4-Benchrotrenyl pyrylium salts as protein organometallic labelling reagents

Article abstract of DOI:10.1016/S0040-4020(99)00947-3

Pyrylium ions are useful reagents to selectively modify the amino group of protein lysine residues. This property was exploited here to label proteins with chromium tricarbonyl complexes in the form of 4-benchrotrenyl pyridinium ions. Kinetic studies of the reaction of a series of benchrotrenyl pyrylium salts with water and n-butylamine were performed and revealed that the overall reactivity of these compounds was highly dependent on their substitution pattern. These compounds could find application in protein X-ray crystallography.

Full text of DOI:10.1016/S0040-4020(99)00947-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 257–263
4-Benchrotrenyl Pyrylium Salts as Protein Organometallic
Labelling Reagents
a
a
b
b,
*
`
´
Bertrand Caro, Franc¸oise Le Guen-Robin, Michele Salmain and Gerard Jaouen
a
´
Laboratoire de chimie organometallique et biologique, UMR CNRS 6509, IUT, BP150, 22302 Lannion Cedex, France
b
´
´
Laboratoire de chimie organometallique, UMR CNRS 7576, Ecole Nationale Superieure de Chimie de Paris,
11, rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France
Received 2 July 1999; accepted 20 October 1999
Abstract—Pyrylium ions are useful reagents to selectively modify the amino group of protein lysine residues. This property was exploited
here to label proteins with chromium tricarbonyl complexes in the form of 4-benchrotrenyl pyridinium ions. Kinetic studies of the reaction of
a series of benchrotrenyl pyrylium salts with water and n-butylamine were performed and revealed that the overall reactivity of these
compounds was highly dependent on their substitution pattern. These compounds could find application in protein X-ray crystallography.
᭧ 1999 Elsevier Science Ltd. All rights reserved.
Introduction
is studied and compared to that of two related 4-benchro-
trenyl pyrylium salts 2 and 3 (Fig. 1).
Pyrylium ions are heterocyclic aromatic compounds that
have been shown to react in a specific manner and under
very mild conditions with protein amino groups by
exchange of oxygen to nitrogen to form positively charged
N-substituted pyridinium adducts.1 We have recently
described the preparation of a series of pyrylium salts,
substituted at the g position by a ferrocenyl, a cymantrenyl,
a benchrotrenyl or a cyclopentadienyl rhenium tricarbonyl
fragment.2 Moreover, the reactivity of the manganese and
the rhenium derivatives with model amines and two proteins
at basic pH has been explored.3
In a preliminary attempt for the use of organometallic
pyrylium ions as heavy atom reagents, we performed
the labelling of a model protein, namely bovine serum
albumin (BSA), with the three 4-benchrotrenyl pyrylium
salts in aqueous solution and observed a different behaviour
of these reagents in relation to their different structural
pattern.
Experimental
We thought that this family of compounds could be poten-
tially attractive reagents for the introduction of heavy atoms
into protein crystals, in the course of protein three-dimen-
sional structure determination by X-ray crystallography.
Actually, preparation of heavy metal derivatives is required
for crystallographic phases determination, when using for
instance the multiple isomorphous replacement (MIR)
method.4 To date, the selection of suitable heavy atom
compounds is still often a trial-and-error process partly
because of the lack of selectivity of the available reagents
for particular protein sites.
Preparation of the organometallic pyrylium salt 1 was
carried out under an argon atmosphere using conventional
Schlenk techniques. Pyrylium salts 2 and 3, pseudobases 5
and 6 and N-butylpyridinium salts 8 and 9 were synthesised
according to literature procedures.2 Solvents were dried and
distilled according to standard procedures. 1H NMR spectra
were recorded on a Bruker AC 200 spectrometer operating
at 200 MHz. Infrared spectra were recorded on a Bomem
MB100 FT-spectrometer in a 1 mm-pathlength CaF2 micro-
cell (Spectratech). UV–visible spectra were recorded on a
Safas uv/mc2 spectrophotometer. Bovine serum albumin
(crystalline grade) was purchased from Serva. Citrate–
phosphate (0.1 M, pH 4, 5 and 6), phosphate (0.1 M, pH 7)
and borate (0.1 M, pH 8 and 9) buffers were prepared from
double-distilled water.
We describe herein the preparation under mild conditions of
[2,6-diphenyl-4-benchrotrenyl pyrylium]BF4 1. Its beha-
viour towards nucleophiles such as water and n-butylamine
* Corresponding author. Tel.: ϩ33-1-43-260061; e-mail: jaouen@ext.
jussieu.fr
Synthesis
Abbreviations: Bct, benchrotrenyl; BSA, bovine serum albumin; CR,
coupling ratio; CY, coupling yield; EHMO, extended Hu¨ckel molecular
orbital; MIR, multiple isomorphous replacement.
