejected from
(
bortezomib, 1, Fig. 1) for the clinical treatment of multiple
myeloma has led to a surge of activities in proteasome research.
PS341 is a highly active proteasome inhibitor, but treatment with
PS341 results in severe side effects. It is not clear yet whether this
is the result of proteasome blockade or because of other factors
that interact with the compound. The development of new and
potentially more active or selective proteasome inhibitors may
provide information on the mode of PS341 and open the way to
develop more proteasome inhibitor based therapies in oncology.
2
the ER (endoplasmatic reticulum) due to improper folding. These
Bio-organic synthesis, Leiden Institute of Chemistry, Leiden University,
P.O. Box 9502, 2300 RA, Leiden, The Netherlands E-mail: h.s.overkleeft@
chem.leidenuniv.nl; Fax: +31-71-5274307; Tel: +31-71-5274342
†
These authors contributed equally to this work
Fig. 1 Proteasome inhibitors at the basis of this study.
1
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Of interest also is the study of the role of the individual
proteasomal proteolytic activities, which can be furthered greatly
by the use of well-defined inhibitors. The mammalian proteasome
differs from the corresponding prokaryotic particles in that the
latter has only one type of catalytic activity. The overall shape of
the prokaryotic 20S proteasome highly resembles the eukaryotic
made is that the potency of peptide vinyl sulfone 4 to inhibit
the proteasome approaches, or even surpasses that of bortezomib
(1) and epoxomicin (2). This is remarkable, since the parent
compound ZL VS (3) is a much weaker inhibitor. Indeed, in
3
general oligopeptides containing epoxyketone or boronic acid
warheads are found to be more potent proteasome inhibitors than
their counterparts that have the same amino acid sequence but
are equipped with a Michael acceptor. This observation raises the
question whether recombining structural features of peptide-based
proteasome inhibitors would lead to more potent compounds.
To address this question, we set out to scramble compounds 1–
4 to arrive at a number of new C-terminally modified peptides,
and assessed these on their proteasome inhibitory activity. We
identified three distinct structural features. These are (a) the
modified amino acid at the C-terminus, being boronic acid, vinyl
sulfone and epoxyketone, (b) the amino acid sequence, being
the trileucine, the epoxomicin tetrapeptide and the bortezomib
pharmacophore, and (c) the presence (as in 4) or absence (as in 3) of
the lipophilic N-terminal extension. These considerations allowed
us to synthesise the panel of 15 compounds listed in Table 1.
2
0S proteasomes, such as the mammalian ones. It has C symmetry
2
and is assembled from two outer rings each containing seven
identical a-subunits, and two inner seven-subunit rings containing
4
identical and catalytic b-subunits. Eukaryotic proteasomes have
evolved to an extent that each a-subunit in one outer ring has a
unique sequence, as is the case with the inner b-rings. Remarkably,
four of the b-subunits lost their proteolytic activities, whereas the
5
three remaining subunits have a diverged substrate preference.
Based on fluorogenic substrate assays the substrate preference is
loosely defined as caspase-like for the b1 subunit (cleaving after
acidic residues), trypsin-like for b2 (cleaving after basic residues)
and chymotrypsin-like for b5 (cleaving after neutral hydrophobic
residues). However, many studies demonstrate that the subunits
are much more promiscuous with respect to the amino acid residue
at the cleavage site.
An important asset in these studies is the use of covalent and
Results and discussion
6
irreversible inhibitors such as the natural product epoxomicin (2),
7
the synthetic Michael acceptor ZL
3
VS (3), and their labelled
The preparation of all compounds follows the same general
strategy: first the synthesis of the (N-terminally extended) amino
acid sequence and then coupling of these to the leucine derived
warheads. As an example, the synthesis of compounds 7, 11
and 16 is depicted in Scheme 1. Briefly, Fmoc-based solid
phase peptide synthesis using HMPB functionalised MBHA resin
gained 18, followed by cleavage using 1% trifluoroacetic acid
in dichloromethane provides partially protected oligopeptide 19,
7–10
(
radio tag, affinity tag, fluorescent tag) counterparts.
Despite
these studies the exact substrate preference of the individual cat-
alytic activities, and the evolutionary benefit that results from the
diversification, is not fully understood. The same holds true for the
role of yet another proteasome particle, the immunoproteasome,
which is formed upon challenge of the mammalian immune system
and which contains three different catalytic subunits, namely b1i
11
7
(
LMP2), b2i (MECL1) and b5i (LMP7). A much sought after
which was condensed with either leucine vinyl sulfone 24 or
6
research goal in immunology is to establish the impact of the
immunoproteasome, in relation to the constitutively expressed
proteasome, on the generation of specific oligopeptides that can
be sequestered by the major histocompatibility complex class I
leucine epoxyketone 25 using BOP as the condensating agent.
Acidic removal of the tBu group afforded the target compounds
7 and 11 after HPLC purification. Target compound 16 was
obtained by coupling of succimidyl ester 22 with leucine boronic
12
10
pathway for presentation to the immune system. To aid these
studies, several research groups are involved in the development
of compounds that selectively target one catalytic subunit of either
the constitutive proteasome or the immunoproteasome.
ester 26, followed by acidic deprotection and HPLC purification.
In the design of our compounds we decided to exclude a set of
compounds that have the azapyracyl moiety in the bortezomib
sequence replaced by the adamantane-spacer moiety. Such a
set of compounds would likely resemble to a large extent the
trileucine derivatives in their inhibition profile. We further decided
to leave the pinanediol protection on the boronic ester, which
stems from the enantioselective preparation of the leucine building
block, in place. It has been reported that boronic esters of this
nature have the same activity and specificity as their unprotected
The pool of proteasome inhibitors reported to date encompasses
numerous structurally diverse compounds. A large category within
this pool has in common that they are peptide-based compounds
equipped with an electrophilic trap at the C-terminus. C-terminal
modifications include, next to boronic acid (as in 1), epoxyke-
1,13
tone (2), vinyl sulfone (3), aldehyde and other electrophiles.
10
The mechanism of inhibition is similar in all examples: the c-
hydroxyl of the N-terminal threonine within the active site of
the catalytic subunits reacts with the electrophilic trap to form
a covalent and (in most cases) irreversible bond. Some control
over subunit specificity can be achieved by altering the nature
of the amino acid residues. Several years ago we demonstrated
that N-terminally extended, hydrophobic peptide vinyl sulfones,
such as the adamantane containing compound 4, are much more
counterparts, and deprotection, for which there is no literature
precedent (in fact there is no reliable literature synthesis of the
drug bortezomib), proved to be detrimental in our hands.
