Facile Cu(ii)-mediated conjugation of thioesters and thioacids to peptides and proteins under mild conditions

Article abstract of DOI:10.1039/c8ob00536b

The bioconjugation of peptide derivatives such as polypeptides, peptide-based probes and proteins is a vibrant area in many scientific fields. However, reports on metal-mediated chemical methods towards native peptides especially non-engineering protein modification under mild conditions are still limited. Herein, we describe a novel Cu(ii)-mediated strategy for the conjugation of thioesters/thioacids to peptides under mild conditions with high functional group tolerance. Based on this strategy, polypeptides, even peptide-based fluorescent probes, can be efficiently constructed. Finally, the selective modification of lysine residues of native Ub with thioesters could be realized and complete conjugation of Ub could be achieved even under equivalent Cu(ii). These promising results could greatly expand Cu(ii)-mediated reaction strategies on chemical biology and molecular imaging.

Full text of DOI:10.1039/c8ob00536b

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Facile Cu(II) mediated conjugation of thioesters and thioacids to
peptides and proteins under mild conditions
Yao Suna†, Zhenbin Lyub†, Zhiqiang Wang b,c, Xiaodong Zengb, Hui Zhoub, Fuchun Xu d, Ziyang Chenb,
Yuling Xua, Ping Xu*c, Xuechuan Hong*b,d
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The bioconjugation of peptide derivatives such as polypeptides, peptide-based probes and proteins is a vibrant area for
many scientific fields. However, the reports on metal-mediated chemical methods towards native peptides especially non-
engineering proteins modification under mild conditions are still limited. Herein, we describe a novel Cu(II)-mediated
strategy for conjugation of thioesters/thioacids to peptides under mild conditions with high functional group tolerance.
Based on this strategy, polypeptides, even peptide-based fluorescent probes can be efficiently constructed. Finally, the
selective modification of Lysine residues of native Ub with thioesters could be realized and complete conjugation of Ub
could be achieved even under equivalent Cu(II). These promising results could greatly expand Cu(II)-mediated reaction
strategies on chemical biology and molecular imaging.
www.rsc.org/
genetic engineering and can reduce protein expression levels.20ꢀ
21
Introduction
Therefore, developing novel and efficient metalꢀmediated
reactions towards native polypeptides especially proteins under
The development of new peptides modification strategies is a
vibrant area in many scientific fields, such as chemical biology,
molecular imaging and biomedicine.1ꢀ6 Over the last decade,
mild conditions with excellent functional group tolerance and
freeꢀengineering of the protein target can greatly expand metalꢀ
mediated reaction strategies in conjugation toolbox.
transition metalꢀmediated polypeptides
and
proteins
conjugation with small molecules has been regarded as a
powerful and efficient tool for the study and manipulation of
a) The conjugation of thioester/thioacid to amino acids or polypeptides
H
Cu(OAc)2/HOBt
PBS/DMF(1:1)
H2N
R1
R3
N
R1
R3
SR
biological function of protein in vitro and in vivo.7ꢀ11
A
+
R2
O
R2
O
pioneered work is the Cu(I)ꢀcatalyzed (3+2) cycloaddition
chemistry which was reported by Sharpless and Meldal in the
early 2000’s.12ꢀ13 However, the cytotoxicity caused by Cu(I)ꢀ
mediated generation of reactive oxygen species (ROS) from O2
would damage the biological systems, which heavily hampered
the widespread utilization of Cu(I) in living system.14ꢀ17 On the
other hand, the presence of many other transition metals beyond
Cu(I), such as palladium and ruthenium could also serve as
alternative sources in vitro and in vivo.18ꢀ19 Although these
methods have been regularly utilized for many biological
applications, most of them require varying degrees of protein
polypeptides/fluorescence peptide-based
probes
b) The conjugation of thioesters/thioacid to native protein
O
R
R
SR
NH
NH2
Cu(OAc)2/HOBt
PBS/DMF
+
O
native protein
Scheme. new strategy for Cu(II)/HOBt-mediated conjugation
thioesters/thioacids to native peptide derivatives.
