Modular Total Synthesis of Farnesyl Analogues of Cell Wall Precursors Lipid i and II Containing the Staphylococcus aureus Pentaglycine Bridge Modification

Article abstract of DOI:10.1021/acs.joc.0c01004

A scalable and modular total synthesis of 3-lipid I and 3-lipid II was accomplished by a novel route involving an efficient solid phase synthesis of the peptide fragment and an effective chemoenzymatic attachment of the second sugar moiety. The generality of this route was further documented by the synthesis of an analogue bearing the pentaglycine interpeptidic bridge modification characteristic for the human pathogen Staphylococcus aureus.

Full text of DOI:10.1021/acs.joc.0c01004

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Note
Modular Total Synthesis of Farnesyl Analogs of Cell Wall Precursors
Lipid I and II, Containing the S. aureus Pentaglycine Bridge Modification
Lukas Wingen, Marvin Rausch, Tanja Schneider, and Dirk Menche
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01004 • Publication Date (Web): 22 Jun 2020
Downloaded from pubs.acs.org on June 26, 2020
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Page 1 of 11
The Journal of Organic Chemistry
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Modular Total Synthesis of Farnesyl Analogs of Cell Wall Precursors
Lipid I and II, Containing the S. aureus Pentaglycine Bridge
Modification
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Lukas M. Wingen,a Marvin Rausch,b,c Tanja Schneider,b and Dirk Menchea,*
aKekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany, bInstitute for
Pharmaceutical Microbiology, University Clinic Bonn, University of Bonn, 53115 Bonn, Germany. cGerman Center for
Infection Research (DZIF), partner site Bonn-Cologne, Bonn.
Supporting Information Placeholder
HO
R
O
O
O
O
P
O
P
O
AcHN
H
O
O
O
2
N
HO
N
O
OH OH
H
O
3-Lipid I R, R'
:
= H
HO
HO
HO
AcHN
R'
O
O
O
NH
N
H
3-Lipid II
R
:
=
R'
= H
HN
O
3-Lipid I (S. aureus): R = H
R'
=
O
O
O
OH
H
H
N
N
N
NH2
H
N
N
O
H
H
O
O
ABSTRACT: A scalable and modular total synthesis of 3-lipid I and 3-lipid II was accomplished by a novel route involving an
efficient solid phase synthesis of the peptide fragment and an effective chemoenzymatic attachment of the second sugar moiety.
The generality of this route was further documented by the synthesis of an analog bearing the pentaglycine interpeptidic bridge
modification characteristic for the human pathogen S. aureus.
The molecular machinery of bacterial cell wall biosynthesis
represents one of the prime targets for antibacterial
intervention. Antibiotics are able to interfere with the
biosynthesis pathway at various stages, either by inhibiting
enzyme function or interaction with essential cell wall building
blocks. Hence, blocking the essential cell wall precursors lipid I
or II (1, 2, Figure 1)can trigger diverse malfunctions within the
bacterial cell resulting in an imperfect or altered bacterial cell
wall ultimately leading to bacterial death.1 The central
membrane-anchored building blocks lipid I (1) and lipid II (2)
also play pivotal roles in bacterial cell physiology and constitute
functional scaffolds for coordination of cell wall biosynthesis,
cell division and organization of enzymatic machineries.1
Interpeptidic variations of these building blocks have been
described for specific pathogens (see Figure 1)2 and these
modifications are increasingly being recognized of high
biochemical importance by allowing for peptidic cell wall
crosslinks, relevant for structural integrity.3 However, detailed
structural and functional studies are severely hampered by the
limited supply of natural components. Although, various
progress in the synthesis of cell wall precursors and also total
syntheses of natural lipid II as well as analogs has been
reported,4-9 the supply issue could not be resolved to meet the
high demand for these compounds. Synthetic efforts suffer from
limited scalability, lengthy solution phase sequences, in
combination with reproducibility and purification issues and
could not be adopted to access interpeptidic analogs.
Furthermore, structural studies with the authentic full-length
building blocks are complicated by unfavorable
physicochemical
properties,
including
aggregation,
precipitation and complex spectroscopic characteristics, which
are mainly due to the lipophilic undecaprenyl tails.10,11 In recent
years, it has become more and more apparent that these tails can
be dramatically shortened and existing biochemical studies with
truncated analogs, such as 3 and 4, suggest these surrogates are
mostly compatible with the authentic molecules.10-14 Herein, we
report an efficient and scalable route towards previously
synthesized7,8,12 farnesyl analogs 3-lipid I (3) and 3-lipid II (4)
by a convergent and modular strategy, involving a reliable and
scalable peptide solid phase synthesis, an improved synthesis of
the central pyrophosphate moiety and
a
late-stage
chemoenzymatic attachment of the second carbohydrate
(GlcNAc). Furthermore, we describe the successful application
of these strategies for the synthesis of a pentaglycine derivative
5 specific for S. aureus, which presents the first chemical
synthesis of an interpeptide containing lipid analog. These
routes are highly convergent, concise, and scalable enabling a
reliable access to sufficient amounts of 3, 4 and 5 for
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biochemical and structural studies and thus offer the potential related pentapeptide analog bearing a methyl ester at the
1
2
3
4
5
6
7
8
to further explore the biological roles of these key precursors of
cell wall biosynthesis and to arrive at a better understanding of
the antibiotic binding modes.
terminal D-Ala-carboxylate, which relied on attachment of this
first amino acid to a Sasrin resin.11 However, only limited
experimental and spectroscopic details are provided. Therefore,
we decided to design a novel route. As shown in Scheme 1, our
final optimized approach relied on initial preparation of the
tetrapeptide D-Ala-L-Lys-D--Glu-L-Ala (17) on solid support
and subsequent attachment of the remaining D-Ala in solution
phase. This approach was crucial to avoid unfavorable
epimerizations that were otherwise observed during attachment
of the required TMSE protecting group on the terminal D-Ala-
carboxylate of a corresponding pentapeptide. Also, in contrast
to the reported route, 2-chlorotrityl chloride (2CTC) resin was
chosen to allow for facile peptide cleavage under mild
conditions (hexafluoroisopropanol, HFIP)15, which would also
be compatible with the silyl protecting groups chosen for the
two terminal carboxylates (D-Ala, L-Glu) and the side chain
amine of lysine. Notably, this identical choice of protecting
groups would also allow for a concomitant removal at a later
stage of the synthesis. Peptide coupling on solid support was
then effected using the Fmoc procedure. Consequently, after
attachment of Fmoc protected D-alanine with suitable
occupancy,16 the resulting solid bound amino acid 12 was
elaborated after capping of the unreacted 2CTC-functionalities
with acetic anhydride and N-methylimidazole. Following
repetitive coupling and deprotection steps on solid support,
peptide 16 was synthesized using the appropriate
fluorenylmethoxycarbonyl (Fmoc) amino acid building blocks
13, 14, and 15, which were accessible from the respective amino
acids using standard protection reactions, i.a. Steglich
esterifications with 2-(trimethylsilyl)ethanol and cleavage
procedures, mainly involving trifluoroacetic acid for tert-
butylcarbamate (BOC) removal.17 After cleavage from the resin
with HFIP, peptide 17 was coupled with amine 18, which
likewise contained a silyl protecting group to allow for a joint
removal of all silyl protecting groups (vide infra). Finally, the
Fmoc group was removed by piperidine in DMF giving desired
pentapeptide 7 in a reliable fashion and high yield (59%) over
eleven steps. The purity and yield compared favorably to the
previous reported (15%).11
As shown in Figure 1, the targeted 3-lipid I architectures are
characterized by an N-acetyl-muramyl-pentapeptide linked to a
farnesyl chain via a pyrophosphate bridge. Our retrosynthetic
strategy was based on three components, a peptide segment 7
and 8 together with a suitably functionalized monosaccharide 6
and terpene 9. For attachment of the second sugar (GlcNAc)
required for 4, a chemoenzymatic approach was envisioned.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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40
41
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46
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R
HO
O
O
O
O
AcHN
O
P
O
H
O
P
O
O
N
7
HO
N
O
OH OH
H
O
3
Lipid I
Lipid II
1
(
): R, R' = H
HO
GlcNAc
O
R'
O
O
NH
N
H
HO
): R =
2
(
HO
AcHN
R' = H
HN
O
R' = Gly-Gly-Gly-Gly-Gly: S. aureus
L-Ala-L-Ala: E. faecalis
OH
N
L-Ala/Ser-L-Ala: S. pneumoniae
D-Asp: E. faecium
H
O
R
HO
O
O
O
O
AcHN
O
P
O
H
O
P
O
O
N
2
HO
N
O
OH OH
H
O
3-Lipid I
3-Lipid II
3
(
): R, R' = H
HO
R'
GlcNAc
O
O
O
NH
N
H
HO
): R =
4
(
HO
AcHN
R' = H
HN
O
3-Lipid I (S. aureus)
5
(
): R = H
OH
N
R' = Gly-Gly-Gly-Gly-Gly
H
O
Ph
O
O
O
O
H
O
O
N
TMSEO
NH2
HN
O
O
P OBn
+
O
HO
O
OBn
6
O
O
NH
R'
+
N
H
HN
O
HO
P O
OTMSE
2
O
N
Scheme 1. Solid Phase Synthesis of Pentapeptide 7
OH
H
9
O
Gly-Gly-Gly-Gly-Gly-Teoc
7
R' = Teoc
8
Figure 1. Natural lipid I/II (1, 2), the sesquiterpene analogs 3, 4
and the novel S. aureus analog 5 and retrosynthetic approach
relying on three components.
Glucosamine derivative 6 was synthesized using a slightly
modified, previously reported procedure. Various more
technical modifications and optimizations, in particular in the
acetalization step, led to a considerable increase of the overall
yield to 60% over six steps.10 An exchange of reported DMF to
MeCN in an intermediate acetalization resulted in an increased
yield of 81% to 96% and additionally facilitated the work up
due to the easier removable MeCN.10 For details of these
optimizations, see SI section.
With 6 in hand a solid phase supported peptide coupling
strategy was investigated for peptide 7. The group of Walker
has previously reported a solid phase synthesis of a closely
2
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The Journal of Organic Chemistry
Ph
O
Ph
O
O
O
O
O
O
O
7
( ), PyBOB
HOBt, DIPEA
R-NH2
O
O
11
HO-D-Ala-Fmoc (
DIPEA
)
1
2
3
Cl
O
AcHN
AcHN
O
P
OBn
O
P
OBn
PS
PS
O
Fmoc
2CTC
10
81%
HO
O
HN
O
N
6
OBn
20
OBn
H
R
12
1. 10% Pd-C, H2
2. AcOH/H2O
4
5
6
7
8
9
10
11
12
13
14
15
16
17
a) Acetic anhydride, N-Methylimidazole
b) = 20% Piperidine/DMF
c) = HBTU, HOBt, Aminoacid, DIPEA
a)
b)
Farnesyl
phosphate (
CDI
HO
HO
HO
HO
O
O
O
O
PS = Polystyrene
9
)
c)
b)
13
)
HO-L-Lys(N-Teoc)-Fmoc (
O
O
O
P
AcHN
AcHN
Cl
O
P
O
O R'
O
P
OH
HN
O
HN
R
O
Cl
2CTC =
14
OH OH
c) TMSE-O-D-Glu(OH)-Fmoc (
)
OH
22
21
R
b)
TBAF
40% (4 steps)
15
HO-L-Ala-Fmoc (
)
c)
MurG, UDP-GlcNAc
55%
HFIP
PS
2CTC D-Ala-L-Lys(N-Teoc)-D--
Glu(O-TMSE)-L-Ala-Fmoc
3
4
82%
16
O
18
)
TMSE-O-D-Ala-NH2
(
H
N
TMSE-O-D-Ala-D-Ala-L-
Lys(N-Teoc)-D--Glu(O-
TMSE)-L-Ala-Fmoc
HO-D-Ala-L-Lys(N-
Teoc)-D--Glu(O-
TMSE)-L-Ala-Fmoc
b)
PyBOB, HOBt, DIPEA
R
R'
=
O
=
7
O
95%
76%
O
19
2
17
Si
Si
O
O
NH
N
O
H
Using previously reported procedures,12,18,19 peptide 7 was then
coupled with carbohydrate fragment 6 using benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOB),
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HN
Si
O
O
N
1-hydroxybenzotriazole
(HOBt)
and
N,N-
H
O
diisopropylethylamine (DIPEA) yielding the respective
glycopeptide 20 in high yield (81%, Scheme 2). However, in
contrast to a described procedure10, a joint removal of the
benzyl groups and the acetal moiety could not be effected with
Pd/C and hydrogen. Therefore, a two-step protocol was used
involving hydrogenolysis of the benzyl groups followed by
acetal cleavage under slightly acidic conditions (HOAc/H2O).13
Resulting phosphate 21 was then connected with farnesyl
phosphate 9 after activation with carbonyldiimidazole (CDI).
