Β-1,2,3-triazolyl-nucleosides as nicotinamide riboside mimics

Article abstract of DOI:10.1080/15257770902865415

The synthesis of a series of pyridine- and piperidine-substituted 1,2,3-triazolides linked to a riboside moiety is described. The presence of a triazolide substituent on the pyridine moiety permitted the facile reduction of the latter under mild hydrogena

Full text of DOI:10.1080/15257770902865415

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β-1,2,3-Triazolyl-Nucleosides as
Nicotinamide Riboside Mimics
E. J. Amigues a , E. Armstrong a , M. Dvorakova a , M. E. Migaud a
M. Huang a
&
a School of Chemistry and Chemical Engineering , Queen's University
Belfast , Belfast, Northern Ireland
Published online: 30 Mar 2009.
To cite this article: E. J. Amigues , E. Armstrong , M. Dvorakova , M. E. Migaud & M. Huang (2009)
β-1,2,3-Triazolyl-Nucleosides as Nicotinamide Riboside Mimics, Nucleosides, Nucleotides and Nucleic
Acids, 28:3, 238-259, DOI: 10.1080/15257770902865415
To link to this article: http://dx.doi.org/10.1080/15257770902865415
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Nucleosides, Nucleotides and Nucleic Acids, 28:238–259, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770902865415
β-1,2,3-TRIAZOLYL-NUCLEOSIDES AS NICOTINAMIDE RIBOSIDE
MIMICS
E. J. Amigues, E. Armstrong, M. Dvorakova, M. E. Migaud, and M. Huang
School of Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast, Northern
Ireland
The synthesis of a series of pyridine- and piperidine-substituted 1,2,3-triazolides linked to a
2
riboside moiety is described. The presence of a triazolide substituent on the pyridine moiety permitted
the facile reduction of the latter under mild hydrogenation conditions. These analogues were
modelled as to define their similarity to nicotinamide riboside and quantify their ability to bind
NAD-dependent protein deacetylases.
Keywords Nucleoside; nucleotide; sirtuin; inhibition
One family of protein deacetylases catalyzes the removal of the acetate
moiety from acetylated lysine residues on proteins, such as histones, us-
ing nicotinamide adenine dinucleotide (NAD) as a co-substrate.[1] Upon
catalysis, in addition to the deacetylated protein being released, NAD
is converted into 2ꢁꢁ/3ꢁꢁ-O-acetylated-adenosine diphosphate ribose and
Received 3 February 2009; accepted 4 March 2009.
M. E. Migaud and M. Huang share joint authorship this article.
The authors thank Professor R. Marmorstein and Ms. B. Sanders (from the Wistar Institute,
Philadelphia, USA) for carrying out the preliminary inhibition studies and extensive discussions, and
the EPSRC/MRC GR0100461 and the EC-FP6-EST program, which financially supported this work.
Address correspondence to M. E. Migaud and M. Huang, School of Chemistry and Chemical
Engineering, David Keir Building, Queen’s University Belfast, Stranmillis Road, Belfast, BT9 5AG,
Northern Ireland. E-mail: m.migaud@qub.ac.uk and m.huang@qub.ac.uk
238

β-1,2,3-Triazolyl-Nucleosides
239
SCHEME 1 Currently proposed mechanisms of deacetylation catalyzed by NAD-dependent protein
deacetylases.
nicotinamide. In cells, these deacetylases are regulated by endogenous
nicotinamide which acts as a non-competitive enzyme inhibitor.[2,3] Con-
sidering that regulation of such NAD-dependent deacetylases is implicated
in numerous biological processes including life-span expansion and p53
expression in eukaryotes,[4] identifying structural and chemical features
that molecules should possess in order to attain potent inhibition is the first
step to developing a robust program toward drug discovery targeting such
enzymes.[5,6]
Two mechanisms can be proposed to explain the early steps of the NAD-
dependent enzyme catalyzed deacetylation (Scheme 1). The first proposed
pathway results in the formation of a stabilized oxycarbocation reaction
intermediate, hence is defined as the SN1 mechanism. Such intermediate
has been observed in enzymatic reactions involving a nicotinamide-riboside
bond cleavage.[7,8] The second predicted pathway involves a transition
state intermediate resulting from the nucleophilic attack of the carbonyl
group from the acetyl-lysine moiety and the simultaneous breakage of the
nicotinamide bound via an SN2 mechanism.[9] As a starting point in our
search for chemical scaffolds that could lead to NAD-dependent deacetylase
inhibition, we decided to investigate the possibilities of mimicking the
increased distance between the nicotinamide and the C1ꢁꢁ-carbon of the
nicotinamide riboside of NAD occurring for either of these mechanisms.
While many nucleoside analogues, mimicking both the adenosine and
the nicotinamide moieties, have been synthesized thus far, compounds
simulating the elongation of the nucleosidic bond between the C1ꢁꢁ of
the northern riboside and the nitrogen of the pyridine ring, have been
under less scrutiny.[10, 11] Developing an effective synthetic programme of
work that would allow the identification of inhibitors of NAD-dependent

240
E. J. Amigues et al.
TABLE 1 Optimization of the 1,3-dipolar cycloaddition reaction conditions
Product
n-ethynylpyridine
Reaction conditions
Yield
6a
5a: n = 2
A
B
C
A
B
C
A
B
C
A
B
A
B
A
B
41%
–
–
6b
6c
5b: n = 3
5c: n = 4
85%
47%
<5%
15%
0%
0%
-
31%
79%
50%
–
8a
8b
8c
5a: n = 2
5b: n = 3
5c: n = 4
12%
A: CuSO4 (0.2 eq.), sodium ascorbate (0.4 eq.), tBuOH/H2O, 40◦C.
B: CuSO4 (0.1 eq.), sodium ascorbate (1.0 eq.), H2O, 100◦C.
C: CuI (1.5 eq.), DBU (15 eq.), toluene, 90◦C.
deacetylases requires a reliable computational process which allows for
the early elimination of unsuitable synthetic targets. To this end, a small
series of modified nucleosides and nucleotides have been synthesized and
evaluated as putative inhibitors of yHst2, a yeast NAD-dependent histone
deacetylase, while a wider range of analogues were screened in silico against
various yHSt2-crystal structures for comparison. Nucleosidic analogues 1a-c
(Scheme 2) possess a pyridine bounded to the riboside ring via a 1,2,3-
triazolide moiety, thus providing additional distance between the riboside
C1ꢁꢁ carbon and pyridine ring. Triazolide linkers were chosen for their
ease of accessibility as well as their ability to enter conjugation with other
aromatic moieties such as pyridine. We thought that as pyridine possessed
an electron-withdrawing ability, the actual electron density over the tria-
zolide ring would potentially match that of the electron density observed
during the breaking of the ribose-nicotinamide glycosidic bond. Further
modifications of these triazolide-containing nucleoside mimics offered the
piperidine derivatives 2a-c (Scheme 2). Finally, a versatile synthetic sequence
allowed access to the nucleotide parents.
We aimed at gaining easy access to fully and partially deprotected
nucleoside analogues to facilitate further chemical modifications such as
selective 5ꢁ-O-phosphorylation. We thus prepared the triazolide adducts via
two distinct synthetic sequences. The fully deprotected nucleoside pyridine
mimic series 1a-c was easily obtained in three steps from the commercially
available β-1-O-acetyl-2,3,5-tri-O-benzoate riboside 3[12] by simple Lewis
acid-catalyzed substitution of acetyl group with azide which afforded β-
1-azido-2,3,5-tri-O-benzoate riboside 4. Direct triazolide formation from 4

