Multi-gram scale synthesis of a bleomycin (BLM) carbohydrate moiety: exploring the antitumor beneficial effect of BLM disaccharide attached to 10-hydroxycamptothecine (10-HCPT)

Article abstract of DOI:10.1039/c8nj06191b

The “tumor-seeking” role of bleomycin (BLM) disaccharide has been demonstrated to serve as a promising tool for cancer diagnosis and a potential ligand for targeted therapy. However, these practical applications are often hampered by the lack of BLM disaccharide. Herein, an efficient multi-gram synthesis of peracetylated BLM disaccharide 20 is achieved by a TMSOTF-mediated glycosidation coupling manner in 43.6% overall yield in terms of benzyl galactoside. The critical innovation of the synthetic strategy is that inexpensive benzyl galactoside was first adopted to prepare an l-gulose subunit 3 as a glycosyl acceptor, with a much shorter route in 73.0% yield, and a 3-O-carbamoyl-mannose donor 4 was achieved in 47.2% yield by lowering the amount of dibutyltin oxide, and merging aminolysis and selective deacetylation into a one-pot reaction. Next, the incorporation of BLM disaccharide into 10-hydroxycamptothecin (10-HCPT), a non-specific model compound, to form conjugate 1 could significantly improve the antitumor activity and display obvious selectivity toward cancerous and normal cells in comparison with 10-HCPT. Moreover, BLM disaccharide itself was non-cytotoxic, clearly indicating the importance and potential of BLM disaccharide in solving the targeted antitumor therapy of cytotoxic drugs.

Full text of DOI:10.1039/c8nj06191b

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Multi-gram scale synthesis of a bleomycin (BLM)
carbohydrate moiety: exploring the antitumor beneficial
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effect
of
BLM
disaccharide
attached
to
10-hydroxycamptothecine (10-HCPT)
MaoLin Li1#, Weiping Huang1#, Zhilin Jiang2#, Yonghui Shi3, Sisi Yuan1, Kaishuo Fu1,
YongJun Chen4, Li Zhou5* and Wen Zhou1*
1 School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, University town,
Waihuan Rd, Panyu, Guangzhou, 510006, China
2Puer University, Puer, 665000, Yunan, China
3Department of Pharmacy, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
510120, Guangdong, China
4South China Research Centre for Acupuncture and Moxibustion, Medical College of Acu-Moxi and
Rehabilitation, Guangzhou University of Chinese Medicine, University town, Waihuan Rd, Panyu,
Guangzhou, 510006, China
5College of Science, Hunan Agricultural University, Changsha, Hunan, 410128, China.
# These authors contributed equally to the work.
 Correspondent. E-mail: zhoulizss@163.com; zhouwen60@126.com
Abstract: The “tumor-seeking” role of bleomycin (BLM) disaccharide has been demonstrated to
be severed as a promising tool for cancer diagnosis and a potential ligand for targeted therapy,
however, these practical applications are often hampered by lack of BLM disaccharide. Herein,
an efficient multi-gram synthesis of peracetylated BLM disaccharide 20 is achieved by a
TMSOTF-mediated glycosidation coupling manner in 43.6% overall yield in terms of benzyl
galactoside. The critical innovation of the synthetic strategy is that inexpensive benzyl
galactoside was first adopted to prepare L-gulose subunit 3 as a glycosyl acceptor, with a much
shorter route in 73.0% yield, and 3-O-carbamoyl-mannosely donor 4 was achieved in 47.2 %
yield by lowering the amount of dibutyltin oxide, and merging aminolysis and selective
deacetylation into one-pot reaction. Next, incorporation of BLM disaccharide into
10-hydroxycamptothecin (10-HCPT), a non-specific model compound, to form conjugate 1 could
significantly improve the antitumor activity and display obvious selectivity toward cancerous and
normal cells in comparison with 10-HCPT, moreover, BLM disaccharide itself was non-cytotoxic,
clearly indicating the importance and potential of BLM disaccharide in solving the targeted
antitumor therapy of cytotoxic drugs.
Key works: BLM disaccharides, multi-gram synthesis, 10-hydroxycamptothecin, targeting
property, antitumor activity.

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1. Introduction
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“Aerobic glycolysis” is an emerging hallmark that cancer cells, even in the presence of oxygen,
are capable of reprogramming their glucose metabolism to confer the requirements of
uncontrollable cell proliferation and growth [1]. Significant alterations of activated oncogenes and
mutant tumor suppressors in tumor cells give rise to glycolytic fueling, for the purpose of
avoidance of cytostatic controls and attenuation of apoptosis. Selectively blocking the pathway of
glycolytic fueling even the glycolysis switch is a therapeutic approach to killing cancer cells with
high specificity. Glyco-conjugates, just like clinically used anticancer drugs coupled with various
mono- or disaccharides, [2] pronouncedly improve their targeting, selectivity and efficacy towards
cancer cells.
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Bleomycins (BLMs) such as Bleomycin A5 (Fig.1) are a family of glycopeptide-derived
antitumor antibiotics for treating several types of cancers. Their antitumor activity originates from
[3]
the ability to effect DNA cleavage in cancer cells.
Apart from this, the beneficial effects of
BLM are largely dependent on the ability of carbohydrate moiety to recognize tumor cells [4-7]. As
[4, 5]
demonstrated by the in vitro cell experiments that micro-bubbles
and dyes [6, 7] were used to
explore the bio-functions of BLM disaccharide, the tumor-seeking property of BLM was driven by
its unique carbohydrate moiety. Structurally, BLM is regarded as a modular system, the aglycon is
a tumoricidal domain while the BLM disaccharide composed of two unusual sugars, L-gulose and
3-O-carbamoyl-D-mannose, is responsible for the targeting property. Deglyco BLM is much less
[4]
cytotoxic than BLM toward cultured cancer cells,
further emphasizing the importance of the
disaccharide moiety to effect efficiently on tumor cells. Again, exploring the effect of adding the
BLM disaccharide to different positions of deglycoBLM, the potential role of the carbohydrate
moiety in mediating the targeting property of BLM is underscored.[4]
The unique targeting of BLM disaccharide fragment, holding great potential in targeted
antitumor therapy and drug delivery, arouse our keen interest to develop a practicable multi-gram
scale synthetic route, in hope to solve its source limitation and extend the clinical applications.
Although a large amount of synthetic methods of BLM disaccharide have been reported for
decades, summarized in Fig. 2, the scale of the vast majority is still on milligram range due to the
unreasonable synthetic strategies. Dissecting its structure from retro-synthetic analysis,

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theoretically, the BLM carbohydrate motif can be derived from the globe deprotection of the
appropriately protected disaccharides, which are achieved by a diastereospecific glycosidation
coupling of L-gulopyranoside and 3-O-carbamonylmannose derivatives. Accordingly, BLM
disaccharide precursor was first synthesized using the bromide-mediated coupling of suitably
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protected L-
Metal Binding Domain
H2N
H2N
O
H
NH2
O
H
N
NH2
N
N
CH3
O
O
NH2
O
H
HO
O
NH
CH3
N
S
N
S
N
H
CH3HN
O
O
H
N
H
N
CH3HO
N
H
H
HO
NH
OH
DNA Binding Domain
O
O
OH
OH
OH
O
O
OH
O
NH2
Bleomycin A5
Disaccharide Domain
Fig. 1 Structure of the representative member of BLM family (Bleomycin A5)
gulopyranoside or 1,6-dehydrated gulopyranoside as an acceptor and appropriately protected
3-O-carbamonyl-mannose as a donor, unfortunately, acetyl located in 3-O-carbamonyl group
cause big trouble in the following deacetylation.[9] Synthesis of 3-O-carbamonyl-mannose
derivatives as donors is relatively simple, and mannose as a starting material is attractive due to
inexpensive cost and no chiral inversion, while gulose is a rare naturally occurring
monosaccharide that becomes inaccessible to direct preparation, incurring the great challenges in
constructing the gulose motif. Numerous continued efforts have been made to develop more
efficient approaches to gaining the BLM disaccharide and its derivatives,[10-15] however, the
construction of gulose subunit is a major bottleneck constraint. Generally, selective inversion of
one or several hydroxyl configurations is preferred from cheap and easy to obtain monosaccharide
including mannose, glucose, galactose and so on. The most popular strategy is that epimerization
of D-mannose at C5 is carried out to prepare different properly protected L-gulose derivatives, in
[10]
combination with accomplishing the selectivity between C2 and C3 hydroxyl groups.
Also,
using an oxidation-reduction sequence to fulfill inversion of stereochemistry at C5, a novel

