Synthesis of hydrophilic Fischer carbene complexes as organometallic marker and PEGylating agent for proteins

Article abstract of DOI:10.1016/j.jorganchem.2005.07.042

Syntheses of Fischer carbene complexes with various hydrophilic tethers (4, 6a, 6b, 6c, 8a, 8b, 14a, 14b, 15 and 19) are described. The water-soluble carbene complex, 14b, was used to label and pegylate bovine serum albumin (BSA) without affecting its conformation. The red complex, 19, underwent a sharp color change to yellow on reaction with BSA, a feature that is potentially useful for developing assay methods.

Full text of DOI:10.1016/j.jorganchem.2005.07.042

Journal of Organometallic Chemistry 690 (2005) 5581–5590
www.elsevier.com/locate/jorganchem
Synthesis of hydrophilic Fischer carbene complexes as
organometallic marker and PEGylating agent for proteins
a
a
b
b
Debasis Samanta , Sudeshna Sawoo , Subrata Patra , Manju Ray ,
c
a,
*
`
Michele Salmain , Amitabha Sarkar
a
Department of Organic Chemistry, Indian Association for the Cultivation of Science, Kolkata 700032, India
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata 700032, India
b
c
Ecole Nationale Superieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Moleculaires (UMR CNRS 7576),
11 rue Pierre et Marie Curie 75231 Paris cedex 05, France
Received 31 May 2005; accepted 4 July 2005
Available online 22 August 2005
Abstract
Syntheses of Fischer carbene complexes with various hydrophilic tethers (4, 6a, 6b, 6c, 8a, 8b, 14a, 14b, 15 and 19) are described.
The water-soluble carbene complex, 14b, was used to label and pegylate bovine serum albumin (BSA) without affecting its confor-
mation. The red complex, 19, underwent a sharp color change to yellow on reaction with BSA, a feature that is potentially useful for
developing assay methods.
ꢀ 2005 Elsevier B.V. All rights reserved.
Keywords: Water-soluble Fischer carbene complex; Bioorganometallics; PEGylation; Protein labelling
1. Introduction
However, Fischer carbene complexes are generally
hydrophobic and insoluble in aqueous medium. This
presents a serious bottleneck for their adaptation to
biological applications. Mixtures of solvents such as
water/acetonitrile have been used [6] but proteins tend
to loose their conformational integrity (hence catalytic
function) in such mixed medium [7] and might thus
interfere with assay results. This prompted us to design
and develop synthesis of water-soluble Fischer carbene
complexes that are biocompatible. To impart hydrophi-
licity to a Fischer carbene core, it appeared to us, one
could incorporate a sugar moiety [8], an ionic group like
NMeþ3 or an oligoethylene glycol/polyethylene glycol
(OEG/PEG) tether (Chart 1). The present study deals
with the last two possibilities.
Non-radioactive labels such as fluorescent, chemi- or
bio-luminiscent labels are being used extensively for
detection of biomolecules in nanogram or microgram
quantities and are replacing the traditional and accurate
but intrinsically hazardous radioactive labels [1]. Re-
cently, Jaouen and others have explored a new method
for labeling protein and other biomolecules with transi-
tion metal carbonyl compounds such as Arene-Cr(CO)3
[2] and Fischer carbene complexes [3,6], etc. Fischer car-
bene complexes [4] are especially attractive because of
their extraordinarily high reactivity towards pendent
amino groups of biomolecules and characteristic strong
IR signals [5] at 1900–2100 cmꢀ1 in the IR absorption
where no organic molecule absorbs.
It may be pertinent to mention that incorporation of
polyethylene glycol (peg) to the Fischer carbene complex
would also constitute a method for the ÔpegylationÕ of a
protein [9]. Pegylation is the process incorporating PEG
chains to a molecule [10]. Pegylated drugs or proteins
*
Corresponding author. Tel.: +91 3324734971; fax: +91
3324732805.
E-mail address: ocas@iacs.res.in (A. Sarkar).
0022-328X/$ - see front matter ꢀ 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2005.07.042

5582
D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
OEt
CH2OH
O
OEt
(CO)5W
MeI
(CO)5W
Br
Br
CH2O
1. BuLi
OH
H
2. W(CO)6
+
NaBH3CN
NMe3+ I -
4 : 50%
-
NMe2
3 : 60%
3. Et3O BF4
OH
OH
NMe2
NH2
2 : 80%
1
R1
Substituted oligo / Polyethylene glycol
R2
n
O
NMe3+I-
O
CH2OH
Sugar
Scheme 1. Synthesis of carbene complex with ionic group.
Ionic Group
Chart 1. Hydrophilic units that could render water solubility.
characterized by spectroscopic methods. The IR spec-
trum showed bands at 1915 and 2065 cmꢀ1, which are
characteristic of a Fischer carbene complex. In the 13C
NMR spectrum, the peak at 321.0 ppm was assigned
to the carbene carbon, typical of a Fischer carbene
complex.
Our initial attempts to quaternize carbene complex 3
with MeI in a common organic solvent like ether, ben-
zene, or pet ether did not meet with success. Therefore,
we dissolved the complex in large excess of MeI and stir-
red the mixture at room temperature. After 48 h, an or-
ange precipitate was deposited (Scheme 1) which was
identified as the quaternized salt 4 by 1H and 13C
NMR and IR spectroscopy. In the proton NMR spec-
trum, position of N-methyl group shifted downfield
from 3.03 to 4.02 ppm after quaternization. The corre-
sponding 13C NMR signal of the N-methyl carbon was
similarly deshielded (from 40.4 to 57.9 ppm). The IR
spectrum showed characteristic absorption at 1942 and
2072 cmꢀ1, typical of a carbene complex.
exhibit increased stability (more resistant to proteolysis),
decreased immunogenicity and increased circulating life
[11]. In some cases peg conjugation was also found to
confer targeting properties to the disease site such as tu-
mor masses by passive diffusion [12]. Effectiveness of
pegylation has been demonstrated by the discovery of
pegylated drugs such as PEG-IFN-a2 which, in combi-
nation with ribavirin, is considered as the Ôgold stan-
dardÕ of therapy for hepatitis C [13]. In the present
study, we report synthesis of relatively hydrophilic
Fischer carbene complexes several of which feature
PEG/OEG groups.
