Deuterium and tritium labelling of N-acyl-L-homoserine lactones (AHLs) by catalytic reduction of a double bond in the layer-by-layer method

Article abstract of DOI:10.1002/ejoc.201300084

N-Acyl-L-homoserine lactones (AHLs) are signalling molecules found in Gram-negative bacteria that enable bacteria to communicate with each other through quorum sensing and with their eukaryotic host cells through inter-kingdom signalling. Very little is known about inter-kingdom signalling mechanisms. Deuterium- and tritium-labelled AHLs were synthesized in an effort to detect the cellular distribution and monitor the membrane transport of AHLs. Most tritium labelling methods use tritium gas, which is difficult to handle, however, here we present a novel, gas-free method with which to obtain deuterium- and tritium-label terminally unsaturated AHLs through catalytic reduction of the double bond. This uncommon double bond reduction employs either sodium borohydride-[²H] or sodium borohydride-[³H] and is performed in the presence of palladium(II) acetate, which acts as a catalyst in the unique layer-by-layer system. Moreover, detailed NMR analysis of the resulting isotopologues is presented. A novel, gas-free method of deuterium- and tritium-labelling of terminally unsaturated AHLs is described. The catalytic reduction of the double bond by sodium borohydrides is performed in the presence of palladium(II) acetate, which acts as a catalyst in the unique layer-by-layer system. Copyright

Full text of DOI:10.1002/ejoc.201300084

Job/Unit: O30084
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FULL PAPER
Isotopic Labelling
D. Jakubczyk, C. Merle,
G. Brenner-Weiss, B. Luy,
S. Bräse* ........................................... 1–9
Deuterium and Tritium Labelling of N-
Acyl-l-homoserine Lactones (AHLs) by
Catalytic Reduction of a Double Bond in
the Layer-by-Layer Method
Keywords: Isotopic labeling / Lactones /
Isomers / Layered compounds / Regioselec-
tivity / Reduction
A novel, gas-free method of deuterium-
and tritium-labelling of terminally unsatu-
rated AHLs is described. The catalytic re-
duction of the double bond by sodium
borohydrides is performed in the presence
of palladium(II) acetate, which acts as a
catalyst in the unique layer-by-layer system.
0000, 0–0
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Job/Unit: O30084
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D. Jakubczyk, C. Merle, G. Brenner-Weiss, B. Luy, S. Bräse
FULL PAPER
DOI: 10.1002/ejoc.201300084
Deuterium and Tritium Labelling of N-Acyl- -homoserine Lactones (AHLs) by
L
Catalytic Reduction of a Double Bond in the Layer-by-Layer Method
Dorota Jakubczyk,[a,b][‡] Christian Merle,[a,c] Gerald Brenner-Weiss,[b] Burkhard Luy,[a,c] and
Stefan Bräse*[a,d]
Keywords: Isotopic labeling / Lactones / Isomers / Layered compounds / Regioselectivity / Reduction
N-Acyl-
L
-homoserine lactones (AHLs) are signalling molec-
however, here we present a novel, gas-free method with
which to obtain deuterium- and tritium-label terminally un-
saturated AHLs through catalytic reduction of the double
bond. This uncommon double bond reduction employs either
sodium borohydride-[2H] or sodium borohydride-[3H] and is
performed in the presence of palladium(II) acetate, which
acts as a catalyst in the unique layer-by-layer system. More-
over, detailed NMR analysis of the resulting isotopologues is
presented.
ules found in Gram-negative bacteria that enable bacteria to
communicate with each other through quorum sensing and
with their eukaryotic host cells through inter-kingdom sig-
nalling. Very little is known about inter-kingdom signalling
mechanisms. Deuterium- and tritium-labelled AHLs were
synthesized in an effort to detect the cellular distribution and
monitor the membrane transport of AHLs. Most tritium label-
ling methods use tritium gas, which is difficult to handle,
catheters or ventilation tubes. AHLs also enable communi-
cation between microorganisms and their eukaryotic host
cells (inter-kingdom signalling).[8] Notably, AHLs are
known to upregulate proinflammatory cytokine expression,
which often modulates the host’s immune response and
leads to bacterial pathogenesis.[9] N-(3-Oxododecanoyl)-l-
homoserine lactone (1, 3OC12-HSL; Figure 1) is the most
prominent AHL.[10] However, other AHL derivatives – with
acyl chains of varying lengths – also exhibit diverse bio-
logical activities.[2,4] Contrary to the adverse effects re-
ported in the literature, 1 might also support immune sys-
tem defence in the host.[5,6] The host’s defence against bac-
teria or bacterial biofilms relies on phagocytic cells, particu-
larly polymorphonuclear neutrophils (PMN), and their ca-
pacity to infiltrate infected sites then subsequently recog-
nize and eliminate pathogens.[11] Very little is known about
the detailed mechanism of the interaction between AHL
and host cells. Therefore, deuterium and tritium labelled
AHLs were synthesized to investigate the cellular uptake of
AHLs and their cellular distribution.
Introduction
N-Acyl-l-homoserine lactones (AHLs) are signalling
molecules found in Gram-negative bacteria. These quorum
sensing molecules and their role in bacterial communication
have been studied extensively,[1–5] particularly in the context
of virulence factor production and biofilm formation. Bio-
film formation is the “common cause of persistent infec-
tions” in chronic and destructive inflammatory processes
such as chronic sinusitis, implant-associated osteomyelitis,
and wound infection.[6,7] Biofilm formation is common in
P. aeruginosa infection. It is the leading cause of morbidity
and mortality in patients with cystic fibrosis, patients with
open, nonhealing wounds and patients requiring indwelling
[a] Karlsruhe Institute of Technology, Institute of Organic
Chemistry,
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
Fax: +49-721-608-8581
E-mail: braese@kit.edu
Homepage: www.ioc.kit.edu/braese
[b] Karlsruhe Institute of Technology, Institute of Functional
Interfaces,
Hermann von Helmholtz-Platz 1, 76344 Eggenstein-
Leopoldshafen, Germany
[c] Karlsruhe Institute of Technology, Institute for Biological
Interfaces 2,
Hermann von Helmholtz-Platz 1, 76344 Eggenstein-
Leopoldshafen, Germany
[d] Karlsruhe Institute of Technology, Institute for Toxicology and
Genetics,
Figure 1. Structure of the N-(3-oxododecanoyl)-l-homoserine lact-
one (3OC12-HSL; 1).
