Chemical modification of recombinant hirudin with palmitic acid in mixed aqueous-organic solutions

Article abstract of DOI:10.1016/j.procbio.2018.11.022

In this study, chemical modification of recombinant hirudin variant-2 (HV2) with palmitic acid (PAL) was proposed as an alternative approach to circumvent the limitations of PEGylation. To facilitate a sufficient contact of the hydrophilic HV2 to the hydrophobic PAL, thereby improving the reaction specificity to achieve the desired mono-PAL-HV2 with high retained bioactivity, the reaction was developed in mixed aqueous-organic solutions. Compared with HV2 conjugation with PAL-benzotriazole (PAL-BTA) in mixed aqueous-NMP solution, the conjugation of HV2 with PAL-N-hydroxysuccinimide (PAL-NHS) in mixed aqueous-DMSO solution could improve the site-specific conjugation of one PAL molecule to a particular lysine residue. Furthermore, the reaction mixture of the latter was further purified by preparative liquid chromatography. Three mono-PAL-HV2 isomers were obtained and retained 36%, 4% and 89% of the in vitro anticoagulant activity of unmodified HV2, respectively. One of the mono-PAL-HV2 isomers, namely, mono-PAL-HV2-3, was isolated with the highest selectivity and exhibited the highest in vitro anticoagulant bioactivity. Modification site analysis of mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2. This study presented a successful PAL modification in which site-specific reaction was improved to achieve the desired mono-PAL-HV2 with highly retained bioactivity in mixed aqueous-organic solutions.

Full text of DOI:10.1016/j.procbio.2018.11.022

Accepted Manuscript
Title: Chemical modification of recombinant hirudin with
palmitic acid in mixed aqueous-organic solutions
Authors: Shu-Chang Wang, Xu-Dong Wang, Xin-Nan Teng,
Zhi-Long Xiu
PII:
S1359-5113(18)31379-5
DOI:
Reference:
https://doi.org/10.1016/j.procbio.2018.11.022
PRBI 11511
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
5 September 2018
28 November 2018
30 November 2018
Please cite this article as: Wang S-Chang, Wang X-Dong, Teng X-Nan,
Xiu Z-Long, Chemical modification of recombinant hirudin with palmitic
acid in mixed aqueous-organic solutions, Process Biochemistry (2018),
https://doi.org/10.1016/j.procbio.2018.11.022
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Chemical modification of recombinant hirudin with palmitic acid in
mixed aqueous-organic solutions
Shu-Chang Wang a, Xu-Dong Wang a, Xin-Nan Teng, Zhi-Long Xiu*
School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,
Dalian 116024, P R China
aThese two authors made an equal contribution to this work.
*Corresponding author:
Prof. Dr. Zhi-Long Xiu, E-mail: zhlxiu@dlut.edu.cn, Tel/ Fax: +86-411-84706369.
Graphical abstract

Highlights
 Recombinant hirudin variant-2 (HV2) was conjugated with palmitic acid (PAL)
 The selectivity of this reaction was improved in mixed aqueous-DMSO solution
 Three mono-PAL-HV2 isomers was efficiently separated by prep-HPLC
 Mono-PAL-HV2-3 showed the highest selectivity and the highest in vitro bioactivity
 The modification site of mono-PAL-HV2-3 was identified as Lys 27 of HV2
Abstract
In this study, chemical modification of recombinant hirudin variant-2 (HV2) with palmitic
acid (PAL) was proposed as an alternative approach to circumvent the limitations of PEGylation.
To facilitate a sufficient contact of the hydrophilic HV2 to the hydrophobic PAL, thereby
improving the reaction specificity to achieve the desired mono-PAL-HV2 with high retained
bioactivity, the reaction was developed in mixed aqueous-organic solutions. Compared with
HV2 conjugation with PAL-benzotriazole (PAL-BTA) in mixed aqueous-NMP solution, the
conjugation of HV2 with PAL-N-hydroxysuccinimide (PAL-NHS) in mixed aqueous-DMSO
solution could improve the site-specific conjugation of one PAL molecule to a particular lysine
residue. Furthermore, the reaction mixture of the latter was further purified by preparative liquid
chromatography. Three mono-PAL-HV2 isomers were obtained and retained 36%, 4% and 89%
of the in vitro anticoagulant activity of unmodified HV2, respectively. One of the
mono-PAL-HV2 isomers, namely, mono-PAL-HV2-3, was isolated with the highest selectivity
and exhibited the highest in vitro anticoagulant bioactivity. Modification site analysis of
mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2. This

study presented a successful PAL modification in which site-specific reaction was improved to
achieve the desired mono-PAL-HV2 with highly retained bioactivity in mixed aqueous-organic
solutions.
Keywords: Recombinant hirudin; Chemical modification; Fatty acid-conjugation; Long-chain
fatty acids; Palmitic acid; Mixed aqueous-organic solutions
1. Introduction
Hirudin, a single-chain cyclic peptide composed of 65 amino acids (molecular weight (MW),
~ 7000 Da), is by far the most potent thrombin inhibitor [1]. Recently, recombinant hirudin has
been developed for therapeutic applications due to the limited source of natural hirudin. Two
recombinant hirudins, including lepirudin and desirudin, have been approved by the FDA.
However, recombinant hirudin is associated with inherent disadvantages, including short
circulation half-life in vivo, strong immunogenicity, and induction of bleeding [2]. PEGylation is
an effective strategy to improve the therapy efficacies of protein and peptide drugs [3, 4], as well
as to overcome such disadvantages of the recombinant hirudin [5-9]. However, PEGylation still
had several limitations after four decades of research [10-13]: (1) The relatively high molecular
weight of PEG tends to reduce the biological activity of the precursor proteins and peptides. (2)
The PEG chain and mono-methoxy terminus of PEG molecules can induce the generation and
activation of anti-PEG antibodies, leading to inflammation, autoimmune diseases, and other side
effects. (3) During the in vivo degradation process, low molecular weight PEG molecules leads
to renal toxicity, while high molecular weight PEG molecules are difficult to be metabolized,
resulting in aggregation in the liver and thus causing hepatotoxicity after long-term use.

