Bicyclic enol cyclocarbamates inhibit penicillin-binding proteins

Article abstract of DOI:10.1039/c6ob01664b

Natural products form attractive leads for the development of chemical probes and drugs. The antibacterial lipopeptide Brabantamide A contains an unusual enol cyclocarbamate and we used this scaffold as inspiration for the synthesis of a panel of enol cyclocarbamate containing compounds. By equipping the scaffold with different groups, we identified structural features that are essential for antibacterial activity. Some of the derivatives block incorporation of hydroxycoumarin carboxylic acid-amino d-alanine into the newly synthesized peptidoglycan. Activity-based protein-profiling experiments revealed that the enol carbamates inhibit a specific subset of penicillin-binding proteins in B. subtilis and S. pneumoniae.

Full text of DOI:10.1039/c6ob01664b

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M. Witte, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB01664B.
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Bicyclic enol cyclocarbamates inhibit Penicillin-binding proteins
Paul Dockerty,a Jerre G. Edens,a Menno B. Tol,b Danae Morales Angeles,b Arnau Domenech,c Yun
Liu,a Anna K. H. Hirsch,a Jan-Willem Veening,c Dirk-Jan Scheffers,b Martin D. Witte*a
Abstract: Natural products form attractive leads for the development of chemical probes and drugs. The antibacterial
lipopeptide Brabantamide A contains an unusual enol cyclocarbamate and we used this scaffold as inspiration for the
synthesis of a panel of enol cyclocarbamate containing compounds. By equipping the scaffold with different groups, we
identified structural features that are essential for antibacterial activity. Some of the derivatives block incorporation of
hydroxycoumarine carboxylic acid-amine D-alanine into the newly synthesized peptidoglycan. Activity-based protein-
profiling experiments revealed that the enol carbamates inhibit a specific subset of penicillin-binding proteins in B. subtilis
and S. pneumoniae.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
growth. A scaffold that triggered our interest was the enol
Introduction
cyclocarbamate. These proline-derived 5,5-fused and 5,7-fused
bicyclic ring systems are found in lipopeptides that have been
Microorganisms produce
a large variety of secondary
isolated from Pseudomonas extracts, such as Brabantamide A
1a
(Figure 1A).7,8 Compounds containing this scaffold,
metabolites for communication, nutrient uptake and self-
defense. These structurally diverse compounds often have
interesting properties, like antimicrobial and cytotoxic activity.
Because of these properties, natural products and parts of
(
)
including natural product 1a and (semi-)synthetic derivatives
1b9 and 2a10 potently inhibit lipoprotein-associated
phospolipase A2 (Lp-PLA2),
a
mammalian group VII
their scaffold have served as starting point for the
development of drugs1 and chemical probes.
2,3
phospholipase A2 that belongs to the serine-hydrolase
superfamily.
Examples of
natural product based drugs include antibiotics like beta-
lactams, which inhibit cell wall synthesis, and tetracyclines and
aminoglycosides, which block protein synthesis. The natural
products vibrolactone, epoxomicin and cyclophellitol have
been converted into tools to study serine hydrolases,4 the
proteasome and glycosidases, respectively.5 Further examples
of probe molecules derived from metabolites are fluorescent
penicillin and moenomycin A analogues, which have been
prepared to study the transpeptidation and transglycosylation
step during peptidoglycan synthesis, respectively.3,6 The vast
majority of the isolated natural products have not yet been
exploited for probe and drug development, while
derivatization of many of these scaffolds may also lead to
interesting research tools or lead compounds. Of particular
interest are natural products that display antibacterial activity.
The rising resistance to commonly applied antibiotics
necessitates the identification of leads that inhibit bacterial
Besides being Lp-PLA2 inhibitors, enol cyclocarbamate
containing lipopeptides, like 1a, have also been shown to
affect bacterial, fungal and oomycete growth by targeting
specific pathways in these microorganisms.11-13 Brabantamide
A (1a) increases phospholipase D activity in oomycetes. Mode
of action studies with reporter strains that carry a firefly
luciferase gene fused to a promoter that is induced by
antibiotics that block the most important biosynthetic
pathways in bacteria indicated that the bactericidal activity of
lipopeptide 1a is caused by inhibition of the peptidoglycan
synthesis pathway and/or cell membrane stress.14 Although 1a
has surfactant-like properties, non-specific disruption of the
cell membrane has been excluded as the sole mode of action,
since lipopeptide 1a did not permeabilize model membranes
at biologically relevant concentrations.12
Despite the fact that it has been shown that the enol
cyclocarbamate is essential to potently inhibit the mammalian
Lp-PLA2, it has not yet been established if the enol
cyclocarbamate is required for the observed activity in bacteria
and oomycetes. Neither have the molecular targets of
molecules containing this scaffold been identified in bacteria.
We aimed to investigate this by preparing a series of enol
cyclocarbamate derivatives 2–4 (Figure 1B-D) that vary in the
substitution pattern and by studying the antibacterial activity
of the resulting compounds. To determine the role of the 5,5-
fused ring system on the biological activity of the scaffold and
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Figure 1. (A) Structure of the natural product Brabantamide A 1a and its deglycosylated form 1b. The enol cyclocarbamate scaffold is depicted in grey. (B) N-Boc-Proline derived
enol cyclocarbamates 2a–i containing various chains. (C) Monocyclic enol carbamates 3a–c (E and Z isomers). (D) N-Boc-4-hydroxyproline derived enol cyclocarbamates 4a–e.
to probe the impact of the substitution pattern, we refluxing the intermediates in anhydrous methanol.16 In a
synthesized monocyclic analogues 3a–c (Figure 1C) and second step, the β-keto esters were reacted with the amine of
substituted derivatives 2a–i and 4a–e (Figure 1B–D), interest to form β-keto amides 7a–d
, 10, 13 and 17a–b.
respectively. To circumvent possible decomposition of the acid Refluxing β-keto ester with hexylamine or benzylamine in
6
and base-labile enol cyclocarbamate during synthesis,15 we THF did not result in the desired β-keto amide products 7b and
developed a novel route in which the enol cyclocarbamate is 7c, but afforded the corresponding enamine products instead.
introduced at a late stage in the synthesis and that allows The same, undesired products were obtained when the β-keto
straightforward functionalization of this labile scaffold in the esters were reacted with the amine in the presence of sodium
final step with copper-catalyzed click chemistry. With the methoxide17 or titanium isopropoxide.18 Traces of the desired
panel of molecules synthesized, we identified the structural β-keto amides could be obtained by treating the amine with
features that are required for antibacterial activity. Using trimethylaluminum to generate the dimethylaluminum amide
metabolic labeling and activity-based protein profiling, we in situ.19 We therefore turned our attention to DABAL-Me3, the
reveal that these enol cyclocarbamates interfere with adduct of 1,4-diazabicyclo[2.2.2]octane (DABCO) and
peptidoglycan synthesis and that they inhibit Class A high trimethylaluminum.20,21 Activating the amine in this fashion
molecular weight penicillin-binding proteins.
improved the yield of the β-keto amide formation reaction and
products 7a–d 10 13 and 17a–b were obtained in moderate
,
,
to reasonable yields. The final steps of the synthesis entailed
the deprotection and formation of the enol cyclocarbamate.
Although removal of the Boc-protecting group with hydrogen
chloride in diethyl ether and subsequent cyclization of the
precipitated hydrochloride salt with carbonyldiimidazole (CDI)
gave the enol cyclocarbamates as separable mixture of E and Z
isomers, the moderate yield prompted us to devise a novel
two-step one-pot procedure. Deprotecting β-keto amides 7a–
Results and discussion
Chemical synthesis
The synthesis of the panel of enol carbamate derivatives
commenced with condensing Meldrum’s acid to Boc-protected
building blocks
5
,
8
,
11 and 15a-b (Scheme 1A-D) using N,N’-
(DCC) and 4-
dicyclohexylcarbodiimide
(dimethylamino)pyridine (DMAP). Filtration and subsequent
acid-base extraction gave the crude Meldrum’s acid derivative
intermediates. Attempts to directly convert these into β-keto
amides using five equivalents of amine in refluxing toluene
resulted in complex mixtures and the β-keto amide was
therefore installed using a two-step procedure. First, the
Meldrum’s acid intermediates were smoothly converted into
d
,
10,
13
and
17a–b
with
trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in CH2Cl2 was directly
followed by cyclization with CDI affording the
lipocyclocarbamates 2a–d
column chromatography, the Z-isomers were obtained as pure
products. Large-scale synthesis of 3a 3c and 4b revealed that a
, 3a, 3c and 4a–b. After silica gel
,
minor isomer was also formed and for 3c both isomers were
the corresponding β-keto esters
6, 9, 12 and 16a–b by
isolated. NOE (Nuclear Overhauser Effect) experiments with
2 | J. Name., 2012, 00, 1-3
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Scheme 1. Synthesis of enol carbamates starting from (A) Boc-proline (B) Boc-glycine (C) Boc-sarcosine (D) N-Boc-4-hydroxyproline. Reagents and conditions: (i) Meldrum’s acid,
DCC, DMAP; (ii) MeOH, reflux (yield over 2 steps: 54–85%); (iii) DABCO, AlMe3, amine (dodecylamine for 7a, 10, 13, 17a and 17b; hexylamine for 7b; benzylamine for 7c; propargyl
amine for 7d) (yield: 36–73%); (iv) TMSOTf then CDI (yield over 2 steps: 34 – 55%); (v) CuSO4, sodium ascorbate, alkyl azide (dodecyl azide for 2e, 4-phenylbenzyl azide for 2f, 6-
azidohexanol for 2g, phytyl azide for 2h; biotin azide for 2i; benzyl azide for 4c; 2-(2-(2-azidoethoxy)ethoxy)ethanol for 3b and 4d; 3-azido-N,N-dimethylpropan-1-amine for 4e)
(yield: 29–68%); (vi) NaH, Br-R (benzyl bromide for 15a; propargyl bromide for 15b) (yield: 90%).
these isomers confirmed that the Z enol carbamate is formed media based MIC (minimum inhibitory concentration) assay.22
preferentially. Moreover, the chemical shifts for the isomers Since lipopeptide 1a was shown to be particularly potent
are in accordance with the values reported in the literature for against Gram-positive bacteria,12,23 we initially focused on
similar structures.15 The diversity of the panel of enol discriminating between active and non-active compounds on
cyclocarbamates was increased further via a copper-catalyzed the Gram-positive model bacterium Bacillus subtilis 168. Cells
click reaction. Alkyne 2d was reacted with dodecyl azide, 6- were cultured in the presence of serial dilutions of the
azidohexanol, phytyl azide, 4-phenylbenzyl azide and biotin compound for 20 hours, after which both the optical density
azide to obtain compounds 2e–i that differ in chain. Reacting (OD600) and resazurin were used to evaluate the activity of the
Z-lipocyclocarbamate 4b with benzyl azide, 2-(2-(2- compound. Metabolically active bacteria convert resazurin
azidoethoxy)ethoxy)ethanol and 3-azido-N,N-dimethylpropan- into highly fluorescent resorufin and the viability of the
1-amine led to derivatives 4c, 4d and 4e, respectively. Finally, bacteria in the presence of the enol cyclocarbamates can be
Z-lipocyclocarbamate 3a was reacted with (2-(2- determined by comparing the fluorescence intensity of B.
azidoethoxy)ethoxy)ethanol to obtain monocyclic compound subtilis cells treated with compound to the fluorescence
3b
.
intensity of control cells treated with 0.05% DMSO (Figure 2A
and Figure S1).24 Of the synthesized compounds, bicyclic enol
Antibacterial activity of enol carbamates
carbamates 2a
, 2e, 4d and 4e inhibit bacterial growth as
The biological activity of the panel of enol cyclocarbamates 2a-
judged by OD600 and by resazurin, with the respective MIC
i, 3a-c and 4a-e was determined using a standardized liquid-
values being 50 ꢀM for 2a and 2e and 400 ꢀM for 4d and 4e.
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Journal Name
Bacillus subꢀlis 168
Streptococcus pneumo
niae
A
μM (μg/mL)
μM (μg/mL)
μM (μg/mL)
400 (140)
400 (173)
μM (μg/mL)
>400
RO
H
N
2a
2b
2c
2d
2e
50 (17.5)
>400
2a
2e
3a
3b
3c
>400
>400
>400
4d
4e
>400
N
O
O
O
>400
H
N
>400
4a
>400
>400
N
OH
50 (21.5)
O
4b-Z
4b-E
4c
O
O
4d: R =
4e: R =
2a
O
N
O
2f
2g
2h
2i
>400
>400
>400
>400
N
N
>400
>100C
N
N
H
N
N
4d
400 (232)
400 (212)
N
N
N
4e
N
N
O
O
2e
O
0.4
0.3
0.2
0.1
0
0.6
0.5
0.4
0.3
0.2
0.1
0
control
control
200 μM
150 μM
100 μM
75 μM
B
C
μM
25
10 μM
5 μM
50 μM
-0.1
0
1
2
3
4
5
6
7
8
9
10 11 12
0
1
2
3
4
5
6
7
8
9
10 11 12
ꢀme (h)
Figure 2. (A) MIC determination on B. subtilis including the full scope of lipocyclocarbamates, active compounds were also tested for MIC on S. pneumoniae. Structures of the most
potent derivatives are depicted. Highest concentration tested was 400 ꢀ
aCompound 4c could not be tested at higher concentrations due to its poor solubility in the media.