β-1,2,3-Triazolyl-Nucleosides
241
SCHEME 2 Synthesis of pyridine and piperidine containing 1,2,3-triazolide adducts.
and the appropriately subtituted alkyne-pyridines 5a-c (Scheme 2) yielded
the fully benzoylated nucleoside analogues 6a-c which, once treated with
methanolic sodium methoxide, yielded the triazolide nucleoside mimics
1a-c. The acetonide-protected pyridine 1,2,3-triazolides 8a-c were obtained
from the corresponding azido-riboside 7 and the appropriate alkyne-
pyridines 5a-c (Scheme 2). Only 1,4-disubstituted triazolides were products
of these reactions. Optimization of the reaction conditions (Table 1),[13,14]
such as variation of the type of catalyst, the catalyst ratio, the pH and the
solvent mixture, permitted access to the pyridine triazolides in yields rang-
ing from 10% to 85% (Table 1). Low yields were particularly consistent with
4-ethynylpyridine, 5c. This low conversion could be related to the nature
of the reagent itself; 4-ethynylpyridine was purchased as a chloride salt,
unstable once neutralized. The reaction conditions (100◦C for extended
period of time) were possibly too rigorous for this particular pyridine-
based reagent. Additionally, the triazolide products 6a-c and 8a-c displayed
extremely poor solubility in both organic and hydrophilic solvents including
water and methanol, which rendered their extraction and purification

242
E. J. Amigues et al.
SCHEME 3 Possible chirality and conformation adopted by 2b.
difficult. Finally, DBU-catalyzed reactions were unsuitable for the formation
of the ester protected triazolides as rapid partial debenzoylation occurred
upon work-up. Phosphorylation and access to the protected nucleotides 9a-b
was easily achieved using a chlorodiethylphosphite/tBuOOH combination.
The palladium-catalyzed reduction of 8a-c to yield 10a-c was carried out
in the presence of formic acid under hydrogen atmosphere at balloon
pressure. Unlike the reduction of pyridines, where high hydrogen pressure
is required, triazolide-substituted pyridines were very rapidly reduced under
these mild conditions. It must also be noted that only one diastereoisomeric
piperidine derivative was obtained in each case and that the reduction
was quantitative. Attempts were made at identifying the stereochemistry
at the newly generated chiral center and the overall conformation of
the piperidine ring (Scheme 3). Unfortunately, NOE experiments were
inconclusive and none of the derivatives were crystallizable. Subsequent
removal of the isopropylidene group offered the fully deprotected pyridine
1a-c and piperidine 2a-c substituted triazolides. A similar sequence that also
included a TMSBr-catalyzed deprotection of the 9a-b phosphate triesters
yielded the piperidine nucleotide derivative 11a-b. It is noticeable that the
partially protected piperidines, including the benzoylated triazolide 12b, are
ideal synthetic intermediates for further synthetic manipulations and access
to libraries of modified nucleosides.
a) TMSN3, AlCl3, CH3CN, 95%, b) MeONa, MeOH, 90%, c) acetone,
H2SO4, 91% d) n-ethynylpyridine 5a-c, cycloaddition conditions A-C; e)
Pd/C, MeOH, HCOOH (10% v/v), H2, quant.; f) TFA/H2O, 1/1, v/v,
95–99%; g) MeOH, MeONa, quant.; h) (EtO)2PCl, Et3N, tBuOOH, 90%;
i) TMSBr, DCM, rt, quant.
We then examined these novel triazolides in silico. First, we wished to
establish which of the triazolide series (1a-c versus 2a-c) best mimicked
electronically the early stage of the catalysis, that is, the nicotinamide-
riboside bond breakage. Second, in order to establish a binding affin-
ity pattern, we wished to model each template in the binding site of
yHst2 co-crystallized with carba-NAD (substrate analogue) (PDB: 2OD2),[2]
with an SN1-based cationic reaction intermediate mimic (adenosine 5ꢁ-
diphosphate (hydroxymethyl) pyrrolidinediol, ADP-HPD) (PDB: 2OD7),[2]

β-1,2,3-Triazolyl-Nucleosides
243
with nicotinamide (in the absence (PDB: 1YC5)[15] or the presence of
ADP-HPD (PDB: 2OD9)[2]) or with adenosine diphosphate ribose in the
absence of nicotinamide (PDB: 1SZD).[16]
To study the electronic character and the electronic similarity of
the triazolyl-nucleoside analogues 1a-c and 2a-c to nicotinamide riboside,
geometrical optimizations were performed at B3LYP/6–31G∗ level with
Gaussian03 program (Gaussian Inc., USA; see Chart 1). Comparing the
LUMO orbitals (Figure 1A) of nicotinamide riboside to that of 1a-c and 2a-c,
we observed that the LUMOs of nicotinamide riboside and of the piperidine
derivatives 2a-c, were overwhelmingly localized on the C1ꢁꢁ carbon of the
ribose ꢁrꢁ ing and the nicotinamide or triazolide ring. This indicated that
the C1 carbons of ribosides 2a-c were of similar electrophilicity as that of
the C1ꢁꢁ carbon of nicotinamide riboside. In contrast, the LUMO orbital
distributions of pyridine derivatives 1a-c on the C1ꢁꢁ carbon was found to be
zero. From the analysis of the HOMO orbitals of the piperidine triazolide
derivatives (Figure 1; 2a-c), we observed that the triazolide ring made little
contribution towards the HOMO orbitals, while the HOMO-1 orbitals were
mostly located on the triazolide ring. On comparing the orbital energies of
the triazolide compounds, we found that the energy gap between HOMO
and HOMO-1 orbitals was not noticeable, which made possible electron
transfer from the second highest occupied orbital. In contrast, for the
pyridine derivatives (1a-c), electron transfer occurred from the HOMO
orbitals (Figure 1). Overall, this indicated that nicotinamide riboside shared
more electronic similarities with the piperidine triazole-ribosides than with
the pyridine triazole-ribosides.
A docking study using the Glide program (Schrodinger Inc.) was then
performed to establish whether the 2a-c series could favorably fit into the
active site(s) of yHst2 enzymes. Energy minimization for 2a-c at B3LYP/6-
31G∗ using Gaussian shows that 2c is the most energy-favorable species
among all the piperidine derivatives and the energy difference between two
diastereoisomers for 2a and 2b can be ignored, which is further confirmed
by single point energy calculation at larger basis set B3LYP/6-311+G∗∗ and
solvation effect implied by CPCM solvation model (Supplementary Table).
Nicotinamide and nicotinamide riboside were first docked into the
carba-NAD-binding site of the resolved yHst structures (PDB: 2OD2).[2]
Results show that both nicotinamide and its riboside derivative bind in the
C-pocket with the same orientation as the nicotinamide carbasugar part of
the co-crystallized carba-NAD (Figure 2A and 2B). The triazolyl-nucleosides
1a-c and 2a-c and the phosphorylated species 11a-c were then docked into
the yHst2-carba-NAD-binding pocket. Analogous to nicotinamide riboside,
the piperidine derivatives were also found to bind preferentially in the
conserved C-pocket. In the crystal structure of yHst2 complexed with
carba-NAD or free nicotinamide, the amide group and carboxyl group
of the carboxamide of nicotinamide form favorable H-bond interactions

244
E. J. Amigues et al.
FIGURE 1 Frontier orbitals for triazolide compounds 1a-c and 2a-c and nicotinamide riboside.
with the carboxyl oxygen of Asp118 and the backbone nitrogen of Ile117,
respectively. Docking the series 2a-c showed that either the heterocyclic
nitrogen or the carbon atoms of the piperidine ring could approach the
carboxyl oxygen of Asp118 and the backbone nitrogen of Ile117 (Figure 3)
of the C-pocket.

β-1,2,3-Triazolyl-Nucleosides
245
FIGURE 2 Docked poses of phosphorylated nicotinamide riboside in the pocket C of yHst2 (A)
Conserved pockets C and D are shown by surface representation of yHst2. Docked pose phosphorylated
nicotinamide riboside (yellow) overlap neatly with the northern ribose of NAD+ (orange). Nicotinamide
(blue) is also docked into the C pocket. (B) Interaction of phosphorylated nicotinamide riboside
(colored by element) with conserved residues in pocket C. NAD is shown in green; Phe44 (cyan) forms
hydrophobic contact with the nicotinamide pyridine ring.