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stereoselective build-up of L-gulose subunit from D-mannose was developed.[11] Similarly, a
synthetic route emerge that a linear allylic alcohol derived from a mannose analogue was
envisaged as an equivalent of L-gulose.[12] Additionally, utilizing the contrary stereochemistry at
C2 and C5 of glucose and gulose, 1,6-hydrated L-gulose was explored with an oxidation-reduction
sequence.[13] However, the yields of these multistep synthetic routes are extremely low and only
reach milligram scales. Although the conversion of L-xylose derivative to L-gulose one was
reported in a preparative scale using a thiazole-based homologation strategy, use of mercuric
chloride and inseparable isomers produced in the extension process of L-xylose to L-gulcose
imposed more restrictions on its larger scale preparation. [14] Recently, a novel access to the gulose
subunit was proposed that starting material D-sorbitol applied to prepare gulose moiety is suitable
to the gram synthesis of BLM disaccharide derivatives. However, the crucial factor was neglected,
that the poor selectivity of aldehyde to protect D-sorbitol sharply decrease the total yield, in
considering the large-scale synthesis. [15]
DOI: 10.1039/C8NJ06191B
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AcO
AcO
O
AcO
AcO
HO
HO
HO
OH
O
STol
OH
OH
O
Mannose
HO
OH
C5
Conversion
HO
HO
Ref.13
at
Glucose
steps
9
risation
in
O
Ph
10
at
HO
Epime
C2,
13%
Ref.
C5
O
O
OH
OBn
BnO
BnO
OBn
O
OH
HO
O
AcO
thiaz
OH
O
L-xyose
BnO
le-based
homologat
HO
OH
OH
OH
HO
O
STol
34%,
ion,
HO
HO
OBn
Ref.14
in
Ph
7
steps
OH
Our strategy
OAc
O
OH
O
OAc
HO
O
head-to-tail inversion, Ref.15
5.8%
Benzyl
OBn
OH
AcO
HgOalac
toside
Conversion
at C3
O
AcO
Gulose Acceptor
O
O
HO
O
HO
OBn
HO
HO
OAc
AcO
inversion
OBn
Ref.15
OH
ail
O
steps,
9
H
BzO
H2NOCO
head-to-t
22%,
in
O
O
3-O-carbamoyl-
mannosyl Donor
OBz
HO
O
AcO
HO
BnO
AcO
H2NOCO
HO
O
H
O
OH
O
O
OH
OH
OH
OAc
HO
NH2
O
O
CCl3
(PhO)2OPO
H3CO
HN
OBn
O
BnO
BnO
H2NOCO
HO
HO
O
O
CCl3
HO
OH
HO
HN
Mannose
Fig. 2 Summary of the synthetic routes of two subunits, gulose and 3-O- carbamoyl-mannose, contained in BLM
disaccharide
Taking these reasons mentioned above into accounts, we herein described a practical
multi-gram synthesis of BLM disaccharide (2), derived from the trimethylsilyl

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trifluoromethanesulfonate (TMSOTF)-mediated glycosidation coupling of L-glucose and
3-O-carbamonyl-mannose derivatives 3 and 4 followed by the globe deprotection. Afterwards,
attachment of BLM disaccharide (2) to 10-hydroxyl of naturally occurring 10-HPTC, which is
characteristic of poor water solubility and low selectivity towards cancerous and normal cells, [16]
generated conjugate 1 (Fig. 3), and then the antitumor activity was evaluated to explore the
bio-function of BLM disaccharide, in hope to extend the underlying and practicable application
scope, especially its improvement on the targeting and efficacy of non-targeted drugs.
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O
O
O
N
OH
N
O
O
O
N
O
OH
O
H
HO
OH
OH
OCONH2
O
OH
O
OH
BLM disaccharide (2)
BLM disaccharide-10-hydroxycamptothecin conjugate (1)
Fig. 3, Structure of BLM disaccharide-10-hydroxycamptochecin conjugate (1)
2. Results and discussion
In terms of retro-synthetic analysis and reported procedures [9-14], the synthesis of BLM
disaccharide is generally accomplished by the assembly of appropriately protected L-gulose and
D-mannose derivatives with 1, 2-O-a-glycosidic linkage followed by globe deprotection. The
overall yield and preparation scale are overwhelmingly dependent on the choice of protecting
groups and starting materials. Although staring materials are diversified (Fig.2), among all the
reported protecting groups for synthesizing BLM disaccharide acetyl group was preferred, mainly
originating from its good compatibility with other protecting groups and easy deprotection. [10,17]
Therefore, peracetyated BLM disaccharide (20) was viewed as a crucial intermediate to synthesize
target compounds 1 and 2. L-gulose residue 3 with unmasked 2-OH for incorporation into
precursor BLM disaccharide derivatives as an acceptor was prepared by selective inversion of
3-OH of benzyl galactoside 6 through a series of protection and deprotection operations (Scheme
1). Selective etherification of galactoside 5 with benzyl alcohol directly produced galactoside 6 in
an acceptable yield (70%) at 90 oC, significantly simplifying the previous procedures consisted of
acetylation, selective benzylation and deacetylation.[18, 19] When p-toluene sulfonic acid (TsOH)

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was replaced by camphorsulfonic acid (CSA), reaction of galactoside 6 with benzaldehyde
5
6
dimethyl acetal easily produced acetal 7 in a quantitative yield[10]. Treatment of 7 with
7
8
o
trifluoromethanesulfonic anhydride (Tf2O) and pyridine at -20 C selectively esterified 3-OH
9
followed by in situ addition of Ac2O or AcCl, generating acetylated 8 in one-pot reaction with the
yield of 87%. It was noted that temperature and mole ratio of Tf2O and Ac2O or AcCl were
determinant factors. Since trifluoromethylsulfonyloxyl group (TfO-) is an excellent leaving group,
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tetrabutylammonium acetoxide ((n-Bu)4NOAc) was selected to substitute TfO- for acetoxyl
[21]
(AcO-),
achieving net inversion of configuration to afford gulose derivative 9, which was
quantitatively deacetylated to diol 10 by treatment with sodium methoxide and methanol. The
selective acetylation at 3-OH was mediated by triethylorthoacetate to offer L-gulose subunit 3 [22]
in an exceptional yield, which functioned first as a glycosyl acceptor for coupling of BLM
disaccharide 2, and ultimately as a glycosyl donor for the following synthesis of conjugate 1.
Ph
OH
OH
O
OH
OH
O
Tf2O
BnOH, 90 o
70.0%
C
PhCH(OCH3)2
OBn
O
O
OH
O
OBn
Py, Ac2O
or AcCl
HO
HO
HO
(+)Camphorsulfonic acid
100%
HO
-20 o
C
HO
87%
HO
S
5
6
7
Ph
1.5eq,CH(OC2H5)3
OBn
Ph
Ph
Ph
O
O
O
O
O
O
O
O
NaOCH3
quant.
(n-Bu)4NOAc
86%
O
O
OBn
O
O
OBn
H2O
97.6%
TfO
OAc
OH
OH
OAc
OH
10
AcO
OAc
OBn
3
9
8
Scheme 1. Synthesis of L-gulose unit 3
Mannose was widely applied to synthesize precursor D-mannose derivatives as starting
material.[4,10,23] Our modified synthetic route to donor D-mannose subunit, which was based on the
method previously proposed by Boger D.H. et al., [10] was illustrated in Scheme 2. Key strategic
elements of the approach included selective benzylation of 3-OH of methylmannoside 12, along
with simultaneous formation of carbamoyl at C3 and release of acetyl at anomeric carbon of
p-nitrophenylcarbonate 16 using ammonolysis, leading to significant increase in the overall yield.
Starting material methylmannoside 12, which was prepared according to the previous
procedure,[24] was refluxing with a catalytic amount of dibutyltin oxide (Bu2SnO), differing in the
amount that 1.1 equivalence of Bu2SnO was required for the formation of intermediate
2,3-O-dibutylstannylene in the previous report,[10, 25] to regulate the selectivity of the following