Hermann von Helmholtz-Platz 1, 76344 Eggenstein-
Leopoldshafen, Germany
Isotope-labelled compounds are employed in several im-
portant applications. Compounds labelled with stable iso-
topes are often used to probe mechanisms in organic chem-
istry and organism metabolism.[12–14] 2H (deuterium) and
[‡] Present address: John Innes Centre, Biological Chemistry
Department, Norwich Research Park,
Norwich, NR4 7UH, United Kingdom
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300084.
2
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Deuterium and Tritium Labelling of N-Acyl-l-Homoserine Lactones
3H (tritium) are most frequently used in organic chemistry tailed NMR studies were performed by using 2D edited
because they exhibit the greatest relative mass differences in HSQC spectroscopy and 1D 13C experiments with varying
comparison to their canonical isotope. Deuterium and tri- decoupling schemes.
tium labelled reagents are commercially available, safe and
relatively inexpensive. Labelling substrates with deuterium
or tritium can provide very important mechanistic and
stereochemical insights into chemical and enzymatic reac-
Results and Discussion
tions. However, most labelling methods require the use of
hydrogen gas, expensive metal-based catalysts, high tem-
Synthesis and Labelling
peratures or microwave irradiation, and caustic reagents,
The aim of our work was to develop a novel synthetic
such as strong acids, bases, reductants or oxidants. Hydro-
route to deuterated and tritiated AHLs. These AHLs were
gen (deuterium or tritium) gas is particularly undesirable
analyzed by detailed NMR studies, which were employed
because it is both difficult to handle (requires sophisticated
apparatus) and explosive. Among the hydride reductants,
sodium borohydride (NaBH4) is especially attractive be-
cause of its low cost, mild nature and ease of handling.
to measure their distribution within cells. The substrates for
the labelling reactions (6a–d) were prepared through known
procedures (Scheme 1).[4,39,40] Commercially available ter-
minally unsaturated carboxylic acids 2a–d were first con-
verted into their corresponding acyl chlorides 4a–d using
However, its application was found to be limited mostly to
the reduction of polar, carbonyl-containing functional
oxalyl chloride; these reactions were completed in very
groups.[15] In 1962, Brown described the reduction of simple
good yield. The amidation reaction with l-homoserine lact-
one hydrobromide (5) was first carried out with commer-
cially available decanoyl chloride to give N-decanoyl-l-
homoserine lactone in 86% yield. The intermediates 4a–d
alkenes by NaBH4, which generates hydrogen gas in situ.[16]
Subsequently, the generated hydrogen gas reduces the alk-
enes under the repeated catalysis of reduced metals.[16,17]
Subsequently, Brown and others tested modifications to this
reacted with l-homoserine lactone hydrobromide in the
method, including combining sodium borohydride and
presence of triethylamine to give the terminally unsaturated
other hydride reagents with palladium, rhodium, platinum,
AHLs 6a–d in very good yields (Scheme 1).
osmium, nickel and indium salts.[18–23] Simagina et al. ex-
plored the effects of both the active component and the
support on catalysts during the hydrolysis of NaBH4.[24]
The activity of the supported metals in the reduction of
sodium borohydride decreases in the order Rh Ͼ Pt ≈ Ru
ϾϾ Pd, regardless of the nature of the support (γ-Al2O3, a
Sibunit carbon material, or TiO2). Nanodispersed platinum
metal powders were found to dramatically increase the rate
of hydrogen generation. In 2009, Tran et al.[15] presented a
modification to this method, which relied on the direct use
of palladium metal instead of metal salts, and the addition
of acetic acid to catalyse sodium borohydride hydrolysis.
Although a considerable amount of work has explored
H/D exchange reactions with transition metals,[25–29] and
the introduction of deuterium along with palladium(II)
salts and sodium borodeuteride,[30–35] little has been done
to adapt the selective borohydride-metal hydrogenations
into isotopic labelling reactions.[36,37] In 1978,
Schwarzmann[37] used potassium [3H]borohydride in the
catalytic hydrogenation of unsaturated gangliosides and
Scheme 1. The synthesis of terminally unsaturated N-acyl-l-homo-
serine lactones 6a–d.
A method to label terminally unsaturated AHLs with
other sphingolipids. Potassium borotritide was dissolved in deuterium through catalytic reduction of the double bond
1 m NaOH solution in the presence of palladium(II) chlor- using sodium borohydride was performed in a modified
ide, which functions as a precatalyst. This method was also layer-by-layer system (see the Supporting Information). In
applied to the catalytic reduction of the keto group in this reaction, the sodium borohydride is hydrolysed by ace-
sphingosines using NaB[3H4] in a unique layer-by-layer sys- tic acid, and released hydrogen reduces palladium(II) acet-
tem.[38] However, to the best of our knowledge, no precise ate to the metallic palladium. The reaction is autocatalytic
deuterium labelling studies on this or related methods have because formed palladium nanoparticles support hydrolysis
been reported. Therefore, a detailed investigation of deuter- of sodium borohydride to generate hydrogen gas in situ,
ium and tritium labelling through the modified catalytic re- which, in turn, reduces palladium salt. The gas is further
duction of double bonds using sodium borodeuteride and utilised in the hydrogenation of the provided alkene. The
sodium borotritide is presented herein. Furthermore, this reaction was performed on four terminally unsaturated
methodology is applied to the isotopic labelling of N-acyl- AHL derivatives 6a–d with varying chain lengths
l-homoserine lactones. To confirm the labelled position, de- (Scheme 2).
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D. Jakubczyk, C. Merle, G. Brenner-Weiss, B. Luy, S. Bräse
FULL PAPER
palladium(II) acetate (one equivalent) did not have a sig-
nificant effect on the deuterium distribution. The length of
the acyl chain did not influence the reaction (Table 1, en-
tries 8–10). Further development of the method was carried
out on the AHL derivative with the shortest acyl chain 6a
(Scheme 2 and Table 1). The reaction failed when the sub-
strate and catalyst solutions were localized to the same
layer. Reducing the reaction time (from 24 to 16 h) under
the same conditions (0.2 equiv. of catalyst, 1 equiv. of
NaBD4, nondeuterated solvents) resulted in similar deuter-
ium distributions with nearly identical yields. The use of
deuterated solvents and excess sodium borohydride resulted
in the formation of heavier isomers (d3 and higher). The
ability to incorporate more than two deuterium atoms in
the labelled compound is an advantage of the method, par-
ticularly in the context of detecting the labels in a biological
system. However, from a chemical point of view, the label-
ling reaction is less selective. The reaction selectivity was
increased by eliminating the acidic deuterium atoms, which
are primarily derived from [D4]acetic acid. This change in
selectivity was achieved by diluting a solution of NaOD
in deuterated water with MeOD (to 0.33 m). Reducing the
amount of sodium borodeuteride to 2 equiv. also enhanced
the selectivity, yielding isotopologue-d2 in 54% deuteration
with only three more isomers (Table 1, entry 4). Total elimi-
nation of deuterated water resulted in a decreased deuter-
ium content. Moreover, acetic acid removal from the reac-
Scheme 2. Deuterium labelling of terminally unsaturated AHLs
6a–d through Pd(OAc)2-catalysed reduction of the double bond by
sodium borodeuteride (see Table 1). R = H or D.