ꢀme (h)
M
.
Growth curves of B. subtilis (B) and S. pneumoniae (C) in the presence of 2a. Note: Light scattering is observed at higher concentrations of 2a presumably due to aggregate
formation. Dissociation of the aggregates over time causes a drop in optical density (OD). The depicted ODs are corrected for light scattering by adjusting the OD at the first time
point with the respective OD in the absence of cells. For clarity at these low OD values, the optical density was plotted on a linear scale.
The corresponding monocyclic analogues 3a–c do not affect 3b
, which is derived from inactive compound 3a and
the viability of B. subtilis, indicating that the 5,5-fused bicyclic structurally related to 4d, does not show activity in the MIC
ring system is indispensable for potent inhibition of bacterial assay, and it thus underlines the importance of the 5,5-fused
growth. Besides the bicyclic enol carbamate, also the bicyclic scaffold for the antibacterial activity.
substitution pattern has a large effect on the activity. Active We subsequently tested the activity of 2a
,
2e
,
4d and 4e on
pathogen
compounds 2a, 2e, 4d and 4e all bear a long, linear alkyl chain the Gram-positive opportunistic human
and replacing this group by smaller and more polar Streptococcus pneumoniae, which is annually responsible for
substituents leads to a significant reduction in antibacterial killing more than 1 million people and in which antibiotic
activity. Hexyl amide derivative 2b, benzyl amide 2c and resistance is on the rise. Culturing S. pneumoniae strain D39
propargyl amide 2d do not show any activity under the with these compounds for 20 hours revealed that only
conditions used. Interestingly, the activity of propargyl amide compounds 2a or 2e had an effect on the viability of this
2d can be restored by reacting it with dodecylazide to form bacterium, with the MIC value being 400
derivative 2e. However, other hydrophobic groups, such as the We then studied the bacterial growth to determine whether
4-phenylbenzyl in 2f and the phytyl in 2h, do not restore the the active compounds 2a 2e 4d and 4e are bacteriostatic,
activity and the introduction of more polar substituents, such bactericidal or bacteriolytic. Several compounds that showed
as the hexylalcohol in 2g and the biotin in 2i, also leads to to be inactive in the MIC assay (3c 4a and 4b) were used in
ꢀ
M.
,
,
,
inactive compounds. Substituents at other positions of the these experiments as controls. Starting with an inoculum of
bicyclic ring system result in remarkable behavior. Both benzyl OD600 0.02 for B. subtilis orOD595 0.04 for S. pneumoniae strain
ether 4a and propargyl ether 4b do not show any activity and D39, with or without the compounds, the optical density was
functionalization of propargyl ether 4b with benzyl azide, as in monitored over time. As expected, monocyclic derivative 3c
4c also leads to an inactive compound. However, equipping 4b and bicyclic benzyl ether 4a, which are inactive in the MIC
with a polyethylene glycol (PEG), as in 4d, or with a tertiary assay, do not inhibit bacterial growth when evaluated at the
amine, as in 4e, results in compounds with moderate activity. same concentrations as the active compounds (compare
Although the amphiphilic nature of these compounds may growth curves Figure 2B, C and Figure S2 with Figure S3).
account for part of the activity, it is likely that the activity of 4d Bicyclic propargyl ether 4b, which was inactive in the MIC
is not solely caused by its surfactant-like properties. This assay at 400
ꢀ
reasoning is reinforced by the fact that PEGylated monocyclic concentration. The cells started to grow after prolonged
M, did slow down growth at higher
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incubation, which corroborates the results of the MIC assays. allows monitoring of de novo biosynthesis.26,27 Culturing B.
Low concentrations of the active enol carbamates 2a 4d and subtilis with HADA for 5 minutes results in strong fluorescent
,
4e also cause a delay in growth of B. subtilis and S. labeling of the septum, as has previously been reported (Figure
pneumoniae (compare growth curves Figure 2B, C and Figure 3A). Incorporation of HADA is completely blocked when the
S2 with Figure S3), but bacterial growth is completely blocked bacteria are pre-incubated with penicillin G for 20 minutes
at higher concentrations of 2a, 4d and 4e. The prolonged lag (Figure 3B). A similar picture was observed when we treated B.
phase observed in the growth curves of S. pneumoniae subtilis with bicyclic dodecylamide derivative 2a (Figure 3C),
cultured with a low concentration of enol carbamate is typical bicyclic PEGylated derivative 4d (Figure 3F), bicyclic dodecyl
for bacteriolytic agents25 and time-lapse microscopy on S. triazole analogue 2e (Figure S5) or bicyclic dimethyl
pneumoniae grown on semi-solid media surfaces containing 2a aminopropane derivative 4e (Figure S5) prior to the addition of
confirm the bacteriolytic activity of the enol carbamates HADA. All these compounds inhibit fluorescent labeling of the
(Figure S4).
septum, while inactive compounds, such as bicyclic
propargylamide 2d (Figure 3D), and compounds that only
delayed the bacterial growth, such as bicyclic propargyl ether
4b (Figure 3E), did not affect fluorescent labeling
Quantification of the fluorescence intensity per cell confirmed
that the decrease in incorporation of HADA is significant
(Figure S6) and live/dead cell assays28 demonstrated that the
decreased incorporation is not caused by membrane
disruption and subsequent cell lysis (Figure S7). We obtained
Identification of the targets of the enol carbamates
Our next interest was to determine the mode of action and to
identify the cellular targets of the synthesized enol
carbamates. Large differences were observed when bacteria
were labeled with hydroxycoumarine carboxylic acid-amine D-
alanine (HADA). This fluorescent substrate analogue is
incorporated into newly synthesized peptidoglycan and thus
Figure 3. HADA labeling in B. subtilis 168 (A-F) and S. pneumoniae (G-L) treated with different enol carbamates. (M) Structures of the enol cyclocarbamate derivatives used. Cells
were incubated with 100 ꢀ of enol carbamate for 20 minutes and then with HADA (0.5 m ) for 5 minutes. 0.05% DMSO/H2O and 100 ꢀ Penicillin G were used as controls.
M
M
M
Representative fluorescence and bright-field micrographs of B. subtilis cells treated with (A) 0.05% DMSO/H2O, (B) Penicillin G, (C) 2a, (D) 2d, (E) 4b and (F) 4d. For uncropped
images see supporting information Figure S5. Representative fluorescence and bright-field micrographs of S. pneumoniae cells treated with (G) 0.05% DMSO/H2O, (H) Penicillin G,
(I) 2a, (J) 2d, (K) 4b and (L) 4d. For uncropped images see supporting information Figure S8.
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Journal Name
A
B
Inhibitor
[μM]
2a
500
+
2a
200
2a
150
+
2d
500
+
2d
200
2d
150
Inhibitor
2a
500
+
2a
500
+
-
[μM]
-
-
+
Bocillin FL (45nM)
+
+
Bocillin FL
kDa
kDa
75
PBP4
75
168
PBP4-null
C
Inhibitor
[μM]
2a
2a
2a
75
+
2d
150
+
penG
4b
-
200 150
100
150
D
Bocillin FL
+
+
PBP1a/b
PBP2a/b/x
H
N
N
H
N
B
F
O
N
S
O
F
O
N
O
Bocillin FL
O
- Na+
O
PBP3
Figure 4. Competition experiments with Bocillin FL. (A) Cells of Bacillus subtilis 168 were treated with indicated amount of compound for 60 min, lysed and subsequently the cell
lysates were incubated with Bocillin FL (45 n ) for 30 minutes. The proteins were resolved on SDS-PAGE and analyzed by fluorescence scanning (15% SDS-PAGE gel); (B) SDS-PAGE
labeling experiment using B. subtilis 168 wild-type and a PBP4-null strain confirms PBP4 as one of the targets of the enol cyclocarbamates; (C); Cells of Streptococcus pneumoniae
M
were treated with indicated amount of compound for 60 minutes, lysed and the cell lysates were subsequently incubated with Bocillin FL (45 n
were resolved on SDS-PAGE and analyzed by fluorescence scanning (15% SDS-PAGE gel) (D) Structure of Bocillin FL.
M) for 30 minutes. The proteins
similar results when we cultured S. pneumoniae D39 with 4d and 4e, and to lesser extend 2e also block labeling of PBP4
HADA in the presence and absence of the compounds (Figure (Figure S9 and S10).
3G-L and Figure S8). Also in this strain, compounds 2a and 4d We subsequently studied if the enol carbamates also target
block fluorescent labeling.
PBPs in S. pneumoniae D39 using the same activity-based
In S. pneumoniae and B. subtilis, penicillin-binding proteins protein-profiling experiment as described for B. subtilis. The
(PBPs) incorporate HADA6,29 and other fluorescent
30-32
D-amino dodecyl amide 2a, propargyl amide 2d and monocyclic
acids (FDAAs)
onto the stempeptide of lipid II and analogue 3c were added to a mid-log culture of S. pneumoniae
peptidoglycan and the decrease in HADA labeling suggests that D39, before the cells were lysed and reacted with Bocillin FL.
the enol carbamates inhibit this process. To establish which Incubating lysates from cells that were treated with 4% DMSO,
PBPs are inhibited by the enol carbamates, we performed as a control, results in three fluorescent bands with a
competitive activity-based protein-profiling experiments. Cells molecular weight of 79, 73 and 45 kDa. These bands
were incubated with the enol carbamates for 60 minutes prior correspond with PBP1a/1b, PBP2a/2b/2x and PBP3,
to cell lysis and the addition of Bocillin FL, a fluorescent respectively. Addition of 2d or 3c to the medium does not alter
penicillin derivative that labels the majority of the PBPs in B. the labeling profile, but incubating S. pneumoniae with dodecyl
subtilis (Figure 4). Fluorescent scanning of the gels revealed amide 2a reduces the labeling intensity of the PBP1a/1b and
that propargyl amide 2d does not inhibit labeling of PBPs, but PBP3 bands by 40 and 60%, respectively (Figure 4C and Figure
dodecyl amide 2a blocks labeling of a Bocillin FL-sensitive PBP S11). Concomitantly, a new fluorescent band (around 40–43
(Figure 4A) with an approximate molecular weight of 70 kDa in kDa) appears when cells are incubated with 2a or 4b (Figure
concentration-dependent manner. The molecular weight of 4C). It has been reported that some antibiotics increase the
this PBP could correspond to PBP3, PBP4, or PBP4A. sensitivity of S. pneumoniae PBPs to proteolysis and this band
Complemented strains that express PBP-GFP fusions and PBP- therefore could be formed by proteolytic cleavage of one of
null strains were used to determine which of these PBPs is the PBPs reacting with 2a. Alternatively, the band could be
inhibited by the enol carbamates.33,34 The PBP-GFP fusion caused by aberrant running of a PBP that reacted with both
proteins migrate differently on the SDS-PAGE due to the Bocillin FL and 2a 35
The addition of phenylmethylsulfonyl
.
increase in molecular weight and as a consequence the Bocillin fluoride (PSMF), a broad-spectrum serine hydrolase inhibitor,
FL labeling pattern will alter. In null strains, the fluorescent during the incubation steps does not block the formation of
band of the respective PBP will be absent when labeling these the band (Figure S11). The biological relevance of this labeled
cells with Bocillin FL. Labeling of PBP-GFP strains did not result protein will have to be determined.
in noticeable differences, but labeling of the PBP4-null strain The identified Class A high-molecular weight PBPs, which have
with Bocillin FL, gave a pattern that corresponds with wild type glycosyltransferase and transpeptidase activity, are not
B. subtilis treated with 2a, indicating that this compound essential for B. subtilis and S. pneumoniae viability. Deletion of
inhibits PBP4 (Figure 4B). Profiling experiments with the PBP4 in B. subtilis36 and PBP1a or PBP1b in S. pneumoniae37,38
complete panel of enol carbamates revealed that compounds does not affect growth and cell-wall synthesis and inhibition of
6 | J. Name., 2012, 00, 1-3
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these PBPs by enol cyclocarbamates cannot explain the Chemicals
View Article Online
DOI: 10.1039/C6OB01664B
ARTICLE
antibacterial activity of the compound class. To elucidate if Resazurin sodium salt was purchased from BD Biosciences.
inhibition of the PBPs does affect the incorporation of HADA, Syto9 and Propidium Iodide were purchased from Life
we treated the B. subtilis PBP4 null strain with HADA. PBP4 has Technologies. All the other chemicals were purchased from
been recently identified as one of the primary enzymes Sigma-Aldrich. The synthesized enol cyclocarbamate
responsible for the global incorporation of unnatural D-amino compounds were dissolved in DMSO and were stored at -20
acid derivatives in the peptidoglycan of B. subtilis.39 Contrary °C.
to enol carbamates 2a and 4d, which completely block septal
incorporation of HADA in wild type B. subtilis, septal labeling of Bacterial strains
the PBP4-null strain with HADA is not altered (Figure S12). The B. subtilis strain 168 was cultured in lysogeny broth (LB). The
latter observation corroborates the results of Fura et al., who strain was first grown in LB overnight at 37 °C before the MIC
showed that FITC-D-lysine is incorporated at the septum of a assay or the optical density (OD600) assay. The MIC assay itself
PBP4-null strain.39 These results clearly demonstrate that the was performed in cation adjusted Mueller-Hinton medium.
observed decrease in septal labeling in B. subtilis is not caused B. subtilis PBP4-null strain was constructed by transformation
by exclusive inhibition of PBP4. This conclusion is reinforced by of chromosomal DNA from strain PS2022 (pbpD::Erm) to B.
the observation that labeling of the septum can be inhibited by subtilis 168 with selection for erythromycin resistance. Correct
treating PBP4-null cells with 2a (Figure S12).
chromosomal insertion was verified by PCR and the absence of
We therefore hypothesize that enol carbamates 2a and 4d do PBP4 in a Bocillin-FL PBP profile.36
not only inhibit PBP4 in B. subtilis, but that they also inhibit S. pneumoniae D39 strain (serotype 2) was grown at 37 °C in
other enzymes in the peptidoglycan synthesis pathway, these C+Y medium with 2% acid (pH 6.8) until a mid-exponential
being PBPs that are not labeled by Bocillin FL or other enzymes phase (OD595 of 0.4).40 The cells were centrifuged for 2 minutes
that are directly or indirectly involved in the synthesis of lipid II at 20,800 rcf and the cell pellet was resuspended in a volume
and the peptidoglycan. This hypothesis is supported by of fresh medium containing 14.5% glycerol (v/v) that would
unpublished data from our lab, which revealed that antibiotics result in an OD595 of 0.4. The cells were then aliquoted and
that inhibit enzymes upstream of the PBPs block incorporation stored at -80 °C. These mid- exponential phase cell stocks will
of HADA.
be referred to as T2 cells.