The inhibition potential of the panel of compounds was assessed
in competition assays employing lysates of the murine EL4
cell line (expressing both the constitutive proteasome and the
immunoproteasome) in combination with fluorophore containing
peptide vinyl sulfone 5. In a first set of experiments, cell lysates
were incubated for one hour with each of the 15 compounds at
0, 0.1, 1, 10 and 100 lM final concentration, prior to treatment
with 0.1 lM final concentration of MV151 (5). The samples were
denatured and resolved by SDS-PAGE and the wet gel slabs were
scanned on a fluorescence scanner (Fig. 2). Lysates that have been
active proteasome inhibitors than their truncated counterparts (for
8
instance, ZL
3
VS (3)). This gain in activity was accompanied by
a loss in subunit selectivity, a finding we capitalized upon by the
development of the broad-spectrum cell permeable proteasome
10
label MV151 (Bodipy TMR-Ahx
3
L VS, 5). One observation we
3
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Table 1 Panel of synthesized proteasome inhibitors
3
4
9
13
14
10
6
7
8
2
15
16
17
11
12
9
exposed to the fluorescent label only display four bands (three
strong bands and one weaker band), that we have previously
demonstrated to correspond to the six proteasome active sites
analogue, PS341 1. This selectivity is abolished when keeping the
boronic ester in place but substituting the peptide sequence, as
in 13–16. In general, the vinyl sulfone is the weakest electrophilic
trap in each series, and the boronic ester the strongest, but there
are some interesting differences. For instance, epoxyketone 10 is
a more potent inhibitor for each proteasome subunit than the
boronic ester 14. At the onset of our experiments we assumed that
10
(
specified as depicted in Fig. 2). The ability of the 15 compounds
to inhibit the proteasome activities is reflected by disappearance
of labelling. Ten compounds from the panel of 15 (namely, 2, 4,
7
, 9, 10, 11, 13–16) proved to be potent proteasome inhibitors,
14
with most or all labelling abolished at 1 lM. Vinyl sulfone
derivatives 3, 6 and 8, and epoxyketone 12 appear to be much
weaker inhibitors. Boronic ester 17 is a weak inhibitor of the b2
and b2i subunits, while potently targeting the remaining subunits.
As the next experiment we repeated the competition experiment
but with the difference that inhibitor concentrations were ranged
from 0 to 500 nM (Fig. 3, compounds 6, 8 and 12 were excluded
since these proved to be hardly effective in the first experiments
employing higher final concentrations). These results corroborate
our earlier findings and allow the assessment of some more subtle
differences between the different inhibitors. The most obvious
result is that boronic ester 17 hardly targets b2 and b2i, a finding
that corresponds to the reported specificity of the unprotected
elongation of a given proteasome inhibitor with the Ada(Ahx)
3
N-terminal cap leads to a more potent compound. This holds true
to some extend, compare, for instance, vinyl sulfones 3 and 4, vinyl
sulfones 6 and 7, epoxyketones 9 and 10, and epoxyketones 2 and
11. However the potency of ZL
3
-boronic ester 13 (its unprotected
3
counterpart has been described in the literature and is known as
MG262) belies the generality of this trend. Indeed, the potency of
this compound is bettered by boronic ester 15 only. Compound
15 is more potent than its N-terminally extended analogue 16
as well. When we performed both competition assays on human
embryonic kidney HEK293T cell lysates, which expresses the
constitutive proteasome only, we found the same general trends
(data not shown).
1
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Scheme 1 Synthesis of compounds 7, 11 and 16.
Fig. 2 Competition assays in EL4 lysates. Lysates were incubated for one hour at 0, 0.1, 1, 10 and 100 lM final concentration. Residual proteasome
activity was labelled with 0.1 lM MV151 for one hour.
Conclusions
instance, while it is true that hydrophobic extension in most
cases contributes to the potency, the most potent compound
from the series presented here proved to be the boronic ester 15
bearing the epoxomicin tetrapeptide sequence without N-terminal
extension. We believe compound 15 to be the most potent peptide-
based proteasome inhibitor reported to date. Possibly, such broad-
spectrum proteasome inhibitors might find clinical application
In conclusion, we have demonstrated that scrambling of structural
elements of known proteasome inhibitors is a viable strategy
to arrive at potent new proteasome inhibitors. Prediction of
the potency of a putative peptide-based inhibitor is not as
straightforward as we considered at the onset of our study. For
This journal is © The Royal Society of Chemistry 2007
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Fig. 3 Competition assays in EL4 lysates. Lysates were incubated for one hour at 0, 25, 50, 100, 200, 300, 400 and 500 nM final concentration. Residual
proteasome activity was labelled with 0.1 lM MV151 for one hour. nd = not determined.
as an alternative for bortezomib. For instance in cases where
bortezomib resistance occurs, an event which is thought to be
linked to regulation in one way or another of those proteasome
activities that are left unmodified by bortezomib (bortezomib
has a labelling profile similar to 17 and leaves the b2 and b2i
subunits untouched at clinical doses). Current investigations in our
laboratory are aimed at further unravelling the subunit preferences
of our panel of compounds, with the aim to arrive at subunit-
specific inhibitors.
Alltima C18 column was used. The applied buffers were A:
O, B: MeCN and C: 1.0% aq. TFA. Optical rotations were
H
2
measured on a Propol automatic polarimeter (sodium D line, k =
7
8
589 nm). ZL
3
VS (3), Ada-Ahx
3
L
3
VS (4), bortezomib pinanediol
10
6
ester (8), epoxomicin (2), Boc-leucine-vinyl-(methyl)–sulfone
(35) and (Boc-leucinyl)–methyloxirane (37) were synthesised as
7
6
described in the literature.
Experimental
All reagents were commercial grade and were used as received
unless indicated otherwise. Tol (Tol)(purum), ethyl acetate (EA)
Scheme 2
(
(
puriss.), diethyl ether (Et
puriss.) were obtained from Riedel–de Ha e¨ n and distilled prior to
2
O) and light petroleum ether (PE)
use. Dichloroethane (DCE), dichloromethane (DCM), dimethyl
˚
formamide (DMF) and dioxane (Biosolve) were stored on 4A
Z-leu
2
-OMe (27)
molecular sieves. Methanol (MeOH) and N-methylpyrrolidone
(
(
NMP) were obtained from Biosolve. Tetrahydrofuran (THF)
Biosolve) was distilled from LiAlH prior to use. Reactions
Z-Leu-OH (5.6 g, 21.5 mmol) and HCl·Leu-OMe (3.92 g,
4
2
1.5 mmol, 1 equiv.) were dissolved in 80 mL DCM and put
were monitored by TLC-analysis using DC-alufolien (Merck,
Kieselgel60, F254) with detection by UV-absorption (254 nm),
spraying with 20% H
under an argon atmosphere. PyBOP (11.2 g, 21.5 mmol, 1 equiv.)
and DiPEA (7.3 mL, 43 mmol, 2 equiv.) were added and the
reaction mixture was stirred for 3 h. The reaction mixture was
2
SO in ethanol followed by charring at
4
◦
∼
150 C, by spraying with a solution of (NH
4
)
6
Mo
7
O
24·4H
2
O
washed with sat. aq. NaHCO
organic phase was dried over MgSO
3
, 1 M HCl and brine, and the
and concentrated. Flash
−
1
−1
(
25 g L ) and (NH
4
)
4
Ce(SO
4
)
4
·2H
2
O (10 g L ) in 10% sulfuric
4
◦
acid followed by charring at ∼150 C or spraying with an aqueous
column chromatography (PE → 10% EA/PE, v/v) gave the title
solution of KMnO
4
(7%) and KOH (2%). Column chromatog-
1
compound (5.7 g, 14.5 mmol, 68%). H NMR (200 MHz, CDCl
3
):
raphy was performed on Screening Devices (0.040–0.063 nm).
