Polyfluorophenyl Ester-Terminated Homobifunctional Cross-Linkers for Protein Conjugation

Article abstract of DOI:10.1055/s-0036-1590974

Along with N -hydroxysuccinimidyl, p -nitrophenyl, and phenylseleno esters, tetra- and penta-fluorophenyl esters were comparatively evaluated in term of their reactivity and hydrolytic stability. Their homobifunctional cross-linkers were prepared to conju

Full text of DOI:10.1055/s-0036-1590974

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Polyfluorophenyl Ester-Terminated Homobifunctional Cross-
Linkers for Protein Conjugation
Jian Wang◊
Ru-Yan Zhang◊
Ya-Cong Wang
Xiang-Zhao Chen
Xu-Guang Yin
Jing-Jing Du
Ze Lei
Ling-Ming Xin
Xiao-Fei Gao
Zheng Liu*
Jun Guo*
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Key Laboratory of Pesticide & Chemical Biology of Ministry of
Education, CCNU-uOttawa Joint Research Centre, Hubei Inter-
national Scientific and Technological Cooperation Base of Pesti-
cide and Green Synthesis, College of Chemistry, Central China
Normal University, Wuhan, Hubei 430079, P. R. China
liuz1118@mail.ccnu.edu.cn
jguo@mail.ccnu.edu.cn
◊Equal contributions from these authors
Published as part of the Cluster Recent Advances in Peptide and Protein Synthesis
Received: 30.04.2017
Accepted after revision: 03.07.2017
Published online: 08.08.2017
important.3 Because of their operational and structural
simplicity, homobifunctional cross-linkers stand out as
valuable tools for bioconjugation practitioners.4
DOI: 10.1055/s-0036-1590974; Art ID: st-2017-w0310-c
The most important component of a homobifunctional
cross-linker is its terminal reactive functional group, which
establishes the method and mechanism for conjugation.
Given the surface availability of amino groups in many pro-
teins, and because lysine is one of most popular handles for
conjugation, various activated esters have been synthesized
and employed for a wide range of amide-bond-forming
conjugations. Homobifunctional N-hydroxysuccinimidyl
Abstract Along with N-hydroxysuccinimidyl, p-nitrophenyl, and phe-
nylseleno esters, tetra- and penta-fluorophenyl esters were compara-
tively evaluated in term of their reactivity and hydrolytic stability. Their
homobifunctional cross-linkers were prepared to conjugate proteins
with small molecules, including carbohydrates, fluorescent dyes, and
poly(ethylene glycol) monomethyl ether. The conjugations proceeded
under mild conditions, affording the corresponding protein conjugates
with good efficiency.
(NHS) esters are widely used, and some are commercially
Key words protein conjugation, cross-linking, carbohydrates, dyes,
polyethylene glycol, linking group
available. Unfortunately, NHS esters have a short half-life, of
the order of hours at physiological pH values, which occa-
sionally results in lower conjugation efficiencies. In addi-
tion, for homobifunctional cross-linkers, a two-step conju-
gation procedure is generally required for effective hetero-
conjugation without the formation of a homodimer or
polymer. But the half-NHS ester, the first-step intermediate,
tends to hydrolyze and to degrade rapidly during purifica-
tion.5
4
Protein conjugation has a variety of applications in life-
science research and assay development. The desirable lev-
1
el of conjugation of small molecules with proteins depends
on the particular application. For the preparation of a semi-
2
synthetic vaccine, a high degree of conjugation is needed
to obtain multivalency of the hapten and to ensure suffi-
cient immunogenicity, whereas for conjugation with an an-
tibody or an enzyme, a low to moderate degree of conjuga-
tion might be optimal to permit retention of the biological
activity of the protein. However, a high efficacy of incorpo-
ration is always desirable, especially in cases where the
small molecule is precious, such as complex oligosaccha-
rides derived from challenging multistep syntheses. There-
fore, choosing an effective protein-conjugation strategy is
To increase the stability of half-esters, Bundle and co-
workers developed
a homobifunctional p-nitrophenyl
(PNP) ester that has been frequently used in syntheses of
6
neoglycoproteins. However, the PNP ester is sometimes too
unreactive to achieve a high degree of conjugation. We have
described a homobifunctional phenylseleno ester (SePh es-
7
ter) cross-linker that demonstrates greater hydrolytic sta-
bility and greater reactivity, permitting highly efficient
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J. Wang et al.
