Synthesis of new biocarrier-nucleotide systems for cellular delivery in bacterial auxotrophic strains

Article abstract of DOI:10.1016/j.tet.2014.09.096

In search for a delivery approach for thymidine monophosphate (TMP) in bacterial cells, we have synthesized a series of conjugates of TMP with biotin having an oxymethyleneoxy ester, a carboxy ester, and different carboxamide linkers between the carboxyl group of biotin and the 3′-OH group of TMP. The synthetic strategy starts from 5′-O-(dibenzylphosphate)-thymidine having the linkers already connected at the 3′-position. Likewise, kanamycin A was linked at the 3′-position of TMP using a carbamoyl or thioethyl carbamoyl group. None of the conjugates were able to sustain growth of a ΔthyA, ΔphoA Escherichia coli strain.

Full text of DOI:10.1016/j.tet.2014.09.096

Tetrahedron 70 (2014) 8843e8851
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of new biocarrierenucleotide systems for cellular delivery
in bacterial auxotrophic strains
Swarup De , Elisabetta Groaz , Mohitosh Maiti , Val eꢀ rie Pezo c, Philippe Marli eꢁ re c,
a
a
a
b,
a,c,
*
Piet Herdewijn
a
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
CEA, DSV, IG, Genoscope, 2 rue Gaston Cr ꢀe mieux, 91057 Evry Cedex, France
ISSB, G ꢀe nopole genavenir6, Equipe X ꢀe nome, 5 rue Henri Desbru ꢁe res, 91030 Evry Cedex, France
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2014
Received in revised form 24 September 2014
Accepted 30 September 2014
Available online 8 October 2014
In search for a delivery approach for thymidine monophosphate (TMP) in bacterial cells, we have syn-
thesized a series of conjugates of TMP with biotin having an oxymethyleneoxy ester, a carboxy ester, and
0
different carboxamide linkers between the carboxyl group of biotin and the 3 -OH group of TMP. The
0
synthetic strategy starts from 5 -O-(dibenzylphosphate)-thymidine having the linkers already connected
0
0
at the 3 -position. Likewise, kanamycin A was linked at the 3 -position of TMP using a carbamoyl or
thioethyl carbamoyl group. None of the conjugates were able to sustain growth of a
cherichia coli strain.
DthyA, DphoA Es-
Keywords:
Thymidine monophosphate
Biotin
Ó 2014 Elsevier Ltd. All rights reserved.
Kanamycin A
Carboxamide linker
Oxymethyleneoxy linker
Carbamoyl linker
1
. Introduction
last approach, which resulted in the first in vivo replication of an
unnatural DNA base pair, its universal applicability to all unnatural
bases and types of backbone modifications is yet unpredictable.
In a previous paper, we discussed our interest in the de-
velopment of a delivery tool for active uptake of nucleotides in
Creating artificial genetic systems capable of supporting Dar-
winian evolution is of prominent importance to synthetic biology,
not only to achieve expansion of the genetic alphabet but specially
1
4
for the genetic reprogramming of engineered microorganisms. The
bacterial cells based on a nutritional selection approach. The
in vivo synthesis of xeno nucleic acids (XNAs), however, is thought
to entail high intracellular consumption of unnatural nucleotide
building blocks, which will thus need to be provided to the host
cell, as most modified nucleosides are poorly phosphorylated by
natural kinases. Nucleotides, however, suffer from low cell-
penetrating ability and generally undergo fast enzymatic de-
phosphorylation before being taken up. Their bacterial uptake is
further complicated by the complex multi-layered wall structure,
which makes prokaryotic cells commonly more difficult to pene-
trate compared to eukaryotic membranes.
general concept consists in linking the information system to an
extracellular nutrient essential for the cell, thus taking advantage of
natural transport pathways. A biocleavable covalent bond is
designed to maintain the conjugate intact during cellular uptake,
but allows facile release of the nucleotide intracellularly. Building
on previous studies, thymidine monophosphate is selected as ar-
5
chetypal nucleotide system. The relevant TMP conjugate is sup-
plied within the nutrient medium to a bacterial strain deleted for
the ThyA gene encoding for thymidilate synthase, which is known
to unconditionally require thymine or thymidine for growth.
Because of the novelty of our delivery approach, we decided to
investigate a large diversity of biological carriers, such as peptides
and vitamins. We describe here the synthesis of biotineTMP con-
jugates. The appeal of this choice is due to the fact that biotin-
negative mutants are also well-established auxotrophs and can
Thus far, efforts have focused on overexpression of endogenous
2
kinases to broad enzymeesubstrate specificity, and more recently,
on the transfection of Escherichia coli (E. coli) with a plasmid
encoding an algal nucleotide triphosphate transporter to enhance
3
triphosphates uptake. Despite the ground-breaking value of this
6
,7
provide additional functional evidence.
Expanding upon this idea, we also wish to prove the same
concept using an inverse selection method. As in most cases,
*
Corresponding author. E-mail address: Piet.Herdewijn@rega.kuleuven.be
(
P. Herdewijn).
http://dx.doi.org/10.1016/j.tet.2014.09.096
040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
0

8844
S. De et al. / Tetrahedron 70 (2014) 8843e8851
antimicrobial compounds penetrate prokaryotic cell membranes
effectively through relatively non-specific transport systems, we
describe here also the synthesis of conjugates of nucleotides with
kanamycin A. Aminoglycosides (AGs) are clinically relevant drugs,
which show broad-spectrum antibacterial activity against both
gram-negative and gram-positive species, binding irreversibly to
the prokaryotic ribosome and mainly interfering with peptide
During the last step, formation of side products resulting from
conjugate decomposition was observed, pointing at instability of
this type of linker under hydrogenation conditions.
Different biotineTMP analogues with carboxyamido-type
linkers 10 (Scheme 2) and 13 (Scheme 3) were prepared adopting
previously optimized coupling and debenzylation procedures,
0
which required access, respectively, to 3 -O-amino thymidylate 8
8
,9
0
elongation at the 30S subunit. Although the detailed mechanism
by which AGs penetrate into the bacterial cytoplasm remains un-
clear, their uptake is known to be a multistep energy requiring
process that can be accumulated against a concentration gradi-
(obtained in four steps from thymidine) and 3 -O-aminoxymethyl
4
thymidylate 11 (obtained in six steps from thymidine). In general
good to excellent isolated yields of products were obtained and
linker cleavage did not occur in the last step.
10,11
0
ent.
In this case, the observation of toxicity would ensure up-
The 3 -carboxy ester conjugate 15 was also prepared similarly,
take and processing of the nucleotide by the cell.
starting from 5 in 65% overall yield over two steps (Scheme 4).
As route 2 (Fig.1) proved to be the most profitable for generating
new biotin conjugates, we chose a similar strategy to connect TMP
2
. Results and discussion
0
to kanamycin A by means of a 3 -carbamoyl linker, which gave
access to conjugates 22 and 24 (Fig. 2), and also led to a structural
reassignment of the aminoglycosidic unit.
Our retrosynthetic analysis for the planned carriereTMP target
compounds is shown in Fig. 1. It becomes apparent that those
conjugates can be accessed by either linking the carrier unit at the
As cationic amino functionalities of aminoglycosides are con-
sidered to be a crucial structural requirement for enhanced up-
0
0
3
1
-position of thymidine followed by 5 -O-phosphorylation (route
), or vice versa (route 2).
15
take, we sought to retain all four amino groups of rings IeIII in
kanamycin A. To construct the relevant conjugates efficiently, we
exploited the differences in chemical reactivity between the pri-
mary hydroxy and amino functionalities present in kanamycin A.
00
We started by replacing the primary hydroxyl group at the 6 -po-
sition of 16 with a thioethanolamino group, which would serve for
the formation of the desired carbamoyl linkage with TMP. The
16
synthesis of compound 19 has been reported by Arya et al.,
however, we adopted here a shorter synthetic route by modifying
17
a more recent procedure by Tor et al. (Scheme 5). To begin the
synthesis, we first needed to selectively protect all four amino
groups of kanamycin A. Among the several protecting strategies
described in the literature, we opted for the classical Boc protection,
which gave compound 17 in excellent yield. Considering the good
leaving group properties of triisopropylbenzenesulfonyl chloride
00
TIBS-Cl), compound 17 was selectively sulphonylated at the 6 -
(
primary hydroxyl group to its corresponding sulfonate 18, which
upon further treatment with 2-aminoethane thiol hydrochloride in
the presence of cesium carbonate, gave compound 19.
0
The introduction of a carbamate linker at the 3 -position of
nucleosides is possible starting from commercially available iso-
1
8
cyanates such as phenyl isocyanates, however, this approach ap-
pears unsuitable for macromolecules containing unprotected
polyhydroxyl and polyamino functionalities. Another commonly
Fig. 1. Retrosynthetic analysis of carriereTMP conjugates.