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reaction, and then in situ addition of tetrabutylammonium bromide and benzyl bromide promoted
the generation of methyl 3-O-benzyl mannoside 13 in 86.2% yield, which was further acetylated
to produce acetylated 14 with 3% H2SO4 in Ac2O. Applying 10% Pd/C not Pt (OH)2/C [10]as a
catalyst, benzyl group of 14 under the atmosphere of hydrogen was removed to provide tetraacetyl
15 in a quantitative yield, followed by reaction with 4-nitrophenyl carbonochloridate (pNPCOCl)
in the presence of triethylamine (Et3N) and dimethylaminopyridine (DMAP) to generate precursor
carbamyl 16. Next, compound 16 was subjected to aminolysis and selective deacetylation with 7N
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ammonia (gas) in methanol to yield carbamyl 3-O-carbamoyl-2, 4, 6-tri-O-acetyl-α-D-mannose
(17) in one-pot reaction, differing in ammonolysis of PNPCO- and selective release of Ac- at
anomeric carbon with aniline [10] or ammonia [15] separately. With extension of the reaction time,
other acetyls were also released from carbamoyl 17. The α-glycosyl diphenylphosphate of
compound 17 proceed smoothly to produce donor suitably protected mannose derivative 4[15]for
next coupling.
OH
OH
O
OAc
OAc
O
OH
OH
OH
OH
i)Bu2SnO,
toluene 115 o
3%H2SO4
in Ac2O
10%Pd/C,H2
99.0%
AcCl,CH3OH
98.2%
C
O
O
HO
HO
AcO
BnO
HO
BnO
HO
HO
ii)K2CO3,
TBAB,BnBr
86.2%
96%
OH
OAc
O
O
13
14
11
12
PNP=
O2N
OAc
OAc
OAc
O
OAc
OAc
O
OAc
OAc
O
7N NH3(g)
in CH3OH
OAc
O
PNPCOCl, Et3N
DMAP,92.0%
PO(Ph)2Cl
AcO
H2NOCO
AcO
HO
AcO
H2NOCO
AcO
PNPOOCO
85.1% 0 o
C
n-BuLi, THF
O
O
OAc
OH
OAc
-78 o
C
75%
Ph
P
O
15
16
17
4
O
Ph
Scheme 2. Synthesis of D-mannose derivative 4
With two building blocks appropriately protected L-gulose and D-mannose subunits 3 and 4 in
hand, we embarked on the assembly of naturally occurring BLM disaccharide 2. As described in
scheme 3, the only α-1, 2-linkage assembly of compounds 3 and 4 was mediated by TMSOTF at 0
oC to afford disaccharide 18. Considering the stability of benzyl group exposed to various
conditions, acetal hydrolysis with acetic acid and in situ acetylation of 18 in the presence of Ac2O
and pyridine smoothly generated hexaacetylate 19, which was further debenzylated under
hydrogen atmosphere to produce the crucial intermediate 20 in 99.3% yield. When debenzylation
of 18 was first applied, no trance of debenzylated product was observed, possibly influenced by
the presence of acetal group.

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OBn
O
5
6
7
8
OBn
OAc
OH
O
Ph
OAc
O
OAc
O
O
AcO
AcO
O
OAc
OAc
OAc
OAc
OAc
OAc
O
NaOCH3
O
Pd/C,H2
99.3%
3% H2SO4,
TMSOTF, 0 o
C
OAc
OAc
O
O
O
Dowex 50 H+
75%
2
3
4
+
Ac2O
85.6%
OCONH2
OCONH2
OCONH2
70.3%
OAc
OAc
OAc
9
19
20
18
10
11
12
13
14
15
16
17
18
19
20
21
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59
60
Scheme 3. Synthesis of BLM disaccharide 2
To confirm whether the steric configuration of carbohydrate moiety we prepared was in
accordance with naturally occurring the BLM disaccharide, target disaccharide 2 from the entire
deacetylation of 20 was used to analyze its specific rotation. As we expected, 2 had the similar
optical property, bearing the value of [α]D25 64.9o (c, 0.5, H2O), to BLM disaccharide ever reported
([α]D25 65.8o (c, 0.5, H2O). [9]
PO(OPh)2
O
O
O
O
NH2
22
DMSO, 56.2%
O
NHCBZ
NaOCH3
OAc
O
OH
O
OAc
O
AcO
PO(OPh)2Cl,
HO
AcO
NHCBZ
OAc
OAc
OH
OH
O
OAc
OAc
O
O
HO
O
1
20
10%Pd/C,
91%
OAc
OH
OAc
O
72.1%
O
n-BuLi
O
TMSOTF
80%
THF, -78 o
C
OCONH2
OCONH2
OCONH2
OAc
OH
OAc
24
10%Pd/C,
22
23
25
81.2%
N(CH2CH3)3
O
N
O
O
O
N
O
N
O
O
OH
O
O
N
OH
OH
O
O
Cl3COCOOCCl3
O
N
N
HO
O
N
H
93%
NO2
OAc
O
NaOCH3
Dowex 50 H+
AcO
21
O
OAc
OAc
O
O
HO
O
OAc
O
10-hydroxycamptochecin(10-HCPT)
22
O2N
OCONH2
21
OAc
26
Scheme 4. Synthesis of BLM disaccharide-10-HCPT conjugate 1
To confirm the beneficial property of the BLM disaccharide, non-specific drug
10-hydroxycamptochecin (10-HCPT, 21), a DNA topoisomerase I (Topo I) inhibitor, [17] was
selected as a model compound to conjugate with BLM disaccharide. As illustrated in scheme
4, construct 20 was activated with PO(OPh)2Cl and n-Butyllithium (n-BuLi), yielding the
only α-glycosyl diphenyl phosphate 23 in 72.1% yield. The latter as a glycosyl donor was
then coupled with CBz (benzoxycarbonyl) protected 2-(2-aminoethoxy) ethan-1-ol, which
was prepared according to the previous procedure,[7] to yield linker coupled disaccharide 24.
A hydrogenolysis procedure was applied to remove CBz- of compound 24 and in situ
treatment with activated 10-HCPT 22[26] to afford conjugate 26. Disappointingly, when 26

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+
was treated with sodium methoxide and Dowex 50 H , no conjugate 1 was found. An
alternative approach proved successful that disaccharide 24 was first deacylated to remove all
the protecting groups with NaOMe followed by direct amidation with compound 22 in the
absence of alkali to yield BLM disaccharide-10-HCPT conjugate 1 in an acceptable yield.
Next, the cell-based anticancer activity of compound 1 was investigated to understand the
effect of BLM disaccharide on the non-specific drug. The lead compound 10-HPCT (21) was
selected as a positive control. Two cancerous cell lines including HCT-116 (human colorectal
cell), HepG2 (Human liver cell) and one normal cell HEK-293T (human embryonic kidney)
were used as in vitro cell models to screen all the tested compounds. As illustrated in Table 1,
the IC50 values of target conjugate 1 against HCT-116 and HepG2 were 0.5, 0.2 μ M
respectively, displaying approximately 10-fold more active than the lead compound 21.
Replacing BLM disaccharide of compound 1 with peracetyated BLM disaccharide to obtain
compound 26 caused a sharp drop in inhibiting the activity of cancerous cells, which was
comparative to 10-HPCT, implying that BLM disaccharide moiety was beneficial to an
increase in antitumor effects. The observation was in accordance with the previous report
regarding the antitumor activity of BLM. [4] Interestingly, BLM disaccharide (2) and linker
coupled BLM disaccharide (25) themselves displayed no cytotoxicity toward the cancerous
and normal cells (IC50>100μM), and no synergistic effect on the anticancer activity was
observed when co-administrated with10-HPCT (21). The results emphasized that attachment
of BLM disaccharide to cytotoxic drug 21 was capable of enhancing the anticancer activity,
possibly ascribing to the recognition of BLM disaccharide to some targets on the surface of
Table 1. The antitumor activity of all the compounds tested against human tumor cell lines HCT-116 and
HepG2 and normal cell line HEK-293T.a
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds
In vitro antitumor activity (IC50, μmolL-1)
HCT-116
HepG2
2.1
HEK-293T
7.6
21 (10-HPCT)
4.1
2
>100
>100
2.7
>100
>100
1.7
>100
>100
9.2
25
26

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1
0.5
3.5
0.2
3.1
22.3
5.7
25+21b
6
7
8
a The in vitro antitumor activity of all the tested compound against two cancer cells and one normal cell was
9
measured by the MTT assay after 48 h of incubation and indicated as the half maximal inhibitory
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
concentration (IC50,μmolL-1), and all the data were obtained from three independent experiments; 25+21
indicated that compounds 25 and 21 were co-administrated.
tumor cells, not the antitumor activity of BLM disaccharide itself. Although there were no
direct evidences to be found and clear mechanisms to be proposed, one or more cell surface
receptors associated with glucose transports in tumor cells may be regarded as one of the
most reasonable explanations.[4] Selectivity of conjugate 1 toward cancerous cells (IC50=0.5
μM against HCT-116; IC50=0.2μM against HepG2) and normal cell HEK-293T (IC50=22.3
μM) gave a clear hint that the markedly different expression of the action targets of BLM
disaccharide existed, and this phenomenon was testified by the previous experiments that the
different uptake ability of cancer and normal cells for BLM disaccharide was measured with
[4, 5]
[6, 7]
micro-bubbles
and dyes.
Taken together, it could be concluded that BLM
disaccharide was a targeting molecule as a vehicle or a ligand to selectively deliver cytotoxic
drugs to tumor cells.
3. Conclusion
In summary, an economic and practical multi-gram synthesis of a BLM disaccharide
derivative has been developed to explore the role of BLM carbohydrate moiety in improving
the antitumor ability to 10-HCPT. The light spots of the synthetic strategy are that
inexpensive benzyl galactoside was first applied to prepare L-gulose subunit 3 as a glycosyl
acceptor, with a much shorter route in 73.0% overall yield, and 3-O-carbamoyl-mannosely
donor was achieved in 47.2% overall yield by lowering the amount of dibutyltin oxide and
combining aminolysis and selective deacetylation into one-pot reaction. Afterwards, BLM
disaccharide was incorporated into non-specific antitumor drug 10-HCPT to form conjugate
1, which can selectively target to cancer cells, clearly indicating that BLM disaccharide
indeed holds great potential in improving the targeting effect on non-specific drugs.
4. Experimental section