2. Results and discussion
2.1. Synthesis of hydrophilic Fischer carbene complexes
To prepare a water-soluble carbene complex, it was
thought that hydrophobicity of part A of the complex
can perhaps be compensated by a strong hydrophilic
group like an ionic group, or OEG/PEG groups in part
B or part C (Fig. 1).
Our initial attempt was to incorporate an ionic group
NMeþ3 . We, therefore, synthesized carbene complex 3
from m-bromo-N,N-dimethylaniline 2 that was obtained
from m-bromoaniline 1 (Scheme 1). The amino group
was placed at the m-position rather than o- or p- so that
electron density from nitrogen does not get delocalized
towards the M@C bond and makes quarternization a
difficult proposition. Aromatic ring, although a hydro-
phobic fragment, was chosen to attach directly with car-
bene part, since it makes synthesis easier and the color
change from red to yellow would facilitate monitoring
of carbene aminolysis. The carbene complex 3 was fully
However, solubility of complex 4 in water is poor
(<0.05 mg/ml), making the complex practically unsuit-
able for reaction in aqueous medium. Discouraged, we
decided to explore introduction of ethylene glycol units
of different chain lengths (oligo/polyethylene glycol) to
impart hydrophilicity to such carbene complexes.
We started with a readily available and relatively
inexpensive triethylene glycol derivative. The carbene
complex 6a was prepared from the corresponding salt
5a [14] (Scheme 2) in the usual manner using acetyl chlo-
ride in dichloromethane at ꢀ40 ꢁC to generate the
unstable acetoxy carbene, which was allowed to react
with monomethoxytriethylene glycol to furnish the de-
sired product 6a as a dark red liquid. In spite of the
hydrophilicity of triethylene glycol part the complex
was not significantly water-soluble. The carbene com-
+
O-NEt4
Part B
O
O Me
(CO)5W
1. CH3COCl / -40˚
C
n
OR1
(CO)5W
Part A
2.
H
O
Me
O
C
n
(CO)5W
X
-40 ˚
C
0 ˚
C
X
R2
5a : X=H
5b : X=NMe2
6a : X=H, n=3, 38%
6b : X=NMe2, n=3, 30%
6c : X=NMe2, n=11 (avg.), 25%
Part C
R1 = Alkyl
R2 = Alkyl, Aryl
Scheme 2. Synthesis of carbene complex with one oligo/poly ethylene
glycol unit.
Fig. 1. Fischer carbene complex.

D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
5583
+
O-NEt4
O
O Me
n
˚
1. CH3COCl / - 40
C
(CO)5W
(CO)5W
CH3
2.
CH3
H O
O Me
n
n=3, 40%
n=6, 30%
˚
7
- 40 to 0
C
Scheme 3. Synthesis of carbene complex with one oligoethyleneglycol unit that contain no aromatic moiety.
HO
OH
Ph3P/CBr4
H
O
H3C
O
O
O
CH3
n
Br
HO
11a : n=3, 55%
O CH3
Na
88 0C
n
n
9a : n=3
9b : n(avg)=7
10a : n=3, 60%
10b : n(avg)=7, 50%
11b : n(avg)=7, 50%
OCH3
OCH3
C
(CO)5W
C
(CO)5W
13
PBr3
Br
O
CH3
O
CH3
CH
O
O CH3
0 0C
n
50 % NaOH
2
/
TBAB C6H6
12a : n=3, 80%
12b : n(avg)=7, 50%
n
14a : n=3, 40%
14b : n(avg)=7, 30%
8a
50 % NaOH
C H
O
O 3 CH3
TBAB/
6
6
(CO)5W
C
CH
O
O CH3
2
3
15, 25%
Scheme 4. Synthesis of carbene complexes containing oligo/poly (ethylene glycol) tether.
plex 6b featuring a polar NMe2 group on the aryl ring
also did not show any improvement in solubility in
water.1
When a polyethylene glycol unit was incorporated,
the carbene complex (6c) displayed only partial solubil-
ity in water. This modification permitted use of carbene
complex in dilute aqueous solution, but we were typi-
cally interested to make a complex that will have much
greater solubility in water so that reactions can be stud-
ied at higher concentration of the complex.
To achieve this goal, it was decided to do away with
the hydrophobic aromatic part altogether. To this end,
carbene complex 8a with one triethylene glycol unit
was synthesized (Scheme 3). It was characterized fully
by 1H, 13C NMR and IR spectroscopic techniques.
Appearance of characteristic peaks at 1934 and
2069 cmꢀ1 confirmed the formation of a metal carbene
complex. A singlet at 4.89 ppm in proton NMR spectra
was assigned to the protons of methyl group directly at-
tached to carbene part. In the 13C NMR spectrum, the
carbene carbon was identified with the peak at
331.8 ppm. The complex 8a, disappointingly, is practi-
cally insoluble in water. The complex 8b with one hexa-
ethylene glycol unit, prepared in the same manner,
showed some improvement in aqueous solubility
(<5 mg/ml).
In our attempt to incorporate two OEG/PEG units
units on one molecule of Fischer carbene complex, we
preferred part C (Fig. 1) rather than part B because ami-
nolysis – displacement of alkoxy group with amino
group – displaces the glycol chain in part B while part
C remains intact. It was readily perceived that the
phase-transfer catalyzed bis-allylation protocol devel-
oped in our laboratory was an ideal method to introduce
two desired chains easily on one molecule under mild
condition [15]. The compound 12a, where both allylic
halide as well as triethylene glycol unit present, was
synthesized (Scheme 4). First, triethylene glycol mono-
methyl ether was brominated with CBr4/triphenylphos-
phine [16] to afford the bromo compound 10a. The
bromo compound 10a was subsequently treated with
the monoalkoxide of cis-2-buten-1,4-diol to generate
the alcohol 11a. Bromination of the alcohol 11a with
PBr3 [17] afforded compound 12a featuring one triethyl-
ene glycol unit and one allylic bromide. Using our pro-
cedure, methoxymethyl carbene complex 13 was treated
with the bromo compound 12a in presence of 50%
NaOH and tetrabutylammonium bromide phase transfer
1
Our attempts to quaternize 6b or 6c in the usual way with methyl
iodide were not successful.