[2,6-Diphenyl-4-benchrotrenylpyrylium]BF4 1. To
mixture of triethylorthobenzoate Cr(CO)3 (636 mg,
a
0040–4020/00/$ - see front matter ᭧ 1999 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)00947-3

258
B. Caro et al. / Tetrahedron 56 (2000) 257–263
Figure 1.
2 mmol) obtained by thermolysis of commercially available
triethylorthobenzoate and Cr(CO)6 in dibutyl ether/THF
mixture and 1-phenyl 1-(trimethylsilyloxy)ethylene
(400 mg, 2.1 mmol) in CH2Cl2 was added BF3(Et2O)
(0.58 mL, 3.8 mmol) at 0ЊC. The solution was magnetically
stirred for 1 h, diluted with diethyl ether and filtered to give
the blue-green solid 1 (510 mg, 51%). 1H NMR (acetone-d6,
d in ppm/TMS) 8.95 (2H, s, H pyrylium) 8.57 (4H, dt,
J1ˆ6.8, J2ˆ1.7, Ho Ph) 7.85 (6H, m, Hm,p Ph) 7.24 (2H, d,
Jˆ6.5, Ho Bct) 6.45 (1H, t, Jˆ6.4, Hp Bct) 5.94 (2H, t, Jˆ7,
Hm Bct). IR (MeCN, n in cmϪ1) 1984, 1923 (CxO).
UV–visible (MeCN, lmax in nm, e in L molϪ1 cmϪ1) 602,
4500.
Physico-chemical studies
1H NMR study of the reaction of 1 with butylamine.
Compound 1 (11 mg, 0.02 mmol) was dissolved in
0.75 mL of CD3CN. Butylamine (0.004 mL, 0.04 mmol)
was added with a micropipette. The solution turned dark
1
orange immediately. The H NMR spectrum was recorded
immediately, then after 18 h.
IR and visible studies of the reaction of 1, 2 and 3 with
butylamine. A fresh 0.01 M solution of 1 was prepared in
MeCN. A stock 0.02 M solution of butylamine was prepared
in MeCN. For visible spectroscopic studies, 0.01 mL of
each solution were mixed and volume was brought up
to 1 mL with MeCN or CHCl3 ([1]finalˆ1×10Ϫ4 M;
[n-BuNH2]finalˆ2×10Ϫ4 M). The same procedure was
applied for compounds 2 and 3. For IR studies, 0.1 mL
of each solution were mixed and volume completed
to 1 mL with MeCN or CHCl3 ([1]finalˆ1×10Ϫ3 M;
[n-BuNH2]ˆ2×10Ϫ3 M).
Pseudobase 4. Pyrylium salt 1 (400 mg, 0.75 mmol) was
dissolved in an acetone:water 4:1 mixture and K2CO3
was added. The solution turned red. After hydrolysis,
extraction with diethyl ether, and purification by prepara-
tive TLC, compound 4 was isolated as an orange solid
(248 mg, 71%).1H NMR (CDCl3, d in ppm/TMS) 8.10
(2H, d, Jˆ7.4, Ho Ph–CO–CyC) 7.95 (2H, d, Jˆ7.5, Ho
Ph–CO) 7.53 (6H, m, Hm,p–Ph) 5.67 (2H, d, Jˆ6.1, Ho
Bct) 5.50 (1H, t, Jˆ5.9, Hp Bct) 5.38 (2H, t, Jˆ6.1, Hm
Bct) 4.66 (2H, s, CH2). IR (CH2Cl2, n in cmϪ1) 1968,
1893 (CxO) 1689, 1659, 1599 (CyO, CyC). UV–
visible (MeCN, lmax in nm, e in L molϪ1.cmϪ1) 442,
3500.
Hydrolysis of 1, 2 and 3. Fresh 0.01 M solutions of 1, 2
and 3 were prepared in MeCN. To 0.01 mL of pyrylium
solution were added 0.99 mL of buffer pH 4, 5, 6, 7 or 8
([pyrylium]finalˆ1×10Ϫ4 M), except for 1 for which 0.7 mL
of buffer were added to 0.01 mL of pyrylium solution and
0.29 mL of MeCN. Absorption monitoring at 550 (2 and 3)
or 600 nm (1) was immediately started.
