Synthesis and characterization of glycosylated nitrogen mustard derivatives and their interaction with bovine serum albumin

Article abstract of DOI:10.1007/s11164-012-0727-2

To improve the specificity of nitrogen mustards towards tumor cells, glucose-nitrogen mustard, fructose-nitrogen mustard, and lactose-nitrogen mustard were prepared as three novel glycosylated nitrogen mustard derivatives by esterification of bis(2-chloro

Full text of DOI:10.1007/s11164-012-0727-2

Res Chem Intermed (2013) 39:1949–1956
DOI 10.1007/s11164-012-0727-2
Synthesis and characterization of glycosylated nitrogen
mustard derivatives and their interaction with bovine
serum albumin
•
Shan Li He-ping Li Long Qin
•
Received: 13 May 2012 / Accepted: 10 July 2012 / Published online: 21 July 2012
Ó Springer Science+Business Media B.V. 2012
Abstract To improve the specificity of nitrogen mustards towards tumor cells,
glucose-nitrogen mustard, fructose-nitrogen mustard, and lactose-nitrogen mustard
were prepared as three novel glycosylated nitrogen mustard derivatives by esteri-
fication of bis(2-chloroethyl)carbamic chloride (BCC) with glucose, fructose, and
lactose, respectively. BCC was synthesized from bis(2-chloroethyl)amine hydro-
chloride and triphosgene. The topic products were characterized by infrared (IR)
and mass spectrometry (MS), and their interaction with bovine serum albumin was
investigated by measuring fluorescence spectra in tris(hydroxymethyl)aminometh-
ane hydrochloride (Tris–HCl) buffer solution at physiological conditions.
Keywords Glycosylated ester ꢀ Nitrogen mustards ꢀ Synthesis ꢀ BSA
Introduction
The nitrogen mustards are an important group of alkylating agents that have been
commonly used in clinical cancer chemotherapy for more than 30 years [1]. Like
almost all other anticancer drugs, their clinical efficacy has been limited by their
toxicity to normal tissues [2]. Thus, it is important for pharmacochemists to reduce
their side-effects and prolong their duration of activity [3, 4]. Many works have
reported that it is possible to improve their targeting of tumor cells by linking them
with different drug carriers such as lectins, glycoproteins, and lipids.
Carbohydrates are the most ubiquitous chemicals in living systems, being
present in most cell surfaces, and have been traditionally associated with energy
production and as building blocks [5]. However, it has been observed that they
S. Li ꢀ H. Li (&) ꢀ L. Qin
College of Chemical and Biologic Engineering, Changsha University of Science and Technology,
Changsha 410004, China
e-mail: lihp@csust.edu.cn
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S. Li et al.
play a more pervasive and sophisticated role in drug delivery to specific sites. The
chemical structures of carbohydrates contain many hydroxyl groups. In some
cases, it has been indicated that linking of antitumor drugs to carbohydrates can
simultaneously improve the hydrophilic nature of the drugs. So, significant effort
has been invested in the development of carbohydrate-based cell-specific drugs.
In recent years, various glycoside anticancer agents, such as bleomycins,
epipodophyllotoxin, and cardioactive glycosides, containing carbohydrate moieties
have been reported [6].
To improve the specificity of nitrogen mustards towards tumor cells, three
glycosylated nitrogen mustard derivatives were synthesized, and their interaction
with BSA was studied spectroscopically.
Experimental
Apparatus and reagents
Fourier-transform IR (FTIR) spectra were recorded from KBr pellets using a Nicolet
Avatar FT/IR-360 spectrophotometer. MS spectra were recorded using a HP6890/
5973 Agilent 1,100 liquid mass spectrometer or Agilent 5973–6890 gas chroma-
tography (GC)/MS system. Fluorescence spectra were measured using a PerkinEl-
mer LS/45 fluorophotometer. Thin-layer chromatography (TLC) analysis was
performed on glass sheets coated with Merck silica gel GF254, and compounds were
visualized by I2 vapor.
Chemicals used included bis(2-chloroethyl)amine hydrochloride (98 %; Aladdin
Reagent), triphosgene (99 %; Aladdin Reagent), glucose (AR; National Pharma-
ceutical Group Chemical Reagent), fructose (BR; National Pharmaceutical Group
Chemical Reagent), and lactose (AR; National Pharmaceutical Group Chemical
Reagent). All other chemical reagents were of analytical grade and were distilled
before use.
