In situ formation of N-trifluoroacetoxy succinimide (TFA-NHS): One-pot formation of succinimidyl esters, N-trifluoroacetyl amino acid succinimidyl esters, and N-maleoyl amino acid succinimidyl esters

Article abstract of DOI:10.1021/jo201686e

A method for the in situ formation of N-trifluoroacetoxy succinimide (TFA-NHS) and its application in the formation of succinimidyl esters is presented. The developed method provides N-trifluoroacetyl and N-maleoyl amino acid succinimidyl esters from a variety of amino acids using a one-pot, high-yielding protocol. Investigations into the formation of an N-maleoyl amino acid succinimidyl ester supported the proposal of a revised reaction mechanism, and contributed to the optimization of the reaction conditions.

Full text of DOI:10.1021/jo201686e

Note
pubs.acs.org/joc
In Situ Formation of N-Trifluoroacetoxy Succinimide (TFA-NHS): One-
Pot Formation of Succinimidyl Esters, N-Trifluoroacetyl Amino Acid
Succinimidyl Esters, and N-Maleoyl Amino Acid Succinimidyl Esters
Nicholas M. Leonard* and Jarmila Brunckova
ADD Organic Operations (Department 04KA, Building AP-8B) Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064-6016, United States
S
* Supporting Information
ABSTRACT: A method for the in situ formation of N-
trifluoroacetoxy succinimide (TFA-NHS) and its application in
the formation of succinimidyl esters is presented. The
developed method provides N-trifluoroacetyl and N-maleoyl
amino acid succinimidyl esters from a variety of amino acids
using a one-pot, high-yielding protocol. Investigations into the
formation of an N-maleoyl amino acid succinimidyl ester
supported the proposal of a revised reaction mechanism, and
contributed to the optimization of the reaction conditions.
commercially available and need to be synthesized and isolated
uccinimidyl esters are an essential functional group in
Sorganic synthesis. Because their stability upon isolation and prior to use, adding an additional step to the overall formation
of the desired succinimidyl ester.16
their reactivity with various nucleophiles, succinimidyl esters
have been utilized in peptide synthesis,1 bioconjugations,2
N-Trifluoroacetoxy succinimide (TFA-NHS) has also been
natural product synthesis,3 intramolecular amide formation,4
used for the synthesis of succinimidyl esters. While the
the synthesis of combinatorial libraries,5 DNA sequencing,6 and
preparation and use of this reagent has been known for some
time,17 TFA-NHS is not commercially available, and except for
the work of Rao and co-workers (Scheme 1),5 has been used
sparingly for the formation of succinimidyl esters.2,18 Also,
TFA-NHS has to be prepared and isolated prior to use, and
current conditions requiring excess trifluoroacetic anhydride
(TFAA) in refluxing benzene17 or toluene19 are undesirable for
large scale processes.
molecular imaging.7 In addition, N-maleoyl amino acid
succinimidyl esters are used extensively as heterobifunctional
linkers in diagnostic immunoassays.8
While there are a number of methods for the formation of
succinimidyl esters,3c,9 the most commonly utilized is the
treatment of a carboxylic acid with a carbodiimide and N-
hydroxysuccinimde. The use of carbodiimides on large scale,
however, is impractical because of the creation of stoichiometric
waste and the requirement of toxic or expensive reagents. N,N′-
Dicyclohexylcarbodiimide (DCC), the most commonly used
carbodiimide, is a low melting solid that is difficult to handle
and a known sensitizer.10 In addition, the dicyclourea
byproduct of the coupling reaction is often difficult to separate
from the desired ester or the subsequent reaction mixtures.