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conjugation with carbohydrates or peptides. However, due
to its poor solubility in aqueous system, the SePh ester is
not suitable for conjugation of hydrophobic molecules, such
as aflatoxin B1, a highly toxic contaminant in agricultural
PBS buffer/DMF
3:1 (v/v)
X
H
Ph
H2N
N
+
Ph
1.2 mM
Ph
Ph
O
rt
O
1
1.0 mM
2
3
8
commodities. Therefore, there is no ‘universal’ activated
O
ester that is capable of efficiently conjugating all types of
small molecules with proteins. More activated esters need
to be comprehensively studied to meet the demands of con-
jugation in various situations.
F
O N
1-PFP X =
O
F
F
1
-NHS X =
O
F
1-SePh X =
SePh
F
F
As part of a research program toward efficient conjugate
7,9
chemistry for glycoconjugates and protein conjugates and
10
1-TFP X =
O
F
F
their applications in fully synthetic vaccines, we report
the use of homobifunctional cross-linkers terminated by a
O
1
-PNP X =
NO2
2
(
,3,5,6-tetrafluorophenyl (TFP) or a pentafluorophenyl
PFP) ester. Our interest in polyfluorophenyl esters arose
from the successful application of TFP and PFP esters in
F
Scheme 1 Reaction between phenethylamine (2) and the activated es-
ters 1-NHS, 1-SePh, 1-PNP, 1-PFP, and 1-TFP
11
12
peptide and glycopeptide syntheses. It has been report-
ed that TFP esters are as reactive as NHS esters, but demon-
strate greater hydrolytic stability. In addition, Huang’s
group reported that pentafluorophenol (PFP-OH), the by-
product of conjugation, is less nucleophilic than NHS and
that PFP-OH does not interfere with the desired product,
13
Consequently, we prepared the corresponding cross-linkers
and 6 (Scheme 2). An adipic acid backbone was chosen
as a spacer because of its nonimmunogenic character.
15
16
5
17
14
F
which is sensitive to nucleophilic attack. In spite of these
advantages, however, TFP and PFP esters have been rarely
used in protein conjugation, particularly in cross-linkers.
We began our investigation by comparing the reactivi-
ties of TFP and PFP esters to those of an NHS ester, a PNP
ester, and an SePh ester at various pH values. As shown in
Scheme 1, 1-NHS, 1-SePh, 1-PNP, 1-TFP, and 1-PFP were
chosen as model compounds, and their reactions with (2-
phenylethyl)amine (2) under mixed solvent/buffer condi-
tions [3:1 v/v 10 mM phosphate-buffered saline
F
OH
F
EDC
F
O
O
TFP-OH
TFPO
PFPO
( )4
5 (84%)
OTFP
OPFP
O
O
HO
( )4
OH
O
O
4
F
(
)4
F
F
OH
F
6
(85%)
EDC
(PBS)/DMF] were examined. 1-TFP and 1-PFP showed a
F
greater reactivity than 1-SePh or 1-PNP, but less than that
of 1-NHS (Figure 1). 1-PFP was slightly more reactive than
PFP-OH
Scheme 2 Preparation of homobifunctional cross-linkers 5 and 6
1-TFP. On the other hand, the hydrolytic stabilities were
also evaluated (see Supporting Information). The order of
stability of the five activated esters was 1-NHS > 1-PFP > 1-
TFP > 1-SePh > 1-PNP.
Collectively, in contrast to NHS and PNP esters, TFP and
PFP esters showed a better balance of reactivity and stabili-
ty, displaying a potential for use in protein conjugation.
