Synthesis of Tritium Labelled and Photoactivatable N-Acyl- L -homoserine Lactones: Inter-Kingdom Signalling Molecules

Article abstract of DOI:10.1002/ejoc.201300800

N-Acyl-L-homoserine lactones (AHLs) are signal molecules that are synthesized in nature by Gram-negative bacteria. They allow communication between bacteria (quorum sensing) and between microorganisms and their eukaryotic host cells (inter-kingdom signalling). However, very little is known about the mechanisms of the latter system. Therefore, tritium labelled photoactivatable AHLs were synthesized to identify specific receptors in immune cells (e.g., human polymorphonuclear neutrophils, PMN) that bind to AHL. A photoaffinity label - a diazirine group - was chosen as the smallest possible photoactivatable group that can be activated by light irradiation. The resulting highly active carbene will bind covalently to the closest chemical structure in the pocket of the receptor. The diazirine label was introduced into the AHL molecule according to a literature method. To isolate the labelled receptor from the cellular protein moiety, the AHL was additionally labelled with tritium. During the development of an effective isotopic labelling method, a simple route to hydrogen isotope incorporation into the AHLs was proposed and explored in detail. According to the new protocol, the photoactivatable diazirine-AHL was labelled with deuterium and tritium by a postsynthetic catalytic exchange of the hydrogen with its isotopes, using deuterium or tritium labelled water along with catalytic amounts of metal salts under mild basic conditions.

Full text of DOI:10.1002/ejoc.201300800

FULL PAPER
DOI: 10.1002/ejoc.201300800
Synthesis of Tritium Labelled and Photoactivatable N-Acyl- -homoserine
L
Lactones: Inter-Kingdom Signalling Molecules
Dorota Jakubczyk,[a,b][‡] Gerald Brenner-Weiss,[b] and Stefan Bräse*[a,c]
Keywords: Synthetic methods / Isotopic labeling / Biosensors / Photochemistry / Molecular modeling
N-Acyl-
L
-homoserine lactones (AHLs) are signal molecules
pocket of the receptor. The diazirine label was introduced
into the AHL molecule according to a literature method. To
isolate the labelled receptor from the cellular protein moiety,
the AHL was additionally labelled with tritium. During the
development of an effective isotopic labelling method, a sim-
ple route to hydrogen isotope incorporation into the AHLs
was proposed and explored in detail. According to the new
protocol, the photoactivatable diazirine-AHL was labelled
with deuterium and tritium by a postsynthetic catalytic ex-
change of the hydrogen with its isotopes, using deuterium or
tritium labelled water along with catalytic amounts of metal
salts under mild basic conditions.
that are synthesized in nature by Gram-negative bacteria.
They allow communication between bacteria (quorum sens-
ing) and between microorganisms and their eukaryotic host
cells (inter-kingdom signalling). However, very little is
known about the mechanisms of the latter system. Therefore,
tritium labelled photoactivatable AHLs were synthesized to
identify specific receptors in immune cells (e.g., human poly-
morphonuclear neutrophils, PMN) that bind to AHL. A pho-
toaffinity label – a diazirine group – was chosen as the small-
est possible photoactivatable group that can be activated by
light irradiation. The resulting highly active carbene will
bind covalently to the closest chemical structure in the
defence of the host.[7,10] The host defence against bacteria
or bacterial biofilms relies on phagocytic cells polymorpho-
nuclear neutrophils (PMN) and their capacity to infiltrate
into infected sites to recognize and eliminate pathogens.[11]
However, very little is known about the detailed mechanism
of the interaction between AHL and host cells.
