One-step iodination of the diazocyclopentadien-2-ylcarbonyl group - A new and convenient preparation of effective radiolabelled photoaffinity probes

Article abstract of DOI:10.1039/b104945n

A detailed study devoted to direct iodination of the photoactivatable diazocyclopentadien-2-ylcarbonyl (Dcp) group is presented. The iodination does not influence the high carbene reactivity of the Dcp-generated carbene. It was shown that the Dcp substituent forms 4-mono-, 5-mono- and 4,5-diiododerivatives upon iodination under oxidative conditions (76, 20 and 4%, respectively, when DcpOMe 2 is iodinated). Photolysis of the individual products of iodination in cyclohexane resulted in rather high insertion into non-activated CH bonds, without noticeable loss of iodine. Syntheses of new phospholipid and ganglioside membrane probes are also described which incorporate the Dcp function via a labile ester bond. A [¹²⁵I]-Dcp-phosphatidylcholine probe exhibiting high specific radioactivity (～500 Ci mmol^-1) was easily prepared at yields of 90% (on the starting Na¹²⁵I), by using peracetic acid as an oxidant. Furthermore, it was successfully used for photolabelling of the integral protein hemagglutinin in a well-characterised influenza virus model. In summary, the Dcp group is efficient for labelling a wide variety of molecules, and as such, it provides a new tool for exploring a diverse range of biological systems.

Full text of DOI:10.1039/b104945n

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One-step iodination of the diazocyclopentadien-2-ylcarbonyl
group—a new and convenient preparation of effective radiolabelled
photoaffinity probes
1
Elena L. Vodovozova, Ekaterina V. Tsibizova and Julian G. Molotkovsky*
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow V-437, 117997 Russia
Received (in Cambridge, UK) 5th June 2001, Accepted 18th July 2001
First published as an Advance Article on the web 28th August 2001
A detailed study devoted to direct iodination of the photoactivatable diazocyclopentadien-2-ylcarbonyl (Dcp) group
is presented. The iodination does not influence the high carbene reactivity of the Dcp-generated carbene. It was
shown that the Dcp substituent forms 4-mono-, 5-mono- and 4,5-diiododerivatives upon iodination under oxidative
conditions (76, 20 and 4%, respectively, when DcpOMe 2 is iodinated). Photolysis of the individual products of
iodination in cyclohexane resulted in rather high insertion into non-activated CH bonds, without noticeable loss of
iodine. Syntheses of new phospholipid and ganglioside membrane probes are also described which incorporate the
Dcp function via a labile ester bond. A [125I]-Dcp-phosphatidylcholine probe exhibiting high specific radioactivity
(∼500 Ci mmolϪ1) was easily prepared at yields of 90% (on the starting Na125I), by using peracetic acid as an oxidant.
Furthermore, it was successfully used for photolabelling of the integral protein hemagglutinin in a well-characterised
influenza virus model. In summary, the Dcp group is efficient for labelling a wide variety of molecules, and as such,
it provides a new tool for exploring a diverse range of biological systems.
then becomes significantly more complex. Obviously, this is the
reason why to this day, the most commonly used probes for
Introduction
Because photoaffinity labelling (PAL) provides direct evidence
PAL are derivatives of commercially available aryl azides that
of spatial proximity of molecules, it is a widely used method in
can be easily iodinated under oxidative conditions. However, it
studies of structure and function of biological systems.1 The
is well documented that nitrenes generated by aryl azides
photochemically labile groups incorporated in a natural ligand
upon photolysis, undergo rapid ring expansion to long-lived
are converted by irradiation into highly reactive intermediates
dehydroazepines which are most reactive with nucleophiles,
(carbene, nitrene or biradical), which are capable of forming a
such as amine and thiol residues.1
covalent bond with the nearest fragment of a biomolecule.
The carbene-generating diazocyclopentadien-2-ylcarbonyl
However, as with any probing technique, PAL is not deprived
(Dcp) group was suggested for PAL by Nielsen et al.,8a with
of shortcomings, and improvements are needed. Therefore,
especial application to studies of biological membranes rich in
the search for new photoaffinity groups with a wide range of
criteria is required, these criteria include high reactivity, a
short lifetime of the excited species, biocompatible irradiation
non-activated hydrocarbon residues. This small-sized and avail-
able labelling group appears attractive to us: DcpOH 1 (Scheme
1) is prepared by a simple 3-step synthesis and, in the absence of
conditions, avoiding intramolecular rearrangement into less
UV light, is rather stable under a range of physicochemical
reactive intermediates after photolysis, minimal size, stability of
covalent bonds with target molecule, possibility of the reliable
detection of cross-linking products, dark stability, and last but
not least availability.1 None of the hitherto known labels fulfil
all requirements. Recently, experiments performed to test the
efficiency of PAL for a number of labels for peptides2 and
DNA3 demonstrated the superiority of the carbene-generating
3-(trifluoromethyl)-3-phenyldiazirine (TFD) group, that was
originally developed by Brunner and co-workers.1c
To enhance the sensitivity of PAL, γ- and strong β-emitters
(
125I, 131I, 32P etc.) are usually used. However, probes bearing
such isotopes exhibit a short shelf life. A solution to this prob-
lem would be to synthesise their precursors that can be stored
for a long time, and then perform radiolabelling immediately
before use. In order to facilitate analysis of the PAL products, it
is also desirable to introduce a radioisotope into the photo-
affinity group itself. The electrophilic substitution with 125I
under oxidative conditions for aromatic residues activated with
any electron-donor substituent is well-known.4 For a number of
photoaffinity groups, e.g., fluorophenyl azide,5 TFD,6 and benzo-
phenone,7 functionalisation methods were developed that allow
the introduction of 125I, although the syntheses of the probes
Scheme 1 a) CH2N2; b) NaI (10 equiv.), chloroamine T (12 equiv.),
MeOH; c) hν, λ > 300 nm, 3 min, cyclohexane.