The first reactions on substrates 6a–d were carried out
over 16–24 hours with 0.2 equivalents of palladium(II) acet-
ate and 1 equiv. of sodium borodeuteride. The first results
gave mainly monodeuterated isotopologues with the follow-
ing deuterium distribution: 32% d2, 54% d1, 14% d0.
Mono- and di-deuterated isomers were observed in nearly
equal ratios (20–30%) with very good yields (74–83%). Re-
versing the layer system (starting from a frozen sodium
borodeuteride solution) decreased the selectivity (six iso-
topologues were observed). The degree of deuteration in-
creased when deuterated reagents were used. However, be-
cause of H/D exchange, especially from [D4]AcOH or D2O,
more isomers were observed (Table 1, entries 2–3). An ex-
cess of sodium borohydride (2–3 equiv.) resulted in a higher
number of heavier isotopologues. The addition of excess
Table 1. Representative results of deuterium labelling of 7a–d and 9a–c through Pd(OAc)2 catalysed reduction of the double bond by
NaBD4.
Entry
n
R
Product
Reactions conditions
Time Yield Deuterium distribution[a] [%] of isomer-dn
[h]
[%]
d5
d4
d3
d2
d1
d0
Substrate
1
2
3
4
1
1
1
1
D
D
D
D
7a
7a
7a
7a
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH/H2O,
1 equiv. NaBD4, 1 m NaOHaq
0.2 equiv. Pd(OAc)2, THF, AcOH-d4, MeOD/D2O, 19
1 equiv. NaBD4, 1 m NaOD in D2O
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOD/D2O,
24
80
6
10
22
20
24
18
–
95
95
85
17
16
–
21
20
7
26
36
19
21
20
54
15
8
–
–
–
–
–
–
20
4 equiv. NaBD4, 1 m NaOD in D2O
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOD, 2 equiv. 4.5
NaBD4, 30% NaOD in D2O diluted in MeOD to
0.33 m
20
5
1
D
7a
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOD, 2 equiv.
NaBD4, 30% NaOD in D2O diluted in MeOD to
0.15 m
2
80
–
13
21
54
12
–
–
6
7
1
1
D
D
7a
7a
0.2 equiv. Pd/C in THF, AcOH; 2 equiv. NaBD4
1.5
80
0
–
–
–
–
–
–
13
–
16
–
11
–
60
–
0.2 equiv. Pd(PPh3)4, THF, AcOH, MeOH 2 equiv. 17
NaBD4, 30% NaOD in D2O diluted in MeOH to
0.33 m
8
3
4
6
–
–
D
D
D
H
D
7b
7c
7d
9a
9b
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH/H2O,
1 equiv. NaBD4, 1 m NaOHaq
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH/H2O,
1 equiv. NaBD4, 1 m NaOHaq
0.2 equiv. Pd(OAc)2, THF, AcOH-d4, 2 equiv.
NaBD4, 1 m NaOH in D2O
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH, 4 equiv. 13
22
22
22
83
74
94
77
–
–
3
–
–
6
18
23
24
–
35
36
33
–
27
30
20
–
14
11
8
–
–
–
–
–
9
–
10
11
12
12
–
–
NaBH4, 0.33 m NaOHaq
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH, 1 equiv. 16.5 58
NaBD4, 30% NaOD in D2O diluted in MeOH to
0.33 m
–
13
52
28
7
13
–
T
9c
0.2 equiv. Pd(OAc)2, THF, AcOH, MeOH 1 equiv.
18
77
–
–
84[b] 16[b]
–
–
–
NaBT4, 0.33 m NaOHaq
[a] Determined by ESI-TOF MS. [b] t3/t2 rather than d3/d2.
4
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Deuterium and Tritium Labelling of N-Acyl-l-Homoserine Lactones
tion medium caused the reaction to fail. This fact con- afforded a good yield (64%).[41] The addition of trimethyl
firmed the crucial role of acetic acid as an initiator through orthoformate enabled the efficient removal of water without
the hydrolysis of sodium borohydride. Considerably reduc- the use of a Dean–Stark trap.[42] The intermediate 8 was, in
ing the reaction time to 4.5 hours resulted in higher selectiv- turn, labelled with deuterium or tritium through catalytic
ity (Table 1, entry 4). Further time decreases (15 min) led to double bond reduction using sodium borodeuteride or so-
slightly lower degrees of deuteration and lower yields. The dium borotritide, respectively (Scheme 3 and Table 1). Al-
optimal reaction time was 2 hours. Furthermore, addition- kene 8 was reduced by nonlabelled sodium borohydride in
ally diluting sodium deuteroxide to 0.15 m afforded the 77% yield (Table 1, entry 11). Deuteration of compound 8
highest selectivity (Table 1, entry 5). However, the complete gave similar results to the deuteration of products 7a–d, and
removal of sodium hydroxide or sodium deuteroxide al- the best reduction result using sodium borodeuteride was
lowed only minimal substrate conversion. This confirmed obtained under similar conditions to those leading to prod-
that the NaOH (water/methanol) solution is a very conve- ucts 7a–d. Dilution of sodium deuteroxide with MeOH to
nient solvent system for sodium borohydride. To make so- 0.33 m allowed the desired double deuterated isomer to be
dium borodeuteride hydrolysis more effective, the acetic obtained in 52% yield (Table 1, entry 12). In all cases, the
acid was frozen as a separate layer. Unfortunately, this level of deuteration of the terminal carbon was found to be
change resulted in a less selective deuterium distribution. higher than for the penultimate carbon atom.