This phosphate 9, in turn, was obtained in good yield (71%)
using a procedure reported by Wessjohann.20 As a proof of the
synthetic design, all silyl protecting groups could then be
removed in a joint fashion using tetrabutylammonium fluoride.
For final purification of derived target compound 3-lipid I (3),
an efficient three step procedure was developed, involving gel
After having established this sequence, we focused on adopting
and applying these procedures also for the synthesis of an
interpeptide bridge containing analog. As first target we chose
the modification found in S. aureus, bearing five glycine
residues attached to the side chain of lysine. This synthetically
challenging modification was considered the most interesting
and important among the interpeptidic modifications described
for specific pathogens.2 As shown in Scheme 3, our successful
solid-phase route towards 8 similarly relied on attachment of
the second D-Ala amino acid to a chlorotrityl resin and stepwise
elaboration using Fmoc protected amino acids. In contrast to the
sequence above, a modified lysine building block was utilized
with the Fmoc group now being at the side chain amine, while
the -amine was protected in an orthogonal fashion with an
allyloxycarbonyl (alloc). After coupling and removal of the
terminal Fmoc group, four glycine residues (24) were then
attached in an iterative fashion using standard coupling and
deprotection protocols. Encouraged by the success of the silyl
protecting groups above, the final glycine moiety was then
protected as a Teoc group. Having confirmed this route by a test
cleavage from the resin, the resulting heptapeptide 26 was then
further elaborated. After cleavage of the alloc protecting group
with tetrakis(triphenylphosphine)-palladium(0) (Pd(PPh)4) and
phenylsilane (PhSiH3),21 amino acids 14 and 15 were attached,
before the resulting nonapeptide 28 was cleaved from the resin
giving 29 in excellent yield (83%) over these nineteen steps on
the solid support. Finally, remaining (TMSE) protected D-
alanine 18 was again coupled in solution, which proceeded
again uneventfully. In total, desired peptide 8 was obtained in
high overall yield (40%) over 21 steps starting from 10,
demonstrating the general suitability of the established
strategies. Notably, this sequence is variable and should be
applicable to other pathogen specific interpeptidic analogs.2
filtration
(Sephadex®
LH-20),
an
ion
exchange
chromatography (Dowex® 50WX8) to substitute residual
tetrabutylammonium counterions with ammonium and a final
HPLC separation. Following this sequence, desired 3-lipid I (3)
was obtained in high purity and good yield (40%) over four
steps from 20. In total, 3-lipid I was obtained in pure form in 16
steps (longest linear sequence from commercial 11) with an
excellent overall yield (19%), which compared favorably to the
previous routes. This sequence proved well scalable and a first
batch of 11 mg of highly pure 3-lipid I was readily obtained.
Finally, β-(1-4)-linked N-acetylglucosamine was attached
chemoenzymatically using the glycosyltransferase MurG to
access 3-lipid II (4). This conversion proceeded with useful
degrees of conversion (55% yield).
Scheme 2. Late Stage Synthesis to Lipid I/II Analogs 3, 4
Scheme 3. Synthesis of Peptide 8 using Solid Phase Support
3
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a), b)
In summary, we have reported an efficient total synthesis of 3-
c)
23
)
HO-L-Lys(N-Fmoc)-Alloc (
lipid I and II analogs 3 and 4 7,8,12,14, that relied on a novel, high
1
b)
PS
2
2CTC D-Ala-L-Lys(N-
(Gly)5Teoc)-N-Alloc
26
yielding and scalable solid phase supported synthesis.
PS
2CTC D-Ala-
24
c) HO-Gly-Fmoc (
b)
)
Fmoc
12
Following
these
sequences
these
important
3
4
5
6
4
simplified/shortened bacterial cell wall building blocks can be
obtained in a reliable and scalable fashion which will help to
resolve the key supply issue and facilitate biochemical studies
and structural analyses of Lipid II antibiotic complexes.
Furthermore, we reported the first total synthesis of a lipid I
analog 5 bearing the pentaglycine peptide modification found
in S. aureus. Additionally, we showed that both Lipid I analogs
were accepted by MurG and converted to the respective Lipid
II analogs. Synthesis of further novel Lipid I/II analogs and
utilization of these building blocks as chemical tool compounds
for biochemical studies and structural investigations may now
be approached.
Pd(PPh)4, PhSiH3
ge
cleava
25
HO-Gly-Teoc (
)
c)
O
c)
TMSE-O-D-Glu(OH)-
14
test
O
Fmoc (
)
HFIP
O
NH HN
b)
NH HN
O
HN
15
c) HO-L-Ala-Fmoc (
)
7
O
O
8
OH
O
PS
O
2CTC D-Ala-L-Lys(N-
(Gly)5Teoc)-D--Glu(O-
TMSE)-L-Ala-Fmoc
NH HN
9
28
NH
O
O
O
Si
27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HFIP
83%
c) TMSE-O-D-
18
Ala-NH2
(
)
TMSE-O-D-Ala-D-Ala-
L-Lys(N-(Gly)5Teoc)-
D--Glu(O-TMSE)-L-
HO-D-Ala-L-Lys(N-
(Gly)5Teoc)-D--Glu(O-
TMSE)-L-Ala-Fmoc
PyBOB, HOBt,
DIPEA, 56%
Ala-Fmoc
O
H
30
29
N
O
NH2
O
EXPERIMENTAL SECTION
O
b)
87%
H
Si
N
O
O
NH
N
H
NH
General experimental methods: All reagents were purchased
from commercial suppliers (Sigma-Aldrich, TCI, Acros,
AlfaAesar, Iris Biotech) in the highest purity grade available
and used without further purification. Anhydrous solvents
(THF, DCM, MeCN, Et2O, toluene) were obtained from a
solvent drying system MB SPS-800 (MBraun) and stored over
molecular sieves (3 or 4 Å). The reactions in which dry solvents
were used were performed under an argon atmosphere in flame-
dried glassware. TLC monitoring was performed with silica gel
60 F254 pre-coated polyester sheets (0.2 mm silica gel,
Macherey-Nagel) and visualized using UV light and staining
with a solution of CAM (1.0 g Ce(SO4)2), 2.5 g (NH4)6Mo7O24,
8 mL conc. H2SO4 in 100 mL H2O) and subsequent heating.
For column chromatography, silica gel (pore size 60 Å, 40-63
μm) obtained from Merck or Aldrich was used. Compounds
were eluated using the stated mixtures under a positive pressure
of air. Solvents for column chromatography were distilled prior
to use. For ion exchange chromatography Dowex® 50WX8,
100–200 mesh by Acros was used. Gel permeation
chromatography was performed using SephadexTM LH–20
purchased from GE Healthcare. All NMR spectra were
recorded on Bruker spectrometers in deuterated solvents
obtained from Deutero and Carl Roth. Spectra were measured
at room temperature and chemical shifts are reported in ppm
relative to (Me)4Si (δ= 0.00 ppm) and were calibrated to the
O
O
O
O
HN
NH
Si
O
a) Acetic anhydride,
N-Methylimidazole
b) = 20% Piperidine/DMF
c) = HBTU, HOBt, Aminoacid,
DIPEA
O
O
N
N
H
H
O
O
NH
8
Si
As shown in Scheme 4, the novel peptide 8 was then used in an
analogous three component coupling sequence to complete the
first total synthesis of an interpeptidic 3-lipid I analog 5, in an
overall yield of 8% in its longest linear sequence over 26 steps
starting from 11, again confirming the general usefulness of
these strategies.22
Scheme 4. Late Stage Synthesis to 3-Lipid I (S. aureus) 5
Ph
O
Ph
O
O
O
O
O
O
O
8
( ), PyBOB
HOBt, DIPEA
R-NH2
O
O
AcHN
AcHN
O
P
OBn
O
P
OBn
86%
HO
O
HN
O
6
OBn
31
OBn
R
1. 10% Pd-C, H2
2. AcOH/H2O
Farnesyl
phosphate (
CDI
HO
HO
HO
HO
O
O
O
O
9
)
O
O
O
P
AcHN
1
AcHN
O
P
O
O
R'
O
P
OH
residual signal of undeuterated solvents. Data for H-NMR
HN
O
HN
R
O
OH OH
OH
33
32
spectra are reported as follows: chemical shift (multiplicity,
coupling constants, number of hydrogens, assignment, see SI
for compound numbering). Abbreviations used are: s (singlet),
d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet),
br (broad). Structural assignments were made with additional
information from gCOSY, gHSQC, gHMBC, NOESY and
TOCSY experiments. Optical rotation data were measured
using an Anton Paar modular compact polarimeter MCP 150,
with either a 10 mm cuvette (0.2 ml) or a 100 mm cuvette (0.7
ml), using a LED (589 nm) as a light source. High-resolution
mass spectrometry (HRMS) spectra were recorded on a Thermo
Fisher Scientific LTQ Orbitrab XL spectrometer using a linear
ion trap and a quadrupole mass filter. Semipreparative and
analytical HPLC analyses were performed on Knauer
Wissenschaftliche Gerate GmbH systems. The solvents for
HPLC were purchased in HPLC grade.
R
TBAF
22% (4 steps)
R'
R
=
O
O
H
N
5
2
=
O
O
H
Si
N
O
O
NH
N
NH
H
O
O
O
O
HN
O
NH
Si
O
N
N
H
H
O
O
NH
O
Si
CONCLUSION
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HO-L-Lys(N-Teoc)-Fmoc (13): Fmoc-L-Lys(N-BOC)-OH
[α]25
Rf 0.17 (10% EtOAc/cyclohexane).
= +31° (c = 2.00 in
퐷
1
1
2
3
4
5
6
7
8
(1.50 g, 3.20 mmol) was dissolved in CH2Cl2 (20 mL) and TFA
(20 mL) was added. The mixture was stirred for 20 min at rt.
