One-pot synthesis of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate (SMCC)

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Organic Preparations and Procedures
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One-pot Synthesis of Succinimidyl-4-
(N-maleimidomethyl)cyclohexane-1-
carboxylate (SMCC)
Nicholas M. Leonard a & Jarmila Brunckova a
a ADD Organic Operations (Dept. 04KA, Building AP-8B), Diagnostics
Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park,
60064-6016, Illinois
To cite this article: Nicholas M. Leonard & Jarmila Brunckova (2012): One-pot Synthesis of
Succinimidyl-4- (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), Organic Preparations and
Procedures International: The New Journal for Organic Synthesis, 44:2, 180-183
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Organic Preparations and Procedures International, 44:180–183, 2012
Copyright © Taylor & Francis Group, LLC
ISSN: 0030-4948 print / 1945-5453 online
DOI: 10.1080/00304948.2012.657938
One-pot Synthesis of Succinimidyl-4-
(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC)
Nicholas M. Leonard and Jarmila Brunckova
ADD Organic Operations (Dept. 04KA, Building AP-8B), Diagnostics Division,
Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6016
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (2, SMCC) has found
utility in numerous areas of chemistry and biotechnology. The well-established chemos-
elective reactivity of SMCC has promoted its use as a heterobifunctional linker in
immumoassays,1 radio-labeling for tumor imaging,2–4 therapeutic agent delivery,5,6 the
immobilization of oligonucleotides on glass surfaces,7 and more recently, in boron neutron
capture therapy.8 The high demand for SMCC has resulted in numerous methods for its
preparation, although to date, a concise, high-yielding synthesis has not been reported.
SMCC was first synthesized by Yoshitake and co-workers in a three-step process that
hinged on an acetic acid/sodium acetate promoted cyclization, and provided the desired
product in 21% overall yield from trans-4-(aminomethyl)cyclohexane carboxylic acid (1).9
In 1991, Nielsen and Buchardt reported the formation of SMCC through the one-pot, DCC
promoted esterification/cyclization of trans-4-(aminomethyl)cyclohexane carboxylic acid
(1) in 75% yield.10
O
N
O
NH2
(i)
O
N
O
C
O
H
CO2
O
1
2
i.) Maleic anhydride, trifluoroacetic anhydride, N-hydroxysuccinimide, sym-collidine,
DMF, 0°C to rt, 92%
However, the method of Nielsen and Buchardt10 was unable to produce analogous
aliphatic substrates were not obtainable in reasonable yields. Adamczyk and Johnson
Submitted September 24, 2011.
Address correspondence to Nicholas M. Leonard, McDonnell Boehnen Hulbert & Burghoff,
LLP, 300 South Wacker Drive, Chicago IL 60606. E-mail: leonard@mbhb.com
180