Similarly, these compounds may inhibit peptidoglycan
synthesis directly or indirectly in S. pneumoniae and we are MIC determination
currently identifying these additional targets.
MIC determination was carried out in 96 well plate following
the CLSI guidelines.22
B. subtilis strain 168 was grown in cation adjusted Mueller-
Hinton broth at 35 °C in the presence of a serial dilution of the
compound during 20 hours. The OD600 was measured with a
BioTEK PowerWave microplate reader, before resazurin (0.015
mL of a 0.01% (wt/vol) solution water) was added. The plate
was incubated at 37 °C for 20 minutes. The fluorescence
emission at 585 nm (excitation 571 nm) was measured. To
determine the relative viability, the observed fluorescence
emission was corrected for background fluorescence and
divided by the fluorescence emission observed for control
cells.
Conclusions
In conclusion, we developed a synthetic route that yielded a
panel of enol cyclocarbamates containing compounds in four
or five steps. By studying the biological activity of these
compounds in a MIC assay, we showed that the bicyclic
scaffold and the dodecyl alkyl chain are indispensable for
antibacterial activity. Modifications on the bicyclic headgroup
are tolerated, but small changes in the functional group can
lead to complete loss of activity. Growth-curve experiments,
time-lapse microscopy and metabolic labeling with HADA
revealed that enol cyclocarbamates inhibit peptidoglycan
synthesis (i.e., they are bacteriolytic). Using activity-based
protein-profiling experiments, we demonstrated for the first
time that compounds containing an enol cyclocarbamate
inhibit a specific subset of PBPs in two Gram-positive species
of bacteria, B. subtilis and S. pneumoniae. While inhibition of
these PBPs may contribute to the inhibition of HADA
incorporation, the enol cyclocarbamates likely also target
other enzymes involved in peptidoglycan synthesis. This makes
the scaffold an attractive starting point in the search for novel
antibiotics.
S. pneumoniae was grown in cation adjusted Mueller-Hinton
supplemented with 5% sheep blood for the MIC assay.
Growth curves
B. subtilis. The OD curves were carried out in 96-well
microtiter plates in triplicates. Cells were first cultures to mid-
exponential phase (OD=0.2 to 0.7), then diluted to OD=0.02
and incubated in LB broth in microtiter plates with different
concentrations of the compounds and the adequate controls.
The suspensions were incubated at 30 °C in a microplate
reader BioTEK PowerWave under agitation to favor aeration
(cycles of 9 minutes agitation/1 minutes no agitation). Growth
(OD600) was measured every 10 minutes and the resulting
values were plotted against the time to obtain the optical
density curves.
Experimental
S. pneumoniae. T2 cells of strain D39 were diluted 100-fold
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and were grown until OD 0.4 in C+Y with 2% acid. For the dried glassware under an atmosphere of nitrogen (unless
assay, this pre-culture was diluted 100-fold in C+Y and otherwise specified) using dry solvents. Reaction temperature
incubated in microtiter plates with or without different refers to the temperature of the oil bath. Solvents were taken
concentrations of the antimicrobials. Growth (OD595) was from a MBraun solvent purification system (SPS-800). All other
measured every 10 minutes in a Tecan Infinite F200 PRO with reagents were purchased from Sigma Aldrich and Acros and
at least three replicates for each condition.
used without further purification unless noted otherwise.
Trimethylsilyl trifluoromethanesulfonate was stored under a
Microscopy
nitrogen atmosphere in a dry Schlenk flask. TLC analysis was
Inhibition of incorporation of the fluorescent
D
-amino-acid performed on Merck silica gel 60/Kieselguhr F254, 0.25 mm.
analogue HADA into peptidoglycan in live B. subtilis was Compounds were visualized using either ninhydrine stain
determined using a wide-field fluorescence microscope. B. (ninhydrin (1.5 g) and AcOH (3 mL) in n-butanol (100 mL)) or a
subtilis 168 was diluted from an overnight culture to an OD600 KMnO4 stain (K2CO3 (40 g), KMnO4 (6 g), H2O (600 mL) and 10%
of 0.02 in casein hydrolysate medium41 and grown at 37 °C NaOH (5 mL)). Flash chromatography was performed using
until an OD600 of 0.2, at which point the compounds were SiliCycle silica gel type SiliaFlash P60 (230 – 400 mesh) as
added to the medium. Cells were incubated for 20 minutes obtained from Screening Devices or with automated column
with compound, followed by 5 minutes labeling with HADA chromatography using a Reveleris flash purification system
(0.5 mM
), after which cells were fixed with 70% ethanol. purchased from Grace Davison Discovery Sciences.1H- and 13C-
Bacteria were imaged under a Nikon Ti-E inverted microscope NMR spectra were recorded on a Varian AMX400 or a Varian
equipped with a CFI Plan Apochromat DM 100× oil objective, 400-MR (400 and 100.59 MHz, respectively) using CDCl3 or
using appropriate filter sets for the dyes used. Digital images DMSO-d6 as solvent. Chemical shift values are reported in ppm
were recorded using a Hamamatsu Orca Flash 4.0 (V2) camera with the solvent resonance as the internal standard (CDCl3: δ
1
and prepared using Adobe Photoshop.
7.26 for H, δ 77.06 for 13C, DMSO-d6 δ 2.50 for H). Data are
reported as follows: chemical shifts (δ), multiplicity (s = singlet,
d = doublet, dd = double doublet, ddd = double double
Activity-based protein profiling using Bocillin-FL
- B. subtilis 168 was diluted from an overnight culture to an OD doublet, td = triple doublet, t = triplet, q = quartet, b = broad,
of 0.1 and cultured until OD 0.3 (usually 2 hours), cells (4 mL m = multiplet), coupling constants J (Hz), and integration. High-
per lane) were collected by centrifugation (5 minutes, 20 800 resolution mass spectra (HRMS) were recorded on a Thermo
rcf) and resuspend in Mueller Hinton and incubated with or Scientific LTQ Orbitrap XL. Remark: When recording the 13C
without compounds during 60 minutes (2.5 % DMSO), cells NMR of the Boc-protected compounds (
6, 7a-d, 9, 10, 12, 13,
were then collected by centrifugation (5 minutes, 15 000 rcf) 15a-b, 16a-b and 17a-b) at 25 °C in DMSO-d6 mixtures of
and resuspended in cold PBS and lysed in the presence of rotamers and enol-keto tautomers are observed in the NMR
lysozyme (0.2 mg/mL, 37°C, 20 minutes) and DNAse (0.1 spectra and the chemical shifts of these mixtures are reported
mg/mL).
in the experimental section. While recording the 13C spectra at
- Lysates from B. subtilis 168 and B. subtilis PBP4-null strains 75 °C does solve this issue in part, it comes at the cost of a
were prepared in the following way: cells were diluted from an reduced sensitivity (several carbonyl peaks are not observed in
overnight culture to an OD of 0.1 and cultured until OD 0.3 these spectra). We included both the 13C NMR spectra
(usually 2 hours), washed two times with PBS, lysozyme (0.5 recorded at 25 °C and at 75 °C in the Supporting Information
1
mg/mL) was added and cells were sonicated (10 seconds pulse, for the majority of the compounds. For the H NMR of these
10 seconds stop (10 times) in order to control the compounds, measuring the spectra in DMSO-d6 at 75 °C
temperature), lysates were then flash-frozen using liquid reduced the presence of rotamers, but it resulted in a poor
nitrogen. Lysates (19 μL, 1 mg/mL protein content) was resolution (multiplicity of the peaks is difficult to observe). In
incubated with or without compound for 60 minutes.
CDCl3 the multiplicity could be observed, but with presence of
- S. pneumoniae was cultured to mid-exponential phase, cells rotamers; for the sake of comparison and clarity, the majority
(4 mL per lane) were collected by centrifugation (5 minutes, 20 is reported in DMSO-d6.
800 rcf) and resuspend in PBS and incubated with or without
compounds during 60 minutes (2.5 % DMSO), cells were then General procedure A for O-alkylation of 4-hydroxy Boc-
lysed in the presence of lysozyme (0.2 mg/mL, 37°C, overnight) Proline. A solution of 4-hydroxy Boc-Proline (1 eq) in dry THF
and DNAse (0.1 mg/mL).
was treated with sodium hydride (2.2 eq, 60% in mineral oil).
The resulting mixture was stirred at 0 °C for 1 h and the
For both strains: Lysates were then incubated with Bocillin-FL corresponding alkyl bromide (1.1 eq) was added. The reaction
(45 n ) during 30 minutes at 37°C. Laemmli sample buffer (SP) mixture was stirred until complete consumption of the starting
containing dithiothreitol (DTT) was added and the proteins material was observed and then acidified to pH 3 by the
were resolved on a 12% SDS-PAGE and fluorescence was addition of 2 HCl and subsequently the reaction mixture was
M
M
visualized using a Typhoon scanner.
extracted with diethyl ether (3 x 20 mL). The combined organic
layers were washed with brine (2 x 20 mL), dried over Na2SO4,
and concentrated under reduced pressure. The crude material
Synthetic procedures
General remarks. All reactions were performed using oven- was purified by silica gel flash column chromatography using
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ARTICLE
ethyl acetate: pentane: AcOH as eluent.
concentration of the reaction mixture was 0.05 M. (Sodium
ascorbate was added from a 40 m
General procedure B for the synthesis of β-keto ester CuSO4 was added from a 200 m
M
stock solution in water,
stock solution in water)
M
compounds. To a solution of N-Boc-protected Amino-Acid (1 When the reaction reached completion, ethyl acetate was
eq) in dry THF was added N,N'-dicyclohexylcarbodiimide (DCC) added and the crude mixture was washed with H2O. The
(1.1 eq) at 0 °C. The reaction mixture was stirred for 2 minutes organic layer was dried over Na2SO4, concentrated under
and 4-dimethylaminopyridine (DMAP) (1.5 eq) and Meldrum´s reduced pressure and purified by silica gel flash column
acid (2,2-dimethyl-1,3-dioxane-4,6-dione) (1.2 eq) were added. chromatography using ethyl acetate: pentane and/or MeOH:
The reaction mixture was stirred overnight at room DCM as eluent. The Boc-protection of trans-4-hydroxy-L-
temperature. Diethyl ether (30 mL) was then added to the proline was realized following a procedure by Zhang et al.42
reaction mixture and the solution was stirred for 10 minutes.
After filtration over paper filter, the filtrate was washed with N-Boc Proline β-ketoester 6. Compound
an aqueous Na2CO3 solution (60 mL), the aqueous solution was according to the general procedure B using N-Boc-
then acidified to pH 3 with 2 HCl and the product was (2.15 g, 10 mmol, 1 eq) DCC (2.25 g, 11 mmol, 1.1 eq), DMAP
6
was prepared
L
-proline
M
extracted with diethyl ether (4 x 30 mL). The organic layer was (2.16 g, 15 mmol, 1.5 eq) and Meldrum’s acid (1.59 g, 11
dried over Na2SO4 and concentrated under reduced pressure. mmol, 1.1 eq) in THF (40 mL) to obtain the Meldrum’s acid
The crude intermediate was dissolved in MeOH (20 mL) and intermediate and subsequent refluxing of this intermediate in
stirred at reflux overnight. The crude mixture was finally MeOH to obtain 6. Flash chromatography using ethyl acetate:
concentrated under reduced pressure and purified by silica gel pentane (1: 4) as eluent yielded 6 (1.9 g, 70 % yield) as a yellow
flash column chromatography using ethyl acetate: pentane as oil. Rf [silica, ethyl acetate: pentane (1: 4)] = 0.30. 1H NMR (400
eluent.