0
Initially, we decided to follow route 1 (Fig. 1) by applying the
same optimized conditions employed for the synthesis of pepti-
applied method to generate carbamates is the activation of the 3 -
19
OH group with CDI, but we felt that the strictly anhydrous con-
ditions required by this protocol would not be compatible with the
poor solubility of most aminoglycosides. A solution was found by
deeTMP conjugates connected though an oxymethyleneoxy ester
4
0
bond. Coupling of biotin at the 3 -position of methylene thio-
2
0
methyl thymidine derivative 1, obtained in turn from thymidine in
0% yield over two steps, and subsequent removal of the 5 -silyl
group, proceeded efficiently giving compound 3 in good yield;
choosing as activating/leaving group 4-nitrophenyl carbonate,
0
6
which is electrophilic enough to react with amino functionalities,
whilst still reasonably stable in aqueous media. Compound 20 was
prepared in a series of straightforward steps, involving phosphor-
ylation at the 5 -position of thymidine after selective pro-
tectionedeprotection at the 3 - and 5 -position with a MMTr and
benzoyl group, respectively, and final 3 -OH activation with 4-
nitrophenyl chloroformate in a controlled way in the presence of
pyridine/DCM.
With both coupling units now available, we proceeded to react
3 -O-(4-nitrophenylcarbonate)eTMP 20 with N-Boc kanamycin A
however, further phosphorylation caused oxidation at the sulfur
III
V
0
atom of biotin, during the conversion step from P to P , giving
undesired sulfoxide and sulfone by-products (Scheme 1A). Various
mild conditions were attempted to attain selective phosphorus
0
0
0
1
2
oxidation, including dilute solutions of I
2
,
m-chloroperbenzoic
acid (MCPBA),13 and (1S)-(þ)-(10-camphorsulfonyl) oxaziridine
14
but none gave the desired product. Switching to the alternative
0
0
disconnection, 5 -O-(dibenzylphosphate)-thymidine 5 was there-
fore prepared in four steps and subsequently subjected to coupling
with biotin, leading to the formation of the desired compound 7
after deprotection (Scheme 1B). However, we found that the
Pummerer rearrangement of compound 5 to 6 was less effective
than in the previous route, probably due to side reaction of the
electrophilic sulfur intermediate at the nucleophilic phosphate
center.
cysteamine 19 (Scheme 6A).
An initial attempt to synthesize 21 using previously optimized
conditions in the presence of triethylamine as base and DCM/DMF
(1:1) as solvent, afforded the desired conjugate in poor yield. Re-
placement of the solvent system with a mixture of 1,4-dioxane/
water (3:1), surprisingly resulted in an improved yield (90%) (Table
1). Removal of the dibenzyl phosphate protection of 21 was

S. De et al. / Tetrahedron 70 (2014) 8843e8851
8845
O
N
O
N
O
N
O
N
A.
NH
NH
NH
NH
BnO
BnO
O
O
O
O
P
O
O
a
b
TBDPSO
TBDPSO
HO
O
71%
O
O
O
O
8
4%
O
O
O
O
S
R
O
R
O
R
O
O
O
1
2
3
4
B.
O
O
O
+
NH
2
Et NH
3
NH
-
O
O
NH
P
O
N
O
BnO
BnO
O
-
O
BnO
BnO
O
P
O
N
O
P
O
N
O
O
O
S
c
a
d
O
4
O
OH
4, 7; R =
H
6
1%
51%
72%
H
O
O
HN
NH
R
S
O
O
5
6
7
0
ꢀ
Scheme 1. Synthesis of 3 -O-methyleneacyloxy-TMP biotin conjugate 7. Reagents and conditions: (a) (i) SO Cl , DCM, 0 C to rt, 2 h, (ii) biotin, DBU, DCM, rt, 24 h; (b) Et N$3HF, THF,
2
2
3
2 3
rt, 72 h; (c) DMSO, Ac O, AcOH, rt, 48 h; (d) 10% Pd/C (Degussa), Et N, MeOH, rt, 16 h.
0
Scheme 2. Synthesis of 3 -O-(carboxamide)-TMP biotin conjugate 10. Reagents and conditions: (a) biotin, DCC, DMAP, DCM/DMF, rt, 24 h; (b) 10% Pd/C (Degussa), MeOH, rt, 24 h.
0
Scheme 3. Synthesis of 3 -O-methyloxycarboxamide-TMP biotin conjugate 6. Reagents and conditions: (a) biotin, DCC, DMAP, DCM/DMF, rt, 24 h; (b) 10% Pd/C (Degussa), Et
3
N,
MeOH, rt, 24 h.

8846
S. De et al. / Tetrahedron 70 (2014) 8843e8851
0
6
-amino group of kanamycin A is known to be the most reactive
0
00
21,22
following the series 6 -NH
2
>1-NH
2
>3-NH
2
>3 -NH
2
.
When
we reacted directly compound 20 with kanamycin A 16 utilizing
the general protocol for carbamate formation (Scheme 6B), in-
termediate 23 was obtained in good yield. Subsequent hydro-
genolysis in the presence of 20% Pd(OH) /C and preparative RP-
2
HPLC purification yielded compound 24 in good yield and high
purity.
Although the reactivity order of the various hydroxyl and
amino groups of kanamycin A is well documented, we thought
necessary to confirm the structures of the final analogues. The
peak assignment for almost all proton and carbon signals of
kanamycin AeTMP conjugates 22 and 24 was unambiguously
determined (Table 2, Supplementary data), as well as the align-
0
0
0
0
ment of the carbamoyl linkage between the 9 and 6 positions
Scheme 4. Synthesis of 3 -ester-TMP biotin conjugate 15. Reagents and conditions: (a)
biotin, DCC, DMAP, DCM/DMF, rt, 24 h; (b) 10% Pd/C (Degussa), MeOH, rt, 24 h.
(Fig. 3A and B), by means of two-dimensional NMR spectroscopy
ꢀ
in D
2
O solution at 25 C. The presence of a distinctive signal at
157.5 ppm in the carbonyl region of the carbon NMR spectrum,
proton integration ratio along with HRMS mass spectrometry
confirmed the formation of a single carbamate linkage at the ex-
1
31
pected positions. While performing the H and P NMR spectro-
scopic analysis of compound 22 (Fig. 3C and D), the appearance of
ꢀ
a minor conformation was observed at low temperature (5 C),
ꢀ
which disappeared when the temperature was raised (50 C), in-
dicating the presence of a single conformer. This phenomenon
may arise due to the formation of rotamers, as the inherent flex-
ibility around glycosidic bonds of kanamycin AeTMP conjugates
2
3
allows them to easily adopt an array of conformations.
In order to test the bacterial uptake of all synthetized biotin and
kanamycin A conjugates as a source of TMP, an E. coli W2666
ꢁ
thyA(ꢁ) strain [spontaneous strepto R mutant of CBO129 (F
W1485 leu thyA dcoB or C supE)] was employed. Its genotype is E.
coli W2666 DthyA DphoA. This strain is deleted for thyA and phoA
genes, which, respectively, encode for thymidylate synthase and
alkaline phosphatase, in order to block all pathways that could lead
to the formation or degradation of TMP inside the bacterial cells. All
compounds were preventively submitted to LC/MS analysis to ex-
clude potential sample contamination, but no traces of thymine
could be observed below detection limit. LB medium and LB agar
Fig. 2. Structures of kanamycin AeTMP conjugates 22 and 24 and key HMBC corre-
lations used for structure determination. Plain arrows (
) indicate HMBC correla-
tions with the carbamate (OCONH) linker (cf. Fig. 3A/B) whilst dotted arrows (
show HMBC correlation used for other peak assignments.
)
ꢀ
were sterilized by autoclaving (120 C, 30 min). Thymine (positive
control), TMP (negative control), and all compounds were dissolved
in MilliQ water and sterilized through a 0.2
mm sterile syringe filter
(
3
VWR). A single colony of the mutant strain was grown overnight at
7 C in LB medium (3 mL). Cells were then harvested by centri-
ꢀ
fugation, washed and resuspended in fresh LB medium. The bac-
terial cells were then diluted 1:1000 in fresh LB medium before
streaking on LB agar plates containing 20
and grown overnight at 37 C. Unfortunately, no colony formation
m
g/mL of each compound
ꢀ
(compounds 7, 10, 13, 15) or toxicity (compounds 22, 24) was
observed.
Scheme 5. Synthesis of kanamycin A cysteamine 19. Reagents and conditions: (a)
ꢀ
Boc
Cs CO
2
2
O, Et
3
N, DMF/H
2
O, 50 C, 6 h; (b) TIBSCl, pyridine, rt, 16 h; (c) HCl$NH
2
CH
2
CH
2
SH,
ꢀ
3
, DMF, 25 C, 16 h.
3. Conclusion
achieved by catalytic hydrogenolysis using an excess of 20%
Pd(OH) /C to avoid potential catalyst poisoning due to the sulfide
moiety. Without further purification, the N-Boc protected in-
termediate 21 was converted to compound 22 upon treatment with
aq TFA, in good yield and high purity after preparative RP-HPLC
purification.