After removing the solvent under reduced pressure DMF
(20 mL) and diisopropylethylamine (2.72 ml, 16.0 mmol) were
added. 2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (1.09 g,
3.84 mmol) was dissolved in DMF (6 mL) and transferred to
the reaction solution. After stirring for 2 h at rt the solvent was
removed under reduced pressure and the crude product was
purified by flash chromatography (eluting first with EtOAc,
then with 10% MeOH/CH2Cl2/0.1% AcOH) to yield 1.79 g
(3.81 mmol, 99%) of a colorless solid. The spectroscopic data
were in agreement with those previously reported.10
MeOH). H NMR (CD3OD, 700 MHz): δ 4.25 – 4.18 (m, 2H,
H-2), 4.09 (q, 1H, J = 7.2 Hz, H-4), 1.44 (s, 9H, 3xCH3-Boc),
1.33 (d, 3H, J = 7.2 Hz, H-5), 1.04 – 1.00 (m, 2H, H-1), 0.06 (s,
9H, Si(CH3)3). 13C{1H} NMR (CD3OD, 176 MHz): δ 175.2 (C-
3), 157.9 (C-6), 80.5 (C-7), 64.4 (C-2), 50.8 (C-4), 28.7
(3xCH3-Boc), 18.2 (C-1), 17.7 (C-5), –1.5 (3xCH3). HRMS
(ESI) m/z: [M + Na]+ Calcd for C13H27NO4SiNa 312.1597;
Found 312.1602.
TMSE-O-D Ala-Boc 18.I (220 mg, 760 µmol) was dissolved in
20% TFA/CH2Cl2 (4.5 mL). After stirring for 1 h at rt, toluene
(15 mL) was added and the solvent was removed under reduced
pressure to yield 144 mg (760 µmol, quant.) of a colorless solid.
[α]2퐷5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Rf 0.16 (10% MeOH/CH2Cl2).
= +1.2° (c = 0.82 in
TMSE-O-D-Glu(OH)-Fmoc (14): HO-D-Glu(O-tBu)-Fmoc
(1.50 g, 3.53 mmol) and DMAP (86.1 mg, 705 µmol) were
dissolved in 20 mL EtOAc and cooled to 0 °C. 2-
(trimethylsilyl)ethanol (758 µL, 5.29 mmol) and DCC (1 M in
CH2Cl2, 4.23 mL, 4.23 mmol) were added. After stirring for 2 h
at rt, the reaction was filtered over celite and the residue was
washed with EtOAc (2x20 mL). The solvent was removed
under reduced pressure and the crude product was purified by
flash chromatography (15% EtOAc/cyclohexane) to yield
TMSE-O-D-Glu(O-tBu)-Fmoc 14.I (1.77 g , 3.37 mmol, 96%)
[α]25
1
MeOH). H NMR (CD3OD, 500 MHz): δ 4.35 – 4.31 (m, 2H,
H-2), 4.00 (q, 1H, J = 7.2 Hz, H-4), 1.51 (d, 3H, J = 7.2 Hz, H-
5), 1.09 – 1.06 (m, 2H, H-1), 0.07 (s, 9H, Si(CH3)3). 13C{1H}
NMR (CD3OD, 126 MHz): δ 171.7 (C-3), 65.8 (C-2), 50.0 (C-
4), 18.2 (C-1), 16.6 (C-5), –1.6 (3xCH3). HRMS (ESI) m/z: [M
+ H]+ Calcd for C8H20NO2Si 190.1258; Found 190.1263.
Tetrapeptide 17: Functionalization of the resin (10 to 12): A
frit containing syringe was loaded with 2-chlorotrityl chloride
resin (280 mg, initial loading 2.1 mmol/g). The resin was
swelled for 20 min in 3 mL CH2Cl2. The solvent was removed
and a solution of Fmoc-D-Ala-OH (91.5 mg, 0.29 mmol) and
DIPEA (201 µL, 1.16 mmol) in 2 mL CH2Cl2 was added. The
syringe was shaken for 1 h at rt. After draining the solvent, the
resin was washed with DMF (5 x 1 min shaking with 2 mL).
The initial resin loading 2.1 mmol/g was reduced in this step to
1.0 mmol/g.
as a colorless solid. Rf 0.32 (15% EtOAc/cyclohexane).
=
퐷
+14° (c = 1.98 in MeOH). 1H NMR (CD3OD, 700 MHz): δ 7.79
(d, 2H, J = 7.5 Hz, Harom.), 7.66 (t, 2H, J = 8.3 Hz, Harom.), 7.38
(t, 2H, J = 7.2 Hz, Harom.), 7.30 (t, 2H, J = 8.0 Hz, Harom.), 4.38
– 4.32 (m, 2H, H-10), 4.23 – 4.19 (m, 4H, H-2, H-4, H-11), 2.33
(t, 2H, J = 7.0 Hz, H-6), 2.15 – 2.07 (m, 1H, H-5), 1.92 – 1.83
(m, 1H, H-5), 1.44 (s, 9H, 3xCH3-Boc), 1.04 – 0.96 (m, 2H, H-
1), 0.04 (s, 9H, Si(CH3)3). 13C{1H} NMR (CD3OD, 176 MHz):
δ 173.8 (C-7), 173.7 (C-3), 158.6 (C-9), 145.3 (Carom.), 145.2
(Carom.), 142.6 (Carom.), 128.8 (Carom.), 128.2 (Carom.), 128.1
(Carom.), 126.3 (Carom.), 126.2 (Carom.), 120.9 (Carom.), 81.8 (C-8),
68.0 (C-10), 64.6 (C-2), 54.8 (C-4), 48.4 (C-11), 32.6 (C-6),
28.4 (3xCH3-Boc), 27.8 (C-5), 18.2 (C-1), –1.5 (3xCH3).
HRMS (ESI) m/z: [M + H]+ Calcd for C29H40NO6Si 526.2619;
Found 526.2598.
a) Capping of free reaction sites: A solution of DMF (900 µL),
N-methylimidazol (600 µL) and acetanhydride (300 µL) was
added to the resin and the syringe was shaken for 45 min at rt.
The reaction solution was removed and the resin was washed
with DMF (5 x 1 min shaking with 2 mL).
b) Deprotection (Removal of Fmoc): The resin was shaken
5 min at rt with 20% piperidine/DMF (2 mL). The deprotection
solution was removed and the resin was loaded again with 20%
piperidine/DMF (2 mL) and was shaken for 15 min at rt. After
removal of the deprotection solution, the resin was washed with
DMF (4 x 1 min shaking with 2 mL), CH2Cl2 (3 x 1 min shaking
with 2 mL) and again with DMF (3 x 1 min shaking with 2 mL).
TMSE-O-D-Glu(O-tBu)-Fmoc 14.I (1.00 g, 1.90 mmol) was
dissolved in CH2Cl2 (30 mL). 2,6-lutidine (4.42 mL,
38.0 mmol) and trimethylsilyl trifluoromethanesulfonate
(TMSOTf, 3.44 mL, 19.0 mmol) were added at 0 °C and stirred
for 30 min. The reaction was stirred for further 30 min at rt
followed by slowly addition of aq. sat. NH4Cl (30 mL). The
aqueous phase was extracted with EtOAc (3x40 mL). The
organic phase was washed with 30% AcOH/water, dried over
MgSO4 and the solvent was removed under reduced pressure to
yield 14 (866 mg, 1.84 mmol, 97%) as a colorless solid. The
spectroscopic data were in agreement with those previously
reported.10
c) Coupling: The resin was loaded with a solution of amino acid
(for
Fmoc-L-Lys(N-Teoc)-OH,
Fmoc-D-Glu(OH)-TMSE
1.5 equiv and Fmoc-L-Ala-OH 4 equiv), HBTU (same equiv as
the used amino acid), HOBt (same equiv as the used amino
acid) and DIPEA (twice the equiv of the used amino acid) in
DMF (2 mL). The resin was shaken 40 min at rt, washed with
DMF (3 x 1 min shaking with 2 mL) and the resin was loaded
again with a fresh reaction solution of the same amino acid and
was shaken for 40 min. After this double coupling procedure,
the resin was washed with DMF (5 x 1 min shaking with 2 mL).
TMSE-O-D-Ala-NH2
(18):
HO-D-Ala-Boc
(1.50 g,
7.93 mmol) and DMAP (194 mg, 1.59 mmol) were dissolved in
EtOAc (40 mL) and cooled to 0 °C. 2-(trimethylsilyl)ethanol
(1.70 mL, 11.9 mmol) and DCC (1 M in CH2Cl2, 9.51 mL,
9.51 mmol) were added. After stirring for 2 h at rt, the reaction
was filtered through celite and the residue was washed with
EtOAc (2x20 mL). The solvent was removed under reduced
pressure and the crude product was purified by flash
chromatography (10% EtOAc/cyclohexane) to yield TMSE-O-
D-Ala-Boc 18.I (2.27 g, 7.84 mmol, 99%) as a colorless solid.
Cleavage from the solid phase: A solution of 20% 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) in CH2Cl2 (3 mL) was added to
the dry resin and shaken for 3 h at rt. The solvent was collected
and the resin was washed further 2 x with 20% HFIP/CH2Cl2.
The solvent was removed under reduced pressure and the
residue dissolved in tBuOH/water and lyophilized to yield
219 mg (82%, 229 µmol) of the tetrapeptide as a colorless solid.
5
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The Journal of Organic Chemistry
(Si(CH3)3), –1.7 (Si(CH3)3). HRMS (ESI) m/z: [M + Na]+ Calcd
= –9.0° (c = 0.44 in
Page 6 of 11
[α]2퐷5
Rf 0.19 (10% MeOH/CH2Cl2).
1
1
for C51H82N6O12Si3Na 1077.5191; Found 1077.5195.
MeOH). H NMR ((CD3)2SO, 700 MHz): δ 12.48 (br, s, 1H,
COOH), 8.25 (d, 1H, J = 7.7 Hz, NH(Glu)), 8.17 – 8.13 (m, 1H,
NH(Ala)), 7.90 – 7.88 (m, 3H, 2x Harom., NH(Lys)), 7.75 – 7.69
(m, 2H, Harom.), 7.47 (d, 1H, J = 7.9 Hz, NH(Ala)), 7.41 (t, 2H,
J = 7.4 Hz, Harom.), 7.32 (t, 2H, J = 7.4 Hz, Harom.), 6.95 – 6.93
(m, 1H, NH(Teoc)), 4.28 – 4.24 (m, 3H, H-4, H-12), 4.22 – 4.16
(m, 4H, H-8, H-13, H-10, H-2), 4.14 – 4.10 (m, 2H, H-23,), 4.01
– 3.98 (m, 2H, H-20), 2.93 – 2.89 (m, 2H, H-18), 2.22 – 2.13
(m, 2H, H-6), 1.99 – 1.92 (m, 1H, H-7), 1.81 – 1.73 (m, 1H, H-
7), 1.61 – 1.55 (m, 1H, H-15), 1.47 – 1.43 (m, 1H, H-15), 1.36
– 1.32 (m, 2H, H-17), 1.24 (d, 3H, J = 7.2 Hz, H-14), 1.24 (d,
3H, J = 7.2 Hz, H-25), 1.22 – 1.17 (m, 2H, H-16), 0.95 – 0.93
(m, 2H, H-24), 0.91 – 0.88 (m, 2H, H-21), 0.01 (s, 9H,
Si(CH3)3), -0.00 (s, 9H, Si(CH3)3). 13C{1H} NMR ((CD3)2SO,
176 MHz): δ 174.0 (C-1), 172.4 (C-9), 171.5 (C-22), 171.1 (C-
5), 170.8 (C-3), 156.0 (C-19), 155.3 (C-11), 149.3 (Carom.),
140.4 (Carom.), 127.3 (Carom.H), 126.8 (Carom.H), 125.0 (Carom.H),
119.8 (Carom.H), 65.4 (C-12), 62.3 (C-23), 61.0 (C-20), 51.9 (C-
4), 51.5 (C-8), 49.6 (C-10), 47.2 (C-2), 46.4 (C-13), 39.8 (C-
18), 31.8 (C-15), 31.0 (C-6), 28.9 (C-17), 26.9 (C-7), 22.3 (C-
16), 18.4 (C-14), 17.1 (C-25), 16.9 (C-21), 16.5 (C-24), –1.4
((Lys)Si(CH3)3), –1.5 ((Glu)Si(CH3)3). HRMS (ESI) m/z: [M +
H]+ Calcd for C43H66N5O11Si2 884.4279; Found 884.4279.