Synthesis of bis(2-chloroethyl)carbamic chloride (BCC)
BCC was prepared according to literature [7]. The main procedure was as follows:
To a solution of 0.3625 g (1.2 mmol) triphosgene in CH2Cl2 (20 mL), triethyl-
amine (1.0 mL) and bis(2-chloroethyl)amine hydrochloride 0.5194 g (3 mmol) in
CH2Cl2 (10 mL) was added dropwise during 40 min and stirred for an additional
1.5 h in an ice-water bath. Methanol was used as quencher, and formation of
reaction products was monitored by TLC at Rf 0.42 (1:2 petroleum ether:ethyl
acetate). Further purification was not necessary, and the product was used directly
in the next step.
Synthesis of glucose-nitrogen mustard (GNM)
To a mixture containing 0.2970 g (1.5 mmol) glucose in water (10 mL) and
0.6120 g (3 mmol) BCC in CH2Cl2 (20 mL) was added triethylamine to keep pH in
123

Glycosylated nitrogen mustard derivatives
1951
the range 8–10. The reaction was stirred at room temperature for 8 h while being
monitored by TLC. After reaction completion, the mixture was separated. The
organic layer was washed with HCl (0.1 mmol L-1) and saturated brine, dried over
Na2SO4, and evaporated. The product was further purified by flash chromatography
on silica gel as needed, and a slightly yellowish, fragrant syrup was obtained. MS
m/z: 500.9 [M-18]?. IR (KBr)/cm-1: 3,600–3,400 (mO–H), 2,966 (mC–H), 1,739
(mC=O), 1,441 (mC–N), 1,300–1,041 (mC–O–C), 763 and 670 (mC–Cl).
Synthesis of fructose-nitrogen mustard (FNM)
Fructose (0.5402 g, 3.0 mmol) in water (10 mL) and 0.6120 g (3 mmol) BCC in
CH2Cl2 (20 mL) were mixed in a flask, and triethylamine was added to keep pH in
the range 8–10. The reaction was stirred at room temperature overnight. After
reaction completion, the aqueous solution was removed. The organic layer was
washed with HCl (0.1 mmol L-1) and saturated brine, dried over Na2SO4, and
evaporated. The terminal product was purified by flash chromatography on silica
gel, and a yellowish, fragrant syrup was obtained. MS m/z: 317 [M-CH2OH]?.
IR (KBr)/cm-1: 3,660–3,485 (mO–H), 2,966 (mC–H), 1,731 (mC=O), 1,466 (mC–N),
1,300–1,041 (mC–O–C), 767 and 671 (mC–Cl).
Synthesis of lactose-nitrogen mustard (LNM)
A mixture containing 0.5400 g (1.5 mmol) lactose in water (10 mL) and 0.6120 g
(3 mmol) BCC in CH2Cl2 (20 mL) was adjusted to alkaline (pH in the range 8–10)
using triethylamine. After stirring for 24 h at room temperature, the mixture was
separated. The organic layer was washed with HCl (0.1 mmol L-1) and saturated
brine, dried over Na2SO4, and evaporated. The result was purified like GNM, and a
yellow oil with unpleasant smell was obtained. MS m/z: 680.7 [M]?. IR (KBr)/
cm-1: 3,485 (mO–H), 2,962 (mC–H), 1,748 (mC=O), 1,433 (mC–C, mC–N), 1,300–1,200
(mC–O–C), 767, 661 (mC–Cl).
Interaction of BSA with GNM, FNM, and LNM
Stock solution of BSA (6 lM) in aqueous Tris-HCl buffer solution (100 mL) of pH
7.4 was prepared, while stock solutions of the products were prepared in dehydrated
alcohol because of their lower solubility in water. For interaction studies with
protein, 3.0 mL aqueous solution of BSA (6 lM) was titrated with various
concentrations of the compounds ranging from 0 to 70 lL; the total volume of
alcohol did not exceed 100 lL. The presence of 100 lL alcohol did not induce
major structural changes in BSA. Each solution was mixed thoroughly before
spectral measurements at room temperature.
Fluorescence measurements were taken by exciting the protein solution at
288 nm. An excitation wavelength of 288 nm was applied to selectively excite
tryptophan residues in protein.
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S. Li et al.
Results and discussion
Synthesis of GNM, FNM, and LNM
To improve the specificity of nitrogen mustards towards tumor cells and research
their biological activity further, the present article focused on synthesis and
characterization of three glycosylated nitrogen mustard derivatives. The synthetic
route is shown in Scheme 1.
