Furan-based acetylating agent for the chemical modification of proteins

Article abstract of DOI:10.1016/j.bmc.2014.12.053

We have synthesized a furan-based acetylating agent, 2,5-bisacetoxymethylfuran (BAMF) from carbohydrate derived 5-hydroxymethylfurfural (HMF) and studied its acetylation activity with amines and cytochrome c. The results show that BAMF can modify proteins in biological conditions without affecting their structure and function. The modification of cytochrome c with BAMF occurred through the reduction of heme center, but there was no change in the coordination property of iron and the tertiary structure of cytochrome c. Further analysis using MALDI-TOF-MS spectrometer suggests that BAMF selectively targeted lysine amino acid of cytochrome c under our experimental conditions. Kinetics study revealed that the modification of cytochrome c with BAMF took place at faster rates than aspirin.

Full text of DOI:10.1016/j.bmc.2014.12.053

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Bioorganic & Medicinal Chemistry 23 (2015) 791–796
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Furan-based acetylating agent for the chemical modification
of proteins
Sudipta De a, Tarun Kumar b, Ashish Bohre a, Laishram R. Singh b, Basudeb Saha a,
⇑
a Laboratory of Catalysis, Department of Chemistry, University of Delhi, North Campus, Delhi 110007, India
b Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, North Campus, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a furan-based acetylating agent, 2,5-bisacetoxymethylfuran (BAMF) from carbohy-
drate derived 5-hydroxymethylfurfural (HMF) and studied its acetylation activity with amines and cyto-
chrome c. The results show that BAMF can modify proteins in biological conditions without affecting their
structure and function. The modification of cytochrome c with BAMF occurred through the reduction of
heme center, but there was no change in the coordination property of iron and the tertiary structure of
cytochrome c. Further analysis using MALDI-TOF-MS spectrometer suggests that BAMF selectively
targeted lysine amino acid of cytochrome c under our experimental conditions. Kinetics study revealed
that the modification of cytochrome c with BAMF took place at faster rates than aspirin.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 2 September 2014
Revised 5 December 2014
Accepted 19 December 2014
Available online 30 December 2014
Keywords:
Furan-based acetylating agent
Lysine residues
Cytochrome c
N-acetylation
Protein modification
1. Introduction
Nt-acetylated proteins are more stable in vivo than non-acetylated
proteins.10 Also the N-terminus of a protein has unique pH-
Protein aggregation and misfolding is a serious concern in the
recent time. Many neurodegenerative diseases including Alzhei-
mer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease
(HD), amyotrophic lateral sclerosis (ALS), and prion diseases are
associated with this.1,2 Hydrophobic interaction between amino
acids in different globin chains causes hemoglobin polymerization
and results sickle cell disease (SCD).3 Sometimes abnormal change
in protein structure can lower the solubility of protein in the body
fluid which causes supersaturation. Example includes sickle cell
disease where sickle hemoglobin aggregates due to structural
change in b-globin unit.4 To overcome this problem, the only
remedy is the modification of protein with proper modifying agent.