As the convergent coupling of complex fragments is impor-
tant to rapidly generate a variety of conjugates, we decided to
further explore the versatility of this reaction process. Since
a single aminomethyl group is present in kanamycin A, the
regioselective formation of a mono-carbamate linker seemed
feasible without prior protection of the other amino groups. The
In summary, we have accomplished the synthesis of a variety of
2
biotineTMP and kanamycin AeTMP conjugates in a straightfor-
0
ward manner starting from previously optimized 3 -modified TMP
intermediates and featuring in turn an oxymethyleneoxy ester,
a carboxy ester, a carboxamide, a carbamoyl, and a thioethyl car-
bamoyl group. On the basis of preliminary biological screening
none of the conjugates showed functional activity in E. coli mutants
lacking thymine. However, this might be due to several factors, for
instance high concentrations needed for substantial growth, which
are currently under further study. As additional evidence, bio-
tineTMP conjugates are undergoing biological evaluation using E.
coli mutants lacking biotin.

S. De et al. / Tetrahedron 70 (2014) 8843e8851
8847
0
ꢀ
Scheme 6. Synthesis of 3 -carbamoyl kanamycin AeTMP conjugates 22 and 24. Reagents and conditions: (a) Et
O (8:2), rt, 24 h; (c) TFA, thioanisole, H O, rt, 5 h.
3
N, 1,4-dioxane/water, 0 C to rt, 18 h; (b) 20% Pd(OH)
2
/C, H
2
, EtOH/
H
2
2
4
4
. Experimental section
.1. General
Table 1
Reaction circumstances for the synthesis of compounds 21 and 23 from 20
Entry Solvent
Temp
rt
Time (h) Yield of 21a (%) Yield of 23a (%)
For all reactions, analytical grade solvents were used. Kanamy-
cin A$H SO was purchased from SigmaeAldrich (containing
kanamycin B as a 5% impurity). Kanamycin A$H SO salt was
1
2
3
DCM/DMF (1:1)
DMF
Dioxane/H
60
48
48
55
90
d
42
82
2
4
rt
ꢀ
2
4
2
O (3:1) 0 C to rt 16
transformed into its free amino form by treatment with Amberlite
IRA-400 ion exchange resin. All moisture-sensitive reactions were
carried out using oven-dried glassware (135 C) under a nitrogen or
a
Isolated yield.
ꢀ
argon atmosphere. Reaction temperatures are reported as bath
temperatures. Pre-coated aluminum sheets (254 nm) were used for
Fig. 3. (A) Key HMBC correlation for compound 22; (B) key HMBC correlation NMR spectrum of compound 24; (C) and (D) temperature-dependent NMR ( H and 31P) study of
1
2
compound 22. All NMR spectra were recorded in D O.

8848
S. De et al. / Tetrahedron 70 (2014) 8843e8851
TLC. Compounds were visualized with UV light (
l
¼254 nm).
under reduced pressure. The crude residue was purified by column
chromatography on silica gel (gradient CH Cl /MeOH, 99:1, v/v;
96:4, v/v; 93:7, v/v) to give 3 (0.49 g, 84%) as a white solid. H NMR
Products were purified by flash chromatography on ICN silica gel
2
2
ꢂ
1
6
3e200, 60 A. All final compounds were purified by preparative RP-
HPLC using a gradient of H O and MeCN, both contain either
0 mmol TEAB or 0.1% TFA as eluant buffer as mentioned. H, C,
and P NMR spectra were recorded on 300, 500, and 600 MHz
2
(300 MHz, DMSO-d
6
)
d
¼11.34 (s, 1H, NH-Thy), 7.68 (s, 1H, H-6), 6.42
1
13
0
5
(s, 1H, NH Bio), 6.37 (s, 1H, NH Bio), 6.01 (app t, J¼7.0 Hz, 1H, H-1 ),
31
0
5.30 (dd, J¼20.2, 6.6 Hz, 2H, OCH
2
O), 5.13 (t, J¼5.1 Hz, 1H, 5 -OH),
0
Bruker spectrometers. Final compounds were characterized using
4.39e4.36 (m, 1H, H-3 ), 4.33e4.28 (m, 1H, H-g Bio), 4.15e4.10 (m,
1
0
0
2
D NMR (H-COSY, HSQC, HMBC, TOCSY, NOESY) techniques. The H
1H, H-f Bio), 3.93e3.90 (m, 1H, H-4 ), 3.61e3.56 (m, 2H, H-5 and H-
13
00
and C chemical shifts were referenced to residual solvent signals
relative to TMS as internal standard wherever applied. Coupling
constants J [Hz] were directly taken from the spectra. Splitting
patterns are designated as s (singlet), d (doublet), t (triplet), q
5 ), 3.13e3.06 (m, 1H, H-e Bio), 2.82 (dd, J¼12.4, 5.1 Hz, 1H, H-h
0
Bio), 2.57 (d, J¼12.4 Hz, 1H, H-h Bio), 2.36 (t, J¼7.4 Hz, 1H, and H-
0
00
3
a Bio), 2.27e2.18 (m, 2H, H-2 and H-2 ), 1.77 (s, 3H, CH ), 1.66e1.45
13
(m, 4H, H-b and H-d Bio), 1.42e1.29 (m, 2H, H-c Bio); C NMR
(
quartet), m (multiplet), and br (broad). High-resolution mass
(75 MHz, DMSO-d
6
)
d
¼172.4 (OCO), 163.6 (C-4), 162.7 (NHCONH),
0
spectra were acquired on a quadrupole orthogonal acceleration
time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Mil-
ford, MA). Samples were infused at 3
150.5 (C-2), 135.9 (C-6), 109.5 (C-5), 87.4 (OCH
2
O), 84.8 (C-1 ), 83.6
0 0 0
(C-4 ), 79.4 (C-3 ), 61.1 (C-5 ), 61.0 (C-f Bio), 59.2 (C-g Bio), 55.3 (C-e
0 0
Bio), 40.2 (C-h, h Bio, merged with DMSO), 36.9 (C-2 ), 33.3 (C-
mL/min and spectra were
obtained in positive (or negative) ionization mode with a resolution
of 15,000 (FWHM) using leucine enkephalin as lock mass.
3
a Bio), 28.0 (C-c Bio), 27.9 (C-d Bio), 24.2 (C-b Bio), 12.2 (CH eThy);
HRMS for C21
30
H N
4
O
8
S [MꢁH]ꢁ calcd: 497.1711, found: 497.1715.
0
0
0
0
4
.2. 5 -O-(tert-Butyl diphenyl silane)-3 -O-(bio-
4.4. 5 -O-(Dibenzylphosphate)-3 -O-(methylthiomethyl)-thy-
tinmethyloxyester)-thymidine (2)
midine (6)
A 1 M solution of SO
2
Cl
2
in CH
2
Cl
2
(2.07 mL, 2.07 mmol) was
Acetic anhydride (10.9 mL) and acetic acid (3.5 mL) were added
to a stirred solution of 5 (2.40 g, 4.78 mmol) in DMSO (15.5 mL). The
reaction mixture was stirred at room temperature for 30 h, and
then it was concentrated under reduced pressure. The residue was
added to a stirred solution of 1 (0.935 g, 0.107 mmol) in dry CH
2
Cl
2
ꢀ
(
20 mL) at 0 C. The reaction mixture was allowed to warm slowly
to room temperature over 2 h, then it was concentrated using
ꢀ
a rotary evaporator (bath temperature 10e15 C) under reduced
neutralized with saturated aq NaHCO
was extracted with ethyl acetate (3ꢂ170 mL). The combined or-
ganic extracts were washed with brine, dried over Na SO , filtered,
3
(230 mL), and the mixture
0
pressure to give a 3 -O-chloromethylthymidine derivative as a light
yellowish foam, which was used without further purification.
