Scalable and cost-effective synthesis of a linker for bioconjugation with a peptide and a monoclonal antibody

Article abstract of DOI:10.1055/s-0033-1340980

An efficient, scalable, and cost-effective synthesis of a linker employed in a bioconjugation process with a peptide and a monoclonal antibody is presented. Several routes were investigated that resulted in the identification of a short synthesis to a key acid intermediate from inexpensive and readily available starting materials. The final coupling of this acid with an aniline to afford the desired linker has been optimized to produce multi-gram quantities of material for clinical studies. The very limited purifications needed for both intermediates and final product make this route amenable to scale. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0033-1340980

PAPER
▌1399
paper
Scalable and Cost-Effective Synthesis of a Linker for Bioconjugation with a
Peptide and a Monoclonal Antibody
Synthesis of a Linker for Bioconjugation with a Peptide
Javier Magano,*a Brian G. Conway,a Douglas Farrand,b Michael Lovdahl,b Mark T. Maloney,a Mark J. Pozzo,c
John J. Teixeira,a John Rizzo,d David Tumeltyd
a
Chemical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, CT 06340, USA
Fax +1(860)7158593; E-mail: Javier.Magano@Pfizer.com
b
Analytical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, CT 06340, USA
c
Biotherapeutics Pharmaceutical Sciences, Pfizer Worldwide Research & Development, 700 Chesterfield Parkway West, Chesterfield,
MO 63017, USA
d
CovX Research, 9381 Judicial Drive, San Diego, CA 92121, USA
Received: 24.01.2014; Accepted after revision: 20.02.2014
degradation, and the stabilization of substances in blood,
Abstract: An efficient, scalable, and cost-effective synthesis of a
among others.2 Bioconjugation has also been employed in
linker employed in a bioconjugation process with a peptide and a
drug delivery systems for some oncology treatments, in
which the active ingredient is connected to the carrier
monoclonal antibody is presented. Several routes were investigated
that resulted in the identification of a short synthesis to a key acid
intermediate from inexpensive and readily available starting mate- through a linker or spacer. This complex can then bind to
rials. The final coupling of this acid with an aniline to afford the de-
sired linker has been optimized to produce multi-gram quantities of
material for clinical studies. The very limited purifications needed
desired site of action.3 Examples of carrier molecules are
for both intermediates and final product make this route amenable
to scale.
its specific receptor contained on the cancer cell and the
net result is the delivery of the therapeutic molecule to the
monoclonal antibodies (mAb)4 and synthetic polymers.5
During the implementation of a project in our laborato-
ries, we were requested to prepare multi-gram quantities
of linker 1 (Figure 1).
Key words: linker, bioconjugation, peptide, monoclonal antibody,
imide, azetidinone
O
O
Biotechnology-based pharmaceuticals or biopharmaceuti-
cals have received considerable attention over the past
few years. The term ‘bioconjugation’ is applied to the co-
valent derivatization of biomolecules, a process in which
two or more molecules come together to afford a complex
that showcases the combined properties of each one of its
individual components.1 This process has found applica-
tions for the minimization of the immunogenicity of poly-
peptides, the protection of substances prone to enzymatic
O
O
N
O
O
N
N
H
O
N
H
O
1
Figure 1 Structure of linker 1
This material is employed to tether a peptide and an mAb
to provide bioconjugate drug substance 3 in a process de-
SH
O
O
O
O
peptide
O
O
N
O
N
N
H
O
N
N
H
O
N
H
O
S
O
peptide
1
2
H2N
O
O
O
O
N
H
N
O
mAb
H
O
N
N
H
O
N
H
mAb
O
S
peptide
3
Scheme 1 Conjugation process between linker 1, a peptide, and a monoclonal antibody (mAb) to provide bioconjugate drug substance 3
SYNTHESIS 2014, 46, 1399–1406
Advanced online publication: 17.03.2014
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0033-1340980; Art ID: SS-2014-M0809-OP
© Georg Thieme Verlag Stuttgart · New York

1400
J. Magano et al.
PAPER
O
picted in Scheme 1. A thiol group of a cysteine residue on
the peptide undergoes Michael addition on the maleimide
moiety of 1 to form a new carbon–sulfur bond and provide
intermediate 2. In a second step, the amino group of a ly-
sine residue on the mAb reacts with the azetidinone func-
tionality on 2 to generate a new amide bond and afford
bioconjugate 3.
O
O
O
N
O
O
N
N
O
N
H
H
O
1
During the early stages of the project, our medicinal
chemistry group had prepared small amounts of 16
through the reaction of commercially available acid 46–8
and aniline 5 in the presence of N-(3-dimethylaminopro-
pyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) as
coupling agent followed by chromatography in 70–78%
yield (Scheme 2).9 After further project progression, larg-
er quantities of 1 were required to support additional clin-
ical studies and the cost associated with the purchase of
acid 4 made this route prohibitively expensive. Therefore,
our group decided to investigate the in-house preparation
of 4 and optimize the reaction conditions of the last amide
bond formation step to generate linker 1.10
O
O
O
O
N
N
O
OH
+
5
H
O
4
O
O
O
CO2R
N
OH
+
H2N
O
O
7
6
O
O
O
O
O
O
O
O
O
O
+
+
CO2R
H2N
OH
N
N
N
O
OH
H2N
OH
+
H
O
H2N
O
9
10
11
4
5
8
commercially available
Scheme 3 Retrosynthetic analysis for linker 1
1. EDC⋅HCl, DIPEA
CH2Cl2, r.t.