Among commercially available carbodiimide reagents, 1-ethyl-
3-(3-dimethylamino-propyl)carbodiimide hydrochloride
(EDAC) is an attractive alternative to DCC, as it forms a
water-soluble urea byproduct that can be removed by aqueous
workup. However, the use of EDAC is undesirable in large scale
pharmaceutical applications as it creates toxic aqueous waste
streams.11 Succinimidyl ester formation without carbodiimides
can be accomplished with activated derivatives of N-hydroxy-
succinimide, and the use of the carbonate,12 oxalate,13
phosphonate,14 and uronium salts15 of N-hydroxysuccinimide
have been reported.3c,9 However, these NHS derivatives are not
N-Maleoyl amino acid succinimidyl esters are frequently used
as heterobifunctional linkers in bioconjugation chemistry.7a,8
Buchardt and Nielsen reported the synthesis of N-maleoyl
amino acid succinimidyl esters through the DCC promoted
cyclization/activation of an in situ formed N-maleamic acid
(Scheme 1).20 This method, however, is plagued by low yields
and the failure of chained aliphatic substrates to provide the
desired products. As demonstrated by Eggleston and Paterson,
TFA-NHS conditions can also provide N-maleoyl amino acid
succinimidyl esters, but optimal results are obtained when the
N-maleamic acids are isolated prior to use (Scheme 1).21 The
need for isolation of the TFA-NHS reagent and the N-
maleamic acid results in a three-reaction process for the
formation of N-maleoyl amino acid succinimidyl esters. Because
of the current limitations in structural diversity and step
Received: August 17, 2011
Published: September 27, 2011
© 2011 American Chemical Society
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Scheme 1. Available Methods for the Formation of Nitrogen-
Functionalized Amino Acid Succinimidyl Esters
Scheme 2. Formation of Succinimidyl Esters and N-
Trifluoroacetyl Amino Acid Succinimidyl Esters
economy, a more efficient method for the formation of N-
maleoyl amino acid succinimidyl esters would find utility in the
synthesis of heterobifunctional linkers for bioconjugation
chemistry.
after concentration of the reaction mixture and trituration of
the resulting residue with ethyl acetate.16
While optimizing the synthesis of a heterobifunctional linker,
conditions for the in situ generation of TFA-NHS were
discovered. A 1:1 mixture of commercially available trifluoro-
acetic anhydride (TFAA) and N-hydroxysuccinimide (NHS)
was observed to convert carboxylic acids to succinimidyl esters
and promote the formation of N-maleamides (Scheme 1).
Herein, we report the use of in situ formed TFA-NHS as a
general method for the single-flask formation of succinimidyl
esters, N-trifluoroacetyl amino acid succinimidyl esters, and N-
maleoyl amino acid succinimidyl esters in high yield from
commercially available (or readily available) amino acid starting
materials.
A variety of carboxylic acids were subjected to reaction
conditions with NHS, TFAA, and pyridine (Scheme 2). Unless
otherwise specified, 2 equiv of NHS and TFAA were required,
and all products were obtained in high purity without
chromatography. Depending on substrate solubility, the
reaction solvent was DMF or a 2:1 mixture of dichloro-
methane/pyridine.22 Heterobifunctional linkers 2b8a and
2d8b−d were isolated in good yield, and an alternative workup
involving concentration of the reaction mixture and trituration
of the resulting residue could be implemented, thereby
eliminating the need for aqueous workup. Treatment of
fumaric acid and tetramethylene glutaric acid provided the
corresponding succinimidyl esters 2c and 2e in excellent yield.
The presence of two carboxylic acid functionalities in these
substrates required the use of five equivalents of NHS, TFAA,
and pyridine. Fumaric acid succinimidyl ester 2c was isolated
N-Trifluoroacetyl amino acid succinimidyl esters were also
synthesized with in situ generated TFA-NHS. Upon treatment
of 6-aminocaproic acid with 2 equiv of TFAA and NHS, the
one-pot protection of the amine and activation of the carboxylic
acid provided 2f (Scheme 2). Amino-substituted benzoic acid
derivatives also provided N-trifluoroacetyl amino acid succini-
midyl esters 2g and 2h in good yield. Rao and co-workers
reported the formation of N-trifluoroacetyl amino acid
succinimidyl esters to require 6 equiv of preformed and
isolated TFA-NHS (Scheme 1).5
The in situ formation of TFA-NHS was further developed for
the formation of N-maleoyl amino acid succinimidyl esters
(Scheme 3). Initial reactions were carried out with isolated N-
maleamic acids, however, it was soon discovered that the N-
maleamic acids could be formed in situ. The optimized process
provides N-maleoyl amino acid succinimidyl esters 3f−j from
readily available amino acids (1f−j) in a single reaction flask
(Scheme 3). Four equivalents of TFAA and NHS are required
for complete conversion, and sym-collidine was found to be the
optimal base.21 This transformation provides efficient access to
costly commercially available N-maleoyl amino acid succini-
midyl esters 3f23 and 3i,24 which serve as heterobifunctional
linkers in bioconjugate chemistry.7a 8a The described in situ
,
TFA-NHS-mediated method provides access to products that
were not obtainable with the method of Buchardt and Nielsen20
(Scheme 1), and allows for the one-pot formation of products
which previously required the three-step process of Eggleston
and Paterson21 (Scheme 1).
