Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification

Article abstract of DOI:10.1021/acs.orglett.6b00983

ortho-Phthalaldehyde (OPA) and its derivatives are found to react chemoselectively with amino groups on peptides and proteins rapidly and tracelessly under the physiological condition via formation of phthalimidines, which provides a novel and promising approach when performing bioconjugation on native proteins. The notable advantages of this method over the existing native protein lysine-labeling approaches include a traceless process, a self-reacting, specific and fast reaction, ease of operation, and the ability to use nonhydrolyzable reagents. Its applications have been effectively demonstrated including conjugation of peptides and proteins, and generation of an active PEGlyated l-asparaginase.

Full text of DOI:10.1021/acs.orglett.6b00983

Letter
pubs.acs.org/OrgLett
Traceless and Chemoselective Amine Bioconjugation via
Phthalimidine Formation in Native Protein Modification
Chun Ling Tung, Clarence T. T. Wong, Eva Yi Man Fung, and Xuechen Li*
Department of Chemistry, The State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, China
S
* Supporting Information
ABSTRACT: ortho-Phthalaldehyde (OPA) and its derivatives are found to react chemoselectively with amino groups on
peptides and proteins rapidly and tracelessly under the physiological condition via formation of phthalimidines, which provides a
novel and promising approach when performing bioconjugation on native proteins. The notable advantages of this method over
the existing native protein lysine-labeling approaches include a traceless process, a self-reacting, specific and fast reaction, ease of
operation, and the ability to use nonhydrolyzable reagents. Its applications have been effectively demonstrated including
conjugation of peptides and proteins, and generation of an active PEGlyated L-asparaginase.
tyrosine.7,8 Furthermore, extensive washing and purification
ative protein modification has found many applications in
Nstudying the functions of the intracellular proteins such as steps are needed to remove the NHS residue before performing
biological assays. Some traceless acyl donors are being used,
such as isothiocyanates and isocyanates, which however, require
a higher pH (>9) condition.9 Generally speaking of the methods
for native protein labeling at amino group sites, one often
encounters the problem of instability or low reactivity of the acyl
donor reagents or generation of byproducts. In addition, the
reaction condition is also critical to the effectiveness of protein
bioconjugation, wherein pH, solvents, and temperature vary for
different labeling reagents.3
During the course of our previous work in the development of
a three-component reaction of ortho-phthalaldehyde (OPA),
amines, and isonitriles to generate 1-carboxamide isoindoles,
before an optimal condition (2 equiv NaHSO3 as additive in
DMSO/H2O) was found, the N-substituted isoindolin-1-ones
(phthalimidines) were observed as the major or sole product in
all the organic solvents tested.10 Indeed, such a reaction of OPA
and amines to form phthalimidines was well documented, first
reported by Thiele in 1909,11a and it has been used to construct
small molecules with the isoindolinone skeleton under the Lewis
or Brønsted acid catalyzed condition generally with low to
modest yields.11 The reaction proceeds likely via formation of an
iminoaldehyde followed by an energetically favorable [1,5]-H
sigmatropic rearrangement of the aldehydic proton as the key
immunostaining and immunolocalization and developing
therapeutic biologics such as polyethylene glycol (PEG)−
protein conjugate drugs, antibody−drug conjugates (ADC),
carbohydrate−protein conjugate vaccines, and so on.1 Thus,
new approaches and strategies for effective protein bioconjuga-
tion are continually being developed.2
Native protein modification often involves the conjugation
chemistry to take place at the nucleophilic side chain of the
amino acids present within a protein under the physiological
conditions, among which lysine and cysteine are the most
common labeling sites.3 As the thiol group of cysteine is a
superior nucleophile, there are a handful of functional groups
capable of chemoselectively reacting with the N-terminal or
internal cysteine residues.2,4 Nevertheless, most cysteine
residues on native proteins are engaged in forming disulfides
with other cysteine residues that are important for function,
protein folding, and stability. Conjugation at the ε-amine group
of the lysine residue and the N-terminus involves using acyl
donors as the labeling reagent.3,5
One of most common methods is to use N-hydroxysuccini-
mide (NHS) esters, which were introduced 30 years ago.6
Various homobifunctional NHS esters have been used to
conjugate dyes, biotin, PEG, and drugs onto native proteins.3
However, studies have revealed that the reaction of NHS esters
is not completely chemoselective toward amino groups and
NHS esters could also react with histidine, serine, and
Received: April 5, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b00983
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
step of the reaction pathway.12 Nevertheless, the reaction
between OPA and amines in the absence of external reagents
under physiological conditions with which to label and modify
native proteins has not been explored previously.
characterized with 1H and 13C NMR spectra as a phthalimidine
derivative. Since phthalimidines could be formed under the PBS
buffer at a neutral pH from the reaction of OPA with primary
amines, this would be an ideal condition for performing peptides
and proteins modification. Therefore, we chose this condition as
our main focus to study its bioconjugation potentials. To study
the concentration effect on the rate of OPA and peptide
conjugation, time course LC−MS studies of the pentapeptide at
different substrate concentrations were conducted (Figure S2).