2. chromatography
70–78%
O
O
O
O
AcOH, 115 °C
18 h
O
+
N
OH
O
O
H2N
OH
O
O
N
O
O
O
64% (5 g scale)
40% (50 g scale)
O
9
N
N
H
O
N
H
6
8
O
1
O
Scheme 2 Medicinal chemistry preparation of linker 1 via amide
bond formation between acid 4 and aniline 5
PhMe
reflux
CO2H
8 +
9
X
N
H
6
CO2H
12
The retrosynthetic analysis for 1 (Scheme 3) shows that
the most logical initial disconnections are the two amide
bonds. Thus, after generating fragments 4 and 5,11 acid 4
can be further broken down into acid 6 and amino ester 7.
Acid 6 then comes from the reaction between maleic an-
hydride (8) and β-alanine (9) whereas 7 can be produced
from the Michael addition of amino alcohol 10 to acrylate
11.
insoluble in
reaction medium
Scheme 4 Initial attempts to prepare acid 6
With acid 6 on hand, we turned our attention to the prep-
aration of the second coupling partner, amino ester 7. Sev-
eral approaches were tried, which are described in
Scheme 5. 2-(2-Aminoethoxy)ethanol (10), which already
possesses the desired amino functionality found in 1, was
treated with Boc2O to afford the protected intermediate 13
in excellent yield.13 The subsequent Michael addition with
ethyl acrylate (14) could not be reproduced after follow-
ing the same reaction conditions that have been employed
by other groups for very similar substrates.14 Even though
the desired product could be detected by mass spectrome-
try analysis, the reaction did not proceed to completion
and some decomposition was observed in the presence of
catalytic sodium hydride or Na metal and a large excess of
acrylate.
Acid 6 was prepared starting from maleic anhydride (8)
and β-alanine (9) following a known literature procedure
(Scheme 4).12 Even though a fair yield (64%) was ob-
tained on small scale, when the reaction was scaled up to
50 grams, the need to remove residual acetic acid by slur-
rying in ethyl acetate caused the yield to drop consider-
ably (40%). Despite the modest yield, this approach
allowed us to generate enough material to meet our imme-
diate needs. An alternative protocol carried out in toluene
at reflux stalled upon generating intermediate 12 due to its
low solubility in the reaction medium.
Synthesis 2014, 46, 1399–1406
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of a Linker for Bioconjugation with a Peptide
1401
O
CO2Et
14
Boc2O, K2CO3
O
O
X
BocHN
OH
H2N
OH
O
Me2CO–H2O, r.t.
93–96%
NaH, THF, r.t.
BocHN
O
OEt
10
13
15
NaOH, Bu4NHSO4
Br
13 +
15
CO2Et
CH2Cl2, reflux
15%
16
O
O
O
19
PhMe, reflux
CO2t-Bu
O
CO2t-Bu
O
N
OH
O
+
10
X
N
O
2 h
base, THF, r.t.
O
90%
O
O
20
18
17
base: NaH, KOt-Bu, DBU, LiHMDS
Scheme 5 Failed attempts to generate amino ester 7
A second approach involved the reaction between alcohol droxy ester 22 in 80% yield. Tosylation of 2223 followed
13 and ethyl 3-bromopropionate (16) to give ester 15. A by displacement of the tosylate group with phthalimide
screen of conditions was carried out (Et3N, 70 °C,15 (24)24 provided intermediate 20. The cleavage of the
K2CO3 in acetone, 70 °C,16 K2CO3 in DMF, r.t. or 70 °C,17 phthalimido group was carried out with hydrazine
K2CO3 in MeCN, 70 °C, KOt-Bu in t-BuOH, r.t.,18 NaOH hydrate25 to afford amine 7 in almost quantitative yield.
in n-Bu4NHSO4 in CH2Cl2, r.t. or reflux,19 DIPEA in Methyl hydrazine and methyl amine could also be used
CH2Cl2, reflux,20 NaOH and n-Bu4NBr in toluene, r.t., for this transformation, but lower purities were obtained
NaOH and n-Bu4NBr in MTBE, r.t.) but only the combi- with these two reagents. On the other hand, the use of con-
nation of sodium hydroxide and tetrabutylammonium bi- centrated ammonium hydroxide in a sealed reactor led to
sulfate in dichloromethane at reflux gave the desired
product albeit in very low yield (15%) after chromatogra-
K2CO3, MeCN
O
O
BnBr
+
phy. When sodium hydride in tetrahydrofuran was em-
ployed,21 the desired product was also detected by mass
spectroscopy analysis but, due to the low conversion, it
was not isolated.
H2N
OH
Bn2N
OH
reflux, 3.5 h
97%
26
10
CO2t-Bu
25
19
CO2t-Bu
O
Bn2N
O
NaOH (50%), CH2Cl2
n-Bu4NBr, r.t., 18 h
Finally, a third approach was pursued that involved the
Michael addition of phthalimido alcohol 1822 to tert-butyl
acrylate (19), but all the conditions that were tried led to
complex mixtures.
27
quant.