2
3
4
5
6
7
8
9
Pentapeptide 7: Fmoc-L-Ala-γ-D-Glu(O-TMSE)-L-Lys(N-
Teoc)-D-Ala-D-Ala-O-TMSE 19 (100 mg, 94.7 µmol) was
dissolved in 20% piperidine/DMF (1 mL). After stirring for
30 min at rt, the solvent was removed under reduced pressure.
The crude product was purified by flash chromatography (10%
MeOH/CH2Cl2) to yield 60.3 mg (72.4 µmol, 76%) of a
colorless solid. The spectroscopic data were in agreement with
those previously reported.10
HO-Gly-Teoc (25): Glycine (250 mg, 3.33 mmol) was
dissolved in 50% dioxane/water (8 mL) and NEt3 (1.38 mL,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9.99 mmol)
and
N-(2-
trimethylsilyl)ethoxycarbonyloxy)succinimide
(950 mg,
3.66 mmol) were added. The solution was stirred on at rt and
diluted with water (20 mL). Citric acid 0.5 M (aq.) was added
until the pH turned 4. The aqueous phase was extracted with
diethylether (3x40 mL) and the combined ether layer was
washed twice with water (15 mL). The organic phase was dried
over MgSO4 and the solvent was removed under reduced
pressure to yield 714 mg (3.26 mmol, 98%) of a colorless solid.
Rf 0.41 (10% MeOH/0.1% AcOH/CH2Cl2). 1H NMR (CD3OD,
500 MHz): δ 4.18 – 4.14 (m, 2H, H-4), 3.80 (s, 2H, H-2), 1.02
– 0.99 (m, 2H, H-5), 0.05 (s, 9H, Si(CH3)3). 13C{1H} NMR
(CD3OD, 126 MHz): δ 173.7 (C-1), 159.4 (C-3), 64.2 (C-4),
43.0 (C-2), 18.6 (C-5), –1.5 (3xCH3). HRMS (ESI) m/z: [M –
H]– Calcd for C8H16NO4Si 218.0854; Found 218.0851.
Fmoc protected pentapeptide 19: To a solution of Fmoc-L-
Ala-γ-D-Glu(O-TMSE)-L-Lys(N-Teoc)-D-Ala-COOH
17
(25.0 mg, 28.7 µmol), PyBOB (22.1 mg, 42.4 µmol) and HOBt
(5.73 mg, 42.4 µmol) in DMF (0.5 mL) was added dropwise a
solution of H2N-D-Ala-O-TMSE 18 (7.49 mg, 39.6 µmol),
DIPEA (24.0 µL, 141 µmol) in DMF (0.5 mL). After stirring
for 25 min at rt the solvent was removed under reduced pressure
and the crude product was purified by HPLC (85% AcN/water,
retention time 14.6 min, using a KNAUER Eurospher II 100-5
C18; 5 µm; 250 x 16 mm + precolumn 30 x 16 mm, 265 nm) to
yield 28.3 mg (26.8 µmol, 95%) of a colorless solid. Rf 0.34
Peptide 27 and Peptide 29: a) Capping of free reaction sites:
A solution of DMF (450 µL), N-methylimidazol (300 µL) and
acetanhydride (150 µL) was added to the resin and the syringe
was shaken for 45 min at rt. The reaction solution was removed
and the resin was washed with DMF (5 x 1 min shaking with
2 mL).
b) Deprotection (Removal of Fmoc): The resin was shaken
5 min at rt with 20% piperidine/DMF (2 mL). The deprotection
solution was removed and the resin was loaded again with 20%
piperidine/DMF (2 mL) and was shaken for 15 min at rt. After
removal of the deprotection solution, the resin was washed with
DMF (4 x 1 min shaking with 2 mL), CH2Cl2 (3 x 1 min shaking
with 2 mL) and again with DMF (3 x 1 min shaking with 2 mL).
[α]25
1
(10% MeOH/CH2Cl2).
= +10° (c = 0.60 in MeOH). H
퐷
NMR ((CD3)2SO, 700 MHz): δ 8.24 (d, 1H, J = 7.7 Hz,
NH(Glu)), 8.16 (d, 1H, J = 7.5 Hz, NH(D-Ala)), 8.14 (d, 1H, J
= 7.5 Hz, NH(D-Ala)), 8.00 (d, 1H, J = 7.3 Hz, NH(Lys)), 7.89
(d, 2H, J = 7.4 Hz, Harom.), 7.75 – 7.69 (m, 2H, Harom.), 7.47 (d,
1H, NH(L-Ala)), 7.41 (t, J = 7.4 Hz, 2H, Harom.), 7.32 (t, 2H, J
= 7.4 Hz, J = 7.5 Hz, Harom.), 6.94 (t, 1H, J = 5.4 Hz, NH(Teoc)),
4.30 (q, 1H, J = 7.5 Hz, H-6), 4.24 (d, 2H, J = 7.0 Hz, H-16),
4.21 – 4.07 (m, 12H, H-12, H-8, H-4, H-14, H-2, H-17, H-28),
4.01 – 3.99 (m, 2H, H-25), 2.92 – 2.90 (m, 2H, H-23), 2.21 –
2.12 (m, 2H, H-10), 1.95 – 1.90 (m, 1H, H-11), 1.80 – 1.75 (m,
1H, H-11), 1.59 – 1.54 (m, 1H, H-20), 1.47 – 1.44 (m, 1H, H-
20), 1.35 (p, 2H, J = 6.8 Hz, H-22), 1.28 (d, 3H, J = 7.3 Hz, H-
18), 1.23 (d, 3H, J = 7.5 Hz, H-30), 1.19 (d, 3H, J = 7.0 Hz, H-
19), 1.26 – 1.15 (m, 2H, H-21), 0.94 – 0.88 (m, 6H, H-1, H-26,
H-28), 0.01 (s, 9H, Si(CH3)3), 0.01 (s, 9H, Si(CH3)3), 0.00 (s,
9H, Si(CH3)3). 13C{1H} NMR ((CD3)2SO, 176 MHz): δ 172.2
(C-14), 171.9 (C-3), 171.3 (C-5), 171.0 (C-27), 171.0 (C-7),
171.0 (C-9), 156.2 (C-24), 155.4 (C-15), 143.6 (Carom.), 140.6
(Carom.), 127.5 (Carom.H), 126.9 (Carom.H), 125.2 (Carom.H), 120.0
(Carom.H), 65.5 (C-16), 62.4 (C-28), 62.3 (C-2), 61.2 (C-25),
52.7 (C-8), 51.6 (C-12), 49.7 (C-14), 47.6 (C-4), 47.4 (C-6),
46.5 (C-17), 39.7 (C-23), 31.3 (C-20), 31.1 (C-11), 29.1 (C-22),
26.9 (C-11), 22.5 (C-21), 18.5 (C-30), 17.9 (C-19), 17.3 (C-26),
16.7 (C-29), 16.6 (C-1), 16.6 (C-18), –1.6 (Si(CH3)3), –1.6
c) Coupling: The resin was loaded with a solution of amino acid
(for Alloc-L-Lys(N-Fmoc)-OH, Fmoc-D-Glu(OH)-TMSE
1.5 equiv and Fmoc-Gly-OH, Teoc-Gly-OH, Fmoc-L-Ala-OH
4 equiv), HBTU (same equiv as the used amino acid), HOBt
(same equiv as the used amino acid) and DIPEA (twice the
equiv of the used amino acid) in 2 mL DMF. The resin was
shaken 40 min at rt washed with DMF (3 x 1 min shaking with
2 mL) and the resin was loaded again with a fresh reaction
solution of the same amino acid and was shaken again for
40 min. After this double coupling procedure, the resin was
washed with DMF (5 x 1 min shaking with 2 mL).
Cleavage from the solid phase: A solution of 20% 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) in CH2Cl2 (3 mL) was added to
the dry resin and shaken for 3 h at rt. The solvent was collected
and the resin was washed further 2 x with 20% HFIP/CH2Cl2.
The solvent was added slowly to cold ether resulting in
precipitation and after centrifugation the supernatant solvent
was discarded and the precipitate dissolved in tBuOH/water and
lyophilized to yield 133 mg (114 µmol, 83%) of the
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Page 7 of 11
The Journal of Organic Chemistry
nonapeptide 29 as a colorless solid. After the glycine couplings (Carom.H), 120.1 (Carom.H), 65.7 (C-12), 62.6 (C-33), 61.9 (C-
1
2
3
4
5
6
7
8
and before the alloc removal a test cleavage from the resin was
performed with 15.0 mg to yield 9.10 mg of the alloc protected
heptapeptide 27.
30), 52.3 (C-4), 51.7 (C-8), 49.8 (C-10), 47.6 (C-2) 46.6 (C-13),
43.5 (C-28), 42.1 (C-24), 42.1 (C-22), 42.0 (C-26), 42.0 (C-20),
38.4 (C-18), 31.9 (C-15), 31.3 (C-6) 28.7 (C-17), 27.1 (C-7),
22.6 (C-16), 18.6 (C-14), 17.3 (C-35), 16.8 (C-31), 16.8 C-34),
–1.4 (Si(CH3)3), –1.5 (Si(CH3)3). HRMS (ESI) m/z: [M + H]+
Calcd for C53H79N10O16Si2 1167.5220; Found 1167.5216.
Alloc removal: The peptide containing resin was dried at HV
and flushed with argon. CH2Cl2 (0.5 mL) were added and the
suspension was stirred 15 min at rt. Then, phenylsilane
(591 µL, 4.79 mmol) in 1 mL CH2Cl2 and Pd(PPh3)4 (57.6 mg,
49.9 µmol) in 3 mL CH2Cl2 were added and the reaction was
stirred for 2 h at rt. The suspension was filled back to a syringe
containing a frit and the solvent was drained. The resin was
washed with CH2Cl2 (6 x 1 min shaking with 3 mL), DMF (4 x
1 min shaking with 3 mL) and again with CH2Cl2 (4 x 1 min
shaking with 3 mL). Before the next coupling the resin was
swollen again with 2 mL of DMF for 20 min at rt and the
solvent was drained.
Peptide 30: Fmoc-L-Ala-γ-D-Glu(O-TMSE)-L-Lys((Gly)5-
Teoc)-D-Ala-COOH 29 (60.0 mg, 51.3 µmol), PyBOB
(40.1 mg, 76.9 µmol), HOBt (10.4 mg, 76.9 µmol) and H2N-D-
Ala-O-TMSE 18 (13.6 mg, 71.8 µmol) were dissolved in DMF
(2 mL) and DIPEA (34.9 µL, 205 µmol) was added
immediately. After stirring for 40 min at rt the solvent was
removed under reduced pressure and the crude product was
dissolved in a minimum DMF and added to ice cold diethyl
ether. The formed precipitate was filtered and washed with cold
ether and dichloromethane, redissolved in MeOH and the
solvent was removed under reduced pressure to yield 38.5 mg
(28.7 µmol, 56%) of a colorless solid. Rf 0.27 (10%
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Characterterization data for compound 27 (testcleavage): Rf
[α]25
0.30 (20% MeOH/CH2Cl2).