HRMS were recorded on a LTQ Orbitrap (Thermo Finnigan). H-
d = 7.52 (d, 1 H, J = 7.7 Hz), 7.32–7.15 (m, 5 H), 6.31 (d, 1 H,
1
J = 8.4 Hz), 5.08–4.90 (m, 2 H), 4.60–4.18 (m, 2 H), 3.59 (s, 3 H),
1
3
and C-APT-NMR spectra were recorded on a Jeol JNM-FX-
00 (200/50), Bruker DPX-300 (300/75 MHz), Bruker AV-400
400/100 MHz) equipped with a pulsed field gradient accessory
13
1
.70–1.35 (m, 6 H), 0.95–0.68 (m, 6 H). C NMR (50.1 MHz,
2
(
CDCl
3
): d = 172.7, 172.5, 155.9, 136.0, 127.9, 127.5, 127.3, 66.2,
52.9, 51.5, 50.3, 41.1, 40.4, 24.3, 24.1, 22.2, 21.7, 21.4.
or a Bruker DMX-600 (600/150 MHz) with cryoprobe. Chemical
shifts are given in ppm (d) relative to tetramethylsilane as internal
Z-Leu
2
-OH (28)
1
3
standard. Coupling constants are given in Hz. All presented C-
APT spectra are proton decoupled. UV spectra were recorded
on a Perkin Elmer, Lambda 800 UV/VIS spectrometer. For RP-
HPLC purifications a BioCAD “Vision” automated HPLC system
Z-Leu
2
-OMe (27), (5.7 g, 14.5 mmol) was dissolved in a mixture of
61 mL dioxane, 21.7 mL MeOH and 4.78 mL 4 M NaOH in H O
(19.1 mmol, 1.32 equiv.) and stirred for 3.5 h. The reaction mixture
was acidified to pH 2 with 1 M HCl and concentrated. The residue
2
(
PerSeptive Biosystems, inc.) equipped with a semi-preparative
1
420 | Org. Biomol. Chem., 2007, 5, 1416–1426
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1
was dissolved in EA, and washed with H
extracted with EA (2×), and the combined organic layers were
dried over MgSO and concentrated. Crystallisation from EA/PE
yielded the title compound (5.03 g, 13.3 mmol, 92%). H NMR
200 MHz, CDCl
2
O. The water layer was
compound, which was used without any further purification. H
NMR (200 MHz, CD
3
OD): d = 4.51–4.34 (m, 2 H), 3.25–3.05
4
(m, 6 H), 2.22–2.10 (m, 6 H), 1.93 (s, 2 H), 1.82–1.20 (m, 39 H),
1
13
1.01–0.83 (m, 12 H). C NMR (50.1 MHz, CD
3
OD): d = 176.0,
(
3
): d = 7.40–7.25 (m, 5 H), 6.65 (d, 1 H, J =
175.7, 174.8, 173.8, 53.0, 51.9, 51.7, 43.7, 41.7, 40.2, 37.8, 36.9,
36.6, 33.8, 30.1, 30.0, 27.5, 26.7, 25.9, 22.1, 21.9.
6
4
.2 Hz), 5.48 (m, 1 H), 5.1 (s, 2 H), 4.65–4.50 (m, 1 H), 4.38–
1
3
.19 (m, 1 H), 1.81–1.43 (m, 6 H), 1.05–0.80 (m, 6 H). C NMR
): d = 175.8, 172.9, 156.5, 136.1, 128.4, 127.6,
27.8, 67.0, 53.3, 50.8, 41.0, 24.7, 24.4, 22.7, 21.7.
(50.1 MHz, CDCl
3
1
HMPB-MBHA resin (29)
-Methylbenzhydrylamine (MBHA) functionalized polystyrene
4
−
1
resin (0.56 g, 0.9 mmol g , 0.5 mmol) was washed with NMP
3×) followed by addition of a preactivated mixture of 4-(4-
hydroxymethyl-3-methoxyphenoxy)–butyric acid (HMPB) linker
0.36 g, 1.5 mmol, 3 equiv.), BOP (0.67 g, 1.5 mmol, 3 equiv.) and
(
(
DiPEA (0.55 mL, 3 mmol, 6 equiv.) in NMP. After 2 h of shaking,
the resin was washed with NMP (3×), MeOH (3×) and DCM
Scheme 4
(
3×), dried and used as such.
Ada-Ahx
3
-Leu
2
-OH (30)
Ac(Me)-Ile -Thr(tBu)-OH (31)
2
Resin 29 (0.5 mmol) was transferred to a flask, coevaporated with
DCE (2×), and condensed with Fmoc-Leu-OH (0.53 g, 1.5 mmol,
Resin 29 (1 mmol) was transferred to a flask, coevaporated
with DCE (2×), and condensed with Fmoc-Thr(tBu)-OH (1.2 g,
3 mmol, 3 equiv.) under the influence of DIC (0.51 mL, 3.3 mmol,
3.3 equiv.) and DMAP (18 mg, 0.15 mmol, 15 mol%) in DCM
for 2 h. The resin was filtered and washed with DCM (2×),
followed by a second condensation cycle. The loading of the
3
3
2
equiv.) under the influence of DIC (0.26 mL, 1.67 mmol,
.3 equiv.) and DMAP (10 mg, 0.075 mmol, 15 mol%) in DCM for
h. The resin was filtered and washed with DCM (2×), followed
by a second condensation cycle. The loading of the resin was
−
1
−1
determined to be 0.42 mmol g (0.93 g, 0.39 mmol, 78%) by
spectrophotometric analysis. The obtained resin was submitted to
four cycles of Fmoc solid-phase synthesis with Fmoc-Leu-OH and
Fmoc-Ahx-OH (3×), respectively, as follows: (a) deprotection with
piperidine/NMP (1/4, v/v, 20 min), (b) wash with NMP (3×), (c)
coupling of Fmoc amino acid (1.2 mmol, 3 equiv.) in the presence
of BOP (0.53 g, 1.2 mmol, 3 equiv.) and DiPEA (0.4 ml, 2.3 mmol,
resin was determined to be 0.55 mmol g by spectrophotometric
analysis. The obtained resin was submitted to two cycles of Fmoc
solid-phase synthesis with Fmoc-Ile-OH and Fmoc(Me)–Ile-OH,
respectively, as follows: (a) deprotection with piperidine/NMP
(1/4, v/v, 20 min), (b) wash with NMP (3×), (c) coupling of Fmoc
amino acid (2.5 mmol, 2.5 equiv.) in the presence of BOP (1.1 g,
2.5 mmol, 2.5 equiv.) and DiPEA (0.5 ml, 3 mmol, 3 equiv.) in
NMP and shake for 2 h, (d) wash with NMP (3×) and DCM (3×).
Couplings were monitored for completion by the Kaiser test. After
Fmoc deprotection of the resin bound tripeptide, acetyl chloride
(0.3 ml, 4 mmol, 4 equiv.) and DiPEA (0.66 mL, 4 mmol, 4 equiv.)
in DCM were added, and the resin was shaken for 2 h. After
washing with DCM (3×) the resin was subjected to mild acidic
cleavage (TFA/DCM, 1/99 v/v, 10 min, 3×) and the collected
fractions were coevaporated with Tol (2×) to give the crude title
compound, which was used without any further purification.