Protein modification is of major interest in chemical biology. In
this regard, post-translational modification (PTM) is essential for
regulating the function, stability, localization and targeting of
many eukaryotic proteins.5,6 Phosphorylation, farnesylation, ubiq-
uitination, glycosylation, acetylation, formylation, amidation,
sumoylation, biotinylation are the examples of PTM. Many chemi-
cal methods have been developed for such purpose by carefully
balancing the reactivity and selectivity.7–9 Site-selective chemical
modification of a protein requires an efficient reaction and an
interesting molecule to attach. Experimental facts have proved that
dependent reactivity and is thus an attractive target for single-site
modification.11 Blocking of the N-terminus by Nt-acetylation
potentially prevents N-terminal ubiquitination, and thus stabilizes
the protein.12,13 Lysine contains a primary amine group which is
protonated under biological pH, but it can still react as a nucleo-
phile. Acetylation of lysine groups in proteins has been extensively
used for modifying enzymatic properties, immunological
reactivity, and proteolytic digestion patterns. Lysine acetylation
has emerged as a major post-translational modification for his-
tones in modulating chromatin-based transcriptional control. It is
a reversible posttranslational modification (PTM), which neutral-
izes the positive charge of this amino acid, changing protein func-
tion in diverse ways.14,15 Lysine acetylation is also important for
p53 functions and interactions and for microtubule stabilization.16
Enormous number of enzymatic acetylating agent is known
these days for chemical acetylation of biomolecules. However, only
a few numbers of chemical acetylating agents is reported. Acetic
anhydride is the simplest example of acetylating agent which can
modify protein molecules by N-terminal acetylation of lysine resi-
dues.17,18 But it is very hard to control its action due to its rapid
reaction in aqueous environment. High reactivity also decreases
the selectivity towards the target molecules. Aspirin is currently
one of the most frequently used drugs which has been shown to
acetylate proteins and biomolecules such as hemoglobin, DNA, RNA
and histones, as well as several plasma constituents, including
⇑
Corresponding author. Tel.: +91 011 2766 6646; fax: +91 011 2766 7794.
E-mail addresses: bsaha@chemistry.du.ac.in, sahab@purdue.edu (B. Saha).
http://dx.doi.org/10.1016/j.bmc.2014.12.053
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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S. De et al. / Bioorg. Med. Chem. 23 (2015) 791–796
O
O
NaBH4 was dissolved in 3 mL distilled water. Then NaBH4 solution
was added drop wise to the HMF solution with continuous stirring.
The reduction process was very fast. After the complete reduction,
the product (BHMF) was isolated by extracting with ethyl acetate
(4 ꢀ 20 mL). Here the aqueous phase was saturated with NaCl for
the quantitative extraction of BHMF. After evaporation of ethyl
acetate, BHMF was obtained as white solid product (0.64 g, 100%
yield) and characterized by NMR studies. 1H NMR (400 MHz,
NH
O
O
O
+
NH2
O
BAMF
Lysine residue
50 mM PO43- buffer
pH 7.4, 37 o
C
CDCl3):
d
6.16 (s, 2H, 2CH), 4.50 (s, 4H, 2CH2). 13C NMR
(100 MHz, CDCl3): d 154.0, 108.5, 57.4.
NH
O
HO
+
OH
O
N
H
2.3. Synthesis of 2,5-bisacetyloxymethylfuran (BAMF)
O
BHMF
Modified protein
In a 10 mL round bottom flask 0.512 g (4 mmol) BHMF was dis-
solved in 1.89 mL (20 mmol) of acetic anhydride. Then sodium ace-
tate (0.066 g, 0.8 mmol) was added into the mixture as a catalyst
and it was stirred for 6 h at room temperature. After the reaction,
2 mL of distilled water was added into the reaction mixture very
slowly to consume the excess acetic anhydride. Reaction product
was isolated by extracting with dichloromethane (5 ꢀ 8 mL). After
evaporation of dichloromethane BAMF was obtained as white solid
product (0.611 g, 72% yield). 1H NMR (400 MHz, CDCl3): d 6.35 (s,
2H, 2CH), 5.01 (s, 4H, 2CH2), 2.06 (s, 6H, 2CH3). 13C NMR
(100 MHz, CDCl3): d 170.5 (C@O), 150 (C–O), 111.4 (CH), 57.9
(CH2), 20.7 (CH3). Melting point = 62 °C.
Scheme 1. Modification of lysine residues in protein leading to adduct formation
via amidation reaction.
hormones and enzymes.19 Aspirin is reported as an antisickling agent
which modifies sickle hemoglobin through bLys-82 acetylation.20
Walder et al. reported a bromo aspirin derivative, acetyl-3,5-dib-
romosalicylic acid (dibromoaspirin), as an antisickling agent which
demonstrated potential acetylating ability for intracellular hemoglo-
bin in vitro.21 The bromoaspirin effectively targets the amine groups
of Hb S unit to get acetylated, which increases the oxygen affinity to
exert the antisickling effects. But sometimes the use of aspirins can
cause fatal gastrointestinal bleeding, hemorrhagic strokes, nephro-
toxicity, and adverse effects on the central nervous system. Thus
investigations for new acetylating agents will remain continue in
the context of suitable application with minimum side-effects.