Next, we attempted to connect 5 and 6 to various small
molecules to prepare the half-esters 11–14 (Table 1). Four
7
substrate amines [Tn antigen (7), 3-(acetylamino)-3-
deoxy-D-glucopyranosylamine (N-GluNAc; 8),9a N-(7-nitro-
7
2,1,3-benzoxadiazol-4-yl)ethane-1,2-diamine (NBD; 9),
Figure 1 Reaction curves (yields of 3 versus time) of amide bond formation between (2-phenylethyl)amine (2) and the activated esters 1-NHS, 1-SePh,
-PNP, 1-PFP, and 1-TFP at various pH values. The yields were estimated by calculation of the analytic HPLC integrations for 1 and 3. Because of hydro-
lysis, the highest yield of 3 from 1-NHS was slightly lower than those from 1-TFP or 1-PFP.
1
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Table 1 Synthesis of Half-Esters 11–14
In summary, the reactivities and stabilities of five types
of activated ester were evaluated. Homobifunctional cross-
linkers terminated by tetra- or pentafluorophenyl esters
showed good efficiency in the preparation of protein conju-
gates. Under similar conditions, pentafluorophenyl esters,
such as 11-PFP (15f) and 11-PFP (15g), showed better in-
corporation efficiencies than tetrafluorophenyl esters, such
as 11-TFP (15a), 11-TFP (15b), 12-PFP (16b), or 12-TFP
O
O
ArO
( )4
OAr
O
O
Ar = TFP or PFP
R
R
NH2
N
( )4
OAr
DMF, i-Pr2NEt
H
a
Entry
1
R–NH
2
Product
Yield (%)
(
16a). The information gained in this study should be help-
OH
O
HO
HO
ful in considering the types of cross-linkers that might meet
the demands of protein conjugation in various situations.
1
1-TFP
11-PFP
50
47
AcHN
O
NH2
O
7
Tn
Funding Information
OH
We are grateful to the National Key Research and Development Pro-
gram of China (2017YFA0505200), the National Natural Science Foun-
dation of China (21402058 and 21272084), and the self-determined
research funds of CCNU from the college’s basic research and opera-
tion of MOE (CCNU16A02001) for support of this research. Assistance
from the Program of Introducing Talents of Discipline to Universities
O
HO
HO
NH2
12-TFP
46
52
2
3
1
2-PFP
3-TFP
AcHN
8
N-GluNAc
of China (111 program, B17019) is also acknowledged
N
oaitn
a
l
Natuarl
Secince
F
o
u
n
d
oaitn
of
C
h
n
i
a
2(
1
4
0
2
0
5
8
N) oaitn
a
l
Natuarl
Secince
F
o
u
n
d
oaitn
of
C
h
n
i
a
2(
1
2
7
2
0
8
4
C)
e
nrta
l
C
h
n
i
a
N
orm
a
l
Unveiytrsi
1(
6
A
0
2
0
0
1)
O
N
N
1
75
74
NH2
O2N
H3C
N
H
13-PFP
Supporting Information
9
NBD
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1590974.
S
u
p
p
ortioIgnfmr oaitn
S
u
p
p
o
nrtogI
i
f
rm oaitn
NH2
O
0 m-PEG
1
1
4-TFP
4-PFP
78
80
4
n
References and Notes
1
a
(1) Means, G. E.; Feeney, R. E. Bioconjugate Chem. 1990, 1, 2.
Isolated yield.
(
(
(
2) Jones, L. H. Nat. Chem. 2015, 7, 952.
3) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876.
4) Hermanson, G. T. Bioconjugate Techniques; Elsevier Academic:
Amsterdam, 2013, 3rd ed. 275.
and poly(ethylene glycol) monomethyl ether (m-PEG;
0) ] were prepared according to the known procedures
18
1
and treated with five equivalents of cross-linker 5 or 6 in
dry DMF, to give the corresponding half-esters 11–14 in
moderate to good yields. The half-esters were purified by
(5) (a) Izumi, M.; Okumura, S.; Yuasa, H.; Hashimoto, H. J. Carbo-
hydr. Chem. 2003, 22, 317. (b) Broecker, F.; Hanske, J.; Martin, C.