6
equiv.) in NMP and shaken for 2 h, (d) wash with NMP (3×)
and DCM (3×). Couplings were monitored for completion by the
Kaiser test. After deprotection of the resin bound pentapeptide,
adamantylacetic acid (0.23 g, 1.2 mmol, 3 equiv.), PyBOP (0.63 g,
1
.2 mmol, 3 equiv.), DiPEA (0.4 mL, 2.34 mmol, 6 equiv.) in NMP
were added, and the resin was shaken for 2 h. After washing with
NMP (3×) and DCM (3×) the resin was subjected to mild acidic
cleavage (TFA/DCM, 1/99 v/v, 10 min, 3×) and the collected
fractions were coevaporated with Tol (2×) to give the crude title
Scheme 3
This journal is © The Royal Society of Chemistry 2007
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9
mmol, 3 equiv.) for 15 min. To this solution, phenylalanine
methyl ester·HCl (0.712, 3.3 mmol, 1.1 equiv.) dissolved in 30 mL
DMF was added, and the reaction mixture was stirred for 1 h. Sat.
aq. NaHCO
Et
3
was added and the water layer was extracted with
2
O (3×). The combined organic layers were dried over MgSO
4
and concentrated. The crude product was purified by column
chromatography (Tol → 40% EA in Tol v/v) to yield the title
1
compound (0.6 g, 2.1 mmol, 70%). H NMR (200 MHz, CDCl
3
):
d = 9.36 (d, 1H, J = 1.5 Hz), 8.71 (d, 1H, J = 2.6 Hz), 8.46 (m,
1
3
1
5
H), 8.29 (d, 1H, J = 7.7 Hz), 7.25 (m, 5H), 5.14–5.04 (m, 1H),
1
3
.74 (s, 3H), 3.35–3.15 (m, 2H). C NMR (50.1 MHz, CDCl
3
): d =
Scheme 5
-Thr(tBu)-OH (19)
71.2, 162.4, 147.2, 144.0, 143.7142.5, 135.5, 128.9, 128.4, 126.9,
3.1, 52.2, 37.8.
Ada-Ahx
3
-Ile
2
HMPB-MBHA resin 29 (2 g, 0.75 mmol g−1 resin, 1.5 mmol)
was coevaporated with DCE (2×) and condensed with Fmoc-
Thr(tBu)-OH (1.8 g, 4.5 mmol, 3 equiv.) under the influence of
DIC (0.7 mL, 4.5 mmol, 3 equiv.) and DMAP (10 mg, 75 lmol)
for 2 h. The resin was filtered, washed with DCM (3×) and
subjected to a second condensation cycle. The loading of the
resin was 0.67 mmol g , as determined by spectrophotometric
analysis. Next, the resin was subjected to five cycles of Fmoc solid-
phase synthesis with Fmoc-Ile-OH (2×) and Fmoc-Ahx-OH (3×),
respectively as follows: (a) deprotection with piperidine/NMP
Pyrazine-2-carbonylphenylalanine (34)
Methyl ester 33 (0.24 g, 0.85 mmol) was dissolved in 3 mL dioxane,
1
.1 mL MeOH and 0.22 mL 4 M NaOH. The reaction mixture
was stirred for 1 h before 0.8 mL 1M KHSO was added, and the
solution was concentrated. The residue was taken up in H O/brine
4
2
(
1/1, v/v), and this solution was extracted with EA (3×). The
−
1
combined organic layers were dried over MgSO and concentrated,
4
yielding the title compound in 69% yield (0.16 g, 0.59 mmol).
LC/MS analysis: R
10.16 min (linear gradient 10 → 90% B
in 20 min), m/z 272.0 [M + H] , 543.1 [2M + H] . H NMR
200 MHz, CDCl
f
+
+
1
(
1/4, v/v, 15 min), (b) washing with NMP (3×) and DCM (3×), (c)
(
3
): d = 9.37 (s, 1H), 8.76 (s, 1H), 8.54 (s, 1H),
.21 (d, 1H, J = 8 Hz), 7.3–7.19 (m, 5H), 5.18–5.07 (m, 1H), 3.42–
.20 (m, 2H). C NMR (50.1 MHz, CDCl
coupling of the Fmoc amino acid (4.5 mmol, 3 equiv.) by shaking
the resin for 2 h in the presence of HCTU (3 equiv., 4.5 mmol),
DiPEA (6 equiv., 9 mmol) and NMP as solvent, (d) washing with
NMP (3×) and DCM (3×). Couplings were monitored by the
Kaiser test for completion. This resin (1.7 g, 0.75 mmol) was
deprotected using piperidine/NMP (1/4, v/v, 15 min), washed
with NMP (3×) and DCM (3×) and condensed with Ada-OH
8
3
1
1
3
3
): d = 174.0, 164.7,
48.7, 145.6, 144.7, 144.6, 137.9, 130.3, 129.5, 127.9, 54.9, 38.2.
(
0.44 g, 2.25 mmol, 3 equiv.) using HCTU (0.93 g, 2.25 mmol,
3
2
equiv.) and DiPEA (0.75 mL, 4.5 mmol, 6 equiv.) in NMP for
h. The resin was washed with NMP (3×) and DCM (3×), before
being subjected to mild acidic cleavage (TFA/DCM, 1/99 v/v,
1
5 min, 3×). The fractions were collected and concentrated in the
presence of Tol, yielding the title compound (505 mg, 0.55 mmol,
1
7
3
0
1
3
1
3%). H NMR (200 MHz, CD
3
OD): d = 4.48–4.23 (m, 4 H),
.25–3.08 (m, 6 H), 2.35–2.12 (m, 7 H), 2.04–1.10 (m, 52 H), 1.00–
.83 (m, 12H). C NMR (50.1 MHz, CD
73.6, 75.1, 68.8, 59.0, 51.8, 43.7, 40.1, 38.0, 37.9, 37.6, 36.9, 36.6,
3.7, 30.1, 30.0, 28.8, 27.5, 26.6, 26.0, 25.7, 21.1, 16.1, 15.9, 11.4,
1.2.
1
3
3
OD): d = 175.8, 173.5,
Scheme 7
Ac(Me)–Ile -Thr-LeuVS (6)
2
Pyrazine-2-carbonylphenylalanine methyl ester (33)
7
Boc-leucine-vinyl-(methyl)-sulfone (35) (87 mg, 0.3 mmol) was
stirred in TFA/DCM (1/1 v/v, 1 mL) until TLC analysis indicated
complete deprotection. The reaction mixture was concentrated in
the presence of Tol (2×) before being dissolved in DCM and put
Pyrazine-2-carboxylic acid (32) (0.37 g, 3 mmol) was dissolved in
DMF (30 mL), put under an argon atmosphere and preactivated
with BOP (1.46 g, 3.3 mmol, 1.1 equiv.) and DiPEA (1.53 mL,
Scheme 6
1
422 | Org. Biomol. Chem., 2007, 5, 1416–1426
This journal is © The Royal Society of Chemistry 2007

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under an argon atmosphere. Ac-(Me)–I
.45 mmol, 1.5 equiv.), BOP (0.2 g, 0.45 mmol, 1 equiv.), DiPEA
0.12 ml, 0.76 mmol, 2.5 equiv.) were added, and the mixture was
stirred for 12 h, before being concentrated in vacuo. The crude was
dissolved in TFA/DCM (1/1 v/v, 2 mL) and stirred at ambient
temperature for 30 min before being concentrated in the presence
2
-T(tBu)-OH 31 (337 mg,
4.90–4.82 (m, 1H), 4.68–4.59 (m, 1H), 3.25–3.13 (m, 2H), 2.89 (s,
1
3
0
(
3H), 1.64–1.49 (m, 1H), 1.45–1.30 (m, 2H), 0.93–0.81 (m, 6H). C
NMR (100 MHz, CDCl
): d = 170.0, 163.0, 147.5, 147.2, 144.1,
3
143.8, 142.9, 136.0, 129.3, 128.9, 128.8, 127.3, 54.5, 47.9, 42.6,
2
0
◦
42.5, 38.4, 24.5, 22.4, 21.7. [a]
D
−12 (c = 0.2, MeOH). HRMS:
+
calcd for C22
H
28
O
4
N
4
SH 445.19040 found 445.19031.