In the process of this research, we found that the sugars had poor solubility in
water, dimethylformamide, and ethanol. However, water was chosen as the best
solution for its low boiling point, nontoxicity, easy separation with dichloromethane
compared with dimethylformamide, and easy reaction with BCC (unlike ethanol).
O
CCl3OCOOCCl3
NEt3
C
.
HCl
Cl
N(CH2CH2Cl)2
(BCC)
NH(CH2CH2Cl)2
1)
O
CH2CH2Cl
CH2CH2Cl
O
C
O
N
O
CH2CH2Cl
O
C
(aq)
Glucose
NEt3
N
OH
(BCC)
2)
CH2CH2Cl
OH
OH
(GNM)
OH
O
O
(aq)
Fructose
OH
(BCC)
3)
CH2CH2Cl
CH2CH2Cl
NEt3
C
N
OH
OH
O
(FNM)
O
C
N(CH2CH2Cl)2
O
OH
OH
OH
O
O
(aq)
Lactose
NEt3
OH
N(CH2CH2Cl)2
O
(BCC)
4)
C
O
O
OH
(LNM)
OH
Scheme 1 Synthesis of GNM, FNM, and LNM
123

Glycosylated nitrogen mustard derivatives
1953
Moreover, when dimethylformamide was applied in this reaction, the whole system
was dark-colored at the end of this step. Tetrabutylammonium bromide was used as
phase-transfer catalyst to promote the reaction, but we found that it was difficult to
remove it completely. In this step, HCl was prepared as byproduct. To make the
reaction reach completion and to improve productivity, the reaction should be
carried out under alkaline conditions.
Characterization of GNM
GNM was prepared by means of esterifying reaction of glucose and BCC. Whether
the reaction occurs can be determined from the characteristic bands of ester
absorption peaks in IR spectra. The glucose spectrum presents the following bands:
O–H stretching vibration at 3,264 cm-1, C–H stretching vibration at 2,941 and
2,884 cm-1, C–C stretching vibration at 1,437–1,323 cm-1, and C–O at
1,160–1,017 cm-1, while the GNM spectra display new absorption peaks at 1739,
1300–1158, 763, and 670 cm-1, respectively, corresponding to C=O, C–O–C
(characteristic band of ester), and C–Cl stretching vibration. The molecular weight
of GNM is 518, whereas we measured [M-18]? = 500.9; this difference may be
caused by loss of molecular H2O. Moreover, the CH2OH fragment was not found by
MS, further illustrating that the reaction happened as desired. From the IR spectra
and MS it can be concluded that synthesis of GNM was successful.
Characterization of FNM
Comparison of the IR spectra between FNM and fructose reveals that the reaction
occurred. The fructose spectrum presents the following bands: O–H stretching
vibration at 3,393 cm-1, C–H stretching vibration at 2,938 cm-1, and C–C
stretching vibration at 1,407 cm-1, while the FNM spectra display new absorption
peaks at 1731 cm-1, 1300–1161 cm-1, 767, and 671 cm-1, corresponding to C=O,
C–O–C, and C–Cl stretching vibration. The molecular weight of FNM is 348,
whereas we measured [M-CH2OH]? = 317 and [CH2OH ? 1]? = 32 by MS; this
may be because of instability of the glycosylated derivative under high
temperature and additionally proves that BCC was grafted onto C1–OH of
fructose. From the IR spectra and MS it can be concluded that the synthesis of
FNM was successful.
Characterization of LNM
The lactose IR spectrum presented O–H stretching vibration at 3,528–3,379 cm-1
C–H stretching vibration at 2,895 cm-1, C–C stretching vibration at 1,407 cm-1
,
,
and C–O at 1,160–1,062 cm-1. In comparison with the lactose IR spectrum, the
LNM spectra display new absorption peaks at 1,748 cm-1, 1,300–1,155 cm-1, 767,
and 671 cm-1, corresponding to C=O, C–O–C, and C–Cl stretching vibration. The
molecular weight of LNM is 680, and we measured [M]? = 680.7 by MS. From all
these results, we conclude that the product is obtained as desired.