DOI: 10.1039/b104945n
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This journal is © The Royal Society of Chemistry 2001
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Table 1 1H NMR (500 MHz, C6D12, 25 ЊC, TMS; δ/ppm, J/Hz) and MS (EI, 70 eV) data for the compounds 2–5
Compound
H-3
H-4
H-5
CH3
m/z (%)
2
6.62
5.91
6.79
3.69
s (3H)
149 (100, Mϩ Ϫ H),
dd (1H)
J3,4 = 3.15
J3,5 = 1.95
6.66
d (1H)
J3,5 = 2.20
6.51
d (1H)
J3,4 = 3.42
6.70
dd (1H)
J4,5 = 4.64
J4,3 = 3.15
—
dd (1H)
J5,3 = 1.95
J5,4 = 4.64
6.91
d (1H)
J5,3 = 2.20
—
121 (40, Mϩ Ϫ H Ϫ N2);
C7H6N2O2 (Mϩ) requires 150.0469
3
4
5
3.70
s (3H)
276 (90, Mϩ),
233 (100, Mϩ Ϫ N2 Ϫ Me);
C7H5N2IO2 (Mϩ) requires 275.9367
276 (85, Mϩ),
6.17
3.70
s (3H)
d (1H)
J4,3 = 3.42
—
233 (100, Mϩ Ϫ N2 Ϫ Me);
C7H5N2IO2 (Mϩ) see above
402 (62, Mϩ),
—
3.70
s (3 H)
s (1 H)
359 (90, Mϩ Ϫ N2 Ϫ Me);
C7H4N2I2O2 (Mϩ) requires 401.8333
conditions, such as heat (>150 ЊC), mild bases and acids
(including peracetic acid),8b and oxidising conditions. We
synthesised 14C/3H-labelled phospholipid probes with the
Dcp function localised at the ω-position of a fatty acyl chain.
Photolabelling yielded carbene insertion into CH bonds of
cyclohexane or constituents of model membranes upon
photolysis at λ > 300 nm.9 The probes were applied in studies of
the membrane topography of cytochrome P-450.10 In further
studies of the Dcp group properties, we have found that it can
be iodinated easily under oxidative conditions, the ability to
generate carbene remaining high.11 The present work is a
detailed study on the iodination of the Dcp function including
aspects on its photolysis. We also report on the syntheses of
new lipid Dcp-probes, preparation of [125I]-Dcp-phosphatidyl-
choline of high specific radioactivity, and its application
for PAL of the membrane-embedded proteins in the well-
characterised influenza virus model.
Results and discussion
Oxidative iodination of the Dcp group, and its photochemical
properties
To facilitate the resolution of individual compounds by HPLC,
methyl ester 2, which is the smallest Dcp probe was used in a
study of the iodination products (Scheme 1). Previously, we
used chloroamine T (CAT) fixed on the polystyrene surface
(Iodo-Beads®, Pierce, U.S.A.)4b for iodination of the Dcp ester
11 (Scheme 3).11 But in the case of hydrophobic substances,
most of the product remained adsorbed on the polymeric
matrix, even in methanol. We have elevated the yield of target
product treating ester 2 with excess quantities of NaI and CAT.
Subsequent HPLC gave three individual products, 4-mono- (3),
5-mono- (4) and 4,5-diiododerivatives (5) (Scheme 1), charac-
terised by 1H NMR and EI MS (Table 1).
Iodination was accompanied by significant changes of UV
spectra: initial ester 2 exhibited λmax at 314 nm (EtOH), whereas
iodinated products 3, 4, and 5 showed the shifted λmax values of
322, 316, and 328 nm, respectively. Measurements of ε values
for 2–5 were complicated by their high volatility, but they
cannot differ significantly from the corresponding values found
for methyl 11-(Dcp-oxy)undecanoate 11 (λmax at 314 nm, ε 15000
in EtOH) and for the mixture of its iodination products (λmax at
326 nm, ε 16700, EtOH).11 Both acid 1 and Dcp-amino deriv-
atives are iodinated easily giving similar mixtures of mono- and
diiododerivatives, irrespective of the molar ratio NaI–Dcp sub-
strate within the 0.5–10 range (data not shown). The capability
of diazocyclopentadiene to undergo electrophilic substitution
reactions is known,12a but, to our knowledge, the direct halo-
genation of Dcp has not been reported.
Fig. 1 Photolysis of Dcp-substrates (6 mM) in cyclohexane: (a)
DcpOMe 2, λ > 300 nm; (b) iodinated ester 2 (3 ϩ 4 ϩ 5), λ > 320 nm.
only.13 Also, of particular importance is the stability of the CI
bond. Previously, photodeiodination which reduces the sensi-
tivity of PAL and leads to artefacts connected with unspecific
trapping of iodine radicals by biomolecules was observed for
some iodoaryl azides.5,14 In this connection, we had to examine
the photochemical properties of the Dcp group itself.
The chemistry of cyclopentadienylidene, carbene generated
upon photoactivation of diazocyclopentadiene, appears to be
well established. Its ground state is a triplet state, but the chem-
istry in solution results from the singlet state, and CH insertion
proceeds with high efficiency.12b,c The course of Dcp photolysis
is shown in Fig. 1a. A solution of ester 2 in cyclohexane was
irradiated for 3 min. The solution was not concentrated,
because the volatility of products might lead to disproportion-
ation losses, instead C6D12 (10% v/v) was added, and 1H NMR
with double resonance analysis was performed. TLC showed
the most abundant compound was rather non-polar, and also
obtained were less polar minor components (their ratio varied
depending on the procedure of evaporation and resolution),
1
The photochemical reactivity of iodinated Dcp is interesting,
because heavy atoms are known to promote intersystem cross-
ing from excited singlet states to undesirable triplet states. The
subsequent reactions may be restricted to hydrogen abstraction
with a little tar at the start. The H NMR spectrum of the
mixture remained the same upon storage for several days at 4
ЊC, thus allowing us to attribute the structures 6a or 6b to the
only abundant product of photolysis, besides tar (Scheme 1,
2222
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Fig. 2). The cyclohexyl proton H1 resonance at a rather low field
(δ 3.55 ppm) was completely confirmed by double resonance at
the adjacent cyclohexyl protons in high field (δ 1.53 ppm); these
spectra were taken in CD3OD. ESI MS also evidenced a
cyclohexane insertion product.