In the present work we report the synthesis of a furan-based
acetylating compound 2,5-bisacetyloxymethylfuran (BAMF) from
5-hydroxymethylfurfural (HMF) which is a dehydration product
of hexose sugars.22,23 Although BAMF was first isolated as a natural
product from an ethyl acetate extract of the terrestrial Streptomy-
ces species,24 the chemical synthesis of this compound has not
been reported yet. We have shown that this compound acetylates
primary amine groups in the biological pH range. The preliminary
results have motivated us to further study its efficiency in the acet-
2.4. Acetylation of cyclohexylamine
In a 10 mL glass vial 19.8 mg (0.2 mmol) cyclohexylamine and
21.2 mg (0.1 mmol) BAMF were taken. To it 1 mL of 50 mM potas-
sium phosphate buffer (pH = 7.4) was added and the reaction mix-
ture was stirred at 37 °C for 6 h. After the completion of reaction,
the aqueous phase was saturated with NaCl and the product was
isolated by extracting with ethyl acetate. Product was characterized
by 1H NMR (Fig. S3) and yield of acetylated cyclohexylamine was
calculated by using mesitylene as an external standard. Yield = 82%.
ylation of cyclohexylamine and L-lysine (Scheme 1). Cytochrome c
2.5. Acetylation of
L-lysine
and lysozyme were selected as model proteins because of their dif-
ferent chemical characteristics, high content and easy accessibility
of surface lysine residues, hydrophobicity indices and extensive
structural information. Cytochrome c also plays a key role in the
energy production in mitochondria and also has so many
enzymatic activities in animals. BAMF has successfully modified
both the proteins without changing their structure and activity.
In a 10 mL glass vial 29.2 mg (0.2 mmol)
L
-lysine and 21.2 mg
(0.1 mmol) BAMF were taken. To it 1 mL of 50 mM potassium
phosphate buffer (pH = 7.4) was added and the reaction mixture
was stirred at 37 °C for 6 h. Due to high solubility of lysine and
its amide in aqueous phase, it was not possible to characterize
the product by 1H NMR. In this case UV–vis spectroscopy was used
since the lysine amide shows an absorbance at 215 nm, while free
lysine does not show any absorbance in this range (Fig. S4). Absor-
bance of free BAMF was subtracted from the acetylated lysine.
2. Experimental
2.1. Materials
2.6. Preparation of protein samples
5-Hydroxymethylfurfural, cytochrome c (from bovine heart)
and lysozyme (from chicken egg white) were purchased from
Sigma–Aldrich. Sodium borohydride and sodium acetate were pur-
chased from SRL, India. Acetic anhydride, ethyl acetate and dichlo-
romethane were supplied by S D Fine-Chem, India. Potassium
dihydrogen phosphate, dipotassium hydrogen phosphate and
potassium chloride were obtained from Merck. Guanidinium chlo-
ride (GdmCl) was the ultrapure sample from MP Biomedicals.
Unless otherwise stated, all chemicals were used without further
purification and distilled water was used as aqueous phase.
Lysozyme and cytochrome c solutions were dialyzed exten-
sively against 0.1 M KCl at pH 7.0 at ꢁ4 °C. Protein stock solutions
were filtered using 0.22-lm millipore filter paper. All the proteins
gave a single band during polyacrylamide gel electrophoresis. Con-
centration of the protein solutions was determined experimentally
using the molar absorption coefficient (
e
) values (3.9 ꢀ 104 Mꢂ1 cmꢂ1
at 280 nm for lysozyme, and 1.06 ꢀ 105 Mꢂ1 cmꢂ1 at 410 nm for
cytochrome c). The concentration of GdmCl stock solution was
determined by refractive index measurements. All solutions for
optical measurements were prepared in the desired degassed
buffer. For desired pH range, 50 mM phosphate buffer (pH 7.4)
was used. Since pH of the protein solution may change on the
addition of co-solvents, pH of each solution was also measured
after the denaturation experiments.