Introduction
N-Acyl-l-homoserine lactones (AHLs) are synthesized in
nature by bacteria such as Pseudomonas aeruginosa and me-
diate bacterial cell-to-cell communication (quorum sens-
ing).[1,2] Quorum-sensing molecules and their role in bacte-
rial communication have been extensively studied[3–7] in the
context of biofilm formation, which causes chronic, de-
structive infections. Recently, several studies have shown
that signalling by these molecules is not restricted to bacte-
ria, but that they also interact with eukaryotic cells in a
phenomenon termed “inter-kingdom signalling”.[8] N-(3-
Figure 1. Structure of the N-(3-oxododecanoyl)-l-homoserine lac-
tone (3OC12-HSL).
Oxododecanoyl)-l-homoserine lactone
1 (3OC12-HSL,
Figure 1) is the most biologically active derivative in this
class.[9] In contrast to the adverse effects reported in litera-
ture, 3OC12-HSL might also support the immune system
Isotope-labelled compounds have several important ap-
plications. Compounds labelled with hydrogen isotopes
have application in mechanistic studies in organic chemis-
try,[12] as well as in many other branches of science such as
in the investigation of models of biological systems[13,14] or
metabolism and in metabolism-mediated toxicity studies.[15]
However, most labelling methods require the use of hydro-
[a] Karlsruhe Institute of Technology, Institute of Organic
Chemistry,
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
E-mail: braese@kit.edu
http://www.ioc.kit.edu/braese/english/index.php
[b] Karlsruhe Institute of Technology, Institute of Functional gen gas, expensive metal-based catalysts, aggressive rea-
gents, higher temperatures, or microwave irradiation.[16]
Interfaces,
Hermann von Helmholtz-Platz 1,
Therefore, new, mild postsynthetic labelling strategies are
still strongly desired.
76344 Eggenstein-Leopoldshafen, Germany
[c] Karlsruhe Institute of Technology, Institute of Toxicology and
Genetics,
Affinity labelling is a biochemical technique that is used
for the investigation of structural and functional properties
of biological systems. The method involves labelling of an
active site of proteins (e.g., receptors) by a small molecule
bearing a group that is capable of specifically binding to
Eggenstein-Leopoldshafen, Germany
[‡] 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.201300800.
592
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 592–597

Tritium Labelled N-Acyl-l-homoserine Lactones
the active site, and a second group that is chemically reac- method demonstrates a straightforward and very target-ori-
tive.[17–19] One of the most important variants of this ented approach to the bi-labelling of a highly biologically
method is photoaffinity labelling. In this approach, a func- active molecule, 3OC12-HSL.
tional group that is chemically unreactive in the dark is con-
verted into a reactive intermediate upon photolysis that
binds irreversibly to the biological receptor.[20] Photoactiv-
atable reagents must possess a photoactive group and must
Results and Discussion
be easily detectable by virtue of having either a radioactive
The first step of the synthesis of N-(3-oxo-5-diazirinedo-
isotope or a fluorophore moiety. However, the latter usually decanoyl)-l-homoserine lactone (9) was based on the syn-
contain several combined aromatic groups, which change thesis of β-ketodecanoic acid (6). For this purpose, two
the structure of the ligand dramatically. Recent studies un- methods were tested (Scheme 1). In the first, bis(trimethylsi-
covered a drastic difference in activity between the alkynyl- lyl) malonate (BSM, 2) was acylated with octanoyl chloride
and azido-tagged 3OC12-HSLs and their unmodified ana- (3) by using a mild base such as triethylamine and lithium
logues.[2,21] Therefore, the most convenient label is the ra- bromide, in good yield (52%).[28] Complexation of the lith-
dioactive isotope, particularly tritium. Importantly, this ra- ium cation by BSM 2 increased the acidity of the α-carbon
dioactive isotope of hydrogen does not change the structure protons thus making them easier removed. As a conse-
of the ligand, which preserves its specificity towards a target quence, the use of strong bases such as metal alkoxides,
receptor. Moreover, the photoactivatable reagents must be metal amides, or alkyllithium reagents was avoided.[29]
bifunctional, i.e., must be linked reversibly to the biological
In the second method, a two-step synthesis was started
ligand (for photoaffinity labelling) or to the biological mac- from acylation of methyl acetate 4 by octanoyl chloride (3)
romolecule such as nucleic acids or lipids (for cross-link- in the presence of lithium diisopropylamide (LDA), with
ing), before photolysis. After activation by light, the photo- very good yield (Scheme 1).[30–32] The obtained methyl 3-
lytic intermediate reacts covalently within the site before it oxodecanoate (5) was further hydrolysed under strong basic
can dissociate.[22] Several photoactivatable groups are conditions. However, even the use of a strong base such as
known, including azido, diazo, and azo groups, diazonium lithium hydroxide and heating under reflux for two days,
ions, benzophenone moieties or diazirines. The latter yielded only 13% of the desired product 6. Thus, the first
groups are found to be the most convenient because of their method was chosen as a more efficient and convenient ap-
chemical stability, selective photolytic activity, and small proach.