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General procedure. All chemicals were of reagent grade quality or better, obtained from
commercial suppliers, and used without further purification. Solvents were used as received or
dried over molecular sieves. All the preparations were carried out using standard Schlenk
techniques. All the key intermediates and products were confirmed by electrospray ionization
mass spectrometry (ESI-MS), recorded on AB Sciex triple TOF 5600+ system, and all the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
structures of products were also confirmed by H NMR and 13C NMR, recorded in a Bruker
Avance 400 (1H at 400 MHz, 13C at 100 MHz), and Chemical shifts were reported in parts per
million using the residual solvent peak as internal standard (CDCl3=7.26 ppm for 1H NMR and
77.16 ppm for 13C NMR, CD3OD=3.31 ppm for H NMR and 48.8 ppm for 13C NMR,
1
CD3SOCD3=2.50 ppm for H NMR and 39.6 ppm for 13C NMR). The optical rotation was
1
recorded in an Autopol VI (Rudolph research analytical). The purity, analyzed with C18-column
run on Agilent Technologies 1260 infinity II, was more than 95%,
4.1. Synthesis of benzyl 4, 6-O-benzylidene --D-galactopyranoside (7)
10 g (37.0 mmol) of 1-O-benzyl-β-galactopyranoside and 0.86 mg (3.7 mmol) of
camphorsulfonic acid (CSA) were dissolved in 40 mL of dry CHCl3, and 6.75 mL (44.43 mmol)
of benzaldehyde dimethyl aetal was added dropwise. The reaction mixture was stirred and
refluxed for 2 h and poured into a mixture of 500 mL of CH2Cl2 and 100 mL of saturated aq
NaHCO3. The organic layer was washed twice with water (100 mL) and once with brine (100 mL),
and then concentrated under reduced pressure to afford white residue, which was further
recrystallized with 50 mL of AcOEt to give 13.1g of compound 7 as a white solid. Yield 99.0 % ;
1
TLC Rf=0.30 (DCM: MeOH (V/V) =50:1); H NMR (400 MHz, Chloroform-d) δ 7.55-7.30 (m,
10H), 5.56 (s, 1H), 5.01 (d, J = 12.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.39 (s, 1H), 4.36 (d, J =
5.6 Hz, 1H), 4.20 (t, J = 2.1 Hz, 1H), 4.10 (d, J = 12.4 Hz, 1H), 3.82 (t, J = 8.3 Hz, 1H), 3.71-3.66
(m, 1H), 3.47 (s, 1H); 13C NMR (100 MHz, Chloroform-d) δ 137.49, 137.07, 129.28, 128.53,
128.28, 128.23, 128.03, 126.42, 101.65, 101.48, 75.33, 72.69, 71.83, 70.92, 69.18, 66.78
4.2. Synthesis of benzyl 2-O-acetyl-3-O-tfyl-4, 6-O-benzylidene --D-galactopyranoside (8)
To a solution containing 12 g of compound 7 (33.5 mmol) dissolved in dry CH2Cl2 (100 mL)
and dry pyridine (6 mL) was added slowly 6.5 mL of Tf2O (38.5 mmol) at -20℃. Prior to addition
of acetyl chloride (4.76 mL, 67.0 mmoL), the reaction continued to gently stirred for 2 h at -20℃.

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The reaction mixture was stirred for another 1 h at the same temperature and then poured into a
mixture of 1L of AcOEt and 80 mL of 1N HCl. The organic layer was washed with 400 mL and
150 mL of brine, and dried over anhydrous Na2SO4 and filtered, and then concentrated under
diminished pressure to give crude residue as yellow oil, which was further purified by column
chromatography over silica gel with petroleum ether (PE) and ethyl acetate (EA) (V/V=3/1) to
afford 15.5 g of compound 8 as a white solid. Yield, 87.0% ; TLC Rf=0.34 (PE:EA=3:1); 1H NMR
(400 MHz, Chloroform-d) δ 7.55 -7.27 (m, 10H), 5.60 (s, 1H), 5.54 (t, J = 8.8 Hz, 1H), 4.96 (d, J
= 10.0 Hz, 1H), 4.89 (m, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.67 (d, J = 12.0 Hz, 1H), 4.56 (d, J = 8.0
Hz, 1H), 4.45 (t, J = 3.6, 5.2 Hz, 1H), 4.41 (s, 1H), 4.10 (d, J=12.0 Hz, 1H), 3.51 (s, 1H), 2.07 (s,
3H); 13C NMR (100 MHz, Chloroform-d) δ 168.82, 136.75, 136.72, 129.25, 128.49, 128.27,
128.03, 127.81, 126.14, 101.00, 99.16, 83.73, 74.10, 70.32, 68.59, 67.68, 65.75, 20.49.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.3. Synthesis of benzyl 2, 3-O-diacetyl-4,6-O-benzylidene --L-gulopyranoside (9)
10 g of compound 8 (18.8 mmoL) and 11.4 g of tetrabutylammonium aceate (37.6 mmol) were
taken up in 70 mL of dry DMF. The reaction was heated up to 50 ℃ and stirred for 4 hours. After
compound 8 was completely consumed by TLC analysis, the resulting result was diluted with a
mixture of 500 mL of AcOEt and 100 mL of water. The organic layer was concentrated under
reduced pressure to afford a reddish-yellow residue, which was further purified by column
chromatography over silica gel with a mixture PE and EA (V/V=2:1) as an eluent to produce 7.2 g
1
of compound 9 as a white solid. Yield, 86.0%; TLC Rf=0.40 (PE:EA=2:1); H NMR (400 MHz,
Chloroform-d) δ 7.53-7.14 (m, 10H), 5.55(s, 1H), 5.39 (t, J=2.1Hz, 1H), 5.27 (t, J=5.2, 3.2Hz, 1H)
4.99-4.93 (m, 2H), 4.68 (d, J =12.8 Hz, 1H), 4.39 (d, J =12.8 Hz, 1H), 4.11-4.08 (m, 1H), 4.01 (s,
1H), 3.77 (s, 1H), 2.12 (s, 3H), 2.02 (s, 3H); 13H NMR (100 MHz, Chloroform-d) δ 169.33,
137.48, 137.28, 129.13, 128.29, 128.19, 127.60, 127.47, 126.27, 101.21, 97.44, 83.73, 74.29,
70.11, 69.43, 69.01, 68.00, 66.02, 20.80.
4.4. Synthesis of benzyl 4, 6-O-benzylidene --L-gulopyranoside (10)
To a solution containing compound 9 (5 g, 11.31mmol) dissolved in 20 mL of dry methanol
was added sodium methoxide (61.0 mg, 1.1 mmol), and the reaction mixture was stirred at room
temperature for 1h until compound 9 was completely consumed under the monitor of TLC. After
completion, 500 mg of resin Dowex 50 was added and gently stirred for another 15 mins, and
filtered through a pad of celite, and then concentrated under diminished pressure to afford 4.1 g of