Therefore, there is an urgent need for other strategies to circumvent these limitations of
PEGylation for protein and peptide drugs, including recombinant hirudin.
Alternatively, chemical modification of proteins and peptides drugs with long-chain fatty acids
(LCFAs, usually the number of carbon atoms >12), such as palmitic acid, is appealing more and
more attention. LCFAs is more advantageous than PEG in multiple aspects [14-23]: (1)
Generally, chemical modification with LCFAs does not significantly reduce the activity of
prodrugs due to the relatively low molecular weights of LCFAs. (2) LCFAs are important
constituents of cell-membrane phospholipids and adipose tissues, thus LCFA-modification can
improve the lipid-solubility of drugs, the intestinal mucosal permeability, and the binding of
drugs to the receptors, and decrease the risk of antibody generation and in vivo degradation of the
drugs. (3) LCFAs can reversibly bind to human serum albumin (HSA) in the body, and the
complex of LCFA and HSA is resistant to renal clearance due to the large size of the molecules
in transmembrane transport, thereby significantly increasing the in vivo half-life of the drug. (4)
In some cases, LCFAs can also induce the drugs to form homo-polymers, further extending their
half-life in vivo. Recently, four fatty acid-modified drugs developed by Novo Nordisk Co.,
namely, Insulin detemir [20], Insulin degludec [21], Liraglutide [22] and Semaglutide [23],
have been approved by the FDA. However, reports on the chemical modification of proteins and
peptides with LCFAs were still limited compared with those on PEGylation. Especially, the
chemical modification of recombinant hirudin with LCFAs has not yet been reported to date.
To obtain the LCFA-recombinant hirudin conjugate efficiently, two issues are to be addressed:
(1) Recombinant hirudin dissolves easily in water but poorly in organic solvents. In contrast,
LCFA dissolves easily in organic solvents but poorly in water. Thus, it is necessary to generate
an environment to facilitate a sufficient contact of the hydrophilic recombinant hirudin to the

hydrophobic LCFA. (2) Similar to PEGlyation, site-specific conjugation is more advantageous
than random conjugation for the chemical modification of recombinant hirudin with LCFAs.
Recently, several site-specific fatty acid-conjugation strategies have been developed in previous
studies [15-17]. Unfortunately, commonly-used site-specific fatty acid-conjugation strategies
may be not applicable to recombinant hirudin due to its unique structure [5-9], which has already
been discussed in our previous report [7]. Therefore, fatty acid conjugation of recombinant
hirudin at the ε-amine group of lysine residues is still a commonly-employed strategy. However,
recombinant hirudin has multiple lysine residues, its conjugation with LCFA would also result in
various conjugates (mono-LCFA and multi-LCFA conjugated forms and their positional
isomers). Moreover, these positional isomers with conjugated LCFA at different lysine residues
of recombinant hirudin would also exhibit different bioactivities compared to the unmodified
parent compound. Thus, another challenge is to improve the reaction specificity to achieve the
desired mono-LCFA-recombinant hirudin with high retained bioactivity. A possible strategy to
solve both issues is to develop a suitably mixed aqueous-organic solution system for the
chemical modification of recombinant hirudin with LCFAs. The advantages of the system
include both the improved reaction specificities of PEGylation of proteins or peptides through
inducing their conformation changes by organic solvents and the good solubilities of both
hydrophilic and hydrophobic substrates in mixed aqueous-organic solutions revealed in previous
studies [6, 24-27]. In a perfectly mixed aqueous-organic solution system, we expect that: (1)
Both the hydrophilic recombinant hirudin and the hydrophobic LCFA can be dissolved into the
system easily; (2) Under the prerequisite of not significantly reducing the biological activity of
recombinant hirudin, the organic solvent in this system can induce its conformation
rearrangement to change the solvent-accessible surface area of the lysine residues, thereby