[N-Butyl-2,6-diphenyl-4-benchrotrenylpyridinium]BF4
7. To a blue solution of 1 in CH2Cl2 (140 mg, 0.26 mmol)
was added an excess of n-butylamine. The red solution
was diluted with diethyl ether and the pyridinium salt
Reaction of BSA with 1. To 0.9 mL of a 50 mM solution of
BSA at pH 9.0 was added 0.1 mL of a 1×10Ϫ3 M solution of
1 in MeCN (1:protein ratioˆ2:1). Reaction was monitored
by visible spectroscopy.
1
7 precipitated as red crystals (120 mg, 80%). H NMR
(CD3CN, d in ppm/TMS) 7.99 (2H, s, H pyridinium)
7.70 (10H, s, Ho,m,p Ph) 6.31 (2H, d, Jˆ6.8, Ho Bct) 5.90
(1H, t, Jˆ6.7, Hp Bct) 5.69 (2H, t, Jˆ6.7, Hm Bct) 4.28 (2H,
m, NCH2) 1.37–0.77 (4H, m, CH2–CH2) 0.40 (3H, t, Jˆ7.0,
CH3). IR (CH2Cl2, n in cmϪ1) 1980, 1907 (CϵO) 1619
Labelling of BSA with pyrylium salts 1, 2 and 3
Solutions of BSA (3.3 g/L) were prepared in buffers of pH
ranging from 5 to 9. The exact concentration was measured
spectroscopically (e280 ˆ 35; 700). Fresh 0.01 M solutions
of pyrylium salts 1, 2 and 3 were prepared in acetonitrile. To
(CyC). UV–visible (MeCN, lmax in nm,
e
in
L molϪ1 cmϪ1) 487, 3000.

B. Caro et al. / Tetrahedron 56 (2000) 257–263
259
Scheme 1. Preparation of complex 1.
0.45 mL of protein solution was added 0.05 mL of pyrylium
solution (initial [pyrylium]/[protein]ϳ20). Mixtures were
stirred for 24 h at room temperature and protein conjugates
were purified by dialysis against 10 mM NH4HCO3. Sample
protein concentration was measured by a standard colori-
metric assay.5 Sample pyridinium concentration was
measured by visible spectroscopy taking compounds 7, 8
the expected organometallic pyrylium salt, together with
other uncharacterised products.
To circumvent this problem, we decided to test an alterna-
tive route based on the second strategy. As the reaction of
orthoesters with carbonyl compounds in the presence of
Lewis or Brønsted acids, is a general and inexpensive
route to 2,6- and 2,4,6-substituted pyrylium salts, we tried
to obtain 1 by heating a mixture of triethylorthobenzoate
chromium tricarbonyl complex, acetophenone and BF3 in
acetic anhydride. In these conditions, pyrylium ring
formation occurred but was accompanied with decomplex-
ation probably because of the rather harsh reaction con-
ditions chosen, leading to 2,4,6-triphenylpyrylium salt 10
(Scheme 1).
and
9
as standards (e485ꢀ7†ˆ2; 400; e460ꢀ8†ˆ3; 400;
e462ꢀ9†ˆ4; 100 in H2O/MeCN 99/1). Coupling ratios CR
and the coupling yields CY were deduced from these
measurements.
Results and Discussion
Synthesis of pyrylium salt 1
We found that the use of the more reactive silyl enol ether of
acetophenone allowed milder reaction conditions (0ЊC in
CH2Cl2) and led to the expected complex 1 in good yield
(Scheme 1). To our knowledge, the use of silyl enol ethers in
pyrylium synthesis is unprecedented and could facilitate the
synthesis of unstable pyrylium salts.
To prepare organometallic derivatives of pyrylium salts, one
can envisage two strategies. The first one requires incor-
poration of the organometallic fragment into an oxygenated
heterocyclic derivative, while the second one involves
building of the heterocycle onto an organometallic complex.
In the past, we applied the second route to prepare several 4-
benchrotrenyl-2,6-dimethylpyrylium salts.2a Using the first
approach, we have recently achieved the synthesis of the
first non-metallocenic pyrylium salts.6 Synthesis of [2,6-
diphenyl-4-benchrotrenylpyrylium]BF4 1 was first tried by
the Milaev method, which belongs to the first strategy.7
However, treatment of 2,6-diphenylpyrylium tetrafluoro-
borate with benchrotrenyl lithium, followed by the addition
of trityl cation, led in our hands, to only a small amount of
Reaction of 1 with butylamine in organic solvents
The mechanism of the pyrylium–pyridinium conversion has
been thoroughly studied (Scheme 2).8
Addition of the amine occurs at positions 2 and 6 and yields
the first intermediate 2H-pyran which generally has a very
short life-time. This ring spontaneously opens to lead to the
Scheme 2. General mechanism of the reaction of pyrylium ions with primary amines.