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compound 9 as a white solid in quantitative yield. TLC Rf=0.34 (PE:EA=1:1, V/V); H NMR (400
MHz, Chloroform-d) δ 7.52 (d, J = 6.5 Hz, 2H), 7.42-7.29 (m, 8H) , 5.55 (t, J = 3.9 Hz, 1H), 5.01
(dt, J = 11.7, 3.6 Hz, 1H), 4.79 (dd, J = 8.0, 3.3 Hz, 1H), 4.59 (dd, J = 11.7, 3.1 Hz, 1H), 4.38 (dd,
J = 13.2, 4.9 Hz, 1H), 4.18 (d, J = 3.3 Hz, 1H), 4.14-4.04 (m, 2H), 3.88 (m, 1H), 3.80 (d, J = 6.8
Hz, 1H), 2.51 (m, 1H); 13C NMR (100 MHz, Chloroform-d) δ 137.69, 137.25, 129.11, 128.53,
128.23, 128.02, 126.27, 101.25, 99.25, 99.23, 75.90, 70.91, 69.87, 69.36, 68.88, 65.96.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.5. Synthesis of benzyl 3-O-acetyl-4, 6-O-benzylidene --L-gulopyranoside (3)
To a solution containing 4 g of compound 10 (11.2 mmoL) dissolved in 30 mL of dry DMF was
added 3.06 mL of ethyl orthoacetate (1.5 equiv, 16.8 mmol) and a catalytic amount of
trifluoroacetic acid. The reaction mixture was stirred at room temperature for 1h before addition of
2 mL of water. After completion, the resulting solution was poured into a mixture of 200 mL of
AcOEt and 60 mL of water. The organic layer was washed with 100 mL of brine, and dried over
anhydrous Na2SO4, and then filtered, and ultimately concentrated under diminished pressure to
produce colorless oil, which was further dried in vacuum for 6 h to offer 4.1 g of donor 3 as a
white solid. Yield, 97.6%. TLC Rf=0.25 (PE:EA=2:1, V/V); 1H NMR (400 MHz, Chloroform-d) δ
7.53-7.51 m, 2H), 7.42-7.28 (m, 8H), 5.55 (s, 1H), 5.42 (d, J = 3.7 Hz, 1H) 5.03 (d, J = 12.4 Hz,
1H), 4.84 (d, J = 8.0 Hz, 1H), 4.63 (d, J = 11.6 Hz, 1H), 4.38 (d, J = 12.4 Hz, 1H), 4.11-4.01 (m,
3H), 3.72 (s, 1H), 2.14 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 169.92, 137.41, 137.22,
129.15, 128.49, 128.23, 128.13, 127.96, 126.23, 101.19, 99.68, 74.22, 70.97, 70.88, 69.17, 67.44,
66.09, 20.96; TOF-MS, m/z: [M+ Na+] , Calcd for C22H26NaO7 , 423.1414, Found, 423.1405.
+
4.6. Synthesis of methyl 3-benzyl-α-D-mannopyranoside (13)
20 g of methyl α-D-mannopyranoside (103.1mmol) and 0.1 equiv. of dibutyltin oxide (2.6g,
10.3 mmoL) were taken up in 100 mL of toluene, and refluxed for 1h. After evaporation of the
solvent under diminished pressure, the residue was dried in vacuum for 2 h and re-dissolved in a
mixture of acetonitrile (150 mL) and dimethyl formamide (12 mL) in the presence of K2CO3
(21.3g, 154.6 mmol) and tetrabutylammonium bromide of (3.3 g, 10.3mmol). To the mixture was
added slowly 24.5 mL of BnBr (206.2 mmol), and the resulting mixture was stirred at 80 ℃ for 3
h and poured into 500 mL of AcOEt and 100 mL of water. The organic layer was washed with 100
mL of brine, and dried over anhydrous Na2SO4, and the filter was concentrated under diminished
pressure to afford the crude residue, which was further purified by flash column chromatography

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over silica gel with a mixture of PE and EA (V/V=1:2) to give 25.0 g of mannopyranoside 13 as a
colorless oil. Yield, 86.2%. TLC Rf=0.16 (PE: EA=1:2, V/V); 1H NMR (400 MHz, Chloroform-d)
δ 7.39-7.27 (m, 5H), 4.72 (s, 1H), 4.68-4.65 (d, J =12.0 Hz, 1H), 4.59-4.56 (d, J = 12.0 Hz, 1H),
4.03 (t, J = 9.6 Hz,1H), 3.91 (s, 1H), 3.81 (d, J = 8.0 Hz, 1H), 3.72 (d, J = 12.8 Hz, 1H), 3.66 (d, J
= 9.2Hz, 1H), 3.53 (d, J = 8.0 Hz, 1H), 3.32 (bs, 3H), 3.30 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ137.68, 128.47, 128.25, 128.01, 100.73, 79.60, 72.27, 71.97, 67.94, 65.02, 61.08,
54.84.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.7. Synthesis of 1, 2, 4, 6-tetra-O-acetyl-3-benzyl- α-D-mannopyranoside (14)
14.2 g of compound 13 (50 mmol) was dissolved in 50 mL of 3% H2SO4-Ac2O, and the
solution was stirred at room temperature for 3h. When the reaction ended, and the mixture was
quenched by addition of 100 mL of saturated aq NaHCO3, and then poured into the mixture of 1 L
of AcOEt and 800 mL of saturated NaHCO3. The organic layer was washed with 200 mL of brine,
and dried over anhydrous Na2SO4. The filter was concentrated under reduced pressure to give the
residue, which was dried in vacuum for 3h to afford 20.8 g of compound 14 as a colorless oil.
Yield, 96.0%
1
TLC Rf=0.28 (PE: EA=3:1, V/V); H NMR (400 MHz, Chloroform-d) δ 7.32-7.25 (m, 5H), 6.10
(d, J = 2.0 Hz, 1H), 5.36 (dd, J =2.4, 2.0 Hz, 1H), 5.29 (t, J=10.0 Hz, 1H), 4.68 (d, J =12.4 Hz,
1H), 4.45 (d, J = 12.4 Hz, 1H), 4.23 (m, 1H), 4.09 (dd, J = 12.3, 2.4 Hz, 1H), 3.94 (m, 1H), 3.86
(d, J = 8.4 Hz, 1H), 2.16 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ 170.77, 170.02, 169.55, 168.09, 137.41, 128.45, 127.97, 127.78, 91.06, 74.12,
71.54,70.80, 67.05, 20.91, 20.87, 20.81, 20.76.
4.8 Synthesis of 1, 2, 4, 6-tetra-O-acetyl-α-D-mannopyranoside (15)
To a solution containing 20 g of compound 14 (22.8 mmoL) in 100 mL of ethyl acetate was
added 9.0 g of 10% Pd/C. The reaction mixture was stirred at room temperature overnight under
the atmosphere of H2. The whole process was monitored by TLC. When finished, the resulting
mixture was filtrated to get rid of Pd/C and concentrated under diminished pressure to give 15.7g
of compound 15 as a colorless oil. Yield, 99.0%. TLC, Rf=0.18 (PE:EA=2:1, V/V); 1H NMR (400
MHz, Chloroform-d) δ 6.12 (d, J = 2.0 Hz, 1H), 5.14 (t, J=10.0 Hz, 1H), 5.098 (m, 1H), 4.30 (m,
1H), 4.12 (d, J=2.0 Hz, 1H), 4.09 (m, 1H), 3.98 (m, 1H), 2.41 (brs, 1H), 2.19 (s, 3H), 2.13 (d, J =
2.4 Hz, 6H), 2.08 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 171.15, 170.74, 170.23, 168.07,

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90.43, 71.09, 70.25, 68.77, 68.50, 62.18, 20.90, 20.88, 20.84, 20.74.
5
6
7
8
4.9. Synthesis of 1,2,4,6-tetra-O-acetyl-3-O-(p-nitrophenylcarbamoyl) -α-D-mannopyranoside
(16)
9
To a solution of compound 15 (15 g, 43.1 mmol) dissolved in 90 mL of THF was added 24.4
mg of DMAP (0.2equiv, 8.6mmol), 12.7 mL of triethylamine (92.0 mmol) and 23.9 g of
p-nitrophenyl chloroformate (172.4 mmol). The reaction mixture was stirred at 40℃ for 4h. When
done, the resulting mixture was diluted with 1000 mL of AcOEt and 100 mL of 1N HCl, and then
washed carefully with 100 mL of saturated NaHCO3 and 150 mL of brine in order. The organic
layer was concentrated under reduced pressure to afford yellow residue, which was further
purified by column chromatography over silica gel with a mixture of PE and EA ( V/V, 3:1→2:1)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
as an eluent to afford 19.9 g of a white solid. Yield, 92.0%. TLC Rf=0.30 (PE:EA=3:1, V/V); H
NMR (400 MHz, Chloroform-d) δ 8.29 (d, J=9.2 Hz, 2H), 7.41 (d, J=9.2 Hz, 2H), 6.15 (d, J =2.0
Hz, 1H), 5.48 (dd, J = 3.2, 1.6 Hz, 1H), 5.44 (t, J =10.0 Hz, 1H), 5.21 (dd, J = 10.0, 3.6 Hz, 1H),
4.32 (dd, J = 12.4, 4.8 Hz, 1H), 4.15 (dd, J = 12.4, 2.4 Hz, 1H), 4.12-4.06 (m, 1H), 2.21 (s, 3H),
2.17 (s, 3H), 2.12 (s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 170.66, 170.03,
169.45, 167.92, 155.23, 151.75, 145.71, 125.38, 121.93, 90.69, 74.27, 70.67, 67.57, 65.06, 61.95,
20.83, 20.79, 20.76, 20.74.
4.10. Synthesis of 2,4,6-tri-O-acetyl-3-O-carbamoyl-α-D-mannopyranose (17)
To a solution containing 15.0 g of compound 16 (29.3 mmol) dissolved in 80 mL of dry THF
was added 26 mL of 7 M NH3 in methanol (184.4 mmol), and stirred for 6 h at room temperature
until compound 16 was completely consumed. The reaction mixture was directly concentrated
under diminished pressure to produce yellow oil, which was purified by flash column
chromatography over silica gel with the mixture solvent (PE: EA: Et3N =1:2:0.01,V/V/V) to
1
afford 8.3 g of compound 17 as a white foam. Yield, 85.1%. TLC Rf=0.10 (PE:EA=1:2,V/V); H
NMR (400 MHz, Chloroform-d) δ 5.36-5.22 (m, 4H), 4.73 (brs, 2H), 4.29-4.22 (m, 2H), 4.18-4.07
(m, 2H), 2.16 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 170.87,
170.26, 170.14, 155.50, 92.15, 70.53, 69.80, 68.49, 66.27, 62.58, 20.98, 20.80.
4.11. Synthesis of 2, 4, 6-tri-O-acetyl-3-O-carbamoyl -α-D-mannopyranosyl diphenyl phosphate
(4)
5 g of compound 17 (14.3 mmol) was dissolved in 20 mL of dry THF containing 4Å molecular