H
2 (15 psi), 5% Pd/C
CO2t-Bu
O
H2N
O
In view of the lack of success of the previous approaches,
a new synthetic route was devised that started from dieth-
ylene glycol (21, Scheme 6). As has been previously re-
ported,21 the Michael addition of 21 to tert-butyl acrylate
(19) in the presence of catalytic sodium hydride gave hy-
MeOH, 40 °C, 5 h
96%
7
Scheme 7 Concise, final approach to amine 7 from (2-amino-
ethoxy)ethanol (10)
NaH, THF
O
O
+
CO2t-Bu
HO
OH
HO
CO2t-Bu
r.t., 18 h
2
21
19
22
80%
O
NH
24
TsCl, Et3N, CH2Cl2
0 °C to r.t., 2 h
96%
O
O
CO2t-Bu
TsO
O
K2CO3, DMF
80 °C, 1 h
23
92%
O
NH2NH2⋅H2
O
O
O
N
CO2t-Bu
H2N
CO2t-Bu
2
2
MeOH, r.t., 1 h
96%
7
O
20
Scheme 6 First successful route to amine 7
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1399–1406

1402
J. Magano et al.
PAPER
decomposition.22b Even though all the intermediates in 27 in quantitative yield and excellent purity (≥95%, area%).
this route are oils, no chromatographic purification was Therefore, the choice of the right protection for the amino
required for any of them and the crude intermediates group was found to be critical for the successful outcome
could be used without further purification.
of the subsequent Michael addition step. The synthesis of
amine 7 was completed by hydrogenolysis of dibenzyl-
amine 27 in the presence of 5% Pd/C.24
In addition to this route from diethylene glycol (21), our
synthetic efforts eventually translated into a successful
and shorter route from 2-(2-aminoethoxy)ethanol (10) With the syntheses of both acid 6 and amine 7 accom-
that became the route of choice (Scheme 7). Thus, the plished, an investigation of the reaction conditions to cou-
amino group in 10 was protected with benzyl bromide ple them was performed. Only EDC and
(25) to provide intermediate 26 in excellent yield.26
A
propylphosphonic anhydride (T3P) provided the desired
screen was then performed to determine the optimal con- product 28, even though in very low yield after chroma-
ditions for the Michael addition of 26 to tert-butyl acrylate tography (Scheme 8). Other coupling reagents such as
(19). A base screen in tetrahydrofuran as solvent gave (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexa-
mostly unreacted alcohol (LiHMDS, DBU) or decompo- fluorophosphate
(PyBOP),
O-(benzotriazol-1-yl)-
sition (KOt-Bu). Based on some published work on simi- N,N,N′,N′-tetramethyluronium hexafluorophosphate (HB-
lar substrates,27 our attention was focused on phase- TU), and N,N′-diisopropylcarbodiimide (DIC) gave de-
transfer catalysis. Several catalysts were tested (n- composition. Interestingly, when 1,1′-carbonyldi-
Bu4NBr,
n-Bu4NOH,
n-Bu4HSO4,
BnMe3NOH, imidazole (CDI) was employed, the product resulting
BnEt3NCl, MeBu3NCl, 0.1 equiv) with 50% sodium hy- from the Michael addition of imidazole to 28 was ob-
droxide (5 equiv) and tert-butyl acrylate (1.5 equiv) in di- tained.
chloromethane at room temperature. Even though all the
A literature search revealed that this type of coupling is
catalysts generated the desired product to a great extent,
usually implemented by utilizing a preactivated acid such
tetrabutylammonium bromide gave the cleanest impurity
as 30 (Scheme 9).
profile with less than 6% of unreacted alcohol. Further op-
We therefore prepared this material according to a modi-
timization of the reactions conditions led to the use of 5
fied literature procedure in a two-step, one-pot process via
the reaction between maleic anhydride (8) and β-alanine
(9) in N,N-dimethylformamide at 60 °C to give acid 6
which, without isolation, underwent reaction with N-hy-
equivalents of tert-butyl acrylate (19), 0.2 equivalent of
tetrabutylammonium bromide, and 5 equivalents of 50%
sodium hydroxide in 10 volumes of dichloromethane,
which resulted in complete conversion to Michael adduct
droxysuccinimide in the presence of N,N′-dicyclohexyl-
carbodiimide (DCC) as coupling reagent.28 An ethanol
O
O
1. peptide coupling
conditions
slurry provided material of excellent purity in 64% yield.
The subsequent coupling with amine 7 was carried out in
dichloromethane with 4-methylmorpholine (NMM) as
base to afford tert-butyl ester 28 in 80% yield after a silica
gel plug to remove polar impurities.29 Other base and sol-
vent combinations (Et3N–DMF,30 aq NaHCO3 in
MeOH31) also provided the desired product but in lower
purities. The synthesis of 4 was completed after tert-butyl
O
CO2t-Bu
N
N
O
6 +
7
H
2. chromatography
O
28
EDC⋅HCl, DIPEA, CH2Cl2: yield = 20%
T3P, DIPEA, MeCN: yield = 13%
Scheme 8 Initial amide bond formation conditions for the coupling
of acid 6 and amine 7
O
N
OH
O
29
O
O
O
O
O
N
DMF, 60 °C
2 h
N
OH
8 +
9
N
O
DCC, r.t., 18 h
64% (2 steps)
O
O
O
6
30
1. NMM, CH2Cl2
r.t., 18 h
O
O
CO2t-Bu
O
N
N
H
O
30 +
7
2. silica gel plug
80%
O
O
28
O
1. TFA–CH2Cl2 (1:1), r.t., 1 h
CO2H
O
N
N
O
H
2. MTBE–EtOAc (1:1)
88%
O
4
Scheme 9 Final coupling conditions between acid 6 and amine 7 and tert-butyl ester cleavage to give acid 4
Synthesis 2014, 46, 1399–1406
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of a Linker for Bioconjugation with a Peptide
1403
ester cleavage with 1:1 trifluoroacetic acid–dichlorometh- ing materials. Optimized conditions have also been found
ane, which gave a much cleaner reaction than anhydrous for the coupling between acid 4 and aniline hydrochloride
4 M hydrochloric acid in 1,4-dioxane. After removing the 5 with HATU as coupling agent. Due to the lack of chro-
solvent under vacuum, the addition of 1:1 MTBE–EtOAc matographic purifications, this route has been easily
caused acid 4 to crystallize in 88% yield and ≥95% purity scaled up to produce multi-hundred grams of non-GMP
(area%).
material for preliminary testing and over 60 g under
cGMP conditions for clinical studies.