improving the reaction specificity to achieve the desired mono-LCFA-recombinant hirudin with
high retained bioactivity.
In this study, the chemical modification of recombinant hirudin variant-2 (HV2) with palmitic
acid (PAL) (Fig. S1), one of the most commonly used LCFAs for fatty acid conjugation of
proteins and peptides in previous studies [14-16], was carried out in mixed aqueous-organic
solutions. Firstly, palmitic acid benzotriazole (PAL-BTA) and palmitic acid
N-hydroxysuccinimide (PAL-NHS) were synthesized and characterized for amine conjugation.
Secondly, the conjugation of HV2 with PAL-BTA or PAL-NHS in different mixed
aqueous-organic solution was investigated and the optimal condition for the conjugation was
determined. Finally, the reaction mixture of HV2 modified with PAL-NHS was isolated and
purified using preparative liquid chromatography. Three mono-palmitic acid modified HV2
(mono-PAL-HV2) isomers were obtained and the bioactivity was further characterized.
2. Materials and Methods
2.1. Materials
HV2 (>95%, detected by HPLC) was purchased from Chongqing Kerun Bio-Pharmaceutical
Co., Ltd. (Chongqing, China). Thrombin (Sigma-T4648), fibrinogen (Sigma-F8630), acetonitrile
(HPLC grade) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis,
WA, USA). Palmitic acid, benzotriazole, N-hydroxysuccinimide, dicyclohexylcarbodiimide
(DCC), triethylamine (TEA), dimethyl sulfoxide (DMSO), tetrahydrofuran, N-methylpyrrolidone
(NMP) and other chemicals were of analytical grade.
2.2 The synthesis of PAL-BTA

PAL (MW=256.42 Da, 5 mmol) and BTA (MW=119.12 Da，5 mmol) were dissolved in ether
(5 mL), and then DCC (MW=206.18 Da, 5 mmol), dissolved in ether, was added dropwise in an
ice-water bath. The reaction was carried out under nitrogen atmosphere at -4°C for 2.5 h, and
then at 20°C for 2.5 h. After the reaction, the reaction mixture was centrifuged at 12,000  g for
30 min to remove the white precipitate dicyclohexylurea (DCU), the supernatant was collected,
and the solvent was removed by evaporation. The product was then dissolved in 5 mL acetone.
Subsequently, crystallization was performed at -20°C to obtain a white needle-like solid, and the
product was then dried in vacuum for 5 h to obtain the white crude product of PAL-BTA. The
molecular formula of the reaction is shown in Fig. 1A.
The crude product of PAL-BTA was purified by an LC3000 semi-preparative HPLC system
(Beijing Chuang Xin Tong Heng Science and Technology Co., Ltd) with a YMC preparative
column (YMC-Pack ODS-A, 20×250 mm, 10 µm). The elution conditions for purification were
as follows: isocratic elution of mobile phase: 40% B (mobile phase A: ddH2O, mobile phase B:
acetonitrile) for 40 min at a flow rate of 15.0 mL/min, and ultraviolet (UV) detection at 254 nm,
the loading volume was 20 μL.
The purity of PAL-BTA were analyzed using a Waters 600-2707-2487 HPLC system (Waters,
Massachusetts, USA). The analytical condition was as follows: C18 column (KP100-5C18 (250
mm × 4.6 mm, 5 μm, Kromasil, Sweden)), a linear gradient of mobile phase 20%–80% B in 30
min (mobile phase A: pure water, mobile phase B: acetonitrile) at a flow rate of 1 mL/min, and
UV detection at 254 nm was utilized. 1H NMR were recorded on Bruker 500 MHz spectrometer,
and the samples were dissolved in deuterated acetone (C3D6O).
2.3 The synthesis of PAL-NHS

Similar to the synthesis of PAL-BTA, the synthesis of PAL-NHS was also carried out by DCC
condensation. Palmitic acid (2 g), N-hydroxysuccinimide (0.9425 g), and DCC (1.77 g) were
added into a flask with a molar ratio of 1:1:1, with the presence of nucleophilic acylation catalyst
4-dimethylaminopyridine (DMAP, 9.5 mg). Subsequently, the solvent tetrahydrofuran (15 mL)
was added in an ice-water bath, and the reaction was carried out in an ice-water bath (0°C) for 20
min, and at room temperature in nitrogen atmosphere for 12 h with constant stirring. After the
reaction, the mixture was chilled at 4°C for 1 h and centrifuged at 12,000  g for 30 min to
remove the white precipitate, dicyclohexylurea (DCU), followed by the crystallization of the
supernatant at -20°C to obtain a white needle-like crystal. The crystal was then dried in vacuum
for 5 h to obtain the white crude product of PAL-NHS. The equation of the reaction was shown
in Fig. 1B.
The purification of PAL-NHS was essentially the same to that of PAL-BTA with minor
modifications. Briefly, the product was purified by isocratic elution with a mobile phase
containing 90% of mobile phase B (acetonitrile) for 30 min, and the eluant was detected by UV
at 215 nm.
The characterization of PAL-NHS was essentially the same to that of PAL-BTA with minor
modifications. Briefly, the product was purified by isocratic elution with a mobile phase
containing 90% of mobile phase B in 30 min, and the eluant was detected by UV at 215 nm. The
1H NMR spectrums were recorded by Bruker 500 MHz spectrometer, and the samples were
dissolved in deuterated dimethyl sulfoxide (DMSO-d6).
2.4 The modification of HV2 with PAL-BTA
For the modification of HV2 with PAL-BTA (Fig. 1A), the influence of the organic solvent
types (aqueous-NMP and aqueous-DMSO), the ratios of aqueous to organic solvents (1:5, 1:4,