MHz, DMSO-d6, 75 °C):
3H), 3.59 (d, J = 4.0 Hz, 2H), 3.47 - 3.27 (m, 2H), 2.29 - 2.03 (m,
General procedure C for the synthesis of β-ketoamide 1H), 1.93 - 1.70 (m, 3H), 1.37 (s, 9H). 13C NMR (101 MHz,
δ = 4.31 (dd, J = 8.8, 4.9 Hz, 1H), 3.64 (s,
compounds. The amidation of unactivated esters was based DMSO-d6, 25°C, mixture of rotamers, 25 °C):
on the procedure described by Novak et al.20 A solution of 1,4- 167.53, 167.46, 153.94, 153.02, 79.30, 64.99, 51.99, 46.71,
diazabicyclo[2.2.2]octane (DABCO) (1.2 eq) in dry toluene was 46.52, 45.47, 45.25, 39.52, 30.77, 29.02, 28.15, 27.90, 24.04,
δ = 202.96,
cooled to 0 °C and trimethylaluminium (2.4 eq, 2 m in toluene) 23.17; IR ν
max/cm-1: 2977, 1752, 1694, 1392, 1366, 1318, 1258,
was added dropwise. The mixture was stirred for 4 hours at 0 1162, 1119, 1013, 772 cm-1; HRMS: (ESI+) Calculated mass
°C. A solution of alkyl amine (1.2 eq) in THF was added and the [M+Na]+ C13H21NO5Na = 294.1312, found: 294.1315.
reaction mixture was heated to 40 °C and stirred for 2 h.
Subsequently the corresponding β-keto ester (1 eq) dissolved Dodecyl β-ketoamide 7a. β-Ketoamide 7a was prepared
in THF was added and the reaction mixture was stirred according to the general procedure C by reacting DABCO (98
overnight at reflux. The reaction mixture was diluted with mg, 0.87 mmol, 1.2 eq) with trimethylaluminium (0.87 mL,
diethyl ether (15 mL), quenched by adding 2
and washed with 2 HCl (2 x 20 mL). The organic layer was produce DABAL in situ, subsequently adding dodecylamine
finally dried over Na2SO4, concentrated under reduced (161 mg, 0.87 mmol, 1.2 eq) in THF (2 mL) to activate the
pressure and purified by silica gel flash column amine and finally adding the corresponding β-ketoester (200
chromatography using ethyl acetate: pentane as eluent mg, 0.73 mmol, 1 eq) in THF (2 mL). Flash chromatography
M HCl dropwise 1.74 mmol, 2.4 eq, 2 M in toluene) in toluene (2 mL) to
M
6
affording the β-ketoamide compounds.
using ethyl acetate: pentane (2: 3) as eluent furnished 7a (141
mg, 45 % yield) as a yellow oil. Rf [silica, ethyl acetate: pentane
General procedure D for the synthesis of enol cyclocarbamate (2: 3)] = 0.25. 1H NMR (400 MHz, DMSO-d6, 75 °C):
δ
= 7.74 (bs,
compounds. A solution of the corresponding β-ketoamide (1 1H), 4.39 - 4.30 (m, 1H), 3.37 - 3.32 (m, 4H), 3.12 - 3.03 (m,
eq) in DCM was cooled to °C before trimethylsilyl 4H), 2.15 - 2.07 (m, 1H), 1.97 - 1.88 (m, 1H), 1.83 - 1.71 (m,
0
trifluoromethanesulfonate (TMSOTf) (2 eq) was added. The 2H), 1.47 - 1.36 (m, 11H), 1.33 - 1.21 (m, 18H), 0.87 (t, J = 6.6
reaction mixture was stirred for 3-4 hours. TLC analysis Hz, 3H). 13C NMR (101 MHz, DMSO-d6, 25°C, mixture of
showed that all starting material had been consumed and rotamers): δ = 204.57, 204.11, 165.87, 165.52, 154.13, 153.38,
therefore 1,1'-carbonyldiimidazole (CDI) (1.5 eq) was added 79.37, 79.29, 65.67, 65.24, 47.64, 47.43, 47.03, 46.80, 32.36,
and the reaction mixture was stirred overnight. The reaction 31.75, 30.07, 29.79, 29.44, 29.17, 28.84, 28.52, 28.30, 27.34,
mixture was directly applied on silica gel column and flash 26.79, 23.37, 22.54, 15.01, 14.37; IR ν
max/cm-1: 3254, 2921,
column chromatography using ethyl acetate: pentane as 2851, 1786, 1701, 1627, 1545, 1467, 1381, 1219, 978 cm-1;
eluent afforded the lipocyclocarbamate compound.
HRMS: (ESI+) Calculated mass [M+Na]+ C24H44N2O4Na
447.3193, found: 447.3197.
=
General procedure E for the synthesis of triazole compounds
via Huisgen Azide-Alkyne Cycloaddition. To a solution of the Hexyl β-ketoamide 7b. β-Ketoamide 7b was prepared
enol cyclocarbamate (1 eq) and corresponding azide (1 eq) in according to the general procedure C by reacting DABCO (98
tert-butanol: MeOH: H2O (1: 2: 1, v/v/v) was added sodium mg, 0.87 mmol, 1.2 eq) with trimethylaluminium (0.87 mL,
ascorbate (30 mol%) and CuSO4 (20 mol%). The final 1.74 mmol, 2.4 eq, 2
M
in toluene) in toluene (2 mL) to
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produce DABAL in situ, subsequently adding hexylamine (114 317.1472, found: 314.1474.
μL, 0.87 mmol, 1.2 eq) in THF (2 mL) to activate the amine and
finally adding the β-ketoester
6 (200 mg, 0.73 mmol, 1 eq) in Dodecyl derivative 2a. Enol cyclocarbamate 2a was prepared
THF (2 mL). Flash chromatography using ethyl acetate: according to the general procedure D using the corresponding
pentane (2: 3) as eluent yielded 7b (169 mg, 68 % yield) as a β-keto amide 7a (100 mg, 0.24 mmol, 1 eq), TMSOTf (86 μL,
yellow oil. Rf [silica, ethyl acetate: pentane (2: 3)] = 0.20. 1H 0.47 mmol, 2 eq) and CDI (81 mg, 0.35 mmol, 1.5 eq) in DCM
NMR (400 MHz, DMSO-d6, 75 °C): δ = 7.75 (bs, 1H), 4.39 - 4.30 (1 mL). Flash chromatography using ethyl acetate: pentane (3:
(m, 1H), 3.41-3.21 (m, 2H), 3.12 - 3.04 (m, 4H), 2.16 - 2.06 (m, 2) as eluent yielded 2a (29 mg, 35 % yield) as a colorless oil. . Rf
20
1H), 1.97 - 1.89 (m, 1H), 1.84 - 1.71 (m, 2H), 1.47 - 1.35 (m, [silica, ethyl acetate: pentane (3: 2)] = 0.22. [α]D = -39.6 (c =
9H), 1.34 - 1.24 (m, 6H), 0.87 (t, J = 7.5 Hz, 3H). 13C NMR (101 0.308, CHCl3); 1H NMR (400 MHz, CDCl3):
δ
= 6.60 (bs, 1H), 5.18
MHz, DMSO-d6, 25°C, mixture of rotamers):
δ = 204.28, 203.87, (s, 1H), 4.49 (app t, J = 8.0 Hz, 1H), 3.73 - 3.61 (m, 1H), 3.36 -
165.69, 165.31, 153.85, 153.12, 79.11, 64.93, 47.27, 47.11, 3.26 (m, 2H), 2.29 - 2.20 (m, 1H), 2.20 - 2.12 (m, 1H), 2.12 -
46.51, 40.16, 39.52, 31.09, 29.03, 28.21, 27.99, 26.13, 23.07, 2.02 (m, 1H), 1.77 - 1.65 (m, 2H), 1.58 - 1.50 (m, 2H), 1.34 –
22.19, 22.15, 14.03 ; IR
cm-1; HRMS: (ESI+) Calculated mass [M+Na]+ C18H33N2O4Na = CDCl3):
363.2254, found: 363.2254. 31.90, 31.43, 29.63, 29.63, 29.61, 29.58, 29.51, 29.33, 29.28,
26.94, 26.37, 22.67, 14.10; IR
max/cm-1: 3257, 2921, 2851,
ν
max/cm-1: 2924, 2854, 1459, 1377, 754 1.23 (m, 18H), 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (101 MHz,
δ
= 163.40, 156.05, 154.30, 99.66, 63.14, 46.00, 39.75,
ν
Benzyl β-ketoamide 7c. β-Ketoamide 7c was prepared 1786, 1701, 1627, 1545, 1467, 1381, 1219, 978 cm-1; HRMS:
according to the general procedure C by reacting DABCO (98 (ESI+) Calculated mass [M+H]+ C20H35N2O3 = 351.2642 found:
mg, 0.87 mmol, 1.2eq) with trimethylaluminium (0.87 mL, 1.74 351.2645; Calculated mass [M+Na]+ C20H34N2O3Na = 373.2462,
mmol, 2.4 eq, 2
M in toluene) in toluene (2 mL) to produce found: 373.2463.
DABAL in situ, subsequently adding benzylamine (95 μL, 0.87
mmol, 1.2 eq) in THF (2 mL) to activate the amine and finally Hexyl derivatve 2b. Enol cyclocarbamate 2b was prepared
adding β-ketoester
6
(200 mg, 0.73 mmol, 1 eq) in THF (2 mL). according to the general procedure D using the corresponding
amide (S)-tert-butyl-2-(3-(hexylamino)-3-
Flash chromatography using ethyl acetate: pentane (1: 1) as β-keto
eluent afforded 7c (155 mg, 62 % yield) as brown oil. Rf [silica, oxopropanoyl)pyrrolidine-1-carboxylate 7b (100 mg, 0.29
ethyl acetate: pentane (1: 1)] = 0.38. 1H NMR (400 MHz, mmol, 1 eq), TMSOTf (106 μL, 0.58 mmol, 2 eq) and 1,1'-
DMSO-d6, 75 °C): δ = 8.30 (bs, 1H), 7.41 - 7.17 (m, 5H), 4.43 - carbonyldiimidazole (81 mg, 0.44 mmol, 1.5 eq) in DCM (1.5
4.21 (m, 3H), 3.53 - 3.29 (m, 4H), 2.20 - 2.04 (m, 1H), 2.02 - mL). Flash chromatography using ethyl acetate: pentane (4: 1)
1.87 (m, 1H), 1.82 - 1.71 (m, 2H), 1.38 (s, 9H). 13C NMR (101 as eluent furnished 2b (28 mg, 35 % yield) as a yellow oil. Rf
20
MHz, DMSO-d6, 25°C, mixture of rotamers):
167.21, 166.91, 154.42, 140.45, 129.69, 128.69, 128.26, 80.44, 0.612, CHCl3) 1H NMR (400 MHz, CDCl3):
66.35, 48.22, 47.82, 43.71, 30.14, 29.51, 29.28, 25.28, 24.39; IR (s, 1H), 4.51-4.45 (m, 1H), 3.74 - 3.61 (m, 1H), 3.35 - 3.19 (m,
max/cm-1: 2944, 2869, 1475, 1384, 1174 cm-1; HRMS: (ESI+) 3H), 2.18 - 2.12 (m, 1H), 2.11 - 2.02 (m, 1H), 2.28-2.20 (m, 1H),
Calculated mass [M+Na]+ C19H27N2O4Na = 347.1965, found: 1.76 - 1.64 (m, 1H), 1.57 - 1.47 (m, 2H), 1.34 - 1.25 (m, 6H),
347.1967. = 163.46,
0.87 (t, J = 5.3 Hz, 3H). 13C NMR (101 MHz, CDCl3):
156.24, 154.32, 99.91, 63.25, 46.12, 39.81, 31.58, 31.56, 29.67,
Propargyl β-ketoamide 7d. β-Ketoamide 7d was prepared 26.73, 26.49, 22.65, 14.12. IR
max/cm-1: 3293, 2929, 1792,
according to the general procedure C by reacting DABCO (246 1694, 1535, 1463, 1391, 1217, 1030, 976 cm-1; HRMS: (ESI+)
mg, 2.2 mmol, 1.2 eq) with trimethylaluminium (2.2 mL, 4.4 Calculated mass [M+H]+ C14H23N2O3
267.1703 found:
mmol, 2.4 eq, 2 in toluene) in toluene (4 mL) to produce 267.1710.
