Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications

Article abstract of DOI:10.1039/d0cc02722g

Two types of pyridoxal analogs, azido pyridoxal (PL-N3) and carboxyl pyridoxal (PL-COOH), were developed as novel bifunctional bioorthogonal molecules. These molecules showed fast imine formation with hydrazinyl groups and stable covalent linkages via azido/carboxyl groups, and thus were of great use for site-specific peptide and protein modifications.

Full text of DOI:10.1039/d0cc02722g

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Conformational control of nonplanar free base porphyrins:
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DOI: 10.1039/D0CC02722G
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Bifunctional Pyridoxal Derivatives as Efficient Bioorthogonal
Reagents for Biomacromolecule Modifications
Xianxian Mao, Wei Li, Shiyu Zhu, Juan Zou, Hongyan Tian, Yuting Duan, Yuntao Wang, Jiayue Fei
and Xiaojian Wang*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Two types of pyridoxal analogs, azido pyridoxal (PL-N
carboxyl pyridoxal (PL-COOH), were developed as novel (Scheme 1, Fig. 1a): azido pyridoxal (PL-N
3
) and we reported the design and synthesis of two types of PLP analogs
, 1) and carboxyl
3
bifunctional bioorthogonal molecules. These molecules pyridoxal (PL-COOH, 2 and 3). Azido pyridoxal 1 was designed to
showed fast imine formation with hydrazinyl groups and react with alkynes, forming a stable covalent linkage and thus
stable covalent linkages via azido/carboxyl groups, and thus suitable for a wide range of crosslinking applications. PL-COOHs
was of great use for site-specific peptide and protein were designed with the carboxyl group bridged with linkers of
modifications.
different lengths, which replaces phosphate group of PLP for the
more stable amide linkage, extending the application scope of
such molecules. To the best of our knowledge, PL-COOH 3 was
previously reported as an enzyme cofactor or protein antagonist,
but never explored as a biorthogonal reagent.9
In the fields of chemical biology and biotechnology, a number of
bioorthogonal tools have been developed to modify biological
molecules such as nucleic acids, proteins and lipids to achieve the
1
purpose of labeling and/or tracking of these molecules. Among
a large number of bioorthogonal reactions, the hydrazone/oxime
ligation is one of the earliest and most widely used reactions.2
Francis group has extensively studied the transamination reaction
at the N-terminus of proteins with various compounds, which
yielded a ketone or aldehyde group at that end of the protein for
3
the further labeling via aldoxime condensation. Kool et al
reported the reaction kinetics between a variety of different
carbonyl compounds and hydrazines, revealing the impact of
4
functional groups at different substitution cites.
Scheme 1 Design strategy of PL analogs
The coenzyme pyridoxal-5’-phosphate (PLP) is a unique
example of aldehydes that has been used for bioconjugation due
to its fast kinetics and relatively stable imine products. Hsein et
The conversion from the phosphate of PLP to azide or
carboxyl might have a negative effect on the activity, as it was
reported that the phosphate group may catalyze the imine
formation as an internal proton source.5
speculate that this might not be a major defect, because it was
reported that the 5'-deoxypyridoxal had only a slightly weaker
5
al utilized PLP to label the amino dendrimer with imine
6
formation. Wang et al reported a simple two-step aqueous
b, 10
However, we
protocol which successfully modified different proteins with PLP
7
through a phosphoramide linkage.
However, one major
limitation of PLP is the poor stability of phosphoramide bond
bridging PLP and amino compounds of interest, such as proteins
and peptides, which makes PLP unsuitable for a variety of
1
1
reactivity than PLP when reacting with glycine, and PLP is one of
the most reactive aldehyde reported.4 To further evaluate the
design, DFT calculations were employed to estimate the reactivity
d
8
applications, especially ones involving acidic conditions. Herein,
3
of PL-N and PL-COOHs, comparing to PLP, using methyl amine as
a model reactant (see Supporting Information for calculation
details). The analysis of rate-determine dehydration step yielded
small differences of activation free energies among the four (0.06
kcal/mol for 1 vs PLP, 0.1 kcal/mol for 2 vs PLP and -0.53 kcal/mol
for 3 vs PLP), indicating small effects of the substitutions on
Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering,
Nanjing Tech University, Nanjing 211816, China.
Email: ias_xjwang@njtech.edu.cn
Electronic Supplementary Information (ESI) available: Experimental details and
supplementary figures.
See DOI: 10.1039/x0xx00000x
12
reactivity.