size.[2,23–27]
The diazirine group was chosen as the most convenient
Our strategy entailed the design and synthesis of a photoactivatable agent. Thus, 3-diazirine-decanoic acid (7)
3OC12-HSL derivative that contained a photoactivatable was prepared according to a procedure described by Husain
group for affinity capture of the receptor and an additional et al.[1] β-Ketodecanoic acid (6) was treated with hydroxyl-
radioactive label that can be used to isolate and identify amine-O-sulfonic acid in liquid ammonia to give an azirid-
the captured receptor from the cellular protein moiety. Our ine intermediate. Oxidation with iodine resulted in the for-
Scheme 1. The synthesis of β-ketodecanoic acid (6).
Scheme 2. Formation of 3-diazirinedecanoic acid (7).
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593

D. Jakubczyk, G. Brenner-Weiss, S. Bräse
FULL PAPER
mation of diazirine 7 in 30% yield (Scheme 2).[1] 3-Diazir- increased by extension of the reaction time (See Scheme 5
inedecanoic acid (7) was subsequently converted into tert- and Table 1, entry 1).
butyl ester 8 with 20% yield (Scheme 3).[1,33]
The second approach relied on the application of the
The ester group of compound 8 was deprotected with phase-transfer catalyst triethylbenzylammonium chloride
trifluoroacetic acid (TFA) and condensed with l-homoser- (TEBA) in a solvent system consisting of a solution of 5%
ine lactone in an EDC-mediated reaction. This gave the de- sodium deuterioxide and ethyl acetate (Table 1).[34] How-
sired N-(3-oxo-5-diazirinedodecanoyl)-l-homoserine lact- ever, even after two days reaction time the deuterium con-
one (9) with 25% yield (Scheme 4).
Deuterium and tritium labelling of 9 requires mild reac- (PTC) conditions were not efficient in this system.
tion conditions. A strong base, for instance, can cleave the Another concept of deuterium labelling of these mo-
tent was not higher than 51%, thus, phase-transfer catalysis
lactone moiety, and elevated temperature can lead to lecules was inspired by previously demonstrated activation
premature decomposition of the diazirine group. Thus, the of the acidic protons in malonic esters by metal salts.[28]
first method tested in this work was based on a postsyn- Metal cations are chelated by the two carbonyl groups and
thetic hydrogen–deuterium(tritium) exchange by deuterated the acidity of the α-protons is enhanced. Tritium labelling
(tritiated) water under mild conditions (mild base, room in this position is beneficial for the stability of the final
temperature). Initial experimental exchange reactions were product because the tritium–carbon bond is much stronger
carried out with lactone 1 by using triethylamine and deu- than a protium–carbon bond. Thus, application of lithium
terium oxide. This reaction yielded only 31% of the mono- bromide to the labelling of substrate 1 increased the deuter-
deuterated isomer. The degree of deuteration was slightly ium content up to 56%. The deuteration degree was still
Scheme 3. Synthesis of tert-butyl 3-oxo-5-diazirinedodecanoate (8); CDI = 1,1Ј carbonyldiimidazole.