With the synthetic route to acid 4 developed, the optimi-
zation of the reaction conditions for the final coupling
with aniline 5 was investigated. In our hands, the amide
bond formation in the presence of EDC·HCl also required
chromatography and the impurity profile was not always
reproducible. In addition, some of the impurities were dif-
ficult to remove by recrystallization or trituration from a
number of solvents. As a consequence, alternative cou-
pling reagents such as O-(7-azabenzotriazol-1-yl)-
All reagents were obtained from commercial sources and used as re-
ceived. Reaction completion and product purity were evaluated by
HPLC or GC using the following conditions: a) HPLC: column:
Zorbax SB-C18 4.6 × 50 mm, 1.8 μm; wavelength: 215 nm; column
temperature: 25 °C; injection volume: 5 μL; eluent: A) H2O (0.2%
HClO4), B) MeCN; gradient: (0 min) A) 90%, B) 10%; (5 min) A)
5%, B) 95%; (10 min) A) 5%, B) 95%; b) GC: column: Agilent HP-
1, 0.2 mm × 12 m, 0.33 μm; initial temperature: 35 °C; final temper-
ature: 290 °C; rate: 30 °C/min. 1H NMR and 13C NMR spectra were
recorded on a 400 MHz spectrometer in either CDCl3 or DMSO-d6
as both solvent and internal standard. Nominal and accurate HRMS
experiments were performed using Thermo Orbitrap FTMS spec-
trometer in the positive and negative mode.
N,N,N′,N′-tetramethyluronium
hexafluorophosphate
(HATU), PyBOP, and DIC were investigated. HATU in
N,N-dimethylformamide gave complete and very clean
reaction after only 30 minutes, whereas PyBOP and DIC
in this same solvent gave a small amount of the desired
product as part of complex mixtures by HPLC. The cou-
pling with HATU in acetonitrile was also tried and it per-
formed equally well than in N,N-dimethylformamide.
2,5-Dioxopyrrolidin-1-yl 3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-
1-yl)propanoate (30)
To a solution of maleic anhydride (8; 86.0 g, 877 mmol) in DMF
(1.03 L) was added β-alanine (9; 78.14 g, 877 mmol). The resulting
Since residual N,N-dimethylformamide was found as a suspension was heated to 60 °C and, after 1 h, a solution was ob-
tained. After 2 h, the mixture was cooled to 0–5 °C and N-hydroxy-
succinimide (29; 126.17 g, 1.09 mol) was added followed by DCC
(361.9 g, 1.75 mol) over 30 min in several portions while the inter-
nal temperature was held below 22 °C. The thick, white slurry was
contaminant after workup, we opted for running the reac-
tion in acetonitrile. The final conditions for this coupling
are shown in Scheme 10.
then stirred at 20 °C for 18 h. The slurry was filtered and the solid
O
O
O
(dicyclohexyl urea) was washed with H2O (1 L) and CH2Cl2 (1 L)
and discarded. To the filtrates was added CH2Cl2 (1 L) and the phas-
es were separated. The aqueous layer was extracted with CH2Cl2
(2 × 300 mL) and the combined organic extracts were washed with
H2O [500 mL; a small amount of brine (50 mL) was added to facil-
itate phase separation], sat. aq NaHCO3 (2 × 300 mL), and dried
(MgSO4). The solvent was removed to give an oily, light tan solid
that was slurried in EtOH (520 mL) for 2 h at 20 °C. The solid was
filtered, washed with EtOH (2 × 75 mL) and dried under vacuum at
30 °C for 72 h to give 186.35 g (64%) of NHS ester 30 as a white
solid;32 HPLC purity: 96.6% (area%); mp 169–171 °C.
O
O
O
N
N
N
O
OH
+
H
H2N
O
4
HCl
5⋅HCl
1. HATU, DIPEA
MeCN, 0 °C to r.t.
2. silica gel plug
3. crystallization from i-PrOAc
83%
O
O
IR (ATR cell): 3088, 2954, 1825, 1783, 1705, 1583, 1446, 1433,
1382, 1325, 1298, 1250, 1211, 1149, 1070, 1049, 998, 956, 901,
836, 813, 786, 767, 756, 696, 651, 634, 596, 561, 544, 528 cm–1.
O
O
N
O
O
N
N
O
N
H
H
1H NMR (400 MHz, DMSO-d6): δ = 2.80 (s, 4 H), 3.05 (t, J = 6.90
Hz, 2 H), 3.75 (t, J = 6.78 Hz, 2 H), 7.05 (s, 2 H).
O
1
13C NMR (100 MHz, DMSO-d6): δ = 25.38, 29.00, 32.67, 134.63,
166.72, 169.93, 170.52.
Scheme 10 Optimized coupling conditions between acid 4 and ani-
line hydrochloride 5 to produce linker 1
HRMS (ESI): m/z [M + H]+ calcd for C11H11N2O6: 267.06116;
found: 267.06147.
After the reaction was complete and an aqueous workup
was performed to remove water-soluble by-products from
the coupling reagent, the crude material was passed
through a silica gel plug to remove baseline impurities.
The addition of i-PrOAc to the resulting oily product
caused the crystallization of linker 1 in 83% yield and
>98% purity (area%).