Even the pentapeptide was at a diluted concentration of 100
μM, a full conversion to the desired product was still observed at
16 min. Not only OPA showed high reactivity, it also had great
chemoselectivity toward primary amines. Four peptides (Ac-
RMF-OH, Ac-DTHC-OH, Ac-WYQ-OH, and H-PFASPLPIP-
OH) containing all the amino acid side chain functional groups
except the lysine amine were treated with OPA for 24 h and no
reaction was observed. It is worth mentioning that the internal
cysteine residue did not react with OPA under this condition
and the N-terminal proline was also inert. These results showed
that OPA reacted exclusively with primary amines in PBS (pH
7.4). It is noted that the N-terminal amine, except N-terminal
Pro, also reacted under this condition. Several proteins including
cytochrome c, lysozyme, ribonuclease A, and BSA have thus
been modified and confirmed by MALDI-TOF (Figures S18−
S23 in the Supporting Information).
In comparison with the widely used NHS chemistry, both
OPA and N-hydroxysuccinimide (NHS) activated ester 1 were
reacted with a model peptide (Ac-SHTHYGAK-OH) for 6 h
(OPA or NHS ester/peptide: 1 mol/1 mol; 50 μM). Along with
a large quantity of the unmodified peptide, 2 singly (MW
1022.4) and 2 doubly (MW 1102.5) modified peptides were
observed under the NHS ester conditions, which probably arise
from nonspecific reactions with other nucleophilic residues like
serine, histidine, and tyrosine in the peptide.7 The reaction with
OPA resulted in a singly modified product (MW 1058.4) with
only trace amount of the unmodified peptide (Figure 1). Next,
At the time, we were pondering whether the reaction of OPA
and amines could well proceed under the aqueous condition,
enabling phthalimidine-mediated native protein labeling. To this
end, the key criteria for biocompatible chemical transformations
have to be met, including aqueous solvent ideally with a neutral
pH, ambient temperature, low reactant concentrations, stability
of the reactant under aqueous conditions, and chemoselective
reaction, all of which make the development of effective
strategies challenging. Herein, we report that OPA and its
derivatives could react chemoselectively with amino groups on
native proteins rapidly under the physiological condition via
formation of phthalimidines, providing an alternative and
promising tool for native protein bioconjugation/labeling. The
notable advantages of this method over the existing protein
lysine-labeling approaches include traceless process, fast and
specific reaction, ease of operation, and using nonhydrolyzable
reagents.
Our studies started with the reaction of OPA with an N-
terminal acetylated pentapeptide (Ac-AFAQK-OH) under
aqueous conditions. The reaction could proceed in water, but
in a very low conversion (12%) after 4 h, as analyzed by RP-
LCMS (Table 1, entry 1). Addition of salts (e.g., NaCl, KCl) did
Table 1. Screening of the Reaction Condition
entry
aqueous solutions
conversion (%)
1
2
H2O
12
10
100 mM NaCl
3
100 mM KCl
19
4
sodium acetate buffer (pH 3.0)
PBS (pH 7.4)
38
5
100
100
100
5
6
borate buffer (pH 10.0)
10 mM Na2HPO4
7
8
10 mM NaH2PO4
9
HEPES buffer (pH 6.8)
10 mM tricine buffer (pH 7.4)
100
100
10
not help improve the conversion (Table 1, entries 2 and 3). As
acidic conditions would be beneficial for the phthalimidine
formation in organic medium,11 sodium acetate buffer (pH =
3.0) was tested but found to be ineffective (Table 1, entry 4). To
our delight, when the PBS (pH 7.4) and borate buffer (pH 10.0)
were tested, a full conversion of the starting peptide to a new
product was observed within 4 h (Table 1, entries 5 and 6).
Interestingly, sodium hydrogen phosphate alone could also
facilitate the reaction (Table 1, entry 7). Other neutral buffers,
such as and HEPES and tricine buffer (Table 1, entries 9 and
10), also worked well for such a reaction. The fact that the
reaction in neutral buffer proceeds much faster than that in
water indicates that the acid−base catalysis is likely involved.