246
E. J. Amigues et al.
FIGURE 3 Docked poses of 11a-c in yHst2 pocket C. Interaction of 11a-c (colored by element) with
conserved residues in pocket C. NAD+ is shown in green, and acetyl-lysine is in magenta. Phe44 (cyan)
forms hydrophobic contact with the triazolide ring.
A good overlap was also observed between the triazolide ring binding
to the C-pocket and that of the pyridine ring of carba-NAD, indicating
that the favorable hydrophobic interaction with Phe44. Figure 4 shows the
interactions of 11a-c with the conserved C-pocket of the NAD-binding site.
Compound 11b presented the highest docking binding affinity with yHst2,
forming H-bond interactions between the piperidine-NH and the carboxyl
oxygen of Asp118. The piperidine 11c offered similar binding affinity as
11b, but weaker H-bond interactions between the piperidine NH and the
backbone amido-nitrogen of Ile117 was observed. Finally, the piperidine
nitrogen in compound 11a was too far to form favorable electrostatic
interactions with the surrounding amino acids (Table 2). As expected,
2a-c ranked lower than its phosphorylated parent series 11a-c (Table 2)
emphasising the importance of the phosphate moiety and its interactions
within the Rossmann fold.
Modeling with structures 1SZD and 2OD7, showed a preference for a
nicotinamide docked into the D-pocket while for all docking experiments,
the nucleosides 2a-c and nucleotides 11a-c showed high scoring in the
C-pocket, except when nicotinamide was present in the D-pocket (PDB:
2OD9). Under these conditions, none of the piperidine derivatives docked

β-1,2,3-Triazolyl-Nucleosides
247
TABLE 2 Docking scores of 2a-c and 11a-c
Target
1SZC
2OD2
2OD9
1SZD
2OD7
2OD9a
Nicotinamide riboside
mononucleotide
11b
11c
−7.44 Cb
−6.68 C
−6.87 C
−6.10 C
−6.96 C
−6.23 C
−5.94 Cc
−5.92 C
−5.11 C
−8.63
−6.82 C −6.83 C −6.94 C −6.44 C
−6.91 C −7.29 C −6.82 C −6.80 C
−6.67 C −7.05 C −6.64 C −6.65 C
−6.63 C −6.37 C −6.19 C −6.76 C
−6.56 C −6.68 C −7.01 C −7.15 C
−5.94 C −5.98 C −5.90 C −6.15 C
−5.44 C −6.02 C −5.56 C −5.66 C
−5.56 C −5.73 C −5.98 C −5.20 C
−5.08 C −5.06 C −4.95 D −4.98 D
−6.67 A
−5.62 A
−6.09 A
−6.40 A
−5.46 C
−5.69 A
−5.68 A
−5.56 A
−5.93 C
−8.77
11a
Nicotinamide riboside
2b
2c
2a
Nicotinamide
ADP-ribose
−9.94
−8.43
−8.30
−9.83
aWith a nicotinamide molecule in the D site
bCapital A, C or D in bold represents that the named compound is docked in the adenosine
monophosphate binding fold or the C or D pockets, respectively.
cCell in red indicates that the docked pose flapped in the pocket.
in the C-pocket but instead docked in the adenosine monophosphate
binding site. These docking experiments also suggested that 11a-c showed
great binding similarities to that of the substrate mimic carba-NAD, thus
indicating that unfortunately they were more likely to be substrate analogues
than transition state mimetics.
Finally, ADP-ribose, which has been shown to be a 100-fold more potent
an inhibitor than nicotinamide,[16] was also docked in the various enzyme
complexes. As expected, it unanimously showed best docked scores com-
pared to the rest of the compounds examined. Therefore, it can be used as
threshold to identify potential nucleotide-based inhibitors using respective
FIGURE 4 Interaction of 11a-c with conserved amino acids in pocket C of yHst2.

248
E. J. Amigues et al.
FIGURE 5 Docked poses of 2a-c (cyan) and 11a-c (green) in pocket C. NAD is in pink. Docked pose of
the phosphorylated nicotinamide riboside is shown in yellow.
protein complex structure (2OD2 for screening of substrate-based templates
and 2OD7 for screening of intermediate-based templates).
In order to relate these empirical results to actual inhibition of yHst2,
compounds 1a, 2a, 11a, and 11b were evaluated for activity. It was felt that
preliminary evaluation of these few compounds would offer sufficient infor-
mation with regard to the validity of the overall approach to such inhibitor
design. A standard inhibition assay (1 µM yHst2 (1-294), 160 µM NAD+, 100
uM acetyl-lysine [saturating concentrations of substrates], reaction time 15
min) was thus used.[2,16] Only the phosphorylated derivative 11a displayed
some level of inhibitory activity with an IC50∼0.5 mM, and was at least
five-fold less potent than nicotinamide.[2] It was surprising to find out that
11b inhibited yHst2 at even higher concentrations (mM). This observation
did not match the calculations. However, it is worth noticing that the
inhibitory activity of 11b was potentiated by the presence of nicotinamide
but yet remained poor, IC50∼5 mM. This modulation was not observed for
11a, thus indicating that these two very similar compounds do not bind
yHst2 in a similar manner. As proposed earlier, it is possible that 11b binds
the adenosine monophosphate binding pocket once nicotinamide is in use.
Alternatively, it is possible that 11b adopted a non-productive conformation
in solution where the amino moiety and the phosphate moiety form a

β-1,2,3-Triazolyl-Nucleosides
249
tight ion pair thus restricting free rotation around the glycosidic linkage.
Considering that most of the compounds synthesized scored higher than
nicotinamide in the docking experiments and yet they were found to
be either non-inhibitors or poor-ones, it will be difficult to reliably use
this method of screening substrate analogues and predict potent yHst2
inhibitors.
In conclusion, we have developed a versatile synthetic sequence which
involved the facile access to triazolide substituted piperidine that gives
access to partially protected pyridine- and piperidine-containing nucleoside
analogues. These routes are easily adapted to the synthesis of the nu-
cleotide parents. Surprisingly, the piperidine series were found to share high
similarities with nicotinamide riboside through frontier molecular orbital
analysis. Docking experiments predicted that the piperidine derivatives
would bind preferentially the C-pocket of yHst2, yet unfortunately, none of
the compounds synthesized offered any relevant level of enzyme inhibition.
Overall, the combined studies show that in silico prediction of the inhibition
of the sirtuin enzymes by highly flexible substrate-like molecules like those
here reported can be deceptive, and that virtual screening proved to be
unhelpful with regards to substrate-based sirtuin inhibitor design.
COMPUTATIONAL EXPERIMENTAL
All of the computational study was carried out on Intel Xeon 5130 × 2
workstations.
Theoretical Calculation
The B3LYP functional and the 6-31G∗ basis set were used in Gaussian
03(Gaussian. Inc) for geometry optimizations of the structures listed in
Chart 1. The frontier molecular orbitals were plotted by GaussView3.0.
CHART 1
Energy calculation for 2a-2c for diastereoisomers of piperidine derivatives
B3LYP/6–31G∗ //
B3LPY/6–311+G∗∗
−989.431678 (4.90)
−989.4296841(6.15)
−989.4277702 (7.35)
−989.4286943 (6.78)
−989.4283452 (6.99)
−989.4394915 (0)
CPCM//
Chiral center 1
B3LPY/6–31G∗
B3LPY/6–31G∗
(S)-2a-piperidine
(R)-2a-piperidine
(S)-2b-piperidine
(R)-2b-piperidine
boat-2c-piperidine
chair-2c-piperidine
−989.1267473(4.64)2
−989.1244254(6.09)
−989.122422 (7.35)
−989.1231952 (6.86)
−989.1227848 (7.12)
−989.134134 (0)
−989.1582874 (5.80)
−989.1556881 (7.43)
−989.1550031 (7.86)
−989.1561039& (7.17)
−989.1558797 (7.31)
−989.1675289 (0)
Note: The chirality at the point of attachment to the triazole (with chirality at the sugar remaining
the same). The values in bracket are relative energies (in kcal/mol) relative to 2c in chair
conformation.