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esters with in situ formed TFA-NHS were all complete within
one hour at 0 °C (Scheme 2), it was unlikely that the observed
intermediate was the N-maleoyl carboxylic acid proposed by
Eggleston and Paterson.21 When the cooled (0 °C) reaction
mixture of N-maleamic acid 4 was quenched 30 min after the
addition of TFAA/NHS, the long-lived intermediate was
isolated and determined to be N-maleamic acid succinimidyl
ester 6 (Scheme 4).22 Further monitoring by HPLC confirmed
the immediate formation of both isomerization byproduct 5
and intermediate 6 upon addition of TFAA/NHS. Also, the
amount of 5 was not observed to fluctuate over the course of
the reaction, suggesting that 5 does not form through the
isomerization of intermediate 6.22
Scheme 3. Formation of N-Maleoyl Amino Acid
Succinimidyl Esters
The HPLC monitoring data and characterization of
compounds 5 and 6 supported the proposal of a revised
reaction mechanism. Treatment of amino acid 1i with maleic
anhydride provides N-maleamic acid 4. Subsequent addition of
TFAA, NHS and sym-collidine promotes the rapid formation of
isomerization byproduct 5 and intermediate 6 (Scheme 5).
Scheme 5. Proposed Reaction Mechanism for the Formation
a
of N-Maleoyl Amino Acid Succinimidyl Ester 3i
Choice of base was found to have a significant impact on the
reaction outcome. When CH2Cl2/pyridine was used as the
solvent for the attempted transformation of N-maleamic acid 4
to 3i, isomerization product 5 was isolated as a brown solid in
94% yield (Scheme 4). Eggleston and Paterson reported the use
Scheme 4. Isolation of Isomerization Product 5 and
Intermediate 6
a
Reagents and conditions: (i) maleic anhydride, DMF, rt, 6 h; (ii)
TFAA (4 equiv), NHS (4 equiv), sym-collidine (6.1 equiv), DMF, 0
°C to rt.
Once the addition is complete, the reaction mixture consists of
primarily 6, and the amount of 5 remains constant. Sterically
hindered esterification of the N-maleamic carboxylic acid of 6 is
slow at 0 °C, so warming of the reaction mixture to room
temperature facilitates esterification and cyclization to form 3i.
Elucidation of the revised reaction mechanism facilitated the
optimization of the reaction conditions. In early studies,
variation in the amount of base, temperature, and method of
TFA-NHS addition provided the desired product (3i)
contaminated with 2−10% of isomerization byproduct 5.
Once the reactive pathways of the reaction were understood,
it was discovered that addition of sym-collidine to in situ
formed 4, followed by slow addition of a solution of TFAA,
NHS, and sym-collidine to the reaction mixture at 0 °C limits
the formation of 5. The optimized procedure allows for the
reproducible isolation of 3i containing less than 1% of
isomerization byproduct 5 in >90% yield on 15 g scale.
In conclusion, a method utilizing the in situ formation of
TFA-NHS for the synthesis of succinimidyl esters has been
presented. This method was shown to facilitate the one-pot
formation of N-trifluoroacetyl and N-maleoyl amino acid
succinimidyl esters in good yield and high purity. In addition,
the characterization of a byproduct and a reactive intermediate
in the formation of N-maleoyl amino acid succinimidyl ester 3i
of less sterically hindered bases to facilitate the formation of
colored byproducts that could not be separated from the
desired product.21 It is plausible that these observed impurities
contain the E-isomer of the N-maleamic acid used in the
reaction.
The isolation of a reaction intermediate provided further
insight into the mechanism of the reaction. It was initially
assumed that the reaction proceeded via the mechanism
proposed by Eggleston and Paterson, in which the mixed
anhydride of the N-maleamic acid and TFA cyclizes to provide
an N-maleoyl carboxylic acid. Subsequent esterification of the
pendent carboxylic acid with an additional equivalent of TFA-
NHS would then provide the N-maleoyl amino acid
succinimidyl ester.21 However, when the formation of 3i was
monitored by HPLC, an intermediate was repeatedly observed
which required overnight stirring at room temperature for full
conversion.22 Considering that the formation of succinimidyl
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66.5, 40.6, 30.7, 29.3, 25.6, 25.5, 24.1; HRMS (ESI) m/z calcd for
C18H22N2O6 362.14779, found 362.14953.
supported the proposal of a revised reaction mechanism, which
facilitated the optimization of the reaction conditions.