Scheme 4. Synthesis of N-(3-oxo-5-diazirinedodecanoyl)-l-homoserine lactone (9).
Scheme 5. Deuterium and tritium labelling of AHLs through exchange reactions.
Table 1. Deuterium and tritium labelling of 1 and 9.
Entry Substrate[a] Reactions conditions
Time [h] Yield [%] Product[a] Deuterium content[b]
(radiochemical yield[c] [%])
1
2
3
4
5
6
1
1
1
1
1
9
Et3N, D2O, MeCN
5% NaOD in D2O/EtOAc, TEBA, 30 °C 24
LiBr, Et3N, D2O, THF
Mg(OAc)2, Et3N, D2O, THF
Mg(OAc)2, Et3N, HTO, THF
Mg(OAc)2, Et3N, HTO, THF
12
93
93
94
98
89
99
10a
10a
10a
10a
10b
11
d1 (41), d0 (59)
d1 (51), d0 (49)
d2 (29), d1 (56), d0 (15)
d3 (6), d2 (79), d1 (6)
22
48
48
48
48
14
[a] See Scheme 5. [b] Determined by ESI-TOF MS. [c] Determined with a liquid scintillation counter.
594
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Tritium Labelled N-Acyl-l-homoserine Lactones
and gas inlet tube with gas bubbler, and anhydrous Et2O (30 mL)
and bis(trimethylsilyl) malonate (2; 267.7 μL, 1.05 mmol) were
added. Triethylamine (152.5 μL, 1.1 mmol) was added dropwise
and a precipitate was formed almost instantly. After stirring for
10 min, the flask was cooled to 0 °C and octanoyl chloride (3;
170.7 μL, 1 mmol) was added (dropwise, slowly). After stirring at
r.t. for 1 h, the reaction was quenched by the addition of cold satd.
aq NaHCO3 (15 mL) and the mixture was stirred for 10 min in an
ice bath. The aqueous layer was separated and acidified to pH 2–
3 by the dropwise addition of cold 1 M HCl. The resulting precipi-
tate was extracted with ethyl acetate and the organic layer was
washed several times with satd. aq NaHCO3. After drying the com-
bined organic solution over anhydrous sodium sulfate, the solvent
was evaporated in vacuo. The desired product 6 (96 mg, 52%) was
lower than expected, probably because of the low solubility
of LiBr. Therefore, the inclusion of magnesium(II) acetate
was tested, resulting a very high deuterium incorporation,
up to 79% of double-deuterated isotopologue (Table 1, en-
try 4). This method was then applied in tritium labelling –
first of substrate 1 and then of lactone 9. As expected, very
good yields and relatively good specific activities were ob-
tained (Scheme 5, Table 1 and experimental section).
Conclusions
The N-(3-oxo-5-diazirinedodecanoyl-[2-3H2])-l-homo-
serine lactone (11) was successfully synthesized in five steps.
It bears a photoactivatable diazirine group and is easily de-
tectable because of its radioactive isotope label. The diazir-
ine moiety is located close to the hydrophilic part of the
molecule and affects the structure of the highly biologically
1
obtained as a colourless solid. Rf = 0.58 (hexane/EtOAc, 1:10). H
NMR (500 MHz, CDCl3): δ = 0.91 (t, 3J = 6.7 Hz, 3 H, CH3),
1.21–1.40 [m, 8 H, CH3(CH2)4], 1.54–1.69 (m, 2 H, CH2CH2CO),
3
2.58 (t, J = 7.4 Hz, 2 H, CH2CO), 3.54 (s, 2 H, COCH2CO) ppm.