= –3.5° (c = 0.28 in MeOH).
퐷
1H NMR ((CD3)2SO, 700 MHz): δ 8.22 – 8.16 (br, m, 2H,
2xNH(Gly)), 8.10 (t, 2H, J = 5.2 Hz, 2xNH(Gly)), 8.04 (s, 1H,
NH(Ala)), 7.73 (t, 1H, J = 5.6 Hz, NH(Lys)), 7.27 – 7.23 (m,
2H, NH(Lys), NH(Teoc)), 5.93 – 5.87 (m, 1H, H-24), 5.29 (dd,
1H, J = 17.2 Hz, 1.5 Hz, H-25), 5.17 (d, 1H, J = 10.5 Hz,
1.2 Hz, H-25), 4.46 (d, 2H, J = 5.2 Hz, H-23), 4.17 – 4.12 (m,
1H, H-2), 4.05 – 4.02 (m, 2H, H-20), 3.98 – 3.96 (m, 1H, H-4),
3.75 (d, 2H, J = 5.7 Hz, H-14), 3.75 (d, 2H, J = 5.7 Hz, H-16),
3.73 (d, 2H, J = 5.8 Hz, H-12), 3.65 (d, 2H, J = 5.9 Hz, H-10),
3.62 (d, 2H, J = 6.0 Hz, H-18), 3.02 (q, 2H, J = 6.7 Hz, H-8),
1.63 – 1.54 (m, 1H, H-5), 1.52 – 1.47 (m, 1H, H-5), 1.40 – 1.34
(m, 2H, H-7), 1.31 – 1.26 (m, 2H, H-6), 1.24 (d, 3H, J = 7.2 Hz,
H-26), 0.93 – 0.91 (m, 2H, H-21), 0.02 (s, 9H, Si(CH3)3).
13C{1H} NMR ((CD3)2SO, 176 MHz): δ 174.0 (C-1), 171.5 (C-
3), 169.7 (C-17), 169.3 (C-15), 169.3 (C-13), 169.0 (C-11),
168.4 (C-9), 156.7 (C-19), 155.7 (C-22), 133.6 (C-24), 116.9
(C-25), 64.4 (23), 61.9 (C-20), 54.4 (C-4), 47.7 (C-2), 43.5 (C-
18), 42.1 (C-16), 42.0 (C-14), 42.0 (C-12), 42.0 (C-10), 38.3
(C-8), 31.8 (C-5), 28.6 (C-7), 22.7 (C-6), 17.6 (C-21), 17.3 (C-
26), –1.4 (Si(CH3)3). HRMS (ESI) m/z: [M + H]+ Calcd for
C29H49N8O12Si 729.3245; Found 729.3241. Characterterization
[α]25
[α]25
1
MeOH/CH2Cl2).
= +7.4° (c = 0.68 in MeOH). H NMR
퐷
((CD3)2SO, 700 MHz): δ 8.24 (d, 1H, J = 7.6 Hz, NH(Glu)),
8.17 – 8.12 (m, 4H, 2xNH-D-Ala, 2xNH(Gly)), 8.08 – 8.04 (m,
2H, 2xNH(Gly)), 8.01 (d, 1H, J = 7.4 Hz, NH(Lys)), 7.89 (d,
2H, J = 7.6 Hz, Harom.), 7.73 – 7.70 (m, 3H, NH(Lyssidechain),
2xHarom.), 7.47 (d, 1H, J = 7.0 Hz, NH-L-Ala), 7.41 (t, 2H, J =
7.4 Hz, Harom.), 7.32 (t, 2H, J = 7.3 Hz, Harom.), 7.22 (t, 1H, J =
6.0 Hz, NH(Teoc)), 4.31 (q, 1H, J = 7.6 Hz, H-6), 4.25 – 4.07
(m, 11H, H-2, H-38, H-16, H-17, H-14, H-12, H-8, H-4), 4.04
– 4.02 (m, 2H, H-35), 3.75 (d, 2H, J = 5.6 Hz, H-31), 3.75 (d,
2H, J = 5.6 Hz, H-29), 3.73 (d, 2H, J = 5.8 Hz, H-27), 3.65 (d,
2H, J = 5.8 Hz, H-25), 3.62 (d, 2H, J = 6.0 Hz, H-33), 3.02 –
2.99 (m, 2H, H-23), 2.22 – 2.12 (m, 2H, H-10), 1.95 – 1.90 (m,
1H, H-11), 1.80 – 1.76 (m, 1H, H-11), 1.60 – 1.56 (m, 1H, H-
20), 1.50 – 1.45 (m, 1H, H-20), 1.39 – 1.35 (m, 2H, H-22), 1.28
(d, 3H, J = 7.3 Hz, H-18), 1.27 – 1.20 (m, 2H, H-21), 1.23 (d,
3H, J = 7.1 Hz, H-40), 1.19 (d, 3H, J = 7.1 Hz, H-19), 0.94 –
0.90 (m, 6H, H-1, H-36, H-39), 0.02 (s, 9H, Si(CH3)3), 0.01 (s,
9H, Si(CH3)3), 0.00 (s, 9H, Si(CH3)3). 13C{1H} NMR
((CD3)2SO, 176 MHz): δ 172.7 (C-13), 172.4 (C-3), 172.0 (C-
5), 171.7 (C-7), 171.5 (C-37), 171.4 (C-9), 169.7 (C-32), 169.3
(C-30), 169.3 (C-28), 169.0 (C-26), 168.4 (C-24), 156.7 (C-34),
155.6 (C-15), 143.8 (Carom.), 140.7 (Carom.), 127.6 (Carom.), 127.1
(Carom.), 125.3 (Carom.), 120.1 (Carom.), 65.7 (C-16), 62.6 (C-38),
62.5 (C-2), 61.9 (C-35), 52.8 (C-8), 51.7 (C-12), 49.8 (C-14),
47.7 (C-4), 47.5 (C-6), 46.6 (C-17), 43.5 (C-33), 42.1 (C-29),
42.1 (C-27), 42.0 (C-31), 42.0 (C-25), 38.4 (C-23), 31.4 (C-20),
31.2 (C-10), 28.8 (C-22), 27.0 (C-11), 22.7 (C-21), 18.7 (C-40),
18.1 (C-19), 17.3 (C-18), 16.8 (C-36), 16.7 (C-1), 16.7 (C-39),
–1.4 (Si(CH3)3), –1.5 (Si(CH3)3), –1.5 (Si(CH3)3). HRMS (ESI)
m/z: [M + Na]+ Calcd for C61H97N11O17Si3Na 1362.6264; Found
1362.6238.
data for compound 29: Rf 0.21 (15% MeOH/CH2Cl2).
= –
퐷
1
2.8° (c = 0.70 in MeOH). H NMR ((CD3)2SO, 700 MHz): δ
8.26 (d, 1H, J = 7.5 Hz, NH(Glu)), 8.19 (br, s, 2H, 2xNH(Gly)),
8.10 – 8.07 (m, 3H, 2xNH(Gly), NH-D-Ala), 7.91 (d, 1H, J =
8.2 Hz, NH(Lys)), 7.89 (d, 2H, J = 7.5 Hz, Harom.), 7.74 – 7.71
(m, 3H, NH(Lyssidechain), 2xHarom.), 7.48 (d, 1H, J = 7.0 Hz, NH-
L-Ala), 7.41 (t, 2H, J = 7.8 Hz, Harom.), 7.32 (t, 2H, J = 7.4 Hz,
Harom.), 7.23 (t, 1H, J = 5.9 Hz, NH(Teoc)), 4.25 – 4.10 (m, 9H,
H-33, H-12, H-13, H-10, H-2, H-4, H-8), 4.04 – 4.02 (m, 2H,
H-30), 3.75 (d, 2H, J = 5.7 Hz, H-24), 3.75 (d, 2H, J = 5.7 Hz,
H-26), 3.73 (d, 2H, J = 5.8 Hz, H-22), 3.65 (d, 2H, J = 5.8 Hz,
H-20), 3.62 (d, 2H, J = 5.9 Hz, H-28), 3.01 (q, 2H, J = 6.4 Hz,
H-18), 2.22 – 2.14 (m, 2H, H-6), 1.98 – 1.93 (m, 1H, H-7), 1.81
– 1.75 (m, 1H, H-7), 1.62 – 1.57 (m, 1H, H-15), 1.47 – 1.46 (m,
1H, H-15), 1.36 (p, 2H, J = 7.1 Hz, H-17), 1.24 (d, 3H, J =
7.0 Hz, H-35), 1.23 (d, 3H, J = 7.3 Hz, H-14), 1.28 – 1.19 (m,
2H, H-16), 0.95 – 0.90 (m, 4H, H-31, H-34), 0.02 (s, 9H,
Si(CH3)3), 0.00 (s, 9H, Si(CH3)3). 13C{1H} NMR ((CD3)2SO,
176 MHz): δ 174.0 (C-1), 172.7 (C-9), 171.7(C-3), 171.3 (C-
32), 171.1 (C-5), 169.7 (C-27), 169.3 (C-25), 169.3 (C-23),
169.0 (C-21), 168.4 (C-19), 156.7 (C-29), 155.6 (C-11), 143.8
(Carom.), 140.7 (Carom.), 127.6 (Carom.H), 127.1 (Carom.H), 125.3
Peptide 8: Fmoc-L-Ala-γ-D-Glu(O-TMSE)-L-Lys((Gly)5-
Teoc)-D-Ala-D-Ala-O-TMSE 30 (35.0 mg, 26.1 µmol) was
dissolved in 20% piperidine/DMF (2 mL). After stirring for
30 min at rt the solvent was removed under reduced pressure.
Cold diethyl ether was added and the resulting precipitate was
filtered, washed with cold diethyl ether, dissolved in MeOH and
the solvent was removed under reduced pressure to yield
25.5 mg (22.8 µmol, 87%) of a colorless solid. Rf 0.53 (22%
[α]25
1
MeOH/CH2Cl2).
