Discovery of a potent and highly β1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones

Article abstract of DOI:10.1039/c1ob06554h

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in

Full text of DOI:10.1039/c1ob06554h

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PAPER
Discovery of a potent and highly b1 specific proteasome inhibitor from a
focused library of urea-containing peptide vinyl sulfones and peptide
epoxyketones†
Wouter A. van der Linden,a Lianne I. Willems,a Tamer B. Shabaneh,b Nan Li,a Mark Ruben,a
Bogdan I. Florea,a Gijs A. van der Marel,a Markus Kaiser,c Alexei F. Kisselev*b and Herman S. Overkleeft*a
Received 9th September 2011, Accepted 5th October 2011
DOI: 10.1039/c1ob06554h
Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show
promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido
structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide
epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the
ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the
proteasome. Compounds 13 and 15 are b5 selective and their potency exceeds that of syringolin A. In
contrast, 5 may well be the most potent b1 selective compound active in living cells reported to date.
1
Introduction
Syringolins form a class of small molecule natural products that
are secreted by strains of the plant pathogen Pseudomonas syringae
pv. syringae (Pss) when these are invading a plant.1 Syringolin A
and B (Fig. 1) are of particular interest as these molecules act upon
the plant proteasome to compromise plant defense mechanisms
against invading pathogens.2,3 The eukaryotic 20S proteasome
contains three catalytically active b subunits, b1, b2 and b5, which
display caspase-like, trypsin-like and chymotrypsin-like activity,
respectively.4 The plant 20S proteasome is homologous to the
mammalian proteasome and syringolins are potent and selective
inhibitors of the human 20S proteasome as well.5 Small molecule
proteasome inhibitors show promising antitumour activity and
bortezomib is used in the clinic against multiple myeloma,
whereas several other proteasome inhibitors are in clinical trials.6–10
Biological evaluation of syringolins on tumour cells has shown
inhibition of cell proliferation and induction of apoptosis.11,12 This
sparked the interest of several organic chemists and lately, the total
syntheses of several syringolins have been published.13–16
Fig. 1 Structure of syringolin A and B.
of syringolins and their analogues. Syringolins contain a 12-
membered lactam core structure the nature of which differs among
the members of this family of compounds. Syringolin A (SylA)
has two (E)-configured double bonds resulting in a strained
system, and syringolin B has only one ring unsaturation. The a,b
unsaturated amide in the ring system is responsible for covalently
and irreversibly inactivating the catalytic Thr1 Og of the 20S
proteasome b subunits via Michael addition.5 SylA inhibits b2 and
b5 potently and b1 at higher concentration.17 The electrophilic
lactam is quite rigid in structure and probably determines to a
large extent the proteasome specificity. Attached to the lactam
core structure is a peptoid fragment, which differs among the
syringolins. SylA and SylB contain a valine-urea-valine moiety at
their pseudo N-terminus. An ureido linkage induces chain reversal
in a peptide, and for this reason has been installed in protease
With the total synthesis of syringolin A and B completed, the
opportunity opened up to perform structure–activity relationships
aGorlaeus Laboratories, Einsteinweg 55, 2300 CC, Leiden, The Netherlands.
E-mail: h.s.overkleeft@chem.leidenuniv.nl; Fax: +31-71-5274307; Tel: +31-
71-5274342
bNorris Cotton Cancer Center, Department of Pharmacology and Toxicol-
ogy, Dartmouth Medical School, Dartmouth College, Lebanon, NH 03756,
USA. E-mail: alexei.f.kisselev@dartmouth.edu
cZMB/Faculty of Biology, Building S03 S02 A55, Universita¨t Duisburg-
Essen, Campus Essen, 45117 Essen, Germany. E-mail: Markus.Kaiser@uni-
due.de; Tel: +49-201-183-4980
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of new compounds. See DOI: 10.1039/c1ob06554h
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Fig. 2 Literature SylA modifications.
inhibitors.18–20 It is also found in several natural peptide-based
compounds with antibiotic activity.21–23 However, the syringolins
are the only examples of proteasome inhibitors that contain this
moiety. Some research has already been conducted (Fig. 2) on
the exocyclic part of syringolins, including a D-AA scan of the
two valines, revealing that the naturally occurring configuration
yields the most potent compounds.16 Addition of aliphatic15 or
hydrophilic16 tails yielded inhibitors with increased potency with
respect to SylA. Addition of a fluorophore was tolerated, yielding
a proteasome probe.24 Hybrids between SylA and glidobactin A
were designed that appear to possess improved potency compared
to SylA.17,25
The proteasome subunit selectivity pattern of an inhibitor is
important for its antitumour activity. Inhibition of b5 is essential,
but coinhibition of b1 or b2 is usually needed to be cytotoxic in cell
lines derived from various cancers.26–28 The structural elements in
syringolins that are responsible for proteasome subunit selectivity
are therefore useful in the design of new proteasome inhibitors.
To determine the effect of the valine-urea-valine motif in the
biological profile of syringolins we incorporated this moiety in
a more conventional linear peptide based proteasome inhibitor
design. In this design, we replaced the unsaturated lactam Michael
acceptor with leucine vinyl sulfone, another Michael acceptor
commonly used to arrive at potent proteasome inhibitors. A
leucine epoxyketone is used as well since this warhead often yields
more potent and selective inhibitors than the vinyl sulfone.29
The decyl chain, improving syringolin A activity,15 is also used in
this library (4 and 8). When the tripeptide structure is compared to
SylA, the distance between the electrophile of the inhibitor and the
urea group is shorter in the tripeptide than in SylA. Therefore, one
more amino acid is incorporated between the urea and electrophile,
yielding tetrapeptide inhibitors (9–16).
The synthesis of the tripeptide vinyl sulfones (VS) commenced
with coupling the TFA·H-LeuVS32 to BocValOH to yield Boc-
protected dipeptide 17 from which the Boc protecting group
was removed to yield dipeptide 18 (Fig. 4). This dipeptide was
reacted with valine tert-butyl ester isocyanate, valine benzylamide
isocyanate or decyl isocyanate to yield three inhibitors 1, 3 and 4.
Peptide 1 was deprotected with TFA to yield 2.
In an attempt to react epoxyketone (EK) TFA·H-Val-Leu-EK
with valine tert-butyl ester isocyanate, no formation of compound
5 was detected. Rather, TFA·H-Val-Leu-EK cyclizes in this basic
reaction medium. As an alternative approach to obtain compound
5, the methyl ester in compound 1915 was transformed to the
hydrazide (20) which was then in situ transformed to the acyl
azide and coupled to TFA·HLeu-EK to arrive at 5, which was
subsequently deprotected to gain 6 (Fig. 4). Compounds 7 and
8 were synthesized by a modified approach. Reacting 2133 with
valine benzylamide isocyanate or decyl isocyanate yielded 22 and
24, which were then Boc-deprotected to yield hydrazides 23 and
25 which were transformed to the acyl azide in situ and coupled to
TFA·H-Leu-EK to obtain 7 and 8.
The synthesis of tetrapeptide vinyl sulfones and epoxyketones
followed a general strategy (Fig. 5). Methyl ester 2634 was
transformed to the corresponding hydrazide 27 by hydrazine
hydrate in methanol. This compound was in situ transformed to
its acyl azide and then coupled to TFA·H-Leu-VS or TFA·H-
Leu-EK to arrive at 28 and 30. 28 was deprotected with TFA in
DCM to give 29, which in the next step was reacted with the three
isocyanates mentioned in the section above to yield tetrapeptides
9, 11 and 12. 9 was deprotected with TFA to yield 10. The same
strategy, employing 30, led to 13–16.
2
Results and discussion
The first set of target compounds for this study are displayed in
Fig. 3. Direct attachment of the leucine-derived electrophilic trap
to the valine-urea-valine moiety yields two tripeptide compounds
(2 and 6). The carboxylic acid functionality is masked as a tert-
butyl ester as its synthetic precursor (1 and 5). A benzylamide cap
is also used (3 and 7), since aromaticity at the N-terminus of a
short proteasome inhibitor could be beneficial for its potency.30,31
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Fig. 3 Panel of synthesized potential proteasome inhibitors.
In a first assessment of inhibitor potency the sixteen compounds
were subjected to a competition assay versus Bodipy-TMR-
epoxomicin (MVB003).35 First, extracts from HEK293T cells were
incubated with a broad concentration range of inhibitor for one
hour, after which remaining proteasome activity was labeled with
MVB003. After SDS-PAGE separation of the proteome, the wet
gel slabs were scanned on a Typhoon fluorescence scanner. Results
are displayed in Fig. 6. Proteasome subunits were assigned based
on earlier work.35
One first obvious conclusion from this broad concentration
scan is that the syringolin A inspired ureido peptide moiety,
coupled to leucine epoxyketone or vinyl sulfone, yields active
proteasome inhibitors. Generally, the inhibitors containing a vinyl
sulfone are less potent than their epoxyketone counterparts, a
phenomenon observed earlier.27,29 A second obvious trend is that
tripeptide inhibitors show some selectivity for b1. The most
distinct selectivity for b1 is displayed by 5 in HEK lysate (Fig.
6). The tetrapeptide inhibitors, on the other hand, appear to
preferentially target b5. Compounds containing the long aliphatic
tail (4, 8, 12, 16) are far less potent than their counterparts lacking
this moiety. This is in sharp contrast to SylA, where the addition
of this tail increased potency dramatically.15
range. In HEK lysate (Fig. 7), several library members indeed are
subunit selective; 9 inhibits b5 by more than 90% at about 1 mM,
at which concentration b1 and b2 appear untouched. Compound
13 fully inhibits b5 at 100 nM, before inhibiting b1 and b2.