1:3 and 1:2, v/v) and the molar ratios of PAL-BTA to HV2 (15:1, 10:1, 5:1 and 3:1) were
investigated and the conversion and yield of mono-PAL-HV2 product were determined to
evaluate the efficiency of the modification. Quantitative analysis was performed using RP-HPLC
and the concentration of the product was calculated by the peak area. The conversion and the
yield of mono-PAL-HV2 product were calculated by the following equations to evaluate the
modification efficiency:
Total PAL-HV2 (mono-, di- and tri-PAL-HV2) (mol)
Conversion (%) =
 100
The initial HV2 (mol)
(1)
Mono-PAL-HV2 (mol)
The initial HV2 (mol)
Yield of mono-PAL-HV2 (%) =
 100
(2)
Each mono-PAL-HV2 isomer (mol)
Total mono-PAL-HV2 isomers (mol)
Selectivity of each mono-PAL-HV2 isomer (%) =
 100
(3)
A typical reaction process was as follows: a 2-mL reaction system was composed of a 1:4
volume ratio of aqueous-NMP as the reaction solution, and the molar ratio of PAL-BTA to HV2
was 15:1. Specifically, 0.25 mL of 4 mg/mL HV2 aqueous solution was aliquoted, and 20 μL of
TEA aqueous solution (diluted 50 folds with distilled water) was added to the HV2 solution to
adjust the pH to 10. Subsequently, 0.13 mL of distilled water was added to the solution.
Thereafter, 0.765 mg of PAL-BTA was dissolved in 1.6 mL of NMP solvent, and then mixed
with the HV2 solutions prepared. The reaction was performed at 25°C for 2 h.
2.5 Modification of HV2 with PAL-NHS

The modification of HV2 with PAL-NHS (Fig. 1B) was essentially the same to that of
PAL-BTA with minor changes. Briefly, PAL-NHS was added to the reaction system with a
batch-wise manner and the reaction time was 24 h. Specifically, the solubility of PAL-NHS in
DMSO, NMP, N,N-dimethylformamide, acetonitrile, acetone, and ether was investigated. A total
of 0.25 mL HV2 aqueous solution (4 mg/mL) was aliquoted, and 20 μL of TEA aqueous solution
(diluted 50 times with distilled water) was added under N2 protection to adjust the pH to 10,
following by the addition of 0.13 mL of distilled water. Thereafter, 0.765 mg of PAL-NHS was
dissolved in 1.6 mL of DMSO, and then added into the reaction system described above in
batches (0.2 mL per 30 min, with a total duration of 4 h), and the mixture was allowed to react at
25°C for 20 h.
2.6 The preparation of mono-PAL-HV2
The reaction was performed with essentially the same procedure to Section 2.5, with minor
changes. Briefly, the reaction solvent was aqueous-DMSO with a volume ratio of 1:4. The molar
ratio of PAL-NHS to HV2 was 3:1, and the mixtures were allowed to react at 25°C for 24 h. The
reaction mixture was first concentrated by ultrafiltration (3 kDa cutoff) to remove small
molecules such as DMSO, and the product was then isolated and purified using a Shimadzu
SPD-M20A liquid chromatograph with a semi-preparative column (YMC-pack Pro C18RS 250
mm × 10 mm, 5 μm). The mobile phase A was ddH2O with 0.1% (v/v) TFA and the mobile
phase B was acetonitrile with 0.1% (v/v) TFA. The liquid preparation conditions were as
follows: the loading volume was 20 μL, the detection wavelength was 215 nm, and the flow rate
was 2 mL/min. The elution conditions were: the column was first equilibrated with 15% B for 10
min, after sample loading, the column was eluted with a linear gradient of 15%-75% B for 60
min, then the column was eluted with 100% B for 10 min, and finally the column was

equilibrated with 15% B for 10 min for the next sample loading. The major conditions of the
high-resolution mass spectrometry were as follows: the heater temperature was 400°C, the sheath
gas flow was 30 arb, the secondary gas flow was 10 arb, the spray current was 3.5 kV, the
capillary temperature was 350°C, and the capillary voltage was 39 V.
2.7 The in vitro anticoagulant activity of mono-PAL-HV2
The in vitro anticoagulant activity of HV2 and mono-PEG-HV2 was evaluated by thrombin
titration assay [28]. The retained bioactivity of mono-PAL-HV2 was calculated according to the
following equation:
Bioactivity of mono-PAL-HV2 (mol)
Retained bioactivity of mono-PAL-HV2 (%) =
 100
Bioactivity of HV2 (mol)
(4)
2.8 Identification of the modification site of mono-PAL-HV2
The modification site of the mono-PAL-HV2 was determined by tryptic peptide mapping (the
combination of trypsinization and LC-MS analysis). A total of 1 mL mono-PAL-HV2 (0.1 mg/L)
dissolved in 50 mM NH4HCO3 solution (pH 7.8) was digested by 50 µL trypsin (1 mg/mL).
After digestion at 37°C for 24 h, the solution was further treated with 100 µL of 0.1 M
DL-Dithiothreitol (DTT) for 30 min. Then the solution was characterized using liquid
chromatography-mass spectrometry (LC-MS) analysis by LTQ Orbitrap XL (Thermo Scientific
Technology Co., Ltd. USA) to identify the digestion fragments of mono-PAL-HV2. The
chromatography column was ZORBAX (RRHD, 2.1 mm × 100 mm, 1.8 µm, Agilent Co., USA).
The mobile phase A was pure water containing 0.1% (v/v) formic acid, and the mobile phase B
was acetonitrile containing 0.1% (v/v) formic acid. The eluant was detected by UV at a