of Tol (2×), yielding Ac(Me)-Ile
2
-Thr-LeuVS (6) as a mixture of
two diastereomers. Semi-preparative RP-HPLC purification of the
major product yielded the title compound as a white solid (1.2 mg,
1
2
lmol, 0.7% isolated yield). H NMR (400 MHz, CD
3
OD): d 6.81
(
1
3
1
dd, J = 14.6, 4.0 Hz, 1H), 6.71 (d, J = 15.2 Hz, 1H), 4.78 (d,
1.2 Hz, 1H), 4.71 (m, 1H), 4.26 (m, 1H), 4.17 (m, 2H), 3.02 (s,
H), 2.95 (s, 3H), 2.12 (s, 3H), 2.05 (m, 1H), 1.88 (m, 1H), 1.71 (m,
H), 1.62–1.26 (m, 6H), 1.19 (m, 1H), 1.18 (d, J = 6.4 Hz, 3H),
2
0
◦
1
.02 (m, 1H), 0.92 (m, 18H). [a]
D
+
−225 (c = 0.024, MeOH).
HRMS: calcd for C27
H
50
O
7
N
4
SH 575.34730, found 575.34769.
Ada-Ahx
3
-Ile -Thr-LeuVS (7)
2
7
Boc-leucine-vinyl-(methyl)-sulfone (35) (76 mg, 0.26 mmol,
1
.1 equiv.) was stirred in TFA/DCM (1/1 v/v, 1 mL) until
Scheme 8
TLC analysis indicated complete conversion of the starting
material. The reaction mixture was concentrated in the presence
of Tol (2×) before being dissolved in DMF and put under an
Z-Leu
3
-2-methyloxirane (9)
argon atmosphere. Ada-Ahx
3
-Ile -Thr(tBu)-OH (19) (217 mg,
2
0
.24 mmol), BOP (117 mg, 0.26 mmol, 1.1 equiv.), and DiPEA
6
(
Boc-leucinyl)-methyloxirane (37) (67 mg, 0.25 mmol) was stirred
(
90 lL, 0.53 mmol, 2.2 equiv.) were added, and the mixture was
in TFA (1 mL) until TLC analysis indicated complete consumption
of the starting material. Tol was added, and the reaction mixture
was concentrated. Z-Leu -OH 28 (104 mg, 0.275 mmol, 1.1 equiv.)
stirred for 12 h, before being concentrated in vacuo. The crude was
dissolved in TFA/DCM (1/1 v/v, 1 mL) and stirred at ambient
temperature for 30 min., before being concentrated in the presence
2
was coevaporated with Tol (2×), dissolved in DCM and put under
an argon atmosphere. PyBOP (150 mg, 0.29 mmol, 1.16 equiv.)
and DiPEA (0.13 mL, 0.75 mmol, 3 equiv.) were added, followed
by the crude TFA·leucinyl-methyloxirane 25. The reaction mixture
of Tol (2×), yielding Ada-Ahx
3
-Ile -Thr-LeuVS (7) as a mixture
2
of two diastereomers. Semi-preparative RP-HPLC purification of
the major product yielded the title compound as a white solid
1
(
(
38.8 mg, 37.5 lmol, 16%). H NMR (300 MHz, CDCl
3
): d = 7.52
was stirred for 1 h. The mixture was washed with H O and brine,
2
m, 2H), 6.86 (dd, J = 4.3, 15 Hz, 1H), 6.65 (d, J = 15 Hz, 1H),
dried over MgSO and concentrated. Crystallisation from EA/PE
4
4
3
3
1
1
5
2
1
.72 (m, 1H), 4.32–4.10 (m, 4H), 3.18 (t, J = 6.9 Hz, 6H), 2.98 (s,
H), 2.29 (t, J = 7.5 Hz, 2H), 2.18 (t, J = 7.4 Hz, 4H), 1.97 (m,
H), 1.93 (s, 2H), 1.83 (m, 2H), 1.73–1.45 (m, 30H), 1.39–1.15 (m,
yielded crude title compound 9, which was further purified by flash
column chromatography (PE → 30% EA/PE, v/v) yielding the
1
title compound (18.7 mg, 44 lmol, 18%). H NMR (200 MHz,
1
3
0H), 0.97–0.89 (m, 18H). C NMR (75 MHz, CDCl
3
): d = 175.2,
CD
4
1
CD
6
3
OD): d = 7.35–7.25 (m, 5H), 5.08 (m, 2H), 4.62–4.38 (m, 2H),
.22–4.05 (m, 1H), 3.24 (d, J = 5.1 Hz, 1H), 2.91 (d, 5.1 Hz,
H), 1.77-1.25 (m, 9H), 0.95-0.83 (m, 18H). C NMR (50.1 MHz,
74.2, 172.7, 172.0, 171.7, 170.1, 147.4, 128.8, 66.5, 59.0, 58.7 (2×),
1.0, 47.8, 42.3, 42.2, 42.1, 38.8, 38.7, 36.4, 35.9, 35.8, 35.3, 32.4,
8.6 (2×), 28.5, 28.4, 26.0, 25.0, 24.9, 24.8, 24.6, 24.4, 22.5, 21.2,
1
3
◦
3
OD): d = 209.5, 175.1, 174.5, 156.0, 129.5, 129.0, 128.8, 67.6,
2
0
9.2, 15.2, 15.1, 11.1, 10.6. [a]
D
−31 (c = 0.2, MeOH). HRMS:
0.1, 54.9, 53,1, 52.7, 51.9, 42.1, 41.9, 40.2, 26.2, 25.8, 25.7, 23.7,
+
calcd for C54
H
95
N
7
O
10SH 1056.67533, found 1056.67658.
◦
2
0
2
3.4, 22.2, 22.0, 24.5, 17.0. [a]
D
−1 (c = 0.2, MeOH). HRMS:
+
calcd for C29
H
45
O
6
N H 532.33811, found 532.33826.