Additionally, sodium borohydride is also known to undergo
hydrolysis upon methanol treatment, which was tested on
substrate 6a, which led to incomplete conversion. The next
modification was tested by keeping sodium borohydride
with methanol in a separate flask. The generated gas was
then added to the second flask with a solution of Pd(OAc)2
through a Teflon pipe. After saturating the precatalyst with
deuterium gas, the substrate solution was added. The re-
sulting deuterium content and the overall yield of the reac-
tion were still lower when compared with the previous
modifications. The use of palladium(0) catalysts, such as
palladium on carbon (10 wt.-% loading, matrix carbon, dry
support) or tetrakis(triphenylphosphine)palladium(0), were
also unsuccessful. In the case of Pd/C, 60% of the substrate
remained in the reaction mixture. The products harboured
low deuterium content (Table 1, entry 6). This confirmed
the importance of the nanosized palladium metal.[24] In
summary, the optimal result was obtained by using
0.2 equiv. Pd(OAc)2, tetrahydrofuran (THF), AcOH,
MeOD, 2 equiv. NaBD4, 30% NaOD in D2O diluted in
Scheme 3. Reduction and deprotection reactions of N-[3-(1Ј, 3Ј-
dioxolane)-11-dodecenoyl]-l-homoserine lactone (8; see Table 1).
MeOD to 0.15 m for 2 hours (Table 1, entry 5). Notably, a
The tritium labelling of 8 was performed by using so-
standard acid-base workup led to very pure products, which
dium borotritide in 0.33 m NaOH solution; the reaction
did not require any further purification.
proceeded in very good yield (77%). However, the total
This uncommon double bond reduction using sodium
radioactivity of tritiated product was only 3.52 mCi (3.5%),
borodeuteride in the presence of palladium(II) acetate was
and the specific activity was 588.6 Ci/mol (Table 1, en-
applied to the labelling of the highly biologically active N-
try 13), which still requires optimisation. However, this re-
(3-oxododecanoyl)-l-homoserine lactone (1). The first two
duction reaction was especially useful for tritium labelling
steps in the synthesis of terminally unsaturated substrate
because of the ease of handling solid sodium borotritide
were carried out according to the previously published pro-
compared with tritium gas. In the last step the acetal groups
cedures,[4,13] and yields were satisfactory (55–60%). The
of all of deuterium-, tritium- and nonlabelled derivatives
first experimental reduction reactions were carried out on
9a–c were deprotected by perchloric acid, giving the corre-
N-(3-oxo-11-dodecenoyl)-l-homoserine lactone. Extending
sponding ketones in excellent yields of ca. 90%
the time of the reaction, use of deuterated reagents, an ex-
(Scheme 3).[43]
cess of sodium borodeuteride or temperature variations,
however, did not yield the desired product. This result could
be explained by the presence of the ketone group, which is
known to be reduced to a hydroxyl group in the presence
NMR Studies
of sodium borohydride. Notably, the potential byproduct
In isotopic labelling studies it is essential to confirm the
N-(3-hydroxydodecanoyl)-l-homoserine lactone was not degree and selectivity of labels within the desired molecule.
isolated from the mixture of products. Therefore, acetal pro- Two main methods, mass spectrometry and NMR spec-
tection of the ketone group was performed by using ethyl- troscopy, can be used for this characterisation. In the case
ene glycol and p-toluenesulfonic acid; the protection step of selectively labelled AHLs, mass spectrometry only pro-
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vides the overall mass of the molecule and cannot be used the typical aliphatic coupling (1JC,D = 19.2 Hz). The relative
to safely identify the deuterated position. We therefore re- yields of all other isotopologues were determined in a sim-
corded 1D 13C{1H} (i.e., a one-dimensional 13C spectrum ilar way by integrating the singlets of the 13C{1H, 2H} spec-
1
with H gated decoupling), 1D 13C{1H,2H} and 2D edited tra while using the 13C{1H} spectra for the identification of
HSQC[44] NMR spectra on the best resolved compound 7a the number of directly attached deuterium atoms. As was
for experimental proof of the deuteration reaction. By com- proven by comparing the integrals of the various aliphatic
paring the various carbon 1D NMR spectra, the 1JC,D cou- carbon atoms, the adjusted recovery delay of 8 s allowed
pling constants and the chemical shifts were used to deter- reliable integration with an accuracy of more than 99%.
mine the deuterium-modified carbon atoms. Only two sig-
For an independent verification of coupling constants
nals in the 13C{1H} experiment of 7a are significantly split and observed coupling patterns in the 1D spectra, 2D
due to deuteration effects, indicating that, indeed, selec- edited HSQC spectra[44] with broadband excitation,[45,46] in-
tively labelled compounds have been obtained. In Figure 2, version,[47,48] and refocusing pulses[49] were recorded to con-
two chemical shift regions (12.4–14.2 ppm and 21.4– firm the degree of deuteration for individual carbon atoms.
22.5 ppm) of 7a are depicted to demonstrate the influence Herein, the magnetisation evolution of a CH2D group acts
of the chemical environment and the degree of deuteration like a CH2 group, i.e., the edited HSQC results in negative
on the chemical shift and multiplicity. In the 1D absorptive signals for the CH2D group, while CH(D2) and
13C{1H,2H} doubly decoupled spectrum (black), each car- CH3 groups lead to positive absorptive signals. The corre-
bon of the particular isotopologue is visible as a singlet with sponding spectrum for 7a is shown in Figure S2 in the Sup-
its own chemical shift value resulting from isotopic shifts. porting Information.
The isotopologue-d2 as the main product of 7a is high-
lighted with slightly thicker lines to illustrate the interpret-
Conclusions
ation of the corresponding spectra. The superposition of
2
the 13C{1H, H} (black line) and the 13C{1H} (red line) 1D
The synthesis of deuterium and tritium labelled AHLs
through catalytic reduction of a double bond by sodium
borohydride, has been shown to be an efficient, mild and
facile labelling method. Moreover, to confirm the degree
and selectivity of the labelling, detailed 2D edited HSQC as
well as 1D 13C NMR experiments were performed. Such
tritium labelled molecules are very helpful tools for the in-
vestigation of biological mechanisms, such as the distribu-
tion of 3OC12 HSL 1 and its ability to cross cellular mem-
branes.
spectra shows the singlet vs. the deuterium triplet due to
Experimental Section
General: Solvents and chemicals used for reactions were purchased
from commercial suppliers. Solvents were dried under standard
conditions; chemicals were used without further purification. All
reactions were carried out under nitrogen in flame-dried glassware.