= +10° (c = 0.99 in MeOH). H NMR
퐷
7
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The Journal of Organic Chemistry
Page 8 of 11
((CD3)2SO, 700 MHz): δ 8.22 – 8.11 (m, 5H, NH(Glu), 2xNH-
analysis. HRMS (ESI) m/z: [M – H]– Calcd for C54H93N7O20Si3P
1
2
3
4
5
6
7
8
D-Ala, 2xNH(Gly)), 8.07 (dt, 2H, J = 11.3, 5.7 Hz, 2xNH(Gly)),
8.01 (d, 1H, J = 7.4 Hz, NH(Lys)), 7.73 (t, 1H, J = 5.5 Hz,
NH(Lyssidechain)), 7.22 (t, 1H, J = 5.9 Hz, NH(Teoc)), 4.29 (p,
1H, J = 7.1 Hz, H-6), 4.20 – 4.08 (m, 7H, H-2, H-4, H-8, H-12,
H-35), 4.06 – 3.99 (m, 2H, H-32), 3.75 (d, 2H, J = 5.5 Hz, H-
28), 3.75 (d, 2H, J = 5.5 Hz, H-26) 3.73 (d, 2H, J = 5.8 Hz, H-
24), 3.65 (d, 2H, J = 5.8 Hz, H-22), 3.62 (d, 2H, J = 6.0 Hz, H-
30), 3.39 (q, 1H, J = 6.9 Hz, H-14), 3.01 (q, 2H, J = 6.9 Hz, H-
20), 2.52 (m. 2H, NH2), 2.20 – 2.14 (m, 2H, H-10), 1.97 –
1.95 (m, 1H, H-11), 1.80 – 1.78 (m, 1H, H-11), 1.61 – 1.56 (m,
1H, H-17), 1.49 – 1.46 (m, 1H, H-17), 1.39 – 1.34 (m, 2H, H-
19), 1.28 (d, 3H, J = 7.3 Hz, H-15), 1.28 – 1.19 (m, 2H, H-18),
1.19 (d, 3H, J = 7.1 Hz, H-16), 1.16 (d, 3H, J = 6.9 Hz, H-37),
0.96 – 0.91 (m, 6H, H-1, H-26, H-33), 0.02 (s, 9H, Si(CH3)3),
0.02 (s, 9H, Si(CH3)3), 0.02 (s, 9H, Si(CH3)3). 13C{1H} NMR
((CD3)2SO, 176 MHz): δ 175.1 (C-13, HMBC) 172.4 (C-3),
172.0 (C-5), 171.8 (C-7), 171.5 (C-34), 171.4 (C-9), 169.7 (C-
29), 169.3 (C-27), 169.3 (C-25), 169.0 (C-23), 168.4 (C-21),
156.7 (C-31), 62.7 (C-35), 62.5 (C-2), 61.9 (C-32), 52.8 (C-8),
51.5 (C-12), 49.8 (C-14), 47.7 (C-4), 47.5 (C-6), 43.5 (C-30),
42.1 (C-26), 42.1 (C-24), 42.0 (C-28), 42.0 (C-22), 38.4 (C-20),
31.4 (C-17), 31.2 (C-10), 28.8 (C-19), 27.1 (C-11), 22.7 (C-18),
20.9 (C-37) 18.0 (C-16), 17.3 (C-15), 16.8 (C-33), 16.8 (C-1),
16.7 (C-36), –1.4 (Si(CH3)3), –1.5 (Si(CH3)3), –1.5 (Si(CH3)3).
HRMS (ESI) m/z: [M + H]+ Calcd for C46H88N11O15Si3
1118.5764; Found 1118.5750.
1274.5526; Found 1274.5608. This free phosphate 21.I
(27.0 mg, 21.2 µmol) was dissolved in 80% AcOH/water
(3 mL) and stirred for 2 d at rt. Mass spectrometry showed full
conversion at this point and toluene (20 mL) was added and the
solvent was removed under reduced pressure to yield 25.1 mg
(21.1 µmol, quant.) of a colorless solid, which was used for the
next reaction without further purification or analysis. HRMS
(ESI) m/z: [M – H]– Calcd for C47H89N7O20Si3P 1186.5213;
Found 1186.5334.
9
Compound 22: Farnesolphosphate 9 (60.0 mg, 200 µmol) was
coevaporated two times from toluene (1 mL) under argon and
dissolved in 50% DMF/THF (1.4 ml). Carbonyldiimidazol
(CDI, 130 mg, 799 µmol) was dissolved in 50% DMF/THF
(1 ml) and added to the farnesolphosphate solution. The
reaction was stirred 2 h at rt and MeOH (35 µL) was added
before stirring for further 45 min. The solvents were removed
under reduced pressure and the synthesized phosphoimidazol
intermediate was coevaporated twice from toluene (1 mL)
under argon before the addition of DMF (1 mL). Mass
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
spectrometry
showed
the
successfully
synthesized
phosphoimidazol intermediate. HRMS (ESI) m/z: [M – H]–
Calcd for C18H28N2O3P 351.1843; Found 351.1835. In the
meantime, 21 (28.0 mg, 23.6 µmol) was coevaporated first
from pyridine (200 µL) and then, twice from toluene (1 mL)
under argon. DMF (1 mL) and the freshly prepared
phosphoimidazole solution were added and the mixture was
stirred 3 d at rt. The solvent was removed under reduced
pressure and the crude product was semi purified by gel
permeation chromatography (Sephadex LH-20, GE Healthcare,
260 x 20 mm, methanol) to yield 26.8 mg (<18.2 µmol, <77%)
of a colorless solid, which was used for the next reaction
without further purification or analysis. Rf 0.80 (4 MeOH/2
CHCl3/0.5 water). HRMS (ESI) m/z: [M – H]– Calcd for
C62H114N7O23Si3P2 1470.6755; Found 1470.6750.
Farnesyl phosphate 9: Trichloroacetonitrile (TCA, 433 µL,
4.32 mmol) was added to farnesol (449 µL, 1.80 mmol). To this
stirred solution was added tetrabutylammoniumphosphate
(TBAP, 0.4 M in acetonitrile, 8.99 mL, 3.60 mmol) over 1 h via
syringe pump. The reaction was stirred for further 7 h at rt and
the solvent was removed under reduced pressure. The crude
product was first purified by flash chromatography (10%
water/20% NH3 (conc.)/Isopropanol) and then percolated
through Dowex® 50WX8 with a 0.025 M NH4HCO3 solution.
Dowex® 50WX8 was washed before usage with 3:1 NH3/water
and 0.025 M NH4HCO3 solution until pH of the eluent turned 8.
The solvent was removed by lyophilisation to yield 425 mg
(1.27 mmol, 71%) of a colorless powder. The spectroscopic
data were in agreement with those previously reported.20
3-Lipid I (3): Protected, semi pure 22 (26.8 mg, <18.2 µmol)
was coevaporated from toluene (1 mL) twice and dissolved in
DMF (0.5 mL). The solution was cooled to 0 °C and a 1 M
solution of tetrabutylammonium fluoride in THF (TBAF,
419 µL, 419 µmol) was added. The reaction mixture was
allowed to warm to rt and was stirred 2 d at rt before removing
the solvent under reduced pressure. The crude product was
purified by gel permeation chromatography (Sephadex LH-20,
Compound 20: H2N-L-Ala-γ-D-Glu(O-TMSE)-L-Lys(N-
Teoc)-D-Ala-D-Ala-O-TMSE 7 (45.6 mg, 54.7 µmol), PyBOB
(32.9 mg, 63.1 µmol), HOBt (8.53 mg, 63.1 µmol) and 6
(27.0 mg, 42.1 µmol) were dissolved in DMF (2 mL) and
DIPEA (35.8 µL, 210 µmol) was added immediately. After
stirring for 45 min at rt the solvent was removed under reduced
pressure and the crude product was purified by flash
chromatography (2.5% MeOH/CH2Cl2) to yield 49.8 mg
(34.2 µmol, 81%) of a colorless solid. The spectroscopic data
were in agreement with those previously reported.10
GE Healthcare, 260
x
20 mm, methanol) and the
tetrabutylammonium counterions were exchanged to
ammonium ions using percolation through Dowex® 50WX8
with a 0.02 M NH4HCO3 solution. Dowex® 50WX8 was
washed before usage with 3:1 NH3/water and 0.02 M NH4HCO3
solution until pH of the eluent turned 8. The solvent was
removed by lyophilisation to yield 20.3 mg (<18.0 µmol) of the
semi purified 3. Final purification was achieved by HPLC (25%
– 50 % NH4HCO3 (0.1% aq.)/methanol, retention time 8.9 min,
using a KNAUER Eurospher II 100-5 C8; 5 µm; 250 x 16 mm
+ precolumn 30 x 16 mm, 205 nm) yielding 10.7 mg
(7.28 µmol, 40% over four steps) of a colorless solid. Rf 0.46 (3
Compound 21: To a solution of glycopeptide 20 (35.6 mg,
24.4 µmol) in MeOH (2.5 mL) was added Pd–C (40.0 mg, Pd-
10%). The reaction vessel was filled with hydrogen. After
stirring for 30 min at rt full conversion was detected by mass
spectrometry and the suspension was filtered over celite. The
precipitate was washed with methanol and the solvent was
removed under reduced pressure to yield 29.0 mg (24.4 µmol,
quant.) of the free phosphate 21.I as a colorless solid, which
was used for the next reaction without further purification or
[α]25
MeOH/3 CHCl3/1 water).
= +59° (c = 0.51 in MeOH). 1H
퐷
NMR (D2O, 500 MHz): δ 5.50 – 5.47 (m, 1H, H-17), 5.46 (t,
1H, J = 7.1 Hz, H-31), 5.22 (t, 1H, J = 6.5 Hz, H-35), 5.20 (t,
1H, J = 7.8 Hz, H-39), 4.54 – 4.49 (m, 2H, H-30), 4.35 (q, 1H,
J = 7.2 Hz, H-4), 4.29 (q, 1H, J = 7.2 Hz, H-12), 4.25 – 4.22 (m,
8
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The Journal of Organic Chemistry
2H, H-6, H-14), 4.20 – 4.10 (m, 3H, H-2, H-10, H-16), 3.98 – (C-35), 119.3 (C-31), 100.1 (C-45), 94.2 (C-17), 78.4 (C-14),