3
(
(
Pyrazine-2-carbonylphenylalanyl)-leucine-vinyl-(methyl)-sulfone
8)
Ada-Ahx –Leu
3
2
-leucinyl-2-methyloxirane (10)
6
Pyrazine-2-carbonylphenylalanine 34 (157 mg, 0.58 mmol) was
preactivated with BOP (282 mg, 0.64 mmol, 1.1 equiv.) and DiPEA
(Boc-leucinyl)–methyloxirane (37) (116 mg, 0.35 mmol) was
stirred in TFA (1 mL) until TLC analysis indicated complete
consumption of the starting material. The reaction mixture was
concentrated in the presence of Tol (2×), dissolved in DCM/DMF
(
0
0.3 mL, 1.7 mmol, 3 equiv.) in DCM. TFA·LeuVS 24 (212 mg,
.7 mmol, 1.1 equiv.) was added, and the mixture was stirred
for 4 h. The reaction mixture was concentrated and subjected to
(19/1, v/v) and put under an argon atmosphere. Ada-Ahx
3
-Leu -
2
flash column chromatography (Tol → EA), yielding two epimers
OH (30) (0.27 g, 0.35 mmol, 1 equiv.), PyBOP (0.2 g, 0.38 mmol,
1.1 equiv.) and DiPEA (0.17 mL, 1.1 mmol, 3 equiv.) were added
and the mixture was stirred for 3 h, before being concentrated
and purified by flash column chromatography (DCM → 10%
(
both 82 mg, 0.18 mmol, 32%). Recrystallisation from PE/acetone
1
yielded the title compound (50 mg, 0.113 mmol, 19%). H NMR
400 MHz, CDCl
(
8
1
3
): d = 9.32 (s, 1H), 8.77 (d, J = 2.0 Hz, 1H),
.59 (s, 1H), 8.50 (m, 1H), 7.43–7.22 (m, 5H), 7.16 (d, J = 8.0 Hz,
H), 6.66 (dd, J = 4.8, 15.2 Hz, 1H), 5.99 (d, J = 15.2 Hz, 1H),
MeOH/DCM, v/v), yielding the title compound (130 mg,
1
0.14 mmol, 41%). H NMR (400 MHz, CD
3
OD): d = 4.58-4.47
This journal is © The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 1416–1426 | 1423

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(
m, 1H), 4.47-4.32 (m, 2H), 3.14 (t, J = 6.3 Hz, 6H), 3.24 (m, 1H),
2
7
1
.92 (d, J = 5.1 Hz, 1H), 2.23 (t, J = 7.4 Hz, 2H), 2.17 (t, J =
.4 Hz, 4H), 1.97–1.89 (m, 5H), 1.77–1.42 (m, 30H), 1.41-1.27 (m,
2
0
◦
2H), 0.99-0.85 (m, 18H). [a]
D
−1.5 (c = 2, MeOH). HRMS:
+
calcd for C51
H
88
N
6
O H 913.67390 found 913.67364
8
Ada-Ahx
3
-Ile
2
-Thr-leucinyl-2-methyloxirane (11)
6
(
Boc-leucinyl)–2-methyloxirane (37) (78 mg, 0.29 mmol,
1
.1 equiv.) was stirred in TFA/DCM (1/1 v/v, 1 mL) until TLC
analysis indicated complete conversion of the starting material
20 min). The reaction mixture was concentrated in the presence
of Tol (2×) before being dissolved in DMF and put under an
argon atmosphere. Ada-Ahx -Ile -Thr(tBu)-OH (19) (236 mg,
.26 mmol), BOP (0.13 g, 0.29 mmol, 1.1 equiv.), and DiPEA
95 lL, 1.1 mmol, 4.4 equiv.) were added, and the mixture was
stirred for 3 h. The crude Ada-Ahx -Ile -Thr(tBu)-leucinyl-2-
methyloxirane (21) was precipitated with EA yielding a mixture
of diastereomers. The major product was purified by semi-
preparative RP-HPLC yielding 21 (27.3 mg, 25.5 lmol, 10%).
tert-Butyl ether 21 was dissolved in TFA/DCM (1/1 v/v) and
stirred for 0.5 h, before being evaporated in the presence of Tol
(
Scheme 9
3
2
0
(
20
◦
3
2
5
0
4
4.3, 52.3, 50.2, 40.2, 38.4, 25.0, 24.8, 23.1, 16.6. [a]
+12 (c =
D
+
.1, MeOH). HRMS: calcd for C23
25.21835.
H
28
O
4
N
4
H 425.21833, found
(
2×) yielding the title compound as a colourless oil (23.6 mg,
Z-Leu
3
-boronic ester (13)
-OH (28) (0.2 g, 0.53 mmol) and HOSu
1
2
2
4
2
7
2
0
1
5
3
1
3.3 lmol, 91%). H NMR (400 MHz, CD
3
OD): d 4.55 (dd, J =
.6, 10.6 Hz, 1H), 4.33 (d, J = 5.2 Hz, 1H), 4.27–4.21 (m, 2H),
.04 (m, 1H), 3.25 (d, J = 5.2 Hz, 1H), 3.14 (t, J = 7.0 Hz, 6H),
.91 (d, J = 5.2 Hz, 1H), 2.24 (t, J = 7.2 Hz, 2H), 2.16 (t, J =
.4 Hz, 4H), 1.94 (m, 3H), 1.91 (s, 2H), 1.82 (m, 2H), 1.74–1.56 (m,
2H), 1.55–1.47 (m, 7H), 1.46 (s, 3H), 1.39–1.11 (m, 11H), 0.94–
Under argon Z-Leu
2
(85.1 mg, 0.73 mmol, 1.4 equiv.) were dissolved in DMF
(1 mL) and DIC (100 lL, 0.64 mmol, 1.2 equiv.) was added. After a
few minutes a fine precipitate was formed. The mixture was stirred
overnight at ambient temperature after which LC/MS analysis
showed complete conversion of Z-Leu
A flame dried two necked reaction flask was put under an ar-
gon atmosphere in which (1R)-4-(1-chloro-3-methyl(butyl)-2,9,9-
1
3
.86 (m, 18H). C NMR (100.2 MHz, CDCl
3
): d = 209.4, 176.0,
2
-OH (28) into Z-Leu -OSu.
2
75.9, 174.0, 173.7, 173.4, 172.1, 68.4, 60.0, 59.6, 59.3, 59.1, 53.0,
1.9, 51.8, 43.7, 40.3, 40.2 (2×), 40.1, 37.9 (2×), 37.6, 37.0, 36.6,
3.7, 30.1 (3×), 27.6, 27.5, 26.6, 26.2, 26.0, 25.9, 23.8, 19.9, 17.0,
2,6
10
trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0 ]decane (152 mg,
2
0
◦
◦
6.0, 15.9, 11.4, 11.2. [a]
D
−17.5 (c = 0.24, MeOH). HRMS:
0.53 mmol, 1 equiv.) was dissolved in THF (5 mL). At −20 C
+
calcd for C55
C
H
95
+
N
7
O
10H 1014.72132 found 1014.72270, calcd for
a LiHMDS solution (1 M in THF, 600 lL, 0.60 mmol, 1.1 equiv.)
55
H
95
N
7
O
10Na 1036.70326, found 1036.70421.
was added dropwise. The mixture was allowed to warm to room
◦
temperature and stirred overnight. At −90 C a HCl solution
(2 M in diethyl ether, 1.50 mL, 3.0 mmol, 5.7 equiv.) was added
(
Pyrazine-2-carbonylphenylalanyl)-leucinyl-2-methyloxirane (12)
◦
dropwise. The mixture was slowly warmed to 0 C (ca. 2 h) and re-
6
◦
(
Boc-leucinyl)-methyloxirane (37) (103 mg, 0.38 mmol) was
cooled to −80 C at which temperature the Z-Leu
2
-OSu described
stirred in TFA (1 mL) until TLC analysis indicated complete
consumption of the starting material. Tol was added, and the reac-
tion mixture was concentrated. Pyrazine-2-carbonylphenylalanine
above and DIPEA (0.90 mL, 5.4 mmol, 1.02 equiv.) were added.