Such endocyclic double bond isomers of substituted
cyclopentadienes were reported to be in thermodynamic equi-
librium, with transformations proceeding via a proton transfer
from C-5 to the adjacent C-atom, and proton-cyclopentadienyl
anion complexes being probable transitional states.15b The
structures and ratio of isomers depend mainly on the character
of substituent(s),15b for methyl cyclopentadienecarboxylate the
product is exclusively or very largely the 1,3-diene (in our case
structure 6b).15a Only minute tar quantities were seen on TLC,
but a comparison of integral resonance intensities in 1H NMR
spectra of compound 2 taken prior and after photolysis in
cyclohexane (6 mM) showed ca. 42% yield of CH insertion
product. For a comparison, photolysis of the 3-(trifluoro-
methyl)-3-phenyldiazirine derivative in cyclohexane gave rise to
several compounds, among which the CH insertion product
accounted for 58% at an initial concentration of 2 mM, and
28% at 30 mM, as determined by GC analysis.6 So, we can note
that the ability of the Dcp-generated carbene to insert into
ordinary CH bond is close to that of the most efficient TFD
label.
Scheme 2 a) hν, λ > 320 nm, 3 min, cyclohexane.
ing radioactivity.11 These results show that the iodinated Dcp
group can be photolysed without considerable loss of iodine.
This could be owing to the longer absorption wavelengths of
Dcp chromophore, leading to a more efficient photoactivation
as compared to that of phenyl azides. The same was suggested
by Weber and Brunner regarding the significant photostability
of the CI bond in iodinated 3-phenyldiazirines upon
photolysis.6
The photolysis course of iodinated Dcp ester 2 (a mixture of
compounds 3, 4, and 5) in cyclohexane was followed with UV
spectra (Fig. 1b). To study the products of the photoreaction,
individual iodinated esters 3, 4, and 5 were photolysed in cyclo-
hexane for 3 min, and then reaction mixtures were analysed as
described above for the ester 2. TLC showed, apart from some
tar as polar tails, highly nonpolar products of the carbene
A comparison of integral resonance intensities in the 1H
NMR spectra of the esters 3–5 taken prior and after photolysis
in cyclohexane (6 mM) showed that cross-linking products
(7ab, 8a), (8ab, 7b) and 9 were formed with the yields of 30, 30,
and 26%, respectively. The CH insertion efficiency for the
iodinated Dcp function appears to be somewhat lower than that
for the non-iodinated one. For a comparison see the data
obtained for methyl 4-azido-2-iodo-3,5,6-trifluorobenzoate
(12%, by preparative TLC)5 and for 2-iodo-4-[3-(trifluoro-
methyl)-3H-diazirin-3-yl]benzyl acetate (48%, by GC/MS)6
at initial concentrations of 2 mM in cyclohexane. Although
further studies are needed to elucidate the details of the photo-
chemistry of the iodinated Dcp group, it may be supposed
because of the sufficiently high yields of CH insertion products
that, upon photoactivation, this function reacts mostly via a
singlet carbene.
1
cross-linking to cyclohexane (ESI MS data). H NMR spectra
of reaction products (see Experimental, and Fig. 3 for represen-
tative spectra) show well-resolved peaks. The double resonance
technique allowed us to propose the following pattern of
photoreaction products (Scheme 2). No evidence of isomers of
diiodide 9 was found in the spectrum of the photolysis products
of ester 5. Such compounds, having up-field resonances of the
ring (substituted) methylene, would be clearly observed. We do
not discuss here possible mechanisms of isomeric transform-
ations that lead to formation of compounds 7–9. This will be
the issue of further studies. Noteworthy is the absence of sig-
nals corresponding to non-iodinated esters 6a (6b), in contrary
to iodinated fluorophenyl azides that yield up to 8% (prepara-
tive TLC) deiodinated product of CH insertion.5 A rather high
stability of the CI bond in the esters 3–5 was confirmed by 20
min photolysis of 125I-labelled 11-(Dcp-oxy)undecanoate 11 in
cyclohexane: subsequent careful washing of the reaction mix-
ture with aqueous NaHSO3 extracted less than 3% of the start-
The existence of the iodinated Dcp label as a mixture of
mono- and diiododerivatives is obviously not of importance
when PAL products should be analysed at the level of large
Fig. 2 1H NMR spectra (C6D12) of DcpOMe 2 prior (upper row) and after (lower row) photolysis in cyclohexane; photolysis was performed till 80%
conversion.
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biomolecules, and could be taken into account only if
smaller molecules (e.g. short peptides, or amino acids) are
under study.
PAL of the membrane proteins of the influenza virus
We tested the ability of radioiodinated Dcp-labelled phospha-
tidylcholine [125I]-12 to probe integral proteins on the well-
studied model of influenza virus.18 The virus envelope is formed
by the lipid bilayer and two glycoproteins, hemagglutinin (HA)
and neuraminidase (NA), packed in spikes protruding into the
outer medium. The HA monomer consists of two SS-bonded
subunits: a small one (HA2) that penetrates deep into the apolar
region of the lipid bilayer, and a large polar HA1. The inner
surface of the viral membrane is lined with matrix protein (M1),
which in turn is in contact with the nucleocapside protein (NP).
In this study, we used a classical strain of the influenza virus,
fowl plaque virus (FPV).18b,c
Syntheses of phospholipid and ganglioside photoprobes
As already noted, a highly photoactive Dcp group is of particu-
lar interest for PAL within the hydrophobic region of biological
membranes. Over the last 25 years, numerous photoactivatable
lipids have been synthesised and applied in structural and func-
tional studies.6,9,16 Most of these lipids are phosphatidylcholines
and sphingolipids (particularly, gangliosides) that carry a
photoaffinity group linked through amide, ester, or ether func-
tion to the ω-position of the fatty acyl chain. A high specific
radioactivity of the probes is important in PAL studies of
membrane embedded proteins, because a great share of the
photoaffinity label immersed into the lipid bilayer is wasted as a
result of cross-linking with surrounding lipids.