2
4
In
a
separate round-bottomed flask, biotin (0.634 g,
Cl (20 mL), and DBU
and concentrated under reduced pressure. The crude residue was
purified by column chromatography on silica gel (gradient hexane/
2
.593 mmol) was suspended in dry CH
2
2
(
0.38 mL, 2.51 mmol) was then added. After 15 min, the solution
EtOAc, 2:1, v/v; 1:1, v/v; 1:3, v/v) to give 6 (1.64 g, 61%) as a colorless
0
1
was added to the crude 3 -O-chloromethylthymidine derivative
foam. H NMR (300 MHz, CDCl
(m, 11H, H-6 and AreH OBn), 6.26 (dd, J¼8.2, 5.8 Hz, 1H, H-1 ),
3
)
d
¼8.22 (br s, 1H, NH), 7.37e7.35
0
(
redissolved in dry CH
kept stirring at room temperature for 24 h. The mixture was then
diluted with CH Cl (100 mL), and 1 M aq acetic acid (4 mL) was
added with vigorous stirring. The aqueous layer was discarded, and
the organic layer was washed with saturated aq NaHCO
(2ꢂ50 mL)
and brine, dried over Na SO , filtered, and concentrated under re-
duced pressure. The crude residue was purified by column chro-
matography on silica gel (gradient CH Cl /MeOH, 99:1, v/v; 98:2, v/
v; 97:3, v/v) to give 2 (0.91 g, 71%) as a colorless foam. H NMR
300 MHz, CDCl
2
Cl
2
, 20 mL), and the reaction mixture was
5.11e5.00 (m, 4H, 2ꢂOCH
2
Ph), 4.64e4.51 (m, 2H, OCH
4.35e4.31 (m, 1H, H-3 ), 4.17e4.09 (m, 3H, H-4 , H-5 and H-5 ),
2
S),
0
0
0
00
2
2
0
00
2.34e2.26 (m, 1H, H-2 ), 2.11 (s, 3H, SCH
3
), 1.92e1.81 (m, 4H, H-2
13
3
and CH
135.6 (d,
3
eThy); C NMR (75 MHz, CDCl
3
)
d
¼163.4 (C-4),150.2 (C-2),
3
2
4
J
C,P¼6.1 Hz, 1C of OCH
2
Ph), 135.3 (C-6), 129.0 (AreC),
0
128.9 (AreC), 128.2 (AreC), 111.5 (C-5), 85.0 (C-1 ), 82.8 (d,
3
2
0
0
2
2
J
J
C,P¼8.2 Hz, C-4 ), 76.3 (C-3 ), 74.1 (OCH S), 69.9e69.8 (2ꢂd,
2
1
2
0
0
C,P¼3.5 Hz, 2ꢂOCH Ph), 67.0 (d, JC,P¼5.7 Hz, C-5 ), 37.5 (C-2 ), 14.0
2
31
(
3
)
d¼11.9 (s, 1H, NH-Thy), 7.68e7.65 (m, 4H, AreH
(SCH
3
), 12.5 (CH
3
eThy); P NMR (121 MHz, CDCl
3
)
d¼ꢁ0.5; HRMS
ꢁ
Ph), 7.51 (s, 1H, H-6), 7.46e7.37 (m, 6H, AreH Ph), 6.94 (s, 1H, NH
31
for C26H N
2
O
8
PS [MꢁH] calcd: 561.1466, found: 561.1461.
0
Bio), 6.91 (s, 1H, NH Bio), 6.27 (dd, J¼8.6, 5.2 Hz, 1H, H-1 ), 5.71 (m,
0
0
0
1
4
4
H, H-g Bio), 4.92 (m, 1H, H-f Bio), 4.59e4.54 (m, 1H, H-3 ),
4.5. 5 -O-(Dibenzylphosphate)-3 -O-(biotinmethyloxyester)-
0
00
0
.38e4.35 (m, 2H, H-5 and H-5 ), 4.12e4.10 (m, 1H, H-4 ),
.03e3.82 (m, 2H, OCH O), 3.21e3.14 (m, 1H, H-e Bio), 2.96e2.82
thymidine (4)
2
0
0
(
(
m, 2H, H-h Bio and H-h Bio), 2.74e2.68 (m, 1H, H-2 ), 2.49e2.28
Following a similar procedure as the one used for the synthesis
of 2, compound 4 was obtained (41.0 mg, 51%) as a colorless foam,
00
m, 2H, H-a Bio), 2.14e2.05 (m,1H, H-2 ),1.83e1.65 (m, 4H, H-b and
), 1.55e1.40 (m, 2H, H-c Bio), 1.11 (s, 9H,
Bu); C NMR (75 MHz, CDCl
¼172.6 (OCO), 165.0 (C-4), 164.3
NHCONH), 151.4 (C-2), 135.4 (AreC), 135.2 (AreC), 134.3 (C-6),
32.6 (AreC), 132.2 (AreC), 130.1 (AreC), 130.0 (AreC), 128.0
H-d Bio), 1.62 (s, 3H, CH
3
starting from 6 (60.0 mg, 0.107 mmol), a 1 M solution of SO
CH Cl (0.13 mL, 0.128 mmol) in dry CH Cl (4 mL), and biotin
(39.1 mg, 0.160 mmol), DBU (0.023 mL, 0.155 mmol) in dry CH Cl
(15 mL). The crude residue was purified by column chromatography
2 2
Cl in
t
13
3
)
d
2
2
2
2
(
1
2
2
0
(
4
AreC), 127.9 (AreC), 111.2 (C-5), 88.1 (OCH
2
O), 85.1 (C-1 ), 84.5 (C-
on silica gel (gradient CH
H NMR (300 MHz, CDCl
2
Cl
2
/MeOH, 99:1, v/v; 97:3, v/v; 93:7, v/v).
¼9.76 (s, 1H, NHeThy), 7.35e7.37 (m,
0
0
0
1
), 81.6 (C-3 ), 64.0 (C-5 ), 62.1 (C-f Bio), 60.3 (C-g Bio), 55.4 (C-e
3
)
d
0
0
Bio), 40.7 (C-h, h Bio), 38.9 (C-2 ), 33.8 (C-a Bio), 28.0 (C-c Bio), 27.9
(
HRMS for C37
11H, H-6 and AreH OBn), 6.81 (br s, 1H, NH Bio), 6.31 (app t,
t
t
0
C-d Bio), 26.9 ( Bu), 24.5 (C-b Bio), 19.2 (1C Bu), 12.1 (CH
3
eThy);
J¼6.6 Hz, 1H, H-1 ), 6.18 (br s, 1H, NH Bio), 5.13e4.99 (m, 6H, OCH
2
O
þ
0
H
48
N
4
O
8
SSi [MþH] calcd: 737.3035, found: 737.3045.
and 2ꢂOCH
2
Ph), 4.53e4.49 (m, 1H, H-3 ), 4.38e4.35 (m, 1H, H-g
Bio), 4.26e4.12 (m, 3H, H-f Bio, H-5 and H-5 ), 4.05e4.03 (m, 1H,
0
00
0
0
4
.3. 3 -O-(Biotinmethyloxyester)-thymidine (3)
H-4 ), 3.16e3.12 (m, 1H, H-e Bio), 2.89 (dd, J¼12.2, 7.2 Hz, 1H, H-h
0
0
Bio), 2.79 (d, J¼12.2 Hz, 1H, H-h Bio), 2.58e2.52 (m, 1H, H-2 ),
00
Triethylamine trihydrofluoride (0.76 mL, 4.67 mmol) was added
2.40e2.27 (m, 3H, H-2 and H-a Bio),1.80 (s, 3H, CH ),1.76e1.69 (m,
3
13
to a stirred solution of 2 (0.86 g, 1.17 mmol) in THF (20 mL) in
a Falcon tube at room temperature. The reaction mixture was
stirred at room temperature for 72 h, and then it was concentrated
4H, H-b and H-d Bio), 1.52e1.43 (m, 2H, H-c Bio); C NMR (75 MHz,
CDCl
¼172.7 (OCO), 164.7 (C-4), 164.2 (NHCONH), 150.7 (C-2),
2
135.5 (d, Ph), 134.5 (C-6), 128.8 (AreC),
3
) d
3
JC,P¼8.0 Hz, 1C of OCH

S. De et al. / Tetrahedron 70 (2014) 8843e8851
8849
0
10PS [MþNa]þ
1
8
2
5
28.7 (AreC), 128.1 (AreC), 111.3 (C-5), 88.1 (OCH
2
O), 84.8 (C-1 ),
(121 MHz, CDCl
calcd: 766.2282, found: 766.2302.
3
)
d
¼ꢁ1.0; HRMS for C34
42 5
H N O
3
0
0
2
3.1 (d, JC,P¼8.1 Hz, C-4 ), 81.3 (C-3 ), 69.9e69.8 (2ꢂd, JC,P¼5.2 Hz,
2
0
ꢂOCH
5.6 (C-e Bio), 40.2 (C-h, h Bio), 38.6 (C-2 ), 33.9 (C-a Bio), 28.2 (C-c
2
Ph), 67.0 (d, JC,P¼5.9 Hz, C-5 ), 62.3 (C-f Bio), 60.2 (C-g Bio),
0
0
0
4.8. 3 -O-(Biotincarboxamide)-thymidine monophosphate
31
Bio), 28.0 (C-d Bio), 24.8 (C-b Bio), 12.5 (CH
3
eThy); P NMR
triethylammonium salt (10)
(
121 MHz, CDCl
3
)
d
¼ꢁ0.4; HRMS for C35
H
43
N
4
O
11PS [MþNa]þ
calcd: 781.2279, found: 781.2288.