Succinimidyl Ester 2b. Representative procedure A was followed
using 1b (6.35 g, 18.6 mmol), N-hydroxysuccinimide (4.29 g, 37.3
mmol), trifluoroacetic anhydride (5.18 mL, 37.3 mmol), and pyridine
(3.00 mL, 37.3 mmol) in DMF (93 mL, 0.20 M) to afford 2b as a
white solid (7.85 g, 94%). Alternative workup: Concentration of the
reaction mixture and trituration of the resulting residue with EtOAc (2
× 100 mL) afforded 2b as a white solid (6.91 g, 83%). Spectral data for
1H NMR was identical to those reported in literature.8b 13C NMR has
not been previously reported: 13C NMR (101 MHz, CDCl3) δ 175.7,
171.0, 169.2, 168.4, 133.9, 45.1, 43.6, 38.7, 36.3, 30.8, 29.8, 28.8, 25.6,
25.6, 24.1.
Succinimidyl Diester 2c. Representative procedure A was
followed using fumaric acid (1c) (0.116 g, 1.00 mmol), N-
hydroxysuccinimide (0.575 g, 5.00 mmol), trifluoroacetic anhydride
(0.70 mL, 5.00 mmol), and pyridine (0.40 mL, 5.00 mmol) in DMF
(5.0 mL, 0.20 M). Alternative workup: Concentration of the reaction
mixture and trituration of the resulting residue with EtOAc (2 × 10
mL) afforded 2c as a white solid (0.308 g, 99%). Spectral data was
identical to those reported in literature.16
EXPERIMENTAL SECTION
■
6-((1r,4r)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-
cyclohexanecarboxamido)hexanoic Acid (1b). To a solution of
3i (6.69 g, 20.0 mmol) in DMF (200 mL, 0.15 M) was added 6-
aminocaproic acid (3.94 g, 30.0 mmol). The resulting mixture was
stirred at rt for 16 h. The reaction mixture was filtered and the filtrate
was concentrated in vacuo to afford a white solid. The solid was
triturated with water (2 × 200 mL) and filtered. The filter cake was
dried at 50 °C under house vacuum for 16 h to provide 1b as a white
solid (6.37 g, 91%): mp 145−148 °C; 1H NMR (400 MHz, DMSO) δ
7.64 (t, J = 5.6 Hz, 1H), 7.01 (s, 2H), 3.23 (d, J = 7.1 Hz, 2H), 2.98
(dd, J = 12.6, 6.7 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.99 (tt, J = 12.0,
3.3 Hz, 1H), 1.73−1.57 (m, 4H), 1.57−1.42 (m, 3H), 1.41−1.30 (m,
2H), 1.30−1.17 (m, 4H), 0.88 (m, 2H); 13C NMR (101 MHz,
DMSO) δ 174.7, 174.4, 171.3, 134.3, 43.8, 43.1, 38.1, 36.1, 33.6, 29.4,
28.4, 28.6, 25.9, 24.2; HRMS (ESI) m/z calcd for C18H26N2O5
350.18482, found 350.18417.
Benzyl 6-(6-(Benzyloxycarbonylamino)hexanamido)-
hexanoate (7). To a suspension of N-benzyloxycarbonyl-6-amino-
hexanoic acid (10.0 g, 37.69 mmol) and 6-amino-hexanoic acid benzyl
ester toluene-4-sulfonic acid25 (14.83 g, 37.69 mmol) in CH2Cl2 (145
mL, 0.26 M) was sequentially added hydroxybenzotriazole (6.11 g,
45.23 mmol), N,N-diisopropylethylamine (16.4 mL, 94.23 mmol) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (8.67 g, 45.23
mmol). After complete addition, the reaction mixture became a clear
solution and was stirred at rt for 18 h. The reaction mixture was
diluted with CH2Cl2 (150 mL) and 1 N HCl (300 mL). The layers
were separated and the organic phase was washed with 1 N HCl (3 ×
300 mL), 1 N NaOH (3 × 200 mL), and saturated aqueous NaCl (2 ×
150 mL). The resulting organic layer was dried (MgSO4) and filtered.