1
A
Inspired by the thiol chemistry in the acylation of coenzyme,
acetylꢀCoA and nonꢀribosomal peptide, the conjugation of
natural peptides with thioesters/thioacids gaining momentum
due to its unique reactivity and selectivity.22ꢀ24 For example,
Philip Garner and Gopi group have reported Cu(II)ꢀpromoted
ligations of peptide thioacids and various amines.23,25ꢀ27
Considering the high stability and low toxicity of Cu(II)
compared with Cu(I), Cu(II) seems to be more favorable for
biological applications.28 Although the Cu(II) salts has been
widely utilized for many scientific fields, Cu(II) metalꢀ
mediated facile conjugation of natural polypeptides especially
proteins with thioesters/thioacids under mild conditions is
a.Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, Hubei
International Scientific and Technological Cooperation Base of Pesticide and
Green Synthesis, Chemical Biology Center, College of Chemistry, Central China
Normal University, Wuhan 430079
b.State Key Laboratory of Virology, Zhongnan Hospital of Wuhan University, Wuhan
University School of Pharmaceutical Sciences, Wuhan 430071, China. E-mail:
xhy78@whu.edu.cn
c. State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing,
Beijing Institute of Radiation Medicine, Beijing102206, China
d.Medical College, Tibet University, Lasa, 850000, China
e. † These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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soughtꢀafter for their broad potential in chemical biology and copper(II) salts such as Cu(OTf)2 and CuSO4. To our delight, all the
molecular imaging. Herein, we demonstrate a novel Cu(II)ꢀ Cu(II) complexes tested produced the desired products in favorable
mediated conjugation approach in a wide range of peptide yields (Table 1, 85% and 77%, Entries 8ꢀ9). We chose the Cu(OAc)2
derivatives (Scheme 1). This reaction tolerates a wide range of as it was inexpensive and commonly available in many standard
functional groups such as ꢀCO2H and ꢀOH under mild reaction laboratories. 1HꢀNMR, 13CꢀNMR and ESIꢀMS of the desired product
conditions and requires no inherent genetic engineering.
3a are included in the ESI.
Results and discussion
O
NH
H2N
O
OMe
BocHN
OMe
Catalyst
Solvent
37 °C
N
+
S(p-tolyl)
H
BocHN
O
N
O
H
3a
Time (h) Yield (%)a
1a
2a
Entry
Catalysts
Solvents
1
2
3
HMDO (1 equiv.)
Cu(OAC)2 (1 equiv.)
56
12
1
86
trace
82
DMF (2 mL)
DMF (2 mL)
Cu(OAC)2 /HOBt (1:1, 1 equiv.) DMF (2 mL)
4
5
HOBt (1 equiv.)
DMF (2 mL)
DMF (2 mL)
12
12
0
HMDO/HOBt (1:1, 1 equiv.)
trace
Table.
polypeptides and tumor targeted peptides
2
Cu(II)/HOBt-mediated conjugation of thioesters/thioacids to
6
7
8
Cu(OAC)2 /HOBt (2 equiv.)
Cu(OAC)2 /HOBt (1 equiv.)
Cu(OTf)2/HOBt (1 equiv.)
DMF (2 mL)
1
1
1
82
80
85
PBS/DMF (1:1,2 mL)
PBS/DMF (1:1,2 mL)
Using the optimized conditions, the construction of
polypeptides and peptideꢀbased fluorescent probes was carried
out (Table 2). As expected, each amino acid tested produced the
desired dipeptides in good to excellent yields (80ꢀ88%) via
Cu(II)/HOBt strategy (Table 2, Entries 1ꢀ3). Furthermore, we
9
CuSO4/HOBt (1 equiv.)