The mixture was slowly warmed to room temperature (ca. 2 h)
and was stirred for another 2 h. After filtration through a path of
hyflo and concentration in vacuo the crude product was purified by
silica gel column chromatography (10% EA/PE → 25% EA/PE)
3
4 (0.11 g, 0.4 mmol, 1.05 equiv.) was coevaporated with Tol
2×) and dissolved in 20 mL DCM and put under an argon
atmosphere. BOP (0.2 g, 0.44 mmol, 1.1 equiv.) and DiPEA
0.2 mL, 1.14 mmol, 3 equiv.) were added, followed by addition
of the TFA·leucinyl-methyloxirane 25. The reaction mixture was
stirred for 1 h before washing with sat. aq. NaHCO and brine.
The organic layer was dried over Na SO and concentrated. Flash
(
affording the title compound as a white solid (197 mg, 0.32 mmol,
1
(
59%). H NMR (400 MHz, CD
3
OD): d = 7.37–7.28 (m, 5H),
6.79 (d, J = 8.1 Hz, 1H), 5.50 (d, J = 6.5 Hz, 1H), 5.12 (d, J =
12.0 Hz, 1H), 5.02 (d, J = 12.2 Hz, 1H), 4.58 (dd, J = 13.4, 7.5 Hz,
1H), 4.27 (dd, J = 8.6, 1.8 Hz, 1H), 4.22–4.13 (m, 1H), 3.10–2.97
(m, 1H), 2.36–2.27 (m, 1H), 2.15 (td, J = 10.5, 5.2, 5.2 Hz, 1H),
2.01 (t, J = 5.4 Hz, 1H), 1.93–1.77 (m, 2H), 1.76–1.55 (m, 5H),
3
2
4
column chromatography (PE → 25% EA/PE) yielded the title
1
compound (8.6 mg, 20 lmol, 5.3% isolated yield). H NMR
1
3
(200 MHz, CDCl
3
): d = 9.35 (d, J = 1.1 Hz, 1H). 8.75 (d, J =
1.55–1.34 (m, 7H), 1.33–1.23 (m, 4H), 0.98–0.79 (m, 21H).
C
2
.2 Hz, 1H), 8.53 (d, J = 1.5 Hz, 1H), 8.37 (d, J = 8 Hz, 1H),
NMR (100 MHz, CD
3
OD): d = 178.4, 175.3, 158.7, 138.0, 129.4
d = 7.31-7.20 (m, 5H), 6.36 (d, J = 7.7 Hz, 1H), 4.91 (dd, J =
(2×), 129.0, 128.8 (2×), 128.6, 84.0, 77.2, 67.7, 55.0, 53.5, 49.4,
6
3
0
1
.9, 8.0 Hz, 1H), 4.60–4.50 (m, 1H), 3.28 (d, J = 5.1 Hz, 1H),
42.0, 41.7, 41.3, 41.1, 39.1, 37.6, 29.9, 27.8, 27.4, 26.7, 25.8, 25.6,
2
0
◦
.20–3.15 (m, 2H), 2.90 (d, J = 4.7 Hz, 1H), 1.60–1.30 (m, 6H),
24.6, 23.7, 23.5, 23.2, 22.5, 22.0, 21.8. [a]
HRMS: calcd for C35
calcd for C35
D
−58 (c = 1, MeOH).
1
3
+
.89–0.82 (m, 6H). C NMR (50.1 MHz, CDCl
3
): d = 207.9, 170.3,
H
56BN
3
O H 626.43349, found 626.43408,
6
+
62.9, 147.5, 144.3, 143.9, 142.7, 136.2, 129.3, 128.6, 127.1, 58.9,
H
56BN
3
O
6
Na 648.41544, found 648.41578.
1
424 | Org. Biomol. Chem., 2007, 5, 1416–1426
This journal is © The Royal Society of Chemistry 2007

View Article Online
Ada-Ahx
3
-Leu
2
-leucinyl-boronic ester (14)
Thr(tBu)-OSu described above and DIPEA (0.90 mL, 5.4 mmol,
3
1.8 equiv.) were added. The mixture was slowly warmed to
Under argon Ada-Ahx
HOSu (35 mg, 0.30 mmol, 2.3 equiv.) were dissolved in DMF
1 mL) and DIC (25 lL, 0.16 mmol, 1.2 equiv.) was added.
After a few minutes a fine precipitate was formed. The mixture was
stirred overnight at ambient temperature after which LC/MS anal-
3
-L
2
-OH (30) (95 mg, 0.13 mmol) and
room temperature (ca. 2 h) and was stirred for another 2 h. After
filtration through a path of hyflo and concentration in vacuo the
crude product was purified by silica gel column chromatography
(
(
PE/EA/MeOH = 3/1/0 → 1/1/0 → 0/1/0 → 0/9/1 v/v/v)
affording Ac(Me)-Ile -Thr(tBu)-Leu-boronic ester (38) as a white
2
ysis showed complete conversion of Ada-Ahx
Ada-Ahx -Leu -OSu. (1R)–4-(1-chloro-3-methyl(butyl)-2,9,
-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0 ]decane (132 mg,
.46 mmol, 3.5 equiv.) was dissolved in THF (4 mL) in a flame
3
-Leu -OH (30) into
2
solid. This material was dissolved in TFA/DCM (1/1 v/v 1 mL).
After 30 min Tol (30 mL) was added and the solvents evaporated.
Purification by RP-HPLC afforded the title compound (9 mg,
3
2
2,6
10
9
0
1
14 lmol, 8%). H NMR (200 MHz, CDCl
3
): d = 7.88 (s, 1H), 7.03
dried two necked reaction flask under an argon atmosphere. At
(
4
2
d, J = 5.10 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.60–4.41 (m, 4H),
◦
−
20 C a LiHMDS solution (1 M in THF, 460 lL, 0.46 mmol,
.27 (d, J = 7.3 Hz, 1H), 4.09 (m, 1H), 3.82 (s, 1H), 2.94 (s, 3H),
3
.5 equiv.) was added dropwise. The mixture was allowed to warm
.87 (m, 1H), 2.32 (m, 1H), 2.14 (s, 3H), 2.02 (m, 2H), 1.96–1.71
◦
to room temperature and stirred overnight. At −90 C a HCl
(
m, 4H), 1.41 (s, 3H), 1.34 (m, 3H), 1.28 (s, 2H), 1.18 (s, 1H),
solution (2 M in diethyl ether, 1.00 mL, 2.0 mmol, 15.4 equiv.)
20
◦
1
.14 (s, 1H), 0.96–0.81 (m, 27H). [a]
D
−360 (c = 0.04, MeOH).
◦
was added dropwise. The mixture was slowly warmed to 0
C
+
HRMS: calcd for C34
calcd for C34
H
61BN
4
O H 649.47061, found 649.47080,
Na 671.45255, found 671.45272.