260
B. Caro et al. / Tetrahedron 56 (2000) 257–263
Table 1. Spectroscopic data of the divinylogous amide resulting from the reaction of 1 with n-butylamine
Compound
d in ppm/TMS (CD3CN)
nmax (cmϪ1) (MeCN)
lmax (nm) (e, cmϪ1MϪ1) (MeCN)
7.94 (2H, d, Jˆ7.5, Ho Ph–CO)
7.51 (3H, s, Hm,p Ph–CO)
7.18 (5H, s, Ho,m,p Ph–CyC)
6.60 (1H, s, H3)
1962, 1885 (CO)
1640 (CO)
453 (12800)
5.70 (2H, d, Jˆ6, Ho Bct)
5.45 (1H, s, H5)
5.31 (1H, t, Jˆ6.2, Hp Bct)
5.15 (2H, t, Jˆ6.3, Hm Bct)
3.77 (2H, br q, NH–CH2)
1.70–0.9 (7H, m, CH2–CH2–CH3)
second intermediate divinylogous amide which is usually
more stable. This divinylogous amide tautomerises into an
imino-enol which rapidly cyclises into the pyridinium.
Tautomerisation has been shown to be the rate-determining
step with aliphatic amines.9 Concurrently, the pyrylium ion
and divinylogous amide can convert into a diketone (also
called pseudobase) in the presence of water.
as indicated by the good resolution of the spectrum (vinylic
protons give narrow singlets).
1
After 18 h, the H NMR spectrum of the solution revealed
the presence of only two species, namely pyridinium 7 and
n-butylamine and no trace of pseudobase 4, indicating that
the conversion of pyrylium 1 to pyridinium 7 was complete.
Upon addition of 2M equiv. of n-butylamine to a solution of
pyrylium ion 1 in CD3CN, the solution turned immediately
to yellow-orange then slowly to red. After 15 min, the H
NMR spectrum of the reaction mixture revealed the
presence of three species, namely n-butylamine, pyridinium
7 (in a weak proportion) and a third compound which was
not readily identified. Chemical shifts and multiplicities for
this compound are reported in Table 1, together with other
spectroscopic data.
The formation of N-butyl-4-benchrotrenyl-2,6-diphenyl-
pyridinium 7 was also monitored in the mid-IR and the
visible spectral ranges. In the 1800–2200 cmϪ1 region,
two nCO bands at 1969 and 1900 cmϪ1 were immediately
detected after mixing 1 and n-butylamine in chloroform
(Fig. 2A). These bands disappeared within 50 min while
two other nCO bands at 1981 and 1914 cmϪ1 simultaneously
appeared. In the visible range, an absorption maximum at
442 nm was detected immediately after mixing 1 and butyl-
amine. The conversion of this intermediate to 7 was char-
acterised with simultaneous bathochrome and hypochrome
effects with a final absorption maximum at 485 nm (Fig. 2B).
A similar behaviour was noticed in MeCN but took place
over a much longer time. Interestingly, the formation of the
divinylogous amide was instantaneous in both solvents.
1
Chemical shifts of the benchrotrenyl group at low field
indicated that this compound was a neutral species. The
presence of two vinylic protons at 6.6 and 5.45 ppm was
consistent either with the divinylogous amide or imino-enol
forms. Three groups of aromatic protons were observed in
the 7–8 ppm region, in particular a doublet for two protons
at 7.95 ppm, characteristic of protons in ortho position of
aromatic ketones (like acetophenone). In contrast, the
phenyl protons of phenylmethyl N-methyl imine appear at
7.5 ppm.10 In conclusion, it seems most probable that the
Kinetics of transformation of 1ϩn-butylamine into 7 was
monitored at 442 nm (in CHCl3) or at 453 nm (in CH3CN).
In all cases, kinetic data were consistent with a first order
reaction rate. Rate constants kobs are reported in Table 2 and
compared to those of the two related 4-benchrotrenyl pyry-
lium salts 2 and 3 and of [2,4,6-triphenylpyrylium]BF4 10
calculated from the data reported by Katritzky and Manzo.11
1
intermediate detected by H NMR was the divinylogous
amide. Moreover, at room temperature there seems to be
only one conformer out of the several possible conformers,
Figure 2. (A) IR study of the reaction of 1 (1 mol) with butylamine (2 mol) in CHCl3. (B) UV–visible study of the reaction of 1 (1 mol) with butylamine
(2 mol) in CHCl3.