Following a similar procedure as the one used for the synthesis
of 7, the triethylammonium salt of compound 10 was obtained as
a white solid (169.0 mg, 82%), starting from 9 (200.0 mg,
0
4
.6. 3 -O-(Biotinmethyloxyester)-thymidine monophosphate
3
0.269 mmol), Et N (0.075 mL, 0.538 mmol), and 10% Pd/C Degussa
triethylammonium salt (7)
1
(100 mg, 50% w/w) in MeOH (20 mL). H NMR (500 MHz, D
2
O)
0
d
¼7.90 (s, 1H, H-6), 6.40 (dd, J¼9.1, 5.7 Hz, 1H, H-1 ), 4.73e4.72 (m,
Pd/C (10%) (Degussa, 15.0 mg, 50% w/w) was added to
0
1
4
H, H-3 ), 4.56e4.54 (m, 1H, H-g Bio), 4.38e4.35 (m, 1H, H-f Bio),
a degassed stirred solution of 4 (30.0 mg, 0.039 mmol) and Et
0.011 mL, 0.079 mmol) in MeOH (10 mL), and the mixture was
subjected to hydrogenation at atmospheric pressure using a bal-
loon filled with H for 24 h. The catalyst was removed by filtration
through a cellulose filter (0.45
3
N
0
0
00
.32 (br s, 1H, H-4 ), 3.92e3.91 (m, 2H, H-5 and H-5 ), 3.32e3.28
(
(
m, 1H, H-e Bio), 2.93 (dd, J¼13.0, 4.9 Hz, 1H, H-h), 2.69 (d, 1H,
0
0
J¼13.0 Hz, H-h Bio), 2.48e2.44 (m, 1H, H-2 ), 2.40e2.34 (m, 1H, H-
2
0
0
2
), 2.15 (t, 2H, J¼7.0 Hz, H-a Bio),1.89 (s, 3H, CH
3
),1.72e1.67 (m,1H,
m
m) and the filtrate was concen-
0
H-b Bio), 1.65e1.60 (m, 2H, H-d Bio), 1.59e1.51 (m, 1H, H-b Bio),
ꢀ
trated under reduced pressure (bath temperature 10e15 C). The
resulting crude residue was purified by RP-HPLC [TEAB (triethy-
2
O/MeCN, 98:2; and TEAB
50 mmol) in H O/MeCN, 50:50]. The collected eluate was freeze-
dried repeatedly until constant mass to obtain the triethylammo-
nium salt of 7 (22.2 mg, 72%) as a white solid. H NMR (500 MHz,
13
1.40e1.34 (m, 2H, H-c Bio); C NMR (125 MHz, D
2
O)
d¼172.8
(ONHCO), 166.9 (C-4), 164.8 (NHCONH), 151.8 (C-2), 137.3 (C-6),
lammonium bicarbonate; 50 mmol) in H
(
0
0
3
0
1
11.4 (C-5), 84.9 (C-3 ), 84.3 (C-1 ), 82.1 (d, JC,P¼8.9 Hz, C-4 ), 63.6
2
2
0
(d, JC,P¼3.9 Hz, C-5 ), 61.4 (C-f Bio), 59.7 (C-g Bio), 54.7 (C-e Bio),
0
0
3
9.2 (C-h, h Bio), 34.8 (C-2 ), 31.8 (C-a Bio), 27.3 (C-c Bio), 27.0 (C-
1
3
1
d Bio), 24.5 (C-b Bio), 11.3 (CH
3
eThy); P NMR (202 MHz, D
2
O)
0
D
2
O)
d
¼7.80 (s, 1H, H-6), 6.28 (app t, J¼7.3 Hz, 1H, H-1 ), 5.46e5.32
ꢁ
d
¼3.4; HRMS for C20
30
H N
5
O
10PS [MꢁH] calcd: 562.1378, found:
0
(
m, 2H, OCH
2
O), 4.63e4.62 (m, 1H, H-3 ), 4.56e4.54 (m, 1H, H-g
5
62.1382.
0
Bio), 4.38e4.36 (m, 1H, H-f Bio), 4.25 (br s, 1H, H-4 ), 3.94e3.88 (m,
0
00
2
5
H, H-5 and H-5 ), 3.29e3.25 (m, 1H, H-e Bio), 2.92 (dd, J¼13.0,
0
0
4
.9. 5 -O-(Dibenzylphosphate)-3 -O-(bio-
0
.0 Hz, 1H, H-h), 2.70 (d, 1H, J¼13.0 Hz, H-h Bio), 2.45 (t, 2H,
tinmethyloxycarboxamide)-thymidine (12)
0
00
J¼7.2 Hz, H-a Bio), 2.42e2.40 (m, 1H, H-2 and H-2 ), 1.88 (s, 3H,
CH
3
), 1.73e1.52 (m, 4H, H-b and H-d Bio), 1.43e1.36 (m, 2H, H-c
Following a similar procedure as the one used for the synthesis
of 9, compound 12 was obtained as a colorless foam (214.0 mg,
13
Bio); C NMR (125 MHz, D
2
O)
d
¼175.5 (OCO), 168.3 (C-4), 164.8
(
(
NHCONH), 152.9 (C-2), 137.0 (C-6), 111.4 (C-5), 88.1 (OCH
2
O), 84.3
7
0
0
6%), starting from 11 (200 mg, 0.365 mmol), biotin (102.6 mg,
.420 mmol), DCC (120.6 mg, 0.584 mmol), and DMAP (1.8 mg,
.0146 mmol) in a mixture of dry DMF (2.0 mL) and dry CH Cl
0
3
0
0
2
C-1 ), 83.8 (d, JC,P¼9.2 Hz, C-4 ), 80.7 (C-3 ), 63.3 (d, JC,P¼3.9 Hz,
0
0
C-5 ), 61.5 (C-f Bio), 59.7 (C-g Bio), 54.8 (C-e Bio), 39.2 (C-h, h Bio),
0
2
2
3
6.6 (C-2 ), 33.1 (C-a Bio), 27.4 (C-c), 27.1 (C-d Bio), 23.4 (C-b Bio),
1
(
(
(
10 mL). H NMR (300 MHz, DMSO-d
br s, 1H, NH), 7.50 (s, 1H, H-6), 7.35e7.35 (m, 10H, AreH OBn), 6.41
br s, 1H, NH Bio), 6.35 (br s, 1H, NH Bio), 6.15 (dd, J¼8.2, 5.9 Hz, 1H,
6
)
d¼11.34 (br s, 1H, NH), 11.00
eThy); 3 P NMR (202 MHz, D
1
O)
d
¼3.4; HRMS for
11.5 (CH
3
O
2
C
21
H
31
N
4
11PS [MꢁH]ꢁ calcd: 577.1375, found: 577.1375.
0
H-1 ), 5.06 (d, J¼8.0 Hz, 4H, 2ꢂOCH
2 2
Ph), 4.87e4.81 (m, 2H, OCH O),
0
0
0
4
.7. 5 -O-(Dibenzylphosphate)-3 -O-(biotincarboxamide)-thy-
4.51e4.50 (m, 1H, H-3 ), 4.33e4.29 (m, 3H, H-g Bio, H-f Bio and H-
0
0
00
midine (9)
4 ), 4.19e4.10 (m, 2H, H-5 and H-5 ), 3.10e3.04 (m, 1H, H-e Bio),
0
2
.81 (dd, J¼12.6, 5.2 Hz, 1H, H-h Bio), 2.57 (d, J¼12.6 Hz, 1H, H-h
0
00
DCC (127.4 mg, 0.618 mmol) was added to a stirred solution of 8
200.0 mg, 0.386 mmol), biotin (108.6 mg, 0.444 mmol), and DMAP
2.0 mg, 0.016 mmol) in a mixture of dry DMF (2.0 mL) and dry
Bio), 2.35e2.28 (m, 1H, H-2 ), 2.17e2.08 (m, 1H, H-2 ), 2.00 (t,
J¼7.0 Hz, 3H, H-a Bio), 1.69 (s, 3H, CH ), 1.63e1.42 (m, 4H, H-b and
(
(
CH
3
1
3
6
H-d Bio), 1.36e1.27 (m, 2H, H-c Bio); C NMR (75 MHz, DMSO-d )
ꢀ
2
Cl
2
(10 mL) at 0 C. The reaction mixture was allowed to warm
d
¼169.6 (ONHCO), 163.6 (C-4), 162.7 (NHCONH), 150.4 (C-2), 135.9
3
slowly to room temperature and stirred for 24 h. All the volatiles
were removed under reduced pressure and the resulting residue
was purified by column chromatography on silica gel (gradient
(d,
J
C,P¼6.9 Hz, 1C of OCH
2
Ph), 135.6 (C-6), 128.5 (AreC), 128.4
0
(AreC), 127.8 (AreC), 109.9 (C-5), 97.0 (OCH
2
O), 84.0 (C-1 ), 82.3 (d,
3
0
0
2
J
C,P¼7.6 Hz, C-4 ), 77.1 (C-3 ), 68.7 (d, JC,P¼5.4 Hz, 2ꢂOCH
2
Ph), 67.1
2
0
CH
7
(
2 2
Cl /MeOH, 99:2, v/v; 97:4, v/v; 94:7, v/v) to give 9 (201.0 mg,
(d, JC,P¼5.0 Hz, C-5 ), 61.0 (C-f Bio), 59.2 (C-g Bio), 55.3 (C-e Bio),
1
0
0
0%) as a colorless foam. H NMR (300 MHz, CDCl
3
þCD
3
OD)
d
¼7.41
39.5 (C-h, h Bio), 36.2 (C-2 ), 32.0 (C-a Bio), 28.1 (C-c Bio), 28.0 (C-
31
d, J¼1.0 Hz, 1H, H-6), 7.39e7.34 (m, 10H, AreH OBn), 6.27 (dd,
3 3
d Bio), 24.9 (C-b Bio), 12.0 (CH eThy); P NMR (121 MHz, CDCl )
0
11PS [MþNa]þ calcd: 796.2388,
J¼8.9, 5.6 Hz, 1H, H-1 ), 5.12e5.00 (m, 4H, 2ꢂOCH
2
Ph), 4.55e4.48
d
¼ꢁ0.9; HRMS for C35
44 5
H N O
0
0
(
4
(
2
m, 2H, H-3 and H-g Bio), 4.33e4.29 (m, 2H, H-4 and H-f Bio),
.23e4.22 (m, 2H, H-5 and H-5 ), 3.21e3.15 (m, 1H, H-e Bio), 2.91
found: 796.2403.