5584
D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
Br
Ag2O
O
O
CH3
H
O
O
H
CH3I
Ag2O
O
H
O
O
CH3
O
6
6
6
17, 70%)
16, 50%)
[Ru]
CH3
6
O
2
O
PPh3
Cl
Cl
Ru
[Ru]
Ph
PPh3
EtO
Grubbs' first generation catalyst
W(CO)5
19, 30%
EtO
W(CO)5
18
Scheme 5. Synthesis of carbene complex 19.
catalyst (5 mol%) at room temperature (Scheme 4). The
diallylated product 14a was obtained as a yellow liquid.
All products were satisfactorily characterized by proton
NMR, 13C NMR and IR spectroscopic techniques. The
characteristic peaks in the 1H NMR spectrum of the final
product 14a are two multiplets in the range of 1.90–
2.45 ppm for the two sets of methylene protons and a
quintet for the methine protons (W@CCH) centered at
4.12 ppm. Also, four olefinic protons of the allyl groups
appear at 5.44–5.67 ppm. A sharp singlet at 4.60 ppm
was assigned to the protons of methoxy group attached
to carbene carbon. The characteristic peaks in 13C
NMR spectra are due to the carbene carbon at 341.3
ppm and two olefinic carbons at around 128 ppm. The
signals due to coordinated carbon monoxide appear at
197.0 and 202.8 ppm. In the IR spectrum, characteristic
sharp signals are observed at 1938 and 2069 cmꢀ1, as
anticipated.
14b was much more soluble in water (55 mg/ml). The
complex 14b was fully characterized by standard spec-
troscopic methods. It was eventually used in protein
modification (vide infra).
However, since no aromatic ring is present in the
water-soluble carbene complex 14b, the complex is
yellow in color that is expected to remain yellow after
reaction with pendent amine groups of proteins. Thus,
the reaction could not be followed by the visual color
change. Similarly in the UV–vis spectrum also, a slight
shift of peak position is expected.
An alternative synthetic strategy to introduce OEG
or PEG tethers in Fischer carbene complexes involved
alkene metathesis, as depicted in Scheme 5. The complex
19 features an aromatic ring adjoining the carbene car-
bon unlike the complexes 14a–b or 15. Hexaethylene
glycol was first monomethylated with methyl iodide in
presence of silver oxide [18] to afford compound 16 fol-
lowed by monoallylation to obtain compound 17.
Alkene metathesis reaction [3] with Fischer carbene
complex 18 by GrubbsÕ first generation catalyst pro-
vided red carbene complex 19 that is partially soluble
in water (solubility 1 mg/ml). This compound exhibited
less solubility (1 mg/ml) in water than complex 14b.
Although the carbene complex 14a contains two
triethylene glycol units, its solubility in water is still
below expectation. Complex 15 featuring three units of
triethylene glycol prepared along the same lines did
not improve the situation. Incorporation of PEG
(MWavg = 350) chains proved beneficial – the complex
Table 1
Solubility comparison of different carbene complexes
Type of oligoethylene glycol (OEG)
Compound
no
No. of repeating
ethylene oxide unit
No. of ionic
groups
No. of aromatic
moiety present
Solubility in water
Not present
Not present
3
0
0
3
3
0
1
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
0
1
1
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
3 mg/ml (0.003 mmol/ml)
2 mg/ml (0.003 mmol/ml)
1 mg/ml (0.001 mmol/ml)
5 mg/ml (0.005 mmol/ml)
4
Triethylene glycol
Triethylene glycol
Triethylene glycol
Triethylene glycol
Triethylene glycol
Hexaethylene glycol
Hexaethylene glycol
Polyethylene glycol
(Mn = 750 for monomethoxy)
Polyethylene glycol
(Mn = 350 for monomethoxy)
6a
6b
8a
14a
15
8b
19
6c
3
3 + 3
3 + 3 + 3
6
6
11 (avg.)
14b
7 + 7 (avg.)
0
0
55 mg/ml (0.057 mmol/ml)

D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
5585
`
A comparison of solubility features vis-a-vis OEG/
PEG content (Table 1) reveals that two PEG
(MWavg = 350) tethers together can impart desired
hydrophilicity to such carbene complexes. Complex 14b
was selected for exploration of anchoring Fischer carbene
complexes on protein surfaces as discussed below.
A
B
2000
1900
1800
2000
1900
1800
2.2. Water-soluble carbene complex for protein
modification
C
A rather robust protein, bovine serum albumin
(BSA), was selected for preliminary studies. It has a to-
tal of 60 pendent lysine residues. The conformation of
BSA is such that some of the lysine amino groups are
exposed while some are located in the interior. The
exposed amino groups would react with the carbene
complex readily. Since a molecule of the carbene
complex 14b possesses two polyethylene glycol units,
reaction with an amino group of the protein would
result in the incorporation of two branches of PEG units
per amine in one operation (Scheme 5). Such mild yet
extremely efficient transformation would make it one
of the choicest methods for pegylation of protein. It
would offer two extra advantages: (i) a heavy metal
atom to aid protein X-ray crystallography is incorpo-
rated; (ii) the organometallic IR label is incorporated
to make a non-radioactive assay feasible.
2000
Fig. 2. IR spectrum of (A) methoxycarbene complex 14b (B) amino-
carbene complex generated by the reaction with butyl amine
(C) protein–carbene conjugate.
BSA
BSA carbene conjugate
1.0
0.5
0.0
Reaction of BSA with carbene complex 14b was per-
formed in borate buffer at pH 9 (Scheme 6) according to
the procedure described by Jaouen and coworkers [6].