2-[2-(Dibenzylamino)ethoxy]ethanol (26)
To a solution of 2-(2-aminoethoxy)ethanol (10; 50.0 g, 475 mmol)
in MeCN (1.25 L) was added benzyl bromide (25; 162.68 g, 951
mmol) followed by K2CO3 (157.74 g, 1.14 mol). The resulting mix-
ture was heated to 50–55 °C for 3.5 h. The mixture was cooled to 20
°C, the solids were filtered off, washed with MeCN (2 × 100 mL),
and the filtrates were concentrated to an oil. The flask was placed in
an ice water bath and aq 1 M HCl (500 mL) was slowly added to
dissolve the oil while the internal temperature was held below 25
°C. The aqueous phase was washed with EtOAc (1 × 200 mL, 1 ×
100 mL) and the organic extracts were discarded. The acidic aque-
ous phase was cooled in an ice water bath and aq 50% NaOH was
slowly added to bring the pH up to 10–11 (30 mL) while the internal
In conclusion, a short and efficient synthetic approach for
the preparation of linker 1 has been developed. An opti-
mized procedure for the preparation of acid 4 afforded this
material in five steps and 65% yield for the longest syn-
thetic branch from inexpensive and readily available start-
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1399–1406

1404
J. Magano et al.
PAPER
temperature was held below 20 °C. The aqueous phase was extract-
ed with CH2Cl2 (4 × 250 mL) and the combined organic extracts
were dried (MgSO4). The solvent was removed under vacuum to
give 136.68 g (97%) of alcohol 26 as a yellow oil that was used in
the next step without further purification; HPLC purity: 97% (area%).
tert-Butyl 3-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido]ethoxy}ethoxy)propanoate (28)
To a solution of amine 7 (18.83 g, 81 mmol) in CH2Cl2 (180 mL)
was added N-methylmorpholine (10.20 g, 101 mmol). The flask
was cooled in a water bath at 10 °C and NHS ester 30 (17.90 g, 67
mmol) was added in small portions while the internal temperature
was held below 20 °C. The cooling bath was removed and the re-
sulting mixture was stirred at 20 °C for 18 h. The cloudy, orange
mixture was cooled in an ice water bath and, when the internal tem-
perature was below 15 °C, 10% aq citric acid (220 mL) was added
to bring the pH down to 3. The layers were separated and the aque-
ous layer was extracted with CH2Cl2 (3 × 100 mL). The combined
organic extracts were dried (MgSO4) and the solvent was removed
to give 32 g of a pale brown oil that was passed through a plug of
silica gel (EtOAc as mobile phase) to give 21.25 g (80%) of ma-
leimido tert-butyl ester 28 as a very thick, colorless oil; HPLC pu-
rity: 95.8% (area%).
IR (ATR cell): 3410, 3061, 3026, 2869, 1601, 1494, 1452, 1367,
1246, 1118, 1058, 1027, 976, 888, 806, 732, 697, 621, 565, 530 cm–1.
1H NMR (400 MHz, CDCl3): δ = 2.63 (br s, 1 H), 2.74 (t, J = 5.90
Hz, 2 H), 3.50–3.55 (m, 2 H), 3.63 (t, J = 6.02 Hz, 2 H), 3.68–3.74
(m, 6 H), 7.26–7.32 (m, 2 H), 7.37 (t, J = 7.53 Hz, 4 H), 7.40–7.45
(m, 4 H).
13C NMR (100 MHz, CDCl3): δ = 53.01, 59.02, 61.57, 69.62, 72.12,
127.02, 128.28, 128.94, 139.38.
HRMS (ESI): m/z [M + H]+ calcd for C18H24NO2: 286.18016;
found: 286.17984.
IR (ATR cell): 3307, 2872, 1704, 1652, 1542, 1445, 1407, 1367,
1251, 1112, 956, 828, 755, 695, 613, 544, 536, 528 cm–1.
tert-Butyl 3-{2-[2-(Dibenzylamino)ethoxy]ethoxy}propanoate
(27)
To a solution of alcohol 26 (26.0 g, 91 mmol) and tert-butyl acrylate
(19; 58.38 g, 455 mmol) in CH2Cl2 (235 mL) was added n-Bu4NBr
(5.87 g, 18 mmol) and aq 50% NaOH (36.44 g, 455 mmol) as a
small stream. The mixture was stirred at 20 °C for 3 h and H2O (200
mL) was added. The layers were separated and the aqueous layer
was extracted with CH2Cl2 (2 × 50 mL). The combined organic ex-
tracts were washed with H2O (50 mL) and dried (MgSO4). The sol-
vent was removed under vacuum to give a yellow oil that partially
solidified due to the presence of residual phase-transfer catalyst.
Heptane (100 mL) was added to the residue and the suspension was
stirred at 20 °C for 1 h. The mixture was filtered through Celite and
the Celite pad was washed with heptane (2 × 25 mL). The filtrates
were concentrated under vacuum to give 36.74 g (97%) of tert-butyl
ester 27 as a pale yellow oil that was used in the next step without
further purification; HPLC purity: 95.9% (area%).
1H NMR (400 MHz, CDCl3): δ = 1.36 (s, 9 H), 2.38–2.49 (m, 4 H),
3.29–3.36 (m, 2 H), 3.41–3.47 (m, 2 H), 3.51 (s, 4 H), 3.64 (t,
J = 6.22 Hz, 2 H), 3.75 (t, J = 7.26 Hz, 2 H), 6.45 (br s, 1 H), 6.63
(s, 2 H).
13C NMR (100 MHz, CDCl3): δ = 28.25, 34.50, 34.68, 36.42, 39.37,
67.00, 69.82, 70.34, 70.38, 80.82, 134.37, 170.00, 170.65, 171.20.