The filtrate was concentrated in vacuo to provide 7 as a white solid
Succinimidyl Ester 2d. Representative procedure A was followed
using 1d8d (3.95 g, 6.85 mmol), N-hydroxysuccinimide (3.15 g, 27.4
mmol), trifluoroacetic anhydride (3.81 mL, 27.4 mmol), and pyridine
(5.54 mL, 68.5 mmol) in DMF (275 mL, 0.025 M). Alternative
workup: Concentration of the reaction mixture and trituration of the
resulting residue with EtOAc (2 × 275 mL) afforded 2d as a white
solid (3.35 g, 73%). Spectral data was identical to those reported in
literature.8d
Succinimidyl Diester 2e. Representative procedure A was
followed using 3,3-tetramethyleneglutaric acid (1e) (0.186 g, 1.00
mmol), N-hydroxysuccinimide (0.575 g, 5.00 mmol), and trifluoro-
acetic anhydride (0.70 mL, 5.00 mmol) in 2:1 CH2Cl2/pyridine (6.0
mL, 0.17 M) to afford 2e as a white solid (0.370 g, 97%): mp 72−75
°C; 1H NMR (400 MHz, CDCl3) δ 2.90 (s, 4H), 2.83 (s, 8H), 1.75 (s,
8H); 13C NMR (101 MHz, CDCl3) δ 169.1, 166.7, 43.4, 38.5, 37.9,
25.6, 23.8; HRMS (ESI) m/z calcd for C17H20N2O8 380.12197, found
380.12287.
N-Trifluoroacetyl Amino Acid Succinimidyl Ester 2f.
Representative procedure A was followed using 6-aminocaproic acid
(1f) (0.131 g, 1.00 mmol), N-hydroxysuccinimide (0.230 g, 2.00
mmol), trifluoroacetic anhydride (0.28 mL, 2.00 mmol), and pyridine
(0.16 mL, 2.00 mmol) in DMF (4.0 mL, 0.25 M) to afford 2f as a
colorless oil which solidified upon standing (0.271 g, 84%). Spectral
data was identical to those reported in literature.26
N-Trifluoroacetyl Amino Acid Succinimidyl Ester 2g.
Representative procedure A was followed using 4-aminobenzoic acid
(1g) (0.137 g, 1.00 mmol), N-hydroxysuccinimide (0.230 g, 2.00
mmol), and trifluoroacetic anhydride (0.28 mL, 2.00 mmol) in 2:1
CH2Cl2/pyridine (6.0 mL, 0.17 M) to afford 2g as a white solid (0.303
g, 88%): mp 220−222 °C; 1H NMR (400 MHz, DMSO) δ 11.74 (br
s, 1H), 8.16 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 8.9 Hz, 2H), 2.90 (s, 4H);
13C NMR (101 MHz, DMSO) δ 170.4, 161.1, 155.0 (q, J = 37.6 Hz),
142.7, 131.4, 121.0, 120.8, 115.5 (q, J = 288.7 Hz), 25.6; HRMS (ESI)
m/z calcd for C13H9N2O5F3 330.04636, found 330.04792.
1
(16.94 g, 95%): mp 74−75 °C; H NMR (400 MHz, CDCl3) δ 7.32
(m, 10H), 5.62 (br s, 1H), 5.10 (s, 2H), 5.08 (s, 2H), 4.92 (br s, 1H),
3.20 (m, 4H), 2.35 (t, J = 7.4 Hz, 2H), 2.13 (t, J = 7.4 Hz, 2H), 1.64
(m, 4H), 1.50 (m, 4H), 1.33 (m, 4H); 13C NMR (101 MHz, CDCl3) δ
173.3, 172.7, 156.4, 136.6, 135.9, 128.5, 128.4, 128.1, 128.1, 128.0,
127.9, 66.5, 66.1, 40.7, 39.1, 36.4, 34.0, 29.6, 29.2, 26.3, 26.2, 25.1,
24.4; HRMS (ESI) m/z calcd for C27H36N2O5 468.26242, found
468.26400.