The product of OPA and amines generated under the
aqueous condition (Table 1, entry 5) was isolated and
Figure 1. LC−MS chromatogram of crude peptide modifications with
NHS ester (1.0 equiv) and OPA (1.0 equiv). The NHS ester resulted in
single and double modifications (top); OPA resulted in a single
modification (bottom). On the basis of a full PDA wavelength 190−400
nm, the ratio of the peaks was calculated by Waters’ Empower system.
the OPA-conjugated product was isolated and analyzed by HCD
MS/MS, which showed the modification to be at the lysine site
forming a phthalimidine (Figure S10). These results indicate
that OPA has a higher degree of selectivity and efficiency toward
primary amines modification compared to the NHS ester.
To allow for attachment of various functional groups onto
proteins, OPA derivatives were synthesized, which included
B
DOI: 10.1021/acs.orglett.6b00983
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
different sizes of PEGs, an alkyne, and a carboxylic acid group
(Scheme 1). The substituent effects on the rate of reactions
Scheme 2. Protein Immobilization
Scheme 1. Synthesis of Functionalized OPA Derivatives
Figure 2. Fluorescent microscopic images of protein immobilization
using an OPA derivative. (A) Bright-field image of rink amide resin
coupled with OPA derivative. (B) Fluorescent image of rink amide
resin coupled with OPA derivative. (C) Bright-field image of rink amide
resin coupled with OPA derivative after conjugated with RFP. (D)
Fluorescent image of rink amide resin coupled with OPA derivative
after conjugated with RFP.
were investigated. The synthesis of OPA derivatives started with
salicylaldehyde derivatives 2a−2c to be refluxed with formic
hydrazide to form 3a−3c, which was then treated with lead(IV)
acetate to yield OPA derivatives 4a−4c. Different functions
groups were attached by simple EDCI-mediated coupling of
compound 6 and thus 8a−8c were synthesized. Then all the
synthetic derivatives were tested with the pentapeptide (Ac-
AFAQK-OH) in the PBS buffer. The reactions of all the
derivatives showed to achieve more than 80% conversion at 16
min (Figure S3 in the Supporting Information). In comparison,
an OPA derivative bearing an alkyne group 8a could be
conjugated to the peptide completely after 32 min, while an
NHS-alkyne 1 only gave a 67% conversion within the same
amount of time. These results showed that OPA has a faster
conversion reacting with amines, and thus, the second order
reaction rate constants of OPA and a model peptide at different
pH conditions were determined. The rate constant of NHS-
alkyne 1 was also compared with OPA-alkyne 8a under the same
condition (Table S2 in the Supporting Information). These
results showed that the conjugation rate of OPA-alkyne (7.49
M−1 s−1) was 5.9-fold faster than that of NHS-alkyne (1.27 M−1
s−1) and the reaction became even faster under the basic
conditions by the second order rate constant. Although the
reaction products are not the same, the results indicate that the
OPA-amine reaction proceeds also faster than other conjugation
and ligation methods.14
Protein immobilization onto a solid support such as resin,
glasses, metal, and nanomaterials have been widely applied for
drug delivery, microarray, and ELISA, etc.13 As such, we
explored the use of this phthalimidine-forming bioconjugation
method to perform protein immobilization. Compound 6
(Scheme 2) was coupled onto Rink amide resin. Red fluorescent
protein (RFP) in PBS solution was allowed to react with the
resin 10 for 30 min. After washing, as seen under the fluorescent
microscope, the RFP was successfully immobilized onto the
solid support in its native state (Figure 2).
PEGylated pharmaceutical proteins exhibit improved clinical
properties including physical and thermal stability, protection
against enzymatic degradation, better solubility, prolonged
blood circulating half-life, reduced immunogenicity, and
reduced toxicity.15 Up to date, nine PEGylated protein drugs
have been approved by the FDA, for six of which PEGylations
are realized using the NHS ester method.16 However, the NHS
ester method has some potential issues, including the instability
of the NHS ester, generation of NHS byproduct which requires
careful purification, and nonspecific reactions at lysine, serine,
tyrosine, and histidine sites.16 In this regard, development of an
improved PEGylation method would be of great value.