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sieve. The temperature was cooled to -78℃, and 13.4 mL of n-BuLi (1.6 M solution in hexane,
21.5 mmol) was added and stirred for 30 mins, and finally diphenyl chlorophosphate (5.9 mL,
28.7 mmol) was added and proceed for another 1h. The resulting reaction mixture was diluted
with 500 mL of AcOEt, and the organic layer was washed with 200 mL of brine, and dried over
Na2SO4, and then filtered and concentrated under reduced pressure to produce the crude residue,
which was further purified by flash column chromatography with the mixture of PE and EA
(V/V=1:1) to afford 6.3 g of compound 4 as a white solid. Yield, 75%. TLC Rf=0.36 (PE:EA=1:1,
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V/V); H NMR (400 MHz, Chloroform-d) δ 7.40-7.13 (m, 10H), 5.89 (d, J = 6.4 Hz, 1H),
5.39-5.24 (m, 3H), 4.92 (brs, 2H), 4.20 (dd, J = 12.4, 4.8 Hz, 1H), 4.12-3.86 (m, 1H), 3.92 (d, J =
12.4 Hz, 1H), 2.21 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ
170.55, 169.77, 169.50, 155.12, 129.97, 125.88, 125.77, 120.26, 120.21, 120.11, 120.06, 96.01,
70.72, 69.06, 65.26, 61.69, 20.77, 20.72, 20.52; TOF-MS, m/z: (M+ Na+), Cacld for
C25H28NLiO13P+, 588.4062, Found, 588.4077 , (M+ Na+), Cacld for C25H28NNaO13P+, 604.1190,
Found, 604.1174.
4.12 Synthesis of benzyl 3-O-acetyl-4,6-O-benzylidene-2-O-(2,4,6-tri-O-acetyl-3-O-carbamoyl-α-
D- mannopyranosyl)--L-gulopyranoside (18)
To a solution of donor 4 (6 g, 10.4 mmol) and acceptor 3 (5.0 g, 12.4 mmol) dissolved in 30 mL
of dry CH2Cl2 in the presence of 4Å molecular sieve was added 2.6 mL of TMSOTf (15.4 mmol)
at -5℃, and the reaction mixture was stirred for 1 h at the same temperature. After completion, it
was poured into a mixture of 100 mL of AcOEt and 20 mL of saturated ammonium chloride. The
organic layer was washed with 100 mL of brine and dried over anhydrous Na2SO4. The filter was
concentrated under diminished pressure to afford a crude residue, which was further purified with
column chromatography over silica gel with a mixture of PE and EA (V/V=1:2) as an eluent to
1
give 5.5 g of compound 18 as a white solid. Yield, 70.3%; TLC Rf=0.36 (PE:EA=1:2, V/V); H
NMR (400 MHz, Chloroform-d) δ 7.51-7.44 (m, 2H), 7.37-7.26 (m, 8H), 5.52 (s, 1H), 5.45 (t, J =
3.5 Hz, 1H), 5.36 (dd, J =3.4, 1.7 Hz, 1H), 5.26 (d, J =1.7 Hz, 1H), 5.16 (t, J =10.1 Hz, 1H), 5.04
(dd, J = 10.2, 3.4 Hz, 1H), 4.96 (d, J = 11.4 Hz, 1H), 4.87 (d, J = 8.3 Hz, 1H), 4.82 (s, 2H), 4.60 (d,
J = 11.4 Hz, 1H), 4.37 (dd, J = 12.6, 1.5 Hz, 1H), 4.20 (dd, J = 11.8, 6.9 Hz, 1H), 4.13 (dd, J = 9.8,
2.1 Hz, 1H), 4.09 (d, J = 2.0 Hz, 1H), 4.07-4.04 (m, 1H), 4.01 (dd, J = 6.0, 1.6 Hz, 1H), 3.99 (dd,
J = 2.9, 1.6 Hz, 1H), 3.73 (d, J = 1.5 Hz, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.69 (s, 3H);

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C NMR (100 MHz, Chloroform-d) δ 170.83, 170.18, 169.94, 169.61, 155.30, 137.33, 137.05,
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129.24, 128.35, 128.19, 128.09, 127.78, 126.37, 101.51, 99.59, 97.76, 74.69, 71.09, 70.45, 70.23,
69.79, 69.38, 69.22, 68.99, 65.97, 65.86, 62.53, 20.81, 20.79, 20.71, 20.16; TOF-MS, m/z: [M +
Na+], Calcd. for C35H41NNaO16+ 754.2318, Found, 754.2305.
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4.13 Synthesis of benzyl 3, 4, 6-tri-O-acetyl-2-O-(2, 4, 6-tri-O-acetyl-3-O-carbamoyl-α-D-
manno- pyranosyl)- -L-gulopyranoside (19)
5 g of compound 18 (6.8 mmol) was dissolved in 20 mL of 3% H2SO4-Ac2O, and the reaction
mixture was stirred for 2 h at room temperature and was poured into a mixture of 60 mL of AcOEt
and 2 mL of triethylamine and 60 mL of water. The organic layer was washed with saturated
NaHCO3 (50 mL) and brine (50 mL) in order, and then dried over anhydrous Na2SO4 for several
hours. The filter was concentrated under diminished pressure to afford crude residue, which was
further purified with column chromatography over silica gel with a mixture PE and EA (V/V=1/2)
1
to give 4.3 g of compound 19 as a white solid. Yield 85.6%; TLC Rf=0.38 (PE:EA=1:2, V/V); H
NMR (400 MHz, Chloroform-d) δ 7.35-7.27 (m, 5H), 5.38 (t, J = 3.7 Hz, 1H), 5.34 (dd, J = 3.4,
1.7 Hz, 1H), 5.25 (d, J = 5.5 Hz, 1H), 5.22 (d, J = 10.1 Hz, 1H), 5.02 (dd, J = 10.2, 3.3 Hz, 1H),
4.94 (d, J = 3.5 Hz, 1H), 4.88 (d, J = 11.3 Hz, 1H), 4.80 (d, J = 8.2 Hz, 1H), 4.72 (s, 2H), 4.61 (d,
J = 11.3 Hz, 1H), 4.27 (dd, J = 12.0, 4.6 Hz, 1H), 4.16 (s, 3H), 4.10-4.05 (m, 1H), 4.04 -4.00 (m,
1H), 3.91 (dd, J = 8.2, 3.8 Hz, 1H), 2.18 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H), 2.07 (s, 6H), 2.06 (s,
3H); 13C NMR (100 MHz, Chloroform-d) δ 170.60, 170.51, 170.19, 169.65, 169.55, 169.52,
155.20, 136.59, 128.48, 128.31, 128.06, 99.78, 98.03, 71.47, 71.09, 70.28, 69.75, 69.36, 69.34,
68.67, 68.16, 65.83, 62.63, 61.70, 20.87, 20.77, 20.70, 20.69; TOF-MS, m/z: [M + Na+], Calcd for
C35H41NNaO16+ 750.2216, Found, 750.2198.
4.14 Synthesis of 3,4,6-tri-O-acetyl-2-O-(2,4,6-tri-O-acetyl-3-O-carbamoyl-α-D-mannopyranosyl)
--L- gulopyranose (20)
To a solution of compound 19 (5.0 g, 6.9 mmol) in 30 mL of ethyl acetate was added a catalytic
amount of Pd/C, and H2 was bubbled into the reaction mixture and stirred overnight. The reaction
mixture was filtered through a pad of celite. The filtrate was concentrated under reduced pressure
to afford 4.4 g of product 20 as colorless oil without purification. Yield, 99.3%; TLC Rf=0.15
1
(PE:EA=1:3, V/V); H NMR (400 MHz, Chloroform-d) δ 5.37 (t, J = 3.6 Hz, 1H), 5.30 (dd, J =
3.3, 1.8 Hz, 1H), 5.25 (d, J = 1.8 Hz, 1H), 5.22 (d, J = 10.2 Hz, 1H), 5.05 (d, J = 8.1 Hz, 1H), 4.99