6-(6-Aminohexanamido)hexanoic Acid (1j). To a solution of 7
(0.870 g, 1.86 mmol) in MeOH/CH2Cl2 (20 mL:20 mL) was added
Pd/C (200 mg). The resulting suspension was stirred under 50 psi of
hydrogen. After 18 h, the mixture was filtered through Celite, and the
filtrate was concentrated in vacuo to provide a 1j as a white solid
1
(0.450 g, 99%): mp 174−176 °C; H NMR (400 MHz, CD3OD/
D2O) δ 3.52 (dt, J = 3.2, 1.6 Hz, 1H), 3.38 (t, J = 6.7 Hz, 2H), 3.17 (t,
J = 7.6 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H),
1.94−1.81 (m, 4H), 1.81−1.68 (m, 4H), 1.64−1.48 (m, 4H); 13C
NMR (101 MHz, CD3OD/D2O) δ 183.6, 176.6, 40.4, 40.2, 38.7, 36.5,
29.5, 27.8, 27.4, 26.8, 26.4, 26.1; HRMS (ESI) m/z calcd for
C12H24N2O3 244.17869, found 244.17926.
Representative Procedure A: Preparation of Succinimidyl
Esters and N-Trifluoroacetyl Amino Acid Succinimidyl Esters.
To a cooled (0 °C) mixture of Z-ε-Ahx−OH (1a) (0.265 g, 1.00
mmol), N-hydroxysuccinimide (0.230 g, 2.00 mmol) and pyridine
(0.32 mL, 4.00 mmol) in CH2Cl2 (5 mL, 0.20 M) was added
trifluoroacetic anhydride (0.28 mL, 2.00 mmol). After addition was
complete, the ice bath was removed and the reaction was stirred at rt
for 1 h. The reaction mixture was diluted with CH2Cl2 (15 mL) and 1
M HCl (20 mL). The layers were separated and the organic layer was
washed with 1 M HCl (2 × 20 mL) and NaHCO3 (2 × 20 mL). The
resulting organic layer was dried (MgSO4) and filtered. The filtrate
was concentrated in vacuo to afford 2a as a clear oil (0.360 g, 99%). 1H
NMR (400 MHz, CDCl3) δ 7.39−7.26 (m, 5H), 5.09 (s, 2H), 4.94 (br
s, 1H), 3.20 (m, 2H), 2.79 (s, 4H), 2.60 (t, J = 7.2 Hz, 2H), 1.76 (m,
N-Trifluoroacetyl Amino Acid Succinimidyl Ester 2h.
Representative procedure A was followed using 2-methoxy-5-amino-
benzoic acid (1h) (0.167 g, 1.00 mmol), N-hydroxysuccinimide (0.230
g, 2.00 mmol), and trifluoroacetic anhydride (0.28 mL, 2.00 mmol) in
2:1 CH2Cl2/pyridine (6.0 mL, 0.17 M) to afford 2h as a white solid
1
(0.264 g, 73%): mp 217−219 °C; H NMR (400 MHz, DMSO) δ
11.41 (br s, 1H), 8.29 (d, J = 2.7 Hz, 1H), 8.02 (dd, J = 9.2, 2.8 Hz,
1H), 7.37 (d, J = 9.2 Hz, 1H), 3.91 (s, 3H), 2.89 (s, 4H); 13C NMR
(101 MHz, DMSO) δ 170.4, 159.9, 157.2, 154.5 (q, J = 37.1 Hz),
129.1, 129.0, 124.0, 115.7 (q, J = 288.4 Hz), 113.9, 113.3, 56.4, 25.6;
HRMS (ESI) m/z calcd for C14H11N2O6F3 360.05692, found
360.05622.
Representative Procedure B: Preparation of N-Maleoyl
Amino Acid Succinimidyl Esters. A suspension of trans-amino-
methylcyclohexane carboxylic acid (1i) (7.86 g, 50.0 mmol) and
2H), 1.54 (dt, J = 13.5, 6.7 Hz, 2H), 1.44 (dt, J = 9.7, 6.8 Hz, 2H); 13
NMR (101 MHz, CDCl3) δ 169.1, 168.4, 156.4, 136.6, 128.4, 128.0,
C
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maleic anhydride (4.90 g, 50.0 mmol) in DMF (250 mL, 0.20 M) was
stirred at rt for 6 h. The resulting clear 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) 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 it was stirred 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 3i as a white solid (15.31 g, 92%): HPLC tR 10.4 min, 99.2%,
(3.20 g, 20.4 mmol) in MeCN (102 mL, 0.20 M) was stirred at rt for
16 h. The suspension was filtered, and the resulting solid was washed
with MeCN (2 × 50 mL). The solid was dried under high vacuum to
afford 4 as a white solid (4.46 g, 86%): HPLC tR 7.0 min, 98.0%, mp
192−194 °C; 1H NMR (400 MHz, DMSO) δ 9.11 (t, J = 5.4 Hz, 1H),
6.43 (d, J = 12.5 Hz, 1H), 6.24 (d, J = 12.5 Hz, 1H), 3.04 (t, J = 6.3
Hz, 2H), 2.13 (tt, J = 12.1, 3.5 Hz, 1H), 1.90 (m, 2H), 1.74 (m, 2H),
1.52−1.36 (m, 1H), 1.26 (m, 2H), 0.95 (m, 2H); 13C NMR (101
MHz, DMSO) δ 176.7, 165.5, 165.4, 132.9, 131.8, 45.2, 42.4, 36.6,
29.3, 28.2; HRMS (ESI) m/z calcd for C12H17N1O5 255.11067, found
255.11031.