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
181
later reported difficulty in reproducing the procedures of Yoshitake and Nielsen, isolating
pure SMCC in only 15–20% yield.11 These undesirable results prompted the develop-
ment of pentafluorophenyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (FMCC)11
as a replacement for SMCC in the synthesis of a heterobifunctional linker.12 Paterson
and Eggleston also cited problems with the previously reported method of Nielsen and
Buchardt, but were able to prepare SMCC using N-trifluoroacetoxy succinimide (TFA-
NHS) as an esterification reagent.13 TFA-NHS has been known for some time,14 and was
previously used for the formation of N-trifluoroacetyl amino acid succinimidyl esters.15
Paterson and Eggleston accomplished the one-pot cyclization/esterification of the isolated
N-maleamic acid of trans-4-(aminomethyl)cyclohexane carboxylic acid (1). This method
provided SMCC in an 89% overall yield, however, the N-maleamic acid intermediate and
the TFA-NHS reagent have to be isolated prior to use, resulting in a three-step overall
process.
During the optimization of the synthesis of a heterobifunctional linker of which SMCC
is an intermediate, we discovered conditions for the in situ generation of TFA-NHS. A 1:1
mixture of commercially available trifluoroacetic anhydride and N-hydroxysuccinimide
promoted the conversion of carboxylic acids to succinimidyl esters and the cyclization of
N-maleamic acids to N-maleamides. The in situ formation of TFA-NHS was applied to the
synthesis of SMCC, and as observed by Nielsen and Buchardt,10 the N-maleamic acid could
be generated in situ. Also, in accordance with the findings of Paterson and Eggleston,13
sym-collidine was determined to be the optimal base. After aqueous work-up and trituration
with diethyl ether, SMCC was reproducibly isolated in >90% yield and >99% purity on
∼15 g scale.
Experimental Section
1H NMR spectra were recorded at ambient temperature at either 300 MHz or 400 MHz on
Varian Mercury, Mercury Plus, Unity Plus or 400-MR spectrometers. Deuterated solvents
were used as received from Sigma-Aldrich and data is reported in δ from TMS as an internal
standard. 13C NMR were recorded at ambient temperature at 100 MHz on a Varian 400-MR
spectrometer and data are reported as δ from TMS as an internal standard. High resolution
mass spectra were acquired using an Agilent G6220A Time-of-Flight Mass Spectrometer
equipped with a dual electrospray ion source operated in the positive ion mode. Mass
correction was accomplished using two internal reference masses. HPLC was performed
on a Waters Alliance 2696 with a Waters 2996 Photodiode Array Detector and Symmetry
C18 (4.6 × 150 mm) analytical column, 98:2 0.1% aqueous TFA/acetonitrile ramped to
0:100 over 20 minutes, 1 mL/min, 230 nm. HPLC samples were prepared as 1 mg in 1
ꢀR
mL of DMF. Melting points were obtained using an Electrothermal Mel-Temp melting
point apparatus and are uncorrected. All reactions, unless specified, were performed under
an atmosphere of nitrogen in glassware that had been flame-dried under vacuum. Unless
otherwise noted, all reagents were commercially obtained. N,N-Dimethylformamide and
diethyl ether were purchased as anhydrous grade from Sigma-Aldrich and used directly
from the bottle.

182
Leonard and Brunckova
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
A suspension of trans-4-(aminomethyl)cyclohexane carboxylic acid (1) (7.86 g, 50.0 mmol)
and maleic anhydride (4.90 g, 50.0 mmol) in DMF (250 mL, 0.20 M) in a 1 L round bottom
flask was stirred at rt for 6 h. The resulting colorless solution was cooled to 0◦C as sym-
collidine (13.9 mL, 105 mmol) was added dropwise (Flask A). In a separate flask (Flask B),
a solution of N-hydroxysuccinimide (23.0 g, 200 mmol) in DMF (250 mL, 0.8 M) in
a 500 mL round bottom flask was stirred at 0◦C as trifluoroacetic anhydride (27.8 mL,
200 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and sym-
collidine (26.4 mL, 200 mmol) was added dropwise. After stirring for 10 min, the solution
in Flask B was added by positive-pressure cannula to Flask A over a period of 1–4 h. Both
flasks were kept at 0◦C for the duration of the addition. After addition was complete, the ice
bath was removed and the reaction mixture warmed to rt overnight. The reaction mixture
was diluted with CH2Cl2 (300 mL) and 1 M HCl (250 mL). The layers were separated and
the organic layer was washed with 1 M HCl (2 × 250 mL). The resulting organic layer
was dried (MgSO4) and filtered. The filtrate was concentrated in vacuo to afford a yellow
solid. The solid was triturated with Et2O (3 × 200 mL) to afford SMCC as a white solid
◦
(15.31 g, 92%): HPLC tR 10.4 min, 99.2%, mp 163–165 C (lit.16 165–167◦C). Spectral
data was identical to those reported in literature:13 1H NMR (400 MHz, CDCl3) δ 6.71 (s,
2H), 3.39 (d, J = 7.0 Hz, 2H), 2.82 (s, 4H), 2.58 (tt, J = 12.2, 3.6 Hz, 1H), 2.16 (m, 2H),
1.79 (m, 2H), 1.73 (m, 1H), 1.54 (m, 2H), 1.06 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
171.1, 170.7, 169.3, 134.2, 43.6, 40.6, 36.3, 29.5, 28.2, 25.8; HRMS (ESI) m/z calcd for
C16H18N2O6 334.11649, found 334.11748.
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