DABAL in situ, subsequently adding propargyl amine (145 μL,
2.2 mmol, 1.2 eq) in THF (3 mL) to activate the amine and Benzyl derivative 2c. Enol cyclocarbamate 2c was prepared
finally adding β-ketoester (500 mg, 1.83 mmol, 1 eq) in THF according to the general procedure D using the corresponding
δ = 205.56, 205.26, [silica, ethyl acetate: pentane (4: 1)] = 0.20. [α]D = -47.1 (c =
δ
= 6.58 (bs, 1H), 5.14
ν
δ
ν
=
M
6
(3 mL). Purification by flash chromatography using ethyl β-keto amide 7c (100 mg, 0.29 mmol, 1 eq), TMSOTf (106 μL,
acetate: pentane (1: 4) as eluent furnished 7d (398 mg, 73 % 0.58 mmol, 2 eq) and CDI (81 mg, 0.44 mmol, 1.5 eq) in DCM
yield) as a yellow oil. Rf [silica, ethyl acetate: pentane (1: 1)] = (1.5 mL). Flash chromatography using ethyl acetate: pentane
1
0.28. H NMR (400 MHz, DMSO-d6, 75 °C):
δ = 8.27 (bs, 1H), (4: 1) as eluent gave 2c (43.1 mg, 55 % yield) as a yellow oil. Rf
20
4.33 (dd, J = 8.9, 5.0 Hz, 1H), 3.96 - 3.84 (m, 2H), 3.41 - 3.30 (m, [silica, ethyl acetate: pentane (1: 1)] = 0.15. [α]D = -33.2 (c =
4H), 2.97 (t, J = 2.3 Hz, 1H), 2.19 - 2.07 (m, 1H), 1.90 (dq, J = 0.476, CHCl3) 1H NMR (400 MHz, CDCl3):
= 7.35 - 7.24 (m,
12.6, 5.9 Hz, 1H), 1.86 - 1.73 (m, 2H), 1.38 (s, 9H). 13C NMR 5H), 6.94 (bs, 1H), 5.20 (s, 1H), 4.52 (d, J = 5.7 Hz, 2H), 4.51 -
(101 MHz, DMSO-d6, 25°C, mixture of rotamers): = 204.27, 4.45 (m, 1H), 3.69 - 3.61 (m, 1H), 3.33-3.26 (m, 1H), 2.29 - 2.19
203.92, 166.01, 165.67, 154.16, 153.40, 88.79, 81.17, 81.13, (m, 1H), 2.19 - 2.11 (m, 1H), 2.10 - 2.00 (m, 1H), 1.75 - 1.64 (m,
79.46, 73.65, 73.58, 65.26, 65.21, 55.28, 47.09, 47.04, 46.96, 1H). 13C NMR (101 MHz, CDCl3):
= 163.48, 156.11, 154.85,
46.82, 29.14, 28.51, 28.44, 28.30, 28.26, 24.29, 23.40. IR 138.42, 128.76, 127.75, 127.48, 99.57, 63.28, 46.14, 43.56,
δ
δ
δ
ν
max/cm-1: 3294, 2977, 1684, 1539, 1399, 1366, 1163 cm-1; 31.54, 26.50. IR max/cm-1: 3297, 2930, 1788, 1693, 1633, 1532,
ν
HRMS: (ESI+) Calculated mass [M+Na]+ C15H22N2O4Na
=
1390, 1218, 972 cm-1.HRMS: (ESI+) Calculated mass [M+H]+
10 | J. Name., 2012, 00, 1-3
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ARTICLE
C15H17N2O3 = 273.1234 found: 273.1240; Calculated mass Calculated mass [M+H]+ C24H24N5O3
[M+Na]+ C15H16N2O3Na = 295.1053, found: 295.1059.
430.1869.
= 430.1873 found:
Propargyl derivative 2d. Enol cyclocarbamate 2d was prepared 6-Hydroxyhexyl-triazolyl derivative 2g. Triazole compound 2g
according to the general procedure D using the corresponding was prepared according to the general procedure E using the
β-keto amide 7d (103 mg, 0.35 mmol, 1 eq), TMSOTf (132 μL, enol cyclocarbamate 2d (15.46 mg, 0.07 mmol, 1 eq) and 6-
0.7 mmol, 2 eq) and CDI (102 mg, 5.25 mmol, 1.5 eq) in DCM azidohexanol (10 mg, 0.07 mmol, 1 eq). Flash chromatography
(2 mL). Flash chromatography using ethyl acetate: pentane (2: using methanol: DCM (1: 24) as eluent yielded 2g (16 mg, 63 %
3) as eluent gave 2d (38.5 mg, 50 % yield) as a white solid. Rf yield) as a colorless oil. Rf [silica, methanol: DCM (1: 24)] =
20
[silica, ethyl acetate: pentane (2: 3)] = 0.21. [α]D = -36.8 (c = 0.10. [α]D20 = -38 (c = 0.450, CHCl3); 1H NMR (400 MHz, CDCl3) :
0.125, CHCl3); 1H NMR (400 MHz, CDCl3):
δ
= 6.78 (bs, 1H), 5.17
δ = 7.56 (bs, 1H), 7.10 (bs, 1H), 5.15 (s, 1H), 4.58 (d, J = 5.6 Hz,
(s, 1H), 4.50 (ddd, J = 8.9, 6.7, 1.7 Hz, 1H), 4.25 - 3.93 (m, 2H), 2H), 4.48 (app t, J = 8.1 Hz, 1H), 4.32 (t, J = 7.0 Hz, 2H), 3.81 -
3.66 (dt, J = 11.4, 7.8 Hz, 1H), 3.31 (ddd, J = 11.3, 8.9, 4.4 Hz, 3.48 (m, 3H), 3.30 (ddd, J = 11.9, 8.8, 4.3 Hz, 1H), 2.40 - 1.99
1H), 2.29 - 2.20 (m, 2H), 2.20 - 2.12 (m, 1H), 2.12 - 2.02 (m, (m, 3H), 2.04 - 1.83 (m, 2H), 1.80 - 1.62 (m, 1H), 1.62 - 1.47 (m,
1H), 1.83 - 1.56 (m, 2H). 13C NMR (101 MHz, CDCl3):
156.29, 155.77, 99.36, 79.97, 71.83, 63.60, 46.47, 31.83, 29.53, 156.36, 155.73, 99.48, 77.16, 63.59, 62.83, 50.61, 46.48, 35.64,
26.84. IR
max/cm-1: 3286, 2924, 1795, 1693, 1636, 1529, 1217, 32.72, 31.85, 30.51, 26.83, 26.49, 25.4. HRMS: (ESI+)
δ δ = 164.01,
= 163.46, 2H), 1.45 - 1.28 (m, 4H). 13C NMR (101 MHz, CDCl3):
ν
1029, 974 cm-1; HRMS: (ESI+) Calculated mass [M+H]+ Calculated mass [M+H]+ C17H26N5O4
C11H13N2O3 = 221.0921 found: 221.0921; Calculated mass 364.1979.
[M+Na]+ C11H12N2O3Na = 243.0740, found: 243.0741
= 364.1972 found:
Phytyl triazolyl derivative 2h. Triazole compound 2h was
Dodecyl-triazolyl derivative 2e. Triazole compound 2e was prepared according to the general procedure E using the enol
prepared according to the general procedure E using the enol cyclocarbamate 2d (29 mg, 0.13 mmol, 1 eq) and phytyl azide
cyclocarbamate 2d (17.9 mg, 0.08 mmol,
dodecylazide (17.3 mg, 0.08 mmol,
1
eq) and (42 mg, 0.13 mmol, 1 eq). Flash chromatography using ethyl
1
eq). Flash acetate: pentane (9: 1) as eluent yielded 2h (33 mg, 47 % yield)
chromatography using ethyl acetate: pentane (7: 3) and then as a colorless oil. . Rf [silica, ethyl acetate: pentane (9: 1)] =
20
methanol: DCM (1: 24) as eluent yielded 2e (22 mg, 65 % yield) 0.09. [α]D = -45.6 (c = 0.250, CHCl3); 1H NMR (400 MHz,
as a colorless oil. Rf [silica, methanol: DCM (1: 24)] = 0.15. CD3OD):
[α]D20 = -35 (c = 0.5, CHCl3); 1H NMR (400 MHz, CDCl3):
δ = 7.99 (bs, 1H), 5.50 - 5.44 (m, 1H), 5.35 (bs, 1H),
= 7.61 5.01 (t, J = 7.5 Hz, 2H), 4.61 (t, J = 8.0 Hz, 1H), 4.54 (s, 2H), 3.62
δ
(bs, 1H), 7.10 (bs, 1H), 5.15 (bs, 1H), 4.58 (bs, 2H), 4.47 (t, J = (dt, J = 10.9, 7.8 Hz, 2H), 3.37 - 3.26 (m, 1H), 2.32 - 2.02 (m,
9.2, 6.8 Hz, 1H), 4.29 (t, J = 7.2 Hz, 2H), 3.64 (dt, J = 11.6, 7.8 4H), 1.82 (d, J = 6.0 Hz, 3H), 1.79 - 1.64 (m, 1H), 1.63 - 1.24 (m,
Hz, 1H), 3.28 (ddd, J = 11.6, 8.9, 4.3 Hz, 1H), 2.27 - 1.99 (m, 14H), 1.23 - 1.08 (m, 4H), 0.90 (t, J = 6.7 Hz, 12H). 13C NMR
3H), 1.93 - 1.82 (m, 2H), 1.75 - 1.62 (m, 1H), 1.34 - 1.17 (m, (101 MHz, CD3OD):
18H), 0.86 (t, J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3):
δ = 159.31, 158.57, 144.99, 144.72, 118.88,
118.37, 64.80, 49.00, 47.02, 40.70, 40.54, 38.51, 38.49, 38.47,
δ
=
163.48, 155.89, 155.20, 99.04, 63.09, 50.49, 46.01, 35.15, 38.45, 38.43, 38.38, 37.93, 37.85, 37.74, 37.65, 33.94, 33.75,
31.86, 31.38, 30.20, 29.56, 29.48, 29.34, 29.29, 28.97, 26.51, 33.06, 32.33, 29.15, 27.36, 26.50, 26.12, 26.10, 25.91, 25.89,
26.35, 22.64, 14.08. IR
1694, 1545, 1400, 1218, 1026, 845, 760 cm-1; HRMS: (ESI+)
ν
max/cm-1: 3277, 2922, 2853, 1789, 25.49, 23.50, 23.10, 23.02, 20.20, 20.14, 20.08, 16.36. IR
ν
max/cm-1: 3385, 2936, 1790, 1694, 1640, 1533, 1217, 1028,
Calculated mass [M+H]+ C23H38N5O3
432.2961.
=
432.2969 found: 973, 761 cm-1. HRMS: (ESI+) Calculated mass [M+H]+
C31H52N5O3 = 542.4064 found: 542.4053.
Biphenyl triazolyl derivative 2f. Triazole compound 2f was Biotin-PEG derivative 2i. Triazole compound 2i was prepared
prepared according to the general procedure E using the enol according to the general procedure using the enol
E
cyclocarbamate 2d (20.3 mg, 0.09 mmol, 1 eq) and 4- cyclocarbamate 2d (27 mg, 0.12 mmol, 1 eq) and biotin azide
phenylbenzyl azide (19.35 mg, 0.09 mmol, 1 eq). Flash (44 mg, 0.12 mmol, 1 eq). Flash chromatography using
chromatography using ethyl acetate: pentane (7: 3) and then methanol: DCM (3: 17) as eluent yielded 2i (15.72 mg, 20 %
methanol: DCM (1: 24) as eluent yielded 2f (27 mg, 68 % yield) yield) as a colorless oil. Rf [silica, methanol: DCM (3: 17)] =
as a colorless oil. Rf [silica, methanol: DCM (1: 24)] = 0.17. 0.21. [α]D20 = -40.1 (c = 0.5, CHCl3) 1H NMR (400 MHz, CD3OD) :
20
1
[α]D = -31 (c = 0.250, CHCl3); H NMR (400 MHz, CDCl3):
δ
=
δ = 7.98 (s, 1H), 7.94 (s, 1H), 5.26 (s, 1H), 4.65 - 4.53 (m, 4H),
7.57 (t, J = 8.2 Hz, 4H), 7.43 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 8.8 4.53 - 4.44 (m, 3H), 4.30 (dd, J = 7.9, 4.4 Hz, 1H), 3.88 (t, J = 5.0
Hz, 3H), 7.11 (bs, 1H), 5.53 (s, 2H), 5.15 (bs, 1H), 4.60 (bs, 2H), Hz, 2H), 3.61-3.57 (m, 10H), 3.53 (t, J = 5.5 Hz, 2H), 3.35 (q, J =
4.46 (app t, J = 8.0 Hz, 1H), 3.64 (dt, J = 11.4, 7.8 Hz, 1H), 3.29 5.5 Hz, 2H), 3.24 - 3.14 (m, 1H), 2.92 (dd, J = 12.7, 5.0 Hz, 1H),
(td, J = 11.4, 8.8, 4.3 Hz, 1H), 2.25 - 1.96 (m, 3H), 1.75 - 1.62 2.70 (d, J = 12.7 Hz, 1H), 2.34 - 2.04 (m, 6H), 1.82 - 1.53 (m,
(m, 1H). 13C NMR (101 MHz, CDCl3):
140.23, 133.39, 128.82, 128.63, 127.78, 127.61, 127.09, 63.11, 176.14, 166.12, 165.96, 159.18, 158.62, 97.78, 71.56, 71.49,
54.14, 46.01, 35.14, 31.37, 26.3. IR
max/cm-1: 3286, 1790,1692, 71.46, 71.26, 70.57, 70.35, 68.14, 64.78, 63.37, 61.63, 57.02,
1640, 1488, 1215, 1022, 909, 757, 727 cm-1. HRMS: (ESI+) 51.47, 49.64, 49.00, 47.02, 41.07, 40.36, 36.74, 35.65, 32.36,
δ δ =
= 155.87, 155.24, 141.73, 4H), 1.54 - 1.36 (m, 2H). 13C NMR (101 MHz, CD3OD):
ν
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29.76, 29.50, 27.38, 26.85. IR
ν
max/cm-1: 3364, 2936, 1799, mixture of rotamers):
δ
= 6.99 - 6.73 (m, 1H), 4.60 - 3.96 (m,
1691, 1632, 1512, 1078, 931, 711 cm-1. HRMS: (ESI+) 4H), 3.39 (d, J = 10.7 Hz, 2H), 3.31 - 3.17 (m, 2H), 2.25 (m, 1H),
Calculated mass [M+H]+ C29H45N8O8S
665.3075.