250
E. J. Amigues et al.
Docking
Docking studies were performed using the program Glide (version 4.5)
(Schrodinger, Inc. http://www.schrodinger.com/) through Mastro graphic
interface for triazolyl-nucleosides and their phosphorylated derivatives.
Conformational flexibility was considered by extensive conformational a
search implemented in Glide. Best docked poses were minimized with the
OPLS forcefield and scored using the GlideScore scoring function.
Protein Structures Used in Docking Studies
The available crystal structure of SIR2 complex can be classified as those
in complex with a free nicotinamide inhibitor (1YC5), a substrate (1SZC:
Sir2/Carba-Nicotinamide-Adenine-Dinucleotide, 2OD2), an intermediate
(2OD9: yHst2/ADP-HPD + Nicotinamide), or a product (1SZD: APR,
2OD7).
Experimental
1
General: The H, 13C and 31P NMR spectra were recorded in CDCl3,
D2O, CD3OD or (CD3)2SO on a Bruker AC 300 MHz spectrometer or
1
a Bruker AMX 500 spectrometer. HMQC and H-1H COSY experiments
1
were performed on a Bruker AMX 500 spectrometer. TMS (0 ppm, H
NMR), CDCl3 (77 ppm, 13C NMR) and triethyl phosphate (0.2 ppm, 31P
NMR) were used as internal references. The chemical shifts (δ) are reported
in p.p.m. (parts per million) and the coupling constants (J values) are
recorded in Hz. The hydrogen and carbon assignments were made when
1
2D experiments (1H-1H COSY and H-13C HMQC experiments) allowed
it. Mass spectra were obtained on a LCT premier Micromass Technologies
Waters (ASEP Belfast).
Benzoic acid (2R,3R,4R,5R)-3,4-dibenzoyl-5-azido-tetrahydro-furan-2-
ylmethyl ester 412 (azido-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl). Aluminium
trichloride (336 mg, 2.5 mmol) and azidotrimethylsilane (2.65 cm3,
20 mmol) were added to a solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-
ribofuranoside 3 (1.0 g, 2.0 mmol) in dry CH3CN (12 cm3) under magnetic
stirring. The resulting reaction mixture was stirred for 2 hours. The crude
reaction mixture was then concentrated under reduced pressure. The
residue was then partitioned between water (50 cm3) and CHCl3 (50 cm3).
The aqueous layer was extracted with CHCl3 (50 cm3 × 3). The combined
organic layers were then dried (MgSO4), filtered and concentrated under
reduced pressure to afford the crude azido derivative as colorless oil (0.97g,
99%).
δH (500 MHz, CDCl3) δ 8.20–7.89 (6H, m, Ar), 7.62–7.32 (9H, m, Ar),
5.85 (1H, dd, J 4.9, 6.5, H-3), 5.68 (1H, d, J 1–2 1.6, H-1), 5.58 (1H, dd, J 1–2
ꢁ
1.6, J 2–3 4.8, H-2), 4.81- 4.75 (2H, m, H-4; H-5), 4.56 (1H, dd, J 4–5 5.4, J 5–5

β-1,2,3-Triazolyl-Nucleosides
251
13.2, H-5ꢁ); δC (125 MHz, CDCl3) δ 166.2 (C = O), 165.2 (C = O), 165.1 (C
= O), 133.7 (Ar), 133.6 (Ar), 133.2 (Ar), 129.8 (Ar), 129.7 (Ar), 129.5 (Ar),
128.8 (Ar), 128.7 (Ar), 128.5 (Ar), 128.4 (2, Ar), 93.3 (C-1), 79.9, 75.3, 71.5,
63.8 (C-5); HRMS (ES) Calcd for C26H21N3O7Na (M+Na+) 510.1277 found
510.1257.
(2R,3R,4S,5R)-2-Azido-5-hydroxymethyl-tetrahydro-furan-3,4-diol
13.[17] To a suspension of the azido precursor (benzoic acid (2R,3R,
4R,5R)-3,4-dibenzoyl-5-azido-tetrahydro-furan-2-ylmethyl
ester
4,
1.99 mmol, 0.97g) in MeOH (20 cm3), was added sodium methoxide
(0.2 mmol) and the resulting reaction mixture was stirred overnight.
The crude reaction mixture was neutralized with Dowex (H+), filtered
and concentrated to dryness. The residue was then partitioned between
H2O and CHCl3. The aqueous layer was washed with CHCl3 and then
freezed-dried to afford the crude product as an oil (0.3 g, 86%).
δH (500 MHz, D2O) 5.21 (1H, d, J 1–2 1.2, H-1), 4.09 (1H, dd, J 2–3 4.7,
J
3–4 6.9, H-3), 3.94 (1H, appdt, J 4–5 3.0, J 3–4 6.3, H-4), 3.88 (1H, dd, J 1–2 1.2,
ꢁ
ꢁ
J 2–3 4.5, H-2), 3.74 (1H, dd, J 4–5 3.0, J 5–5 12.5, H-5), 3.56 (1H, dd, J 4–5 5.8,
J 5–5 12.5, H-5ꢁ); δC (125 MHz, D2O) 94.9 (C-1), 83.8 (C-4), 74.9 (C-2), 70.5
ꢁ
(C-3), 62.2 (C-5).
((3aR,4R,6R)-6-Azido-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-
yl) methanol 7. To an acetone solution (100 cm3) of the fully deprotected
azido-sugar 13 was added H2SO4(conc.) (5 drops) and the resulting reaction
mixture was stirred overnight. The reaction was then diluted with CHCl3
(100 cm3) and neutralized with NaHCO3, sat. (50 cm3). The aqueous
layer was extracted with CHCl3 (3 × 50 cm3 portions). The combined
organic extracts were dried (MgSO4), filtered concentrated under reduced
pressure to afford the crude product as yellow oil. Purification by flash
chromatography (75/25→50/50, PE/EtOAc, v/v) afforded the pure
product as a clear oil 1.44 g (91%).
δH (500 MHz, CDCl3) δ 5.52 (1H, s, H-1), 4.74 (1, dd, J 1–2 1.0, J 2–3 6.0,
H-2), 4.50 (1H, d, J 2–3 5.9, H-3), 4.23 (1H, dt, J 3–4 0.9, J 4–5 4.8, H-4), 3.73
(1H, dd, J 4 -5 4.3, J 5–5 12.2, H-5), 3.66 (1H, dd, J 4–5 5.2, J 5–5 12.2, H-5ꢁ),
2.48 (1H, br s, OH), 1.47 (3H, s), 1.29 (3H, s); δC (125 MHz, CDCl3) δ
112.9 (C(CH3)2), 97.8 (C-1), 88.4 (C-4), 85.8 (C-3), 81.6 (C-2), 63.4 (C-5),
26.4 (CH3), 24.8 (CH3); HRMS (ES) Calcd for C8H12N3O4 (M-H-) 214.0828
found 214.0835.
ꢁ
ꢁ
ꢁ
Typical Procedure for Triazole Formation (8a-c)
To a stirred solution of isopropylidene protected sugar azide 7 (0.5M;
typically; 105 mg) and n-ethynyl-pyridine (0.5M) in H2O/tBuOH (1/1, v/v)
was added a solution of CuSO4×5H2O (0.2 eq.) and sodium ascorbate
(0.4 eq.) in water. The resulting reaction mixture was stirred overnight at
40◦C. The crude reaction mixture was extracted with three equal amounts
of EtOAc, combined, dried (MgSO4), filtered and evaporated to afford