We obtained the probe [125I]-12 with high specific radioactiv-
ity (∼500 Ci mmolϪ1; 90% yield from the starting Na125I) apply-
ing peracetic acid as an oxidant.4c In contrast, when labelling
Dcp-bearing substances with even tracer amounts of iodine
We have prepared a set of Dcp-labelled photoaffinity lipids
for subsequent radioiodination, particularly phosphatidyl-
choline 12 (PC) and gangliosides 16 and 17 designed for the
depth-dependent membrane probing (Scheme 3). To make the
analysis of PAL products easier, it is desirable to have a labile
bond between photoaffinity group and the rest of the probe
molecule. Since PAL deals most often with proteins as target
biomolecules, the ester bond, being much more susceptible to
alkaline hydrolysis than the peptide, is suitable here. First, the
ω-substituted fatty acyl methyl ester 11 was synthesised. 11-
Aminoundecanoic acid was deaminated with HNO2 in water to
yield 11-hydroxyundecanoic acid then esterified by methanol,
and treated with Tf2O to give triflate † 10, which was further
reacted with DcpOH 1 to form the ester 11. By its partial
saponification, 11-(Dcp-oxy)undecanoic acid was obtained,
and then subjected to esterification of lysophosphatidyl-
choline following the established procedure,17 to give Dcp-PC
probe 12.
The long-chain ganglioside 16 was synthesised by acylation
of lysoganglioside GM1 with p-nitrophenyl ester 13 in DMSO.
To obtain the short-chain probe 17, p-nitrophenyl ester 15 was
prepared, starting from the iodoacetic acid through its conden-
sation with p-nitrophenol and subsequent treatment by DcpOH
1. Notice the high yields of gangliosides 16 and 17. This might
be due to using DMSO as a solvent for the total conversion of
lysoganglioside via reaction with activated esters 13 and 15.
This is followed by a gel filtration (instead of adsorption
or reversed phase chromatography) to purify target products
present at micromole quantities.
Scheme 3 a) HNO2; b) MeOH–HCl; c) Tf2O–Py; d) 1–Et3N; e) KOH,
iPrOH–H2O; f ) HCl; g) lysophosphatidylcholine, DCC, 4-amino-
pyridine; h) CF3COONp–Py; i) lysoGM1–Et3N; j) HONp, DCC.
† The IUPAC name for triflate is trifluoromethanesulfonate.
Fig. 3 1H NMR spectra (C6D12) of 5-iodo-DcpOMe 4 prior (upper row) and after (lower row) complete photolysis in cyclohexane.
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Manchester, UK) system equipped with a nanospray inter-
face. IR spectra were recorded on a Specord 751R spectrometer.
Mps were measured on a Kofler hot-stage apparatus and are
uncorrected. Elemental analysis were carried out only for the
crystalline substances.
Analytical TLC was done on precoated plates (silica gel 60
F254, RP-8254, RP-18254) from Merck. Column chromatography
was carried out with Merck silica gel 60 (40–63 µm), RP-8 or
RP-18 (25–40 µm); gel filtration—with Sephadex LH-20 from
Pharmacia. HPLC was performed isocratically on an Altex 334
solvent delivery system equipped with a model 153 UV detector
(254 nm). Slab PAGE was carried out on a home-made
apparatus.
Photolyses were carried out in a Pyrex glass (2 mm walls)
tube under stirring in an argon atmosphere using a medium
pressure mercury lamp (30 W, emission max 365 nm) at the
distance of 7 cm. To cut off any irradiation below 320 nm, the
light was filtered through saturated aqueous CuSO4 (1 cm
layer).
Solvents were purified and dried by standard procedures; all
evaporations were performed in vacuo at temperatures less than
40 ЊC. Dcp acid 1,8b p-nitrophenyl trifluoroacetate,21a peracetic
acid4c were synthesised as described earlier. Ester 2 was
obtained by the treatment of acid 1 with freshly prepared and
twice distilled into ether CH2N2. Lysophosphatidylcholine
(LPC) was prepared from egg PC by phospholipase A2
hydrolysis,22 lyso-GM1—by hydrolysis of the natural ganglio-
side GM1.23 Na125I (100 mCi mlϪ1 in NaOH) was purchased
from “Izotopy” (Sankt-Peterburg, Russia). All works with
radioactivity were performed in a C-type laboratory. For quan-
titation of 125I, a γ-counting system Compugamma 1282 from
LKB Wallac was used. Autoradiographies were carried out with
RX Fuji Medical X-ray films.
Fig. 4 Photoaffinity labelling of the virus proteins with a Dcp
phospholipid probe. Fowl plague virus suspension (0.1 mg protein) was
incubated with the probe [125I]-12 (0.16 nmol, 80 µCi) for 3 h at 37 ЊC,
then irradiated for 5 min at λ > 320 nm. After delipidation with organic
solvents, the material was resolved by 12.5% SDS-PAGE under
reducing conditions.19 Gel was stained with 0.05% Coomassie brilliant
blue (left lane), followed by autoradiography (right lane; exposure time
24 h at Ϫ60 ЊC). Protein standards (kDa) were run in parallel.
(no-carrier-added, nca, 0.5 nmol of Na125I with specific radio-
activity >2000 Ci mmolϪ1, 1 mCi) by means of CAT, high yield
of radioiodination (48% for the lipid [125I]-12) could be
achieved only if the molar ratio NaI–CAT–Dcp substrate was
raised to 1 : 10 : 100 (data not shown), similarly to radioiodin-
ation of tyrosine.4d Obviously, the use of peracetic acid should
enhance the specific radioactivity of the [125I]-Dcp-carry-
ing probes near to the level of nca. However, the need of
scrupulous manipulations with subnanomolar quantities of
the Dcp substrate is the main obstacle here. The purification
of radioiodinated probe from noniodinated precursor, com-
plicates the procedure by necessity of HPLC (TLC does not
provide separation), and therefore separation was not carried
out.
Fowl plague virus (influenza type A virus) was grown in
chicken fibroblasts and purified as described earlier.18c
All manipulations with Dcp derivatives were carried out
under yellow light.
Iodination of the Dcp group, and its photolysis
Methyl esters of 1-diazo-4-iodocyclopenta-2,4-dien-2-
ylcarboxylic acid 3, 1-diazo-5-iodocyclopenta-2,4-dienyl-2-
carboxylic acid 4 and 1-diazo-4,5-diiodocyclopenta-2,4-dien-2-
ylcarboxylic acid 5. A suspension of chloroamine T trihydrate
(2.49 g, 8.83 mmol) in methanol (7 ml) was added under stirring
to a solution of ester 2 (112 mg, 0.73 mmol) and NaI (1.1 g, 7.3
mmol) in methanol (3 ml). In a few seconds, the resulting brown
solution became yellow, and was stirred for 10–20 min extra.