Compound 5 is very selective for the b1 subunit. Compared to
NC001, a very selective tetrapeptide epoxyketone, compound 5 is
only slightly less potent in HEK lysate.26,36 The benzyl amides
in this library are generally less selective than their tert-butyl
counterparts; 11 coinhibits b2 before complete b5 inhibition is
reached. Benzyl amide 7 coinhibits b5 much earlier than the
selective tert-butyl ester 5. The difference in selectivity between
13 and 15, however, is less pronounced.
The parent compound, SylA, inhibits b5 and b2 in the low
micromolar range and only inhibits b1 at higher concentration
(Fig. 7). In this respect, the activities of the tetrapeptide inhibitors
of this library in general more resemble that of SylA than the
activities of the tripeptides do. Interestingly, there are several
compounds in this library that display more potent proteasome
inhibition than the parent compound, SylA. Compounds 13 and
15 are the most potent inhibitors in this study; compared to
SylA in HEK lysate (Fig. 7), these two compounds inhibit b5 at
about 100-fold lower concentration. The most potent and selective
compounds were also analysed for their inhibitory capacities
against purified rabbit 26S proteasome with subunit specific
fluorogenic peptides. To better compare these results with the
competition assay gels from Fig. 7, the gel bands were quantified
and plotted against inhibitor concentrations. From these plots,
To gain better insight in subunit selectivity of the most potent
inhibitors, the competition assay was repeated in a more detailed
concentration series (Fig. 7). Tripeptide vinyl sulfones 2–4 as well
as 8 and 12 were omitted because the inhibitory effect of these
compounds falls mostly outside the more diluted concentration
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Fig. 4 Synthesis of tripeptide vinyl sulfones and epoxyketones.
the IC50 values were calculated and the results are summarised in
Table 1. The IC50 values found with the fluorogenic peptide assay
are higher than those found in the competition assay, but one
should be careful when comparing IC50 values obtained with two
very different experimental setups. The subunit selectivity trends
found with the fluorogenic peptide assay, however, in most cases
agree with the results from the competition assay. In both settings,
5 is a selective b1 proteasome inhibitor, while 15 is a selective b5
inhibitor. In the fluorogenic substrate assay, compound 13 shows
onset of inhibition of the b1 and b2 sites when b5 is targeted, while
in the competition assay this compound appears more selective.
We have no explanation for this discrepancy yet. The differences
could be caused by the presence of other proteasome forms in
cell lysate (i.e. PA200 and PA28 activated proteasomes) or post-
translational modifications that affect active site specificity and
that are either species or tissue specific and may be lost during
preparation of proteasomes from muscle.
of these compounds and compared to literature subunit selective
proteasome inhibitors NC001 and NC005-VS.26,36 Living HEK
cells were incubated with the inhibitors for 4 h. after which re-
maining proteasomal activity was labeled by cell permeable probe
MVB003. The results are summarised in Table 2. Interestingly, 5
shows fivefold higher potency in living HEK cells than NC001,
while NC001 is slightly more potent in cell extract. This difference
could be ascribed to better cell permeability of 5, possibly due to
its smaller size than NC001. Compound 5 is the most potent and
selective b1 inhibitor to date. Compounds 13 and 15 do inhibit the
proteasome in living HEK cells and therefore are cell permeable,
but their b5 selectivity is much lower than observed in HEK
extracts and NC005-VS is much more b5 selective in cells than
13 and 15.
Having evaluated a small library of proteasome inhibitors
containing tri- and tetrapeptides with the valine-urea-valine motif,
a dramatic shift in selectivity from b1 to b5 is observed when
the ureido linkage is moved one place in the molecule. We chose
a tetrapeptide vinyl sulfone scaffold in which the ureido-linkage
Compounds 5, 13 and 15 were evaluated on their proteasomal
inhibition capacities in living HEK cells to assess cell permeability
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Fig. 5 Synthesis of tetrapeptide vinyl sulfones and epoxyketones.
Table 1 Apparent IC50 (mM) values calculated from semi log plots of residual proteasome activity against inhibitor concentration. Either band intensities
from each lane of the competition assay gels in Fig. 7 were quantified and used as input, or 26S proteasomes, purified from rabbit muscles, were incubated
with different concentrations of inhibitors for 30 min at 37 ◦C followed by measuring remaining activity with fluorogenic peptides (Suc-LLVY-AMC, b
5, Ac-LPnLD-AMC, b1, Ac-RLR-AMC, b2)
Quantified competition assay gels
Fluorogenic peptide assay
b5
b1
b2
b5
b1
b2
Compound
IC50 (mM)
IC50 (mM)
>30
5
~50
>50
2.9
0.23
4.2
0.17
0.016
0.48
0.008
0.5
0.46
1.4
0.52
>50
>50
>50
1.3
2.8
2.4
>50
25
0.33
>50
>50
>50
>50
>50
2.1
0.69
5.9
0.28
>50
6.6
>30
6
7
>10
1.0
1.2
>30
>10
>30
9
10
11
0.64
0.3
18
>0.3
10
>0.3
13
14
15
0.013
0.24
>0.3
16
SylA
NC001
1.3
>50
>50
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Fig. 6 Competition assay in HEK293T lysate. Lysates (15 mg) were incubated with indicated end concentration of inhibitor for 1 h at 37 ◦C. Residual
proteasome activity was labeled by MVB003 (0.5 mM end concentration) for 1 h at 37 ◦C). Upper band b2 (ꢀ), middle band b1 (ꢁ), lower band b5 ( ).
᭹
Fig. 7 Competition assay in HEK293T lysate. Lysates (15 mg) were incubated with indicated end concentration of inhibitor for 1 h at 37 ◦C. Residual
proteasome activity was labeled by MVB003 (0.5 mM end concentration) for 1 h at 37 ◦C. N.D. not determined. Upper band b2 (ꢀ), middle band b1 (ꢁ),
lower band b5 ( ).
᭹
is ‘shifted’ through the molecule to arrive at two more potential
proteasome inhibitors with the urea after the first or second amino
acid in the peptide instead of the third (Fig. 8). The Fmoc group
in 34 and 3237 was removed by DBU.38 Dipeptide 35 was coupled
to Fmoc-Val-OH and again deprotected with DBU to arrive at
tripeptide 37. TFA·H-Leu-VS was converted to its isocyanate
by phosgene and DiPEA in DCM15 and subsequently reacted
with amine 33 which resulted in tetrapeptide 40. Amine 33 was
bition. Apparently, the place of the ureido-linkage in the peptide
inhibitor determines its activity and selectivity for proteasome
subunits. The lack of activity could also be the result of ‘inversed’
amino acid side chain configuration caused by chain reversal due
to the ureido linkage. Substitution for D-amino acids at P2-4 for
40 or P3 and P4 for 42 might restore activity of these scaffolds.
3
Conclusions
39
converted to the isocyanate by phosgene and sat. aq. NaHCO3
and reacted with amine 18 which resulted in tetrapeptide 42.
Compounds 40 and 42 were subjected to the competition assay in
HEK lysate versus MVB003 (Fig. 9). Only at high concentrations
(100 mM), these two compounds show limited proteasome inhi-
Incorporating the exocyclic valine-urea-valine motif, which is
found in syringolin A, into a vinyl sulfone or epoxyketone
oligopeptide results in a new set of potent proteasome inhibitors.
In general, epoxyketone containing inhibitors are more potent
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Fig. 8 Synthesis of two potential tetrapeptide proteasome inhibitors with ureido-linkage after P1 or P2.
Fig. 9 Competition assay in HEK lysate (15 mg protein). Lysates were incubated with indicated end concentration of inhibitor for 1 h at 37 ◦C. Residual
proteasome activity was labeled by MVB003 (0.5 mM end concentration) for 1 h at 37 ◦C. Upper band b2 (ꢀ), middle band b1 (ꢁ), lower band b5 ( ).
᭹
than their vinyl sulfone counterparts, but this increase in potency
is often accompanied by a decrease of subunit selectivity, a
phenomenon observed earlier.27,29,36 The position of the ureido
linkage with respect to the electrophilic trap has a profound
effect on proteasome subunit selectivity. A short distance, as in
compounds 1–4 and 5–8 results in preference for the b1 subunit
of the proteasome. Tripeptide epoxyketone 5 is a potent and very
selective inhibitor of the b1 subunit. In living cells, this compound
is fivefold more potent than NC001 and is the most potent b1
selective compound known to date. Further optimisation of this
compound, for example by changing the P2 valine for a norleucine
residue or changing the tert-butyl for a smaller methyl group,
might make this molecule even more selective for b1. When an
extra amino acid is incorporated in between the ureido linkage
and the warhead, as in inhibitors 9–12 and 13–16, a preference for
the b5 subunit is observed, with tetrapeptide epoxyketones 13 and
15 being quite selective inhibitors of the b5 site. When the ureido
linkage is incorporated at different places in the peptide, two more
compounds are obtained, 40 and 42, but both are hardly active
as a proteasome inhibitors. Tetrapeptide epoxyketones 13 and 15
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Table 2 Apparent IC50 (mM) values calculated from semi log plots
of residual proteasome activity against inhibitor concentration. Band
intensities from each lane of competition assay gels in living cells
(Supplementary Information S2) were quantified and used as input. N.I.
no inhibition
obtained by literature procedures. NC001 and NC005-mvs were
obtained as described.26,36
General procedure formation of isocyanates A15. Amine or
amine HCl salt (1 equiv.) was dissolved in DCM and DiPEA (2.2
equiv.) was added. This solution was slowly added to a solution
of triphosgene (0.35 equiv.) or phosgene (1.1 equiv., 20 wt-% sln
in Tol.) and the mixture was stirred for an additional 10 min after
addition and used in the next step without any purification.