B. Caro et al. / Tetrahedron 56 (2000) 257–263
261
Table 2. Reaction of pyrylium ions (1 mol) with butylamine (2 mol);
kinetic data
stability in water. Hydrolysis of pyrylium ions shows a
pseudo-first order kinetics, and pH-dependent kobs values
have been measured. Rate constant kobs is the sum of the
pseudo-first-order constant of formation kf of pseudobase
and kr of its reversion to pyrylium (Eq. (1)). The rate
constants of the forward and the reverse reactions kf and kr
can be calculated by applying the following empirical equa-
tions:
Compound
104 kobs (sϪ1
)
Reference
CHCl3
MeCN
1
2
3
10
10
nd
nd
4.72
0.58
Instantaneous
68
0.32
This worka
This workb
This workb
11c
kobsˆkfϩkr
ꢀ1†
ꢀ2†
ꢀ3†
a At 20ЊC.
b At 25ЊC.
Kˆkf=kr
c At 25ЊC, calculated from the published values of 2.95×10Ϫ4 and
0.2×10Ϫ4 sϪ1 (correction factor: 1.6) to take into account that BFϪ4 was
the counter-anion instead of ClOϪ4 :
kfˆꢀ1=ꢀ1ϩ1=K††kobs
Rate of formation of 7 was roughly twice as high as that of
N-butyl-2,4,6-triphenylpyridinium. A strong solvent effect
was observed for the rate of reaction of 1 and 10 (rates
approximately 15 times faster in CHCl3 than in MeCN).
This indicates that the rate-determining step is identical in
the uncomplexed and complexed series. This rate-determin-
ing step is accompanied by a conformational change from
an s-cis, s-trans to an s-cis, s-cis conformation. The Cr(CO)3
fragment could destabilise the s-cis, s-trans conformation
The behaviour of pyrylium ion 1 in water was studied by
visible spectroscopy. In the range of pH studied (from 4 to
8), fast and complete conversion of 1 to pseudobase 4 was
observed, as evidenced by comparison of the UV–visible
spectrum of the aqueous solutions at infinite time with that
of authentic 4. The conversion obeyed first-order kinetics
and rate constants kobs are reported in Table 3 together with
those of pyrylium ion 10.
because of sterical constraints, inducing an increase of kobs
This effect is nevertheless modest.
.
It appears that, in the range of pH studied, uncomplexed
pyrylium ion 10 appears kinetically more resistant than 1
to attack by water. It has been established that the rate-
determining step of the pyrylium/pseudobase inter-conver-
sion was the oxodienol to diketone conversion.13b In this
viewpoint, it means that the conversion to diketone is
slightly favoured in the case of 1. This could be related to
the electron-withdrawing property of the Cr(CO)3 group that
may enhance the acidity of the enol and favour conversion
to the ketonic form (for a discussion on the electron-with-
drawing effect of the Bct group, see Ref. 14). Previous
experimental and theoretical studies in the phenylpyruvic
acid (Ph–CH2–CO–COOH) series showed that the Bct
fragment destabilised the enolic system in alpha by altera-
tion of the conjugation between the aromatic ring and the
enol double bond.15
Much more noticeable was the effect of the nature of the
substituents on the pyrylium ring. Hence, the rate of forma-
tion of 8 resulting from the reaction of 2 with n-butylamine
was comparatively very fast (Table 2). The lower steric
crowding brought by the two methyl groups in positions 2
and 6 and the resulting decreased conjugation of the s-cis,
s-trans form could favour the isomerisation in s-cis, s-cis
and therefore cyclisation. Addition of a CH2–CH2 bridge to
2 to give 3 decreased the rate of reaction, which could be
due to an increase of the steric crowding in the transition
state.
Behaviour of pyrylium ions 1, 2 and 3 in aqueous
medium
0
A pKa of 4.4 was measured for the 2,4,6-triphenylpyrylium
Hydrolysis studies of pyrylium salts at different pH is well-
documented.12,13 In aqueous medium, pyrylium ions equili-
brate with their pseudobase via 2H-pyran and oxodienol
intermediates (Scheme 2). The overall pyrylium–pseudo-
base equilibrium is pH-dependent and characterised by an
ion/pseudobase equilibrium.13b This relatively good
stability of 10 in water was explained by the mesomeric
effect of the phenyl substituents which stabilises the
pyrylium ring. Conversely, total conversion of 1 to 4 in
the same range of pH seems surprising since the Cr(CO)3
fragment has long been known to stabilise positive charges
at the benzylic position.16 In the pyrylium series, we have
ϩ
0
apparent dissociation constant Ka ˆ[pseudobase][H ]/
[pyrylium]. This constant is a measurement of pyrylium
Table 3. Hydrolysis of 1, 2, 3 and 10 at 20ЊC (kinetic parameters)
1a
10b
2c
3a
pH
kobs (minϪ1
)
kobs (minϪ1) (pH)
kobs (minϪ1
)
kf (minϪ1
)
kr (minϪ1
)
K
kobs (minϪ1
)
kf (minϪ1
)
kr (minϪ1
)
K
4.1
5.1
6.1
7.0
8.1
0.2
0.01 (4.28)
0.075 (4.90)
0.045
0.32
2.04
6.07
fast
0.033
0.32
2.04
6.07
–
0.012
2.8
n.d.
n.d.
n.d.
