Synthesis of a homotrifunctional conjugation reagent based on maleimide chemistry

Article abstract of DOI:10.1016/j.tetlet.2006.02.032

A novel homotrifunctional conjugation reagent, 1,3,5-tris-(N- maleimidomethyl)benzene has been synthesized in high yield with minimum purification. The reactivity of this compound was examined by using l-cysteine as a nucleophile.

Full text of DOI:10.1016/j.tetlet.2006.02.032

Tetrahedron Letters 47 (2006) 2619–2622
Synthesis of a homotrifunctional conjugation reagent based
on maleimide chemistry
Omar K. Farha, Richard L. Julius and M. Frederick Hawthorne*
Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East,
Los Angeles, CA 90095-1569, USA
Received 14 November 2005; revised 3 February 2006; accepted 6 February 2006
Available online 23 February 2006
Abstract—A novel homotrifunctional conjugation reagent, 1,3,5-tris-(N-maleimidomethyl)benzene has been synthesized in high
yield with minimum purification. The reactivity of this compound was examined by using L-cysteine as a nucleophile.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
conjugation reagents involving both intramolecular and
intermolecular cross-linking has been an invaluable tool
for the study of proteins. Homobifunctional conjugation
reagents have been used as molecular rulers5 to allow the
determination of the distance between two cross-linked
amino acid residues.6 This information can be pertinent
to the determination of the tertiary and quaternary struc-
ture of proteins,7 and is especially valuable for proteins
that are difficult to crystallize8 or to refine low-resolution
crystal structures.9 These intramolecular cross-linking
distances are also useful to probe the conformational
states of an allosteric enzyme10 in addition to character-
izing transmembrane transport channels11 and hormone
receptors.12 Intermolecular protein cross-linking has
shown similar efficacy in elucidating protein–protein
interactions,13 improving both the pharmacodynamics14
and potency15 of protein therapeutics as well as facilita-
ting the development of enzyme–antibody conjugates for
enzyme immunoassay.16
Maleimide-containing compounds are a widely utilized
class of substrates employed in chemical and biological
applications due to their reactivity in Michael-addition
reactions and their dienophilic nature.1 This reactivity
is exemplified by the fact that the maleimido group is
the reagent of choice for functionalizing thiols, including
the cysteine residues of a protein.2 In addition of being
an excellent Michael-acceptor, maleimides are also
highly chemoselective, reacting 103 times faster with a
thiol group than an amine group at neutral pH and
below.3 Consequently, maleimides are model reagents
for use in a biological setting. In the preponderance of
proteins or peptides, free thiols are uncommon, provid-
ing ideal targets for modification at a defined site.
Modern techniques of peptide synthesis or protein
engineering allow facile incorporation of a cysteine
residue at a defined location in a peptide or protein.4
Such cysteines may be located on the protein surface
allowing for straightforward site-specific attachment
with predictable stoichiometry without significantly
altering biological activity.
Similarly, homotrifunctional conjugation reagents have
been used to characterize the drug binding domains of
ATP-binding cassette transporters17 and to define the
catalytically active form of membrane enzymes.18 How-
ever, the usefulness of current tris-maleimido com-
pounds is hindered by inefficient synthetic routes and
the utilization of a nucleophilic nitrogen as the central
core atom.19 In an effort to expand upon these successes,
The exploitation of purposely introduced as well as
wild-type thiol functionality through multifunctional
a
versatile, stable trishomofunctional conjugation
Keywords: Maleimide; Cysteine; Diels–Alder; Mitsunobu; Thiol;
Homotrifunctional conjugation reagent; 1,3,5-Tris(hydroxymethyl)-
benzene; 1,3,5-Tris-(N-maleimidomethyl)benzene.
reagent based upon maleimide chemistry was developed
and is described here.
*
Corresponding author. Tel.: +1 310 825 7378; fax: +1 310 825
5490; e-mail: mfh@chem.ucla.edu
Several literature precedents exist for N-alkylation of a
variety of imides under Mitsunobu conditions.20 The
Both authors contributed equally to this work.
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.032