The solvent was evaporated partly in vacuo followed by filtra-
tion from the precipitate (iodoamine T and NaCl). The filtrate
was desalted on a RP-8 column eluted with methanol–water,
4 : 6, then 1 : 1, 7 : 3, and pure methanol. A product of iodin-
ation (61 mg, ∼30%), in aliquots, was further separated into
individual substances by HPLC (7 µm silica SGX Tessek
4 × 250 mm column, 0.2% ether in hexane, 1 ml minϪ1): the
retention times for the products 3, 4, 5 are 32, 20, 15 min,
respectively.
Following the established procedure, PC probe [125I]-12 was
incorporated into the outer leaflet of the FPV membrane (the
molar ratio probe–FPV phosphatidylcholine, ~0.01),18c and
photolysed. Then unbound lipids were removed by extraction
with organic solvents, and the residue was analysed by PAGE,
where the behaviour of viral proteins is well known.18,19 As
shown in Fig. 4, the only labelled protein was the membrane
anchor HA2. This pattern coincides completely with the estab-
lished organisation of the viral envelope.18
The first application of the radioiodinated probe 12 for a
study of mitochondrial ATP synthase ensemble in membrane
gave new interesting data.20
It should be said in conclusion that the Dcp group, as shown
in this study, is an efficient photoaffinity label. This, together
with other advantages of the group, the convenience of intro-
ducing 125I in one step just before use, the small size, and avail-
ability, make it a promising tool in biological studies.
Photolysis of the ester 2 in cyclohexane. A solution of ester 2
in cyclohexane (6 mM) was irradiated (without CuSO4 filter: λ
> 300 nm) to yield compound 6a (or 6b); δ (C6D12) 6.63 (1 H, dt,
J3,4 or J5,4 5.38, J3,5 or J5,3 1.47, 3-H or 5-H), 6.04 (1 H, dt, J4,3 or
J4,5 5.38, J4,5 or J4,3 1.70, 4-H), 3.65 (3 H, s, Me), 3.55 (1 H, tt,
Jax,ax 11.98, Jax,eq 3.25, cyclohexyl-H), 2.99 (2 H, t, J5,4 or J3,4
1.47, 5-H or 3-H); δ(CD3OD) 6.82 (1 H, dt, J3,4 or J5,4 5.63, J3,5
or J5,3 1.47, 3-H or 5-H), 6.42 (1 H, dt, J4,3 or J4,5 5.63, J4,5 or J4,3
1.47–1.70, 4-H), 3.97 (3 H, s, Me), 3.64 (1 H, tt, Jax,ax 11.67,
Jax,eq 3.18, cyclohexyl-H), 3.38 (2 H, t, J5,4 or J3,4 1.47, 5-H or 3-
Experimental
Absorption spectra were recorded with a LKB Ultrospec II
4050 spectrophotometer. 1H NMR spectra were registered on a
Bruker WM-500 spectrometer at 500 MHz and are reported in
δ-units with tetramethylsilane as the internal standard. J values
are given in Hz. Mass spectra were measured on a Varian MAT
44 apparatus at an ionisation potential of 70 eV, or a Finnigan
MAT 9005 (ESI), or a MSVK instrument (252Cf-plasma desorp-
tion tof, “Elektron” company, Sumy, Ukraine). Exact mass
determinations were performed on a Q-tof (Micromass,
H), 1.50–1.63 (m, cyclohexyl); m/z (ESI) 207.2 (100%, Mϩ
H), 229.2 (20, Mϩ ϩ Na); C13H18O2 (Mϩ) requires 206.1307.
ϩ
J. Chem. Soc., Perkin Trans. 1, 2001, 2221–2228
2225

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Photolyses of the iodinated esters 3, 4 and 5 in cyclohexane.
The photolyses were performed at λ > 320 nm and yielded
compounds 7a: δ(C6D12; assignments of resonances are given in
accordance with the initial numeration of C-atoms in the
diazocyclopentadiene ring) 6.89 (1 H, t, J 1.23, 3-H), 3.13 (2 H,
d, J 1.22, 5-H); 7b: 6.61 (1 H, dd, J 1.47, J 2.20, 5-H), 6.13 (1 H,
dd, J 1.23, J 5.63, 3-Heq), 6.05 (1 H, dd, J 2.20, J 5.63, 3-Hax);
8a: 6.95 (2 H, m, 3-H and 4-H), 5.08 (1 H, t, J 1.22, 5-H); 8b:
6.91 (1 H, m, 3-H), 6.68 (1 H, dd, J4,3 2.20, J4,1 1.22, 4-H), 3.20
(1 H, m, 1-H). Multiplets in the spectra of the photolysed esters
3 and 4 at 3.63–3.65 ppm obviously are sets of methyl singlets,
and the cyclohexyl 1-H resonance (tt) seems to be hidden under
them. Compound 9: δ(C6D12) 6.57 (1 H, s, 3-H), 3.65–3.63 (1 H,
m, presumably 1-H), 3.64 (3 H, s, Me). MS data (ESI) for the
photolysed ester 3 (compounds 7a ϩ 7b ϩ 8a): m/z 387.3
(100%, Mϩ ϩ Na ϩ MeOH), 301.4 (40, Mϩ Ϫ OMe); C13H17IO2
(Mϩ) requires 332.0273; photolysed ester 4 (compounds 8a ϩ
8b ϩ 7b): m/z 387.3 (100), 301.4 (50); photolysed ester 5 (com-
pound 9): m/z 456.8 (12, Mϩ Ϫ H), 428.5 (18, Mϩ ϩ H Ϫ OMe);
C13H16I2O2 (Mϩ) requires 457.9239.
(15900); m/z (252Cf ) 334 (100%, Mϩ); C18H26N2O4 (Mϩ) requires
334.1932.