Quantified competition assay gels
b5
b1
b2
Compound
IC50 (mM)
General procedure formation of isocyanates B39. HCl·H-
ValNHBn42 was dissolved in 1 : 1 DCM : sat. aq. NaHCO3 at 0 ◦C.
The layers were allowed to separate and phosgene (1.2 equiv.,
20 wt-% in Tol.) was added to the DCM layer and stirring was
continued for 10 min after which the mixture was extracted with
DCM (3¥). The organic layers were combined and dried over
Na2SO4 and concentrated. The resulting crude isocyanate was
used without further purification.
5
6.7
0.065
0.34
0.31
0.16
N.I.
>10
>10
0.23
0.063
N.I.
NC001
13
>10
<0.05a
<0.05a
0.058
15
NC001
a About 80% inhibition of activity observed at 50 nM.
are the most potent compounds in this library, more potent than
the parent compound SylA. The subunit selectivity pattern of 15
shows similarities to that of SylA, but displays higher potency,
indicating that the antitumour activity of this compound should
be assessed. The activities of the tetrapeptide inhibitors in this
study resemble those of compounds Z-L3-EK and Z-L3-VS in
terms of selectivity. Z-L3-EK and Z-L3-VS have a slight preference
for b5 while the tetrapeptides in this study appear to possess more
b5 selectivity.29 Translating the findings of this study back to the
case of syringolin A meets difficulties. One could envision that the
molecular basis of selectivity of 5, 13 and 15 could be unraveled
with the crystal structures of these compounds in complex with
20S proteasomes.5,40,41
General procedure for azide coupling. Hydrazide or hydrazide
HCl (1 equiv.) was dissolved in DMF : EA 3 : 1 and cooled to -30 ◦
C. tBuONO (1.1 equiv.) and HCl (4 M in dioxane, 2.8 equiv.) were
added and the resulting mixture stirred for 3 h. TFA·H-Leu-EK
or TFA·H-Leu-VS (1.1 equiv.) in DMF was added followed by
DiPEA (5 equiv.) and the mixture was allowed to warm to RT
overnight. The mixture was diluted with EA and washed with H2O
(3¥). The organic layer was dried over Na2SO4 and concentrated
before flash column chromatography.
Boc-Val-Leu-VS (17). Boc-Val-OH (359 mg, 1.65 mmol, 1.1
equiv.) was dissolved in EA. HBTU (683 mg, 1.8 mmol, 1.2
equiv.), DiPEA 0.87 mL, 5.25 mmol, 3.5 equiv.) and TFA·LeuVS
(1.5 mmol, 1 equiv.) were added and the mixture stirred for 4 h.
The mixture was washed with 1 M HCl (3¥), sat. aq. NaHCO3
(3¥) and brine before being dried over Na2SO4. The residue was
purified by column chromatography (30% EA : PE→60% EA : PE)
to yield the title compound (587 mg, 1.5 mmol, quant.). 1H-NMR
NMR (400 MHz, CDCl3): d 6.88 (d, J = 6.6 Hz, 1H), 6.79 (dd,
J1 = 15.1, J2 = 5.3 Hz, 1H), 6.52 (d, J = 15.1 Hz, 1H), 5.33 (d, J =
7.5 Hz, 1H), 4.87–4.62 (m, 1H), 3.86 (dd, J1 = 8.3, J2 = 7.1 Hz,
1H), 2.89 (s, 3H), 2.15–2.00 (m, 1H), 1.70–1.56 (m, 1H), 1.40 (s,
9H), 0.96–0.81 (m, 12H). 13C NMR (100 MHz, CDCl3): d 171.63,
155.89, 147.63, 129.16, 79.80, 60.25, 47.65, 42.62, 42.48, 30.17,
28.15, 24.50, 22.63, 21.65, 19.27, 17.85.
4
Experimental
4.1 Synthesis
All reagents were commercial grade and were used as received
unless indicated otherwise. Toluene (Tol.) (purum), ethyl acetate
(EtOAc) (puriss.), and light petroleum ether (PetEt) (puriss.) were
obtained from Riedel-de Hae¨n or Biosolve and were distilled prior
to use. Dichloromethane (DCM), dimethylformamide (DMF),
˚
and dioxane were stored on 4 A molecular sieves. Tetrahydrofuran
(THF) was distilled from LiAlH4 prior to use. Reactions were
conducted under an argon atmosphere. Reactions were monitored
by TLC analysis by using DC-fertigfolien (Schleicher & Schuell,
F1500, LS254) with detection by UV absorption (254 nm),
spraying with 20% H2SO4 in ethanol or (NH4)6Mo7O24C4H2O (25 g
L-1) and (NH4)4Ce(SO4)4C2H2O (10 g L-1) in 10% sulfuric acid
followed by charring at ~150 ◦C or by spraying with an aqueous so-
lution of KMnO4 (7%) and KOH (2%). Column chromatography
was performed on Screening devices (0.040–0.063 nm). LC/MS
analysis was performed on a LCQ AdvantageMax (Thermo
Finnigan) equipped with an Gemini C18 column (Phenomenex).
HRMS were recorded on a LTQ Orbitrap (Thermo Finnigan). 1H-
and 13C-APT-NMR spectra were recorded on a Bruker DPX-300
(300/75 MHz) or Bruker AV-400 (400/100 MHz) equipped with
a pulsed field gradient accessory. Chemical shifts are given in ppm
(d) relative to tetramethylsilane as an internal standard. Coupling
constants are given in Hz. All presented 13C-APT spectra are
proton decoupled. TFA.H-Leu-EK2 and TFA.H-Leu-VS32 were
TFA·H-Val-Leu-VS (18). Boc-Val-Leu-VS (17) was stirred in
1 : 1 DCM : TFA for 30 min before coevaporation with toluene
(3¥) yielded the title compound, which was immediately used in
the next reaction without further purification.
tBuO-Val-urea-Val-Leu-VS (1). A solution of TFA·H-Val-
Leu-VS (18, 305 mmol, 1 equiv.) and DiPEA (111 ml, 671 mmol,
2.2 equiv.) in DCM was added to the isocyanate of valine tert-
butyl ester (obtained from HCl·H-Val-OtBu (64 mg, 305 mmol, 1
equiv.) employing general procedure A). The remaining solution
was stirred for 1 h and concentrated. The residue was dissolved
in EA and washed with 1 M HCl (2¥), sat. aq. NaHCO3 and
brine before being dried over Na2SO4. Column chromatography
(40% EA : PE→70% EA : PE) yielded the title compound (119 mg,
243 mmol, 80%). LCMS: Rt 10.28 min (linear gradient 10 → 90%
ACN + 0.1% TFA, 15 min). 1H NMR (400 MHz, CDCl3): d (ppm)
7.42 (d, J = 7.98 Hz, 1H), 6.80 (dd, J1 = 15.06, J2 = 5.76 Hz, 1H),
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6.70 (d, J = 15.16 Hz, 1H), 6.34 (d, J = 7.99 Hz, 1H), 6.00 (d, J =
8.97 Hz, 1H), 4.76–4.67 (m, 1H), 4.24 (dd, J1 = 8.82, J2 = 5.29 Hz,
1H), 4.03 (t, J = 8.40, 1H), 2.93 (s, 3H), 2.13–1.94 (m, 2H), 1.73–
1.58 (m, 1H), 1.47 (s, 9H), 1.46–1.38 (m, 2H), 0.97–0.84 (m, 18H).
13C NMR (100 MHz, CDCl3): d (ppm) 219.35, 172.97, 172.34,
158.28, 147.04, 129.88, 81.59, 60.16, 58.69, 48.02, 42.66, 42.53,
31.37, 30.76, 28.04, 24.60, 22.56, 22.08, 19.47, 18.99, 18.56, 17.98.
HRMS calcd. for [C23H44N3O6S1]+ 490.29453, found 490.29445.
29.52, 29.32, 27.06, 24.66, 22.75, 22.64, 22.25, 19.52, 18.82, 14.06.
HRMS calcd. for [C24H48N3O4S1]+ 474.33600, found 474.33595.
tBuO-Val-urea-Val-NHNH2 (20). Methyl ester 19 15 (234 mg,
708 mmol, 1 equiv.) was dissolved in MeOH. Hydrazine hydrate
(1 ml, 21 mmol, 30 equiv.) was added and the mixture refluxed
overnight. Coevaporation with toluene (4¥) yielded the title
compound which was used without further purification. 1H NMR
(400 MHz, CD3OD): d 6.41 (dd, J1 = 12.3, J2 = 9.1 Hz, 1H),
4.17–4.00 (m, 1H), 3.99–3.84 (m, 1H), 2.06 (dt, J1 = 12.1, J2 = 6.8
HO-Val-urea-Val-Leu-VS (2). 1 was stirred in 1 ml TFA for
45 min before being coevaporated to yield the title compound in
quantitative yield. LCMS: Rt 6.19 min (linear gradient 10 → 90%
Hz, 1H), 2.02–1.89 (m, 1H), 1.46 (s, 9H), 1.01–0.85 (m, 12H). 13
C
NMR (100 MHz, CD3OD): d 173.87, 173.48, 160.37, 82.45, 60.25,
60.18, 59.27, 59.19, 32.58, 32.11, 28.32, 28.21, 19.67, 19.59, 18.53,
18.05.