2.2. Synthesis of 2,5-bishydroxymethylfuran (BHMF)
In a 50 mL round bottom flask 0.63 g (5 mmol) HMF was dis-
solved in 10 mL distilled water. In another flask, 0.19 g (5 mmol)

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S. De et al. / Bioorg. Med. Chem. 23 (2015) 791–796
793
2.7. Modification of cytochrome c and lysozyme by BAMF
of lysozyme was then plotted against the different concentrations
of BAMF used.
Acetylation of amino groups of cytochrome c and lysozyme was
performed in the following way. Cytochrome c and lysozyme
2.13. ESI-MS analysis
(50 lM both) were incubated overnight with different concentra-
tions of BAMF (0.3 mM, 0.6 mM, 0.9 mM and 1.2 mM) at 37 °C in
50 mM phosphate buffer at pH 7.4. Protein samples modified in
this manner were used for the subsequent experiments. The ratio
of protein/BAMF was varied as per needed throughout the experi-
ment. For the reference experiment the protein (cytochrome c) was
treated with aspirin under the same conditions.
ESI-MS was performed on native and BAMF-modified peptides
using a Waters Corp. (QTOF) II MS. 5
l
L of the sample solutions
m i.d. reverse-phase
were separated by HPLC on a 15 cm ꢀ 75
l
C18 column using a 20 min linear gradient of 5–100% acetonitrile
in 0.1% formic acid and at a flow rate of 0.2 mL minꢂ1
.
2.14. MALDI-TOF-MS analysis
2.8. Tryptic digestion for mass spectroscopic analysis
Protein molecular ions were analyzed in linear, positive ion
mode using a SCIEX TOF/TOF 4800 Plus Analyzer (Applied Biosys-
The pH of native and modified cytochrome c was adjusted to
7.5–9.0 by adding 10 mM ammonium bicarbonate containing
1 mM CaCl2. The lyophilized trypsin was solubilized in 1 mM EDTA
and added to the protein solution to a final concentration of ꢁ1:50
trypsin/protein (w/w). After overnight incubation at 37 °C, the
samples were cooled to room temperature, and the pH was
adjusted to 3–4 with formic acid to stop the digestion process.
tems). The trypsin digested mixtures (13
lL) were acidified with
0.7 L of 10% (v/v) trifluoroacetic acid (TFA). A C4 ZipTip was
l
washed with 50% (v/v) aqueous acetonitrile containing 0.1% (v/v)
TFA and then equilibrated with 0.1% (v/v) TFA solution. The acidi-
fied sample (10
washed with 0.1% (v/v) TFA. Finally, the sample was eluted with
75% (v/v) aqueous acetonitrile containing 0.1% (v/v) TFA (1.2 L).
lL) was extracted with the prepared ZipTip and
l
2.9. Thermal denaturation study
The prepared samples were mixed 1:10 (vol/vol) with a saturated
solution of sinapinic acid in 50% aqueous acetonitrile/0.1% aqueous
Thermal denaturation studies of cytochrome c were carried out
in a Jasco V-660 UV–visible spectrophotometer equipped with a
Peltier-type temperature controller at a heating rate of 1 °C per
minute. This scan rate was found to provide adequate time for
equilibration. Each sample was heated from 20 to 85 °C. The
change in absorbance with increasing temperature was followed
at 400 nm for cytochrome c. About 650 data points of each transi-
tion curve were collected. Measurements were repeated three
times. After denaturation, the protein sample was immediately
cooled down to measure reversibility of the reaction.
trifluoroacetic acid (1:1, vol/vol) and 1 lL spotted onto the stain-
less steel MALDI target plate and allowed to dry before the analy-
sis. Using an acceleration voltage of 25 kV and a laser intensity of
2500 V, each spot was analyzed a minimum of three times, accu-
mulating spectra composed of approximately 2500 laser shots in
total.