The evaporation of solvents and concentration of reaction mixtures
were performed in vacuo at 50 °C with a Heidolph Rotary Evapo-
rator, Laborota 4000. Thin-layer chromatography (TLC) was car-
ried out on silica gel plates (Kieselgel 60, Merck) with visualisation
by iodine vapour and the Seebach solution.[50] Normal-phase silica
gel (Silica gel 60, 230–400 mesh, Merck) was used for flash
chromatography. IR and Raman spectra were recorded with a
Bruker Vertex 80 FTIR spectrometer (Bruker Optik GmbH,
Ettlingen, Germany) with a single reflection “Golden Gate” dia-
mond ATR sampling unit (Specac, UK). 1H and 13C NMR spectra
were recorded with a Bruker-ACS-60, Ultrashield 500 Plus spec-
trometer. High-resolution mass spectra (HRMS) were determined
with a Finnigan MAT90. ESI-TOF mass spectra were recorded
with an ESI-TOF Mariner system (Applied Biosystems).
Figure 2. 13C{1H, 2H} (black) and 13C{1H} (red) spectra of a 90 μm
sample of 7a in CD2Cl2. The highlighted lines indicate the signal
of the main isotopologue-d2 with the corresponding 1JC,D coupling
in the 13C{1H} (red) spectrum. Altogether eight isotopologues can
be identified in the spectra with their corresponding relative yield
(see the Supporting Information). The relaxation delay was ad-
justed to 8 seconds to allow reliable integration of the carbon NMR
spectra.
Preparation of Acyl Chlorides. General Procedure I: To a solution
of 9-decenoic acid (1 equiv.) in hexane (15 mL per 1 mmol) at room
temperature, oxalyl chloride (2 equiv.) was added dropwise over
10 min, under a nitrogen atmosphere. The reaction temperature
was then increased to 40–45 °C, and the reaction was continued for
17 h. After this time, the solvent was evaporated and the crude
6
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Deuterium and Tritium Labelling of N-Acyl-l-Homoserine Lactones
product was dried by a high vacuum pump to give 9-decenoyl
chloride, which was used in the next step without further purifica-
tion.
sulfate solution and saturated sodium chloride solution. The water
solution was washed one more time with ethyl acetate. After drying
the organic solution over anhydrous sodium sulfate, the solvent was
evaporated in vacuo. The crude residue was purified by flash col-
umn chromatography (hexane/ethyl acetate, 2:3) to give the desired
product as a white solid.
9-Decenoyl Chloride (4d): Yield 257 mg (68%); Rf = 0.76 (hexane/
ethyl acetate, 5:1). 1H NMR (500 MHz, CDCl3): δ = 1.25–1.44 (m,
8 H, 4ϫCH2), 1.65–1.69 (m, 2 H, CH2CH2COCl), 1.71–1.76 (m,
2 H, CH2=CHCH2), 1.97–2.26 (m, 2 H, CH2COCl), 4.94–5.03 (dd,
N-(Pentanoyl-d2)-L-homoserine Lactone (7a): Yield 16 mg (86%); Rf
1J = 17.2, 2J = 2.1 Hz, 2 H, CH2=CHCH2), 5.77–5.86 (m, 1 H, = 0.2 (hexane/ethyl acetate, 2:3). H NMR (500 MHz, CDCl3): δ =
1
CH2=CHCH2) ppm. 13C NMR (125 MHz, CDCl3): δ = 24.2 (s, C- 0.80–1.10 (m, 1.8 H, CDH2), 1.22–1.39 (m, 1 H, CDHCDH2),
1
2
3), 28.3 (s, C-4), 28.8 (m, C-5), 29.0 (m, C-6), 29.5 (m, C-7, CH2),
33.7 (m, C-8), 47.1 (s, C-2), 114.2 (s, C-10), 138.9 (s, C-9), 169.6 (s,
C-1) ppm. MS (ESI-TOF): m/z (%) = 211 (100) [M+ + Na].
1.50–1.69 (m, 2 H, CH2CH2CO), 2.13 (dddd, J = 20.3, J = 11.5,
3J = 8.8, 4J = 5.0 Hz, 1 H, 3α-H), 2.18–2.38 (m, 2 H, COCH2),
1
2
3
2.86 (ddd, J = 13.9, J = 8.6, J = 5.8 Hz, 1 H, 3β-H), 4.28 (ddd,
2
3
1
2
1J = 11.3, J = 9.3, J = 5.9 Hz, 1 H, 4α-H), 4.47 (td, J = 9.0, J
Preparation of Terminally Unsaturated N-Acyl-L-homoserine Lact-
1
2
3
= 1.0 Hz, 1 H, 4β-H), 4.55 (ddd, J = 11.6, J = 8.6, J = 5.8 Hz,
ones. General Procedure II: To a stirred solution of l-homoserine
lactone hydrobromide (5) (1 equiv.) in CH2Cl2 (20 mL per 1 mmol)
at 0 °C and triethylamine (2.4 equiv.) was added. The mixture was
stirred at 0 °C for 0.5 h then the alkenoyl chloride (1 equiv.) was
added dropwise over 5 min. The reaction mixture was allowed to
come to room temperature and was stirred for 4 h. The solution
was evaporated to dryness and the residue was redissolved in ethyl
acetate and sequentially washed with 1 m sodium hydrogen carbon-
ate solution, 1 m potassium hydrogen sulfate solution and saturated
sodium chloride solution. After drying over anhydrous sodium
sulfate, the solvent was removed by rotary evaporation to leave the
crude product, which was subsequently purified by column
chromatography on silica with ethyl acetate.
1 H, 2-H), 6.00–6.19 (m, 1 H, NH) ppm. 13C NMR (125 MHz,
1
1
CDCl3): δ = 13.5 (quint, J = 19.1 Hz, CЈ-5, CHD2), 22.1 (t, J =
19.1 Hz, CЈ-4, CHD), 27.4, 30.7, 35.9, 49.3, 66.1, 173.7, 175.5 ppm.
MS (ESI-TOF): m/z (%) = 188 (100) [M+ + H], 189 (39), 190 (24),
187 (22); Deuterium distribution: 13% d4, 21% d3, 54% d2, 12%
d1.