Succinimidyl Ester 5. To a cooled (0 °C) solution of 4 (0.255 g,
1.00 mmol), N-hydroxysuccinimide (0.575 g, 5.00 mmol), and
pyridine (1.60 mL, 20.0 mmol) in CH2Cl2 (10 mL, 0.10 M) was
added trifluoroacetic anhydride (0.70 mL, 5.00 mmol). The resulting
orange reaction mixture was warmed to rt over 24 h. The reaction
mixture was diluted with CH2Cl2 (20 mL) and 1 M HCl (20 mL). The
layers were separated and the organic layer was washed with 1 M HCl
(2 × 20 mL) and saturated aqueous NaHCO3 (2 × 20 mL). The
organic layer was dried (MgSO4) and filtered. The filtrate was
concentrated in vacuo to provide 5 as a brown solid (0.424 g, 94%):
HPLC tR 9.3 min, 81.0%, mp 93−95 °C; 1H NMR (400 MHz,
CDCl3) δ 7.15 (d, J = 15.5 Hz, 1H), 6.99 (d, J = 15.5 Hz, 1H), 6.41 (t,
J = 5.9 Hz, 1H), 3.26 (t, J = 6.4 Hz, 2H), 2.87 (s, 4H), 2.83 (s, 4H),
2.60 (tt, J = 12.2, 3.4 Hz, 1H), 2.18 (m, 2H), 1.89 (m, 2H), 1.66−1.48
(m, 3H), 1.08 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 170.6, 169.2,
168.9, 162.4, 160.8, 140.8, 124.3, 45.6, 40.4, 36.9, 29.3, 28.1, 25.6, 25.6;
HRMS (ESI) m/z calcd for C20H23N3O9 449.14343, found 449.14471.
Succinimidyl Ester 6. To a cooled (0 °C) solution of 4 (0.638 g,
2.50 mmol) and N-hydroxysuccinimide (1.15 g, 10.0 mmol) in DMF
(15 mL, 0.17 M) was added sym-collidine (1.33 mL, 10.0 mmol). The
reaction mixture was stirred at 0 °C for 10 min, and trifluoroacetic
anhydride (1.40 mL, 10.00 mmol) was added dropwise. The resulting
reaction mixture was stirred at 0 °C for 30 min. The reaction mixture
was diluted with CHCl3 (50 mL) and 1 M HCl (50 mL). The layers
were separated and the organic layer was washed with 1 M HCl (2 ×
50 mL). The organic layer was dried (MgSO4) and filtered. The filtrate
was concentrated in vacuo to provide a white solid. The white solid
was triturated with chloroform (25 mL) to provide 6 as a white solid
(0.258 g, 29%): HPLC tR 8.8 min, 98.4%, mp 186−187 °C; 1H NMR
(400 MHz, DMSO) δ 14.99 (br s, 1H), 9.08 (t, J = 5.6 Hz, 1H), 6.44
(d, J = 12.5 Hz, 1H), 6.24 (d, J = 12.5 Hz, 1H), 3.07 (t, J = 6.3 Hz,
2H), 2.81 (s, 4H), 2.70 (tt, J = 12.1, 3.5 Hz, 1H), 2.01 (m, 2H), 1.80
(m, 2H), 1.51 (m, 1H), 1.44 (m, 2H), 1.07 (m, 2H); 13C NMR (101
MHz, DMSO) δ 170.9, 170.2, 165.5, 165.4, 132.5, 132.0, 44.9, 39.6,
36.1, 28.7, 28.0, 25.5; HRMS (ESI) m/z calcd for C16H20N2O7
352.12705, found 352.12723.
mp 158−160 °C (lit.27 165−167 C). Spectral data was identical to
̊
those reported in literature:21 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.