=
665.3065 found: 1.58 – 1.33 (m, 11H), 1.31 – 1.05 (m, 18H), 0.86 (t, J = 6.7 Hz,
3H). 13C NMR (101 MHz, DMSO-d6, 25°C, mixture of rotamers):
δ = 201.10, 200.58, 166.11, 165.80, 154.74, 80.69, 80.43,
N-Boc-N-propargyl glycine 8. A solution of Boc-Gly-OH (1.75 g, 79.95, 77.10, 74.83, 74.57, 69.05, 57.67, 55.84, 55.53, 55.08,
10 mmol, 1 eq) in dry THF (40 mL) was treated with NaH (0.88 48.14, 48.07, 36.87, 31.66, 29.39, 29.32, 29.18, 29.06, 28.25,
g, 60% in mineral oil, 22 mmol, 2.2 eq.). The resulting mixture 28.07, 26.64, 25.62, 22.47, 14.32. IR ν
max/cm-1: 3312, 1925,
was stirred at 0° C for 1 hour and propargyl bromide (80 wt. % 1854, 1704, 1454, 1367, 1247, 1163, 945 cm-1. HRMS: (ESI+)
in toluene, 1.23 mL, 11 mmol, 1.1 eq.) was added. After Calculated mass [M+Na]+ C24H42N2O4Na = 445.3037, found:
overnight stirring at room temperature, TLC analysis showed 445.3039.
incomplete conversion and an additional amount of NaH (0.5
equiv.) and propargyl bromide (0.5 equiv.) were added at 0° C. Propargyl functionalized monocyclic derivative 3a. Enol
The reaction was stirred overnight at room temperature upon cyclocarbamate 3a was prepared according to the general
which full conversion of starting material was observed. The procedure D by reacting the appropriate β-keto amide 10 (51
reaction mixture was then acidified to pH 3 by addition of 2
M
mg, 0.12 mmol, 1 eq), TMSOTf (45 μL, 0.24 mmol, 2 eq) and
HCl and subsequently was extracted with diethyl ether (3 x 50 CDI (35 mg, 0.18 mmol, 1.5 eq) in DCM (0.8 mL). Flash
mL). The combined organic layers were washed with brine (3 x chromatography using ethyl acetate: pentane (3:7) gave Z-3a
100 mL), dried over Na2SO4, and concentrated under reduced (10.4 mg, 25 % yield) and E-3a (4.2 mg, 10 % yield) as colorless
pressure. The crude material was purified by silica gel flash oil. Z-isomer: Rf [silica, ethyl acetate: pentane (3: 7)] = 0.19. 1H
chromatography using ethyl acetate: pentane: AcOH (10: 189: NMR (400 MHz, CDCl3):
1) as eluent to give (2.12 g, 99 %) as a light brown oil. Rf 1H), 4.38 (d, J = 2.1 Hz, 2H), 4.19 (d, J = 2.5 Hz, 2H), 3.54 - 3.17
[silica, ethyl acetate: pentane: AcOH (10: 189: 1)] = 0.15. (m, 2H), 2.40 (t, J = 2.5 Hz, 1H), 1.66 - 1.46 (m, 2H), 1.43 - 1.10
HRMS: (ESI+) Calculated mass [M+Na]+ C10H15NO4Na (m, 18H), 0.88 (t, J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3):
δ = 6.57 (bs, 1H), 5.22 (t, J = 2.0 Hz,
8
=
δ
236.0893, found: 236.0893. Spectral data were in accordance = 183.74, 162.99, 148.20, 100.37, 74.74, 47.55, 39.72, 33.65,
with the literature.43
31.90, 29.61, 29.57, 29.52, 29.33, 29.28, 26.94, 22.67, 14.10. IR
max/cm-1: 2924, 2854, 1801, 1693, 1623, 1454, 1154, 1054 cm-
was prepared 1. HRMS: (ESI+) Calculated mass [M+H]+ C20H33N2O3 = 349.2486
using 2-((tert- found: 349.2482. E-isomer: Rf [silica, ethyl acetate: pentane (3:
butoxycarbonyl)(prop-2-yn-1-yl)amino)acetic acid = 5.57 (s, 1H), 5.42 (t, J
(2.12 g, 9.9 7)] = 0.55. 1H NMR (400 MHz, CDCl3) :
ν
N-Boc-N-propargyl β-ketoester 9. β-Ketoester
9
according to the general procedure
B
8
δ
mmol, 1 eq) DCC (2.22 g, 10.8 mmol, 1.1 eq), DMAP (1.65 g, = 2.3 Hz, 1H), 4.79 (d, J = 2.6 Hz, 2H), 4.17 (d, J = 2.5 Hz, 2H),
13.5 mmol, 1.5 eq), Meldrum’s acid (1.56 g, 10.8 mmol, 1.1 eq) 3.28 (q, J = 6.7 Hz, 2H), 2.34 (t, J = 2.6 Hz, 1H), 1.55 - 1.45 (m,
in THF (50 mL) and subsequent refluxing with MeOH. Flash 2H), 1.35 - 1.20 (m, 18H), 0.88 (t, J = 6.7 Hz, 4H). 13C NMR (101
chromatography using ethyl acetate: pentane (1: 11.5) as MHz, CDCl3):
eluent gave title compound (2.3 g, 85 % yield) as a yellow oil. 39.65, 33.67, 29.78, 29.50, 29.43, 27.09, 22.84, 14.27. IR
Rf [silica, ethyl acetate: pentane (1: 11.5] = 0.20. H NMR (400
MHz, CDCl3, mixture of rotamers):
= 4.25 (s, 1H), 4.18 (s, 2H), (ESI+) Calculated mass [M+H]+ C20H33N2O3 = 349.2486 found:
δ = 165.45, 157.91, 153.58, 97.33, 74.35, 49.35,
9
1
ν
max/cm-1: 2923, 2852, 1803, 1691, 1619, 1055 cm-1. HRMS:
δ
4.10 (s, 1H), 3.75 (s, 3H), 3.60 - 3.28 (app m, 2H), 2.26 (t, J = 2.5 349.2474.
Hz, 1H), 1.64 - 1.36 (m, 9H). 13C NMR (101 MHz, DMSO-d6,
25°C, mixture of rotamers): δ = 199.45, 171.29, 167.77, 154.72, PEG-functionalized monocyclic derivative 3b. Triazole
80.58, 80.42, 80.24, 80.05, 79.97, 75.15, 74.90, 56.05, 55.75, compound 3b was prepared according to the general
52.42, 48.12, 46.29, 37.75, 37.54, 37.05, 36.92, 28.36, 28.27, procedure E using the enol cyclocarbamate 3a-Z (4 mg, 0.012
28.16. IR
1161, 872, 774 cm-1. HRMS: (ESI+) Calculated mass [M+Na]+ 1.4 eq). Flash chromatography using ethyl acetate: pentane
ν
max/cm-1: 3281, 2978, 1698, 1453, 1394, 1368, 1248, mmol, 1 eq) and polyethylene glycol azide (3 mg, 0.017 mmol,
C13H19NO5Na = 292.1155, found: 292.1159.
(3:2) then methanol: DCM (1: 199) as eluent yielded 3b (3.6
mg, 60 % yield) as a yellow oil. Rf [silica, methanol: DCM (1:
1
N-Boc-N-propargyl β-ketoamide 10. β-Ketoamide 10 was 199)] = 0.11. H NMR (400 MHz, CDCl3) :
δ = 7.95 (s, 1H), 6.55
prepared according to the general procedure C by reacting 1,4- (t, J = 6.1 Hz, 1H), 5.14 (s, 1H), 4.62 (s, 2H), 4.57 (t, J = 4.9 Hz,
DABCO (60 mg, 0.53 mmol, 1.2 eq) with trimethylaluminium 2H), 4.40 (s, 2H), 3.88 (t, J = 4.9 Hz, 2H), 3.76 (t, J = 4.5 Hz, 2H),
(0.53 mL, 1.06 mmol, 2.4 eq, 2
mL) to produce DABAL in situ, subsequently adding - 1.47 (m, 2H), 1.31 - 1.20 (m, 18H), 0.88 (t, J = 6.6 Hz, 3H). 13
dodecylamine (98 mg, 0.53 mmol, 1.2 eq) in THF (1 mL) to NMR (101 MHz, CDCl3) : = 163.29, 153.61, 148.99, 100.11,
activate the amine and finally adding the corresponding β-keto 77.16, 72.58, 70.58, 70.34, 69.26, 61.85, 50.49, 48.64, 39.83,
ester (120 mg, 0.44 mmol, 1 eq) in THF (1 mL). Flash 38.96, 32.07, 29.80, 29.76, 29.70, 29.51, 29.47, 27.11, 22.85,
M in toluene) in toluene (1.5 3.64 (s, 4H), 3.59 (t, J = 4.5 Hz, 2H), 3.30 (q, J = 6.8 Hz, 2H), 1.59
C
δ
9
chromatography using ethyl acetate: pentane (2: 3) as eluent 14.28. IR ν
max/cm-1: 3286, 2911, 2836, 1761, 1654, 1621, 1512,
afforded 10 (68 mg, 36 % yield) as a yellow oil. Rf [silica, ethyl 1442, 1367, 1211, 1021 cm-1. HRMS: (ESI+) Calculated mass
acetate: pentane (2: 3)] = 0.26. 1H NMR (400 MHz, CDCl3, [M+H]+ C26H46N5O6 = 524.3443 found: 524.3434; Calculated
12 | J. Name., 2012, 00, 1-3
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mass [M+Na]+ C26H46N5O6Na = 546.3262, found: 546.3252.
ARTICLE
C18H33N2O3 = 325.2485 found: 325.2482. E-isomer: Rf [silica,
ethyl acetate: pentane (7: 3)] = 0.58. 1H NMR (400 MHz,
N-Boc sarcosine β-ketoester 12. β-Ketoester 12 was prepared CDCl3):
according to the general procedure using N-(tert- 2H), 2.97 (s, 3H), 1.58 - 1.43 (m, 2H), 1.39 - 1.10 (m, 18H), 0.87
butoxycarbonyl)-sarcosine 11 (1.89 g, 10 mmol, 1 eq) DCC (t, J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3):
= 165.65,
δ = 5.55 (s, 1H), 4.66 (d, J = 2.5 Hz, 2H), 3.36 - 3.18 (m,
B
δ
(2.26 g, 11 mmol, 1.1 eq), DMAP (1.83 g, 15 mmol, 1.5 eq), 158.07, 154.38, 96.77, 77.16, 52.04, 39.59, 32.05, 30.57, 29.82,
Meldrum’s acid (1.59 g, 11 mmol, 1.1 eq) in THF (50 mL) and 29.77, 29.76, 29.71, 29.67, 29.48, 29.42, 27.08, 22.82, 14.25 IR
subsequent refluxing with MeOH. Flash chromatography using
ethyl acetate: pentane (2: 3) as eluent gave title compound 12 1. HRMS: (ESI+) Calculated mass [M+H]+ C18H33N2O3 = 325.2485
(2.45 g, 55 % yield) as a yellow oil. Rf [silica, ethyl acetate: found: 325.2481; Remark: NOE experiment confirmed the E
ν
max/cm-1: 2934, 2851, 1805, 1689, 1629, 1459, 1151, 1046 cm-
pentane (2: 3)] = 0.25. 1H NMR (400 MHz, DMSO-d6, 75°C):
4.12 (s, 2H), 3.66 (s, 3H), 3.55 (s, 2H), 2.79 (s, 3H), 1.38 (s, 9H). proton for each isomer).