Thus, a solution of BSA (100 lM, 1 ml) and carbene
complex (6000 lM, 1 ml) was incubated in borate buffer
(pH 9) at 25 ꢁC for 6 h. The reaction mixture was
applied to a gel filtration column (35 · 1.5 cm) packed
with sephadex G-50 to separate protein carbene conju-
gate from unreacted carbene complex. Two clearly sep-
arated yellow bands were thus observed; the upper band
corresponded to the unreacted carbene complex and the
lower band corresponded to the protein-carbene
conjugate.
-0.5
190
200
210
220
230
240
250
Wavelength (nm)
Fig. 3. CD spectrum of: BSA (black curve); BSA–carbene conjugate
(red curve).
The product 20, a protein conjugated with several
branched PEG as well as IR active organometallic label,
was characterized by IR (Fig. 2), UV–vis (Fig. 4A) and
W(CO)5
H
NH2
N
rPEG
Borate
Buffer
CH
OCH3
+
Protein
NH2
(CO)5W
Protein
NH2
rPEG
rPEG
pH = 9
rPEG
CH
20
PEG tether
14b n(avg)=7
rPEG =
O
O
CH3
n
Scheme 6. Reaction of protein (BSA) with carbene complex for PEGylation and labeling.

5586
D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
361
b
A
B
0.4
0.2
0.0
346
a
a
0.016
0.008
b
400
500
300
200
600
400
wavelength / nm
Wave length / nm
Fig. 4. (A) UV–vis spectra of methoxycarbene complex 14b (curve a) and BSA–carbene conjugate (curve b) (B) UV–vis spectra of carbene complex
19 (curve a) and BSA–carbene conjugate (curve b).
CD spectra (Fig. 3). A characteristic shift of the wave-
number of the prominent mco band from 1934
(Fig. 2A) to 1926 cmꢀ1 (Fig. 2C) for 20 was observed.
The shift of these bands towards the lower wavenumbers
as compared to compound 14b was consistent with the
higher electron-donating ability of aminocarbenes. A
similar change was observed when the reaction was per-
formed with butyl amine rather than BSA (Fig. 2B).
There is no appreciable change in the CD spectrum of
BSA after reaction with carbene complex (Fig. 3) imply-
ing that the conformation of BSA remained practically
unaltered in spite of the organometallic conjugate.
Quantification of the number of amino carbene
adducts per protein molecule was done by the method
described by Jaouen and others [6]. First, the concentra-
tion of protein was measured by the colorimetric assay
described by Bradford [19]. The concentration of amino-
carbene complex was then determined spectroscopically,
taking the adduct generated in situ by the reaction of
carbene complex 14b with an excess of n-butylamine
[e(346 nm) = 5200 l molꢀ1 cmꢀ1] as standard. It was
thus estimated that 18 out of 60 amino groups of BSA
were converted to aminocarbenes.
In the similar way, we performed reaction of red com-
plex 19 with protein BSA in the borate buffer at pH 9 to
study the reaction more easily by naked eye as well as by
UV–vis spectroscopy. Our preliminary experiment
showed a clear change of color from red to yellow within
4 h after reaction with protein BSA. In the UV–vis spec-
tra, the characteristic peak of ethoxy carbene at 417 nm
vanished completely after reaction (Fig. 4B) unlike yel-
low carbene complex 14b (Fig. 4A). In the IR spectra
corresponding shift of peak position from 1934 to
1924 cmꢀ1 was observed.
BSA in aqueous buffer to provide aminocarbene biocon-
jugates. (2) The W(CO)5 moiety may serve as a useful IR
marker for non-radioactive assay. This strategy of incor-
poration of heavy metal atom like tungsten into protein
molecules could also aid protein crystallography. (3)
Attachment of the carbene complex simultaneously
accomplishes facile and efficient pegylation of the pro-
tein, a method that should find wide application in drug
research.
4. Experimental
General considerations: All reactions were carried out
under an atmosphere of argon. Ether and benzene were
distilled under argon from sodium and benzophenone.
Dichloromethane was dried over P2O5 in the usual
way. Compound 5a [20], 5b [20], 7 [20], 13 [21], 18 [22]
were synthesized following reported procedures. All
chemicals were purchased from commercial suppliers
(Aldrich, Strem, Merck) and used as received. Infrared
spectra were recorded on a Shimadzu FTIR-8400 spec-
trometer and absorptions are expressed in cmꢀ1. The
1H and 13C NMR spectra were obtained on a Bruker
AC300 spectrometer. Elemental analyses were per-
formed using a Carlo-Ebra 1100 automatic analyzer.
UV–vis spectra were recorded on a Varian Cary 50
Bio spectrophotometer. The spectral background
absorption was subtracted by using the UV–vis spectra
of the same solvent mixture.
4.1. Synthesis of m-bromo-N,N-dimethylaniline (2)
To a stirred solution of m-bromoaniline (430 mg,
2.5 mmol) in acetonitrile, formaline (1 ml) was added
by cooling. Stirring was continued for 3 min. Sodium
cyanoborohydride (250 mg, 4 mmol) was added por-
tionwise and the mixture was stirred for another
25 min. Glacial acetic acid was added drop-wise until
solution became neutral. Stirring was continued for
another 45 min with the occasional addition of acetic
acid to keep the pH neutral. Solvent was evaporated
3. Conclusion
In conclusion, we have described herein the following
significant results: (1) This is the first report of water-sol-
uble, stable Fischer carbene complexes with PEG/OEG
tethers that readily react with the free amino groups of

D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
5587
out, 2 (N) KOH was added to make pH of the solu-
4.4. General procedure for the synthesis of carbene
complex (6a–c), (8a–b)
tion near 9. Extracted with ethyl acetate (3 times),
dried over sodium sulphate, solvent was evaporated
and the residue was subjected to flash column chroma-
tography using ethyl acetate:pet ether (1:9) as eluting
solvent to obtain the pure product 2 as colorless li-
quid (400 mg, 80%). 1H NMR (CDCl3): d 2.71 (s,
6H), 6.41 (d, 1H, J = 6 Hz), 6.59 (m, 2H), 6.85 (t,
1H, J = 6 Hz). 13C NMR (CDCl3): d 40.5, 111,
115.2, 119.2, 130.3. Anal. Calc. For C8H10BrN: C,
48.02; H, 5.05. Found C, 47.89; H, 5.10%.