0.0022
0.008
–
n.d.
0.36
2.31
0.27
0.47
0.64
1.67
–
–
–
–
0.003
0.027
0.14
0.73
0.0008
0.019
0.14
0.148 (7.34)
0.39 (8.04)
0.73
–
a In H2O/MeCN 7/3.
b Ref. 13.
c In H2O/MeCN 99/1.

262
B. Caro et al. / Tetrahedron 56 (2000) 257–263
1
Table 4. Reaction of BSA with pyrylium salts 1, 2 and 3 (measurement of
the coupling ratio CR and the coupling yield CY; reaction conditions: see
Section 2)
shown by H NMR and EHMO calculations, that enhanced
delocalisation of the positive charge occurred from the
pyrylium ring to the Bct fragment.2b The lower stability of
1 in water could be explained by the nature of the medium.
In the constraint molecule 1, the presence of the Cr(CO)3
fragment could disturb the solvation of the pyrylium ring by
water. To partly prevent this desolvation process, 1 could
adopt a conformation in which the benchrotrenyl and pyry-
lium rings are less conjugated and could contribute to an
overall destabilisation of the complexed pyrylium in water.
However, complementary work, including determination of
activation parameters would be necessary to fully under-
stand this difference of stability.
pH
5.1
6.1
7.0
8.1
9.0
1
[BSA] (mM)
[pyridinium] (mM)
CRa
28
55
1.9
9
26
41
38
53
137
5.3
24
326
8.0
36
399
18.4
84
847
16.0
73
CY (%)b
2
[BSA] (mM)
[pyridinium] (mM)
CR
37
50
64
53
46
294
8.0
38
350
7.2
35
443
6.9
48c
497
9.4
47
420
9.2
45
CY (%)
For comparison purposes, we also studied the behaviour of
the two other 4-benchrotrenyl pyrylium ions 2 and 3 in
water in the same range of pH. Compound 2, with two
methyl substituents in positions 2 and 6, showed complete
conversion to 5 at pH 5 and above, as evidenced by the
visible spectrum of the final species. At pH 4.1, approxi-
mately 26% of pyrylium ion 2 remained at equilibrium
(Kˆ‰5Š=‰2Šˆ2:8), indicating that hydrolysis was a reversible
process as previously noticed. Partial reverse conversion of
pseudobase 5 to pyrylium ion 2 was indeed observed at pH
3
[BSA] (mM)
[pyridinium] (mM)
CR
42
48
58
64
60
227
5.5
26
217
4.5
22
385
6.6
33
502
7.8
39
422
7.0
34
CY (%)
a CRˆ[pyridinium]/[BSA].
b CYˆCR/([pyrylium]/[BSA])initial×100.
c ([2]/[BSA])initialˆ14.
4.1 with a first-order rate constant kobs of 0.034 minϪ1
.
protein, is a very convenient model to study amine-reactive
protein labelling agents because of its rather high content in
lysines (59). The reaction of BSA at pH 9 with 2 M equiv. of
1, (30:1 amine:pyrylium molar ratio), was monitored spec-
troscopically. Immediately after mixing both compounds,
the solution turned yellow-orange (lmaxˆ471 nm). This
was followed by a shift towards the high wavelengths within
7 h, together with a hypochrome effect, very similar to what
had been observed during the reaction of 1 with n-butyl-
amine (Fig. 2B). The maximum of absorption reached
484 nm, which was very close to that of 7. This conversion
showed first order kinetics with a rate constant kobs of
1.3×10Ϫ4 sϪ1. In these conditions, formation of BSA–pyri-
dinium adduct was quantitative.
Kinetic data of the hydrolysis of 2 were consistent with a
first-order reaction rate and rate constants kobs are also
reported in Table 3. At pH 5 and above, hydrolysis of 2
was faster than that of 1, probably because of the presence
of the two methyl substituents instead of the bulky phenyl
groups.