PBS/DMF (1:1,2 mL)
1
77
a Isolated yield based on the thioester
Table. 1 Optimization of the Cu(II)/HOBt-mediated dipeptide conjugation
To achieve this goal, the conjugation of NꢀBocꢀPheꢀS(pꢀ
tolyl)thioester (1a) with
successfully extended this strategy to obtain tetrapeptide (3e
)
LꢀTrpꢀOMe (2a) in DMF was initially
and pentapeptide (3f) conjugated products with satisfactory
yields (Table 2, Entries 4ꢀ5). All these products were verified
by ESIꢀMS. Interestingly, the reaction was completed within 6
min when thioesters were replaced by thioacetic acid (Table 2,
Entry 6). This result is consistent with previous literature
reports that thioacetic acid exhibits higher reactivity than
thioesters.26,30 Recently, the development of peptideꢀbased
probes represents an emerging field in medical chemistry,
chemical biology and molecular imaging due to their favorable
excretion pharmacokinetics and good biocompatibility.31ꢀ36
Therefore, we explored the preparations of peptideꢀbased
fluorescent probes via Cu(II)/HOBt strategy. Very pleasingly,
the conjugation reaction between the Cy5.5 thioacid (see ESI)
and various tumor targeting peptides (RGD, JMV594, AE105
and dimerꢀRGD) proceeded smoothly and provided the desired
imaging probes (3h-3k) in good yield within 0.5 h. (Table 2,
Entries 7ꢀ10, Figure S2ꢀS6). Our methodology was effective
with high functional group tolerance such as CO2H and OH,
which could provide an alternative for the construction of
peptideꢀbased probes for in vitro and vivo bioꢀimaging in the
future besides the gold standard maleimide and NHS ester
chemistry.31
performed as a model reaction (Table 1). It is noteworthy that
HMDO can promote the reaction individually in a good yield (86%)
in an extended time (56 h) (Table 1, Entry 1). As a result, its further
applications for polypeptide and protein conjugation was largely
limited. We therefore investigated a Cu(II) salt (1 eq) as a new
catalytic system to improve the efficiency of this conjugation.
Surprisingly, only a trace amount of the desired product was
observed after 12 h (Table 1, Entry 2). 1ꢀHydroxybenzotriazole
(HOBt) was widely used as a common coupling additive in the
standard coupling reaction.29 Inspired by this, we anticipated that
HOBt could be helpful for the copperꢀmediated conjugation. To our
delight, the reactions can be completely converted to the desired
product within 1 h under the combination of Cu(OAc)2 and HOBt
(1:1, 1eq) based on HPLC analysis (Table 1, Entry 3). In a control
experiment, no desired product was observed in the presence of
HOBt solely or the mixture of HMDO/HOBt (Table 1, Entries 4ꢀ5).
Furthermore, the excess amount of HOBt did not affect the reaction
yield (Table 1, Entry 6). We speculated that Cu(II) would be the
activation of the thioester/thioacid, allowing the formation of a
transient HOBt ester inꢀsitu, and this catalytic system also exhibited
high biocompatibility (Figure S1). Ideally, the conjugation reaction
should be carried out in aqueous medium for potential applications
which aimed at proteins or other biomolecules. Thus, the novel
Cu(II)/HOBtꢀmediated peptide conjugation was efficiently
accomplished in phosphate buffered solution (PBS) and DMF (1:1,
vol/vol) in 80% yield (Table 1, Entry 7). Encouraged by this
promising result, the scope of the reaction was tested with various
Finally, the modification of native proteins was attempted.
The potential of Cu(II)/HOBtꢀmediated conjugation of
thioesters/thioacids with Ubiquitin (Ub, comprises 76 amino
acid residues, 7 lysine residues, exact mass=8564.7 Da) was
evaluated. CbzꢀLꢀPhe thioester (100 eq), Cu(OAc)2 (1 eq) and
HOBt (10 eq) were incubated with Ub in PBS and DMF (1:1
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vol/vol) (Table 3, Entry 1). The conversion of Ub was with trypsin and analyzed by LCꢀMS/MS. The MS and MS2
monitored by MALDIꢀTOFꢀMS spectrum. Surprisingly, no spectra revealed the possible multiple conjugation sites such as
conjugation product was observed by MALDIꢀTOFꢀMS even N terminus and all the εꢀamino groups of lysine residue (K6,
after the reaction was continued for
6 h (Figure S7). K11, K29, K33, K48 and K63 except K27 (Figure S17ꢀS22).
Considering the poor solubility of the aromatic thioester, the This result strongly suggested that the conjugation was not
proportion of DMF was increased in the mixture solvents from specific and both the lysine residue and N terminus of Ub could
50% to 70%. An anticipated partial conversion of Ub to monoꢀ be modified. There are several factors that might have affected
labeled product was observed by the mass spectrum analysis the conjugation yield and sites. Herein, we hypothesized that
(Table 3, Entry 2, Figure 1a, m/z 8846.6614). It was difficult to the nonꢀsiteꢀspecific modification of Ub is probably due to the
completely consume Ub even the amount of substrates (100 eq high reactivity and small steric hindrance of CH3COSH during
to 200 eq) and Cu(II) (1 eq to 2 eq) was increased in an the conjugation. Based on these results, the selective
extended reaction time (6 h to 16 h). (Table 3, Entry 3ꢀ4, Figure modification of Lys residues of native Ub with thioester could
S8). To determine the selectivity of the conjugation of Ub be realized and complete conjugation of Ub could be achieved
protein, the Ub adduct was digested with trypsin. The resulting even under equivalent Cu(II).
peptides were analyzed by liquid chromatography electrospray
ionization tandem mass spectrometry (LCꢀMS/MS, Figure S9).