1-O-Acyl-2-O-[11-(1-diazocyclopenta-2,4-dien-2-yl-2-carb-
onyloxy)undecanoyl]-sn-glycero-3-phosphocholine 12. Aqueous
KOH (5%, 0.4 ml) was added to a solution of methyl ester 11
(25 mg, 75 µmol) in propan-2-ol (16 ml), and the mixture was
incubated at rt (about 14 h) till trace amounts of the DcpOH 1
appeared (TLC control). After neutralisation with 1 M HCl
and evaporation, the residue was suspended in H2O (1 ml), acidi-
fied with 1 M HCl to pH 2–3 and extracted with CHCl3 (3 × 0.5
ml). The extracted material was subjected to silica gel column
chromatography (gradient of a mixture iPrOH–AcOH, 10 : 1,
in CHCl3, 0 5%), then on a RP-18 column (MeOH–H2O,
7 : 3, then 9 : 1) to yield the TLC pure 11-(Dcp-oxy)undecanoic
acid (8.2 mg, 34%); UV spectrum is similar to that of the methyl
ester 11.
The LPC (5 mg, 10 µmol) was converted to the trifluoro-
acetate form by co-evaporation with trifluoroacetic acid (5 µl)
in CHCl3 (0.5 ml). After drying overnight under high vaccum,
the resulting LPC salt and 11-(Dcp-oxy)undecanoic acid
(3.2 mg, 10 µmol) were dissolved in dry CHCl3 (0.4 ml), a sus-
pension of 4-aminopyridine (5 mg, 53 µmol) in CHCl3 (0.6 ml),
and DCC (5 mg, 25 µmol) in CCl4 (25 µl) were added, and the
mixture was stirred for 24 h at rt under an argon atmosphere.
After filtration and concentration in vacuo, the reaction mixture
was purified by gel filtration (1 × 20 cm column) in a mixture
CHCl3–MeOH, 1 : 1, followed by chromatography on a RP-18
column (MeOH–CHCl3–H2O, 10 : 1 : 1, then 10 : 1 : 0.25) to
yield pure (TLC) phospholipid 12 (3.5 mg, 44%); δ(CDCl3) 7.03
(1 H, br dd, J5Ј,4Ј 4.57, J5Ј,3Ј 1.83, 5Ј-H), 6.82 (1 H, br t, 3Ј-H),
6.07 (1 H, br dd, J4Ј,5Ј 4.27, J4Ј,3Ј 3.35, 4Ј-H), 5.21 (1 H, m), 4.38–
4.52 (4 H, br m), 4.24 (2 H, t, J 6.71, DcpOCH2), 3.92–4.17
(4 H, br m), 3.42 (9 H, s), 2.26–2.36 (4 H, m), 1.64–1.78 (6 H,
m), 1.22–1.36 (∼36 H, m), 0.89 (3 H, t, J 6.71, Me); m/z (ESI)
Syntheses of phospholipid and ganglioside photoprobes
11-Hydroxyundecanoic acid. A solution of NaNO2 (483 mg,
7 mmol) in H2O (2 ml) was added to a solution of
11-aminoundecanoic acid (1 g, 5 mmol) in H2O–AcOH, 4 : 1
(5 ml), then the mixture was heated at 100 ЊC for 1 h and
allowed to cool to rt during several hours. After acidification
with 1 M HCl to pH 3.0, the mixture was extracted with CHCl3
(3 × 5 ml). The extract was evaporated, dried in vacuo, and the
residue was subjected to silica gel column chromatography
(gradient of a mixture iPrOH–AcOH, 10 : 1, in CHCl3,
0
10%) to yield 11-hydroxyundecanoic acid (382 mg, 38%);
mp 64–65 ЊC (from H2O) (lit.,21b 65–66 ЊC); νmax/cmϪ1 3620
(OH), 2933 and 2860 (CH ), 1700 (C᎐O), 1226 and 1200
᎐
2
(OHCOOH); δ(CDCl3) 3.64 (2 H, t, HOCH2), 2.35 (2 H, t,
CH2COOH), 1.64 (2 H, m, HOCH2CH2), 1.55 (2 H, m,
CH2CH2COOH), 1.29 (12 H, m, (CH2)6).
ϩ
ϩ
798.504 (100%, M1 ϩ H), 820.519 (34, M1 ϩ Na), 836.477
(58, M1ϩ ϩ K); C41H72N3PO10 (M1ϩ) requires 797.5015 [(M1ϩ ϩ
H) requires 798.5093]; 826.546 (70, M2ϩ ϩ H), 848.545 (15, M2
ϩ
ϩ
ϩ
ϩ
ϩ Na), 864.515 (30, M2 ϩ K); C43H76N3PO10 (M2 = M1
ϩ
2CH2) requires 825.5328 [(M2ϩ ϩ H) requires 826.5406].
Methyl 11-hydroxyundecanoate. AcCl (5 ml) was added
under stirring to MeOH (50 ml). This mixture was added
immediately to 11-hydroxyundecanoic acid (200 mg, 1 mmol).
After 3.5 h at 40 ЊC, the reaction mixture was evaporated, and
the residue was purified by silica gel column chromatography
(CHCl3–MeOH, 9 : 1) to give TLC pure oily product (115 mg,
53%).
p-Nitrophenyl 11-(1-diazocyclopenta-2,4-dien-2-ylcarbonyl-
oxy)undecanoate 13. A solution of p-nitrophenyl trifluoro-
acetate (5.8 mg, 24 µmol) in dry pyridine (200 µl) was added to
the 11-(Dcp-oxy)undecanoic acid (4 mg, 12 µmol) and stirred
for 5 h at rt. After evaporation, the reaction mixture was puri-
fied by silica gel column chromatography (gradient of ethyl
acetate in toluene, 0 2%) to yield yellow oily product 13 (2.7
mg, 50%); m/z (252Cf ) 441.6 (60%, Mϩ); C23H27N3O6 (Mϩ)
requires 441.1960.
Methyl 11-(trifluoromethylsulfonyloxy)undecanoate 10.