13C NMR (101 MHz, DMSO-d6, 25°C, mixture of rotamers):
199.72, 199.54, 167.35, 167.28, 155.21, 154.78, 79.07, 78.90, N-Boc-trans-4-benzyloxy-
58.14, 57.51, 51.90, 51.87, 45.91, 45.79, 39.52, 35.17, 35.10, 15a was prepared according to general procedure A using N-
27.95, 27.77. IR -proline (500 mg, 2.16 mmol, 1 eq), NaH
max/cm-1: 2979, 1692, 1459, 1391, 1362, 1241, Boc-trans-4-hydroxy-
1169, 871 cm-1. HRMS: (ESI+) Calculated mass [M+Na]+ (190 mg, 4.75 mmol, 2.2 eq, 60% in mineral oil) and benzyl
C11H19NO5Na = 268.1155, found: 268.1155. bromide (283 L, 3.6 mmol, 1.1 eq) in THF (12 mL). Flash
chromatography using ethyl acetate: pentane: AcOH (120: 79:
δ
=
and Z isomers (with a typical chemical shift of the alkene
δ
=
L
-proline 15a. The title compound
ν
L
ꢀ
N-Boc sarcosine β-ketoamide 13. β-Ketoamide 13 was 1) as eluent gave 15a (619 mg, 90 % yield) as a yellow oil. Rf
prepared according to the general procedure C by reacting 1,4- [silica, ethyl acetate: pentane: AcOH (120: 79: 1)] = 0.15. 1H
DABCO (660 mg, 5.83 mmol, 1.2 eq) with trimethylaluminium NMR (400 MHz, DMSO-d6, 75° C):
(5.83 mL, 11.66 mmol, 2.4 eq, 2 in toluene) in toluene (10 4.40 (m, 2H), 4.33 - 4.09 (m, 2H), 3.64 - 3.36 (m, 2H), 2.42 -
mL) to produce DABAL in situ, subsequently adding 2.28 (m, 1H), 2.18 - 1.98 (m, 1H), 1.38 (s, 9H). 13C NMR (101
dodecylamine (1.07 g, 5.83 mmol, 1.2 eq) in THF (4 mL) to MHz, DMSO-d6, 25°C, mixture of rotamers): = 174.55, 174.07,
δ = 7.59 - 7.09 (m, 5H), 4.65 -
M
δ
activate the amine and finally adding the corresponding β-keto 154.11, 153.59, 138.73, 138.67, 128.71, 128.04, 127.93, 79.46,
ester 12 (1.19 g, 4.85 mmol, 1 eq) in THF (3 mL). Flash 76.90, 76.13, 70.45, 70.37, 58.11, 57.84, 52.23, 51.90, 36.19,
chromatography using ethyl acetate: pentane (1: 3) as eluent 35.46, 28.52, 28.33. HRMS: (ESI+) Calculated mass [M+H]+
afforded 13 (972.5 mg, 50 % yield) as a yellow oil. Rf [silica, C17H24NO5 = 322.1649 found: 322.1651 Spectral data were in
ethyl acetate: pentane (1: 3)] = 0.21. 1H NMR (400 MHz, accordance with the literature.42,43
DMSO-d6, 75°C):
3.19 - 2.98 (m, 2H), 2.77 (s, 3H), 1.45 - 1.34 (m, 11H), 1.33 - N-Boc-trans-4-propargyloxy-
1.13 (m, 18H), 0.87 (t, J = 6.2 Hz, 3H). 13C NMR (101 MHz, 15b was prepared according to general procedure A using N-
DMSO-d6, 25°C, mixture of rotamers): = 201.04, 200.69, Boc-trans-4-hydroxy- -proline (755 mg, 3.27 mmol, 1 eq), NaH
165.27, 165.02, 155.15, 154.85, 78.87, 78.62, 58.12, 57.56, (288 mg, 7.19 mmol, 2.2 eq, 60% in mineral oil) and propargyl
47.83, 47.79, 39.52, 38.67, 35.08, 35.00, 31.32, 29.09, 29.04, bromide (535 L, 3.6 mmol, 1.1 eq, 80 wt. % in toluene) in THF
28.96, 28.77, 28.75, 27.95, 27.78, 26.37, 22.11, 13.89. IR (18 mL). Flash chromatography using ethyl acetate: pentane:
δ = 7.79 (bs, 1H), 4.11 (s, 2H), 3.28 (s, 2H),
L
-proline 15b. The title compound
δ
L
ꢀ
ν
max/cm-1: 1928, 1851, 1709, 1458, 1371, 1245, 1169, 940 cm-1. AcOH (80: 119: 1) as eluent afforded 15b (781 mg, 90 % yield)
HRMS: (ESI+) Calculated mass [M+Na]+ C22H42N2O4Na
445.3037, found: 445.3039.
=
as a slightly yellow oil. Rf [silica, ethyl acetate: pentane: AcOH
1
(80: 119: 1)] = 0.19. H NMR (400 MHz, DMSO-d6, 75° C):
δ =
12.26 (bs, 1H), 4.25 (app p, 1H), 4.17 (d, J = 2.4 Hz, 2H), 4.12 (t,
Methyl functionalized monocyclic derivative 3c. Enol J = 7.7 Hz, 1H), 3.51 - 3.40 (m, 2H), 3.28 (t, J = 2.4 Hz, 1H), 2.36
cyclocarbamate 3c was prepared according to the general - 2.27 (m, 1H), 2.09 - 1.97 (m, 1H), 1.39 (s, 9H). 13C NMR (101
procedure D by reacting the β-keto amide 13 (209 mg, 0.52 MHz, DMSO-d6, 25°C, mixture of rotamers): δ = 174.53, 174.07,
mmol, 1 eq), TMSOTf (193 μL, 1.02 mmol, 2 eq) and CDI (145 154.05, 153.54, 80.74, 79.51, 77.55, 76.66, 75.89, 58.00, 57.72,
mg, 0.75 mmol, 1.5 eq) in DCM (3 mL). Flash chromatography 56.19, 56.13, 52.03, 51.69, 35.91, 35.17, 28.49, 28.30. HRMS:
using ethyl acetate: pentane (7:3) then ethyl acetate: pentane (ESI+) Calculated mass [M+Na]+ C13H19NO5Na = 292.1155,
(7:3) gave Z-3c (81.34 mg, 48 % yield) and E-3c (34.86 mg, 21 % found: 292.1162 Spectral data were in accordance with the
yield) as colorless oil. Z-isomer: Rf [silica, ethyl acetate: literature.44
pentane (7: 3)] = 0.19. 1H NMR (400 MHz, CDCl3):
1H), 5.15 (s, 1H), 4.28 (s, 2H), 3.41 - 3.24 (m, 2H), 2.99 (s, 3H), N-Boc-trans-4-benzyloxy-
1.62 - 1.45 (m, 2H), 1.46 - 1.03 (m, 18H), 0.87 (t, J = 6.7 Hz, 3H). Ketoester 16a was prepared according to the general
13C NMR (101 MHz, CDCl3):
= 163.45, 153.72, 148.92, 99.73, procedure using (2S,4R)-4-(benzyloxy)-1-(tert-
δ = 6.63 (bs,
L
-proline β-ketoester 16a. β-
δ
B
77.16, 50.54, 40.15, 39.84, 34.34, 32.04, 30.72, 29.78, 29.76, butoxycarbonyl)pyrrolidine-2-carboxylic acid 15a (544 mg, 1.69
29.72, 29.67, 29.65, 29.56, 29.48, 29.44, 29.39, 27.09, 27.00, mmol, 1 eq) DCC (380 mg, 1.86 mmol, 1.1 eq), DMAP (367mg,
22.82, 14.25. IR
1159, 1034 cm-1. HRMS: (ESI+) Calculated mass [M+H]+ eq) in THF (6.5 mL) to obtain the Meldrum’s acid intermediate,
ν
max/cm-1: 2931, 2849, 1807, 1699, 1621, 1450, 2.55 mmol, 1.5 eq), Meldrum’s acid (269 mg, 1.86 mmol, 1.1
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ARTICLE
Journal Name
which was converted into 16a by subsequent refluxing in 47.43, 35.11, 34.28, 31.73, 29.48, 29.42, 29.14, 28.46, 28.25,
MeOH. Flash chromatography using ethyl acetate: pentane (3: 26.78, 22.53, 14.38 IR ν
max/cm-1: 3245, 2927, 1696, 1401, 1161,
17) as eluent furnished 16a (345 mg, 54 % yield) as a brownish 1094, 739 cm-1. HRMS: (ESI+) Calculated mass [M+Na]+
oil. Rf [silica, ethyl acetate: pentane (3: 17)] = 0.17. 1H NMR C31H50N2O5Na = 553.3612, found: 553.3611.
(400 MHz, DMSO-d6, 75° C) : δ = 7.40 - 7.24 (m, 5H), 4.53 (d, J =
12.2 Hz, 2H), 4.48 (d, J = 12.0 Hz, 2H), 4.40 (t, J = 8.2 Hz, 1H), N-Boc-trans-4-propargyloxy-
L
-proline β-ketoamide 17b. β-
4.18 - 4.12 (m, 1H), 3.65 (s, 3H), 3.61 - 3.54 (m, 1H), 3.49 - 3.41 Ketoamide 17b was prepared according to the general
(m, 1H), 2.38 - 2.28 (m, 1H), 2.06 - 1.99 (m, 1H), 1.38 (s, 9H). procedure C by reacting DABCO (489 mg, 4.36 mmol, 1.3 eq)
13C NMR (101 MHz, DMSO-d6, 25°C, mixture of rotamers):
δ
=
with trimethylaluminium (4.36 mL, 8.72 mmol, 2.6 eq, 2 M in
203.59, 168.39, 153.94, 139.23, 129.31, 129.23, 128.50, toluene) in toluene (8 mL) to produce DABAL in situ,
128.34, 80.62, 80.49, 77.55, 76.66, 70.84, 70.76, 64.89, 64.60, subsequently adding dodecylamine (808 mg, 4.36 mmol, 1.3
52.95, 52.87, 52.74, 46.06, 46.05, 35.45, 28.81, 28.80. IR eq) in THF (4 mL) to activate the amine and finally adding the
ν
max/cm-1: 2924, 2854, 1743, 1611, 1455, 1377, 1240, 1030, corresponding β-ketoester 13b (1.09 g, 3.09 mmol, 1 eq) in
861, 755 cm-1. HRMS: (ESI+) Calculated mass [M+Na]+ THF (4 mL). Flash chromatography using ethyl acetate:
C20H28NO6Na = 378.1911, found: 378.1911.
pentane (3: 17) as eluent gave 17b (391 mg, 63 % yield) as a
1
yellow oil. Rf [silica, ethyl acetate: pentane (3: 17)] = 0.28. H
N-Boc-trans-4-propargyloxy-
prepared according to the general procedure B using (2S,4R)-1- (m, 1H), 4.24 - 4.17 (m, 1H), 4.16 (s, 2H), 3.57 - 3.50 (m, 1H),
(tert-butoxycarbonyl)-4-(prop-2-yn-1-yloxy)pyrrolidine-2- 3.48 - 3.39 (m, 1H), 3.35 (d, J = 8.6 Hz, 2H), 3.31 - 3.26 (m, 1H),
L-proline β-ketoester 16b. was NMR (400 MHz, DMSO-d6, 75°C): δ = 7.77 (s, 1H), 4.48 - 4.34
carboxylic acid 16b (781 mg, 2.9 mmol, 1 eq) DCC (642 mg, 3.13 - 3.03 (m, 2H), 2.32 - 2.21 (m, 1H), 2.15 - 1.94 (m, 1H),
3.19 mmol, 1.1 eq), DMAP (532 mg, 4.36 mmol, 1.5 eq) and 1.42 - 1.35 (m, 11H), 1.31 - 1.21 (m, 18H), 0.87 (t, J = 6.5 Hz,
Meldrum’s acid (460 mg, 3.19 mmol, 1.1 eq) in THF (16 mL) to 3H). 13C NMR (101 MHz, DMSO-d6, 25 °C, mixture of
obtain the Meldrum’s acid intermediate and subsequent rotamers): δ = 204.42, 203.93, 165.85, 165.47, 154.35, 153.41,
refluxing of this intermediate in MeOH to obtain 16b. Flash 80.71, 80.67, 79.80, 77.65, 77.61, 76.84, 75.88, 64.00, 63.80,
chromatography using ethyl acetate: pentane (1: 4) as eluent 56.07, 52.48, 52.12, 47.56, 47.31, 34.79, 33.96, 31.75, 29.51,
gave 16b (669 mg, 71 % yield) as a yellow oil. Rf [silica, ethyl 29.45, 29.20, 29.17, 28.47, 28.25, 26.80, 22.55, 14.39. IR
1
acetate: pentane (1: 4)] = 0.22. H NMR (400 MHz, DMSO-d6,
ν
max/cm-1: 3311, 2923, 2853, 1649, 1547, 1394, 1162, 1130,
75 °C)
δ
= 4.35 (t, J = 8.2 Hz, 1H), 4.23 - 4.19 (m, 1H), 4.17 (d, J 975 cm-1. HRMS: (ESI+) Calculated mass [M+Na]+ C27H46N2O5Na
= 2.4 Hz, 2H), 3.65 (s, 3H), 3.61 - 3.49 (m, 2H), 3.49 - 3.42 (m, = 501.3299, found: 501.3299.
2H), 3.29 (t, J = 2.1 Hz, 1H), 2.36 - 2.22 (m, 1H), 2.13 - 1.86 (m,
1H), 1.38 (s, 9H). 13C NMR (101 MHz, DMSO-d6, 25 °C, mixture trans-4-benzyloxy-
L
-proline
derivative
4a.
Enol
of rotamers):
δ = 203.06, 167.82, 167.75, 154.47, 153.36, cyclocarbamate 4a was prepared according to the general
80.72, 80.68, 80.13, 80.06, 77.67, 77.64, 76.78, 75.89, 64.29, procedure D by reacting the appropriate β-keto amide 17a
64.10, 63.90, 56.04, 52.50, 52.45, 52.34, 52.19, 52.15, 49.03, (112.5 mg, 0.19 mmol, 1 eq), TMSOTf (71 μL, 0.39 mmol, 2 eq)
45.61, 45.38, 34.73, 34.68, 33.90, 33.80, 28.47, 28.43, 28.28, and CDI (54 mg, 0.29 mmol, 1.5 eq) in DCM (1 mL). Flash
28.21, 28.18, 25.86, 25.82. IR ν
max/cm-1: 2978, 1695, 1396, chromatography using ethyl acetate: pentane (3: 7) as eluent
1367, 1162, 1255, 1086, 772 cm-1. HRMS: (ESI+) Calculated afforded 4a (39 mg, 44 % yield) as a colorless oil. Rf [silica,
20
mass [M+Na]+ C16H23NO6Na = 348.1418, found: 348.1419.
ethyl acetate: pentane (1: 1)] = 0.55. [α]D = -55 (c = 0.040,
CHCl3); 1H NMR (400 MHz, CDCl3):
L-proline β-ketoamide 17a. β- (bs, 1H), 5.16 (s, 1H), 4.75 (dd, J = 10.7, 6.0 Hz, 1H), 4.52 (s,
δ = 7.40 - 7.27 (m, 5H), 6.56
N-Boc-trans-4-benzyloxy-
Ketoamide 17a was prepared according to the general 2H), 4.38 (t, J = 4.9 Hz, 1H), 3.86 (dd, J = 12.5, 4.9 Hz, 1H), 3.40
procedure C by reacting DABCO (116 mg, 1.04 mmol, 1.2 eq) (d, J = 12.5 Hz, 1H), 3.35 - 3.25 (m, 2H), 2.39 (dd, J = 13.3, 6.0
with trimethylaluminium (1.05 mL, 2.08 mmol, 2.4 eq, 2
toluene) in toluene (3 mL) to produce DABAL in situ, - 1.22 (m, 18H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,
subsequently adding dodecylamine (192 mg, 1.04 mmol, 1.2 CDCl3) : = 163.18, 155.87, 153.49, 136.98, 128.63, 128.16,
M in Hz, 1H), 1.72 (dd, J = 13.3, 4.7 Hz, 1H), 1.58 - 1.47 (m, 2H), 1.27
δ
eq) in THF (3 mL) to activate amine and finally adding the 127.68, 100.13, 79.62, 71.57, 61.84, 52.68, 39.73, 38.08, 31.90,
corresponding β-keto ester 16a (328 mg, 0.87 mmol, 1 eq) in 29.63, 29.61, 29.58, 29.51, 29.33, 29.28, 26.94, 22.67, 14.10. IR
THF (3 mL). Flash chromatography using ethyl acetate:
pentane (1: 1) as eluent yielded 17a (304 mg, 60 % yield) as a 1374, 1215, 1094, 1026, 979 cm-1. HRMS: (ESI+) Calculated
yellow oil. Rf [silica, ethyl acetate: pentane (1: 1)] = 0.15. 1H mass [M+H]+ C27H41N2O4 = 457.3061 found: 457.3063.