E.; Baek, J. Y.; Wahlbrink, A.; Wojcik, F.; Hartmann, L.;
Rademacher, C.; Anish, C.; Seeberger, P. H. Nat. Commun. 2016,
either silica gel chromatograph or trituration with Et O (see
2
7
, 11224. (c) Möginger, U.; Resemann, A.; Martin, C. E.;
Supporting Information). The moderate yields of the carbo-
hydrate substrates (Table 2, entries 1 and 2) were mainly
due to the hydrolysis of esters during purification.
Parameswarappa, S.; Govindan, S.; Wamhoff, E.-C.; Broecker, F.;
Suckau, D.; Pereira, C. L.; Anish, C.; Seeberger, P. H.; Kolarich, D.
Sci. Rep. 2016, 6, 20488.
(
(
6) Wu, X.; Ling, C.-C.; Bundle, D. R. Org. Lett. 2004, 6, 4407.
7) Yin, X.-G.; Gao, X.-F.; Du, J.-J.; Zhang, X.-K.; Chen, X.-Z.; Wang, J.;
Xin, L.-M.; Lei, Z.; Liu, Z.; Guo, J. Org. Lett. 2016, 18, 5796.
8) The ratios of aflatoxin B1/BSA are 25:1, 50:1, and 100:1; there
are 3.9, 6.1, and 13.6 alfatoxin B1 molecules loaded per BSA
molecule, respectively. For details, see the Supporting Informa-
tion.
The purified half-esters 11–14 were then coupled with
bovine serum albumin (BSA) and ovalbumin (OVA) by incu-
19
bation for 24 hours in buffer. The average degree of incor-
poration was calculated from the increase in the molecular
weight of the BSA and OVA, as determined by MALDI-TOF
(
20
MS, with sinapinic acid as the matrix. TFP linker 5 and PFP
linker 6 showed a good efficiency, comparable to that of
PNP linker but inferior to that of SePh linker, in the prepara-
tion of neoglycoprotein Tn-BSA (Table 2, entries 1–3 and 6–
(9) (a) Du, J.-J.; Gao, X.-F.; Xin, L.-M.; Lei, Z.; Liu, Z.; Guo, J. Org. Lett.
2
2
016, 18, 4828. (b) Gao, X.-F.; Du, J.-J.; Liu, Z.; Guo, J. Org. Lett.
016, 18, 1166. (c) Gao, X.-F.; Sun, W.-M.; Li, X.-M.; Liu, X.-J.;
Wang, L.-S.; Liu, Z.; Guo, J. Catal. Commun. 2016, 73, 103.
10) Yin, X.-G.; Chen, X.-Z.; Sun, W.-M.; Geng, X.-S.; Zhang, X.-K.;
Wang, J.; Ji, P.-P.; Zhou, Z.-Y.; Baek, D. J.; Yang, G.-F.; Liu, Z.; Guo,
J. Org. Lett. 2017, 19, 456.
2
1
8
). With regards to PEGylation, the TFP ester performed
(
2
2,23
better than NHS ester (entry 15).
Unexpectedly, the PFP
linker was less effective than SePh linker in the conjugation
of hydrophobic NBD derivatives to BSA (entry 13), even
though 40% DMF was employed in the PBS buffer.