2620
O. K. Farha et al. / Tetrahedron Letters 47 (2006) 2619–2622
Mitsunobu method has several advantages including
in situ generation of the reactive species characterized
by a very labile leaving group under mild reaction con-
ditions. However, the scope of N-alkylation of imides
was limited to substrates that have no electrophilic reac-
tion centers, such as phthalimide and succinimide, due
to the requirement of nucleophilic triphenyl phosphine
as a Mitsunobu reactant. Walker overcame this obstacle
by changing the order of reagent addition in a modified
Mitsunobu procedure to produce high yields of N-alkyl-
ated maleimides using alcohols as convenient starting
materials.1,21 However, this method was only applied
to simple alkyl systems.1,21 In this letter, we expand this
simple synthetic approach utilizing Mitsunobu reaction
conditions by combining the reversible Diels–Alder
addition of furan to maleimide22 to serve as a protective
group for the maleimide double bond against nucleo-
philes. This allows the synthesis of the desired homotri-
functional maleimide conjugation reagent (6) derived
from 1,3,5-tris(hydroxymethyl)benzene.
Scheme 1. Reagents: (a) THF, PPh3, and DEAD.
Synthesis of the tris-maleimido derivative, 6, began with
the reduction of trimethylbenzene-1,3,5-tricarboxylate
by lithium aluminum hydride, as shown in Scheme 2,
to produce 2, in 90% yield.26 Subsequent reaction of 2
under Mitsunobu conditions with maleimide resulted
in the production of a red-brown polymeric material
insoluble in all common laboratory solvents. Conse-
quently, a model Mitsunobu reaction was investigated
in which succinimide was substituted for maleimide
(Scheme 2). Succinimide exhibits structural and chemi-
cal features similar to those of maleimide, but without
the ability to undergo Michael addition. The reaction
of succinimide with 2 under Mitsunobu conditions pro-
duced 327 in 77% yield, validating the sensitivity of the
unprotected maleimide double bond to any free nucleo-
philes. The fortuitous precipitation of 3 (and later, 527)
from the reaction mixture eliminated the need for a
laborious chromatographic isolation of product from
spent and excess reagents associated with the Mitsunobu
reaction.28
2. Results and discussion
The majority of literature methods describing the syn-
thesis of N-substituted maleimides involve reaction of
a primary amine with maleic anhydride followed by
subsequent dehydration of the intermediate maleamic
acid.1 This reaction is limited by the required harsh
reaction conditions as well as the necessity of employing
primary amines as starting materials.1,23 Even under
suitable circumstances, yields can be compromised by
the low solubility of the maleamic acid and the weak
nucleophilicity of the amines.1,23 Previous reports of
the synthesis of tris-maleimido amines, tris(2-malemido-
ethyl)amine,19a and tris(4-maleimidophenyl)amine,19b
suffer from low yields, 29% and 12%, respectively. This
is presumably due to difficulties resulting from the ring
closure step as well as the reaction of the nucleophilic
amine core with the maleimide double bond.
Protection of the maleimide double bond was accom-
plished through a Diels–Alder reaction using furan as
a diene and maleimide as a dienophile. Both the endo
and exo isomers of 4 were isolated, however, both were
useful for this application. Production of 4 allowed the
reaction of 2 and 4 without the possibility of nucleo-
phile-initiated polymerization. The reaction of 2 with 4
under the conditions used to produce 3 similarly pro-
duced 5 in 70% yield while no polymerization products
were observed. Heating 5 in anisole for 1 h at the reflux
temperature accomplished the retro-Diels–Alder reac-
tion and led to the isolation of the final product, 6,29
in 82% yield after column chromatography.
Three alternative methods for direct N-alkylation of
maleimides have been proposed. The first, by Lerner
and Schwartz,24 involves generation of the silver or mer-
cury salt of maleimide followed by reaction with an
alkyl bromide. However, this reaction was hindered by
the stability of the salts, which generally led to low yields
and only with simple alkyl chains.1,24 A recent report by
Turnbull describing the direct use of benzyl halides in
the presence of K2CO3 has its own limitation due to
the reactivity of the benzyl halides.25 In an effort to
avoid these problems the method described by Walker
using alcohols as starting materials for direct N-alkyl-
ation of maleimide under mild Mitsunobu conditions
was adopted.1,21
The reactivity and efficacy of the final product 6 in thiol
conjugation was investigated using its reaction with
L-cysteine methyl ester hydrochloride under rigorously
oxygen-free conditions illustrated in Scheme 3. L-Cys-
teine methyl ester hydrochloride was chosen as a simple
representative thiol-containing molecule with peripheral
functional groups suitable for further functionalization
while at the same time providing an idealized demon-
stration of peptide conjugation. Overnight reaction of
6 with L-cysteine methyl ester hydrochloride in 1:1
water/acetonitrile at room temperature afforded the
tris-conjugated 7,30 confirmed by APCI-MS, in greater
1
than 90% yield as determined by H NMR integration.
Using benzyl alcohol in a model system, direct N-alkyl-
ation of maleimide was attempted, as shown in Scheme
1. As previously reported,1 the order of addition of reac-
tants was found to be important to reaction yield. When
to PPh3, initially present, was added DEAD (diethyl
azodicarboxylate), alcohol, and finally maleimide (in
the order given), 1 was isolated at 86% yield.
In summary, production of molecules containing multi-
ple maleimide functionalities using common techniques
is encumbered by a variety of factors, including the sta-
bility and reactivity of the starting materials as well as
by solubility issues. Described here is the facile synthesis
of a novel homotrifunctional conjugation reagent, 6,
based on maleimide chemistry, which necessitates a min-