A
solution of methyl 11-hydroxyundecanoate (115 mg, 0.53
mmol) in dry CHCl3 (6 ml) with pyridine (70 µl, 0.87 mmol) was
added to a solution of Tf2O (144 µl, 0.87 mmol) in CHCl3 (6 ml)
under stirring at Ϫ10 ЊC. After 2 h stirring at 0 ЊC, the reaction
mixture was extracted with H2O (3 × 3 ml), then the organic
phase was filtered through cotton wool, evaporated and dried
in vacuo to yield compound 10 (159 mg, 86%) as an individual
colourless oil as indicated by TLC.
p-Nitrophenyl iodoacetate 14. The solutions of p-nitrophenol
(46 mg, 0.36 mmol) in dry CHCl3 (3 ml) and DCC (80 mg, 0.4
mmol) in CCl4 (0.4 ml) were added at 0 ЊC under stirring to a
solution of iodoacetic acid (56 mg, 0.3 mmol) in CHCl3 (3 ml).
After 1 h stirring, the reaction mixture was allowed to stand at
rt overnight. Then it was filtered through cotton wool, evapor-
ated, redissolved in toluene, filtered once again and evaporated.
The residue was purified by silica gel column chromatography
in toluene with 0.5% AcOH to yield product 14 (51 mg, 55%);
mp 80 ЊC (from EtOH); δ(CDCl3) 8.30 (2 H, d, J5Ј,6Ј = J3Ј,2Ј 9.17,
3Ј-H and 5Ј-H), 7.33 (2 H, d, J2Ј,3Ј = J6Ј,5Ј 9.17, 2Ј-H and 6Ј-H),
3.95 (2 H, s, ICH2CO) (Found: C, 31.24; H, 2.00; N, 4.42; I,
41.00. C8H6NIO4 requires C, 31.29; H, 1.97; N, 4.56; I, 41.33%).
Methyl
11-(1-diazocyclopenta-2,4-dien-2-ylcarbonyloxy)-
undecanoate 11. A solution of ester 10 (159 mg, 0.46 mmol) in
dry acetone (2.6 ml) and triethylamine (80 µl) were added to a
solution of acid 1 (20 mg, 0.15 mmol) in acetone (1 ml). The
reaction mixture was stirred for 40 min at rt, then evaporated,
and the residue was subjected to silica gel column chrom-
atography (gradient of ethyl acetate in heptane, 0 7%) to yield
TLC individual oily product 11 (25 mg, 50%); δ(CDCl3) 7.02
(1 H, dd, J5Ј,4Ј 4.64, J5Ј,3Ј 1.96, 5Ј-H), 6.82 (1 H, dd, J3Ј,4Ј 3.18,
J3Ј,5Ј 1.96, 3Ј-H), 6.07 (1 H, dd, J4Ј,5Ј 4.60, J4Ј,3Ј 3.20, 4Ј-H), 4.24
(2 H, t, J 6.72, DcpOCH2), 3.66 (3 H, s, Me), 1.70 (2 H, t,
J 6.72, CH2COO), 1.61 (2 H, m), 1.38 (2 H, m), 1.29 (12 H, m,
(CH2)6); λmax (EtOH)/nm 215 (ε/dm3 molϪ1 cmϪ1 12400) and 314
p-Nitrophenyl (1-diazocyclopenta-2,4-dien-2-ylcarbonyloxy)-
acetate 15. Triethylamine (28 µl, 200 µmol) was added under
stirring to a solution of Dcp-OH acid 1 (13.8 mg, 0.1 mmol) in
dry acetone (1 ml), followed by the addition of ester 14 (31.5
mg, 0.1 mmol) in acetone (1 ml). The reaction mixture was
2226
J. Chem. Soc., Perkin Trans. 1, 2001, 2221–2228

View Article Online
stirred overnight at rt. After evaporation, the residue was puri-
fied by RP-18 column chromatography (MeOH–H2O, 1 : 1,
then 6 : 4) to yield product 15 (25 mg, 80%); δ(CDCl3) 8.30 (2 H,
d, J5Ј,6Ј = J3Ј,2Ј 9.15, 3Ј-H and 5Ј-H), 7.36 (2 H, d, J2Ј,3Ј = J6Ј,5Ј 9.15,
2Ј-H and 6Ј-H), 7.12 (1 H, dd, J5Љ,4Љ 4.58, J5Љ,3Љ 1.98, 5Љ-H Dcp),
6.97 (1 H, dd, J3Љ,4Љ 3.10, J3Љ,5Љ 2.14, 3Љ-H Dcp), 6.14 (1 H, dd,
J4Љ,5Љ 4.58, J4Љ,3Љ 3.30, 4Љ-H Dcp), 5.04 (2 H, s, OCH2COO); m/z
chemical purity, TLC). After concentration, the residue was
dissolved in CHCl3 (1 ml), stored at Ϫ20 ЊC and used for PAL
the next day. (All manipulations with methanolic solutions were
performed rapidly to avoid perceptible hydrolysis of ester bonds
in nmol of [125I]-12.)
Photolysis of [125I]-12 in the fowl plague virus. A solution of
the probe [125I]-12 (80 µCi) in ethanol (10 µl) was added under
argon to the virus suspensions in phosphate buffered saline, pH
7.0 (0.1 mg protein mlϪ1, 1 ml, pre-bubbled with argon) and
incubated for 3 h at 37 ЊC and gentle rocking. Then the suspen-
sions were irradiated for 5 min at λ > 320 nm (CuSO4 filter) and
ambient temperature, with stirring. After delipidation of the
samples with 3 volumes of CHCl3–MeOH, 1 : 3, v/v (sodium
dodecyl sulfate was added up to 0.4% to some samples, and
they were incubated for 15 min prior to the addition of organic
solvents to achieve more exhaustive delipidation), protein pre-
cipitates (2000g centrifugation) were dried in vacuo, and then
subjected to PAGE according to the established procedure,18c,19
with drying and autoradiography.
(
252Cf ) 315.7 (80%, Mϩ); C14H9N3O6 (Mϩ) requires 315.0551.