The third pyrylium salt studied 3 also possesses two methyl
substituents in positions 2 and 6 and an additional
cyclohexyl ring at positions 4 and 5. Although it is very
close in structure to 2, its behaviour in water was totally
different. In fact, we observed complete hydrolysis of 3 into
6 only at pH 7 and above, while at pH 6 and below,
conversion of 3 was partial. This allowed us to measure
spectrophotometrically an apparent dissociation constant
A series of labelling experiments of BSA was then carried
out with 1, 2 and 3 and initial pyrylium ion: protein around
20:1 at pH 5, 6, 7, 8 and 9. Reaction mixtures were stirred
for 24 h at room temp. and protein conjugates were purified
by dialysis. We first noticed that BSA–pyridinium adducts
were formed in all the conditions tested, as shown by the
visible and IR spectra of the resulting conjugates, that
closely resembled those of 7, 8 and 9.
Ka ˆ10Ϫ5.8 M. The reversibility of the reaction was also
0
confirmed by the partial conversion of 6 to 3 at pH 6 and
below. All these processes obeyed first order kinetics and
rate constants kobs of the conversion of 3 to 6 are displayed in
Table 3. Pyrylium ion 3 appeared kinetically more stable in
water than 1, 2 and 10. This could be due to favourable
steric effects. Moreover, pyrylium 3 is also thermodynami-
0
cally more stable (pKa ˆ5.8). The presence of the additional
fused ring, that blocks the coplanar conformation, could
thus provide an enhanced mesomeric overlap of the pyry-
lium ring with the benzene Cr(CO)3 group. In this case, the
electron-donating ability of the phenyl Cr(CO)3 fragment
towards the pyrylium ring could fully act.
We then measured the coupling ratio CR of each conjugate,
which is defined as the number of benchrotrenyl pyridinium
grafts bound per protein molecule. To do that, protein
concentration was measured by the Coomassie blue
method5 and pyridinium concentration was measured by
visible spectroscopy, taking 7, 8 or 9 as standards, respec-
tively. The coupling yields CY were deduced from these
measurements and the resulting data are reported in
Table 4. We observe that results differ markedly from one
compound to another. The yield of conjugation of 1 to BSA
was found to be dependent on the pH of the reaction
medium, with the highest CR (and CY) reached at pH 8.
For 2 and 3, the influence of pH on the coupling yield was
less significant. Additionally, a variable amount of orange
precipitate was isolated at the end of the reaction of 1 with
In summary, the kinetic behaviour of pyrylium ions 1, 2 and
3 towards n-butylamine and water was found to be very
close to that of the related uncomplexed salts. Their thermo-
dynamic stability in water was found to be highly dependent
on the substitution pattern of the pyrylium ring.
Reaction of pyrylium ions 1, 2 and 3 with BSA
Bovine serum albumin (BSA), a 66 kDa single chain, acidic

B. Caro et al. / Tetrahedron 56 (2000) 257–263
263
BSA. Its IR spectrum showed that this compound was
pseudobase 4, which resulted from the competitive hydro-
lysis of 1, and was insoluble in the reaction medium at the
working concentration.
applied to prepare 5d transition metal complexes (contain-
ing tungsten, rhenium or osmium for instance). These path-
ways will make up our future research in the pyrylium
chemistry field. At the same time, labelling of protein
crystals will be attempted in order to validate our approach.
The mechanism of reaction of pyrylium ions with lysine and
protein lysine residues in water has been previously estab-
lished.1b Initially, a mixture of pseudobase and divinylogous
amide is formed, resulting from the competitive attack of
water and amine. The divinylogous amide is converted to
the pyridinium while the pseudobase is able to slowly
convert into the divinylogous amide or to decompose via
its pseudoanion. The relative rate of these two reactions
varies with the structural pattern of the pyrylium. In the
case where the pseudobase–divinylogous amide conversion
is faster than the decomposition reaction, the pseudobase
reacts with the amine and leads to the pyridinium.
Acknowledgements
We thank the Centre National de la Recherche Scientifique
(CNRS) for financial support.
References
1. (a) O’Leary, M. H.; Samberg, G. A. J. Am. Chem. Soc. 1971, 93,
3530–3532. (b) Katritzky, A. R.; Mokrosz, J. L; Lopez-Rodriguez,
M. L. J. Chem. Soc., Perkin Trans. 2 1984, 875–878. (c) Dill, K.;
Hu, S. H.; Sutharchanadevi, M.; Katritzky, A. R. J. Protein Chem.
1988, 7, 341–348. (d) Fernandez, A.; Katritzky, A. R.; Sutharch-
anadevi, M.; Stevens, B. R. Biochem. Biophys. Res. Commun.
1989, 163, 1356–1363.