1
ACN + 0.1% TFA, 15 min). H-NMR (400 MHz, CD3OD): d
(ppm) 6.78 (dd, J1 = 15.17, J2 = 5.29 Hz, 1H), 6.63 (dd, J1 = 15.18,
J2 1.34 Hz, 1H), 4.73–4.64 (m, 1H), 4.19 (d, J = 4.72 Hz, 1H),
3.99 (d, J = 6.60 Hz, 1H), 2.95 (s, 3H), 2.21–2.09 (m, 1H), 2.08–
1.99 (m, 1H), 1.78–1.62 (m, 1H), 1.60–1.50 (m, 1H), 1.49–1.38 (m,
1H), 1.03–0.84 (m, 18H). 13C NMR (100 MHz, CD3OD): d (ppm)
176.02, 174.72, 160.55, 148.57, 130.92, 60.94, 59.47, 49.14, 43.35,
42.83, 32.18, 32.05, 25.86, 23.37, 21.94, 19.96, 19.75, 18.30, 17.94.
HRMS calcd. for [C19H36N3O6S1]+ 434.23193, found 434.23186.
tBuO-Val-urea-Val-Leu-EK2 (5). Prepared by the general pro-
cedure for azide coupling employing 20 (33 mg, 100 mmol, 1 equiv.)
and TFA·H-Leu-EK (31 mg, 110 mmol, 1.1 equiv.). Purification by
column chromatography (DCM →1% MeOH : DCM, 2¥) yielded
the title compound (22 mg, 47 mmol, 47%). LCMS: Rt 8.93 min
(linear gradient 10 → 90% ACN + 0.1% TFA, 15 min). 1H NMR
(400 MHz, CDCl3): d (ppm) 6.49 (d, J = 7.55 Hz, 1H), 5.51 (d,
J = 8.52 Hz, 1H), 5.39 (d, J = 8.82 Hz, 1H), 4.60 (ddd, J1 = 10.60,
J2 = 7.72, J3 = 3.23 Hz, 1H), 4.26 (dd, J1 = 8.78, J2 = 4.62 Hz, 1H),
4.08 (dd, J1 = 8.56, J2 = 7.07 Hz, 1H), 3.36 (d, J = 4.98 Hz, 1H),
2.88 (d, J = 4.97 Hz, 1H), 2.15–1.93 (m, 2H), 1.76–1.59 (m, 2H),
1.46 (s, 9H), 1.51 (s, 3H), 1.58–1.51 (m, 1H), 0.95–0.82 (m, 18H).
13 C NMR (100 MHz, CDCl3): d (ppm) 208.44, 172.72, 172.24,
157.72, 81.63, 59.27, 59.11, 58.47, 52.48, 50.41, 39.97, 31.49, 31.18,
29.71, 28.08, 25.13, 23.36, 21.33, 19.27, 18.98, 18.10, 17.65, 16.78.
HRMS calcd. for [C24H44N3O6]+ 470.32246, found 470.32233.
Benzylamide-Val-urea-Val-Leu-VS (3). Vinyl sulfone 18
(225 mM, 1 equiv.) and DiPEA (41 ml, 241 mmol, 1.1 equiv.) were
dissolved in DCM and this mixture was added to the isocyanate of
valine benzylamide (obtained from HCl·H-Val-NHBn42 (104 mg,
338 mmol, 1.5 equiv.) employing general procedure B). The
remaining solution was stirred overnight and concentrated. The
residue was dissolved in DCM and washed with H2O (2¥)
and dried over Na2SO4. Column chromatography (DCM→10%
MeOH : DCM yielded the title compound (43 mg, 81 mmol, 36%).
LCMS: Rt 7.53 min (linear gradient 10 → 90% ACN + 0.1% TFA,
15 min). 1H NMR (400 MHz, CDCl3/CD3OD 1/1): d (ppm) 7.36–
7.21 (m, 5H), 6.81 (dd, J1 = 15.13, J2 = 5.17 Hz, 1H), 6.59 (dd,
J1 = 15.12, J2 = 1.49 Hz, 1H), 4.75–4.65 (m, 1H), 4.41 (q, J =
14.94, 2H), 4.02 (d, J = 6.69 Hz, 1H), 3.96 (d, J = 6.64 Hz, 1H),
2.96 (s, 3H), 2.06 (m, 2H), 1.74–1.61 (m, 1H), 1.59–1.37 (m, 2H),
1.02–0.84 (m, 18H).). 13C NMR (100 MHz, CDCl3/CD3OD): d
(ppm) 172.64, 147.33, 128.73, 128.00, 127.02, 126.78, 59.24, 58.95,
47.35, 42.71, 41.92, 41.84, 30.66, 30.25, 24.22, 22.08, 20.90, 18.70,
18.60, 17.14, 17.09. HRMS calcd. for [C26H43N4O5S1]+ 523.29487,
found 523.29478.
HO-Val-urea-Val-Leu-EK (6). 5 (13 mg, 28 mmol) was stirred
with 2 mL TFA for 1 h before being coevaporated with toluene
(3¥) to yield the title compound quantitatively. LCMS: Rt 9.05 min
(linear gradient 10 → 90% ACN + 0.1% TFA, 15 min). 1H-NMR
(400 MHz, CD3OD): d 4.56 (dd, J1 = 10.6, J2 = 3.0 Hz, 1H), 4.20
(d, J = 4.8 Hz, 1H), 4.06 (d, J = 6.6 Hz, 1H), 3.29 (d, J = 5.1 Hz,
1H), 2.95 (d, J = 5.1 Hz, 1H), 2.16 (dq, J1 = 13.6, J2 = 6.8 Hz, 1H),
2.11–1.90 (m, 1H), 1.84–1.22 (m, 3H), 1.48 (s, 3H), 1.16–0.78 (m,
18H). 13C NMR (100 MHz, CD3OD): d 209.58, 176.05, 174.89,
160.41, 60.09, 59.98, 59.39, 53.06, 51.79, 40.25, 32.45, 32.03, 26.25,
23.75, 21.47, 19.79, 19.71, 18.24, 17.86, 17.04. HRMS calcd. for
[C20H36N3O6]+ 414.25986 found 414.25989.
Decyl-urea-Val-Leu-VS (4). Vinyl sulfone 18 (266 mM, 1
equiv.) and DiPEA (100 ml, 585 mmol, 2.2 equiv.) were dissolved
in DCM and this solution was added to 1-isocyanatodecane
(obtained from decyl amine (58 ml, 293 mmol, 1.1 equiv.) employing
general procedure A). The remaining solution was stirred for 1
h and washed with 1 M HCl (3¥) and sat. aq. NaHCO3 and
the solution was dried over Na2SO4. Column chromatography
(40% EA : PE→80% EA : PE) yielded the title compound (85 mg,
Benzylamide-Val-urea-Val-NHNHBoc (22). A solution of H-
Val-NHNHBoc33 (260 mg, 1.12 mmol, 1.2 equiv.) and DiPEA
(203 ml, 1.23 mmol, 1.2 equiv.) in DCM was added to a solution of
valine benzylamide isocyanate (prepared from HCl·H-Val-NHBn
(229 mg, 943 mmol, 1 equiv.) using general procedure B) in DCM.
The resulting mixture was stirred for 2 h. The mixture was washed
with 1 M HCl (2¥) and brine and dried over MgSO4. Column
chromatography (DCM → 5% MeOH : DCM) yielded the title
compound (230 mg, 496 mmol, 53%). LCMS: Rt 7.08 min (linear
gradient 10 → 90% ACN + 0.1% TFA, 15 min), (ESI-MS (m/z):
464.00 (M + H)+). 1H NMR (400 MHz, CD3OD, CDCl3): d 7.89
(t, J = 5.7 Hz, 1H), 7.37–7.18 (m, 5H), 4.43–4.29 (m, 2H), 4.02
(d, J = 6.5 Hz, 1H), 3.96 (d, J = 6.7 Hz, 1H), 2.19–1.91 (m, 2H),
1.44 (s, 9H), 1.00–0.83 (m, 12H). 13C NMR (100 MHz, CD3OD,
CDCl3): d 172.70, 158.11, 137.59, 127.78, 126.82, 126.52, 58.93,
57.01, 42.46, 30.72, 30.39, 27.16, 18.37, 18.27, 16.97.