wavelength of 215 nm. The sample was loaded at 10% B with a sample volume of 20 μL and a
flow rate of 1 mL/min. The column was eluted with a 30 min linear gradient of mobile phase B
from 10% to 30%. In addition, the conditions of the high-resolution mass spectrometry analysis
were described in Section 2.6.
2.9 RP-HPLC analysis of the PAL-conjugated HV2
Reaction mixtures of HV2 were analyzed using a Waters 600-2707-2487 HPLC system. The
chromatography column was LiChrospher 100 RP-18 (250 mm × 4.0 mm, 5 µm, Merck,
Germany). The mobile phase A was pure water with 0.1% (v/v) TFA, and mobile phase B was
pure acetonitrile with 0.1% (v/v) TFA. The eluant was detected by UV detection at a wavelength
of 215 nm. The sample volume was 20 μL, and the flow rate was 1 mL/min. The elution
conditions were as follows: 0-85 min, 15%-100% B (v/v).
2.10 LC-MS analysis of the PAL-conjugated HV2
LC-MS analysis of the reaction mixtures described in Section 2.4 and Section 2.5 were
recorded by LTQ Orbitrap XL (Thermo Scientific Technology Co., Ltd. USA). The
chromatography column was ZORBAX (RRHD, 2.1 mm × 100 mm, 1.8 µm, Agilent Co., USA),
the mobile phase A was ddH2O containing 0.1% (v/v) formic acid, and the mobile phase B was
acetonitrile containing 0.1% (v/v) formic acid. The eluant was detected by UV at a wavelength
of 215 nm. The sample was injected in 100% A with a sample volume of 20 μL and a flow rate
of 1 mL/min. The conditions of the elution were as follows: the column was eluted by a gradient
of 0-60% B in 0-20 min, then the gradient of B was increased to 80% in 20-25 min and the
gradient of B was increased to 100% in 25-30 min. The conditions of the high-resolution mass
spectrometry analysis was described in Section 2.6.

2.11. Data analysis
All data were obtained from three independent replicates and are represented as the mean ±
SD unless otherwise mentioned. The statistical analysis was performed using one-way ANOVA
followed by Tukey’s multiple comparison. A value of Pꢀ<ꢀ0.05 was considered as statistically
significant.
3. Results and Discussion
3.1. Synthesis and characterization of PAL-BTA and PAL-NHS
A DCC condensation process for the synthesis of PAL-BTA and PAL-NHS was established.
Compared with the conventional method for carboxylic acid activation, i.e. the acid chloride
method, DCC condensation is safer and easier to operate and has been widely applied in
pharmaceutical industry. As is shown in Fig. S2 in Supplementary material, the purity of the
PAL-BTA and PAL-NHS purified by preparative liquid chromatography was 93% and 81%,
1
respectively. The structure of PAL-BTA and PAL-NHS was also determined by H NMR (Fig.
1
S3 and S4 in Supplementary material), and the tentative chemical shift assignment in the H
NMR spectrum of PAL-BTA and PAL-NHS was shown in Table S1 in Supplementary
material. These results showed that the desired PAL-BTA and PAL-NHS were successfully
synthesized.
3.2 Modification of HV2 with PAL-BTA and PAL-NHS
PAL-BTA has strong hydrophobicity due to its hydrophobic precursor, palmitic acid, which is
a saturated fatty acid, while HV2 is highly hydrophilic. Therefore, neither pure aqueous solution

nor pure organic solvent can effectively promote their contact, which is a prerequisite for the
modification reaction to be carried out. Hence, establishing a proper reaction system is the key to
promote the reaction. Considering the characteristics of palmitic acid and HV2, a mixed
aqueous-organic solutions reaction system might be a good choice. In addition to the type of the
organic solvent, the ratio of the aqueous fraction to the organic fraction and the molar ratio of
PAL-NHS to HV2 are also key factors influencing the efficiency of the reaction. The factors
leading to the optimal efficiency of the reaction were determined and the results are shown in
Fig. 2.
Preliminary experiments showed that PAL-BTA has desirable solubility in both mixed
aqueous-NMP and mixed aqueous-DMSO solutions, therefore these systems were used for the
modification of HV2 with PAL-BTA. As is shown in Fig. 2A, the efficiency of the conversion
using NMP as organic solvent was higher than that using DMSO at a same ratio of aqueous to
organic solvent, although the yield of mono-PAL-HV2 product using NMP was not significantly
higher than that using DMSO. This indicated that NMP was more advantageous than DMSO for
the modification HV2 with PAL-BTA to some extent. As is shown in Fig. 2B, with the decrease
of the ratio of aqueous to DMSO (v/v), both the conversion and the yield of mono-PAL-HV2
product firstly increased between 1:2 and 1:4 ratios of aqueous to DMSO (v/v) and then almost
reached a plateau between 1:4 and 1:5 ratios of aqueous to DMSO (v/v). Furthermore, as is
shown in Fig. 2C, with the increase of the molar ratio of PAL-BTA to HV2, both the conversion
and the yield of mono-PAL-HV2 product increased. Therefore, the reaction solution system was
finally determined to be a mixed aqueous-NMP (1:4, v/v) solution, with a molar ratio of
PAL-BTA to HV2 of 15:1. The reaction mixtures were characterized by LC-MS and the results
are shown in Fig. S5 in Supplementary material. The LC-MS data (Table 1) showed that three