ν
max/cm-1: 3245, 2922, 2853, 1790, 1694, 1632, 1535, 1455,
NMR (400 MHz, DMSO-d6, 75°C): δ = 7.77 (s, 1H), 7.41 - 7.18
(m, 5H), 4.54 - 4.46 (m, 2H), 4.46 - 4.38 (m, 1H), 4.17 - 4.10 (m, trans-4-propargyloxy-
L
-proline
derivative
4b.
Enol
1H), 3.58 - 3.41 (m, 2H), 3.39 - 3.27 (m, 2H), 2.34 - 2.17 (m, cyclocarbamate 4b was prepared according to the general
1H), 2.11 - 1.99 (m, 1H), 1.37 (s, 9H), 1.31 - 1.22 (m, 18H), 0.92 procedure D by reacting the appropriate β-keto amide 17b
- 0.82 (m, 3H). 13C NMR (101 MHz, DMSO-d6, 25 °C, mixture of (130 mg, 0.27 mmol, 1 eq), TMSOTf (98.8 μL, 0.54 mmol, 2 eq)
rotamers): δ = 204.49, 204.03, 165.91, 165.52, 153.55, 138.63, and CDI (75.4 mg, 0.4 mmol, 1.5 eq) in DCM (1.5 mL). Flash
128.69, 127.97, 79.80, 77.12, 76.19, 70.28, 64.11, 63.90, 52.26, chromatography using ethyl acetate: pentane (from 1:4 to 3:2)
14 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6OB01664B
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as eluent gave E-4b (9.8 mg, 8.8 % yield) and Z-4b (27.8 mg, polyethylene glycol azide (6.1 mg, 0.035 mmol, 1.3 eq). Flash
25.2 % yield) as a colorless oil. The chemical shifts of the chromatography using methanol: DCM (1: 199) as eluent
protons that are characteristic protons for the E and Z isomer yielded 4d (8.3 mg, 52 % yield) as a yellow oil. Rf [silica,
are in correspondence with those that have been reported for methanol: DCM (1: 199)] = 0.11. [α]D20 = -10 (c = 0.180, CHCl3);
the isomers of lipocyclocarbamates intermediates.15 Z-isomer: 1H NMR (400 MHz, CDCl3) :
δ = 7.80 (s, 1H), 6.63 - 6.48 (m, 1H),
20
Rf [silica, ethyl acetate: pentane (3: 2)] = 0.20. [α]D = -39.3 (c 5.16 (s, 1H), 4.73 (dd, J = 10.6, 5.9 Hz, 1H), 4.66 (d, J = 12.4 Hz,
1
= 0.214, CHCl3); H NMR (400 MHz, CDCl3) :
δ = 6.56 (bs, 1H), 1H), 4.62 (d, J = 12.4 Hz, 1H), 4.56 (t, J = 5.0 Hz, 2H), 4.48 (t, J =
5.19 (s, 1H), 4.77 - 4.67 (m, 1H), 4.52 (t, J = 5.4 Hz, 1H), 4.19 (d, 5.4 Hz, 1H), 3.94 - 3.81 (m, 3H), 3.72 (t, J = 4.4 Hz, 2H), 3.62 (s,
J = 2.4 Hz, 2H), 3.90 (dd, J = 12.7, 5.5 Hz, 1H), 3.39 (d, J = 12.7 4H), 3.57 (t, J = 4.5 Hz, 2H), 3.36 (d, J = 12.6 Hz, 1H), 3.29 (q, J =
Hz, 1H), 3.36 - 3.26 (m, 2H), 2.49 (t, J = 2.4 Hz, 1H), 2.42 (dd, J = 6.7 Hz, 2H), 2.41 (dd, J = 13.2, 6.0 Hz, 1H), 1.72 (td, J = 13.2,
13.3, 6.0 Hz, 1H), 1.75 (ddd, J = 13.3, 10.6, 5.3 Hz, 1H), 1.58 - 10.6, 5.1 Hz, 1H), 1.58 - 1.46 (m, 2H), 1.33 - 1.21 (m, 18H), 0.87
1.48 (m, 2H), 1.32 - 1.23 (m, 18H), 0.87 (t, J = 7.1 Hz, 3H). 13C (t, J = 6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3):
NMR (101 MHz, CDCl3): = 163.30, 155.98, 153.47, 114.05, 155.86, 153.54, 124.22, 100.03, 79.89, 72.39, 70.49, 70.19,
δ = 163.24,
δ
100.34, 79.55, 75.54, 61.87, 56.95, 52.75, 39.86, 38.01, 32.03, 69.24, 62.67, 61.81, 61.69, 52.73, 50.38, 39.75, 37.95, 31.89,
29.76, 29.74, 29.71, 29.65, 29.46, 29.42, 27.07, 22.80, 14.24. 29.63, 29.60, 29.57, 29.51, 29.32, 29.28, 26.94, 22.66, 14.10.
IR ν ν
max/cm-1: 3310, 2924, 2854, 1800, 1693, 1626, 1551, 1365, IR max/cm-1: 3299, 2922, 2853, 1789, 1694, 1631, 1537, 1465,
1167, 1091, 981 cm-1. HRMS: (ESI+) Calculated mass [M+H]+ 1375, 1218, 1032 cm-1. HRMS: (ESI+) Calculated mass [M+H]+
C23H37N2O4 = 405.2748 found: 405.2748; Calculated mass C29H50N5O7 = 580.3705 found: 580.3670.
[M+Na]+ C23H36N2O4Na = 427.2567, found: 427.2563 E-isomer:
Rf [silica, ethyl acetate: pentane (3: 2)] = 0.51. [α]D20 = -9.7 (c = (1-(3-(Dimethylamino)propyl)-triazole derivative 4e. Triazole
1
0.064, CHCl3); H NMR (400 MHz, CDCl3):
δ = 5.55 (s, 1H), 5.44 compound 4e was prepared according to the general
(bs, 1H), 5.20 (dd, J = 10.6, 6.0 Hz, 1H), 4.50 (t, J = 5.9 Hz, 1H), procedure E using the enol cyclocarbamate 4b (8 mg, 0.020
4.25 (dt, J = 15.8, 1.6 Hz, 1H), 4.14 (dt, J = 16.1, 1.8 Hz, 1H), mmol, 1 eq) and 3-azido-N,N-dimethylpropan-1-amine (3 mg,
3.93 (dd, J = 12.7, 5.9 Hz, 1H), 3.46 - 3.14 (m, 3H), 2.88 (dd, J = 0.022 mmol, 1.4 eq). Flash chromatography using methanol:
13.7, 6.0 Hz, 1H), 2.44 (app q, J = 2.2 Hz, 1H), 1.61 (ddd, J = DCM (1: 9) as eluent yielded 4e (3 mg, 29 % yield) as a yellow
20
13.7, 10.2, 5.5 Hz, 1H), 1.60 - 1.46 (m, 2H), 1.44 - 1.20 (m, oil. Rf [silica, methanol: DCM (1: 9)] = 0.15. [α]D = -27.4 (c =
18H), 0.88 (t, J = 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3):
1
0.073, CHCl3); H NMR (400 MHz, CDCl3):
δ
=
δ
164.63, 161.65, 156.83, 97.95, 78.74, 77.31, 74.94, 62.71, (bs, 1H), 5.16 (s, 1H), 4.73 (dd, J = 10.8, 6.1 Hz, 1H), 4.66 (d, J =
= 7.65 (s, 1H), 6.54
56.42, 52.80, 39.54, 36.49, 31.89, 29.62, 29.55, 29.52, 29.32, 12.6 Hz, 1H), 4.62 (d, J = 12.8 Hz, 1H), 4.56 - 4.36 (m, 2H), 3.88
29.26, 26.93, 22.66, 14.10. IR ν
max/cm-1: 3288, 2922, 2853, (dd, J = 12.6, 5.5 Hz, 1H), 3.37 (d, J = 12.6 Hz, 1H), 3.33 - 3.26
1792, 1696, 1633, 1545, 1465, 1375, 1216, 1092, 1032, 980 (m, 2H), 2.51 - 2.31 (m, 9H), 2.25 - 2.14 (m, 2H), 1.80 - 1.67 (m,
cm-1. HRMS: (ESI+) Calculated mass [M+H]+ C23H37N2O4
405.2748 found: 405.2742.
=
1H), 1.61 - 1.47 (m, 2H), 1.32 - 1.17 (m, 18H), 0.88 (t, J = 6.8 Hz,
3H). 13C NMR (101 MHz, CDCl3):
= 163.09, 155.89, 153.28,
144.14, 123.66, 100.28, 79.98, 62.71, 61.76, 55.33, 52.70,
Benzyl triazole derivative 4c. Triazole compound 4c was 47.36, 43.97, 39.70, 37.99, 31.89, 29.61, 29.52, 29.33, 29.29,
prepared according to the general procedure E using the enol 26.95, 22.67, 14.10. IR
max/cm-1: 3287, 2922, 2853, 1788,
δ
ν
cyclocarbamate 4b (5 mg, 0.012 mmol, 1 eq) and benzylazide 1694, 1632, 1544, 1465, 1391, 1219, 1091, 1034, 837 cm-1.
(1.53 μL, 0.012 mmol, 1 eq). Flash chromatography using HRMS: (ESI+) Calculated mass [M+H]+ C28H49N6O4 = 533.3810
methanol: DCM (1: 24) as eluent yielded 4c (8 mg, 50 % yield) found: 533.3809.
as a colorless oil. Rf [silica, methanol: DCM (1: 24)] = 0.18.
20
[α]D = -3.8 (c = 0.313, CHCl3); H NMR (400 MHz, CDCl3) :
1
δ =
Acknowledgements
7.45 (bs, 1H), 7.41 - 7.35 (m, 3H), 7.34 - 7.26 (m, 2H), 6.61 -
6.48 (m, 1H), 5.53 (s, 2H), 5.13 (s, 1H), 4.69 (dd, J = 10.7, 5.7
Hz, 1H), 4.60 (s, 2H), 4.45 (t, J = 5.4 Hz, 1H), 3.86 (dd, J = 12.5,
5.3 Hz, 1H), 3.46 - 3.21 (m, 3H), 2.37 (dd, J = 13.3, 5.7 Hz, 1H),
1.84 - 1.64 (m, 1H), 1.60 - 1.44 (m, 2H), 1.37 - 1.12 (m, 18H),
The authors would like to thank Larisa Cortes Tolalpa for
technical assistance and David Popham (Department of
Biological Sciences, Virginia Tech) for the strain PS2022 (which
was used to make the PBP4-null strain); A.K.H.H. is supported
by NWO-ECHO-STIP grant 717.014.004 and by the Dutch
Ministry of Education, Culture, Science (gravitation program
024.001.035). Y.L. is supported by a PhD fellowship from the
Chinese Scholarship Council. J.W.V. is supported by a VIDI
grant from The Netherlands Organisation for Scientific
Research (NWO, 864.12.001) and by an ERC-starting grant
(337399-PneumoCell). D.J.S. is supported by NWO-VIDI grant
864.09.010. M.D.W. is supported by NWO-VENI grant
722.012.003 and a Marie Sklowdoska Curie Career Integration
Grant.
0.87 (t, J = 6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3):
δ = 163.41,
155.94, 153.72, 134.33, 129.39, 129.16, 128.38, 100.17, 80.11,
62.86, 61.98, 54.62, 52.86, 39.97, 38.12, 32.06, 29.79, 29.74,
29.68, 29.49, 29.44, 27.10, 22.84. IR ν
max/cm-1: 3241, 2924,
2853, 1793, 1696, 1635, 1535, 1466, 1390, 1218, 1090, 1049,
984 cm-1. HRMS: (ESI+) Calculated mass [M+H]+ C30H44N5O34
538.3388 found: 538.3384.
=
PEG-triazole derivative 4d. Triazole compound 4d was
prepared according to the general procedure E using the enol
cyclocarbamate 4b (11 mg, 0.027 mmol,
1 eq) and
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