1
179 mmol, 67%). H NMR (400 MHz, CDCl3): d (ppm) 8.28 (d,
J = 7.54 Hz, 1H), 6.88 (dd, J1 = 15.10, J2 = 6.04 Hz, 1H), 6.68
(d, J = 14.98 Hz, 1H), 6.55–6.43 (m, 1H), 5.91–5.83 (m, 1H),
4.77–4.64 (m, 1H), 4.12–4.04 (m, 1H), 3.24–3.09 (m, 1H), 3.05–
2.95 (m, 1H), 2.93 (s, 1H), 2.02–1.85 (m, 1H), 1.72–1.58 (m, 1H),
1.54–1.35 (m, 5H), 1.33–1.21 (m, 16H), 1.02–0.81 (m, 12H). 13C
NMR (100 MHz, CDCl3): d (ppm) 173.49, 158.70, 147.46, 129.73,
60.05, 47.94, 42.73, 42.69, 40.42, 31.87, 31.35, 30.47, 29.64, 29.60,
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Benzylamide-Val-urea-Val-NHNH3Cl (23). 22 (250 mg,
600 mmol) was stirred with 4 ml 4 M HCl in dioxane for 1 h before
coevaporation with toluene (3¥), yielding the title compound
which was used without purification. LCMS: Rt 4.87 min (linear
gradient 10 → 90% ACN + 0.1% TFA, 15 min), (ESI-MS (m/z):
(d, J = 7.4 Hz, 1H), 5.56 (d, J = 8.5 Hz, 1H), 5.03 (t, J = 5.6 Hz,
1H), 4.71–4.43 (m, 1H), 4.10 (t, J = 7.8 Hz, 1H), 3.35 (d, J = 5.0
Hz, 1H), 3.25–3.00 (m, 2H), 2.89 (d, J = 5.0 Hz, 1H), 2.10–1.85 (m,
1H), 1.52 (s, 3H), 1.73–1.36 (m, 3H), 1.40–1.16 (m, 16H), 0.97–0.80
(m, 15H). 13C NMR (100 MHz, CDCl3): d 208.37, 173.26, 158.22,
59.27, 59.15, 52.41, 50.56, 40.75, 40.57, 39.63, 31.88, 31.40, 30.28,
30.20, 29.68, 29.57, 29.40, 29.34, 29.31, 26.95, 25.16, 23.33, 22.66,
21.29, 19.19, 18.23, 16.78, 14.07. HRMS calcd. for [C25H48N3O4]+
454.36393, found 454.36392.
1
364.00 (M - Cl-)+). H NMR (400 MHz, DMSO): d 9.26 (t, J =
5.9 Hz, 1H), 8.31–7.77 (m, 5H), 7.31 (d, J = 8.2 Hz, 1H), 7.18
(d, J = 9.1 Hz, 1H), 5.11 (t, J = 6.3 Hz, 1H), 4.98–4.74 (m, 1H),
2.90–2.54 (m, 2H), 1.98–1.45 (m, 12H).
Benzylamide-Val-urea-Val-Leu-EK (7). Prepared by the gen-
eral procedure for azide coupling employing 23 (39 mg, 100 mmol,
1 equiv.) and TFA·HLeu-EK (31 mg, 110 mmol, 1.1 equiv.). Purifi-
cation by column chromatography (DCM→3% MeOH : DCM)
yielded the title compound (10 mg, 20 mmol, 20%). LCMS: Rt
8.41 min (linear gradient 10 → 90% ACN + 0.1% TFA, 15 min).
1H-NMR (400 MHz, CDCl3 : CD3OD): d 7.43–7.18 (m, 5H), 4.54
(dd, J1 = 10.6, J2 = 3.1 Hz, 1H), 4.37 (dd, J1 = 36.1, J2 = 14.9 Hz,
2H), 4.04 (dd, J1 = 6.7, J2 = 4.9 Hz, 2H), 3.29 (d, J = 5.0 Hz, 1H),
2.91 (d, J = 5.1 Hz, 1H), 2.09–1.93 (m, 2H), 1.80–1.64 (m, 1H),
1.48 (s, 3H), 1.57–1.21 (m, 1H), 0.99–0.85 (m, 18H). 13C NMR (100
MHz, CDCl3, CD3OD): d 208.39, 173.42, 173.29, 158.71, 138.24,
128.21, 127.28, 126.93, 59.28, 58.85, 58.71, 51.96, 50.41, 42.84,
39.06, 31.08, 31.05, 24.94, 22.69, 20.49, 18.74, 18.71, 17.30, 16.05.
HRMS calcd. for [C27H43N4O5]+ 503.32280, found 503.32271.
Boc-Val-Leu-NHNH2 (27). Methyl ester 2634 was dissolved in
50 ml MeOH and hydrazine hydrate (9.7 ml, 200 mmol, 20 equiv.)
was added and the mixture refluxed for 2 h. The reaction mixture
was coevaporated with toluene. (5¥) and the residue used without
further purification. H NMR (400 MHz, CD3OD): d 4.43 (dd,
J = 9.4, 5.6 Hz, 1H), 3.86 (d, J = 7.0 Hz, 1H), 2.03 (dq, J = 13.6,
1
6.7 Hz, 1H), 1.91–1.49 (m, 3H), 1.48 (s, 9H), 1.10–0.79 (m, 12H).
Boc-Val-Leu2-VS (28). Prepared from 27 (344 mg, 1 mmol, 1
equiv.) and TFA·H-Leu-VS (336 mg, 1.1 mmol, 1.1 equiv.) using
the general procedure for azide coupling. Column chromatogra-
phy (Tol. → 80% EA : Tol.) yielded the title compound (362 mg,
1
719 mmol, 72%). H NMR (400 MHz, CDCl3): d 7.39–7.22 (m,
1H), 6.97 (d, J = 6.8 Hz, 1H), 6.85 (dd, J1 = 15.1, J2 = 5.3 Hz,
1H), 6.61 (d, J = 15.1 Hz, 1H), 5.45 (d, J = 6.6 Hz, 1H), 4.85–4.69
(m, 1H), 4.57–4.42 (m, 1H), 3.92 (t, J = 6.7 Hz, 1H), 2.94 (s, 3H),
2.14 (dq, J1 = 13.5, J2 = 6.8 Hz, 1H), 1.76–1.39 (m, 6H), 1.45 (s,
9H), 0.97–0.91 (m, 18H). 13C NMR (100 MHz, CDCl3): d 172.02,
171.57, 156.30, 147.44, 129.36, 80.22, 60.69, 52.16, 47.72, 42.76,
42.71, 40.41, 30.09, 28.25, 24.87, 24.61, 22.83, 22.58, 21.91, 19.23,
17.94.
Decyl-urea-Val-NHNHBoc (24).
A
solution of H-Val-
NHNHBoc33 (237 mg, 1.02 mmol, 1 equiv.) and DiPEA (185 ml,
1.12 mmol, 1.1 equiv.) in DCM was added to a solution of decyl
isocyanate (prepared from decylamine (213 ml, 1.07 mmol, 1.05
equiv.) using general procedure A) in DCM. The resulting mixture
was stirred for 1 h. The mixture was washed with 1 M HCl (2¥)
and brine and dried over MgSO4. Column chromatography (PE →
40% acetone : PE) yielded the title compound (250 mg, 600 mmol,
60%). LCMS: Rt 10.23 min (linear gradient 10 →90% ACN +
0.1% TFA, 15 min), (ESI-MS (m/z): 415.00 (M + H)+). 1H NMR
(400 MHz, CD3OD, CDCl3): d 6.03 (d, J = 8.8 Hz, 1H), 5.98 (t,
J = 5.2 Hz, 1H), 5.56 (t, J = 5.1 Hz, 1H), 4.07 (s, 1H), 3.22–2.99
(m, 2H), 2.09–2.00 (m, 1H), 1.47 (s, 9H), 1.34–1.20 (m, 16H), 0.98
(dd, J1 = 14.5, J2 = 6.8 Hz, 6H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, CD3OD, CDCl3): d 172.75, 158.61, 57.04, 39.53, 31.38,
30.71, 29.71, 29.61, 29.10, 29.06, 28.89, 28.80, 27.43, 26.40, 22.12,
18.52, 17.27, 13.36.
Boc-Val-Leu2-EK (30). Prepared from 27 (344 mg, 1 mmol, 1
equiv.) and TFA·H-Leu-EK (314 mg, 1.1 mmol, 1.1 equiv.) using
the general procedure for azide coupling. Column chromatogra-
phy (20% EA : Tol. → 60% EA : Tol.) yielded the title compound
1
(437 mg, 903 mmol, 90%). H NMR (400 MHz, CDCl3): d 7.59
(d, J = 7.1 Hz, 1H), 7.44 (d, J = 6.9 Hz, 1H), 5.95 (d, J = 9.0 Hz,
1H), 4.70–4.45 (m, 2H), 4.05 (t, J = 8.5 Hz, 1H), 3.45 (d, J = 4.2
Hz, 1H), 2.87 (d, J = 5.0 Hz, 1H), 2.09–1.94 (m, 1H), 1.75–1.30
(m, 6H), 1.51 (s, 3H), 1.45 (s, 9H), 0.98–0.82 (m, 18H). 13C NMR
(100 MHz, CDCl3): d 208.57, 172.02, 155.86, 79.18, 59.67, 59.13,
52.28, 51.33, 50.09, 41.00, 39.16, 30.99, 28.32, 24.83, 24.39, 23.21,
22.75, 22.32, 19.02, 18.38, 16.66.
Decyl-urea-Val-NHNH3Cl (25). 24 (250 mg, 600 mmol) was
stirred with 4 ml 4 M HCl in dioxane for 1 h before coevaporation
with toluene (3¥) yielded the title compound which was used
without purification. LCMS: Rt 7.39 min (linear gradient 10 →
90% ACN + 0.1% TFA, 15 min), (ESI-MS (m/z): 315.00 (M -
tBuO-Val-urea-Val-Leu2-VS (9). 28 (50 mg, 100 mmol, 1
equiv.) was treated with 1 : 1 TFA : DCM for 30 min, coevaporated
with toluene (3¥). The residue was dissolved in DCM and DiPEA
(40 mmol, 244 mmol, 2.4 equiv.) was added and this mixture was
added to a solution of valine tert-butyl ester isocyanate (prepared
from HCl·H-Val-OtBu (23 mg, 110 mmol, 1.1 equiv.) using general
procedure A). The resulting mixture was stirred for 30 min before
being washed with 1 M HCl and sat. aq. NaHCO3 and dried over
MgSO4. Column chromatography (DCM → 1% MeOH : DCM)
afforded the title compound (42 mg, 70 mmol, 70%). LCMS: Rt
8.80 min (linear gradient 10 → 90% ACN + 0.1% TFA, 15 min).