HRMS (ESI): m/z [M + Na]+ calcd for C18H28N2O7 + Na:
407.17887; found: 407.17844.
3-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanami-
do]ethoxy}ethoxy)propanoic Acid (4)
A solution of tert-butyl ester 28 (14.88 g, 39 mmol) in CH2Cl2 (148
mL) was cooled in a water bath at 10 °C and trifluoroacetic acid
(148 mL, 1.97 mol) was added as a small stream. The cooling bath
was removed and the mixture was allowed to warm to 20 °C and
stirred for 1 h. The solvent was removed under vacuum and the oily
residue was coevaporated with toluene (2 × 150 mL). To the result-
ing oil was added 1:1 EtOAc–MTBE (100 mL) and some seeds of
acid 4. The suspension was stirred at 20 °C for 1 h and the solid was
filtered, washed with 1:1 EtOAc–MTBE (10 mL) and dried under
vacuum at 35 °C for 1 h to give 9.66 g of acid 4 as a white solid;
HPLC purity: 99.0% (area%). The filtrates were concentrated and
slurried in 1:1 EtOAc–MTBE (40 mL) at 20 °C for 72 h. The result-
ing solid was filtered, washed with 1:1 EtOAc–MTBE (10 mL) and
dried as for the previous batch to give an additional 1.51 g of acid 4
as a white solid for a combined yield of 88%; mp 72–74 °C; HPLC
purity: 99.0% (area%).
IR (ATR cell): 3027, 2869, 1728, 1601, 1494, 1453, 1392, 1366,
1328, 1251, 1155, 1113, 1074, 1027, 847, 745, 733, 698, 606, 541,
534 cm–1.
1H NMR (400 MHz, CDCl3): δ = 1.46 (s, 9 H), 2.49 (t, J = 6.60 Hz,
2 H), 2.71 (t, J = 6.20 Hz, 2 H), 3.51–3.55 (m, 2 H), 3.55–3.62 (m,
4 H), 3.66 (s, 4 H), 3.70 (t, J = 6.60 Hz, 2 H), 7.20–7.26 (m, 2 H),
7.28–7.34 (m, 4 H), 7.36–7.41 (m, 4 H).
13C NMR (100 MHz, CDCl3): δ = 34.70, 34.86, 34.88, 39.48, 66.44,
69.82, 70.05, 70.32, 134.45, 170.55, 170.86, 174.80.
HRMS (ESI): m/z [M + H]+ calcd for C25H36NO4: 414.26389;
found: 414.26337.
IR (ATR cell): 3294, 3088, 2913, 1691, 1633, 1557, 1496, 1481,
1449, 1415, 1373, 1328, 1315, 1240, 1218, 1128, 987, 948, 922,
841, 762, 697, 644, 618, 549, 536 cm–1.
1H NMR (400 MHz, CDCl3): δ = 2.56 (t, J = 7.50 Hz, 2 H), 2.63 (t,
J = 5.81 Hz, 2 H), 3.38–3.45 (m, 2 H), 3.50–3.56 (m, 2 H), 3.57–
3.66 (m, 4 H), 3.77 (t, J = 5.81 Hz, 2 H), 3.85 (t, J = 7.50 Hz, 2 H),
6.53 (br s, 1 H), 6.70 (s, 2 H).
tert-Butyl 3-[2-(2-Aminoethoxy)ethoxy]propanoate (7)
To a solution of dibenzylamine 27 (35.0 g, 85 mmol) in MeOH (420
mL) was added 5% Pd/C (JM US 2, 14 g) and the mixture was hy-
drogenated in an Atlantis reactor at 40 °C and 15 psi. After 2 h, the
catalyst was filtered off and the filtrates were concentrated to give
18.90 g (96%) of amine 7 as a hazy oil that was used in the next step
without any further purification.
13C NMR (100 MHz, CDCl3): δ = 34.70, 34.86, 34.88, 39.48, 66.44,
69.82, 70.05, 70.32, 134.45, 170.55, 170.86, 174.80.
HRMS (ESI): m/z [M – H]+ calcd for C14H19N2O7: 327.11970;
found: 327.11871.
IR (ATR cell): 2977, 2868, 1727, 1456, 1393, 1367, 1254, 1157,
1113, 847, 755, 589, 564, 547, 536 cm–1.
1H NMR (400 MHz, CDCl3): δ = 1.30 (s, 9 H), 2.36 (t, J = 6.43 Hz,
2 H), 2.71 (t, J = 5.19 Hz, 2 H), 3.36 (t, J = 5.19 Hz, 2 H), 3.43–3.50
(m, 4 H), 3.57 (t, J = 6.64 Hz, 2 H).
13C NMR (100 MHz, CDCl3): δ = 28.18, 36.36, 41.92, 66.98, 70.32,
70.44, 73.58, 80.52, 170.92.
HRMS (ESI): m/z [M + H]+ calcd for C11H24NO4: 234.16998;
found: 234.17009.