mono-PAL-HV2 isomers and a small amount of di-PAL-HV2 were generated in the reaction
mixtures together with the unreacted HV2. In our previous studies on the PEGylation of HV2
[5-9], mono-PEG-HV2 isomers could not be effectively separated by RP-HPLC due to the
polydispersity of PEG; while in this study, three mono-PAL-HV2 isomers were effectively
separated by the same RP-HPLC conditions. These results may provide an effective approach for
the investigation of the effects of different modification sites on the retained bioactivity of the
modified HV2.
For the modification of HV2 with PAL-NHS, preliminary experiments were carried out using
exactly the same reaction conditions with PAL-BTA and undesirable results were obtained.
During the modification reaction, a floc precipitate was generated in the reaction mixture, and
the total content of soluble equivalent HV2 in unmodified and modified forms in the supernatant
was significantly reduced according to HPLC analysis. This phenomenon may be caused by the
presence of PAL-NHS, which might have changed the solvent environment of the reaction
system, inducing the aggregation and precipitation of HV2. Therefore, a suitable
aqueous-organic solution system and an appropriate feeding mode were the key to improve the
HV2 modification reaction with PAL-NHS, on one hand by effectively promoting the contact
between substrates, and on the other hand by controlling the content of PAL-NHS to reach the
balance between more precipitation of HV2 and higher conversion of the reaction. As shown in
Fig. 3A, the reaction conversion using DMSO as organic solvent was higher than that of NMP
under the same conditions. Therefore, mixed aqueous-DMSO solution system was chosen for
further optimization. The effect of aqueous to DMSO (v/v) ratios (1:5, 1:4, 1:3 and 1:2, v/v) were
also investigated, and a relatively optimized aqueous-DMSO (v/v) ratio of 1:4 was chosen
according to the balance of the solubilities of HV2 and PAL-NHS in the mixed aqueous-DMSO

solutions. This is because the solubility of HV2 decreased as the DMSO ratio increased, while in
contrast, the solubility of PAL-NHS decreased as the DMSO ratio decreased. In contrast to the
modification of HV2 with PAL-BTA in mixed aqueous-NMP solution (Fig. 2C), both the
conversion and the yield of mono-PAL-HV2 product both decreased as the molar ratio of
PAL-NHS to HV2 increased for the modification of HV2 with PAL-NHS in mixed
aqueous-DMSO solution (Fig. 3B). This is mainly because the amount of soluble HV2
molecules available for modification decreased as the molar ratio of PAL-NHS to HV2 increased
(Fig. 3B). Therefore, the reaction solution system was determined to be a mixed aqueous-DMSO
(1:4, v/v) solution with a molar ratio of PAL-NHS to HV2 molar ratio of 3:1. The reaction
mixtures were characterized by LC-MS and the results are shown in Fig. S6 in Supplementary
material and Table 1. In contrast to the reaction using PAL-BTA, tri-PAL-HV2 was generated.
As shown in Fig. 2 and 3, the modification of HV2 with PAL-NHS in mixed aqueous-DMSO
solution has a higher efficiency than that using PAL-BTA and NMP as the organic solvent. This
is probably due to the unique feature of DMSO, which is a polar aprotic solvent frequently used
as a solvent to improve the efficiencies of chemical reactions, e.g., the enhanced PEGylation
efficiencies of HV2 and G-CSF in solution system containing DMSO in previous studies [6,
26].As is shown in Fig. 4, both modification of HV2 with PAL-BTA and that with PAL-NHS
generated three mono-PAL-HV2 isomers, but the selectivity of the three isomers in each reaction
were different. The three mono-PAL-HV2 isomers were named as mono-PAL-HV2-1,
mono-PAL-HV2-2, and mono-PAL-HV2-3, respectively (Fig. 4). For PAL-BTA, the selectivity
of three mono-PAL-HV2 isomers were similar (mono-PAL-HV2-1 : mono-PAL-HV2-2 :
mono-PAL-HV2-3 = 33% : 28% : 39%) (Fig. 4A), while their selectivity showed striking
difference for the three mono-PAL isomers derived from PAL-NHS (mono-PAL-HV2-1:

mono-PAL-HV2-2: mono-PAL-HV2-3 = 18% : 23% : 59%) (Fig. 4B). This result may be
caused by the different conformation changes of HV2 induced by the different reaction solution
systems. HV2 used in this study has three lysine residues (Lys 24, Lys 27, and Lys 35) that are
available (Fig. S1A) for the potential amine conjugation. Our previous study showed that the
three lysine residues had similar solvent accessible surface area in aqueous solution, that is,
similar reaction probability, by molecular dynamics simulation [29]. This result is consistent
with the modification of HV2 with PAL-BTA in mixed aqueous-NMP solution, which achieved
a similar selectivity of three mono-PAL-HV2 isomers (Fig. 4A). Interestingly, the modification
of HV2 with PAL-NHS in mixed aqueous-DMSO solution resulted in three mono-PAL-HV2
isomers with dramatically different selectivity (Fig. 4B). A possible explanation is that DMSO
can change the conformation of HV2, resulting in a large difference in the surface area of the
three lysine residues in mixed aqueous-DMSO solution. Similar results were observed during the
PEGylation of proteins and peptides in previous studies [24, 25]. For instance, DMSO changed
the conformation of three peptides, thus improving the selectivity of N-terminal PEGylation [24].
The selectivity of transglutaminase-mediated PEGylation of salmon calcitonin and human
growth hormone was improved in a mixed aqueous-ethanol solution compared to those in
aqueous solution [25]. To our best knowledge, the improved selectivity of the modification of
HV2 with PAL-NHS in mixed aqueous-DMSO solution was the first study in the chemical
modification of proteins and peptides drugs with LCFAs.
3.3. Preparation and characterization of mono-PAL-HV2
On the other hand, the aim of this study was to improve the specificity of the reaction to
achieve the desired mono-LCFA-recombinant hirudin with a high selectivity and a
highly-retained bioactivity. Therefore, it is necessary to investigate the effect of the different