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Cluster
Table 2 Summary of the Loading Efficiencya
e
f
Entry
Half-ester
Product
pHb
Molar ratioc
nd
11.6 (19.4 , 13 )
Efficiency (%)
Loading (%)
g
h
1
2
3
4
5
6
7
8
9
11-TFP
11-TFP
11-TFP
11-TFP
11-TFP
11-PFP
11-PFP
11-PFP
11-PFP
11-PFP
12-TFP
12-PFP
13-TFP
13-PFP
13-PFP
14-TFP
14-TFP
14-PFP
14-PFP
11-TFP
11-PFP
12-TFP
12-PFP
14-TFP
14-TFP
14-PFP
14-PFP
Tn-BSA (15a)
7.5
7.5
7.5
7.0
8.0
7.5
7.5
7.5
7.0
8.0
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
30:1
20:1
10:1
20:1
20:1
30:1
20:1
10:1
20:1
20:1
20:1
20:1
20:1
20:1
40:1
20:1
40:1
20:1
40:1
20:1
20:1
20:1
20:1
20:1
40:1
20:1
40:1
39
41
66
43
41
44
45
55
41
39
35
49
66
62
51
44, (21j)
36
46
37
17
14
11
18
19
14
16
15
20
14
11
15
14
22
15
9
g
h
Tn-BSA (15b)
8.1 (14.6 , 11 )
g
h
Tn-BSA (15c)
6.6 (8.3 , 5 )
8.7
Tn-BSA (15d)
Tn-BSA (15e)
8.2
g
h
Tn-BSA (15f)
13.3 (19.4 , 13 )
g
h
Tn-BSA (15g)
9.0 (14.6 , 11 )
g
h
Tn-BSA (15h)
5.5 (8.3 , 5 )
Tn-BSA (15i)
8.3
7.8
7.1
9.8
13.3
14
13
12
17
22
21
34
15
24
16
25
17
14
11
18
19
29
16
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Tn-BSA (15j)
N-GluNAc-BSA (16a)
N-GluNAc-BSA (16b)
NBD-BSA (17)
i
g
NBD-BSA (18a)
NBD-BSA (18b)
m-PEG-BSA (19a)
m-PEG-BSA (19b)
m-PEG-BSA (20a)
m-PEG-BSA (20b)
sTn-OVA (21a)
Tn-OVA (21b)
12.4 (14 )
20.4i
8.9
14.6
9.2
14.9
3.4 (4.4g)
2.9 (4.4g)
2.2
N-GluNAc-OVA (22a)
N-GluNAc-OVA (22b)
m-PEG -OVA (23a)
m-PEG -OVA (23b)
m-PEG-OVA (24a)
m-PEG-OVA (24b)
3.7
3.8
5.8
3.3
6.0
a
b
c
d
e
f
Protein concentration 0.1 mM.
pH of PBS.
Molar ratio of half-ester to protein.
Number of small molecules incorporated per protein molecule.
The percentage of small molecules incorporated over the half-ester used.
The percentage of lysine in the protein incorporating the small molecule. Only 30–35 of the 59 lysine residues of BSA are usually accessible to coupling by small
molecules; the maximum loading percentage of BSA is 50−59%. There are 20 lysine residues in OVA.
g
h
i
7
Value obtained by using adipic acid phenylselenyl diester cross-linker.7
Value obtained by using adipic acid p-nitrophenyl diester cross-linker.
The half-esters were dissolved in DMF (0.8 mL) and added to PBS (1.2 mL).
Value obtained by using an NHS ester. The degree of PEGylation was determined by the trinitrobenzenesulfonic acid method.
j
21
(
11) For PFP esters used in peptide synthesis, see: (a) Kisfaludy, L.;
Roberts, J. E.; Johnson, R. H.; Mayers, G. L.; Kovacs, J. J. Org.
Chem. 1970, 35, 3563. For TFP esters used in peptide synthesis,
see: (b) Hui, K. Y.; Holleran, E. M.; Kovacs, J. Int. J. Pept. Protein
Res. 1988, 31, 205.
(12) Vetter, D.; Tumelty, D.; Singh, S. K.; Gallop, M. A. Angew. Chem.
Int. Ed. 1995, 34, 60.
(13) Lockett, M. R.; Phillips, M. F.; Jarecki, J. L.; Peelen, D.; Smith, L.
M. Langmuir 2008, 24, 69.
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J. Wang et al.
Cluster
(
14) Miermont, A.; Barnhill, H.; Strable, E.; Lu, X.; Wall, K. A.; Wang,
(c) Yin, Z.; Chowdhury, S.; McKay, C.; Baniel, C.; Wright, W. S.;
Bentley, P.; Kaczanowska, K.; Gildersleeve, J. C.; Finn, M. G.;
BenMohamed, L.; Huang, X. ACS Chem. Biol. 2015, 10, 2364.