252
E. J. Amigues et al.
the crude triazolide derivative. The residue was then purified by flash
chromatography (100/0 → 95/5, CHCl3/EtOH, v/v), to afford the pure
product.
[(3aR,4R,6R,6aR)-2,2-Dimethyl-6-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-
tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-methanol 8a. (0.08g; 41%) δH (500
MHz, CDCl3) δ 8.43 (1H, ddd, J 0.9, 1.7, 4.9), 8.40 (1H, s, CH = C), 8.00
(1H, dt, J 1.0, 8.0), 7.69 (1H, dd, J 1.8, 7.7), 7.17 (1H, ddd, J 1.2, 4.9, 7.5),
6.19 (1H, d, J 1–2 1.9, H-1), 5.33 (1H, dd, J 1–2 1.9, J 2–3 5.9, H-2), 4.99 (1H,
ꢁ
dd, J 3–4 1.7, J 2–3 5.9, H-3), 4.53 (1H, ddd, J 3–4 1.9, J 4–5 3.3, J 4–5 5.1, H-4),
ꢁ
ꢁ
ꢁ
3.78 (1H, dd, J 4–5 3.4, J 5–5 12.3, H-5), 3.67 (1H, dd, J 4–5 5.2, J 5–5 12.3,
H-5ꢁ), 1.57 (3H, s), 1.35 (3H, s); δC (125 MHz, CDCl3) δ 149.5 (Ar), 149.1
(Ar), 147.9 (Ar), 137.0 (Ar), 123.0 (Ar), 122.2 (Ar), 120.3 (CH = C), 113.6
(C(CH3)2), 94.9 (C-1), 89.1 (C-4), 85.3 (C-2), 81.8 (C-3), 62.8 (C-5), 26.9
(C(CH3)2), 25.1 (C(CH3)2); HRMS (ES) Calcd for C15H19N4O4 (M+H+)
319.1406 found 319.1410.
[(3aR,4R,6R,6aR)-2,2-Dimethyl-6-(4-pyridin-3-yl-[1,2,3]triazol-1-yl)-
tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-methanol 8b. (0.11g; 85%) δH (500
MHz, DMSO) δ 9.05 (1H, appd, J 2.2), 8.85 (1H, s, CH = C), 8.56 (1H, dd,
J 1.6, 4.8), 8.23 (1H, appdt, J 2.0, 7.9), 7.50 (1H, dd, J 4.8, 7.5), 6.31 (1H,
d, J 1–2 1.8, H-1), 5.40 (1H, dd, J 1–2 1.9, J 2–3 6.0, H-2), 4.95 (1H, dd, J 3–4 1.9,
J 2–3 6.0, H-3), 4.29 (1H, ddd, J 3–4 1.9, 5.7, 6.0, H-4), (H-5 and H-5ꢁ under
water signal), 1.53 (3H, s), 1.35 (3H, s); δC (125 MHz, DMSO) δ 149.1 (Ar),
146.4, 143.9, 132.6, 126.3, 124.1, 121.5 (CH = C), 112.8 (C(CH3)2), 93.4
(C-1), 88.0 (C-4), 84.0 (C-2), 81.6 (C-3), 61.2 (C-5), 26.7 (C(CH3)2), 25.0
(C(CH3)2); HRMS (ES) Calcd for C15H19N4O4 (M+H+) 319.1406 found
319.1428.
[(3aR,4R,6R,6aR)-2,2-Dimethyl-6-(4-pyridin-4-yl-[1,2,3]triazol-1-yl)-tetra-
hydro-furo[3,4-d][1,3]dioxol-4-yl]-methanol 8c. (0.02 g; 15%) δH (500 MHz,
DMSO) δ 8.96 (1H, s, CH = C), 8.64 (2H, dd, J 1.6, 4.5), 7.83 (2H, dd, J
1.6, 4.5), 6.32 (1H, d, J 1–2 1.8, H-1), 5.41 (1H, dd, J 1–2 or 3–4 1.8, J 2–3 or 3–4
6.0, H-2/H-3), 5.06 (1H, br s, OH), 4.94 (1H, dd, J 1–2 or 3–4 1.8, J 2–3 or 3–4
ꢁ
6.0, H-2/H-3), 4.30 (1H, ddd, J J 3–4 1.8, J 4–5 5.8, J 4–5 5.9, H-4), (H-5
and H-5ꢁ under water signal), 1.52 (3H, s), 1.35 (3H, s); δC (125 MHz,
DMSO) δ 150.4, 144.3, 137.5, 122.8, 119.6 (CH = C), 112.8 (C(CH3)2), 93.5
(C-1), 88.1 (C-4), 84.0 (C-2), 81.6 (C-3), 61.2 (C-5), 26.7 (C(CH3)2), 25.0
(C(CH3)2); HRMS (ES) Calcd for C15H19N4O4 (M+H+) 319.1406 found
319.1402.
Typical Procedure for Pyridine Reduction (10a-c)
Pd/C (10% wt) was added to a solution of triazolide diluted in
MeOH/HCOOH (9/1, v/v). The resulting suspension was stirred vigorously
under an atmosphere of H2 overnight. The crude reaction mixture was
filtered through a pad of celite, which was thoroughly rinsed with MeOH.

β-1,2,3-Triazolyl-Nucleosides
253
The combined filtrates were evaporated to dryness, coevaporated twice with
toluene to afford the crude piperidine derivative quantitatively.
[(4R,6R)-2,2-Dimethyl-6-(4-piperidin-2-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furo[3,4-d][1,3]dioxol-4-yl]-methanol 10a. δH (500 MHz, D2O) δ 8.26 (1H,
s, CH = C), 6.35 (1H, s, H-1), 5.53 (1H, d, J 6.0), 5.01 (1H, d, J 6.0),
4.52–4.47 (2H, m), 3.59 (1H, dd, J 4.8, 12.1), 3.48–3.45 (2H, m), 3.17–3.13
(1H, m), 2.23–2.20 (1H, m), 2.01–1.92 (3H, m), 1.74–1.65 (2H, m), 1.59
(3H, s), 1.40 (3H, s); δC (125 MHz, D2O) δ 144.7 (CH = C), 124.0 (CH
= C), 115.0 (C(CH3)2), 93.9 (C-1), 88.6 (C-4), 84.4 (C-2), 81.7 (C-3),
61.7 (C-5), 52.2, 43.3, 26.2 (2), 24.6, 22.0, 21.9; HRMS (ES) Calcd for
C15H25N4O4 (M+H+) 325.1876 found 325.1880.
[(4R,6R)-2,2-Dimethyl-6-(4-piperidin-4-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furo[3,4-d][1,3]dioxol-4-yl]-methanol 10b. δH (500 MHz, DMSO) δ 8.12
(1H, s br, CH = C), 6.20 (1H, s br, H-1), 5.30 (1H, appd, J 2–3 5.3, H-2), 4.90
ꢁ
(1H, appd, J 1–2 and 3–4 5.7, H-3), 4.21 (1H, appt, J 3–4, 4–5 and 4–5 5.6, H-4), 3.34
(1H, appd, J 5.1), 3.20–3.15 (2H, m), 2.95–2.83 (3H, m), 2.00–1.97 (2H,
m), 1.69–1.65 (2H, m), 1.51 (3H, s), 1.33 (3H, s); δC (125 MHz, DMSO) δ
148.1 (CH = C), 121.7 (CH = C), 113.6 (C(CH3)2), 93.7 (C-1), 88.5 (C-4),
84.8 (C-2), 82.5 (C-3), 62.0 (C-5), 44.4, 32.3, 30.5, 27.6, 25.9; HRMS (ES)
Calcd for C15H25N4O4 (M+H+) 325.1876 found 325.1880.
[(3aR,4R)-2,2-Dimethyl-6-(4-piperidin-3-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furo [3,4-d] [1,3]dioxol-4-yl]-methanol 10c. δH (500 MHz, MeOD) δ 8.19
(1H, s, CH = C), 6.23 (1H, s, H-1), 5.29 (1H, appd, J 2–3 4.4, H-2), 4.92 (1H,
appd, J 1–2 and 3–4 4.0, H-3), 4.23 (1H, s br), 3.36–3.34 (2H, m), 3.17–3.07
(1H, m), 2.81–2.70 (1H, m), 2.06–2.02 (1H, m), 2.01–1.92 (1H, m),
1.77–1.61 (3H, m), 1.52 (3H, s), 1.34 (3H, s); δC (125 MHz, MeOD) δ 148.0
(CH = C), 121.8 (CH = C), 113.9 (C(CH3)2), 94.7 (C-1) 88.8 (C-4), 85.1
(C-2), 82.2 (C-3), 62.1 (C-5), 44.2, 31.4, 28.5, 26.4, 24.6, 22.0; HRMS (ES)
Calcd for C15H25N4O4 (M+H+) 325.1876 found 325.1866.
General Procedure for the Phosphorylation (9a-b)
Dry Et3N (3.0 eq.) was added to a stirred solution of 2,3-O-protected
triazolide (8a-b; typically 0.032 g; 0.1mmol) in dry DCM (5 ml); after 5 min-
utes, diethylchlorophosphite (1.5 eq.) was added; and the resulting reaction
mixture was then stirred for 2 hours. The phosphite triester was oxidized
with tert-butylhydroperoxide (2.0 eq.). After 20 minutes under stirring the
crude reaction mixture was concentrated to dryness. The crude residue was
purified by flash chromatography (100/0→100/5 CHCl3/EtOH, v/v) to
afford the pure phosphate triester.
Phosphoric
acid
(3aR,4R,6R)-2,2-dimethyl-6-(4-pyridin-2-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furo[3,4-d][1,3]dioxol-4-ylmethyl ester diethyl ester
9a. (0.04 g; 90%) δH (500 MHz, CDCl3) δ 8.57 (1H, ddd, J 0.9, 1.7, 4.9),
8.33 (1H, s, CH = C), 8.14 (1H, dt, J 1.0, 7.7), 7.77 (1H, dt, J 1.8, 7.7),
7.23 (1H, ddd, J 1.1, 4.8, 7.5), 6.20 (1H, d, J 1–2 1.3, H-1), 5.53 (1H, dd,