13C NMR (125 MHz, CDCl3): δ = 14.1 (s, CH3), 22.6 (s, C-9, CH2),
active N-(3-oxododecanoyl)-l-homoserine lactone in a neg- 23.4 (s, C-8, CH2), 28.9 (m, C-7, CH2), 29.6 (m, C-6, CH2), 31.6
(s, C-5, CH2), 43.3 (s, C-4, CH2), 47.6 (s, C-2, CH2), 170.8 (s, C-1,
COOH), 209.7 (s, C-3, C=O) ppm. ESI-TOF MS: m/z (%) = 187
(100) [M + H]+.
ligible way. An additional advantage of this approach is the
fact that the tritium label is introduced in the last step of the
synthesis, reducing the time required to handle radioactive
material. The bifunctional molecule is being applied in on- 3-Diazirinedecanoic Acid (7): Anhydrous ammonia (100 mL) was
condensed into a round-bottomed flask. 3-Oxodecanoic acid (6;
3.2 g, 17.2 mmol) was dissolved in a small amount of anhydrous
MeOH (10 mL) and added to the flask, and the mixture was stirred
at –35 to –40 °C for 5 h. The solution was cooled with dry-ice, and
a solution of hydroxylamine-O-sulfonic acid (2.22 g, 19.6 mmol) in
anhydrous MeOH (5 mL) was added over a period of 30 min. The
dry-ice bath was removed, and the mixture was refluxed with stir-
ring at –35 °C for 1 h. The mixture was warmed slowly to room
temperature and stirred overnight, then the ammonia was allowed
to evaporate. The resulting slurry was filtered and the filter cake
was washed with several portions of methanol. The combined solu-
tion was concentrated in vacuo and the crude aziridine residue was
dissolved in CH2Cl2 (10 mL) and treated with triethylamine
(2.96 mL, 21.3 mmol). A solution of iodine (5.8 g, 23 mmol) in
CH2Cl2 (20 mL) was slowly added with stirring until the appear-
ance of a persistent orange-brown colour. The crude residue was
purified by flash column chromatography (hexane/EtOAc, 1:1) to
give the desired product (1.02 g, 30%) as an orange-brown oil. Rf
= 0.39 (hexane/EtOAc, 1:10). 1H NMR (500 MHz, CDCl3): δ =
0.89 (t, 1J = 6.9 Hz, 3 H, CH3), 0.90–1.14 [m, 2 H, CH2(CH2)4],
1.15–1.26 [m, 8 H, CH3(CH2)4], 1.55 (t, 1J = 7.8 Hz, 2 H,
CH2CN2), 2.33 (s, 2 H, CN2CH2CO), 10.10 (s, 1 H, COOH) ppm.
13C NMR (125 MHz, CDCl3): δ = 14.1 (s, C-10, CH3), 22.6 (s, C-
9, CH2), 23.7 (s, C-8, CH2), 25.9 (s, C-7, CH2), 28.9 (s, C-6–5,
CH2), 31.6 (s, C-4, CH2), 32.4 (s, C-2, CH2), 39.6 (s, C-3, CN2),
going photoaffinity labelling studies of a potential receptor
for inter-kingdom signalling. In general, this mild and con-
venient method could be applied in labelling of other bioac-
tive molecules for the investigation of their biological activi-
ties.
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.
Evaporation of solvents and concentration of reaction mixtures
were performed in vacuo at 60 °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 visualization
by iodine vapour and Seebach solution.[35] Normal-phase silica gel
(silica gel 60, 230–400 mesh, Merck) was used for flash chromatog-
raphy. IR and Raman spectra were recorded with a Bruker Vertex
80 FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany)
with a single reflection “Golden Gate” diamond ATR sampling
1
unit (Specac, UK). H and 13C NMR spectra were recorded with
a Bruker-ACS-60, Ultrashield 500 Plus spectrometer. Chemical
shifts are reported as δ parts per million (ppm) values relative to
the CHCl3 signal (1H: δ = 7.26 ppm, 13C: δ = 77.0 ppm). Coupling
constants (J) are given in Hertz [Hz]; s = singlet, d = doublet, t =
triplet, quint. = quintet, sept = septet, m = multiplet, dt = doublet
of triplets, ddd = double doublet of doublets. ESI-TOF mass spec-
tra were recorded with a ESI-TOF Mariner system (Applied Biosy-
stems). UV/Vis spectra were recorded with an Agilent 8453 UV/Vis
Photospectrometer. Absorption was measured in tetrahydrofuran
solution from 200 to 800 nm with a resolution of 1 nm in a 8.5 mm
1 mL UV micro cuvette (Brand GmbH, Wertheim, Germany) at
room temperature. Scintillation counting was carried out with a
Wallac 1409 apparatus by using a “mélange scintillant III” cocktail
from SDS company.