3. Results and discussion
3.1. UV–vis spectroscopic study
2.10. Circular dichroism (CD) study
The simple synthesis of BAMF comprises two steps, (i) reduc-
tion of HMF to BHMF and (ii) acetylation of BHMF to BAMF. To
study the activity of synthesized compound (BAMF) in aminoacet-
ylation reaction we chose a primary amine compound, cyclohexyl-
amine which was treated with BAMF in the same reaction
conditions as that of biological media. The 1H NMR spectrum
(Fig. S3) shows almost quantitative transfer of acetyl groups from
BAMF to cyclohexylamine to form its corresponding amide. After
achieving this success we sought to find out the acetyl group trans-
CD measurements were made in a Jasco J-810 spectropolarime-
ter equipped with peltier controller at 25 °C with six accumula-
tions. Protein concentration used for the CD measurements was
0.5 g/L. Cells of 1.0 cm path length were used for the measure-
ments of near-UV spectra in the range of 240–320 nm. Necessary
blanks were subtracted. The CD instrument was routinely cali-
brated with D-10-camphorsulfonic acid.
ferring ability of BAMF to L-lysine. The UV spectra showed similar
result in the same condition which promoted us to explore the
BAMF for protein modification.
2.11. Extrinsic fluorescence study
Fluorescence spectra of the protein samples were measured in a
Perkin Elmer LS 55 Spectrofluorimeter in a 3 mm quartz cell, with
both excitation and emission slits set at 10 nm. Protein concentra-
To demonstrate the feasibility of our newly prepared BAMF for
the protein modification, we have employed UV–vis spectroscopy
to confirm that the cytochrome c has been modified by BAMF.
The protein cytochrome c was treated by different concentrations
of BAMF in phosphate buffer at pH 7.4 (Fig. 1a). Since heme center
is the most essential part of cytochrome c for folding state,25 we
have concentrated on its detail study. The typical absorption peak
at 410 nm corresponds to the Soret band of heme protein, while a
broad band with maximum at 530 nm is for Fe+3 center in ferricy-
tochrome c. The adduct formation by the BAMF can be assessed by
studying the reduction of heme iron of cytochrome c from Fe+3 to
Fe+2 state. The disappearance of peak at 530 nm and appearance
of new peaks at 522 and 551 nm indicate the reduction of iron cen-
ter in cytochrome c as a result of modification. The same results
were found when cytochrome c was subjected to modify using
homocysteine thiolactone.26 The reduction of the iron center is
occurred most probably due to the change in ligation property.
The Soret band also showed a red shift to 414 nm for BAMF and
tion for all the experiments was 5 lM. For ANS-protein binding
experiments the excitation wavelength was 360 nm, and emission
spectra were recorded from 400–600 nm. ANS concentration was
kept 16 fold higher than that of protein concentration. Necessary
blanks were subtracted for each sample. Each spectrum was
repeated at least three times.
2.12. Activity study of lysozyme
Lysozyme hydrolytic activity was determined turbidimetrically
by measuring the decrease in absorbance at 450 nm of a suspen-
sion of bacterial cells. The effect of BAMF adduct formation on
the function of lysozyme was studied by the treatment of different
concentrations of BAMF (0.3, 0.6, 0.9 and 1.2 mM) in phosphate
buffer at pH 7.4. Each study was done for 1500 s. Specific activity

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S. De et al. / Bioorg. Med. Chem. 23 (2015) 791–796
Cyt c native
0.20
0.15
0.10
0.05
0.00
Cyt c native
a
b
0.5 mM BAMF
1.0 mM BAMF
2.0 mM BAMF
3.0 mM BAMF
4.0 mM BAMF
6.0 mM BAMF
Cyt c modified
0.12
0.08
0.04
0.00
600
650
700
750
800
360
400
440
480
520
560
Wavelength (nm)
Wavelength (nm)
0.4
0.3
0.2
0.1
0.0
Cyt c native
2 mM BAMF
2 mM Aspirin
Cyt c native
2 mM BAMF
2 mM Aspirin
c
d
0.40
0.35
0.30
0.25
0.20
0
600
1200 1800 2400 3000 3600
Time (s)
350
400
450
500
550
600
Wavelength (nm)
Figure 1. (a) UV–vis spectra of modified cytochrome c with different concentration of BAMF. (b) 695 nm band in modified cytochrome c (red line) and native cytochrome c
(black line). (c) UV–vis spectral changes in cytochrome c (50 M) modified by 2 mM BAMF and aspirin. The protein was incubated for overnight at 37 °C, pH 7.4. Soret band of
l
modified protein (red and blue line) and native cytochrome c (black line). (d) Redox kinetics of cytochrome c at 550 nm wavelength treated with 2 mM BAMF and 2 mM
aspirin.