To investigate the potential of our newly developed
bioconjugation method in PEGylating native proteins, we
synthesized an OPA derivative 8b bearing monomethyl ethylene
glycol with average Mn 750 (mPEG750) and reacted it with
cytochrome c as a model protein (Figure S25). The PEGylation
of cytochrome c was performed at a 1:2 lysine to OPA-
mPEG750 ratio, with a final protein concentration of 50 μM in
PBS (pH 7.4). The degree of PEGylation was monitored by
SDS-PAGE and MALDI-TOF at different time points. The
results showed that all the accessible 19 lysine amines on
cytochrome c were conjugated with mPEG750. The mass of the
conjugating protein was increasing due to incomplete
PEGylation from 0.5 to 2 h. After 4 h, a constant mass was
observed as the fully PEGylated cytochrome c product.
Pegaspargase (Oncaspar), a PEGylated form of E. coli L-
asparaginase, is used to treat childhood acute lymphoblastic
leukemia.17 By depleting the asparagine level in blood, the
malignant lymphoid cells that rely on extracellular asparagine
thus undergo apoptosis.18 PEGylation of asparaginase is usually
performed randomly on lysine residues with PEG having an
average mass of 5000 Da using the NHS ester method.16 It was
noted that the PEGylation also took place at serine, tyrosine,
and histidine sites.16 To explore the utility of the OPA-amine
bioconjugation developed here, an OPA derivative equipped
with mPEG (8c) with an average Mn of 5000 was reacted with
E. coli L-asparaginase for 4 h under the same conditions as above.
The PEGylated protein was then purified by size exclusion
column chromatography. The activity of thus-obtained
C
DOI: 10.1021/acs.orglett.6b00983
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
PEGlyated asparaginase was determined by the asparaginase
activity kit (Sigma-Aldrich). In contrast to the native L-
asparaginase, the obtained PEGylated asparaginase retained
46.7% of the initial activity (Figure 3). Next, the OPA-
the University of Hong Kong, Shenzhen Basic Research Grant
(Grant JCYJ20140903112959961), and the CAS Interdiscipli-
nary Innovation Team.
REFERENCES
■
(1) (a) Biju, V. Chem. Soc. Rev. 2014, 43, 744. (b) Takaoka, Y.; Ojida,
A.; Hamachi, I. Angew. Chem., Int. Ed. 2013, 52, 4088. (c) Boutureira,
O.; Bernardes, G. J. L. Chem. Rev. 2015, 115, 2174.
(2) (a) MacDonald, J. I.; Munch, H. K.; Moore, T.; Francis, M. B. Nat.
Chem. Biol. 2015, 11, 326. (b) Chan, A. O.-Y.; Ho, C.-M.; Chong, H.-
C.; Leung, Y.-C.; Huang, J.-S.; Wong, M.-K.; Che, C.-M. J. Am. Chem.
Soc. 2012, 134, 2589. (c) Abbas, A.; Xing, B.; Loh, T.-P. Angew. Chem.,
Int. Ed. 2014, 53, 7491.
́
(3) (a) Basle, E.; Joubert, N.; Pucheault, M. Chem. Biol. 2010, 17, 213.
(b) Spicer, C. D.; Davis, B. G. Nat. Commun. 2014, 5, 4740.
(4) (a) Kurpiers, T.; Mootz, H. D. Angew. Chem., Int. Ed. 2007, 46,
5234. (b) Chudasama, V.; Smith, M. E.; Schumacher, F. F.;
Papaioannou, D.; Waksman, G.; Baker, J. R.; Caddick, S. Chem.
Commun. 2011, 47, 8781. (c) Abad, S.; Nolis, P.; Gispert, J. D.;
Spengler, J.; Albericio, F.; Rojas, S.; Herance, J. R. Chem. Commun.
2012, 48, 6118. (d) Dhar, T.; Kurpiers, T.; Mootz, H. D. Methods Mol.
Biol. 2011, 751, 131. (e) Sejwal, P.; Han, Y.; Shah, A.; Luk, Y. Org. Lett.
2007, 9, 4897.
(5) (a) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7,
876. (b) Chen, X.; Muthoosamy, K.; Pfisterer, A.; Neumann, B.; Weil,
T. Bioconjugate Chem. 2012, 23, 500. (c) Fernandez-Moreira, V.;
Alegre-Requena, J. V.; Herrera, R. P.; Marzo, I.; Gimeno, M. C. RSC
Adv. 2016, 6, 14171.
(6) Lomant, A. J.; Fairbanks, G. J. Mol. Biol. 1976, 104, 243.
(7) Kalkhof, S.; Sinz, A. Anal. Bioanal. Chem. 2008, 392, 305.
(8) Swaim, C. L.; Smith, J. B.; Smith, D. L. J. Am. Soc. Mass Spectrom.