1H NMR (400 MHz, CDCl3/CD3OD 1/1): d (ppm) 6.81 (dd, J1 =
15.13, J2 = 4.91 Hz, 1H), 6.62 (dd, J1 = 15.14, J2 = 1.36 Hz, 1H),
4.74–4.63 (m, 1H), 4.39 (t, J = 7.37, 1H), 4.04 (d, J = 5.13 Hz, 1H),
3.95 (d, J = 5.78 Hz, 1H), 2.97 (s, 3H), 2.16–2.03 (m, 2H), 1.73–1.57
(m, 6H), 1.47 (s, 1H), 1.04–0.88 (m, 24H). 13C NMR (100 MHz,
1
Cl-)+). H NMR (400 MHz, CD3OD, CDCl3): d 4.06 (d, J = 6.2
Hz, 1H), 3.28–3.01 (m, 2H), 2.17–2.05 (m, 1H), 1.56–1.44 (m, 2H),
1.41–1.14 (m, 14H), 0.99 (t, J = 7.1 Hz, 6H), 0.89 (t, J = 6.8 Hz,
3H).
Decyl-urea-Val-Leu-EK (8). Prepared by the general proce-
dure for azide coupling employing 25 (53 mg, 151 mmol, 1 equiv.)
and TFA·H-Leu-EK (47 mg, 166 mmol, 1.1 equiv.). Purification by
column chromatography (DCM → 3% MeOH : DCM and 10%
EA : Tol. → 30% EA : Tol.) yielded the title compound (24 mg,
53 mmol, 35%). LCMS: Rt 11.18 min (linear gradient 10 → 90%
ACN + 0.1% TFA, 15 min). 1H NMR (400 MHz, CDCl3): d 6.84
190 | Org. Biomol. Chem., 2012, 10, 181–194
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CDCl3/CD3OD): d (ppm) 173.00, 172.27, 171.43, 158.72, 146.50,
128.55, 80.62, 59.40, 58.16, 51.38, 47.07, 41.18, 41.06, 39.34, 29.89,
29.78, 26.57, 23.94, 23.84, 21.59, 21.55, 20.09, 19.95, 17.97, 17.80,
16.43, 16.37. HRMS calcd. for [C29H55N4O7S]+ 603.37860, found
603.37866.
19.85, 18.30, 14.45. HRMS calcd. for [C30H59N4O5S]+ 587.42007,
found 587.42017.
tBuO-Val-urea-Val-Leu2-EK (13). 30 (41 mg, 85 mmol, 1
equiv.) was treated with 1 : 1 TFA : DCM for 30 min, coevaporated
with toluene (3¥). The residue was dissolved in DCM and DiPEA
(31 ml, 187 mmol, 2.2 equiv.) was added and this mixture was
added to a solution of valine tert-butyl ester isocyanate (prepared
from HCl·H-Val-OtBu (27 mg, 128 mmol, 1.6 equiv.) using general
procedure A). The resulting mixture was stirred overnight. The
reaction mixture was washed with 1 M HCl (3¥), H2O (3¥)
and dried over Na2SO4. Column chromatography (DCM → 2%
MeOH : DCM yielded the title compound (14 mg, 24 mmol, 28%).
LCMS: Rt 9.84 min (linear gradient 10 → 90% ACN + 0.1% TFA,
15 min). 1H NMR (400 MHz, CDCl3): d (ppm) 8.02 (s, 1H) 6.49
(d, J = 7.5 Hz, 1H), 6.19 (s, 1H), 4.74–4.60 (m, 1H), 4.53 (dd, J1 =
14.7, J2 = 7.4 Hz, 1H), 4.38 (dd, J1 = 9.2, J2 = 4.9 Hz, 1H), 4.25
(t, J = 8.3 Hz, 1H), 3.41 (d, J = 4.8 Hz, 1H), 2.87 (d, J = 4.8 Hz,
1H), 2.09–1.95 (m, 1H), 1.90–1.30 (m, 7H), 1.52 (s, 3H), 1.49 (s,
1H), 1.01–0.74 (m, 24H). 13C NMR (100 MHz, CDCl3): d (ppm)
173.37, 172.83, 171.92, 157.73, 81.41, 59.13, 58.85, 58.11, 52.56,
52.01, 50.32, 40.96, 39.12, 32.42, 31.96, 28.11, 24.92, 24.46, 23.37,
23.09, 22.17, 21.09, 19.25, 18.99, 18.74, 18.07, 16.89. HRMS calcd.
for [C30H55N4O7]+ 583.40653 found 583.40668.
HO-Val-urea-Val-Leu2-VS (10). 9 (21 mg, 35 mmol) was stirred
with 2 ml TFA for 1 h before being coevaporated with toluene (3¥)
1
to yield the title compound quantitatively. H NMR (400 MHz,
CD3OD, CDCl3): d (ppm) 7.94 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 7.5
Hz, 1H), 6.82 (dd, J1 = 15.1, J2 = 4.8 Hz, 1H), 6.59 (dd, J1 = 15.1,
J2 = 1.5 Hz, 1H), 4.78–4.64 (m, 1H), 4.45–4.31 (m, 1H), 4.22 (d, J =
4.9 Hz, 1H), 3.91 (d, J = 5.6 Hz, 1H), 2.98 (s, 3H), 2.29–2.02 (m,
2H), 1.76–1.36 (m, 6H), 1.10–0.80 (m, 24H). 13C NMR (100 MHz,
CD3OD): d 175.91, 175.12, 174.46, 160.92, 148.35, 130.84, 61.26,
59.51, 53.42, 49.16, 43.26, 42.83, 41.44, 32.06, 31.83, 25.96, 25.84,
23.42, 21.91, 21.84, 19.85, 19.74, 18.34, 18.08. HRMS calcd. for
[C25H46N4O7S]+ 547.31600, found 547.31610.
Benzylamide-Val-urea-Val-Leu2-VS (11). 28 (51 mg, 101 mmol,
1 equiv.) was treated with 1 : 1 TFA : DCM for 30 min, co-
evaporated with toluene (3¥). The residue was dissolved in
DCM and DiPEA (37 ml, 222 mmol, 2.2 equiv.) was added and
this mixture was added to a solution of valine benzylamide
isocyanate (prepared from HCl·H-Val-NHBn (35 mg, 144 mmol,
1.4 equiv.) using general procedure B). The resulting mixture was
stirred overnight. Upon quenching with H2O the title compound
precipitated as a white solid (15 mg, 23 mmol, 23%). LCMS: Rt
7.88 min (linear gradient 10 →90% ACN + 0.1% TFA, 15 min).
1H NMR (400 MHz, CDCl3:CD3OD): d 7.37–7.15 (m, 5H), 6.83
(dd, J1 = 15.1, J2 = 5.3 Hz, 6H), 6.59 (dd, J1 = 15.1, J2 = 1.2
Hz, 6H), 4.58–4.24 (m, 4H), 4.08 (dd, J1 = 15.4, J2 = 7.1 Hz,
2H), 2.93 (s, 3H), 2.20–1.88 (m, 2H), 1.80–1.53 (m, 4H), 1.54–
1.13 (m, 2H), 1.19–0.59 (m, 24H). 13C NMR (100 MHz, CDCl3,
CD3OD): d 173.14, 172.46, 172.09, 158.43, 147.20, 137.61, 128.53,
127.92, 126.89, 126.84, 126.65, 59.16, 58.76, 51.60, 47.28, 42.51,
41.78, 41.68, 39.81, 30.97, 30.57, 24.29, 24.06, 22.07, 21.97, 21.01,
20.83, 18.53, 18.47, 17.40, 17.32. HRMS calcd. for [C32H54N5O6S]+
636.37893, found 636.37909.
HO-Val-urea-Val-Leu2-EK (14). 13 (11 mg, 19 mmol) was
stirred with 1 ml TFA for 1 h and coevaporated with toluene (3¥)
1
to yield the title compound quantitatively. H-NMR (400 MHz,
CD3OD): d 4.54 (dd, J1 = 10.7, J2 = 3.0 Hz, 1H), 4.47 (dd, J1 =
8.7, J2 = 6.3 Hz, 1H), 4.20 (d, J = 4.6 Hz, 1H), 4.05 (d, J = 6.4 Hz,
1H), 3.27 (d, J = 5.1 Hz, 1H), 2.94 (d, J = 5.1 Hz, 1H), 2.24–2.12
(m, 1H), 2.10–1.97 (m, 1H), 1.82–1.26 (m, 6H), 1.48 (s, 3H), 1.18–
0.58 (m, 24H). 13C NMR (100 MHz, CD3OD): d 171.60, 61.96,
57.29, 56.33, 50.10, 49.65, 48.90, 39.10, 37.12, 29.42, 29.06, 23.26,
22.75, 20.82, 20.41, 19.27, 18.45, 16.90, 16.77, 15.26, 14.91, 14.10.
HRMS calcd. for [C26H47N4O7]+ 527.34393 found 527.34387.