7
◦
(
ca. 2 h) and re-cooled to −80 C at which temperature the Ada-
+
H
61BN
4
O
7
Ahx
3
-Leu
2
-OSu described above and DIPEA (0.60 mL, 3.6 mmol,
2
7.7 equiv.) were added. The mixture was slowly warmed to room
temperature (ca. 2 h) at which it was stirred for another 2 h. After
filtration through a path of hyflo and concentration in vacuo the
crude product was purified by silica gel column chromatography
Ada-Ahx
3
-Ile -Thr-Leu-boronic ester (16)
2
Under argon Ada-Ahx
3
-Ile -Thr(tBu)-OH (19) (134 mg,
2
(
PE/EA/MeOH = 3/1/0 → 1/1/0 → 0/1/0 → 0/9/1 v/v/v)
0
.15 mmol) and HOSu (29 mg, 0.25 mmol, 1.7 equiv.) were
affording the title compound as a white solid (74 mg, 73 lmol,
dissolved in DMF (2 mL) and DIC (35 lL, 0.23 mmol, 1.5 equiv.)
was added. After a few minutes a fine precipitate was formed.
The mixture was stirred overnight at ambient temperature after
5
2
9%). Further purification by RP-HPLC afforded 14 (20 mg,
1
0 lmol, 16%). H NMR (600 MHz, CD
3
OD): d = 4.61 (dd,
J = 9.6, 5.4 Hz, 1H), 4.37 (dd, J = 9.6, 5.4 Hz, 1H), 4.15 (dd, J =
which LC/MS analysis showed complete conversion of Ada-Ahx
3
-
1
2
0.2, 8.4 Hz, 1H), 3.17 (t, J = 7.2 Hz, 6H), 2.71 (t, J = 7.2 Hz, 1H),
.36 (m, 1H), 2.27 (t, J = 7.2 Hz, 2H), 2.20 (t, J = 7.2 Hz, 4H), 2.15
Ile -Thr(tBu)-OH (19) into Ada-Ahx -Ile -Thr(tBu)-OSu. A flame
2
3
2
dried two necked reaction flask equipped was put under an ar-
gon atmosphere in which (1R)-4-(1-chloro-3-methyl(butyl)-2,9,9-
trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0 ]decane (132 mg,
(
m, 1H), 1.97 (m, 4H), 1.94 (s, 2H), 1.88 (m, 1H), 1.81 (m, 1H),
1
1
3
3
1
4
3
2
2
.79–1.61 (m, 23H), 1.58 (m, 2H), 1.56–1.50 (m, 6H), 1.48 (d, J =
0.2 Hz, 1H), 1.40 (s, 3H), 1.39–1.33 (m, 8H), 1.31 (s, 3H), 0.99 (s,
H), 0.98 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.93 (s,
2,6
10
◦
0
.46 mmol, 3.1 equiv.) was dissolved in THF (4 mL). At −20 C
a LiHMDS solution (1 M in THF, 460 lL, 0.46 mmol, 3.1 equiv.)
1
3
H), 0.90 (s, 3H). C NMR (150 MHz, CD
3
OD): d = 178.5, 176.4,
was added dropwise. The mixture was allowed to warm to room
75.8, 175.0, 173.6, 84.0, 77.2, 55.8, 53.5, 53.4, 51.9, 49.6, 43.8,
3.7, 41.8, 41.7, 41.4, 41.2, 40.2, 40.1, 39.2, 37.9, 37.7, 37.0, 36.6,
6.5, 33.8, 30.2, 30.1, 30.0, 29.9, 27.8, 27.6, 27.5, 27.4, 26.7, 26.6,
6.5, 26.3, 25.9, 25.7, 24.6, 23.6, 23.5, 23.4, 23.3, 23.2, 22.5, 22.1,
◦
temperature and stirred overnight. At −90 C a HCl solution
(2 M in diethyl ether, 1.00 mL, 2.0 mmol, 13.3 equiv.) was added
◦
dropwise. The mixture was slowly warmed to 0 C (ca. 2 h) and
◦
re-cooled to −80 C at which temperature the Ada-Ahx
3
-Ile -
2
2
0
◦
1.9, 21.8, 19.3, 18.8, 17.3, 13.2. [a]
D
−21.7 (c = 0.12, MeOH).
Thr(tBu)-OSu described above and DIPEA (0.60 mL, 3.6 mmol,
4 equiv.) were added. The mixture was slowly warmed to room
+
HRMS: calcd for C57
H
99BN
6
O H 1007.76722, found 1007.76902.
8
2
temperature (ca. 2 h) and was stirred for another 2 h. After
filtration through a path of hyflo and concentration in vacuo the
crude product was purified by silica gel column chromatography
(PE/EA/MeOH = 3/1/0 → 1/1/0 → 0/1/0 → 0/9/1 v/v/v)
Ac(Me)-Ile -Thr-Leu-boronic ester (15)
2
Under argon Ac(Me)-Ile
and HOSu (35 mg, 0.304 mmol, 1.8 equiv.) were dissolved in DMF
1 mL) and DIC (35 lL, 0.23 mmol, 1.4 equiv.) was added. After a
2
-Thr(tBu)-OH (31) (78 mg, 0.17 mmol)
affording Ada-Ahx
3
-Ile -Thr(tBu)-Leu-boronic ester (23) as a
2
(
white solid. This material was dissolved in TFA/DCM (1/1
v/v, 1 mL). After 30 min Tol (30 mL) was added and the
few minutes a fine precipitate was formed. The mixture was stirred
overnight at ambient temperature after which LC/MS analysis
showed complete conversion. (1R)-4-(1-chloro-3-methyl(butyl)–
solvents evaporated. Purification by RP-HPLC afforded the title
1
compound (7 mg, 6.3 lmol, 4%). H NMR (300 MHz, CD
3
OD):
2
,6
10
2
, 9, 9-trimethyl-3, 5-dioxa-4-bora-tricyclo[6.1.1.0 ]decane
d = 4.52 (dd, J = 7.2, 4.5 Hz, 1H), 4.21–4.13 (m, 4H), 3.14 (t, J =
6.9 Hz, 6H), 2.72 (dd, J = 8.4, 6.9 Hz, 1H), 2.36 (m, 1H), 2.25
(t, J = 7.5 Hz, 2H), 2.17 (t, J = 7.5 Hz, 4H), 1.94 (m, 4H), 1.91
(s, 2H), 1.79 (m, 2H), 1.77–1.43 (m, 30H), 1.41–1.26 (m, 15H),
1.23–1.11 (m, 5H), 0.94 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.91
(
168 mg, 0.59 mmol, 3.5 equiv.) was dissolved in THF (5 mL) in a
flame dried two necked reaction flask under an argon atmosphere.
◦
At −20 C a LiHMDS solution (1 M in THF, 550 lL, 0.55 mmol,
3
.2 equiv.) was added dropwise. The mixture was allowed to warm
◦
20
◦
to room temperature and stirred overnight. At −90 C a HCl
(s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.87 (s, 3H). [a]
0.08, MeOH). HRMS: calcd for C61
D
−72.5 (c =
+
solution (2 M in diethyl ether, 1.50 mL, 3.0 mmol, 17.6 equiv.) was
H
106BN
7
+
O
10
H
1108.81670,
106BN O10Na 1130.79864, found
◦
added dropwise. The mixture was slowly warmed to 0 C (ca. 2 h)
found 1108.81820, calcd for C61
1130.79987.
H
7
◦
and re-cooled to −80 C at which temperature the Ac(Me)-Ile
2
-
This journal is © The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 1416–1426 | 1425

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This work was supported by the Netherlands Organization for
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Centre. Thanks to Hans van der Elst for RP-HPLC purifications
and to Kees Erkelens and Fons Lefeber for assistance with NMR.
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426 | Org. Biomol. Chem., 2007, 5, 1416–1426
This journal is © The Royal Society of Chemistry 2007