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Journal Name
The designed compounds were then synthesized as shown in Table 1 The reaction rate (k
Fig. 1b. Briefly, starting from commercially available pyridoxine aldehydes and HP.
1
) and equilibrium constant (Keq) between
View Article Online
DOI: 10.1039/D0CC02722G
hydrochloride, 3 and 4’ hydroxyl groups were first protected as
K
Keq
(M )
1
Compounds
1
3
-1 -1
-1
the cyclic acetal 5 to prevent undesired side reactions. The 5’
hydroxyl group was then either converted into the azido group
with DPPA through Mitsunobu reaction, or into the chloride
which was further substituted by the cyanide group. The acetal
protection was then cleaved with acid, and the cyanide was also
hydrolyzed into carboxyl during the same process. Finally, the
(M s )
2.6 ± 0.1
5.8 ± 0.1
5
5
5
PL-N
3
(2.1 ± 0.5) × 10
(1.2 ± 0.2) × 10
(1.7 ± 0.1) × 10
PL-COOH 3
PLP
PL
2-AMBA
8.5 ± 0.1
a
0.11 ± 0.01
0.15 ± 0.01
0.21 ± 0.01
N/D
a
N/D
N/D
a
2
-FBA
a
N/D: not determined
released 4’ hydroxyl was oxidized into aldehyde with MnO
2
,
Thus, in order to study PL-N
modification, hydrazine containing dye, fluorescein-5-
thiosemicarbazide (FTZ) was first tested as a model molecule. PL-N
was incubated with cyclooctyne modified bovine serum albumin
3
mediated biomacromolecular
yielding azido pyridoxal 1 (PL-N ) and carboxyl pyridoxal (PL-
3
a
COOH) 2. The PL-COOH 3 was prepared by oxidation of reported
carboxyl pyridoxine derivative (see Supporting Information, Fig.
S9).14
3
1
9
(
BSA-FDIBO, 12) to obtain 13 (BSA-FDIBO-PL), which was then
a)
H
O
H
O
H
O
reacted with FTZ in PBS, yielding fluorescein-labled BSA (Fig. 2a). The
reaction mixture was purified via ultracentrification and analyzed
with UV-VIS spectroscopy. As shown in Fig. 2b, the absorption peak
HO
HO
HO
COOH
N3
COOH
N
1
N
2
N
3
b)
OH
HO
HO
O
H
O
O
corresponding to PL-N
and an average of 2.7 PL-N
estimated. The apparent low yield (31.5%) was mostly due to the
undesired modification of PL-N on BSA-FDIBO via imine formation
or nonspecific absorption, which was also observed in the control
experiment with BSA and PL-N (see Supporting Information, Fig.
S13a). Further analysis was conducted with sodium dodecyl sulfate
3
at 390 nm and FTZ at 490 nm was observed,
HO
a
O
b
O
O
c
d
HO
N3
OH
OH
N3
N3
3
and 0.85 FTZ molecules per BSA were
N
N
5
e
N
6
N
N
4
7
1
3
O
OH
O
H
O
f
O
g,h NaO
i
HO
COONa
Cl
CN
COOH
3
N
N
N
N
8
9
10
2
Fig. 1 (a) Structure of designed and synthesized 1 and 2. (b) Synthesis of 1 and 2. polyacrylamide gel electrophoresis (SDS-PAGE, Fig. 2c), and only BSA-
a) DMP, TsOH, acetone, RT, 20 h, 63%. b) DPPA, DBU, THF, RT, 4 h, 97%. c) 1M PL-FTZ showed a strong fluorescent band (column 7), whereas the
HCl, THF, reflux, 4 h, 95%. d) MnO
2
, THF, RT, 6 h, 54%. e) SOCl
2
, CH
2
Cl
2
, RT, 2 h, controls of unmodified BSA with FTZ (column 4) or BSA-FDIBO with
O, FTZ (column 5) had negligible signal.
o
9
1
1%. f) TMSCN, NaF, DMF, 69 C, overnight, 94%. g) 6 M HCl, Reflux, 11 h. h) H
2
M NaOH, 40 oC, 97% over two steps. i) MnO
, RT, overnight, 15%.
2
DMP = 2,2-dimethoxypropane; TsOH = p-toluenesulfonic acid; DPPA =
diphenylphosphoryl azide; DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; SOCl
2
=
thionyl chloride; TMSCN = trimethylsilyl cyanide.