3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{2-[2-(3-oxo-3-{4-
[3-oxo-3-(2-oxoazetidin-1-yl)propyl]phenylamino}prop-
oxy)ethoxy]ethyl}propanamide (1)
Acid 4 (1.00 g, 3.02 mmol) and aniline hydrochloride 5 (0.70 g, 2.75
mmol) were suspended in MeCN (14 mL). The suspension was
cooled in an ice water bath and HATU (1.25 g, 3.30 mmol) and
DIPEA (1.44 mL, 8.24 mmol) were added. The cooling bath was re-
moved and the resulting yellow solution was allowed to warm to 20
°C. After 1 h, the solvent was removed under vacuum to give a yel-
Synthesis 2014, 46, 1399–1406
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of a Linker for Bioconjugation with a Peptide
1405
low oil that was dissolved in CH2Cl2 (40 mL). The organic phase
was washed with H2O (2 × 10 mL), aq 1 M HCl (5 mL), H2O (5
mL), and dried (MgSO4) with a small amount of solid NaHCO3. The
solvent was removed to give 2.9 g of a yellow oil that was passed
through a pad of silica gel (MeCN as mobile phase) to give 1.74 g
of a clear oil. To this oil was added i-PrOAc (40 mL), which caused
the oil to crystallize. The suspension was stirred for 2 h at 20 °C and
the solid was filtered, washed with i-PrOAc (2 × 10 mL) and dried
under vacuum at 40 °C for 18 h to give 1.22 g (84%) of 1 as a white
solid; HPLC purity: 98.6% (area%); mp 98–99 °C.
Vargas, A. J.; Carmona, A. T.; Robina, I.; Lewis, G. K.;
Wang, L.-X. Bioorg. Med. Chem. 2007, 15, 4220.
(9) (a) Ishino, T.; Palanki, M. S. S.; Violand, B. N.; Das, T. K.;
Hodge, T. S.; Levin, N. J.; Parsons, E. K. Patent WO
2012/059873 A2, 2012; Chem. Abstr. 2012, 156, 628793.
(b) Annathur, G. V.; Balu, P.; Finn, R. F.; Huang, J.;
Laurent, O. A.; Levin, N. J.; Luksha, N. G.; Martin, J. P. Jr.;
Moskowitz, H.; Palanki, M. S. S.; Pozzo, M. J.; Waszak, G.
A.; Xie, J. Patent WO 2013/093720 A2, 2013; Chem. Abstr.
2013, 159, 159296.
(10) During the preparation of this manuscript, a synthesis of
linker 1 has been reported from acid 6 and amine 7 (Scheme
11). However, no experimental details, yields, or
information on reaction scale were included. See: Palanki,
M. S. S.; Bhat, A.; Lappe, R. W.; Liu, B.; Oates, B.; Rizzo,
J.; Stankovic, N.; Bradshaw, C. Bioorg. Med. Chem. Lett.
2012, 22, 4249.
IR (ATR cell): 3248, 3081, 2898, 2864, 2162, 1980, 1784, 1698,
1652, 1633, 1604, 1570, 1544, 1516, 1489, 1445, 1411, 1379, 1318,
1265, 1247, 1212, 1140, 1066, 1043, 1028, 1009, 985, 963, 923,
835, 827, 760, 723, 697, 617, 595, 570, 552, 529 cm–1.
1H NMR (400 MHz, CDCl3): δ = 2.38 (t, J = 7.23 Hz, 2 H), 2.58 (t,
J = 5.68 Hz, 2 H), 2.84–2.96 (m, 4 H), 2.98 (t, J = 5.31 Hz, 2 H),
3.30 (q, J = 5.31 Hz, 2 H), 3.47 (t, J = 5.10 Hz, 1 H), 3.51 (t,
J = 5.31 Hz, 2 H), 3.54–3.67 (m, 4 H), 3.72 (t, J = 7.23 Hz, 2 H),
3.78 (t, J = 5.77 Hz, 2 H), 6.32 (t, J = 5.22 Hz, 1 H), 6.63 (s, 2 H),
7.10 (d, J = 8.42 Hz, 2 H), 7.39 (d, J = 8.42 Hz, 2 H), 8.56 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 29.55, 34.47, 34.68, 36.08, 36.75,
37.97, 38.28, 39.32, 67.15, 69.86, 70.20, 70.34, 120.32, 129.15,
134.40, 136.37, 136.67, 165.30, 170.02, 170.11, 170.32, 170.75.
HRMS (ESI): m/z [M + Na]+ calcd for C26H32N4O8 + Na:
551.21233; found: 551.21190.
O
O
1. NHS, DIC, THF, r.t.
+
H2N
N
OH
O
CO2t-Bu
O
2. 15% TFA, CH2Cl2, r.t.
O
7
6
O
O
O
O
N
O
CO2H
H2N
N
N
H
O
5
HBTU, DIPEA, DMF, r.t.
1
O
4
Acknowledgment
Scheme 11 Synthesis of linker 1 from acid 6 and amine 7
The authors thank Dr. David C. Whritenour for helpful suggestions,
Mr. Victor Soliman for collecting the HRMS data, and Groton’s
gram-laboratory staff for their support during the implementation of
this project.
(11) For the large-scale preparation of aniline 5·HCl, see:
Magano, J.; Bock, B.; Brennan, J.; Farrand, D.; Lovdahl, M.;
Maloney, M. T.; Nadkarni, D.; Oliver, W. K.; Pozzo, M. J.;
Teixeira, J. J.; Wang, J.; Rizzo, J.; Tumelty, D. Org. Process
Res. Dev. 2014, 18, 142.
(12) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.;
Clerx, J.; Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem.
Soc. 2005, 127, 2966.
(13) Brennauer, A.; Keller, M.; Freund, M.; Bernhardt, G.;
Buschauer, A. Tetrahedron Lett. 2007, 48, 6996.
(14) (a) Reddy, D. S.; Vander Velde, D.; Aubé, J. J. Org. Chem.
2004, 69, 1716. (b) Hashimoto, M.; Yang, J.; Holman, G. D.
ChemBioChem 2001, 2, 52.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
References
(1) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.;
Academic Press: San Diego, 2008.
(2) Veronese, F. M.; Morpurgo, M. Farmaco 1999, 54, 497.