modification sites on the bioactivity of the three mono-PAL-HV2 isomers. For this purpose, the
reaction mixture derived from the modification of HV2 with PAL-NHS in mixed
aqueous-DMSO solution was firstly isolated and purified (Fig. 5). Three fractions were collected
and identified as mono-PAL-HV2 isomers by MS analysis, whose results showed that three
fractions had the same molecular weight of 7148 Da (Fig. 6A, only one representative
spectrum was shown). Therefore, the three mono-PAL-HV2 isomers corresponded in Fig. 4B
were indicated in Fig. 5. As is shown in Fig. 6B-D, all the three mono-PAL-HV2 fractions
collected had a relatively high HPLC purity (> 90%) and could be used for the subsequent
bioactivity characterization. In most cases, highly concentrated organic solvents can irreversibly
reduce the activity of hydrophilic proteins even if organic solvents are removed in subsequent
steps. While HV2 is a small peptide with high structural stability, which could restore its native
structure after DMSO removal and being re-dissolved into aqueous solution, and remain at
95-97% of the anticoagulant activity in mixed aqueous-DMSO solutions at various DMSO ratios
(10-90%, v/v) in our previous study [6]. Therefore, mono-PAL-HV2 isomers were anticipated to
restore its native structure and bioactivity after being purified into aqueous solution. Similar
results have been observed during the PEGylation of proteins and peptides in previous studies [6,
26]. As is shown in Fig. 7, the in vitro anticoagulant activities of mono-PAL-HV2-1,
mono-PAL-HV2-2, and mono-PAL-HV2-3 retained 36%, 4%, and 89% of the in vitro
anticoagulation activity of unmodified HV2, respectively. In contrast, mono-PEG-HV2 derived
from PEGylation of HV2 with 5 kDa mPEG-SC in mixed aqueous-DMSO solution retained 42%
of the in vitro anticoagulant activity of unmodified HV2 [6]. The results indicated that LCFA
might be more favorable than PEG for the retention of the activity of prodrugs. Interestingly,
mono-PAL-HV2-3, the selectivity of which was the highest (Fig. 4B) among the three

mono-PAL-HV2 isomers, also retained the in vitro anticoagulation activity to the highest level
(Fig. 7). These results showed that the modification of HV2 with PAL-NHS in mixed
aqueous-DMSO solution achieved the desired mono-LCFA-recombinant hirudin with both a
high selectivity and a high in vitro bioactivity.
Furthermore, the modification site of mono-PAL-HV2-3 was identified by the tryptic peptide
mapping (the combination of trypsinization and LC-MS analysis) (Fig. S7 in Supplementary
material). As is shown in Fig. S1A, HV2 had three potential trypsin cleavage sites (Lys24,
Lys27, and Lys35). As is shown in Fig. S7A, after cleavage by trypsin, four fragments (T1
(I1-K24), T3 (C28-K35), T4 (G36-Q65) and T2-PAL (G25-K27-PAL)) were observed and
further identified by MS analysis (Fig. S7B-F). Although the digest fragment of T2 (G25-K27)
with a very low concentration was also observed (Fig. S7A, B), the yield of T2-PAL
(concentration of T2-PAL/(total concentrations of T2-PAL and T2)) was above 90%. These
results indicated that the modification site of mono-PAL-HV2-3 was Lys27, as the observed
digest fragment of T2 might be mainly from the impurities of mono-PAL-HV2-3 (Fig. 6D).
According to the identification of the modification site of mono-PEGylated protein or peptide
conjugated one PEG at the lysine residue in previous studies [30, 31], if one PEG molecule was
conjugated at Lys 27, both T2 and T3 would not be observed due to the shielding effect of the
conjugated PEG molecule. In contrast, T2-PAL and T3 were observed in this study, indicating
that the conjugated PAL had little effect on tryptic digestion due to its relatively small molecular
weight.
Overall, a homogeneous mono-PAL-HV2-3 with highly retained bioactivity and a conjugated
PAL at Lys27, were successfully obtained, and its selectivity in mono-PAL-HV2 isomers were
improved through the modification of HV2 with PAL-NHS in mixed aqueous-DMSO solution.