0
00
0
0
dd, J¼12.8, 4.9 Hz, 1H, H-h Bio), 2.73 (d, J¼12.8 Hz, 1H, H-h Bio),
4.10. 3 -O-(Biotinmethyloxycarboxamide)-thymidine mono-
0
.54e2.48 (m, 1H, H-2 ), 2.14 (t, J¼6.7 Hz, 3H, H-a Bio), 1.94e1.84
phosphate triethylammonium salt (13)
0
0
(
m, 1H, H-2 ), 1.81 (d, J¼0.9 Hz, 3H, CH
H-d Bio), 1.51e1.43 (m, 2H, H-c Bio);
CDCl OD)
þCD
51.3 (C-2), 136.0 (C-6), 135.6 (d, JC,P¼5.7 Hz, 1C of OCH
3
), 1.75e1.61 (m, 4H, H-b and
13
C NMR (75 MHz,
Following a similar procedure as the one used for the synthesis
of 7, the triethylammonium salt of compound 13 was obtained as
a white solid (170.6 mg, 79%), starting from 12 (210 mg,
0.271 mmol), Et N (0.075 mL, 0.543 mmol), and 10% Pd/C Degussa
3
(105 mg, 50% w/w) in MeOH (20 mL). H NMR (500 MHz, D
3
3
d
¼172.8 (ONHCO), 165.1 (C-4), 164.9 (NHCONH),
3
1
(
1
2
2
Ph), 129.4
0
AreC), 129.1 (AreC), 128.5 (AreC), 111.8 (C-5), 85.4 (C-3 ), 85.3 (C-
0
3
0
2
1
), 81.6 (d,
ꢂOCH
J
C,P¼8.3 Hz, C-4 ), 70.5e70.4 (d,
J
C,P¼5.6 Hz,
2
O)
2
0
0
2
Ph), 68.1 (d, JC,P¼5.8 Hz, C-5 ), 62.4 (C-f Bio), 60.6 (C-g Bio),
d
¼7.85 (d, J¼1.1 Hz, 1H, H-6), 6.31 (app t, J¼7.3 Hz, 1H, H-1 ), 4.98
0
0
0
5
5.9 (C-e Bio), 40.6 (C-h, h Bio), 36.4 (C-2 ), 32.8 (C-a Bio), 28.7 (C-c
(s, 2H, OCH
2
O), 4.71e4.70 (m, 1H, H-3 ), 4.57e4.54 (m, 1H, H-g
31
0
Bio), 28.5 (C-d Bio), 25.5 (C-b Bio), 12.4 (CH
3
eThy); P NMR
Bio), 4.39e4.37 (m, 1H, H-f Bio), 4.28e4.27 (m, 1H, H-4 ),

8850
S. De et al. / Tetrahedron 70 (2014) 8843e8851
0
00
3
(
2
1
1
(
1
7
.97e3.90 (m, 2H, H-5 and H-5 ), 3.31e3.27 (m, 1H, H-e Bio), 2.94
4.13. N-Boc kanamycin A (17)
To a stirred solution of kanamycin A 16 (1.8 g, 3.71 mmol) in
0
dd, J¼13.0, 5.0 Hz, 1H, H-h), 2.71 (d, 1H, J¼13.0 Hz, H-h Bio),
0
00
.42e2.39 (m, 1H, H-2 and H-2 ), 2.20 (t, 2H, J¼7.0 Hz, H-a Bio),
.89 (d, J¼1.1 Hz, 3H, CH
.39e1.37 (m, 2H, H-c Bio); C NMR (125 MHz, D
ONHCO), 166.2 (C-4), 164.8 (NHCONH), 151.3 (C-2), 137.2 (C-6),
3
), 1.70e1.54 (m, 1H, H-b and H-d Bio),
a mixture of DMF/H
2
O (4:1, 48 mL), was added di-tert-butyldicar-
1
3
ꢀ
2
O)
d¼172.9
bonate (5.12 mL, 22.29 mmol) and the solution was heated at 60 C
for 5 h and then cooled to room temperature. After removal of all
the volatiles, the resulting residue was washed with hexane
(2ꢂ100 mL) and then with water (3ꢂ100 mL), and finally dried in
0
3
0
11.4 (C-5), 96.8 (OCH
2
O), 84.3 (C-1 ), 83.8 (d, JC,P¼8.7 Hz, C-4 ),
0
2
0
8.4 (C-3 ), 63.4 (d, JC,P¼3.7 Hz, C-5 ), 61.5 (C-f Bio), 59.7 (C-g Bio),
0
0
5
4.8 (C-e Bio), 39.2 (C-h, h Bio), 36.1 (C-2 ), 31.5 (C-a Bio), 27.2 (C-c
vacuo to afford 17 (2.99 g, 91%) as a white solid. R
f
¼0.4 (10% MeOH
3
1
1
Bio), 27.1 (C-d Bio), 24.2 (C-b Bio), 11.2 (CH
3
eThy); P NMR
in DCM). H NMR (300 MHz, DMSO-d
6
)
d
¼6.89 (br s, 1H), 6.58 (br s,
(
202 MHz, D
2
O)
d
¼2.8; HRMS for C21
H
32
N
5
O
11PS [MꢁH]ꢁ calcd:
1H), 6.50 (d, J¼8.6 Hz, 1H), 6.34 (br s, 1H), 5.35 (d, J¼3.8 Hz, 1H),
5.26 (s, 1H), 4.89 (m, 4H), 4.68 (d, J¼6.2 Hz, 1H), 4.19 (t, J¼5.4 Hz,
5
92.1484, found: 592.1480.
1
1
H), 3.82 (m, 1H), 3.61e3.35 (m, 9H), 3.30e3.17 (m, 6H), 3.07 (m,
H), 1.80 (m, 1H), 1.40e1.35 (m, 37H); C NMR (75 MHz, DMSO-d )
6
13
0
0
4
.11. 5 -O-(Dibenzylphosphate)-3 -O-(biotinester)-thymidine
d
¼156.3, 156.1, 155.3, 154.9, 101.1, 97.8, 84.0, 80.3, 77.8, 77.7, 77.2,
(
14)
7
3
8
5.0, 72.9, 72.7, 72.1, 70.5, 70.3, 70.1, 67.4, 60.3, 55.9, 50.0, 49.1, 41.4,
þ
19 [MþH]þ calcd:
4.7, 28.3, 28.2, 28.1; HRMS [ESI ] for C38
85.4550, found: 885.4552.