N-(1-Oxoheptanoyl-d2)-L-homoserine Lactone (7b): Yield 36 mg
1
(84%); Rf = 0.38 (hexane/ethyl acetate, 1:4). H NMR (500 MHz,
CDCl3): δ = 0.70–0.89 (m, 2 H, CDH2), 1.19–1.40 (m, 5.5 H,
CDH2CDH + 2ϫCH2), 1.51–1.69 (m, 2 H, CH2CH2CO), 2.17
1
(dddd, J = 20.4, 2J = 11.5, 3J = 8.8, 4J = 4.5 Hz, 1 H, 3α-H), 2.25
1
(m, 2 H, COCH2), 2.86 (ddd, J = 13.9, 2J = 8.5, 3J = 5.8 Hz, 1
1
2
3
H, 3β-H), 4.30 (ddd, J = 11.3, J = 9.3, J = 5.9 Hz, 1 H, 4α-H),
N-(1-Oxo-9-decenoyl)-L-homoserine Lactone (6d): Yield 288 mg
1
2
1
4.50 (td, J = 8.9, J = 0.8 Hz, 1 H, 4β-H), 4.60 (ddd, J = 11.6,
2J = 8.6, 3J = 6.3 Hz, 1 H, 2-H), 6.25 (d, 1J = 4.4 Hz, 1 H,
NH) ppm. 13C NMR (125 MHz, CDCl3): δ = 13.8 (quint, 1J =
1
(90%); Rf = 0.49 (hexane/ethyl acetate, 2:3). H NMR (500 MHz,
CDCl3): δ = 1.20–1.39 [m, 8 H, COCH2CH2(CH2)4], 1.50–1.1.68
(m, 2 H, COCH2CH2), 2.06 (dd, 1J = 13.9, 2J = 7.3 Hz, 2 H,
1
19.8 Hz, CЈ-7, CHD2), 22.2 (t, J = 19.1 Hz, CЈ-6, CHD), 25.4 (s,
1
2
3
2
COCH2), 2.18 (dddd, J = 18.4, J = 11.5, J = 8.8, J = 4.8 Hz, 1
CЈ-5, CH2), 30.3 (s, CЈ-4, CH2), 31.5 (s, CЈ-3, CH2), 33.9 (s, C-3,
CH2), 36.7 (s, CЈ-2, CH2), 49.2 (s, C-2, CH2), 66.2 (s, C-4, CH2),
174.0 (s, CЈ-1, C=O), 175.7 (s, C-1, C=O) ppm. MS (ESI-TOF):
m/z (%) = 216 (100) [M+ + H], 215 (79), 217 (53), 214 (41), 218
(15). Deuterium distribution: 6% d4, 18% d3, 35% d2, 27% d1, 14%
d0.
H, 3α-H), 2.27 (dt, 1J = 7.3, 2J = 1.4 Hz, 2 H, CH2=CHCH2), 2.91
(ddd, J = 13.9, J = 8.1, J = 5.8 Hz, 1 H, 3β-H), 4.3 (ddd, J =
11.3, 2J = 9.4, 3J = 5.9 Hz, 1 H, 4α-H), 4.49 (dt, 1J = 9.2, 2J =
0.9 Hz, 1 H, 4β-H), 4.59 (ddd, J = 11.6, J = 8.6, J = 5.8 Hz, 1
H, 2-H), 4.80–4.98 (m, 2 H, CH2=CH2CH2), 5.70–5.89 (m, 1 H,
CH2=CHCH2), 6.07 (d, 1J = 4.5 Hz, 1 H, NH) ppm. 13C NMR
(125 MHz, CDCl3): δ = 25.4 (s, CЈ-3, CH2), 28.8 (m, CЈ-4, CH2),
1
2
3
1
1
2
3
N-(1-Oxooctanoyl-d2)-L-homoserine Lactone (7c): Yield 41 mg
1
28.9 (m, CЈ-5, CH2), 29.3 (m, CЈ-4, CH2), 30.6 (s, C-3, CH2), 33.7 (74%); Rf = 0.49 (hexane/ethyl acetate, 1:4). H NMR (500 MHz,
(s, CЈ-8, CH2), 36.2 (s, CЈ-2, CH2), 49.2 (s, C-2, CH2), 66.2 (s, C- CDCl3): δ = 0.79–0.96 (m, 2 H, CDH2), 1.20–1.39 (m, 7.5 H,
4, CH2), 114.2 (s, CЈ-10, CH2), 139.1 (s, CЈ-9,CH), 173.8 (s, CЈ-1, CDH2CDH + 3ϫCH2), 1.40–1.58 (m, 2 H, CH2CH2CO), 2.19
C=O), 175.7 (s, C-1, C=O) ppm. MS (ESI-TOF): m/z (%) = 254
(dddd, 1J = 20.4, 2J = 11.5, 3J = 8.9, 4J = 4.4 Hz, 1 H, 3α-H), 2.15–
3
(100) [M+ + H].
2.31 (m, 2 H, COCH2), 2.88 (ddd, 1J = 13.3, 2J = 8.6, J = 5.9 Hz,
1
2
3
1 H, 3β-H), 4.32 (ddd, J = 11.3, J = 9.3, J = 5.9 Hz, 1 H, 4α-
Preparation of Deuterium and Tritium Labelled N-Acyl-L-homo-
H), 4.50 (td, 1J = 9.1, 2J = 1.0 Hz, 1 H, 4β-H), 4.62 (ddd, 1J =
11.5, J = 8.5, J = 6.0 Hz, 1 H, 2-H), 6.27 (d, J = 4.8 Hz, 1 H,
NH) ppm. 13C NMR (125 MHz, CDCl3): δ = 13.9 (quint, 1J =
serine Lactones through Catalytic Reduction. General Procedure III:
1st Layer: N-(1-Oxo-9-decenoyl)-l-homoserine lactone (6d)
(1 equiv.) was dissolved in THF (2 mL per 0.1 mmol) and frozen
in liquid nitrogen. 2nd Layer: Palladium(II) acetate solution
(0.2 equiv.) in THF (2 mL per 0.02 mmol), acetic acid (500 μL;
8.2 mmol per 0.02 mmol) and methanol (or [D4]methanol)
(1.25 mL per 0.1 mmol) was added and frozen. 3rd Layer: Further
THF (1.5 mL per 0.1 mmol) was added and again frozen. 4th Layer:
Subsequently, sodium borohydride (1–4 equiv.; sodium borodeuter-
ide/sodium borotritide) was dissolved in 0.15–0.33 m water solution
of sodium hydroxide or sodium hydroxide (or sodium deuteroxide)
solution in H2O or in [D4]methanol (1 mL per 0.05 mmol) and
closed tightly in the reaction vessel. The reaction mixture was al-
lowed to come to the room temperature and agitated for 1–16 h.
After that time the mixture was filtered from black palladium resi-
due and the solvent was removed by rotary evaporation. The resi-
due was redissolved in ethyl acetate and washed sequentially with
1 m sodium hydrogen carbonate solution, 1 m potassium hydrogen
2
3
1
1
20.6 Hz, CЈ-8, CHD2), 22.6 (t, J = 19.3 Hz, CЈ-7, CHD), 24.2 (s,
CЈ-6, CH2), 28.9 (m, CЈ-5, CH2), 30.3 (s, CЈ-4, CH2), 31.0 (s, CЈ-3,
CH2), 33.9 (s, C-3, CH2), 36.2 (s, CЈ-2, CH2), 49.3 (s, C-2, CH2),
66.2 (s, C-4, CH2), 174.0 (s, CЈ-1, C=O), 175.7 (s, C-1, C=O) ppm.