The solution of carbene salt (n mmol) in methylene
chloride was cooled to ꢀ40 ꢁC (acetonitrile-dry ice
bath). Freshly distilled acetyl chloride (n mmol) was
added to this solution and stirred for 45 min at
ꢀ30 ꢁC. The color of the solution changed to dark
red. The reaction mixture was again cooled to
ꢀ40 ꢁC, alcohol (1.2n mmol) was added and it was
warmed to 0 ꢁC over a period of 2 h and stirred for
4 h at that temperature. After addition of alcohol the
color of the reaction mixture gradually changed from
dark red to red. The solvent was then evaporated un-
der reduced pressure at room temperature and dried
in vacuum and purification was done by flash column
chromatography.
4.2. Synthesis of carbene complex (3)
In a two-necked round bottom flask, 3-bromo-N,N-
dimethyl aniline (240 mg, 1.2 mmol) was dissolved in
5 ml of dry ether and was cooled to 0 ꢁC. Butyl lithium
(0.93 ml, 1.1 mmol) was added drop-wise to it and stir-
red at 0 ꢁC for 20 min. The lithiated product was added
to 2 ml ethereal suspension of tungsten hexacarbonyl
(357 mg, 1 mmol) at 0 ꢁC. The reaction mixture was stir-
red at 0 ꢁC for 10 min first and then at room tempera-
ture for 15 min. Tungsten hexacarbonyl dissolved
completely with the formation of a red solution. Evapo-
ration of the solvent followed addition of 2 ml of de-
gassed distilled water. MeerweinÕs salt (1.1 mmol) was
added to it in one portion. The complex was extracted
with pet ether and concentrated by removal of solvent.
The crude product was purified by flash column chro-
matography using 1:9 dichloromethane-pet ether as elut-
ing solvent to afford pure product 3 (300 mg, 60%) as a
deep red solid (m.p. 58 ꢁC). IR (CHCl3): 1915,
2065 cmꢀ1 (mCO). 1H NMR (CDCl3): d 1.73 (t, 3H,
J = 8 Hz), 3.03 (s, 6H), 5.05 (q, 2H, J = 8 Hz), 6.84
(m, 2H), 6.95 (m, 1H), 7.28 (m, 1H). 13C NMR (CDCl3):
d 14.9, 40.4, 80.0, 111.4, 113.0, 115.4, 128.6, 149.7,
150.0, 197.5, 203.8, 321.0. Anal. Calc. for C16H15NO6W:
C, 38.35; H, 3.02; N, 2.80. Found C, 38.50; H, 3.11; N,
2.65%.
4.4.1. Complex (6a)
Dark red viscous liquid, 38%. IR (CHCl3): 1938.5,
1
2067.5 cmꢀ1 (mCO). H NMR (CDCl3): d 3.29 (s, 3H),
3.47 (t, 2H, J = 6 Hz), 3.62 (m, 6H), 4.04 (t, 2H,
J = 6 Hz,), 5.01 (t, 2H, J = 6 Hz), 7.3 (m, 3H), 7.5 (m,
2H). 13C NMR (CDCl3): d 59.3, 69.4, 70.9, 71.0, 71.2,
72.2, 83.1, 126.9, 128.4, 132.1, 155.5, 197.6, 203.9,
320.0. Anal. Calc. for C19H20O9W: C, 39.61; H, 3.50.
Found C, 39.75; H, 3.60%.
4.4.2. Complex (6b)
Dark red viscous liquid, 30%. IR (CHCl3): 1938,
2067 cmꢀ1 (mCO). 1H NMR (CDCl3): d 3.01 (s, 6H),
3.38 (s, 3H), 3.63 (m, 8H), 4.12 (t, 2H, J = 4 Hz),
5.06 (t, 2H, J = 4 Hz), 6.82 (m, 2H), 6.98 (m, 1H),
7.25 (m, 1H). 13C NMR (CDCl3): d 40.3, 58.8, 69.0,
70.5, 70.8, 71.8, 82.5, 111.5, 128.5, 149.7, 155.9,
197.4, 203.7, 321.7. Anal. Calc. for C21H25NO9W: C,
40.73; H, 4.07; N, 2.26. Found C, 40.59; H, 4.14; N,
2.35%.
4.4.3. Complex (6c)
4.3. Synthesis of carbene complex (4)
Dark red viscous liquid, 25%. IR (CHCl3): 1938,
2067 cmꢀ1 (mCO). 1H NMR (CDCl3: d 2.92 (s, 6H),
3.29 (s, 3H), 3.58 (m, 50H), 4.25 (t, 2H, J = 5 Hz),
4.95 (t, 2H, J = 5 Hz), 7.01 (m, 4H). 13C NMR (CDCl3):
d 39.7, 58.1, 60.9, 62.3, 62.9, 68.4, 69.9, 71.3, 72.0, 82.1,
110.7, 112.9, 115.1, 128.1, 149.3, 160.2, 196.9, 203.1,
321.1.
Carbene complex 3 (500 mg, 1 mmol) was dissolved
in 5 ml of methyl iodide and the mixture was stirred
for 48 h at room temperature. The product 4 was ob-
tained (320 mg, 50%) as an orange ppt (m.p. 158 ꢁC
(decom)) after filtration and washing several times
with ether and dried under vacuum. IR (CHCl3):
1942, 2071 cmꢀ1 (mCO). 1H NMR (CDCl3: d 1.77 (t,
3H, J = 6 Hz), 4.02 (s, 9H), 5.11 (q, 2H, J = 6 Hz),
7.56 (d, 1H, J = 8 Hz), 7.74 (m, 2H), 8.12 (dd, 1H,
J1 = 2 Hz, J2 = 8 Hz). 13C NMR (CDCl3): d 14.7,
57.9, 80.9, 80.0, 115.2, 120.5, 126.3, 130.9, 145.8,
157.8, 196.4, 202.3, 315.9. Anal. Calc. for C17H18I-
NO6W: C, 31.75; H, 2.82; N, 2.18. Found C, 31.66;
H, 2.92; N, 2.25%.