One future research will be focused on further improving the selectivity and yield of the desired
mono-PAL-HV2-3 in this mixed aqueous-DMSO solution, as well as investigating the effects of
different modification sites on the retained bioactivities of the mono-PAL-HV2 isomers. Another
future research will be focused on evaluating the potential of mono-PAL-HV2-3 to be developed
as a candidate antithrombotic drug, such as biochemical characterization (e.g., thermal stability,
in vitro plasma stability and kinetic constants for its interaction with thrombin), pharmacological
and pharmacokinetic efficacy, thrombolytic effect and the risk of bleeding.
4. Conclusions
In this study, the chemical modification of HV2 with PAL was successfully developed. Firstly,
PAL-BTA and PAL-NHS were successfully synthesized for amine conjugation at the lysine
residues of HV2. Then, conjugation of HV2 with PAL-BTA and PAL-NHS was performed in
mixed aqueous-NMP and aqueous-DMSO solutions, respectively, and the optimized conditions
were determined. Consequently, conjugation of HV2 with PAL-NHS in mixed aqueous-DMSO
solution was superior to its conjugation of HV2 with PAL-BTA in mixed aqueous-NMP
solution, with a more site-specific conjugation of one PAL molecule to a certain lysine residue,
possibly due to the different conformation changes of HV2 induced by the different reaction
solution systems. Furthermore, three mono-PAL-HV2 isomers was efficiently separated from
the reaction mixture of the modification of HV2 with PAL-NHS in mixed aqueous-DMSO
solution. The three mono-PAL-HV2 isomers, namely, mono-PAL-HV2-1, mono-PAL-HV2-2,
and mono-PAL-HV2-3, retained 36%, 4%, and 89% of in vitro anticoagulant activity of
unmodified HV2, respectively, indicating that different modification sites of the three
mono-PAL-HV2 isomers have significant effects on their retained bioactivities. Interestingly,

mono-PAL-HV2-3, with the highest selectivity among the three mono-PAL-HV2 isomers, also
retained the in vitro anticoagulation activity at the highest level. The modification site analysis
of mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2.
Our study demonstrated a successful application in which site-specificity reaction was
improved to obtain a homogeneous mono-PAL-HV2 with high retained bioactivity in mixed
aqueous-organic solutions, which is expected to be extended to long-chain fatty acids
conjugation of other proteins and peptides.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgment
This research was financially supported by the National Natural Science Foundation of China
(Grant No. 21606038 and 81072590), the Fundamental Research Funds for the Central
Universities (DUT18JC36).
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Legends
O
C
H
N
CH3
7
N
N
COOH
+
N
H3C
DCC
N2
N
7
N
PAL-BTA
PAL
BTA
O
(A)
O
C
C
N2
CH3
N
N
+
CH3
7
NH2
7
N
H
N
PAL-HV2
HV2
PAL-BTA
O
C
O
O
DCC
N2
CH3
7
N
OH
N O
COOH
+
H3C
7
O
O
PAL-NHS
PAL
NHS
(B)
O
C
O
O
C
N2
CH3
H2O
+
CH3
7
N
O
NH2
N
7
H
O
PAL-HV2
HV2
PAL-NHS
Fig. 1. (A) The synthesis of palmitic acid benzotriazole (PAL-BTA) and its conjugation with
recombinant hirudin variant-2 (HV2); (B) The synthesis of palmitic acid N-hydroxysuccinimide
(PAL-NHS) and its conjugation with HV2

(C)
(B)
(A)
12
10
8
12
10
8
12
10
8
Conversion
Yield of mono-PAL-HV2
Conversion
Yield of mono-PAL-HV2
Conversion
Yield of mono-PAL-HV2
a
a
a
b
a
b
b
a
c
6
6
b
a
6
a
a
c
a
b
d
c
4
4
4
c
c
2
2
2
0
0
1:2
Ratio of aqueous to NMP (v/v)
1:4
1:3
5:1
3:1
15:1
10:1
1:5
0
NMP
DMSO
Molar ratio of PAL-BTA to HV2
Fig. 2. The effects of organic solvents and molar ratio of PAL-BTA to HV2 on the
modification of HV2 with PAL-BTA. (A) The effect of organic solvents; (B) The effect of the
ratio of aqueous to NMP; (C) The effect of the molar ratio PAL-BTA to HV2. (In addition to the
factors investigated, the other conditions include: reaction temperature: 25°C, pH=10, duration
of reaction: 2 h, the volume ratio of aqueous to organic solvents: 1:4, the molar ratio of
PAL-BTA to HV2: 15:1). Data are expressed as meanꢀ±ꢀstandard deviation (SD) (nꢀ=ꢀ3). Values
with different letters (a, b, c and d) are significantly statistically different from each other
(one-way ANOVA followed by Tukey's multiple comparison, Pꢀꢀ<ꢀ0ꢀ.05). Values without
significant difference in each comparison share the same letter.

(B)
(A)
80
70
60
50
40
30
20
10
0
Conversion
Yield of mono-PAL-HV2
Conversion
60
50
40
30
20
10
0
a
Soluble equivalent HV2
Yield of mono-PAL-HV2
Soluble equivalent HV2
a
a
a
b
b
a
a
b
a
b
b
c
c
c
c
3:1
5:1
Molar ratio oPfAL-NHS to HV2
10:1
NMP
DMSO
Fig. 3. The effe cts of organic solvents (A) and molar ratio of PAL-NHS to HV2 (B) on the
modification of HV2 with PAL-NHS. (In addition to the factors investigated, other conditions
include: Reaction temperature: 25 °C, pH=10, duration of reaction: 24 h, The volume ratio of
aqueous to organic solvents: 1:4, The molar ratio of PAL-NHS to HV2: 3:1). Data are expressed
as meanꢀ±ꢀSD (nꢀ=ꢀ3). Values with different letters (a, b, and c) are significantly statistically
different from each other (one-way ANOVA followed by Tukey's multiple comparison,
Pꢀꢀ<ꢀ0ꢀ.05). Values without significant difference in each comparison share the same letter.

Fig. 4. The RP-HPLC analysis of the reaction mixture of the modification of HV2 with
PAL-BTA (A) and PAL-NHS (B).