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Synthesis of BCC
BCC is an important synthetic intermediate, being crucial for the subsequent steps
in the whole process. It was prepared from bis(2-chloroethyl)amine hydrochloride
and triphosgene in methylene dichloride at 0–5 °C for 2 h in presence of
triethylamine. The nitrogen in BCC acts as a nucleophile with triphosgene to form
a N–C bond. The triphosgene-mediated reaction of BCC outlined here has
advantages over existing methods: the reaction can be carried out without handling
hazardous phosgene, the starting material is commercially available, formation of
SO2 or POCl3 (as occurs with chlorinating agents such as thionyl chloride and
phosphorus pentachloride) is avoided, and a nitrogen atmosphere is not necessary
[8]. The initiator and organic base for this step can be dimethylformamide,
triethylamine, or pyridine; however, triethylamine was chosen because of the easy
isolation of its hydrochloride salt from organic solvents, the lack of dark-colored
impurities, and its suitability for further use. Progress of the reaction was monitored
conveniently by TLC.
Interaction of BSA with GNM, FNM, and LNM
Serum albumins are the most abundant proteins in plasma [9, 10]. It is well known
that BSA is often used as a target protein molecule because of its low cost, ready
availability, and unusual ligand-binding properties [11, 12]. Moreover, the whole
structure of the BSA molecule is similar to that of the human serum albumin (HSA)
molecule, meaning that many studies of BSA can be considered to be consistent
with HSA [13]. To research the interaction of the three glycosylated nitrogen
mustard derivatives with BSA, the quenching intrinsic fluorescence of protein was
used.
The fluorescence spectrum of BSA presents strong emission with maximum at
356 nm when excited at 288 nm. The results showed that, with addition of GNM,
FNM, and LNM, respectively, the intrinsic fluorescence of BSA was gradually
quenched compared with pure BSA solution. It can be concluded that the
environment of tryptophan is highly affected by the interaction with the derivatives.
As many studies have reported on the interaction of small molecules with BSA, it
is possible to estimate the quenching mode by using the Stern–Volmer Eq. (1) [14].
F0=F ¼ 1 þ Kqs0½Qꢁ ¼ 1 þ KSV½Qꢁ;
ð1Þ
where F0 is the fluorescence intensity of BSA solution without quencher at 356 nm,
F is the fluorescence intensity of BSA solution in the presence of quencher at
356 nm, Kq is the biomolecular quenching rate constant, s0 is the fluorophore
lifetime in the absence of quencher, KSV is the Stern–Volmer quenching constant,
and [Q] is the quencher concentration [15, 16]. Figure 1 shows Stern–Volmer plots
of BSA-GNM, BSA-FNM, and BSA-LNM, respectively.
Figure 1 shows that the Stern–Volmer plots were linear and did not change from
linearity with increasing concentration of the three derivatives, to some extent
revealing the occurrence of a single type of quenching, either static or dynamic.
The corresponding plot expressions are listed in Table 1.
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Glycosylated nitrogen mustard derivatives
1955
1.30
GNM
FNM
.........
LNM
1.25
1.20
1.15
1.10
1.05
1.00
0.0
0.5
1.0
1.5
2.0
2.5
[Q] (10-6mol/L)
Fig. 1 Stern–Volmer plot of BSA ? GNM, BSA ? FNM, and BSA ? LNM solutions with different
GNM, FNM, and LNM concentrations
Table 1 Quenching constants of BSA ? GNM, BSA ? FNM, and BSA ? LNM systems calculated
according to Stern–Volmer plots
System
KSV (L/mol)
Kq (L/mol s)
R
BSA ? GNM
BSA ? FNM
BSA ? LNM
7.499 9 104
7.088 9 104
1.1098 9 105
7.499 9 1012
7.088 9 1012
1.1098 9 1013
0.98461
0.99163
0.9938
According to the Stern–Volmer equation, KSV values for the three systems
were obtained. Generally, for most protein molecules, s0 is *10-8 s. Therefore,
Kq could be calculated from the relation KSV = Kqs0. Since the maximum value
of Kq for a diffusion-controlled quenching process with a biopolymer is about
2.0 9 1010 L/mol s [17], the high value of the quenching rate constants imply
that the quenching is static. Table 1 shows that the Kq values of the three system
were all [2.0 9 1010. Thus, it can be concluded that the quenching model of
these novel glycosylated nitrogen mustard derivatives with BSA is static
quenching.
Conclusions
BCC was successfully grafted to glucose, fructose, and lactose, respectively, to
obtain GNM, FNM, and LNM. The products were characterized by IR and MS, and
their biological activity was measured in Tris-HCl buffer solution at pH 7.4. The
quenching model for these novel glycosylated nitrogen mustard derivatives with
BSA is static quenching.
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S. Li et al.
Acknowledgments This project is supported by the Scientific Research Fund of Hunan Provincial
Education Department, Hunan Provincial Natural Science Foundation of China (06JJ2038), and Hunan
Provincial Graduate Innovation Fund of China.
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