4.4.4. Complex (8a)
Yellow viscous liquid, 40%. IR (CHCl3): 1934,
2069 cmꢀ1 (mCO). 1H NMR (CDCl3): d 2.92 (s, 3H),
3.40 (s, 3H), 3.64 (m, 8H), 4.03 (m, 2H), 4.89 (m, 2H).
13C NMR (CDCl3): d 52.1, 58.6, 68.9, 70.6, 71.9, 83.0,
197.1, 203.3, 331.8. Anal. Calc. for C14H18O9W: C,
32.71; H, 3.53. Found C, 32.66; H, 3.60%.

5588
D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
4.4.5. Complex (8b)
4.6.2. Compound (11b)
Colorless liquid, 50%. IR (CHCl3): 3413 cmꢀ1 1H
.
NMR (CDCl3): d 3.36 (s, 3H), 3.58 (m, 30H), 4.10 (d,
2H, J = 6 Hz), 4.18 (d, 2H, J = 6 Hz), 5.69 (m, 1H),
5.82 (m, 1H). 13C NMR (CDCl3): d 58.1, 58.7, 66.4,
69.2, 70.2, 70.4, 71.7, 127.6, 132.7.
Yellow viscous liquid, 30%. IR (CHCl3): 1918,
2070 cmꢀ1 (mCO). 1H NMR (CDCl3): d 2.77 (s, 3H),
3.23 (s, 3H), 3.50 (m, 20H), 3.94 (t, 2H, J = 3 Hz),
4.84 (t, 2H, J = 3 Hz). 13C NMR (CDCl3): d 29.6,
52.3, 58.9, 68.8, 68.9, 70.0, 70.4, 70.8, 71.8, 83.2, 197.1,
203.4, 331.9. Anal. Calc. for C20H30O12W: C, 37.17;
H, 4.68. Found C, 37.25; H, 4.60%.
4.7. General procedure for the synthesis of compound
(12a–b)
4.5. General procedure for the synthesis of (10a–b)
Phosphorous tribromide (n mmol) was added drop-
wise to an ice-cold solution of alcohol 11a or 11b
(n mmol) in dry dichloromethane. Stirring was contin-
ued at 0 ꢁC for 30 min. Methanol was then added
drop-wise at 0 ꢁC. Solvent was evaporated and the resi-
due was subjected to flash column chromatography to
obtain the pure product 12a or 12b.
Carbon tetrabromide (2.4n mmol) was added to 10n
ml dichloromethane solution of oligo/polyethylene
glycol monomethyl ether (2n mmol) under argon atmo-
sphere. Reaction mixture was cooled to 0 ꢁC and triphe-
nyl phosphine (3n mmol) in dichloromethane (2n ml)
was added drop-wise to it. After stirring for 3–5 h the
solvent from reaction mixture was evaporated out.
Ether (5n ml) was added, kept for 5 min and filtered.
The same process (addition of ether, filtration and evap-
oration of solvent) was repeated thrice. The residue was
subjected to flash column chromatography to obtain the
pure product.
4.7.1. Compound (12a)
Colorless liquid, 80%. IR (CHCl3): 2875 cmꢀ1 1H
.
NMR (CDCl3): d 3.27 (s, 3H), 3.61 (m, 12H), 3.98
(d, 2H, J = 6 Hz), 4.14 (d, 2H, J = 6 Hz), 5.70 (m, 1H),
5.85 (m, 1H). 13C NMR (CDCl3): d 26.4, 58.9, 65.9,
69.6, 70.3, 70.4, 70.5, 71.7, 128.1, 131.1. Anal. Calc.
for C11H21BrO4: C, 44.46; H, 7.12. Found C, 44.51; H,
7.19%.
4.5.1. Compound (10)
1
Colorless liquid, 60%. H NMR (CDCl3): d 3.39 (s,
3H), 3.48 (t, 2H), 3.55 (m, 2H), 3.68 (m, 6H), 3.82 (t,
2H). 13C NMR (CDCl3): d 30.2, 58.9, 70.3, 70.4, 71.0,
71.7. Anal. Calc. for C7H15BrO3: C, 37.02; H, 6.66.
Found C, 37.11; H, 6.70%.
4.7.2. Compound (12b)
Colorless liquid, 50%. 1H NMR (CDCl3): d 3.30
(s, 3H), 3.63 (m, 32H), 4.01 (d, 2H), 4.16 (d, 2H), 5.72
(m, 1H), 5.87 (m, 1H). 13C NMR (CDCl3): d 26.4,
58.9, 66.0, 69.6, 70.4, 70.5, 71.8, 128.1, 131.1.
4.5.2. Compound (10b)
1
Colorless liquid, 50%. H NMR (CDCl3): d 3.37 (s,
3H), 3.47 (t, 2H, J = 6 Hz), 3.62 (m, 22H), 3.80 (t, 2H,
J = 6 Hz) 13C NMR (CDCl3): d 30.2, 58.9, 70.4, 70.5,
71.1, 71.8, 128.5,132.1.
4.8. General procedure for the synthesis of carbene
complex (14a–b), (15)
The carbene complex 13 or 8a (n mmol) and tetrabu-
tylamonium bromide (TBAB) (0.05n mmol) in dichloro-
methane (15n mL) was treated with 50% aqueous NaOH
and the halide 12a or 12b (4n mmol). The mixture was
stirred at room temperature under argon. The dichloro-
methane layer was taken, dried over sodium sulphate
and concentrated under reduced pressure. The pure
product was isolated and purified by flash
chromatography.
4.6. General procedure for the synthesis (11a–b)
Sodium metal (1.5n mmol) was added in small pieces
to cis-2-butene-1,4-diol (7n mmol) for 4 h with stirring
and was heated at 88 ꢁC for 1 h. To it the bromo com-
pound 10a, 10b (n mmol) was added drop-wise. Heating
was continued at 88 ꢁC for 5 h. Reaction mixture was al-
lowed to come at room temperature and purified by
flash column chromatography.