(18) Yildiz, I.; Deniz, E.; McCaughan, B.; Cruickshank, S. F.; Callan, J.
F.; Raymo, F. M. Langmuir 2010, 26, 11503.
Q.; Finn, M. G.; Huang, X. Chem. Eur. J. 2008, 14, 4939.
15) Bis(2,3,5,6-tetrafluorophenyl) Adipate (5); Typical Procedure
Adipic acid (1.00 g, 6.84mmol) and EDC (3.15 g, 16.8 mmol)
were dissolved in dry CH Cl (50 mL), and the solution was
(
2
2
allowed cool to 0 °C in an ice bath. 2,3,5,6-tetrafluorophenol
2.50 g, 15.1 mmol) was added, and the solution was warmed to
(19) Protein Conjugates of Half-Esters; General Procedure
BSA or OVA (10 mg) was dissolved in PBS (pH 7.5, 2 mL), and the
appropriate half-ester 11–14 was dissolved in DMF (100 μL).
The ester solution was slowly injected into the protein solution
and the mixture was left for 24 h at rt. The aqueous phase was
then collected, diluted with deionized water, and dialyzed
against five changes of deionized water. The solution was lyo-
philized to afford the protein conjugate as a white solid.
(20) Patel, M. K.; Vijayakrishnan, B.; Koeppe, J. R.; Chalker, J. M.;
Doores, K. J.; Davis, B. G. Chem. Commun. 2010, 46, 9119.
(21) (a) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discov. 2003, 2, 214.
(b) Pelegri-O’Day, E. M.; Lin, E.-W.; Maynard, H. D. J. Am. Chem.
Soc. 2014, 136, 14323.
(
rt and stirred for 6 h. After removal of the solvent under reduced
pressure, the crude product was purified by flash column chro-
matography [silica gel, PE–CH Cl (1:1)] to give a white solid;
2
2
yield: 2.54 g (84%); mp 99.1–99.3 °C.
1
H NMR (600 MHz, CDCl ): δ = 7.01 (t, J = 8.8 Hz, 2 H), 2.77 (s, 4 H),
3
1
9
1
.94 (d, J = 4.8 Hz, 4 H). F NMR (376 MHz, CDCl ): δ = –138.40
3
13
to –140.13 (m), –152.79 to –153.62 (m). C NMR (101 MHz,
CDCl ): δ = 168.90, 145.97 (dtd, J = 248.5, 11.9, 4.2 Hz), 140.53
3
(dddd, J = 250.4, 15.3, 4.8, 2.3 Hz), 129.80–129.23 (m), 103.19 (t,
J = 22.8 Hz), 32.87, 23.84.
(
16) (a) Steger, J.; Graber, D.; Moroder, H.; Geiermann, A.-S.; Aigner,
M.; Micura, R. Angew. Chem. Int. Ed. 2010, 49, 7470. (b) Liu, Y.;
Kang, Y.; Wang, J.; Wang, Z.; Chen, G.; Jiang, M. Biomacromole-
cules 2015, 16, 3995.
17) Cross-linkers that contain heterocyclic motifs proved to be
immunodominant, leading to a reduced immune response to
the haptens targeted, see: (a) Peeters, J. M.; Hazendonk, T. G.;
Beuvery, E. C.; Tesser, G. I. J. Immunol. Methods 1989, 120, 133.
(22) Boccu, E.; Largajolli, R.; Veronese, F. M. Z. Naturforsch., C 1983,
38, 94.
(23) The degree of PEGylation on BSA as determined by the trinitro-
benzenesulfonic acid method is generally higher than that veri-
fied by a more reliable method, see: Sartore, L.; Caliceti, P.;
Schiavon, O.; Monfardini, C.; Veronese, F. M. Appl. Biochem. Bio-
technol. 1991, 31, 213.
(
(b) Buskas, T.; Li, Y.; Boons, G.-J. Chem. Eur. J. 2004, 10, 3517.
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E