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(dd, J = 10.6, 3.5 Hz, 2H), 4.96 (s, 2H), 4.93 (dd, J = 3.7, 1.3 Hz, 1H), 4.28 -4.21 (m, 1H), 4.21-
4.18 (m, 1H), 4.14 (dd, J = 11.3, 5.8 Hz, 1H), 4.11-4.04 (m, 3H), 3.78 (dd, J = 8.1, 3.6 Hz, 1H),
2.18 (s, 3H), 2.12 (s, 3H), 2.12 (s, 3H), 2.07 (s, 6H), 2.04 (s, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 170.72, 170.67, 170.25, 169.53, 155.71, 98.23, 94.36, 73.14, 70.35, 69.93, 69.63,
69.37, 68.78, 68.19, 65.71, 62.61, 62.00, 20.92, 20.87, 20.77, 20.74, 20.69; TOF-MS, m/z: [M +
Na+], Calcd for C25H35NaO18+ 660.1746, Found: 660.1726.
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4.15. Synthesis of BLM disaccharide (2)
To a solution containing 100 mg of compound 20 (0.16 mmol) was dissolved in 2 mL of
methanol was added a catalytic amount of sodium methoxide. The reaction mixture was stirred at
room temperature for 20min before addition of Dowex 50 (H+ form), and filtered, and then
concentrated under diminished pressure to afford 60 mg of BLM disaccharide as a colorless foams.
Yield, 100%. [α]D25 41.3o (c, 0.9, H2O); H NMR (400 MHz, Methanol-d4) δ 5.21 (d, J = 1.9 Hz,
1
1H), 4.92 (dd, J = 8.2, 3.7 Hz, 2H), 4.86 (s, 2H), 4.05-4.03 (m, 2H), 3.89 (t, J =6.0 Hz, 1H),
3.84-3.77 (m, 2H), 3.75-3.64 (m, 7H). 13C NMR (100 MHz, Methanol-d4) δ 159.52, 102.29, 95.37,
76.15, 75.91, 75.16, 74.94, 72.99, 71.18, 70.20, 66.04, 62.72, 62.61. TOF-MS, m/z: [M+Na+],
Calcd. for C13H23NNaO12+, 408.1112, Found: 408.1101.
4.16. Synthesis of activated 10-hydroxycamptochecin (22)
5 g of 10-hydroxycamptochecin (13.8 mmol) in tetrahydrofuran (THF, 200 mL) was treated
with 10.8 g of 4-nitrophenyl chloroformate (54.0 mmol), 20 mL of triethylamine , and the reaction
mixture was allowed to proceed at room temperature for 1 h. After completion, the product was
extracted with 1L of ethyl acetate, and the organic layer was washed with 500 mL of water and
150 mL of brine, and then dried with anhydrous Na2SO4, and the solvent was removed in vacuo to
offer a crude residue, which was recrystallized with ethyl acetate to afford 6.7 g of activated
10-hydroxycamptochecin as a yellow solid. Yield 93%; 1H NMR (400 MHz, Dimethyl
sulfoxide-d6) δ 8.72 (s, 1H), 8.39 (d, J = 12.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 7.96
(d, J = 8.0 Hz, 1H), 7.76 (d, J = 12.0 Hz, 1H), 7.35 (s, 1H), 6.55 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H),
1.87 (t, J = 8.0 Hz, 2H), 0.89 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, Dimethyl sulfoxide-d6))
δ172.92, 157.25, 155.51, 153.51, 151.03, 150.46, 149.29, 146.60, 145.98, 145.69, 131.95, 131.31,
131.16, 128.64, 126.66, 126.01, 125.57, 123.21, 119.78, 119.47, 116.26, 97.33, 72.84, 65.72,
50.73, 30.76, 8.25.

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4.17. Synthesis of 3,4,6-Tri-O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-O-carbamoyl-α-D manno-
6
7
pyranosyl)-β-L-gulopyranosyl Diphenyl Phosphate (23)
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9
To a solution of compound 20 (6 g, 9.3 mmol) containing 4Å molecular sieve in 40 mL of dry
THF at -78℃ was added 8.8 mL of 1.6 M n-BuLi solution in hexane (14.0 mmol). After the
mixture was stirred for 10min, 3.9 mL of diphenyl chlorophosphate (18.8 mmol) was added and
proceeded for another 40 min. The resulting mixture was poured into a mixture of 200 mL of
AcOEt and 100 mL of saturated NH4Cl. The organic layer was washed with 100 mL of brine, and
dried over anhydrous Na2SO4, and filtered, and then concentrated under reduced pressure to yield
the crude residue, which was further purified by flash column chromatography over silica gel with
a mixture PE and EA (V/V=1:1) to afford 6.2 g of compound 23 as a white solid. Yield 72.1 %;
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1
TLC Rf=0.26 (PE:EA=1:2, V/V); H NMR (400 MHz, Chloroform-d) δ 7.31 (m, 4H), 7.19 (m,
6H), 5.74-5.62 (m, 1H), 5.42 (d, J = 3.7 Hz, 1H), 5.28 (s, 1H), 5.25-5.19 (m, 1H), 5.13 (s, 1H),
5.04 (d, J = 9.9 Hz, 1H), 4.98 (d, J = 3.7 Hz, 1H), 4.69 (s, 2H), 4.29 (m, 2H), 4.15-4.02 (m, 4H),
4.01-3.95 (m, 1H), 2.22 (s, 3H), 2.15 (s, 3H), 2.08 (s, 6H), 2.01 (s,3H), 1.98 (s, 3H). 13C NMR
(100 MHz, Chloroform-d) δ 169.58, 169.40, 169.17, 168.44, 168.27, 168.20, 128.83, 128.65,
124.57, 124.53, 119.26, 119.21, 119.04, 118.99, 97.63, 96.38, 96.32, 70.91, 70.81, 70.49, 68.69,
68.55, 68.23, 67.69, 66.55, 64.88, 61.60, 60.15, 19.82, 19.71, 19.63; TOF-MS, m/z: [M+Na+],
Calcd for C37H44NNaO21P +, 892.2036, Found, 892.1998.
4.18 Synthesis of 2-[2-(Benzyloxycarbonylamino)ethyloxy]ethyl
3,4,6-Tri-Oacetyl-2-O-(3,4,6-tri-O-acetyl-2-O-(carbamoyl)-α-D-mannopyranosyl)-α,β-L-gulopyra
nose (24)
To a stirred solution containing 6.0g of compound 23 (6.8 mmol) and 4Å molecular sieve
mixed in 30 mL of dry CH2Cl2 was added 2.6 g of CBz linker of (11.2 mmol). The reaction
mixture was stirred at 0℃ for 30 min before addition of the activated agent TMSOTf (1.3 mL, 9.4
mmol). At the same temperature the reaction mixture was stirred for another 1h and quenched
with adequate Et3N. The resulting solvent was extracted with 250 mL of AcOEt, and the organic
layer was washed with water (300 mL) and brine (150 mL) in order, and dried over anhydrous
Na2SO4. The filter was concentrated under reduced pressure to give a residue, which was purified
by flash column chromatography over silica gel with a mixture of PE and EA (V/V=1/2) to afford