175.8 (s, C-1, COOH) ppm. IR (ATR): ν = 2980, 1710, 1657, 1330,
˜
1218, 1055 cm–1. Raman: ν = 2899, 1693, 1592, 1258, 1045,
˜
881 cm–1. UV (THF): λmax = 343 (ε = 69 M–1 cm–1) nm. ESI-TOF
MS: m/z (%) = 197 (100) [M – H]+.
tert-Butyl 3-Oxo-5-diazirinedodecanoate (8): Solution 1: To a solu-
tion of 3-diazirine-decanoic acid (7; 34 mg, 0.17 mmol) in anhy-
drous THF (2.5 mL) under a nitrogen atmosphere, 1,1Ј-carbonyl-
diimidazole (CDI; 33 mg, 0.2 mmol) was added at room tempera-
ture. The mixture was stirred at room temperature for 4 h. Solution
2: To a solution of mono tert-butyl malonate (32 μL, 0.2 mmol)
in anhydrous THF (2.5 mL) at 0 °C under a nitrogen atmosphere,
isopropylmagnesium chloride (2 M in THF, 187 μL, 0.37 mmol)
was added dropwise. After 30 min at 0 °C the solution was heated
at 50 °C for 30 min and cooled again to 0 °C. Solution 1 was added
3-Oxodecanoic Acid (6): Anhydrous LiBr (powder; 95 mg, 1 mmol)
was transferred to a 100 mL two-neck flask fitted with a septum
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D. Jakubczyk, G. Brenner-Weiss, S. Bräse
FULL PAPER
by using a cannula and the mixture was warmed to room tempera-
ture. After stirring for 16 h, the reaction was quenched by the ad-
dition of 1 M HCl (6 mL) and the aqueous phase was extracted
with ethyl acetate. The combined organic layers were washed with
NaHCO3, brine, dried with Na2SO4 and concentrated under re-
duced pressure. The crude residue was purified by flash column
chromatography (hexane/EtOAc, 10:1) to give the desired product
(10 mg, 20%) as an orange-brown oil. Rf = 0.47 (hexane/EtOAc,
recrystallization (EtOAc with a few drops of hexane) to give the
desired product (11 mg, 99%) as a white solid. Rf = 0.51 (hexane/
EtOAc, 1:3). 1H NMR (500 MHz, CDCl3): δ = 0.87 (t, 3J = 6.5 Hz,
3 H, CH3), 1.19–1.31 (m, 12 H, 6ϫCH2), 1.50–1.68 (m, 2 H,
1
CH2CH2CO), 2.12–2.30 (m, 1 H, 3α-H), 2.52 (t, J = 7.3 Hz, 2 H,
CH2CO), 2.60–2.80 (m, 1 H, 3β-H), 3.47 (s, 0.2 H, COCHDCO),
4.20–4.38 (m, 1 H, 4α-H), 4.40–4.56 (m, 1 H, 4β-H), 4.58–4.69 (m,
1 H, 2-H), 7.61–7.81 (m, 1 H, NH) ppm. 13C NMR (125 MHz,
1
3
1:10). H NMR (500 MHz, CDCl3): δ = 0.89 (t, J = 6.9 Hz, 3 H, CDCl3): δ = 14.1 (s, CЈ-12, CH3), 22.5 (s, CЈ-11, CH2), 23.4 (s, CЈ-
CH3), 0.91–1.12 (m, 2 H, CH2CN2), 1.14–1.32 (m, 10 H, 5ϫCH2),
1.48 (s, 9 H, 3ϫCH3), 2.47 (s, 2 H, CN2CH2CO), 3.38 (s, 2 H,
COCH2CO) ppm. 13C NMR (125 MHz, CDCl3): δ = 14.1 (s, C-12,
10, CH2), 28.9–29.8 (m, CЈ-6–9, CH2), 29.9 (s, C-3, CH2), 43.2
(quint., J = 20.1 Hz, CЈ-2, CD2), 43.9 (s, CЈ-4, CH2), 49.0 (s, C-2,