254
E. J. Amigues et al.
J 1–2 1.3, J 2–3 6.0, H-2), 5.01 (1H, dd, J 3–4 2.2, J 2–3 6.0, H-3), 4.56 (1H,
dt, J 3–4 2.1, J 4–5 5.6, H-4), 4.05–3.96 (6H, m, CH2CH3×2, H-5 and H-5ꢁ),
1.59 (3H, s, C(CH3)2), 1.39 (3H, s, C(CH3)2), 1.24 (6H, ddt, J 1.0, 4.8,
7.1, CH3CH2×2); δC (125 MHz, CDCl3) δ 149.8, 149.5, 148.7, 136.9,
123.1, 121.9, 120.1, 114.0, 93.8 (C-1), 86.7 (d, J 7.9, C-4), 84.5 (C-2), 81.7
(C-3), 66.2 (d, J 5.6, C-5), 64.1 (d, J 5.7, CH2CH3), 26.8 (C(CH3)2), 25.2
(C(CH3)2), 16.0 (d, J 7.4, CH2CH3×2); δP (121 MHz, CDCl3) 0.15 (s);
HRMS (ES) Calcd for C19H28N4O7P (M+H+) 455.1696 found 455.1690.
Phosphoric
acid
(3aR,4R,6R)-2,2-dimethyl-6-(4-pyridin-3-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furo[3,4-d][1,3]dioxol-4-ylmethyl ester diethyl ester
9b. (0.04 g; 85%) δH (500 MHz, CDCl3) δ 9.04 (1H, s, Ar), 8.57 (1H, d, J
4.2, Ar), 8.20 (1H, d, J 7.9, Ar), 8.17 (1H, s, CH = C), 7.35 (1H, dd, J 4.8,
7.9, Ar), 6.23 (1H, d, J 1–2 1.3, H-1), 5.46 (1H, dd, J 1–2 1.3, J 2–3 6.0, H-2),
4.99 (1H, dd, J 3–4 2.0, J 2–3 6.0, H-3), 4.57–4.55 (1H, m, H-4), 4.11–3.97
(6H, m, CH2CH3×2, H-5 and H-5ꢁ), 1.59 (3H, s, C(CH3)2), 1.39 (3H, s,
C(CH3)2), 1.22 (6H, dt, J 0.7, 7.1, CH3CH2×2); δC (125 MHz, CDCl3) δ
149.4, 147.0, 145.1, 133.1, 123.8, 119.7, 114.1, 94.1 (C-1), 86.6 (d, J 8.0,
C-4), 86.5 (C-2), 81.4 (C-3), 66.3 (d, J 5.7, C-5), 64.1 (d, J 5.7, CH2CH3×2),
26.9 (C(CH3)2), 25.1 (C(CH3)2), 16.0 (d, J 6.5, CH2CH3×2); δP (121 MHz,
CDCl3) 0.02 (s); HRMS (ES) Calcd. for C19H28N4O7P (M+H+) 455.1696
found 455.1693.
Phosphoric acid mono-[(2R,3S,5R)-3,4-dihydroxy-5-(4-piperidin-2-yl-
[1,2,3]triazol-1-yl)-tetrahydro-furan-2-ylmethyl] ester 11a. The phosphate
triester 9a (0.08 mmol) was reduced using the procedure for pyridine
reduction as described for 10a-c to afford quantitatively phosphoric acid
(3aR,4R,6R)-2,2-dimethyl-6-(4-piperidin-2-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furo[3,4-d][1,3]dioxol-4-ylmethyl ester diethyl ester 14a as colorless oil
(δH (500 MHz, MeOD) δ 8.34 (1H, s, CH = C), 6.37 (1H, br s, H-1), 5.49
(1H, appd, J 1–2 and 2–3 5.7, H-2), 4.56–4.54 (2H, m), 4.13–4.08 (6H, m,
CH2CH3×2, H-5, H-5ꢁ), 3.52–3.48 (1H, m), 3.25–3.22 (1H, m), 2.29–2.25
(1H, m), 2.11–1.97 (3H, m), 1.86–1.79 (2H, m), 1.61 (3H, s, C(CH3)), 1.43
(3H, s, C(CH3)), 1.33 (6H, appt, J 6.9, CH2CH3×2); δC (125 MHz, MeOD)
δ 145.6, 124.6, 115.2, 95.1 (C-1), 88.1 (d, J 7.7, C-4), 85.8 (C-2), 82.9 (C-3),
68.1, (d, J 5.6, C-5), 65.8 (d, J 6.0, CH2CH3), 53.2, 46.0, 29.8, 27.2, 25.4,
23.1, 16.4 (d, J 6.4, CH2CH3); δP (121 MHz, MeOD) -0.7; HRMS (ES)
Calcd for C19H34N4O7P (M+H+) 461.2165 found 461.2152). The crude
reaction mixture was dissolved in dry DCM and bromotrimethylsilane (6
eq.) was then added. The reaction mixture was monitored by TLC. After
complete conversion the reaction mixture was concentrated to dryness.
The crude residue was then resuspended in water and trifluoroacetic acid
was added. The resulting reaction mixture was stirred for 30 minutes. The
crude reaction mixture was evaporated to dryness, coevaporated twice with
toluene to afford the crude deprotected triazolide 11a.