1
2
3
4
5
6
7
8
3.96 (m, 1H, H-18), 3.92 – 3.84 (m, 2H, H-20), 3.80 (dd, 1H, J
= 9.6, 9.6 Hz, H-15), 3.66 (dd 1H, , J = 9.6, 9.6 Hz, H-19), 3.02
(t, 2H, J = 7.5 Hz, H-26), 2.32 (t, 2H, J = 7.8 Hz, H-8), 2.20 –
2.15 (m, 3H, H-9, H-34), 2.15 – 2.11 (m, 4H, H-33, H-38), 2.04
(t, 2H, J = 7.1 Hz, H-37), 2.02 (s, 3H, CH3-NHAc), 1.93 – 1.89
(m, 1H, H-9), 1.87 – 1.77 (m, 2H, H-23), 1.74 (s, 3H, H-43),
1.72 – 1.70 (m, 2H, H-25), 1.70 (s, 3H, H-42), 1.64 (s, 6H, H-
41, H-44), 1.52 – 1.47 (m, 2H, H-24), 1.46 (d, 3H, J = 7.3 Hz,
H-28), 1.43 (d, 3H, J = 6.8 Hz, H-29), 1.39 (d, 3H, J = 7.2 Hz,
H-22), 1.35 (d, 3H, J = 7.2 Hz, H-21). 13C{1H} NMR (D2O, 176
MHz): δ 179.8 (C-1), 177.6 (C-27), 175.8 (C-11), 175.7 (C-7),
174.1 (C-5), 174.1 (NHAc-C=O), 174.0 (C-3), 173.6 (C-13),
143.2 (C-32), 136.7 (C-36), 133.4 (C-40), 124.4 (C-39), 124.2
(C-35), 119.3 (C-31), 94.7 (C-17), 79.9 (C-15), 78.0 (C-14),
73.0 (C-18), 68.0 (C-19), 63.1 (C-30), 60.3 (C-20), 54.2 (C-10),
54.2 (C-6) 53.4 (C-16), 51.0 (C-2), 49.9 (C-12), 49.6 (C-4), 39.1
(C-26), 38.8 (C-33), 38.8 (C-37), 31.7 (C-8), 30.5 (C-23), 28.1
(C-9), 26.3 (C-25), 25.8 (C-38), 25.5 (C-34), 24.9 (C-42), 22.2
(NHAc-CH3), 22.0 (C-24), 18.6 (C-22), 17.4 (C-21), 17.0 (C-
41), 16.8 (C-28), 16.5 (C-29), 15.7 (C-43), 15.3 (C-44). 31P
NMR (D2O, 284 MHz): δ –10.9 (d, J = 14.0 Hz), –13.4 (d, J =
14.0 Hz). HRMS (ESI) m/z: [M – H]– Calcd for C46H78N7O21P2
1126.4731; Found 1126.4715. The spectroscopic data were in
agreement with those previously reported.12
77.9 (C-15), 75.9 (C-46), 74.0 (C-48), 73.8 (C-18), 72.4 (C-19),
70.3 (C-47), 63.1 (C-30), 61.1 (C-50/C-20), 59.7 (C-50/C-20),
56.1 (C-49), 54.3 (C-6), 53.7 (C-10), 53.5 (C-16), 51.0 (C-2),
50.0 (C-12), 49.6 (C-4), 39.2 (C-26), 38.8 (C-33), 38.8 (C-37),
31.8 (C-8), 30.5 (C-23), 28.2 (C-9), 26.5 (C-25), 25.8 (C-38),
25.6 (C-34), 24.9 (C-42), 22.2 (C-24), 22.2 (NHAc-CH3), 22.1
(NHAc-CH3), 18.7 (C-22), 17.4 (C-21), 17.0 (C-41), 16.9 (C-
28), 16.5 (C-29), 15.7 (C-43), 15.3 (C-44). 31P NMR (D2O,
284 MHz): δ –10.9 (d, J = 20.7 Hz), –13.4 (d, J = 19.2 Hz).
HRMS (ESI) m/z: [M – 2H]2– Calcd for C54H90N8O26P2
9
3H]3– Calcd for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
664.2726; Found 664.2719. [M
–
C54H89N8O26P2 442.5127; Found 442.5117. The spectroscopic
data were in agreement with those previously reported.12
Compound 31: H2N-L-Ala-γ-D-Glu(O-TMSE)-L-Lys((Gly)5-
Teoc)-D-Ala-D-Ala-O-TMSE 8 (21.1 mg, 18.9 µmol), PyBOB
(13.4 mg, 25.7 µmol), HOBt (3,47 mg, 25.7 µmol) and acid 6
(11.0 mg, 17.4 µmol) were dissolved in DMF (1.5 mL) and
DIPEA (11.7 µL, 68.6 µmol) was added immediately. After
stirring for 45 min at rt the solvent was removed under reduced
pressure. Cold EtOAc was added and the resulted precipitate
was filtered off, washed with cold EtOAc, dissolved in MeOH
and the solvent was removed under reduced pressure to yield
26.0 mg (14.9 µmol, 86%) of a colorless solid. Rf 0.27 (10%
[α]25
1
MeOH/CH2Cl2).
= +25° (c = 0.36 in MeOH). H NMR
퐷
3-Lipid II (4): In vitro synthesis of Compound 4 was
performed in a total volume of 4.5 mL containing 1.50 mg
(1.33 µmol) Compound 3, 2 mM UDP-D-GlcNAc, 50 mM NaPi
and 0.5 mM MgCl2, pH 6.5. The reaction was initiated by the
addition of 300 μg of MurG-His6. After incubation for 4 h at
30 °C the reaction was quenched by the addition of 3 mL
MeOH and evaporated to dryness. Dried samples were
dissolved in distilled water for mass spectrometric analysis. The
crude product was purified by HPLC (35% – 50% NH4HCO3
(0.1% aq.)/methanol, retention time 8.0 min, using a KNAUER
Eurospher II 100-5 C8; 5 µm; 250 x 16 mm + precolumn 30 x
16 mm, 205 nm) yielding 0.98 mg (0.73 µmol, 55%) of a
colorless solid. Recombinant MurG-His6 enzyme was
overexpressed and purified as described23 and dialized
[α]25
H2O). 1H NMR (D2O, 700 MHz): δ 5.50 – 5.47 (m, 1H, H-17),
5.46 (t, 1H, J = 6.9 Hz, H-31), 5.23 (t, 1H, J = 6.4 Hz, H-35),
5.20 (t, 1H, J = 6.4 Hz, H-39), 4.63 (d, 1H, J = 8.3 Hz, H-45),
4.51 – 4.49 (m, 2H, H-30), 4.35 (q, 1H, J = 7.1 Hz, H-4), 4.32
– 4.27 (m, 2H, H-12, H-14), 4.24 – 4.20 (m, 2H, H-6, H-10),
4.15 – 4.12 (m, 3H, H-2, H-16), 3.98 – 3.89 (m, 4H, H-18, H-
19, H-20, H-50), 3.86 – 3.81 (m (pt), 1H, H-15), 3.79 – 3.74 (m,
3H, H-20‘,H-49, H-50‘), 3.58 (pt (dd), 1H, J = 8.6, 8.6 Hz, H-
48), 3.46 – 3.91 (m, 2H, H-46, H-47), 2.97 (t, 2H, J = 7.0 Hz,
H-26), 2.34 – 2.33 (m, 2H, H-8), 2.19 – 2.17 (m, 3H, H-9, H-
34), 2.14 – 2.11 (m, 4H, H-33, H-38), 2.07 (s, 3H, CH3-NHAc),
2.05 (t, 2H, J = 7.7 Hz, H-37), 2.01 (s, 3H, CH3-NHAc), 1.93 –
1.89 (m, 1H, H-9‘), 1.86 – 1.82 (m, 1H, H-23), 1.81 – 1.79 (m,
1H, H-23‘), 1.74 (s, 3H, H-43), 1.71 (s, 3H, H-42), 1.69 – 1.66
(m, 2H, H-25), 1.64 (s, 6H, H-41, H-44), 1.47 (d, 3H, J =
6.9 Hz, H-28), 1.46 (d, 3H, J = 6.8 Hz, H-29), 1.45 – 1.39 (m,
2H, H-24), 1.40 (d, 3H, J = 7.2 Hz, H-22), 1.35 (d, 3H, J =
7.2 Hz, H-21). 13C{1H} NMR (D2O, 176 MHz): δ 179.8 (C-1),
177.6 (C-27), 175.7 (C-11), 175.7 (C-7), 174.4 (NHAc-C=O),
174.2 (NHAc-C=O), 174.1 (C-5), 174.0 (C-3), 173.6 (C-13),
143.2 (C-32), 136.7 (C-36), 133.5 (C-40), 124.4 (C-39), 124.2
(CD3OD, 700 MHz): δ 7.49 – 7.47 (m, 2H, Harom.), 7.43 – 7.34
(m, 13H, Harom.), 5.84 (dd, 1H, J = 5.8, 3.6 Hz, H-19), 5.63 (s,
1H, H-22), 5.15 – 5.08 (m, 4H, 2xCH2-Ph), 4.38 (q, 1H, J =
7.1 Hz, H-16), 4.38 (q, 1H, J = 7.1 Hz, H-6), 4.35 – 4.30 (m,
3H, H-4, H-12, H-14), 4.20 – 4.12 (m, 8H, H-2, H-8, H-18, H-
41, H-44), 4.03 (dd, 1H, J = 9.8, 4.0 Hz, H-23), 3.92 – 3.77 (m,
15H, H-17, H-20, H-21, H-23, H-31, H-33, H-35, H-37, H-39),
3.21 – 3.19 (m, 2H, H-29), 2.28 (t, 2H, J = 7.2 Hz, H-10), 2.19
– 2.12 (m, 1H, H-11), 1.91 – 1.87 (m, 1H, H-11‘), 1.85 (s, 3H,
CH3-NHAc), 1.77 – 1.72 (m, 1H, H-26), 1.68 – 1.62 (m, 1H, H-
26), 1.56 – 1.49 (m, 2H, H-28), 1.40 (d, 3H, J = 7.2 Hz, CH3),
1.37 (d, 3H, J = 7.2 Hz, CH3), 1.37 – 1.32 (m, 2H, H-27) 1.35
(d, 3H, J = 7.2 Hz, CH3), 1.34 (d, 3H, J = 6.8 Hz, CH3), 1.02 –
0.94 (m, 6H, H-1, H-42, H-45), 0.05 (s, 9H, Si(CH3)3), 0.04 (s,
9H, Si(CH3)3), 0.02 (s, 9H, Si(CH3)3). 13C{1H} NMR (CD3OD,
176 MHz): δ 175.6 (C=O), 174.9 (C=O), 174.7 (C=O), 174.6
(C=O), 174.5 (C=O), 174.1 (C=O), 173.8 (NHAc-C=O), 173.4
(C=O), 172.9 (C=O), 172.7 (C=O), 172.7 (C=O), 172.1 (C=O),
against 10 mM NaPi buffer, pH 7.0.
= +11° (c = 0.18 in
퐷
171.5 (C=O), 159.4 (C-40), 138.9 (Carom. quart.
,
-Ph), 137.0 (d, J =
-Bn),
6.5 Hz, Carom. quart.-Bn), 137.0 (d, J = 6.5 Hz, Carom. quart.
,
,
130.1 (Carom.), 130.0 (Carom.), 129.9 (Carom.), 129.8 (Carom.), 129.8
(Carom.), 129.3 (Carom.), 129.2 (Carom.), 129.2 (Carom.), 127.3
(Carom.), 102.8 (C-22), 97.9 (C-19), 82.3 (C-21), 78.4 (C-16),
76.5 (C-17), 71.2 (d, J = 6.0 Hz, CH2-Ph), 71.1 (d, J = 6.0 Hz,
CH2-Ph), 69.1 (C-23), 66.0 (C-20), 64.7 (C-2), 64.5 (C-44),
64.5 (C-41), 55.5 (C-18), 55.0 (C-8), 53.2 (C-12), 50.3 (C-14/C-
4/C-6), 50.1 (C-4/C-14/C-6), 49.7 (C-6/C-14/C-4), 45.0 (CH2-
Gly), 44.0 (CH2-Gly), 43.9 (CH2-Gly), 43.8 (CH2-Gly), 43.7
(CH2-Gly), 40.0 (C-29), 32.4 (C-10), 32.2 (C-26), 29.8 (C-28),
28.2 (C-11), 24.1 (C-27), 22.9 (NHAc-CH3), 19.9 (CH3), 18.7
(C-42), 18.5 (CH3), 18.3 (C-45), 18.2 (C-1), 18.0 (CH3), 17.4
(CH3), –1.4 (Si(CH3)3), –1.4 (Si(CH3)3), –1.5 (Si(CH3)3). 31P
NMR (CD3OD, 284 MHz): δ –2.6. HRMS (ESI) m/z: [M + Na]+
Calcd for C78H121N12O25Si3PNa 1763.7503; Found 1763.7525.
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Page 10 of 11
Compound 32: To a solution of glycopeptide 31 (24.2 mg, using a KNAUER Eurospher II 100-5 C8; 5 µm; 250 x 16 mm
1
2
3
4
5
6
7
8
13.9 µmol) in MeOH (2 mL) was added Pd–C (25.0 mg, Pd-
10%). The reaction vessel was filled with hydrogen. After
stirring for 30 min at rt full conversion was detected by mass
spectrometry and the suspension was filtered over celite. The
catalyst was washed with methanol and the solvent was
removed under reduced pressure to yield 19.5 mg (12.5 µmol,
90%) of the free phosphate 32.I as a colorless solid, which was
used for the next reaction without further purification or
+ precolumn 30 x 16 mm, 205 nm) yielding 4.4 mg (3.04 µmol,
22% over four steps) of a colorless solid. Rf 0.38 (3 MeOH/3
[α]25
1
CHCl3/1 water).