CH), 65.8 (s, C-4, CH2), 166.3 (s, CЈ-1, C=O), 174.7 (s, C-1, C=O),
1
CH3), 22.6 (s, C-11, CH2), 23.7 (s, C-10, CH2), 27.9 (s, 3ϫCH3), 206.7 (s, CЈ-3, C=O) ppm. ESI-TOF MS: m/z (%) = 322 (100) [M
28.3 (s, C-9, CH2), 29.2 (s, C-8, CH2), 29.7 (s, C-7, CH2), 31.6 (s, + Na]+, 323 (40), 321 (9); deuterium distribution: 79% d2, 15% d3,
C-6, CH2), 32.5 (s, C-4, CH2), 47.8 (s, C-5, CN2), 50.8 (s, C-2, 6% d1.
CH2), 82.4 [s, C(CH3)3], 165.8 (s, C-1, C=O), 199.3 (s, C-3,
N-(3-Oxododecanoyl-[2-3H2])-
tained according to the general procedure from
L
-homoserine Lactone (10b): Ob-
C=O) ppm. ESI-TOF MS: m/z (%) = 297 (100) [M + H]+.
1
(10 mg,
33.6 μmol), magnesium acetate tetrahydrate (7 mg, 33.6 μmol), tri-
tium oxide (900 μL, 50 mmol, specific activity: 1 mCi/g) and trieth-
ylamine (5.5 μL, 40 μmol) in THF (1 mL). The desired product was
obtained as a white solid. Chemical yield: 8 mg (89%); Radioactiv-
ity = 1.78 μCi (66 kBq); Specific activity = 0.22 mCi/g; Radiochem-
ical yield: 22% Rf = 0.51 (hexane/EtOAc, 1:3).
N-(3-Oxo-5-diazirinedodecanoyl)-l-homoserine Lactone (9): tert-Bu-
tyl 3-oxo-5-diazirine-dodecanoate (8; 7.4 mg, 0.025 mmol) was
stirred in TFA/CH2Cl2 (1:1, 540 μL) for 20 min. After solvent evap-
oration, the resulting β-keto acid (8 mg, 0.025 mmol) was dissolved
in 1,4-dioxane (2 mL) and 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide (EDC; 6.7 mg, 0.035 mmol), hydroxybenzotriazole
(HOBt; 3.4 mg, 0.025 mmol) and l-homoserine lactone hydrobrom-
ide (4.5 mg, 0.025 mmol) were added together with a few drops of
water (1.6 mL) at room temperature. Triethylamine (7 μL,
0.05 mmol) was added and the solution was stirred at room tem-
perature for 16 h. The mixture was evaporated and the residue was
dissolved in ethyl acetate, washed with water, dried with Na2SO4
and the solvent was removed by rotary evaporation. The crude resi-
due was purified by flash column chromatography (hexane/EtOAc,
1:3) to give the desired product (2 mg, 25%) as a yellow oil. Rf =
N-(3-Oxo-5-diazirinedodecanoyl-[2-3H2])-
L-homoserine
Lactone
(11): Obtained according to the general procedure from 9 (1 mg,
3 μmol), magnesium acetate tetrahydrate (1 mg, 4 μmol), tritium
oxide (100 μL, 5.5 mmol, specific activity: 1 mCi/g) and triethyl-
amine (1 μL, 7 μmol) in THF (100 μL). The crude residue was puri-
fied by recrystallization (EtOAc with a few drops of hexane) to give
the desired product as a white solid. Chemical yield (1 mg, 99%);
Radioactivity = 0.14 μCi (5.2 kBq); Specific activity = 0.14 mCi/g;
Radiochemical yield: 14% Rf = 0.65 (hexane/EtOAc, 1:3).