The reactivities of synthesized pyridoxal derivatives were
then studied with 2-hydrazinopyridine (HP) as a model
compound, comparing to the naturally occurring pyridoxal-5'-
1
5
phosphate (PLP) and pyridoxal (PL). Reactions were carried out
in phosphate buffered saline (PBS, pH 7.4) and monitored with
UV-VIS spectrometer, following the peak formation of the
7
corresponding hydrazone. The experimental data of PL-N
3
1 and
PL-COOH 3 fitted the rate equation for a reversible second-order
reaction, and PL fitted the rate equation for a pseudo-first order
reaction (see Supporting Information, Fig. S11,), while experiment
with PL-COOH 2 yielded irreproducible results (data not shown).16
3
As shown in Table 1, The reactivity of PL-N and PL-COOH 3 was
much higher than PL, since the intermolecular hemiacetal was
broken after the substitution of the 5’ hydroxyl group, which was
1
7
consistent with previous report.
Two control bifunctional
aldehydes, 2-(azidomethyl)benzaldehyde (2-AMBA) and 2-formyl
benzoic acid (2-FBA), were also examined, yielding much slower
kinetics. PL-N and PL-COOH 3 were sightly less reactive but still
3
Fig. 2 (a) Modification of BSA with FTZ. (b) UV-vis absorption spectra of BSA-FDIBO
(10.18 μM, black), BSA-FDIBO-PL (10.11 μM, blue), and BSA-PL-FTZ (10.07 μM, red).
(c) SDS-PAGE visualized under UV light (bottom) and after Coomassie-blue stain
(top). Well from left to right: protein ladder (each line: 80 kDa and 60 kDa, from
comparable to PLP, and should be of great use for bioconjugation
applications, because their hydrazone formation rates were
top to bottom), BSA, BSA-FDIBO, BSA∼FTZ, BSA-FDIBO∼FTZ, BSA-FDIBO-PL and
4
d
among the fastest ones, and much higher than the most widely
BSA-PL-FTZ from 2 to 7.
−
1
−1
used Cu-free azide−alkyne cycloadditions (0.057 M
s
for
aza-
−1
−1
dibenzocyclooctyne
dibenzocyclooctyne).
and
0.29
M
s
for
1
8
2
| J. Name., 2012, 00, 1-3
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A major challenge of biomolecule conjugation is to make site-
specific modification, which often involve unnatural amino acids and macromolecules was examined. The BSA-FDIBO was reacted with
To expand the application of PL-N
3
, conjuation between two
View Article Online
DOI: 10.1039/D0CC02722G
nucleic acids. Inspired by the reported work from Li et al,20 a sortase eGFP-PL-N
A (SrtA) mediated reaction protocol was employed to specifically was purified via ultracentrification and analyzed with UV-VIS
modify a protein’s C-terminal into an azide functionality with PL-N spectroscopy. As shown in Fig. 3d, when both eGFP-PL-N and BSA-
to prepare BSA-PL-eGFP (Fig. 3a). The reaction mixture
3
3
.
3
As shown in Fig. 3a, enhanced green fluorescent protein (eGFP) FDIBO were presented in the reaction, the absorption peak
containing a SrtA recognition sequence was expressed and corresponding to eGFP at 485 nm was observed, and an average of
hydrozinolyzed by SrtA.20a The obtained eGFP hydrazide (eGFP- 1.3 eGFP molecules per BSA was estimated. The control experiment
NHNH
eGFP-PL-N
DIBAC modified 5-(2-aminoethylamino)-1-naphthalenesulfonic acid
DIBAC-EDANS, 15) was reacted with eGFP-PL-N to afford eGFP-PL- the phosphoramide bond formed between the phosphate group of
2
) was then reacted with PL-N
3
to afford azide-modified eGFP was performed with BSA-FDIBO and plain eGFP, and minimal, if any,
, a eGFP absorption was observed.
One key limitation of PLP application was the lack of stability of
(
3
, 14). To confirm the successful conjugation of PL-N
3
(
3
EDANS. As shown in Fig. 3b, the emission spectrum of DIBAC-EDANS PLP and the amino group of biomolecules of interest. Thus, we took
partly overlaps with the excitation spectrum of eGFP, making them a advantage of the reaction between the carboxyl pyridoxal PL-COOH
good pair for fluorescence resonance energy transfer (FRET). Upon 3 and the amino residues for the modification of polypeptides at
excitation at 335 nm, an increase of fluorescence signal was observed definded sites. A model peptide with a sequence of RGDYKGGKG was
at 508 nm and approximately 50% of the fluorescent intensity was synthesized via standard Fmoc solid-phase chemistry on Rink Amide
recovered from FRET (Fig. 3c), which was not observed by just mixing MBHA resin (Fig. 4a). The two lysine residues were installed with
DIBAC-EDANS and eGFP in the same cuvette, proving the successful Fmoc-Lys(Boc)-OH (YKG) or Fmoc-Lys(Mtt)-OH (GKG). The Mtt group
conjugation between eGFP-PL-N
3
and DIBAC-EDANS.