4.8.1. Complex (14a)
Yellow viscous liquid, 40%. IR (CHCl3): 1938,
4.6.1. Compound (11a)
1
Colorless liquid, 55%. IR (CHCl3): 3446, 2875 cmꢀ1
.
2069 cmꢀ1 (mCO). H NMR (CDCl3): d 2.02 (m, 2H),
1H NMR (CDCl3): d 3.33 (s, 3H), 3.47 (m, 12H), 4.07
(d, 2H, J = 4 Hz), 4.14 (d, 2H, J = 4 Hz), 5.65 (m, 1H),
5.77 (m, 1H). 13C NMR (CDCl3): d 58.0, 58.6, 66.3,
69.0, 70.1, 70.2, 70.3, 71.5, 127.4, 132.4. Anal. Calc.
for C11H22O5: C, 56.39; H, 9.46. Found C, 56.45; H,
9.41%.
2.29 (m, 2H), 3.36 (s, 6H), 3.53 (m, 24H), 4.0 (d, 4H,
J = 6 Hz), 4.12 (m, 1H), 4.60 (s, 3H), 5.54 (m, 4H).
13C NMR (CDCl3): d 29.4, 58.8, 66.5, 69.5, 70.5, 71.5,
71.8, 128.4, 129.0, 197.0, 202.8, 341.3. Anal. Calc. for
C30H46O14W: C, 44.24; H, 5.69. Found C, 44.33; H,
5.75%.

D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
5589
4.8.2. Complex (14b)
Yellow viscous liquid, 30%. IR (CHCl3): 1938,
71.7, 71.9, 116.8, 134.5. Anal. Calc. for C16H32O7: C,
57.12; H, 9.59. Found C, 56.95; H, 9.49%.
1
2068 cmꢀ1 (mCO). H NMR (CDCl3): d 1.98 (m, 2H),
2.24 (m, 2H), 3.29 (s, 6H), 3.53 (m, 56H), 3.95 (d, 4H,
J = 6 Hz), 4.04 (quint, 1H, J = 6 Hz), 4.55 (s, 3H,
W@COCH3), 5.51 (m, 4H). 13C NMR (CDCl3): d
30.0, 59.3, 66.9, 69.8, 70.7, 70.8, 71.0, 71.9, 72.2, 128.8,
129.5, 197.4, 203.3, 341.6.
4.11. Synthesis of carbene complex (19)
Carbene complex 18 (542 mg, 1 mmol) and com-
pound 17 (168 mg, 1.5 mmol,) was taken in dry dichlo-
romethane under argon atmosphere and GrubsÕ first
generation Ru-based catalyst (20 mol%, 17 mg) was
added to it. The reaction mixture was refluxed for 4 h
at 60 ꢁC. Then solvent was evaporated out and the pure
product 19 (30%) was obtained after purification by
flash column chromatography with MeOH: DCM
(5:95). IR (CHCl3): 1932.5, 2063.7 cmꢀ1 (mCO). 1H
NMR (CDCl3): d 1.71 (t, 3H), 2.58 (m, 2H), 3.38 (s,
3H), 3.63 (m, 24H), 4.01 (d, 2H, J = 6 Hz), 4.09 (t,
2H, J = 6 Hz), 4.76 (q, 2H, J = 6 Hz), 5.76(m, 2H),
6.89 (d, 2H, J = 9 Hz), 7.86 (d, 2H, J = 9 Hz). 13C
NMR (CDCl3): d 14.9, 32.9, 58.8, 66.6, 67.4, 67.5,
69.2, 69.4, 70.4, 70.5, 70.6, 71.5, 71.8, 79.4, 113.6,
114.4, 120.5, 129.7, 131.8, 146.7, 197.6, 203.0, 310.7.
Anal. Calc. for C32H44O14W: C, 45.93; H, 5.26. Found
C, 45.79; H, 5.18%.
4.8.3. Complex (15)
Yellow viscous liquid, 25%. IR (CHCl3): 1932,
1
2067 cmꢀ1 (mCO). H NMR (CDCl3): d 2.08 (m, 2H),
2.31 (m, 2H), 3.37 (s, 9H), 3.63 (m, 38H), 4.01 (d, 4H,
J = 2 Hz), 4.07 (qunt, 1H, J = 2 Hz), 4.94 (t, 2H,
J = 2 Hz), 5.57 (m, 4H). 13C NMR (CDCl3): d 29.6,
58.9, 66.6, 68.9, 69.6, 70.6, 70.8, 71.9, 83.3, 128.6,
129.0, 197.1, 202.9, 339.4. Anal. Calc. for
C36H58O17W: C, 45.67; H, 6.18. Found C, 45.61; H,
6.11%.
4.9. Synthesis of hexaethylene glycol monomethyl ether
(16)
To a solution of hexethylene glycol (5.2 g, 20 mmol)
in 60 ml dichloromethane, silver oxide (5.21 g, 22 mmol)
was added portion-wise. It was stirred for 2 h at room
temperature. Methyl iodide (3.4 g, 24 mmol) was added
drop-wise to it at 0 ꢁC and was stirred for additional
24 h at room temperature. Then filtered through cellite,
filtrate was concentrated under vacuo and the residue
was subjected to flash column chromatography to ob-
tain pure product 16 (50%) as colorless liquid. IR
4.12. Reaction of carbene complex 14b with BSA
Reaction of the representative protein BSA with car-
bene complex 14b was performed in borate buffer (at pH
9). Thus, a solution of BSA (100 lM, 1 ml) and carbene
complex 14b (6000 lM, 1 ml) were incubated for 6 h in
borate buffer at 25 ꢁC. The reaction medium was applied
to a gel filtration column (packed with Sephadex G-50)
to separate protein carbene conjugate from unreacted
carbene complex. Two clearly separated yellow bands
were thus observed; the upper band corresponds to the
unreacted carbene complex and the lower band corre-
sponds to the protein–carbene conjugate. Quantification
of the number of amino carbene adducts per protein
molecule was done by the method described by Jaouen
and others [6]. First, the concentration of protein was
measured by the colorimetric assay described by Brad-
ford [18]. The concentration of aminocarbene com-
plex was then determined spectroscopically, taking the
adduct generated in situ by the reaction of carbene com-
plex 14b with an excess of n-butylamine [e(346 nm) =
5200 l molꢀ1 cmꢀ1] as standard. It was thus estimated
that 18 out of 60 amino groups of BSA were converted
to aminocarbenes.