Benzylamide-Val-urea-Val-Leu2-EK (15). 30 (50 mg, 101 mmol,
1 equiv.) was treated with 1 : 1 TFA : DCM for 30 min, coevap-
orated with toluene (3¥). The residue was dissolved in DCM
and DiPEA (37 ml, 222 mmol, 2.2 equiv.) was added and this
mixture was added to a solution of valine benzylamide isocyanate
(prepared from HCl·H-Val-NHBn (34 mg, 144 mmol, 1.4 equiv.)
using general procedure B). The resulting mixture was stirred for
3 h. The reaction mixture was washed with H2O (3¥) and dried over
Na2SO4. Column chromatography (DCM → 4% MeOH : DCM)
followed by crystallisation (DCM : PE) yielded the title compound
(25 mg, 40 mmol, 40%). LCMS: Rt 8.51 min (linear gradient 10 →
90% ACN + 0.1% TFA, 15 min). 1H NMR (400 MHz, CD3OD):
d 7.35–7.18 (m, 5H), 6.57 (dd, J1 = 8.9, J2 = 6.1 Hz, 1H), 4.58–4.49
(m, 2H), 4.44 (d, J = 15.0 Hz, 1H), 4.33 (d, J = 14.9 Hz, 1H), 4.18–
4.04 (m, 2H), 3.25 (d, J = 5.0Hz, 1H), 2.89(d, J = 5.0Hz, 1H), 2.05–
1.88 (m, 2H), 1.77–1.24 (m, 6H), 1.48 (s, 3H), 0.99–0.83 (m, 24H).
13C NMR (100 MHz, CD3OD, CDCl3): d 207.71, 172.54, 172.42,
172.22, 157.96, 137.61, 127.50, 126.59, 126.20, 58.39, 58.15, 58.08,
51.24, 50.56, 49.71, 42.09, 39.95, 38.11, 30.74, 30.69, 24.17, 23.68,
22.01, 21.45, 20.82, 19.69, 18.06, 16.87, 16.79, 15.34. HRMS calcd.
for [C33H54N5O6]+ 616.40686 found 616.40698.
Decyl-urea-Val-Leu2-VS (12). 28 (50 mg, 100 mmol, 1 equiv.)
was treated with 1 : 1 TFA : DCM for 30 min, coevaporated with
toluene (3¥). The residue was dissolved in DCM and DiPEA
(36 mmol, 220 mmol, 2.2 equiv.) was added and this mixture was
added to a solution of decyl isocyanate (prepared from decylamine
(24 mg, 160 mmol, 1.6 equiv.) using general procedure A). The
resulting mixture was stirred for 3 h before being washed with
1 M HCl (3¥) and H2O (3¥) and dried over Na2SO4. Column
chromatography (DCM → 2% MeOH : DCM) afforded the title
compound (25 mg, 43 mmol, 43%). LCMS: Rt 10.91 min (linear
gradient 10 → 90% ACN + 0.1% TFA, 15 min).1H NMR (400
MHz, CD3OD): d (ppm) 6.82 (dd, J1 = 15.2, J2 = 4.9 Hz, 1H),
6.66 (dd, J1 = 15.2, J2 = 1.5 Hz, 1H), 4.74–4.66 (m, 1H), 4.42 (dd,
J1 = 9.6, J2 = 5.3 Hz, 1H), 3.97 (d, J = 5.8 Hz, 1H), 3.23–3.06 (m,
2H), 3.00 (s, 3H), 2.16–2.02 (m, 1H), 1.80–1.57 (m, 5H), 1.54–1.43
(m, 3H), 1.39–1.27 (m, 14H), 1.08–0.87 (m, 21H). 13C NMR (100
MHz, CD3OD): d 175.43, 174.52, 161.13, 148.39, 130.83, 61.47,
53.45, 43.22, 42.81, 41.27, 41.06, 33.07, 31.87, 31.30, 30.76, 30.70,
30.52, 30.46, 28.02, 26.01, 25.86, 23.74, 23.47, 23.42, 21.90, 21.69,
Decyl-urea-Val-Leu2-EK (16). 30 (49 mg, 101 mmol, 1 equiv.)
was treated with 1 : 1 TFA : DCM for 30 min, coevaporated with
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toluene (3¥). The residue was dissolved in DCM and DiPEA (37 ml,
222 mmol, 2.2 equiv.) was added and this mixture was added to
a solution of decyl isocyanate (prepared from decylamine (24 ml,
121 mmol, 1.2 equiv.) using general procedure A). The resulting
mixture was stirred overnight. The reaction mixture was washed
with 1 M HCl (3¥), H2O (3¥) and dried over Na2SO4. Column
chromatography (DCM → 2% MeOH : DCM yielded the title
compound (16 mg, 29 mmol, 29%). LCMS: Rt 11.30 min (linear
9H), 1.07–0.81 (m, 12H). 13C NMR (100 MHz, CDCl3): d 175.06,
171.77, 80.92, 53.03, 50.39, 43.69, 41.17, 27.46, 24.42, 24.28, 22.93,
22.39, 21.53, 20.95.
Fmoc-Val-Leu2-OtBu (36). H-Leu-Leu-OtBu 35 (586 mg,
1.95 mmol, 93%), Fmoc-Val-OH (695 mg, 2.05 mmol, 1.05 equiv.),
HBTU (850 mg, 2.24 mmol, 1.15 equiv.) and DiPEA (725 ml,
4.39 mmol, 2.25 equiv.) were dissolved in DCM and stirred for
1.5 h. before being washed with 1 M HCl (2¥), sat. aq. NaHCO3
(4¥) and dried overMgSO4. Column chromatography (EA : Tol. 0
→ 30%) yielded the title compound (1.15 g, 1.85 mmol, 95%). 1H
NMR (400 MHz, CDCl3): d 7.73 (d, J = 7.5 Hz, 2H), 7.64–7.52
(m, 2H), 7.37 (dd, J1 = 9.5, J2 = 5.1 Hz, 2H), 7.30–7.22 (m, 2H),
7.15 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.11 (d, J = 9.1
Hz, 1H), 4.66 (dd, J1 = 15.1, J2 = 7.5 Hz, 1H), 4.53 (dd, J1 = 13.5,
J2 = 8.5 Hz, 1H), 4.44 (dd, J1 = 10.1, J2 = 7.3 Hz, 1H), 4.30–4.22
(m, 1H), 4.19 (t, J = 7.2 Hz, 1H), 4.12 (t, J = 8.2 Hz, 1H), 2.15–
1.99 (m, 1H), 1.77–1.46 (m, 6H), 1.43 (s, 9H), 0.94 (t, J = 6.4 Hz,
6H), 0.86 (dd, J1 = 10.1, J2 = 6.2 Hz, 12H). 13C NMR (100 MHz,
CDCl3): d 171.88, 171.45, 171.35, 156.47, 143.82, 141.20, 127.60,
126.99, 125.21, 125.13, 119.86, 81.70, 67.07, 60.31, 51.58, 51.29,
47.09, 41.53, 41.31, 31.46, 27.91, 24.74, 24.60, 22.83, 22.52, 21.96,
19.05, 18.21.
1
gradient 10 → 90% ACN + 0.1% TFA, 15 min). H NMR (400
MHz, CD3OD, CDCl3): d (ppm) 4.55–4.51 (m, 1H), 4.43 (dd, J1 =
8.9, J2 = 5.5 Hz, 1H), 4.03 (d, J = 6.5 Hz, 1H), 3.29 (d, J = 5.0 Hz,
1H), 3.20–2.99 (m, 2H), 2.90 (d, J = 5.0 Hz, 1H), 2.11–1.92 (m,
1H), 1.77–1.32 (m, 6H), 1.49 (s, 3H), 1.39–1.10 (m, 16H), 1.05–
0.78 (m, 21H). 13C NMR (100 MHz, CDCl3, CD3OD): d 208.04,
172.86, 172.33, 158.66, 58.57, 58.53, 51.76, 50.85, 49.99, 40.15,
39.50, 38.69, 31.34, 30.60, 29.65, 29.60, 29.04, 29.00, 28.84, 28.75,
26.34, 24.55, 24.04, 22.54, 22.07, 22.00, 21.11, 20.28, 18.50, 17.02,
15.97, 13.25. HRMS calcd. for [C25H47N4O7S]+ 547.31600 found
547.31610.
H-Val-Leu-OtBu (33). FmocValLeuOtBu37
(956 mg,
1.88 mmol, 1 equiv.) was dissolved in THF and treated
with EtSH (1.4 ml, 18.8 mmol, 10 equiv.) and DBU (28 ml,
188 mmol, 0.1 equiv.) for 2 h and coevaporated with toluene.
Column chromatography (EA : PE : MeOH 50 : 50 : 0 → 100 : 0 : 0
→ 95 : 0 : 5) yielded the title compound (524 mg, 1.83 mmol,
H-Val-Leu2-OtBu (37). Fmoc-Val-Leu-Leu-OtBu 36 (1.15 g,
1.85 mmol, 1 equiv.) was dissolved in THF and treated with
EtSH (1.37 ml, 18.5 mmol, 1 equiv.) and DBU (28 ml, 185 mmol,
0.1 equiv.) for 1.5 h before being concentrated and purified on
column chromatography (EA : PE : MeOH 50 : 50 : 0 → 100 : 0 : 0
→ 95 : 0 : 5) to yield the title compound (672 mg, 1.68 mmol, 91%)
1H NMR (400 MHz, CDCl3): d 7.85 (d, J = 8.8 Hz, 3H), 7.40 (d,
J = 7.7 Hz, 3H), 4.74–4.58 (m, 3H), 4.40 (dd, J = 13.9, 7.9 Hz, 3H),
3.23 (d, J = 4.1 Hz, 3H), 2.36–2.18 (m, 3H), 1.74–1.49 (m, 6H),
1.46 (s, 9H), 0.98 (d, J = 6.9 Hz, 3H), 0.95–0.87 (m, 12H), 0.84 (d,
J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 174.15, 171.75,
171.41, 80.93, 59.87, 51.25, 50.67, 41.00, 40.87, 30.63, 27.60, 24.46,
24.33, 22.64, 22.31, 21.86, 21.83, 19.31, 15.94.