was then selectively removed and the corresponding Lys residue was
modified with PL-COOH 3 to yield peptide Ac-RGDYKGGK(PL)G-NH
RGDPL) via a stable amide linkage. As a comparison, PLP failed to
2
(
modify the peptide through the same procedure, due to the unstable
phosphoramide linkage, and only unmodified model peptide
(
(
peptiRGD) was observed on the liquid chromatography (LC) analysis
Fig. 4b). The obtained RGDPL was able to further react with FTZ, and
1
.5 eq FTZ was enough to convert 96.9% RGDPL into modified
product (HPLC yield), despite the possible competeing reaction
between the free amino group of the other Lys residue and the
installed aldehyde group (Fig. 4c). The high conversion rate, stable
product formation, and good bioorthogonality further proved the
usefulness of PL-COOH 3 as a click reagent.
Fig. 3 (a) Modification of eGFP with DIBAC-EDANS and conjugation between eGFP
and BSA with PL-N . (b) UV/Vis absorption of eGFP (6.6 μM, red) in 1 X PBS and
emission spectra of DIBAC-EDANS 15 (0.73 μM, λex = 335 nm, blue) in methanol.
c) Fluorescence emission spectrum of eGFP (100 nM, black, λex = 335 nm), eGFP-
3
Fig. 4 (a) Modification of peptiRGD with PL-COOH 3 and RGDPL with FTZ. (b) Liquid
chromatogram of peptiRGD with or withour modification: peptiRGD (red, 1.84 min),
RGDPL (blue, 8.02 min) and RGDPLP (black, 1.85 min). (c) Liquid chromatogram of
modification of RGDPL with FTZ: FTZ (red, 11.17 min), RGDPL (black, 8.02 min) and
RGDPL-FTZ (blue, 9.49 min).
(
PL-EDANS (100 nM, red, λex = 335 nm), eGFP∼EDANS (100 nM, blue, λex = 335 nm),
eGFP (100 nM, purple, λex = 490 nm). (d) UV-vis absorption spectra of BSA-
FDIBO∼eGFP (5.23 μM, black), and BSA-PL-eGFP (7.30 μM, red).
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In conclusion, we have reported two novel bifunctional 5 (a) R. G. Wiegand, J. Am. Chem. Soc., 1956, 78, 530V7i;ew(bA)rtMicle. OAn.liGne.
del Vado, J. Donoso, F. Muñoz, G. R. EchevDaOrrI:ia10a.1n0d39F/.DG0.CBCla0n2c7o22, GJ.
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A. Basagoitia, E. Pizarro, R. Goldsmid, J. G. S. Blanco and F. G.
3
bioorthogonal molecules: azido pyridoxal (PL-N ) and carboxyl
pyridoxal (PL-COOH 3). These pyridoxal analogs were easy to
prepare, and had fast reaction kinetics to form stable ligation
3
products. Azido pyridoxal (PL-N ), a molecule containing dual
bioorthogonal functionalities, showed unique properties to mediate
biomacromolecular crossliking with stable triazole linkage and
hydrazone formation, utilizing its azido and formyl group respectively.
The carboxyl pyridoxal (PL-COOH 3) was a useful subsitution of PLP
for modification of amino groups which are abundant in
Blanco, HeIv. Chim. Acta, 1998, 81, 837; (d) G. R. Echevarrı
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(a) E. Groman, Y. Z. Huang, T. Watanabe and E. E. SNELL, Proc. Natl.
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bioorthogonal reactions and for biomacromolecule modifications
and would be of great use for a variety of applications.
This work was supported by grants from the National Natural
Science Foundation of China (21708019), Natural Science Foundation
of Jiangsu (BK20170987), Jiangsu province “Double Plan” and start-
up fund at Nanjing Tech University (39837135). We thank Prof. Lei
Liu and Prof. Yiming Li (Tsinghua University) for providing the
plasmids of SrtA and eGFP. We are grateful to the High Performance
Computing Center of Nanjing Tech University for supporting the
computational resources.
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Conflicts of interest
There are no conflicts to declare.
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