(CHCl3): 3365 cmꢀ1 1H NMR (CDCl3): d 3.33 (s,
.
3H), 3.58 (m, 24H). 13C NMR (CDCl3): d 58.1, 60.7,
69.7, 71.1, 71.9. Anal. Calc. for C13H28O7: C, 52.69;
H, 9.52. Found C, 52.59; H, 9.58%.
4.10. Synthesis of 1-propene hexethyleneglycol
monomethyl ether (17)
In a two-necked round bottom flask NaH in dry THF
(10 mL) was taken and alcohol 16 (2.96 g, 10 mmol) was
added drop-wise at 0 ꢁC. After stirring the reaction mix-
ture for 3 h at room temperature the allyl bromide was
added to it at 0 ꢁC and stirring was continued for addi-
tional 4 h. Then saturated solution of NH4Cl (2 ml) was
added to quench excess NaH. THF was evaporated un-
der vacuum and extracted with ethyl acetate. The col-
lected ethyl acetate layer was dried over sodium
sulphate and concentrated under reduced pressure.
The residue was subjected to flash column chromatogra-
phy using methanol–dichloromethane (2:98) to obtain
pure product as colorless liquid (70%). 1H NMR
(CDCl3): d 2.69 (s, 3H), 3.61 (m, 24H), 3.99 (d, 2H,
J = 6 Hz), 5.20 (m, 2H), 5.88 (m, 1H). 13C NMR
(CDCl3): d 58.76, 69.19, 70.27, 70.34, 70.35, 70.36,
Acknowledgements
The authors wish to thank Professor Abhijit Chak-
rabarti of Saha Institute of Nuclear Physics, Kolkata,
for valuable discussions; Mr. Kalyan Giri of Saha Insti-
tute of Nuclear Physics, for CD measurements. Two

5590
D. Samanta et al. / Journal of Organometallic Chemistry 690 (2005) 5581–5590
[9] (a) A. Abuchowski, J.R. McCoy, N.C. Palczuk, T. van Es, F.F.
Davis, J. Biol. Chem. 252 (1977) 3582;
authors (D.S. and S.S.) thank CSIR, India, for research
fellowships.
(b) O. Kinstler, G. Molineux, M. Treuheit, D. Ladd, C. Gegg,
Adv. Drug Delivery Rev. 54 (2002) 477.
References
[10] F.F. Davis, Adv. Drug Deliery Rev. 54 (2002) 457.
[11] (a) A. Abuchowski, T. van Es, N.C. Palczuk, F.F. Davis, J. Biol.
Chem. 252 (1977) 3578;
[1] (a) J.P. Gosling, Clin. Chem. 36 (1990) 1408;
(b) A. Vessieres, M. Salmain, P. Brossier, G. Jaouen, J. Pharm.
Biomed. Anal. 21 (1999) 625.
(b) C. Delgrado, G.E. Francis, D. Fisher, Crit. Rev. Ther. Drug
Carrier Syst. 9 (1992) 249;
[2] (a) G. Jaouen, A. Vessieres, I.S. Butler, Acc. Chem. Res. 26
(1993) 361;
(c) N.V. Katre, Adv. Drug Deliery Rev. 10 (1993) 91;
(d) A.P. Chapman, Adv. Drug Deliery Rev. 54 (2002) 531.
[12] P. Caliceti, F.M. Veronese, Adv. Drug Delivery Rev. 55 (2003)
1261.
(b) M. Salmain, A. Vessieres, A. Verenne, P. Brossoier, G.
Jaouen, J. Organomet. Chem. 589 (1999) 92.
[3] S. Maiorana, E. Licandro, D. Perdicchia, C. Baldoli, B. Vandoni,
C. Giannini, M. Salmain, J. Mol. Catal. A 204–205 (2003) 167.
[4] (a) W.D. WulfComprehensive Organometallic Chemistry II, vol.
12, Pergamon Press, New York, 1995, p. 469;
(b) J. Barluenga, J. Santamaria, M. Tomas, Chem. Rev. 104
(2004) 2259.
[13] P. Ferenci, Digest. Liver Disease 35 (2003) 601.
[14] J.A. Connor, E.M. Jones, J. Chem. Soc. Chem. Commun. (1971)
570.
[15] Sk.R. Amin, A. Sarkar, Organometallics 14 (1995) 547.
[16] P.J. Kocienski, G. Cernigliaro, G. Feldstein, J. Org. Chem. 42
(1977) 353.
[5] M. Salmain, J.-C. Blais, H.-T. Huy, C. Compain, G. Jaouen, Eur.
J. Biochem. 269 (2001) 5479.
[17] E.J. Corey, D.E. Cane, L. Libit, J. Am. Chem. Soc. 93 (1971)
7016.
[6] M. Salmain, E. Licandro, C. Baldoli, S. Maiorana, H.-T. Huy,
G. Jaouen, J. Organomet. Chem. 617–618 (2001) 376.
[7] (a) A. Zaks, A.M. Klibanov, J. Biol. Chem. 263 (1988) 3194;
(b) T. Asakura, K. Adachi, E. Schwartz, J. Biol. Chem. 253
(1978) 6423.
[18] A. Bouzide, G. Sauve, Tetrahedron Lett. 38 (1997) 5945.
[19] M.M. Bradford, Anal. Biochem. 72 (1976) 248.
[20] E.O. Fischer, R. Auman, Chem. Ber. 101 (1968) 954.
[21] T.R. Hoye, K. Chen, J.R. Vyvyan, Organometallics 12 (1993)
2806.
[8] K.H. Doetz, R. Ehlenz, D. Paetsch, Angew. Chem. Int. Ed. 36
(1997) 2376.
[22] D. Samanta, N. Faure, F. Rondelez, A. Sarkar, Chem. Commun.
(2003) 1186.