2004, 15, 736.
(9) (a) Morales, A. R.; Schafer-Hales, K. J.; Marcus, A. I.; Belfield, K.
D. Bioconjugate Chem. 2008, 19, 2559. (b) Varadarajan, A.; Sharkey, R.
M.; Goldenberg, D. M.; Hawthorne, M. F. Bioconjugate Chem. 1991, 2,
102.
Figure 3. Enzymatic activity (left) and proteolysis stability of the
PEGylated and native L-asparaginase (right).
PEGylated L-asparaginase was able to be resistant to proteolytic
degradation by enzymes including trypsin and Glu-C and
retained 70.0% and 40% of activities respectively; while non-
PEGylated asparaginase lost its activity completely after
enzymatic proteolysis (Figure 3).
In summary, we have demonstrated that the OPA-amine
reaction to form a phthalimidine is an effective biocompatible
chemical transformation, enabling phthalimidine-mediated
native protein bioconjugation. Under the physiological buffer
condition (e.g., PBS buffer, pH 7.4), OPA and its derivatives
efficiently and chemoselectively react with the amino group of
native proteins, which provides a user-friendly and promising
method for native protein modification. This method has the
characteristics of operational simplicity, chemoselective reac-
tion, low reactant concentration, traceless process, and readily
assessable and nonhydrolyzable reagents. Its potential applica-
tions have been demonstrated in functionalization, immobiliza-
tion, and PEGylation of native proteins.
(10) Zhang, Y. F.; Lee, C. L.; Liu, H.; Li, X. Org. Lett. 2012, 14, 5146.
(11) (a) Thiele, J.; Schneider, J. Liebigs Annalen 1909, 369, 287.
(b) Takahashi, I.; Hatanaka, M. Heterocycles 1997, 45, 2475. (c) Kulla,
E.; Zuman, P. Org. Biomol. Chem. 2008, 6, 3771. (d) Cronin, J.R.;
Pizzarello, S.; Gandy, W. E. Anal. Biochem. 1979, 93, 174. (e) Per
́
ard-
Viret, J.; Prange, T.; Tomas, A.; Royer, J. Tetrahedron 2002, 58, 5103.
́
(12) Alajarin, M.; Sanchez-Andrada, P.; Lopez-Leonardo, C.; Alvarez,
A. J. Org. Chem. 2005, 70, 7617.
ASSOCIATED CONTENT
* Supporting Information
■
S
(13) (a) Camarero, J. A. Biopolymers 2008, 90, 450. (b) Wang, T.-H.;
Lee, W.-C. Biotechnol. Bioprocess Eng. 2003, 8, 263. (c) Taylor, R. H.;
Fournier, S. M.; Simons, B. L.; Kaplan, H.; Hefford, M. A. J. Biotechnol.
2005, 118, 265. (d) Besselink, T.; Liu, M.; Ottens, M.; van Beckhoven,
R.; Janssen, A. E.; Boom, R. M. J. Sep. Sci. 2013, 36, 1185.
(14) Saito, F.; Noda, H.; Bode, J. W. ACS Chem. Biol. 2015, 10, 1026.
(15) (a) Abuchowski, A.; van Es, T.; Palczuk, N. C.; Davis, F. F. J. Biol.
Chem. 1977, 252, 3578. (b) Harris, J. M.; Chess, R. B. Nat. Rev. Drug
Discovery 2003, 2, 214.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b00983.
Experimental procedures and characterization data
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: xuechenl@hku.hk.
Author Contributions
(16) Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. Polym. Chem.
2011, 2, 1442.
(17) (a) Graham, M. L. Adv. Drug Delivery Rev. 2003, 55, 1293.
(b) Ortega, J. A.; Nesbit, M. E., Jr.; Donaldson, M. H.; Hittle, R. E.;
Weiner, J.; Karon, M.; Hammond, D. Cancer Res. 1977, 37, 535.
(18) Masurekar, A.; Fong, C.; Hussein, A.; Revesz, T.; Hoogerbrugge,
P. M.; Love, S.; Ciria, C.; Parker, C.; Krishnan, S.; Saha, V. Blood Cancer
J. 2014, 4, e203−1.
C.L.T. and C.T.T.W. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by the National Basic Research
Program of China (Grant 2013CB836900), the Area of
Excellence Scheme of the University Grants Committee
(Grant AoE/M-12/06), Strategic Research Theme on Drug of
D
DOI: 10.1021/acs.orglett.6b00983
Org. Lett. XXXX, XXX, XXX−XXX