68 4
H N O
Following a similar procedure as the one used for the synthesis
of 9, compound 14 was obtained (370.0 mg, 74%) as a colorless
foam, starting from 5 (345 mg, 0.686 mmol), biotin (201.0 mg,
.823 mmol), DCC (198.0 mg, 0.960 mmol), and DMAP (8.0 mg,
.068 mmol) in a mixture of dry DMF (3 mL) and dry CH Cl
15 mL). H NMR (300 MHz, CDCl
4.14. O-TIBS-N-Boc kanamycin A (18)
0
0
(
2
2
To a stirred solution of 17 (2.48 g, 2.81 mmol) in dry pyridine
1
3
)
d
¼10.95 (br s, 1H, NH Thy), 7.40
(
(
50 mL) was added 2,4,6-triisopropylbenzenesulfonyl chloride
5.96 g,19.67 mmol)and the solutionwas stirred at 25 C for 16 h. The
(
s, 1H, H-6), 7.34e7.33 (m, 10H, AreH OCH
2
Ph), 6.76 (br s, 1H, NH
ꢀ
0
Bio), 6.50 (br s, 1H, NH Bio), 6.26 (app t, J¼6.7 Hz, 1H, H-1 ),
reaction mixture was neutralized by adding 1 N HCl and diluted with
water. The aqueous layer was extracted with ethyl acetate
0
5
4
3
(
(
.13e5.00 (m, 5H, H-3 and OCH
2
Ph), 4.51e4.50 (m, 1H, H-g Bio),
0
00
0
.30e4.22 (m, 1H, H-f Bio, H-5 and H-5 ), 4.11 (br s, 1H, H-4 ),
.17e3.15 (m, 1H, H-e Bio), 2.91e2.88 (m, 1H, H-h Bio), 2.78e2.74
(
3ꢂ400 mL). The collected organic fractions were washed with brine,
dried over Na SO , and concentrated in vacuo. The crude residue was
purified by column chromatography over silica gel (3% MeOH in
DCM) to afford 18 (1.91 g, 59%) as awhite solid. R
¼0.48 (10% MeOH in
¼7.28 (s,1H), 6.58 (br s,1H), 5.35
d, J¼3.8 Hz, 1H), 5.04 (m, 2H), 4.38 (m, 2H), 4.14 (m, 3H), 3.71e3.31
m, 12H), 3.18 (t, J¼9.4 Hz, 1H), 2.95 (m, 1H), 2.02 (m, 1H), 1.45e1.41
0 0
m, 1H, H-h Bio), 2.35e2.25 (m, 3H, H-a Bio and H-2 ), 1.97e1.94
2
4
0
0
3
m, 1H, H-2 ), 1.81 (s, 3H, CH ), 1.69e1.60 (m, 4H, H-b and H-
d Bio), 1.46e1.44 (m, 2H, H-c Bio); C NMR (75 MHz, CDCl
13
f
3
)
1
3
DCM). H NMR (300 MHz, CD OD) d
d
¼172.7 (OCO), 164.3 (C-4), 164.2 (NHCONH), 150.5 (C-2), 135.1 (d,
(
(
(
1
8
5
3
J
C,P¼6.1 Hz, 1C of OCH
2
Ph), 134.7 (C-6), 128.5 (AreC), 128.3
0
3
(
AreC), 127.7 (AreC), 111.2 (C-5), 84.1 (C-1 ), 82.4 (d, JC,P¼8.1 Hz,
1
3
m, 37H), 1.28e1.25 (m, 18H); C NMR (75 MHz, CD
3
OD)
d¼159.4,
0
0
2
C-4 ), 74.1 (C-3 ), 69.4 (app t,
J
2
C,P¼4.7 Hz, 2ꢂOCH Ph), 66.8 (d,
59.2, 157.9, 157.7, 155.3, 152.3, 130.7, 124.9, 102.8, 99.8, 85.8, 80.9,
2
0
J
C,P¼4.7 Hz, C-5 ), 61.7 (C-f Bio), 60.0 (C-g Bio), 55.2 (C-e Bio), 40.2
0.6, 80.4, 80.2, 76.8, 74.5, 73.9, 72.3, 71.9, 71.7, 71.6, 69.2, 57.4, 52.2,
0.9, 41.9, 36.0, 35.5, 30.8, 28.9, 28.8, 28.7, 25.2, 25.1, 23.9; HRMS
0
0
(
C-h, h Bio), 36.8 (C-2 ), 33.4 (C-a Bio), 28.0 (C-c Bio), 27.8 (C-
31
3 3
d Bio), 24.3 (C-b Bio), 12.0 (CH eThy); P NMR (121 MHz, CDCl )
þ
þ
[ESI ] for C53
90 4
H N O
21S [MþH] calcd: 1151.5890, found: 1151.5895.
d
¼ꢁ0.7; HRMS for C34
H
41
N
4
O
10PS [MþH]þ calcd: 729.2353, found:
7
29.2354.
4.15. N-Boc kanamycin A cysteamine (19)
0
4
.12. 3 -O-(Biotinester)-thymidine monophosphate triethy-
To a stirred solution of 18 (0.9 g, 0.782 mmol) in DMF (30 mL)
was added 2-mercaptoethylamine hydrochloride (0.67 g,
lammonium salt (15)
5
.86 mmol) followed by cesium carbonate (2.55 g, 7.82 mmol) and
ꢀ
Following a similar procedure as the one used for the synthesis
of 7, the triethylammonium salt of compound 15 was obtained as
a white solid (335.0 mg, 88%), starting from 14 (370.0 mg,
the solution was stirred at 25 C for 16 h. DMF was partly removed
in vacuo and then partitioned between water (150 mL) and ethyl
acetate (300 mL). The aqueous layer was extracted with ethyl ac-
etate (2ꢂ150 mL). The collected organic fractions were washed
0
.686 mmol), Et
3
N (0.141 mL, 1.015 mmol), and 10% Pd/C (Degussa,
1
185.0 mg, 50% w/w) in MeOH (30 mL). H NMR (600 MHz, DMSO-
2 4
with brine, dried over Na SO , and concentrated in vacuo. The
d
6
)
d
¼11.34 (br s, 1H, NH Thy), 7.91 (s, 1H, H-6), 6.51 (br s, 1H, NH
crude residue was purified by column chromatography over silica
0
Bio), 6.40 (br s, 1H, NH Bio), 6.23 (dd, J¼9.0, 5.8 Hz, 1H, H-1 ),
gel (5% MeOH in DCM) to afford 19 (0.45 g, 61%) as an off-white
0
1
5
1
5
.28e5.27 (m, 1H, H-3 ), 4.32e4.30 (m, 1H, H-g Bio), 4.15e4.12 (m,
solid. R
d
f
¼0.38 (10% MeOH in DCM). H NMR (300 MHz, CD
3
OD)
0
0
H, H-f Bio), 4.07 (br s, 1H, H-4 ), 3.90e3.83 (m, 2H, H-5 and H-
¼5.13 (d, J¼3.5 Hz, 1H), 5.08 (d, J¼3.3 Hz, 1H), 4.17 (t, J¼6.7 Hz, 1H),
0
0
), 3.13e3.10 (m, 1H, H-e Bio), 2.83e2.82 (m, 1H, H-h Bio merged
3.72e3.52 (m, 6H), 3.47e3.38 (m, 7H), 3.21 (m, 3H), 3.05 (m, 1H),
0
with triethylamine), 2.58 (d, 1H, J¼12.4 Hz, H-h Bio), 2.36 (t, 2H,
2.90e2.81 (m, 3H), 2.68 (dd, J¼14.5, 6.8 Hz, 1H), 2.12 (m, 1H),
0
13
J¼7.1 Hz, H-a Bio), 2.33e2.29 (m, 1H, H-2 ), 2.21e2.17 (m, 1H, H-
1.46e1.43 (m, 37H); C NMR (75 MHz, CD
3
OD)
d
¼159.4, 159.2,
0
0
), 1.81 (s, 3H, CH
2
3
), 1.66e1.62 (m, 1H, H-b Bio), 1.61e1.55 (m, 2H,
158.1, 157.7, 102.6, 100.6, 85.2, 83.2, 80.6, 80.4, 80.2, 71.2, 74.5, 74.0,
0
H-d Bio), 1.51e1.44 (m, 1H, H-b Bio), 1.39e1.32 (m, 2H, H-c Bio);
73.8, 72.4, 72.1, 72.0, 71.8, 57.0, 52.0, 50.8, 41.8, 40.0, 35.9, 34.7, 31.4,
13
þ
C NMR (150 MHz, DMSO-d
6
)
d
¼172.6 (OCO), 163.9 (C-4), 162.9
30.4, 28.9, 28.8, 28.8, 28.7, 24.2; HRMS [ESI ] for C40
H N
73 5
O
18
S
0
[MþH]þ calcd: 944.4744, found: 944.4733.
(
NHCONH), 150.7 (C-2), 136.2 (C-6), 110.3 (C-5), 83.7 (C-1 ), 83.5 (d,
J
3
0
0
2
0
C,P¼7.4 Hz, C-4 ), 75.5 (C-3 ), 64.2 (d, JC,P¼6.0 Hz, C-5 ), 61.1 (C-f
0
0
0
Bio), 59.3 (C-g Bio), 55.4 (C-e Bio), 39.6 (C-h,h Bio merged with
DMSO), 36.5 (C-2 ), 33.4 (C-a Bio), 28.0 (C-c and C-d Bio), 24.5 (C-
4.16. 5 -O-(Dibenzylphosphate)-3 -O-(N-Boc kanamycin A
0
cysteamine carbamate)-thymidine (21)
3
1
b Bio), 12.2 (CH
HRMS for
47.1279.
3
eThy); P NMR (202 MHz, DMSO-d
H
6
)
d
¼ꢁ0.1;
C
20
29
N
4
O
10PS [MꢁH]ꢁ calcd: 547.1269, found:
To a stirred solution of compound 19 (353 mg, 0.374 mmol) in
1,4-dioxane/water (3:1, 8 mL) was added 20 (250 mg, 0.374 mmol)
5

S. De et al. / Tetrahedron 70 (2014) 8843e8851
8851
ꢀ
3JC,P¼5.3 Hz), 128.5, 128.3, 127.7, 127.6, 127.1, 110.8, 99.9, 99.7, 86.8,
followed by triethylamine (0.052 mL, 0.374 mmol) at 0 C and then
the solution was stirred at room temperature for 16 h. After com-
pletion of the reaction, the mixture was evaporated to dryness in
vacuo and the residue was purified by column chromatography (4%
86.1, 84.6, 82.2, 74.2, 73.7, 72.4, 72.0, 71.5, 71.2, 70.6, 70.4, 69.6, 67.8,
31
67.0, 59.8, 54.2, 50.0, 48.9, 41.0, 36.2, 33.5, 11.5; P NMR (121 MHz,
þ
20P [MþH]þ calcd:
D
2
O)
d
¼ꢁ0.89; HRMS [ESI ] for C43
61 6
H N O
MeOH in DCM) to obtain compound 21 (495 mg, 90%) as a white
1013.3751, found: 1013.3745.