= +21° (c = 0.44 in MeOH). H NMR
퐷
(D2O, 700 MHz): δ 5.50 – 5.48 (m, 1H, H-17), 5.46 (t, 1H, J =
6.7 Hz, H-31), 5.22 (t, 1H, J = 6.7 Hz, H-35), 5.20 (t, 1H, J =
6.7 Hz, H-39), 4.54 – 4.49 (m, 2H, H-30), 4.37 (q, 1H, J =
7.2 Hz, H-14), 4.30 (q, 1H, J = 7.2 Hz, H-4), 4.26 – 4.17 (m,
3H, H-6, H-10, H-12), 4.16 – 4.10 (m, 2H, H-2, H-16), 4.08 (s,
2H, H-52), 4.03 (s, 2H, H-48/H-50), 4.01 (s, 2H, H-48/H-50),
3.98 – 3.96 (m, 1H, H-18), 3.91 (s, 2H, H-46), 3.90 – 3.83 (m,
2H, H-20), 3.82 (s, 2H, H-54), 3.80 (dd, 1H, J = 9.6, 9.6 Hz, H-
15), 3.66 (dd, 1H, J = 9.6, 9.6 Hz, H-19), 3.23 (t, 2H, J = 6.7 Hz,
H-26), 2.37 – 2.28 (m, 2H, H-8), 2.20 – 2.16 (m, 3H, H-9, H-
34), 2.15 – 2.11 (m, 4H, H-33, H-38), 2.04 (t, 2H, J = 7.1 Hz,
H-37), 2.02 (s, 3H, CH3-NHAc), 1.93 – 1.88 (m, 1H, H-9), 1.83
– 1.73 (m, 2H, H-23), 1.74 (s, 3H, H-43), 1.72 – 1.70 (m, 2H,
H-25), 1.71 (s, 3H, H-42), 1.64 (s, 6H, H-41, H-44), 1.56 – 1.51
(m, 2H, H-24), 1.46 (d, 3H, J = 7.2 Hz, H-22), 1.43 (d, 3H, J =
6.8 Hz, H-28), 1.38 (d, 3H, J = 7.2 Hz, H-29), 1.35 (d, 3H, J =
7.2 Hz, H-21). 13C{1H} NMR (D2O, 176 MHz): δ 179.9 (C-1),
177.7 (C-27), 175.7 (C-11), 175.6 (C-7), 174.2 (C-5), 174.1
(NHAc-C=O), 174.0 (C-3), 173.6 (C-13), 172.2 (C-47/C-49/C-
51), 172.1 (C-47/C-49/C-51), 172.0 (C-47/C-49/C-51), 171.0
(C-45), 169.9 (C-53) 143.2 (C-32), 136.7 (C-36), 133.4 (C-40),
124.4 (C-39), 124.2 (C-35), 119.3 (C-31), 94.7 (C-17), 79.9 (C-
15), 78.0 (C-12), 73.0 (C-18), 68.0 (C-19), 63.2 (C-30), 60.3
(C-20), 54.3 (C-10), 54.3 (C-6), 53.4 (C-16), 51.0 (C-2), 49.8
(C-4), 49.5 (C-14), 42.6 (C-48/C-50), 42.5 (C-48/C-50), 42.5
(C-46), 42.4 (C-52), 41.2 (C-54), 39.0 (C-26), 38.8 (C-33), 38.8
(C-37), 31.8 (C-8), 30.7 (C-23), 28.1 (C-9), 27.8 (C-25), 25.8
(C-38), 25.5 (C-34), 24.9 (C-42), 22.4 (NHAc-CH3), 22.2 (C-
24), 18.7 (C-28), 17.4 (C-21), 17.0 (C-41), 16.9 (C-22), 16.6
(C-29), 15.7 (C-43), 15.3 (C-44). 31P NMR (D2O, 162 MHz) δ
= –10.8 (d, J = 15.4 Hz), –13.3 (d, J = 15.4 Hz). HRMS (ESI)
m/z: [M – H]– Calcd for C56H93N12O26P2 1411.5805; Found
1411.5810. [M – 2H]2– Calcd for C56H92N12O26P2 705.2866;
Found: 705.2877.
analysis. HRMS (ESI) m/z: [M
–
H]– Calcd for
9
C64H108N12O25Si3P 1559.6599; Found 1559.6516. This free
phosphate 32.I (16.7 mg, 10.7 µmol) was dissolved in 80%
AcOH/water (3 mL) and stirred for 2 d at rt. Mass spectrometry
showed full conversion at this point and toluene (30 mL) was
added and the solvent was removed under reduced pressure to
yield 15.7 mg (10.7 µmol, quant.) of a colorless solid, which
was used for the next reaction without further purification or
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
analysis. HRMS (ESI) m/z: [M
–
H]– Calcd for
C57H104N12O25Si3P 1471.6286; Found: 1471.6201.
Compound 33: Farnesolphosphate 9 (27.7 mg, 92.1 µmol) was
coevaporated from toluene (1 mL) two times and dissolved in
50% DMF/THF (1.4 ml). Carbonyldiimidazol (CDI, 59.7 mg,
368 µmol) was dissolved in 50% DMF/THF (1 ml) and added
to the farnesolphosphate solution. The reaction was stirred 2 h
at rt and MeOH (16 µL) was added before stirring further
45 min. The solvents were removed under reduced pressure and
the synthesized phosphoimidazol intermediate was
coevaporated from toluene (1 mL) twice before the addition of
DMF (1 mL). Mass spectrometry showed the successfully
synthesized phosphoimidazol intermediate. HRMS (ESI) m/z:
[M – H]– Calcd for C18H28N2O3P 351.1843; Found: 351.1835.
In the meantime, phosphate 32 (15.7 mg, 10.7 µmol) was
coevaporated first from 95 µL pyridine and then twice from
1 mL toluene under argon. 0.5 mL DMF and the freshly
prepared phosphoimidazole solution were added and the
reaction was stirred 3 d at rt. The solvent was removed under
reduced pressure and the crude product was semi purified by
gel permeation chromatography (Sephadex LH-20, GE
Healthcare, 260 x 20 mm, methanol) to yield 10.9 mg
(<6.20 µmol, <58%) of a colorless solid, which was used for the
next reaction without further purification or analysis. Rf 0.62 (4
MeOH/2 CHCl3/0.5 water). HRMS (ESI) m/z: [M – H]– Calcd
for C72H129N12O28Si3P2 1755.7828; Found 1755.7826. [M –
2H]2– Calcd for C72H128N12O28Si3P2 877.3877, Found 877.3879.
3-Lipid II (S. aureus): In vitro synthesis of 3-Lipid II (S.
aureus) was performed in a total volume of 30 μL containing
10 µg Compound 5, 2 mM UDP-D-GlcNAc, 50 mM NaPi and
0.5 mM MgCl2, pH 6.5. The reaction was initiated by the
addition of 2 μg of purified MurG-His6. After incubation for
4 h at 30 °C the reaction was quenched by the addition of 60 µl
MeOH and evaporated to dryness. Dried samples were
dissolved in distilled water for mass spectrometric analysis.
Recombinant MurG-His6 enzyme was overexpressed and
purified as described23 and dialized against 10 mM NaPi
buffer, pH 7.0. HRMS (ESI) m/z: [M – 2H]2– Calcd for
C64H105N13O31P2 806.8263; Found 806.8255. [M – 3H + Na]2–
Calcd for C64H104N13O31P2Na 817.8173; Found 817.8166. [M –
3H]3– Calcd for C64H104N13O31P2 537.5484; Found 537.5475.
3-Lipid I (S. aureus) (5): Protected, semi pure 33 (10.9 mg,
<6.20 µmol) was coevaporated twice from 1 mL toluene under
argon and dissolved in DMF (0.7 mL). The solution was cooled
to 0 °C and a 1 M solution of tetrabutylammonium fluoride in
THF (TBAF, 116 µL, 116 µmol) was added. The reaction
mixture was allowed to warm to rt and was stirred 2 d at rt
before removing the solvent under reduced pressure. The crude
product was purified by gel permeation chromatography
(Sephadex LH-20, GE Healthcare, 260 x 20 mm, methanol) and
the tetrabutylammonium counterions were exchanged to
ammonium using percolation through Dowex® 50WX8 with a
0.02 M NH4HCO3 solution. Dowex® 50WX8 was washed
before usage with 3:1 NH3/water and 0.02 M NH4HCO3 solution
until pH of the eluent turned 8. The solvent was removed by
lyophilisation to yield 8.20 mg (<5.80 µmol) of the semi
purified 5. Final purification was achieved using HPLC (25% –
50 % NH4HCO3 (0.1% aq.)/methanol, retention time 8.1 min,
ASSOCIATED CONTENT
Supporting Information
Copies of NMR spectra and details on the optimized route to 6. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
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The Journal of Organic Chemistry
Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-
Resistant Strains for Cell Wall Synthesis. Chem. Eur. J. 2013, 19,
12104–12112.
Corresponding Author
1
* E-Mail: dirk.menche@uni-bonn.de
2
3
4
5
(14) Lebar, M. D.; May, J. M.; Meeske, A. J.; Leiman, S. A.;
Lupoli, T. J.; Tsukamoto, H.; Losick, R.; Rudner, D. Z.; Walker,
S.; Kahne, D. Reconstruction of Peptidoglycan Cross-Linking
Leads to Improved Fluorescent Probes of Cell Wall Synthesis. J.
Am. Chem. Soc. 2014, 136, 10874–10877.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
6
Financial support was provided by the Deutsche
Forschungsgemeinschaft (DFG, German Research Foundation)–
Project-ID TRR261. We thank Andreas J. Schneider (University of
Bonn) for excellent HPLC support and the group of Prof. Diana
Imhof (University of Bonn) for technical support in solid phase
synthesis.
7
8
9
(15) Bollhagen, R.; Schmiedberger, M.; Barlosb, K.; Grell, E. A
New Reagent for the Cleavage of Fully Protected Peptides
synthesised on 2-Chlorotrityl Chloride Resin. J. Chem. Soc.,
Chem. Commun. 1994, 2559–2560.
(16) 0.5 Equivalents of 11 were used, see experimental section for
details.
(17) see experimental section section for full details.
(18) Liu, C. Y.; Guo, C. W.; Chang, Y. F.; Wang, J. T.; Shih, H.
W.; Hsu, Y. F.; Chen, C. W.; Chen, S. K.; Wang, Y. C.; Cheng, T.
J. R; Ma, C.;Wong, C. R.; Fang, J. M.; Cheng, W. C. Synthesis
and Evaluation of a New Fluorescent Transglycosylase Substrate:
Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl
Moiety. Org. Lett. 2010, 12, 1608–1611.
(19) Chen, L.; Men, H.; Ha, S.; Ye, X. Y.; Brunner, L.; Hu, Y.;
Walker, S. Intrinsic Lipid Preferences and Kinetic Mechanism of
Escherichia coli MurG. Biochemistry 2002, 41, 6824–6833.
(20) Lira, L. M.; Vasilev, D.; Pilli, R. A.; Wessjohann, L. A. One-
pot synthesis of organophosphate monoesters from alcohols.
Tetrahedron Lett. 2013, 54, 1690–1692.
(21) Kato, D.; Verhelst, S. H. L.; Sexton, K. B.; Bogyo, M. A
General Solid Phase Method for the Preparation of Diverse
Azapeptide Probes Directed Against Cysteine Proteases. Org.
Lett. 2005, 7, 5649–5652.
(22) A first batch of 5 could also be converted to the
corresponding lipid II analog, confirming the general usefulness,
also of this chemoen-zymatic approach.
(23) Schneider, T.; Kruse, T.; Wimmer, R.; Wiedemann, I.; Sass,
V.; Pag, U.; Jansen, A.; Nielsen, A. K.; Mygind, P. H.; Raventós,
D. S.; Neve, S.; Ravn, B.; Bonvin, A. M. J. J.; De Maria, L.;
Andersen, A. S.; Gammelgaard, L. K.; Sahl, H. G.; Kristensen; H.
H. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall
Precursor Lipid II. Science. 2010, 328, 1168–1172.
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