1
97%). H NMR (400 MHz, CDCl3): d 7.71 (d, J = 8.5 Hz, 1H),
4.57–4.42 (m, 1H), 3.27 (d, J = 4.0 Hz, 1H), 2.35–2.19 (m, 1H),
2.05 (s, 2H), 1.80–1.49 (m, 3H), 1.46 (s, 9H), 0.99 (d, J = 7.0 Hz,
3H), 0.97–0.89 (m, 6H), 0.85 (d, J = 6.9 Hz, 3H). 13C NMR (100
MHz, CDCl3): d 173.83, 171.91, 81.09, 59.76, 50.58, 41.23, 30.57,
27.57, 24.56, 22.48, 21.51, 19.23, 15.87.
Fmoc-Leu2-OtBu (34). Leucine tert-butyl ester (382 mg,
2.04 mmol, 1 equiv.) was dissolved in DCM and Fmoc-Leu-OH
(757 mg, 2.14 mmol, 1.05 equiv.), HBTU (890 mg, 2.35 mmol, 1.15
equiv.) and DiPEA (759 ml, 4.59 mmol, 2.25 equiv.) were added
and the mixture was stirred for 2 h before being concentrated. The
residue was taken up in EA and washed with 1 M HCl (2¥), sat.
aq. NaHCO3 (4¥) and brine before drying over MgSO4. Column
chromatography (EA : Tol. 0 → 16%) afforded the title compound
(1.09 g, 2.04 mmol, quant.). 1H NMR (400 MHz, CDCl3): d 7.70
(d, J = 7.5 Hz, 2H), 7.56 (t, J = 7.0 Hz, 2H), 7.40–7.29 (m, 2H),
7.23 (t, J = 7.0 Hz, 2H), 7.13 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 7.9
Hz, 1H), 5.99 (d, J = 8.7 Hz, 1H), 4.49 (dd, J1 = 13.8, J2 = 7.9
Hz, 1H), 4.43–4.33 (m, 2H), 4.32–4.24 (m, 1H), 4.20–4.13 (m, 1H),
1.80–1.46 (m, 6H), 1.43 (s, 9H), 0.98–0.83 (m, 12H). 13C NMR (100
MHz, CDCl3): d 172.11, 171.62, 156.05, 143.65, 143.50, 140.97,
127.38, 126.79, 124.89, 119.64, 81.33, 66.80, 53.11, 51.27, 46.84,
41.53, 41.18, 27.69, 24.60, 24.38, 22.77, 22.44, 21.88.
tBuO-Leu-Val-urea-Val-Leu-VS
(42). BocValLeuVS
17
(64 mg, 164 mmol, 1 equiv.) was stirred in 1 : 1 TFA : DCM
for 30 min. before being coevaporated with toluene (3¥). The
residue was dissolved in DCM and treated with the isocyanate
of Val-Leu-OtBu 41 (obtained from H-Val-Leu-OtBu 33 (54 mg,
188 mmol, 1.15 equiv.) employing general procedure B) and
DiPEA (60 ml, 361 mmol, 2.2 equiv.). The mixture was stirred
for 2 h before being washed with 1 M HCl (2¥) and dried over
MgSO4. Column chromatography (MeOH : DCM 1 → 3%)
yielded the title compound (54 mg, 90 mmol, 54%). LCMS: Rt
9.02 min (linear gradient 10 → 90% ACN + 0.1% TFA, 15 min).
1H NMR (400 MHz, CD3OD): d 6.81 (dd, J1 = 15.2, J2 = 5.4
Hz, 1H), 6.68 (d, J = 15.2 Hz, 1H), 6.57–6.39 (m, 1H), 4.83–4.66
(m, 1H), 4.36 (t, J = 7.5 Hz, 1H), 4.15 (t, J = 6.5 Hz, 1H), 4.05
(t, J = 6.4 Hz, 1H), 2.98 (s, 3H), 2.04 (dq, J = 13.0, 6.5 Hz, 2H),
1.71 (td, J1 = 13.3, J2 = 6.6 Hz, 2H), 1.67–1.53 (m, 4H), 1.47
(s, 9H), 1.15–0.82 (m, 24H). 13C NMR (100 MHz, CD3OD): d
174.66, 174.59, 173.20, 148.49, 130.97, 82.56, 60.91, 60.84, 60.00,
52.90, 49.12, 43.34, 42.84, 41.66, 32.63, 32.30, 28.27, 25.91, 25.83,
23.37, 23.29, 22.10, 22.06, 20.00, 18.54, 18.49. HRMS: calcd. for
[C29H55N4O7S]+ 603.37860 found 603.37881
H-Leu2-OtBu (35). Fmoc-Leu2-OtBu 34 (1.09 g, 2.04 mmol,
1 equiv.) was dissolved in THF and EtSH (1.5 ml, 20 mmol,
10 equiv.) and DBU (30 mL, 200 mmol, 0.1 equiv.) were added
and the mixture was stirred for 1.5 h. before being co-evaporated
with toluene. Column chromatography (EA : PE : MeOH 50 : 50 : 0
→ 100 : 0 : 0 → 95 : 0 : 5) yielded the title compound (586 mg,
1
1.95 mmol, 93%). H NMR (400 MHz, CDCl3): d 7.71 (d, J =
8.6 Hz, 1H), 4.46 (dt, J1 = 8.7, J2 = 5.2 Hz, 1H), 3.42 (dd, J1 =
9.6, J2 = 4.2 Hz, 1H), 1.87 (s, 2H), 1.81–1.29 (m, 6H), 1.46 (s,
192 | Org. Biomol. Chem., 2012, 10, 181–194
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tBuO-Leu2Val-urea-Leu-VS
(40). TFA·H-Leu-VS
38
bated with inhibitors for 30 min at 37 ◦C followed by assay of ac-
tivity with fluorogenic substrates Suc-LLVY-amc (chymotrypsin-
like site), Ac-RLR-amc (trypsin-like site), and Ac-nLPnLD-amc
(caspase-like site).43
(220 mmol, 1 equiv.) was transformed to the isocyanate 39
employing general procedure A. H-Val-Leu-Leu-OtBu 37 (97 mg,
242 mmol, 1.1 equiv.) and DiPEA (80 ml, 484 mmol, 2.2 equiv.)
were dissolved in DCM and added to the isocyanate solution
and the resulting mixture was stirred for 2 h, washed with 1
M HCl (2¥) and dried over MgSO4. Column chromatography
(MeOH : DCM 0 → 3%) yielded the title compound (113 mg,
183 mmol, 83%). LCMS: Rt 9.40 min (linear gradient 10 →
Acknowledgements
This work was supported by the Netherlands Organisation for Sci-
entific Research (NWO) and the Netherlands Genomics Initiative
(NGI) by the U.S. National Cancer Institute (5RO1CA124634 and
ARRA Supplement to this Award). Thanks to Hans van der Elst
for HRMS analysis, Kees Erkelens and Fons Lefeber for assistance
with NMR.
1
90% ACN + 0.1% TFA, 15 min). H NMR (400 MHz, CDCl3,
CD3OD): d 7.93 (t, J = 7.9 Hz, 2H), 6.84 (dd, J1 = 15.1, J2 = 5.1
Hz, 1H), 6.53 (d, J = 15.1 Hz, 1H), 6.31 (d, J = 8.0 Hz, 1H), 6.02
(d, J = 8.8 Hz, 1H), 4.55–4.42 (m, 2H), 4.35 (dd, J1 = 14.5, J2 =
8.1 Hz, 1H), 4.13–4.03 (m, 1H), 2.98 (s, 3H), 2.09–1.95 (m, 1H),
1.85–1.50 (m, 9H), 1.47 (s, 9H), 1.10–0.77 (m, 24H). 13C NMR
(100 MHz, CDCl3, CD3OD): d 172.55, 172.15, 171.43, 157.68,
148.96, 127.92, 81.13, 51.14, 50.99, 48.16, 42.58, 41.69, 40.28,
40.11, 30.76, 26.96, 24.12, 23.96, 21.90, 20.96, 20.74, 18.39, 16.92.
HRMS: calcd. for [C30H57N4O7S]+ 617.39425, found 617.39466.
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4.3 Competition experiments in vivo
1 ¥ 106 HEK cells were seeded and grown overnight. 100 ¥ stock
solution of inhibitor was added to 1 ml medium and the cells
were incubated for 4 h at 37 ◦C. MVB003 (100 ¥ stock solution in
DMSO, 5 mM end concentration) was added and incubated for 2 h
at 37 ◦C. Cells were harvested, washed 2¥ with PBS and permeated
in digitonin lysis buffer (4 ¥ pellet volume, 50 mM Tris pH 7.5, 250
mM sucrose, 5 mM MgCl2, 5 mM DTT, 0.025% digitonin) for 15
min on ice and centrifuged at 16.100 rcf. for 20 min at 4 ◦C. The
supernatant containing the cytosolic fraction was collected and the
protein content was determined by Bradford assay (Biorad). 10 mg
protein (dilution with lysis buffer) was boiled in Laemmli’s sample
buffer and resolved as described above. Coomassie Brilliant Blue
staining of the gel verified protein loading.
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