β-1,2,3-Triazolyl-Nucleosides
255
δH (500 MHz, D2O) δ 8.29 (0.7H, s, CH = C), 8.19 (0.3H, br s, H-1), 6.10
(1H, s br), 4.36–4.25 (2H, m), 4.04–4.00 (4H, m), 3.80 (1H, m), 3.43–3.39
(1H, m), 3.14–3.17 (1H, m), 2.18–1.63 (8H, m δC (125 MHz, D2O) δ 144.6,
144.3, 123.8, 123.7, 123.0, 92.8, 92.5, 84.5, 83.3, 83.2, 74.4, 73.8, 69.5, 69.2,
65.9, 65.0 (d, J 5.9), 51.2, 51.1 (2), 44.3, 27.4, 27.3, 20.9, 15.0 (2); δP
(121 MHz, D2O) 0.3; HRMS (ES) Calcd for C12H20N4O7P (M-H+) 363.1070
found 363.1078.
5-O-diethylphosphate-2,3-O-isopropylidene-1-N-(4ꢁ-piperidin-3ꢁꢁ-yl)-
[1ꢁ,2ꢁ,3ꢁ]-triazol-β-D-ribofuranoside 14b. Pd/C (10 %) was added to a
solution of 2,3-O-protected triazolide 9b (0.6 g, 1.32 mmol) in MeOH/
HCOOH (9/1, v/v). The reaction mixture was stirred under an atmosphere
of H2 for 2 days. The reaction mixture was filtered through an AUTOTOP
syringe filter (Whatman) and the solvent was evaporated to dryness. The
residue was coevaporated with toluene to remove residual HCOOH and
then it was dissolved in water and washed with CHCl3. The aqueous layer
was freeze-dried to give pure intermediate (0.30 g, 49%) as a white solid.
1H NMR (DMSO): δ 8.04 (1H, s, CH = C), 6.28 (1H, s, H-1), 5.50 (1H,
d, J 5.9 Hz, H-2), 5.06 (1H, dd, J 5.9, 1.9 Hz, H-3), 4.66–4.60 (1H, m, H-4),
4.20–3.79 (8H, m, H-5, CH2CH3), 3.56 (1H, d, J 12.1 Hz), 3.39 (1H, d, J
13.1 Hz), 3.30–3.18 (1H, m), 3.07 (1H, dd, J 12.0, 1.8 Hz), 2.99 (1H, dd, J
11.8, 2.4 Hz), 2.19–1.95 (2H, m), 1.86–1.68 (2H, m), 1.54 (3H, s, C(CH3)2),
1.36 (3H, s, C(CH3)2), 1.18 (6H, t, J 7.1 Hz, CH2CH3). 13C NMR (DMSO):
δ 148.2 (C = CH), 122.7 (CH = C), 115.0 (C(CH3)2), 94.1 (C-1), 86.5 (d, J
8.3 Hz, C-4), 84.5 (C-2), 81.4 (C-3), 67.5 (d, J 5.4 Hz, C-5), 66.0 (CH2CH3),
47.8, 44.4, 31.2, 28.2, 26.3 (d, J 6.9 Hz, C(CH3)2), 24.6 (C(CH3)2), 21.9, 16.0
(d, J 6.4 Hz, CH2CH3), 15.7 (d, J 6.3 Hz, CH2CH3). 31P NMR (D2O): δ-0.05.
20
[α]D = −38.7◦ (DMSO); HRMS (ES) Calcd for C19H33N4O7PCl (M+Cl−)
495.1781, found 495.1786.
5-O-phosphate-1-N-(4ꢁ-piperidin-3ꢁꢁ-yl)-[1ꢁ,2ꢁ,3ꢁ]-triazol-β-D-ribofurano-
side 11b. TMSBr (275 µL, 2.08 mmol) was added to a solution of
2,3-O-protected triazolide intermediate 14b (160 mg, 0.35 mmol) in dry
DCM and the reaction mixture was stirred overnight. The reaction mixture
was evaporated to dryness. The residue was dissolved in water and Et3N was
used to adjust pH to 7. The aqueous phase was freeze-dried to give pure
product 11b as a triethylamine salt.
1H NMR (DMSO): δ 8.08 (1H, s, CH = C), 6.03 (1H, t, J 4.8 Hz, H-1),
4.54 (1H, dt, J 4.7, 1.5 Hz, H-2), 4.35 (1H, t, J 4.7 Hz, H-3), 4.26 (1H, dt,
J 5.1, 2.7 Hz, H-4), 4.04 (1H, ddd, J 11.6, 4.8, 2.6 Hz, H-5), 3.99–3.95 (1H,
m, H-5), 3.52–3.49 (1H, m), 3.32 (1H, td, J 6.8, 3.7 Hz), 3.21 (1H, tt, J 11.4,
3.9 Hz), 3.04 (1H, t, J 11.9 Hz), 2.93 (1H, dt, J 12.3, 3.2 Hz), 2.11–2.02
(1H, m), 1.96–1.88 (1H, m), 1.80–1.64 (2H, m). 13C NMR (DMSO): δ 148.2
(C = CH), 121.5 (CH = C), 92.8 (C-1), 84.4 (d, J 8.5 Hz, C-4), 75.5 (C-2),
70.7 (C-3), 65.0 (d, J 2.8 Hz, C-5), 47.6, 44.3, 31.1, 27.9 (d, J 10.8 Hz), 21.8

256
E. J. Amigues et al.
20
31P NMR (D2O): δ1.00 [α]D = −15.3◦ (DMSO). HRMS (ES) Calcd for
C12H21N4O7P (M-H+) 363.1075, found 363.1075.
General Procedure for Triazole Formation (6a-c)
Benzoic acid (2R,3R,4R,5R)-3,4-dibenzoyl-5-azido-tetrahydro-furan-2-
ylmethyl ester 4 (100 mg, 1.0 eq) was suspended in water (10 cm3).
Sodium ascorbate (1.0 eq) and L-ascorbic acid (1.0 eq) were added to the
suspension. n-Ethynyl-pyridine (1.0 eq) was added to the reaction mixture.
CuSO4.5H2O (0.1 eq) was added and the resulting reaction mixture was
then heated at 100◦C for 17 hours. The reaction was monitored by TLC (9/1
CHCl3/EtOH, v/v). The reaction mixture was cooled to room temperature
and extracted with EtOAc (30 cm3 × 3). Combined extracts were dried
(MgSO4), filtered and concentrated under reduced pressure to afford the
crude reaction product.
Benzoic
acid
(2R,3R,4R,5R)-3,4-dibenzoyl-5-(4-pyridin-2-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furan-2-ylmethyl ester 6a. (0.04 g, 31%) δH (500
MHz, CDCl3) δ 8.56–8.53 (1H, m, Ar), 8.41 (1H, s, CH = C), 8.15–8.12
(1H, m, Ar), 8.04–7.93 (7H, m Ar), 7.80–7.74 (1H, m, Ar), 7.61–7.47 (4H,
m, Ar), 7.44–7.33 (7H, m, Ar), 7.24–7.21 (1H, m, Ar), 6.50 (1H, d, J 1–2 3.2,
H-1), 6.33 (1H, dd, J 1–2 3.3, J 2–3 5.0, H-2), 6.15 (1H, appt, J 2–3 5.7, H-3),
ꢁ
4.92 (1H, dd, J 3–4 4.2, J 4–5 9.8, H-4) 4.81 (1H, dd, J 4–5 3.75, J 5–5 12.5, H-5),
4.64 (1H, dd, J 4–5 4.65, J 5–5 12.2, H-5ꢁ); δC (125 MHz, CDCl3) δ 166.1 (C
ꢁ
= O), 165.1 (C = O), 165.0 (C = O), 149.7 (Ar), 149.4 (Ar), 148.8 (Ar),
136.8 (Ar), 133.8 (Ar), 133.6 (Ar), 133.2 (Ar), 129.9 (Ar), 129.8 (2, Ar),
129.3, 128.6, 128.5, 128.4, 123.0, 121.4, 120.4 (CH = C), 90.5 (C-1), 81.2
(C-4) 75.3 (C-2), 71.6 (C-3), 63.6 (C-5); HRMS (ES) calcd for C33H27N4O7
(M+H+) 591.1880 found 591.1869
Benzoic
acid
(2R,3R,4R,5R)-3,4-dibenzoyl-5-(4-pyridin-3-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furan-2-ylmethyl ester 6b. (0.06 g, 50%) δH (500
MHz, CDCl3) δ 8.77 (1H, s br), 8.49 (1H, s br), 7.98–7.88 (8H, m, Ar),
7.51–7.42 (3H, m, Ar), 7.34–7.29 (6H, m, Ar), 6.46 (1H, d, J 1–2 3.6, H-1),
6.24 (1H, dd, J 1–2 3.7, J 2–3 5.1, H-2), 6.09 (1H, t, J 2–3 5.35, H-3), 4.86–4.82
(2H, m, H-4 and H-5 or H-5ꢁ), 4.55–4.52 (1H, m, H-5 or H-5ꢁ), δC (125
MHz, CDCl3) δ 166.0 (C = O), 165.1 (C = O), 165.0 (C = O), 149.3 (Ar),
146.9 (Ar), 145.2 (Ar), 133.9 (Ar), 133.7 (Ar), 133.5 (Ar), 133.0 (Ar), 129.8
(Ar), 129.7 (Ar), 129.6 (Ar), 129.1 (Ar), 128.6 (Ar), 128.5 (Ar), 128.5 (Ar),
128.3 (Ar), 118.9 (CH = C), 90.4 (C-1), 81.3 (C-4) 75.2 (C-2), 71.4 (C-3),
63.3 (C-5); HRMS (ES) calcd. for C33H27N4O7 (M+H+) 591.1880 found
591.1863
Benzoic
acid
(2R,3R,4R,5R)-3,4-dibenzoyl-5-(4-pyridin-4-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furan-2-ylmethyl ester 6c. (0.01 g, 12%) δH (500
MHz, CDCl3) δ 8.10 (1H, s, CH = C), 8.06–7.96 (7H, m, Ar), 7.62–7.52
(4H, m, Ar), 7.45–7.38 (6H, m, Ar), 6.54 (1H, d, J 1–2 3.6, H-1), 6.29 (1H,