(3) Le Sann, C. Nat. Prod. Rep. 2006, 23, 357.
(4) (a) Hermentin, P.; Seiler, F. R. Behring Inst. Mitt. 1988, 82,
197. (b) Hermentin, P.; Doenges, R.; Gronski, P.; Bosslet,
K.; Kraemer, H. P.; Hoffmann, D.; Zilg, H.; Streinstraesser,
A.; Schwartz, A.; Kuhlmann, L.; Lüben, G.; Seuler, F. R.
Bioconjugate Chem. 1990, 1, 100.
(5) (a) Pang, Y.; Liu, J.; Wu, J.; Li, G.; Wang, R.; Su, Y.; He, P.;
Zhu, X.; Yan, D.; Zhu, B. Bioconjugate Chem. 2010, 21,
2093. (b) Voit, B. I.; Lederer, A. Chem. Rev. 2009, 109,
5924. (c) Carlmark, A.; Hawker, C.; Hult, A.; Malkoch, M.
Chem. Soc. Rev. 2009, 38, 352. (d) Saha, A.; Ramakrishnan,
S. Macromolecules 2008, 41, 5658. (e) Gao, C.; Yan, D. Y.
Prog. Polym. Sci. 2004, 29, 183. (f) Tomalia, D. A.; Fréchet,
J. M. J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719.
(g) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem.
Rev. 1999, 99, 1665.
(15) Leach, S. G.; Cordier, C. J.; Morton, D.; McKiernan, G. J.;
Warriner, S.; Nelson, A. J. Org. Chem. 2008, 73, 2753.
(16) (a) Cooney, M. J.; Halton, B. Aust. J. Chem. 1996, 49, 533.
(b) Juhász, L.; Docsa, T.; Brunyászki, A.; Gergely, P.;
Antus, S. Bioorg. Med. Chem. 2007, 15, 4048.
(17) (a) Kim, I.-H.; Morisseau, C.; Watanabe, T.; Hammock, B.
D. J. Med. Chem. 2004, 47, 2110. (b) Tachibana, K.;
Imaoka, I.; Yoshino, H.; Kato, N.; Nakamura, M.; Ohta, M.;
Kawata, H.; Taniguchi, K.; Ishikura, N.; Nagamuta, M.;
Onuma, E.; Sato, H. Bioorg. Med. Chem. Lett. 2007, 17,
5573.
(18) Bird, C. W.; Butler, H. I.; Coffee, E. C. J.; James, L. M.;
Schmidl, B. W. C. Tetrahedron 1989, 45, 5655.
(19) Monge, S.; Sélambarom, J.; Roque, J. P.; Pavia, A. A.
Tetrahedron 2001, 57, 9979.
(6) Bradshaw, C.; Sakamuri, S.; Fu, Y.; Oates, B.; Desharnais,
J.; Tumelty, D. Patent WO 2008081418 A1 20080710, 2008;
Chem. Abstr. 2008, 149, 168435.
(7) Acid 4 was purchased from Quanta Biodesign Limited at a
cost of $ 750/g.
(8) (a) Kasagi, N.; Kojima, M.; Hirai, H. Japanese Patent JP
2007277130 A 20071025, 2007; Chem. Abstr. 2007, 147,
474626. (b) Li, H.; Guan, Y.; Szczepanska, A.; Moreno-
(20) Philippon, A.; Degueil-Castaing, M.; Beckwith, A. L.;
Maillard, B. J. Org. Chem. 1998, 63, 6814.
(21) Douelle, F.; Capes, A. S.; Greaney, M. F. Org. Lett. 2007, 9,
1931.
(22) (a) Katoh, A.; Kudo, H.; Saito, R. Heterocycles 2005, 66,
285. (b) Lown, J. W.; Koganty, R. R.; Joshua, A. V. J. Org.
Chem. 1982, 47, 2027.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1399–1406

1406
J. Magano et al.
PAPER
(23) (a) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am.
Chem. Soc. 2005, 127, 3400. (b) Tomita, H. Japanese Patent
JP 2006327984 A 20061207, 2006; Chem. Abstr. 2007, 146,
45865.
(24) Miller, R. J.; Kuliopulos, A.; Coward, J. K. J. Org. Chem.
1989, 54, 3436.
(27) Dupraz, A.; Guy, P.; Dupuy, C. Tetrahedron Lett. 1996, 37,
1237.
(28) Zhou, M.; Ghosh, I. Org. Lett. 2004, 6, 3561.
(29) Milgrom, L. R.; O’Neill, F. Tetrahedron 1995, 51, 2137.
(30) Menger, F. M.; Bian, J.; Seredyuk, V. A. Angew. Chem. Int.
Ed. 2004, 43, 1265.
(25) Houghton, R. P.; Southby, D. T. Synth. Commun. 1989, 19,
3199.
(26) Visintin, C.; Aliev, A. E.; Riddall, D.; Baker, D.; Okuyama,
M.; Hoi, P. M.; Hiley, R.; Selwood, D. L. Org. Lett. 2005, 7,
1699.
(31) Lee, K. J.; Mao, S.; Sun, C.; Gao, C.; Blixt, O.; Arrues, S.;
Hom, L. G.; Kaufmann, G. F.; Hoffman, T. Z.; Coyle, A. R.;
Paulson, J.; Felding-Habermann, B.; Janda, K. D. J. Am.
Chem. Soc. 2002, 124, 12439.
(32) Paterson, M. J.; Eggleston, I. M. Synth. Commun. 2008, 38,
303.
Synthesis 2014, 46, 1399–1406
© Georg Thieme Verlag Stuttgart · New York