11-(1-Diazocyclopenta-2,4-dien-2-ylcarbonyloxy)undecanoyl
GM1 16. Triethylamine (1 µl, 7 µmol) and a solution of
p-nitrophenyl ester of 13 (0.53 mg, 1.2 µmol) in DMSO (50 µl)
were mixed with a solution of lyso-GM1 (1 mg, ∼1 µmol) in
DMSO (50 µl). After 16 h incubation at rt, a drop of water, and
CHCl3–MeOH, 1 : 1 (0.1 ml) were added to the reaction
mixture; the following gel filtration (0.5 × 20 cm column) in a
mixture CHCl3–MeOH, 1 : 1, yielded TLC-pure ganglioside 16
(∼98% on the basis of UV absorption); UV spectrum is similar
to that of the ester 11; δ(CD3OD) (data for the several protons)
7.35 (1 H, br dd, J5Ј,4Ј 4.58, J5Ј,3Ј 1.83, 5Ј-H), 6.97 (1 H, br t (dd,
J3Ј,4Ј 3.4, J3Ј,5Ј 1.8), 3Ј-H), 6.26 (1 H, br dd, J4Ј,5Ј 4.37, J4Ј,3Ј 3.46,
4Ј-H), 5.87 (1 H, dt, J5,4 15.34, J5,6 7.72, 5-H of (E)C᎐C), 5.64
᎐
Acknowledgements
(1 H, dd, J4,5 15.21, J4,3 7.78, 4-H of (E)C᎐C), 4.63, 4.55, and
᎐
4.49 (3 × 1 H, 3 dd, J 7.61, 8.03, and 7.81, 1-H hexapyranoses
This work was supported by the Russian Foundation for Basic
Research (RFBR) (grant no. 00-04-48922). We are grateful to
Dr A. S. Gambaryan, Institute of Poliomielitis, Moscow,
for the kind gift of the influenza virus samples, and Dr A. B.
Tuzikov, Institute of Bioorganic Chemistry, for the helpful
discussions. We thank Dr I. I. Mikhalyov for preparation
of the lysoganglioside, and Dr Bo Ek, The Swedish University
of Agricultural Sciences, Uppsala, for the HRMS spectra.
2
3
β), 4.05 (1 H, m, 2-H), 2.92 (1 H, dd, J 12.20, J 4.42, 3-Heq
NeuAcα), 2.36 (2 H, t, J 7.52, CH2CO), 2.20 and 2.18 (2 × 3 H,
2 s, 2 NAc), 2.11 (1 H, dd ∼ t, J 12.10, 3-Hax NeuAcα), 1.91
(2 H, m, CH2CH2CO), 1.45–1.55 (∼36 H, m), 1.08 (3 H, t,
ϩ
J 7.10, Me); m/z (ESI) 1582.875 (100%, M1 ϩ H), 1604.869
ϩ
(33, M1ϩ ϩ Na), 1620.837 (61, M1ϩ ϩ K); C72H119N5O33 (M1
)
ϩ
requires 1581.7885 [(M1 ϩ H) requires 1582.7963]; 1610.912
(39, M2ϩ ϩ H), 1632.893 (25, M2ϩ ϩ Na), 1648.866 (49, M2ϩ ϩ
ϩ
ϩ
K); C74H123N5O33 (M2 = M1 ϩ 2CH2) requires 1609.8198
[(M2ϩ ϩ H) requires 1610.8276].
References
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Ganglioside 17 was synthesised as described for the probe 16,
from lyso-GM1 (1 µmol) and p-nitrophenyl Dcp-oxyacetate 15
(380 µg, 1.2 µmol); yield 88% (on the basis of the UV absorp-
tion); δ(CD3OD) 7.40 (1 H, br dd, J5Ј,4Ј 4.6, J5Ј,3Ј 1.8, 5Ј-H), 7.09
(1 H, br t, 3Ј-H), 6.29 (1 H, br t (dd, J4Ј,5Ј 4.3, J4Ј,3Ј 3.3), 4Ј-H),
5.89 (1 H, dt, J5,4 15.32, J5,6 7.70, 5-H of (E)C᎐C), 5.65 (1 H,
᎐
dd, J4,5 15.20, J4,3 7.72, 4-H of (E)C᎐C), 4.63, 4.59, and 4.50
᎐
(3 × 1 H, 3 dd, J 7.60, 8.02, and 8.02, 1-H hexapyranoses β),
2
3
4.03 (1 H, m, 2-H), 2.92 (1 H, dd, J 12.20, J 4.40, 3-Heq
NeuAcα), 2.85 (2 H, s, COCH2ODcp), 2.20 (2 × 3 H, br s, 2
NAc), 1.45–1.57 (∼22 H, m), 1.09 (3 H, t, J 7.10, Me); m/z (ESI)
1456.740 (10%, M1ϩ ϩ H), 1478.714 (87, M1ϩ ϩ Na), 1494.701
(90, M1ϩ ϩ K); C63H101N5O33 (M1ϩ) requires 1455.6477 [(M1ϩ ϩ
ϩ
H) requires 1456.6555]; 1484.723 (20, M2 ϩ H), 1506.740
ϩ
(67, M2ϩ ϩ Na), 1522.722 (100, M2ϩ ϩ K); C65H105N5O33 (M2
ϩ
ϩ
= M1 ϩ 2CH2) requires 1483.6790 [(M2 ϩ H) requires
1484.6868].
PAL of the membrane proteins of the influenza virus
Radioiodinated phospholipid probe [125I]-12. A solution of the
phosphatidylcholine 12 (∼1.6 µg, ∼2 nmol) in 10 µl of AcOH
was added to a solution of Na125I (nca; 0.5 nmol, >2000 Ci
mmolϪ1, 1 mCi) in aqueous NaOH (10 µl, pH 10.0) in com-
mercial ampoule, followed by addition of ∼11% peracetic acid
solution in AcOH (5 µl). After short vortexing and 5 min incu-
bation at rt, the reaction was quenched with 10% NaHSO3
(50 µl) and 5 M NaOH (35 µl). Then the mixture was extracted
with a CHCl3–MeOH mixture (2 : 1, 50 µl × 2) by vortexing.
The organic phases were collected and co-evaporated, the resi-
due was dissolved in an MeOH–CHCl3–H2O system, 10 : 1 : 1,
applied on an RP-18 micro-column (200 µl) and washed with
MeOH–H2O (9 : 1, 200 µl, then 3 : 2, 1 ml). Elution with MeOH
(2 ml) yielded ∼0.96 mCi of the probe [125I]-12 (95% of radio-
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