MS2 spectrums were analyzed using pFind 2.0.37 The results of
MS spectrum revealed that the possible monoꢀconjugation site
would be K33 or K48 of peptide fragment of the trypsinized Ub
(Figure S10 and S11). The selectively modification of εꢀamino
groups of lysine residue of Ub with CbzꢀLꢀPhe thioester rather
than N terminus was quite encouraging. Selective monoꢀlabeled
lysine residue product was also achieved by a biotin thioester.
The MS and MS2 spectra indicated that the conjugation sites
were K6, K33 or K48 (Figure S12ꢀS14).
O
O
CbzHN
Cu(OAc)2/HOBT
PBS/DMF
NHCbz
S
NH2
N
H
+
Ub
Ub
partially complete
O
O
Cu(OAc)2/HOBT
N
H
NH2
+
SH
PBS/DMF
100% conversion
Thioesters/Thioacids
Entry
1
Catalyst
Solvents
Time
Product
0 %
6 h
6 h
Cu(OAc)2(1 eq) Phe-S(p-tolyl) 100 eq
HOBt (10 eq)
DMF:PBS=5:5
2
Cu(OAc)2(1 eq) Phe-S(p-tolyl) 100 eq
HOBt (10 eq)
DMF:PBS=7:3
mono-modified
6 h
3
4
Cu(OAc)2(1 eq) Phe-S(p-tolyl) 200 eq
HOBt (10 eq)
DMF:PBS=7:3
DMF:PBS=7:3
mono-modified
mono-modified
Figure. 1 a) MALDI-TOF-MS spectrum for conjugation thioesters to Ub; b) MALDI-
TOF-MS spectrum for conjugation thioacids to Ub.
16 h
Cu(OAc)2(2 eq) Phe-S(p-tolyl) 200 eq
HOBt (10 eq)
Cu(OAc)2(1 eq) CH3COSH 100 eq
HOBt (2 eq)
DMF:PBS=5:5
PBS
6 h
6 h
90 % (mono,dimer
triple modified)
5
6
Cu(OAc)2(1 eq) CH3COSH 100 eq
HOBt (2 eq)
90 % (mono,dimer
modified)
Conclusions
CuSO4(2 eq)
HOBt (4 eq)
CH3COSH 200 eq
DMF:PBS=5:5
6 h
100 % (mono,dimer,triple
tetra and penta modified)
7
In short, we have successfully developed Cu(II)/HOBt
strategy for the facile conjugation of peptides under mild
conditions with excellent functional group tolerance and freeꢀ
engineering technique. Based on this innovative methodology,
polypeptides and peptideꢀbased fluorescent probes have
efficiently been constructed. Furthermore, native proteins such
as Ubiquitin have been successfully conjugated with small
molecules. This strategy could greatly expand the applications
of Cu(II)ꢀmediated reaction strategies in chemical biology and
molecular imaging.
Table. 3 Cu(II)/HOBt-mediated conjugation of thioesters/thioacids to Ub protein.
In order to improve the modification efficiency of Ub,
CH3COSH (MW=76.1 Da, 100 eq) was chosen for this
conjugation reaction. As expected, the result of MALDIꢀTOFꢀ
MS indicated that ~90% of Ub was converted to a mixture of
monoꢀlabeled, dimmerꢀlabeled and tripleꢀlabeled products
under the same condition (Table 3, Entry 5, Figure S15). This
result confirmed the thioacid was more reactive than the
thioester in the protein conjugation. The conjugation was
further carried out in PBS solution. The monoꢀlabeled and
dimmerꢀlabeled were observed in the MALDIꢀTOFꢀMS (Table
3, Entry 6, Figure S16). Increasing the amount of thioacid and
Cu(II), a mixture of 1 to 5 labeled Ub products were obtained
(Table 3, Entry 7, Figure 1b). The Ub conjugate was digested
Acknowledgements
This work was partially supported by grants from NSFC
(21708012, 81773674, 81573383), 111 project B17019, NSFHP
(2017CFA024, 2017CFB711, 2017CFB151, 2016ACA126), the
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