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4.6 g of compound 24 as white foams. Yield, 80%; 1H NMR (400 MHz, Chloroform-d)
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δ7.34-7.30(m, 5H), 5.52-5.49 (m, 1H), 5.36-5.34 (t, J=4.0 Hz, 1H), 5.27-5.25 (m, 2H), 5.36-5.34 (d,
J=8.0 Hz, 1H), 5.12-5.04 (m, 2H), 5.02-4.89 (m, 3H), 4.80 (m, 1H), 4.78-4.70 (brs, 2H), 4.26-4.22 (m,
1H), 4.15-4.02 (m, 5H), 3.98-3.90 (m, 1H), 3.85-3.79 (m, 1H), 3.78-3.70 (m, 1H), 3.69-3.45 (m, 5H),
3.40-3.31 (m, 2H), 2.16 (s, 3H), 2.09 (s, 6H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H); 13C NMR (100
MHz, Chloroform-d) δ 170.60,170.49,170.18, 170.04,169.47,169.42,156.55, 155.31, 136.66, 128.51,
128.16, 128.10, 100.84, 98.13, 71.92, 70.25, 70.19, 70.02, 69.71, 69.68, 69.31, 68.92, 68.71, 68.09,
67.6, 66.62, 65.82, 62.64, 62.01, 61.69, 40.83, 20.86, 20.75, 20.71, 20.68, 20.66;TOF-MS, m/z: [M +
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Na+], Calcd for C37H50N2 Na O21+, 881.2798, Found, 881.2843.
4.19 Synthesis of conjugate 26
To a stirred solution containing 500 mg of compound 24 (1.06 mmol) were dissolved in a
mixture of DMF (10 mL) and methanol (1 mL) was added 50 mg of 10% Pd/C, and the reaction
was stirred for 4 h under atmosphere of H2. After filtered to remove Pd/C, 0.67 g of compound 22
(1.27mmol), triethyamine (0.5 mL) and DMAP (100 mg) were added to the reaction mixture, and
stirred at room temperature for 24 h, and the consumption of compound 24 was analyzed by TLC.
The reactive mixture was diluted with 100 mL of AcOEt and 100 mL of saturated NH4Cl. The
organic phase was washed with 200 mL of water and 100 mL of brine in order, and dried with
anhydrous Na2SO4, and filtered, and then evaporated under diminished pressure to afford yellow
residue, which was purified by flash column chromatography over silica gel to afford 741.9 mg of
conjugate 1 as yellow solid. Yield 81.2%; 1H NMR (400 MHz, Chloroform-d) δ 8.33 (s, 1H), 8.20
(d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.66 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 6.09 (t, J = 8.0 Hz, 1H),
5.74 (d, J = 16.0 Hz, 1H), 5.40 (t, J = 4.0 Hz, 1H), 5.36 -5.23 (m, 5H), 5.09-5.02 (m, 1H), 4.95 (d,
J = 4.0 Hz, 1H), 4.84 (d, J = 12.0 Hz, 1H), 4.75 (s, 2H), 4.27 (m, 1H), 4.17-3.97 (m, 6H),
3.97-3.87 (m, 2H), 3.77-3.60 (m, 5H), 3.54-3.45 (m, 2H), 2.21 (s, 3H), 2.16 (s, 3H), 2.10 (s, 3H),
2.08 (s, 9H), 1.91 (m, J = 2H), 1.04 (t, J = 8.0 Hz, 3H). TOF-MS, m/z: [M + H+], Calcd for
C50H59N4O25, 1115.3458, Found, 1115.3377.
4.20 Synthesis of 2-[2-(amino)ethyloxy]ethyl 2-O-(3-O-Carbamoyl- α -D-mannopyranosyl)-
α,β-L-gulopyranose (25)
To a solution containing 1.0 g of 24 (1.16mmol) dissolved in 10 mL of anhydrous methanol was
added a freshly prepared solution of 5.0 mL of 0.4 M sodium methoxide in methanol. The reaction

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mixture was allowed to stir at room temperature for 30min, and the complete consumption of
starting material was confirmed by TLC. After completion, 800 mg of Dowex 50X resin was
added, and shaken for 10min, and then filtered. To the solution of the crude product in methanol
was added a catalytic amount of Pd/C, and then H2 was bubbled continuously for 1h. The reaction
was monitored with HPLC, when done, the reaction mixture was filtered with Celite and
concentrated under diminished pressure to afford 500 mg of compound 25 as a white foams for
next reaction without purification. Yield, 91.0%; TOF-MS, m/z: [M+Na+], Calcd for
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C17H32N2O13Na, 495.1797, Found, 495.1785.
4.21. Synthesis of BLM disaccharide-10-HCPT conjugate (1)
To a stirred solution containing 500 mg of compound 25 (1.06 mmol) were dissolved in 10 mL
of DMSO, was added 0.67 g of compound 22 (1.2equiv, 1.27mmol). The reaction mixture was
stirred at room temperature for 24h, and the consumption of compound 25 was monitored by TLC.
The solvent DMSO was removed under diminished pressure to afford yellow residue, which was
1
purified by flash column chromatography to afford conjugate 1 as yellow solid. Yield 56.2%; H
NMR (400 MHz, Dimethyl sulfoxide-d6) δ 8.64-8.40 (m, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.80-7.96
(m, 1H), 7.87 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.33 (s, 1H), 7.25 (s, 1H), 6.54 (s,1H), 5.42 (s, 1H),
5.26 (d, J=20.0 Hz, 2H), 4.98-4.36(m,10H), 4.01-3.11 (m, 20H), 1.92 (m, J = 2H), 0.98 (m, 3H).
1H NMR (100 MHz, Dimethyl sulfoxide-d6) δ 173.04, 157.28, 154.69, 152.58, 150.56, 150.12,
149.81,146.36, 145.89, 143.65, 131.53, 131.06, 130.66, 130.32, 129.71, 128.75, 126.78, 123.52,
119.17, 109.26, 97.14, 74.63, 74.19, 73.86, 72.86, 71.54, 70.08, 69.40, 68.47, 67.34, 66.98, 65.71,
64.72, 60.82, 49.07, 41.00, 30.75, 8.25. TOF-MS, m/z: [M+Na+], Calcd for C38H46N4O19Na,
885.2654, Found, 885.2608.
4.22. In vitro antitumor activity test
4.22.1 Cells culture
HCT-116 cells, HepG2 cells and HEK-293 T cells were grown in DEME (Gibco, Thermo
Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, Life technologies) and
1% penicillin-streptomycin mixed antibiotic supplement (Gibco, Life technologies). Cell lines
were maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air.
4.22.2 Assessment of antitumor activity by MTT assay
The tested compounds were dissolved in appropriate amount of DMSO before the experiment

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to obtain the known concentration of solution, from which a definite amount was taken, and
diluted to the various concentrations with DEME. HCT-116, HepG2 and HEK-293T, bearing
period of logarithmic growth, were adjusted to 5*104 cells/mL, and then seeded into 96-well plates.
After cultured for 12 h in 37 °C under a humidified incubator (5% CO2 and 95% air). Cells were
incubated in complete mediums in the absence (negative control) and presence of various
concentrations of compounds tested, respectively, for 48 h. Each compound was arranged three
parallel wells. The supernatant was removed, and then 40μL MTT solution (5 mg/mL) was added
to each well. After re-incubated for another 4 h, 100μL of DMSO was added to each well for
dissolving the formazan crystals. The percentage of cell viability was determined by measuring
the absorbance (Abs) at wavelength of 490 nm using a Multiskan MK3 microplate reader (BioTek
Elx800, USA). The determination of IC50 values was acquired in accordance with linear
regression analysis of the concentration-response curves plotted for each compound tested.
Conflict of interest
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None declared.
Acknowledgments
The work is supported in part by financial support from National Natural Science Foundation of
China (No.21772028, 31360456, 3106052), Start-up Foundation of Guangzhou University of
Chinese Medicine for Young scholar (A1-AFD018171Z) and General Scientific Research project
of Guangzhou (201804010325), and Science and Technology plan of puer city (No. 2014-38). We
also thank professor Tian Xu very much for critical reading our manuscript.
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Multi-gram scale synthesis of a bleomycin (BLM)

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carbohydrate moiety: exploring the antitumor beneficial
effect of BLM disaccharide attached to
10-hydroxycamptothecine (10-HCPT)
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MaoLin Li1#, Weiping Huang1#, Zhilin Jiang2#, Yonghui Shi3, Sisi Yuan1, Kaishuo Fu1,
YongJun Chen4, Li Zhou5* and Wen Zhou1*
1 School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, University town,
Waihuan Rd, Panyu, Guangzhou, 510006, China
2Puer University, Puer, 665000, Yunan, China
3Department of Pharmacy, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
510120, Guangdong, China
4 South China Research Centre for Acupuncture and Moxibustion, Medical College of Acu-Moxi and
Rehabilitation, Guangzhou University of Chinese Medicine, University town, Waihuan Rd, Panyu,
Guangzhou, 510006, China
5College of Science, Hunan Agricultural University, Changsha, Hunan, 410128, China.
# These authors contributed equally to the work.
 Correspondent. E-mail: zhoulizss@163.com; zhouwen60@126.com
OH
OH
O
OH
OH
HO
HO
O
OBn
HO
O
HO
O
N
O
O
O
NH2
O
OH
O
HO
OH
OH
OCONH2
O
N
O
OH
O
OH
O
O
N
O
OH
OH
O
O
N
O
H
OH
O
N
HO
OH
OH
OCONH2
O
OH
HO
O
OH
Conjugate (1) (Specific drug)
10-hydroxycamptothecin (Non-specific drug)