O. K. Farha et al. / Tetrahedron Letters 47 (2006) 2619–2622
2621
Scheme 2. Reagents and conditions: (a) THF, LiAlH4, and H2O; (b) THF, PPh3, DEAD, succinimide; (c) H2O; (d) THF, PPh3, DEAD; (e) anisole, D.
Scheme 3. Reagents: (a) H2O, MeCN, L-cysteine methyl ester hydrochloride.
imum of purification. The application of a Diels–Alder
reaction with furan to protect the maleimide double
bond from nucleophilic attack combined with a modi-
fied Mitsunobu reaction to provide N-alkylation of the
maleimide–furan adduct produced 5. Subsequent ther-
mal cleavage of the furan protecting group afforded
the desired product 6 in good yield. The reactivity of
the maleimide moieties present in 6 was investigated
through its tris-conjugation with L-cysteine methyl ester
hydrochloride, a model for peptide conjugation, which
also provides a suitable reagent for further derivation.
APCI-MS confirmed tris-addition to the maleimide
functions, validating the utility of 6.
Research Fellowship for O.K.F. We would also like to
thank Dr. Mark W. Lee for his helpful suggestions.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.02.032.
References and notes
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Acknowledgments
The authors thank the National Science Foundation for
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1
solid. Mp 165.0–169.5 °C. H NMR (400 MHz, CD2Cl2):
2.69 (12H), 4.57 (6H), 7.18 (3H); 13C NMR (100.6 MHz,
CD2Cl2): 28.2, 41.7, 127.66, 136.8, 176.9; HRMS (EI+) m/z
calcd for C21H21N3O6: 411.1430, found: 411.1430. Com-
pound 5: white solid. Decomposition point 120 °C. 1H
NMR (400 MHz, CD2Cl2): 3.51 (6H), 4.37 (6H), 5.27 (6H),
6.27 (6H), 6.97 (3H); 13C NMR (100.6 MHz, CD2Cl2):
41.7, 46.0, 79.5, 127.7, 134.5, 136.3, 174.5; HRMS (APCI)
m/z calcd for C21H15O6N3Na+ (M+NaÀ3 furan):
428.0853, found: 428.0841.
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29. A typical procedure is as follows. A suspension of 0.25 g
(0.41 mmol) of 5 in 15 mL of anisole was heated at 145 °C
for 1 h. The reaction was cooled to room temperature and
then chromatographed on silica employing hexanes to
remove the anisole followed by EtOAc to elute 6. The
solvents were removed via rotary evaporation and the
product was dried under vacuum to yield 0.13 g (82%) of 6
as a white solid. Mp 173.0–175.0 °C. 1H NMR (400 MHz,
CD2Cl2): 4.60 (6H), 6.70 (6H), 7.16 (3H); 13C NMR
(100.6 MHz, CD2Cl2): 40.8, 127.3, 134.1, 137.1, 170.1;
HRMS (MALDI) m/z calcd for C21H15O6N3Na+:
428.0853, found: 428.0846.
30. The test reaction of 6 with L-cysteine methyl ester
hydrochloride was performed as follows. Solvents were
deoxygenated by using the following method: The sample
container, provided with a magnetic stirrer, was subject to
a partial vacuum that was broken with argon. This process
was repeated four times. A deoxygenated water solution
(1 mL) of L-cysteine methyl ester hydrochloride (40 mg,
0.23 mmol) was added to a deoxygenated solution of 6
(30 mg, 0.074 mmol) in acetonitrile (1 mL) and was stirred
under argon for 12 h. The solution was concentrated via
rotary evaporation and the resulting residue was lyophi-
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3097.
lized to give
a white solid. APCI-MS calcd for
C33H45N3O12S3Cl3H+: 879.3, found: 879.5.