413 nm for aspirin modified cytochrome c respectively (Fig. 1c).
Generally a blue shift of Soret band means unfolding of proteins.27
So in this study protein structure remains in its folding state which
does not cause any aggregation after modification. Another wave-
length from 600 to 800 nm was selected in the UV–vis experiment
to evaluate the sixth iron-heme ligand, Met-80 (Fig. 1b). The peak
at 695 nm remained intact which suggests that there was no
change in the tertiary structure of cytochrome c as well as no loss
of ligand from heme center. The reported compound BAMF was
compared with the existing acetylating agent aspirin in terms of
activity. Figure 1c shows the UV–vis spectra of similarly modified
cytochrome c in both cases when treated with 2 mM BAMF and
2 mM aspirin. To compare the rate of adduct formation by BAMF
and aspirin we studied the reduction kinetics of cytochrome c in
presence of the two compounds. The redox kinetics of heme center
in cytochrome c was performed at 550 nm for 1 h duration. Results
shown in Figure 1d confirms that the rate of adduct formation by
BAMF is many fold higher than aspirin.
also procured near UV CD spectra of lysozyme which showed no
significant change in CD signal upon BAMF treatment as shown
in Figure 2b and hence no perturbation in the tertiary structure
of lysozyme. So by the CD study it is again proved that BAMF has
no such effect on the structure of protein molecules as revealed
by the UV–vis spectroscopy earlier.
3.3. ANS-binding study
Whenever there is structural perturbation in protein, its hydro-
phobic core is exposed. ANS (1-anilino naphthalene-8-sulfonate) is
a fluorescent dye and being hydrophobic in nature it always tends
to bind the hydrophobic core of the protein. After binding with the
hydrophobic core of the protein there is increase in the emission
fluorescence of ANS along with a shift in emission peak from
520 nm to 475 nm, which confirms the exposure of hydrophobic
residues of proteins to outside and hence the unfolding of the pro-
tein. In our study we also sought to find out the effect of BAMF
adduct formation upon protein structure. So to check whether
Nt-acetylation can perturb conformation of cytochrome c, we car-
ried out extrinsic fluorescence study using ANS dye. Figure 3a
shows ANS binding study of cytochrome c treated with different
concentrations of BAMF which revealed no exposure of hydropho-
bic core of cytochrome c and hence no binding of ANS. ANS binding
study was also done to confirm that there was any change in the
structure of lysozyme (Fig. 3b). The same results were obtained
to prove that BAMF has no effect on the proteins for the exposure
of hydrophobic core.
3.2. CD Study
As we have showed that the potential of BAMF to form adduct
with the lysine residues of the protein is very high, we were then
interested in investigating the effect of BAMF on the structure of
cytochrome c. For this, we performed near UV CD measurements
and here we did not find any change in the near UV CD signal of
modified cytochrome c with different concentrations of BAMF
used. The characteristic negative peaks of cytochrome
c at
282 nm and 289 nm of tyrosyl side chains did not show any
change. These results (Fig. 2a) showed that there is no effect of
adduct formation by BAMF on the tertiary structure of cytochrome
c. We were unable to obtain far UV CD spectra because of the high
noise level due to high absorption of BAMF in far UV CD range. We
3.4. Thermal denaturation study
Heat induced denaturation of cytochrome c was carried out to
test the effect of BAMF adduct formation on thermodynamic