MS (ESI-TOF): m/z (%) = 230 (100) [M+ + H], 229 (83), 231 (62),
228 (31). Deuterium distribution: 23% d3, 36% d2, 30% d1, 11%
d0.
N-(1-Oxodecanoyl-d2)-L-homoserine Lactone (7d): Yield 17 mg
1
(94%); Rf = 0.59 (hexane/ethyl acetate, 1:4). H NMR (500 MHz,
CDCl3): δ = 0.87 (m, 2.14 H, CDH2), 1.15–1.35 [m, 11 H,
CDH2CDH(CH2)5], 1.50–1.71 (m, 2 H, CH2CH2CO), 2.12 (dddd,
2
3
4
1J = 20.4, J = 11.5, J = 8.9, J = 5.5 Hz, 1 H, 3α-H), 2.10–2.21
(m, 2 H, COCH2), 2.86 (ddd, J = 14.0, 2J = 8.6, 3J = 5.8 Hz, 1
H, 3β-H), 4.29 (ddd, J = 11.3, J = 9.3, J = 5.8 Hz, 1 H, 4α-H),
4.47 (td, J = 8.9, J = 0.9 Hz, 1 H, 4β-H), 4.56 (ddd, J = 11.6,
1
1
2
3
1
2
1
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7

Job/Unit: O30084
/KAP1
Date: 05-07-13 10:51:48
Pages: 9
D. Jakubczyk, C. Merle, G. Brenner-Weiss, B. Luy, S. Bräse
FULL PAPER
2J = 8.6, 3J = 5.8 Hz, 1 H, 2-H), 6.07 (d, 1J = 4.6 Hz, 1 H, (hexane/ethyl acetate, 1:4). MS (ESI-TOF): m/z (%) = 304 (100)
NH) ppm. 13C NMR (125 MHz, CDCl3): δ = 13.8 (quint, 1J = [M+ + H], 302 (19), 322 (100) [M+ + H2O], 320 (19). Tritium distri-
19.2 Hz, CЈ-10, CHD2), 22.5 (t, 1J = 19.8 Hz, CЈ-9, CHD), 25.4,
29.0, 29.2, 29.4, 30.3, 30.7, 34.2, 36.2, 49.3, 66.1, 173.9, 175.6 ppm.
MS (ESI-TOF): m/z (%) = 280 (100) [M+ + Na], 281 (73), 279 (60),
282 (37), 278 (25), 283 (10). Deuterium distribution: 3% d5, 12%
d4,24% d3, 33% d2, 20% d1, 8% d0.
bution: 84% t3, 16% t2.
Deuteration Analysis by NMR Spectroscopy: 8192 data points were
acquired corresponding to an acquisition time of 390 ms. The 1D
spectra were recorded with 3072 scans and 4 dummy scans. 1H-
WALTZ65 decoupling with an rf-amplitude corresponding to a 90°
pulse of 140 μs was irradiated during acquisition. For 13C{1H,2H}
spectra, 2H-GARP decoupling with an rf-amplitude corresponding
to a 90° pulse of 400 μs was applied in addition. All spectra were
processed with zero filling up to 16384 points and with an ex-
ponential apodization function corresponding to a line broadening
of 1 Hz. The baseline-separated signals were integrated by selecting
the signals manually (see Figure 2). 2D edited HSQC experiments
were acquired with 2048 times 2048 data points corresponding to
acquisition times of 285 ms and 170 ms.[1] The spectra were re-
corded with 4 scans, 16 dummy scans and a relaxation delay of 2
seconds. 1H-GARP decoupling with an rf-amplitude corresponding
to a 90° pulse of 70 μs was irradiated during acquisition. The spec-
tra were processed with zero filling to 4096 times 4096 points and
apodized using a 90° phase-shifted quadratic sine function in the
indirect and an exponential function with additional line broaden-
ing of 1 Hz in the direct dimension.
N-[3-(1Ј,3Ј-Dioxolane)dodecanoyl-d2]-L-homoserine Lactone (9b):
1
Yield 19 mg (58%); Rf = 0.57 (hexane/ethyl acetate, 1:3). H NMR
(500 MHz, CDCl3): δ = 0.75–0.94 (m, 2 H, CDH2), 1.15–1.35 (m,
13 H, CDH2CDH + 6ϫCH2), 1.56–1.77 [m, 2 H, CDH(CH2)6-
1
2
3
4
CH2], 2.14 (dddd, J = 20.3, J = 11.4, J = 8.8, J = 4.8 Hz, 1 H,
1
2
3
3α-H), 2.65 (s, 2 H, COCH2), 2.80 (ddd, J = 14.3, J = 8.6, J =
5.9 Hz, 1 H, 3β-H), 4.01–4.19 (m, 4 H, -OCH2CH2O-), 4.27 (ddd,
1J = 11.2, 2J = 9.3, 3J = 5.9 Hz, 1 H, 4α-H), 4.47 (dt, 1J = 9.1,
1
2
2
1.1 Hz, 1 H, 4β-H), 4.58 (ddd, J = 11.6, J = 8.6, J = 6.3 Hz, 1
H, 2-H), 7.01 (d, 1J = 6.1 Hz, 1 H, NH) ppm. 13C NMR (125 MHz,
1
1
CDCl3): δ = 13.9 (quint, J = 19.2 Hz, CЈ-12, CH2D), 22.5 (t, J =
19.1 Hz, CЈ-11, CHD), 23.7 (s, CЈ-10, CH2), 28.8 (m, CЈ-9, CH2),
29.1 (m, CЈ-8, CH2), 29.3 (m, CЈ-7, CH2), 29.9 (m, CЈ-6, CH2), 30.4
(s, C-3, CH2), 37.5 (s, CЈ-4, CH2), 44.2 (s, CЈ-2, CH2), 49.2 (s, C-
2, CH2), 64.9 (s, -OCCH2CH2O-, CH2), 65.1 (s, -OCCH2CH2O-,
CH2), 65.9 (s, C-4, CH2), 109.6 (s, 1 C, CЈ-3), 169.8 (s, CЈ-1, C=O),
175.2 (s, C-1, C=O) ppm. MS (ESI-TOF): m/z (%) = 344 (100) [M+
+ H], 343 (54), 345 (25), 342 (14). Deuterium distribution: 13% d3,
52% d2, 28% d1, 7% d0.