N-Maleoyl Amino Acid Succinimidyl Ester 3f. Representative
procedure B was followed to afford 3f as a clear oil (300 mg, 97%).
Flask A: 6-Aminocaproic acid (1f) (0.131 g, 1.00 mmol), maleic
anhydride (0.098 g, 1.00 mmol), DMF (5 mL, 0.2 M), and sym-
collidine (0.28 mL, 2.10 mmol). Flask B: N-Hydroxysuccinimide
(0.460 g, 4.00 mmol), trifluoroacetic anhydride (0.56 mL, 4.00 mmol),
sym-collidine (0.53 mL, 4.00 mmol), and DMF (5 mL, 0.8 M).
Spectral data was identical to those reported in literature.7a
N-Maleoyl Amino Acid Succinimidyl Ester 3g. Representative
procedure B was followed to afford 3g as a white solid (0.283 g, 90%).
Flask A: 4-Aminobenzoic acid (1g) (0.137 g, 1.00 mmol), maleic
anhydride (0.098 g, 1.00 mmol), DMF (5 mL, 0.2 M), and sym-
collidine (0.28 mL, 2.10 mmol). Flask B: N-Hydroxysuccinimide
(0.460 g, 4.00 mmol), trifluoroacetic anhydride (0.56 mL, 4.00 mmol),
sym-collidine (0.53 mL, 4.00 mmol), and DMF (5 mL, 0.8 M): mp
195−197 °C (lit.5 194−195 °C). Spectral data was identical to those
reported in literature.7a,21
N-Maleoyl Amino Acid Succinimidyl Ester 3h. Representative
procedure B was followed to afford 3h as a white solid (0.113 g, 92%).
Flask A: 2-Methoxy-5-aminobenzoic acid (1h) (0.060 g, 0.36 mmol),
maleic anhydride (0.035 g, 0.36 mmol), DMF (5 mL, 0.07 M), and
sym-collidine (0.10 mL, 0.76 mmol). Flask B: N-Hydroxysuccinimide
(0.116 g, 1.44 mmol), trifluoroacetic anhydride (0.20 mL, 1.44 mmol),
sym-collidine (0.19 mL, 1.44 mmol), and DMF (1.8 mL, 0.8 M): mp
182−184 °C; 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 2.6 Hz, 1H),
7.58 (dd, J = 9.0, 2.7 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 6.86 (s, 2H),
3.96 (s, 3H), 2.89 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 169.4,
169.3, 160.1, 159.7, 134.5, 133.8, 130.6, 123.9, 114.9, 113.1, 56.7, 25.9;
HRMS (ESI) m/z calcd for C16H12N2O7 344.06445, found 344.06577.
N-Maleoyl Amino Acid Succinimidyl Ester 3j. Representative
procedure B was followed to afford 3j as a white solid (0.384 g, 91%).
Flask A: 1j (0.244 g, 1.00 mmol), maleic anhydride (0.098 g, 1.00
mmol), DMF (5 mL, 0.2 M), and sym-collidine (0.28 mL, 2.10 mmol).
Flask B: N-Hydroxysuccinimide (0.460 g, 4.00 mmol), trifluoroacetic
anhydride (0.56 mL, 4.00 mmol), sym-collidine (0.53 mL, 4.00 mmol),
and DMF (5 mL, 0.8 M): mp 72−74 °C; 1H NMR (400 MHz,
CDCl3) δ 6.68 (s, 2H), 5.78 (br s, 1H), 3.50 (t, J = 7.2 Hz, 2H), 3.25
(dd, J = 12.6, 6.5 Hz, 2H), 2.85 (s, 4H), 2.63 (t, J = 7.1 Hz, 2H), 2.16
(t, J = 7.5 Hz, 2H), 1.78 (q, J = 7.2 Hz, 2H), 1.70−1.50 (m, 6H), 1.46
(m, 2H), 1.30 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 173.0, 171.0,
169.4, 168.6, 134.2, 39.1, 37.8, 36.6, 31.0, 29.0, 28.4, 26.5, 25.9, 25.8,
25.3, 24.4; HRMS (ESI) m/z calcd for C20H27N3O7 421.18490, found
421.18582.
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC chromatograms and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Nicholas.Leonard@abbott.com
■
ACKNOWLEDGMENTS
N.M.L. thanks Prof. Laura L. Anderson (University of Illinois,
Chicago) for helpful discussions and editing.
■
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