Because of the insolubility of 4 in the reaction medium, the
yield of formation of the pyridinium adduct should be
related to the relative rates of formation of pseudobase
and divinylogous amide as a function of pH. It seems prob-
able that the rate of formation of the divinylogous amide
increases with the pH, owing to the deprotonation of amine,
so that maximum yield is reached at basic pH.
´ ´
´ ´
2. (a) Caro, B.; Senechal-Tocquer, M.-C.; Senechal D.; Marrec, P.
Tetrahedron Lett. 1993, 34, 7259–7262. (b) Malisza, K. L.; Top,
´ ´
´ ´
S.; Vaissermann, J.; Caro, B.; Senechal-Tocquer, M.-C.; Senechal,
D.; Saillard, J.-Y.; Triki, S.; Kahlal, S.; Britten, J. F.; McGlinchey,
M. J.; Jaouen, G. Organometallics 1995, 14, 5273–5280.
Conclusions
´ ´
3. Salmain, M.; Malisza, K. L.; Top, S.; Jaouen, G.; Senechal-
´ ´
The reactivity of three 4-benchrotrenyl pyrylium ions with
several molecules was compared. In organic solvents, the
pyrylium salts all reacted with n-butylamine by the well-
known O,N-exchange reaction and yielded the correspond-
ing pyridinium salts. However, rates of reaction were found
to be highly dependent on their substitution pattern and on
the polarity of the solvent, with the conversion of the
divinylogous amide to the pyridinium being the rate-deter-
mining step.
Tocquer, M.-C.; Senechal, D.; Caro, B. Bioconjugate Chem.
1994, 5, 655–659.
4. Branden, C.; Tooze, J. Introduction to Protein Structure;
Garland: New York and London, 1991; pp 269–285.
5. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
´ ´
6. Caro, B.; Robin-Le Guen, F.; Senechal-Tocquer, M.-C.; Pret,
V.; Vaissermann, J. J. Organomet. Chem. 1997, 543, 87.
7. Milaev, A. G.; Okhlobystin, O. Yu. Khim. Geterosikl. Soedin.
1985, 593, 597.
8. Balaban, A. T.; Fischer, G. W.; Dinulescu, A.; Koblik, A. V.;
Dorofeenko, G. N.; Mezhritski, V. V.; Schroth, W. In Adv. Hetero-
cyclic Chem., Suppl. II, Katritzky, A. R. Ed.; Academic Press: New
York, 1982; pp 114–127.
9. Katritzky, A. R.; Mokrosz, J. L.; De Rosa, M. J. Chem. Soc.,
Perkin Trans. 1984, 2, 849–855.
10. Olah, G. A.; Kreienbu¨hl, P. J. Am. Chem. Soc. 1967, 89, 4756.
11. Katritzky, A. R.; Manzo, R. H. J. Chem. Soc. Perkin Trans.
1981, 2, 571–575.
12. Williams, A. J. Am. Chem. Soc. 1971, 93, 2727–2733.
13. (a) Katritzky, A. R.; De Rosa, M.; Grzeskowiak, N. E. J. Chem.
Soc., Perkin Trans. 2 1984, 841–848. (b) Katritzky, A. R.; Leahy,
D. E. J. Chem. Soc., Perkin Trans. 2 1984, 867–873.
In water, the pyrylium ions hydrolysed to their correspond-
ing pseudobase and the rate and extent of hydrolysis were
found to be dependent both on pH and on the substitution
pattern. Pyrylium ion 3 with an additional fused ring
appeared to possess a particularly remarkable thermo-
dynamic stability in water.
Reaction of the e-amino group of some of the lysine residues
of BSA with the pyrylium ions led pyridinium adduct
formation and the yield in pyridinium adducts was found
to be dependent on the pyrylium structure.
´
14. Solladie-Cavallo, A. Polyhedron 1985, 4, 901–910.
Pyrylium salts are very attractive molecules for the side-
chain specific labelling of proteins. Their water-solubility
and reactivity can be tuned by modifying the substituents
(by including sulphonate groups for example)13a and/or by
changing the counter-anion. Moreover, several synthetic
strategies are now available to include a transition metal
complex fragment into a pyrylium structure and could be
´ ´
´ ´
15. Le Bihan, J.-Y.; Senechal-Tocquer, M.-C.; Senechal, D.;
Gentric, D.; Caro, B.; Halet, J.-F.; Saillard, J.-Y.; Jaouen, G.;
Top, S. Tetrahedron 1988, 44, 3565–3574.
16. (a) Holmes, J. D.; Jones; D. A. K.; Pettit, R. J. Organomet.
Chem. 1965, 4, 324–331. (b) Seyferth, D.; Merola, J. M.;
Eschbach, C. S. J. Am. Chem. Soc. 1978, 100, 4124–4131.