Supporting Information (see footnote on the first page of this arti-
cle): Description of the layer-by-layer method and detailed 2D ed-
ited HSQC as well as 1D 13C NMR spectra.
Deprotection of N-[3-(1Ј,3Ј-Dioxolane)alkanoyl]-L-homoserine Lact-
ones. General Procedure IV: To an ice-cooled solution of deuterium
or tritium labelled 9 (1 equiv.) in CH2Cl2 (15 mL per 0.05 mmol),
60% perchloric acid solution (250 μL per 0.05 mmol) was added.
The mixture was stirred at 0 °C for 0.5 h and then at room tempera-
ture for 2 h. The solution was then evaporated to dryness, and the
residue was redissolved in ethyl acetate. The ethyl acetate solution
was washed with cold water and brine. After drying over anhydrous
sodium sulfate, the solvent was evaporated.
Acknowledgments
Financial support from the Helmholtz program Biointerfaces is
gratefully acknowledged. The authors thank Silvia Diabaté from
the Karlsruhe Institute of Technology (KIT). The authors thank
the Institute of Toxicology and Genetics (ITG) for support in work
with radioactive substances. We are very thankful to Horst Geckeis,
Clemens Walther and Peter Kaden from the KIT, Institute for Nu-
clear Waste Disposal (INE) for help with analysis of tritium lab-
elled compounds. Weslee Glenn from Massachusetts Institute of
Technology (MIT), USA / John Innes Centre, Biological Chemistry
Department, Norwich, UK is thanked for correction of the manu-
script.
N-(3-Oxododecanoyl-d2)-L-homoserine Lactone (10b): Yield 13 mg
1
(90%); Rf = 0.54 (hexane/ethyl acetate, 1:3). H NMR (500 MHz,
CDCl3): δ = 0.72–0.97 (m, 2 H, CDH2), 1.16–1.31 [m, 11 H,
CDH2CDH(CH2)5], 1.49–1.69 (m, 2 H, COCH2CH2), 2.24 (dddd,
1J = 20.3, 2J = 12.4, 3J = 8.9 Hz, 4J = 1 H, 3α-H), 2.53 (t, 1J =
1
2
3
7.4 Hz, 2 H, COCH2), 2.76 (ddd, J = 14.6, J = 8.7, J = 6.0 Hz,
1
2
1 H, 3β-H), 3.47 (s, 2 H, COCH2CO), 4.28 (ddd, J = 11.0, J =
9.3, J = 6.0 Hz, 1 H, 4α-H), 4.48 (dt, J = 9.1, J = 1.3 Hz, 1 H,
[1] N. Amara, R. Mashiach, R. D. Amar, P. Krief, S. A. H. Spieser,
M. J. Bottomley, A. Aharoni, M. M. Meijler, J. Am. Chem. Soc.
2009, 131, 10610–10619.
[2] M. Boyer, F. Wisniewski-Dye, FEMS Microbiol. Ecol. 2009, 70,
1–19.
[3] N. Nanting, L. Minyong, J. Wang, B. Wang, Med. Res. Rev.
2009, 29, 65–124.
[4] S. R. Chhabra, C. Harty, D. S. Hooi, M. Daykin, P. Williams,
G. Telford, D. I. Pritchard, B. W. Bycroft, J. Med. Chem. 2003,
46, 97–104.
3
1
2
1
2
3
4β-H), 4.59 (ddd, J = 11.5, J = 8.7, J = 6.7 Hz, 1 H, 2-H), 7.68
(d, J = 5.7 Hz, 1 H, NH) ppm. 13C NMR (125 MHz, CDCl3): δ
1
1
1
= 13.8 (quint, J = 19.3 Hz, CЈ-12, CH2D), 22.5 (t, J = 19.1 Hz,
CЈ-11, CHD), 23.4 (s, CЈ-10, CH2), 28.9 (m, CЈ-9, CH2), 29.1 (m,
CЈ-8, CH2), 29.4 (m, CЈ-7, CH2), 29.9 (m, CЈ-6, CH2), 30.1 (s, C-
3, CH2), 43.9 (s, CЈ-4, CH2), 47.9 (s, CЈ-2, CH2), 49.0 (s, C-2, CH2),
65.8 (s, C-4, CH2), 166.3 (s, CЈ-1, C=O), 174.7 (s, C-1, C=O), 206.7
(s, CЈ-3, C=O) ppm. MS (ESI-TOF): m/z (%) = 322 (100) [M+
+
[5] J. A. Olsen, R. Severinsen, T. B. Rasmussen, M. Hentzer, M.
Givskov, J. Nielsen, Bioorg. Med. Chem. Lett. 2002, 12, 325–
328.
Na], 321 (54), 323 (25), 320 (14). Deuterium distribution: 3% d5,
12% d4,24% d3, 33% d2, 20% d1, 8% d0.
N-(3-Oxododecanoyl-[3H3])-
L-homoserine Lactone (10c): This com-
[6] J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 1999,
284, 1318–1320.
pound was obtained in two steps according to General Procedures
III and IV from 8 (2.6 mg, 7.63 μmol), palladium(II) acetate
(1.7 mg, 7.63 μmol), and acetic acid (50 μL, 0.82 mmol) in a mix-
ture of THF (477 μL) and methanol (99 μL), followed by sodium
borohydride-[3H] (0.288 mg, 7.63 μmol, 100 mCi, 3.7 GBq), which
was dissolved in 0.33 m sodium hydroxide solution (160 μL). Chem-
ical yield 1.8 mg (77%); radioactivity: 3.52 mCi (130.45 MBq); Spe-
cific activity: 588.6 Ci/mol; radiochemical yield 3.5%. Rf = 0.50
[7] J. B. Lyczak, C. L. Cannon, G. B. Pier, Clin. Microbiol. Rev.
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[8] M. Cooley, S. R. Chhabra, P. Williams, Chem. Biol. 2008, 15,
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[9] D. T. Hughes, V. Sperandio, Nat. Rev. Microbiol. 2008, 6, 111–
120.
[10] K. P. Rumbaugh, J. A. Griswold, A. N. Hamood, Microbes In-
fect. 2000, 2, 1721–1731.
8
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

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/KAP1
Date: 05-07-13 10:51:48
Pages: 9
Deuterium and Tritium Labelling of N-Acyl-l-Homoserine Lactones
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Received: January 16, 2013
Published Online: ᭿
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