1
0.66 (hexane/EtOAc, 1:4). H NMR (500 MHz, CDCl3): δ = 0.88
(t, 1J = 7.1 Hz, 3 H, CH3), 1.02–1.14 (m, 2 H, CH2CH2CN2), 1.22–
Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of H and 13C NMR spectra of key compounds.
1
1.39 (m, 8 H, 4ϫCH2), 1.50 (t, J = 8.1 Hz, 2 H, CH2CN2), 2.18–
2.31 (m, 1 H, 3α-H), 2.49 (s, 2 H, COCH2CN2), 2.70–2.89 (m, 1 H,
3β-H), 3.51 (s, 2 H, COCH2CO), 4.12–4.30 (m, 1 H, 4α-H), 4.51
(td, 1J = 8.9, 2J = 1.0 Hz, 1 H, 4β-H), 4.57–4.71 (m, 1 H, 2-H),
7.70–7.89 (m, 1 H, NH) ppm. 13C NMR (125 MHz, CDCl3): δ =
14.1 (s, CЈ-12, CH3), 22.6 (s, CЈ-11, CH2), 23.7 (s, CЈ-10, CH2), 26.6
(s, CЈ-9, CH2), 28.9–29.8 (m, CЈ-8, CH2), 29.9 (m, CЈ-7, CH2), 30.3
(s, C-3, CH2), 31.6 (s, CЈ-6, CH2), 32.5 (s, CЈ-4, CH2),47.7 (s, CЈ-
5, CN2), 48.4 (s, CЈ-2, CH2), 49.2 (s, C-2, CH), 66.0 (s, C-4, CH2),
166.0 (s, CЈ-1, C=O), 175.0 (s, C-1, C=O), 203.1 (s, CЈ-3,
C=O) ppm. ESI-TOF MS: m/z (%) = 323 (100) [M + H]+.
Acknowledgments
Financial support from the Helmholtz program, Bio-interfaces is
gratefully acknowledged. The authors thank Silvia Diabaté from
the Karlsruhe, Germany Institute of Technology (KIT). The Insti-
tute of Toxicology and Genetics (ITG) is thanked for support in
the handling of radioactive substances. The authors are very thank-
ful to Horst Geckeis and Peter Kaden from KIT and the Institute
for Nuclear Waste Disposal (INE) for help with the analysis of
tritium labelled compounds. Dr. Nicole Jung and her group from
KIT, Institute of Organic Chemistry is thanked for sharing their
experience with diazirine synthesis and for being very helpful and
friendly.
Preparation of Deuterium and Tritium Labelled N-(3-Oxoalkanoyl)-
L-homoserine Lactones; General Procedure: To a stirred solution of
N-(3-oxododecanoyl)-l-homoserine lactone 1 (1 equiv.) in THF
(1 mL per 0.037 mmol) was added magnesium acetate tetrahydrate
(1 equiv.), triethylamine (1.2 equiv.) and deuterium oxide or tritium
oxide (1 mL per 0.037 mmol). The mixture was stirred at r.t. for
24–48 h then evaporated to dryness and the residue was redissolved
in ethyl acetate. The ethyl acetate solution was sequentially washed
with 1 M sodium hydrogen carbonate solution, 1 M potassium hy-
drogen sulfate solution, and saturated sodium chloride solution.
After drying over anhydrous sodium sulfate, the solvent was evapo-
rated in vacuo.
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Published Online: November 28, 2013
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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