1
solid. R
f
¼0.45 (10% MeOH in DCM). H NMR (500 MHz, CD
3
OD)
d
¼7.47 (s, 1H), 7.36 (s, 10H), 6.25 (dd, J¼8.6, 5.8 Hz, 1H), 5.14e5.07
0
4
.19. 3 -O-(Kanamycin A carbamate)eTMP (24)
(
m, 6H), 5.05 (br s, 1H), 4.29 (m, 2H), 4.20 (m, 2H), 3.71e3.60 (m,
4
3
5
H), 3.55e3.48 (m, 4H), 3.42e3.32 (m, 6H), 3.19 (t, J¼9.3 Hz, 1H),
Compound 24 was obtained as a white solid (86.7 mg, 88%)
starting from compound 23 (120 mg, 0.118 mmol) following the
.02 (dd, J¼13.9, 2.1 Hz, 1H), 2.75e2.62 (m, 3H), 2.33 (dd, J¼14.2,
13
.5 Hz, 1H), 2.07 (m, 1H), 1.76 (s, 3H), 1.44e1.42 (m, 37H); C NMR
same hydrogenation procedure described for compound 22, using
(
1
125 MHz, CD
3
OD)
d
¼166.18, 159.5, 159.2, 158.0, 157.7, 152.3, 137.2,
31
2
d
0% Pd(OH)
2
/C (24 mg, 0.2 equiv w/w). P NMR (202 MHz, D
2
O)
37.1 (d, 3
J
C,P¼2.3 Hz), 137.0 (d,
3
J
C,P¼2.3 Hz), 130.0, 129.8, 129.3,
ꢁ
20P [MꢁH]ꢁ calcd: 831.2666,
¼ꢁ0.02; HRMS [ESI ] for C29
49 6
H N O
3
1
12.2, 102.7, 100.2, 86.3, 84.4 (d, JC,P¼7.8 Hz), 80.6, 80.5, 80.4, 80.2,
found: 831.2666.
7
7.2, 76.0, 74.7, 74.1, 73.7, 72.5, 72.4, 72.3, 71.8, 71.2 (2ꢂd,
2
2
J
C,P¼5.8 Hz), 68.8 (d, JC,P¼6.1 Hz), 57.2, 50.9, 41.7, 38.2, 35.0, 33.9,
31
Acknowledgements
2
8.9, 28.9, 28.9, 28.8, 28.8, 12.6; P NMR (202 MHz, CD
3
OD)
þ
27PS [MþNa]þ calcd:
d
¼ꢁ0.03; HRMS [ESI ] for
65 98 7
C H N O
We would like to thank Prof. Abram Aertsen and Anirban Ghosh
for kindly providing the bacterial strain, Prof. Jef Rozenski for per-
forming LC/MS analysis and Lia Margamuljana for helping with the
biological test. The research leading to these results has received
funding from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-2013)/ERC
1
494.5861, found: 1494.5873.
0
4
.17. 3 -O-(Kanamycin A cysteamine carbamate)eTMP (22)
To a stirred solution of 21 (200 mg, 0.136 mmol, 1 equiv) in
EtOH/H O (8:2, 15 mL), was added 20% Pd(OH) /C (160 mg,
.8 equiv w/w) and evacuation was then carried out with hydrogen
2
2
ꢀ
Grant agreement n ERC-2012-ADG_20120216/320683.
0
atmosphere replacements (3ꢂ). The reaction mixture was stirred at
room temperature for 24 h under an atmospheric pressure of hy-
drogen. After completion of the reaction, the catalyst was removed
Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2014.09.096.
by filtration through a cellulose filter (0.45
m
m) and the filtrate was
0
concentrated under reduced pressure to obtain crude 3 -O-(N-Boc
kanamycin A cysteamine carbamate)eTMP (w175 mg, quant.). The
so obtained residue was treated without further purification with
References and notes
ꢀ
9
0% aq TFA (5 mL) at 0 C and then the solution was stirred at room
1. Benner, S. A.; Sismour, A. M. Nat. Rev. Genet. 2005, 6, 533e543.
temperature for 5 h. After completion of the reaction, the mixture
was evaporated to dryness in vacuo. The residue was coevaporated
2. Wu, Y.; Fa, M.; Tae, E. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2002,
124, 14626e14630.
three times with toluene to remove residual TFA and to give crude
3. Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Correa, I.
R.; Romesberg, F. E. Nature 2014, 509, 385e388.
0
3
-O-(kanamycin A cysteamine carbamate)eTMP. The crude residue
Oþ2% ACN
O). The collected eluates were
freeze-dried repeatedly until constant mass to afford compound 21
4
5
. De, S.; Groaz, E.; Herdewijn, P. Eur. J. Org. Chem. 2014, 2014, 2322e2348.
. Pezo, V.; Liu, F. W.; Abramov, M.; Froeyen, M.; Herdewijn, P.; Marliere, P. Angew.
Chem., Int. Ed. 2013, 52, 8139e8143.
was purified by preparative RP-HPLC (0.1% TFA in 98% H
and 0.1% TFA in 98% ACNþ2% H
2
ꢁ
2
6. Prakash, O.; Eisenberg, M. A. J. Bacteriol. 1974, 120, 785e791.
7
. Piffeteau, A.; Zamboni, M.; Gaudry, M. Biochim. Biophys. Acta 1982, 688, 29e36.
. Davis, B. D. Microbiol. Rev. 1987, 51, 341e350.
9. Magnet, S.; Blanchard, J. S. Chem. Rev. 2005, 105, 477e498.
0. Taber, H. W.; Mueller, J. P.; Miller, P. F.; Arrow, A. S. Microbiol. Rev. 1987, 51,
39e457.
1. Becker, B.; Cooper, M. A. ACS Chem. Biol. 2013, 8, 105e115.
3
1
(
(
86 mg, 71% over two steps) as trifluoroacetic salt. P NMR
8
ꢁ
19PS [MꢁH]ꢁ
202 MHz, D
2
O)
d¼ꢁ0.02; HRMS [ESI ] for C31
54 7
H N O
1
calcd: 890.2860, found: 890.2852.
4
1
0
0
4
.18. 5 -O-(Dibenzylphosphate)-3 -O-(kanamycin A
12. Semenyuk, A.; F o€ ldesi, A.; Johansson, T.; Estmer-Nilsson, C.; Blomgren, P.;
Br €a nnvall, M.; Kirsebom, L. A.; Kwiatkowski, M. J. Am. Chem. Soc. 2006, 128,
carbamate)-thymidine (23)
12356e12357.
1
3. Sekine, M.; Iimura, S.; Nakanishi, T. Tetrahedron Lett. 1991, 32, 395e398.
Compound 23 was obtained according to the procedure for the
preparation of compound 21 starting from kanamycin A 16
14. Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett. 2000, 2, 243e246.
15. Luedtke, N. W.; Carmichael, P.; Tor, Y. J. Am. Chem. Soc. 2003, 125, 12374e12375.
1
1
1
6. Charles, I.; Xue, L.; Arya, D. P. Bioorg. Med. Chem. Lett. 2002, 12, 1259e1262.
7. Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361e1371.
8. Hirao, I.; Itoh, K.; Nishino, S.; Araki, Y.; Ishido, Y. Carbohydr. Res. 1986, 152,
(
100 mg, 0.206 mmol), compound 20 (138 mg, 0.206 mmol), and
triethylamine (29 L, 0.206 mmol). The crude residue was purified
by column chromatography (IPA/H O/Et N 10:2:1) to give 23 as
a white solid (171 mg, 82%). R
¼0.45 (IPA/H
300 MHz, D O)
¼7.16 (s,1H), 7.09 (s,10H), 6.1 (app t, J¼6.0 Hz,1H),
.21 (d, J¼5.5 Hz, 1H), 5.01 (m, 2H), 4.84 (m, 4H), 4.19e3.89 (m, 4H),
.81e3.08 (m, 14H), 2.99 (t, J¼10.4 Hz, 1H), 2.82 (t, J¼11.3 Hz, 1H),
m
159e172.
2
3
1
19. Suzuki, M.; Sekido, T.; Matsuoka, S.-i.; Takagi, K. Biomacromolecules 2011, 12,
f
2 3
O/Et N 6:2:2). H NMR
1
449e1459.
0. Liu, Q.; Luedtke, N. W.; Tor, Y. Tetrahedron Lett. 2001, 42, 1445e1447.
21. Schepdael, V. A.; Busson, R.; Claes, P. Bull. Soc. Chim. Belg. 1992, 101, 709e718.
(
2
d
2
5
3
2
2
2
2. Sainlos, M.; Belmont, P.; Vigneron, J.-P.; Lehn, P.; Lehn, J.-M. Eur. J. Org. Chem.
2003, 2003, 2764e2774.
13
.31 (m, 1H), 2.04e1.80 (m, 2H), 1.52 (s, 3H), 1.33e1.19 (m, 1H);
C
3. Blount, K. F.; Zhao, F.; Hermann, T.; Tor, Y. J. Am. Chem. Soc. 2005, 127,
NMR (75 MHz, D
2
O)
d¼165.3, 156.8, 151.0, 135.7, 134.8 (d,
9818e9829.