β-1,2,3-Triazolyl-Nucleosides
257
dd, J 1–2 3.3, J 2–3 5.1, H-2), 6.14 (1H, t, J 2–3 5.4, H-3), 4.97–4.91 (2H, m,
H-4 and H-5 or H-5ꢁ), 4.60 (1H, dd, J 4.5, 13.2, H-5 or H-5ꢁ), δC (125 MHz,
CDCl3) δ 165.8 (C = O), 164.9 (C = O), 164.0 (C = O), 133.7 (Ar), 133.6
(Ar), 133.3 (Ar), 129.7 (Ar), 129.6 (Ar), 129.4 (Ar), 128.9 (Ar), 128.5
(Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar), 128.0 (Ar), 119.7 (CH = C), 90.3
(C-1), 81.2 (C-4), 75.0 (C-2), 71.1 (C-3), 63.0 (C-5); HRMS (ES) calcd for
C33H27N4O7 (M+H+) 591.1880 found 591.1824.
Typical Procedure for Acetonide Removal (1a-c and 2a-c)
Trifluoroacetic acid was added to a solution/suspension of triazolide
in water. The resulting reaction mixture was stirred for 30 minutes. The
crude reaction mixture was evaporated to dryness, co-evaporated twice
with toluene to afford the crude deprotected triazolide derivative as the
trifluoroacetate salt quantitatively.
(2R,5R)-2-Hydroxymethyl-5-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furan-3,4-diol 1a. δH (500 MHz, D2O) δ 8.99 (1H, s, CH = C), 8.72 (1H, d, J
5.6), 8.61 (1H, appdt J 1.2, 6.1), 8.35 (1H, d, J 8.1), 7.96 (1H, appt, J 6.7),
6.22 (1H, d, J 1–2 3.8, H-1), 4.74 (1H, dd, J 1–2 3.9, J 2–3 5.8, H-2), 4.43 (1H, t,
J 2–3 and 3–4 5.2, H-3), 4.25 (1H, dt, J 4–5 3.2, J 3–4 5.1, H-4), 3.84 (1H, dd, J 4–5
3.2, J 5–5 12.7, H-5), 3.72 (1H, dd, J 4–5 4.9, J 5–5 12.7, H-5ꢁ); δC (125 MHz,
D2O) δ 147.9, 141.6, 126.5, 126.1, 125.1, 93.0 (C-1), 86.0 (C-4), 75.5 (C-2),
70.6 (C-3), 61.4 (C-5); HRMS (ES) Calcd for C12H14N4O4Na (M+Na+)
301.0913 found 301.0914.
ꢁ
ꢁ
ꢁ
(2R,5R)-2-Hydroxymethyl-5-(4-pyridin-3-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furan-3,4-diol 1b. δH (500 MHz, D2O) δ 9.14 (1H, s), 8.87 (1H, ddd, J 1.4,
2.0, 8.3), 8.72 (1H, s, CH = C), 8.69 (1H, d, J 5.7), 8.08 (1H, dd, J 6.1,
8.0), 6.13 (1H, d, J 3.9, H-1), 4.66 (1H, appd, J 4.5, H-2), 4.38 (1H, t, J
5.2, H-3), 4.19 (1H, dt, J 3.3, 5.1, H-4), 3.79 (1H, dd, J 3.2, 12.7, H-5),
3.67 (1H, dd, J 5.0, 12.7, H-5ꢁ); δC (125 MHz, D2O) δ 143.5, 141.8, 140.7,
138.5, 130.5, 128.2, 123.6, 92.8 (C-1), 85.9 (C-4), 75.4 (C-2), 70.6 (C-3),
61.5 (C-5); HRMS (ES) Calcd for C12H15N4O4 (M+H+) 279.1093 found
279.1092.
(2R,5R)-2-Hydroxymethyl-5-(4-pyridin-4-yl-[1,2,3]triazol-1-yl)-tetrahydro-
furan-3,4-diol 1c. δH (500 MHz, D2O) δ 8.96 (1H, s, CH = C), 8.74 (2H,
appd, J 7.0), 8.38 (2H, appd J 7.0), 6.19 (1H, d, J 1–2 3.8, H-1), 4.42 (1H, t,
J 1–2 and 2–3 5.2, H-3), 4.23 (1H, dt, J 4–5 3.2, J 3–4 5.1, H-4), 3.83 (1H, dd, J 4–5
3.2, J 5–5 12.7, H-5), 3.71 (1H, dd, J 4–5 5.0, J 5–5 12.7, H-5ꢁ); δC (125 MHz,
D2O) δ 147.4, 142.1, 126.2, 123.2, 93.0 (C-1), 86.0 (C-4), 75.5 (C-2), 70.6
(C-3), 61.5 (C-5); HRMS (ES) Calcd for C12H15N4O4 (M+H+) 279.1093
found 279.1085.
ꢁ
ꢁ
ꢁ
(2R,5R)-2-Hydroxymethyl-5-(4-piperidin-2-yl-[1,2,3]triazol-1-yl)-tetra-
hydro-furan-3,4-diol 2a. δH (500 MHz, D2O) δ 8.17 (1H, s, CH = C), 6.04
(1H, d, J 1–2 4.0, H-1), 4.55 (1H, appt J 1–2 and 2–3 4.4, H-2), 4.39 (1H, dd, J

258
E. J. Amigues et al.
2.9, 11.8), 4.28 (1H, t, J 2–3 and 3–4 5.2, H-3), 4.10 (1H, dt, J 4–5 3.4, J 3–4 5.1,
ꢁ
ꢁ
ꢁ
H-4), 3.70 (1H, dd, J 4–5 3.2, J 5–5 12.7, H-5), 3.58 (1H, dd, J 4–5 5.0, J 5–5
12.7, H-5ꢁ), 3.35 (1H, d, J 12.9), 3.04 (1H, dt, J 2.6, 12.5), 2.11–2.09 (1H,
m), 1.93–1.80 (3H, m), 1.65–1.51 (2H, m); δC (125 MHz, D2O) δ 163.2 (q,
J 35.7 Hz, C = O), 144.5 (CH = C), 123.5 (CH = C), 116.7 (q, J 291.3
Hz), 92.6 (C-1), 85.8 (C-3), 75.3 (C-2), 70.6 (C-4), 61.5 (C-5), 52.2, 45.3,
28.4, 21.9; HRMS (ES) Calcd for C12H21N4O4 (M+H+) 285.1563 found
285.1560.
(2R,5R)-2-Hydroxymethyl-5-(4-piperidin-3-yl-[1,2,3]triazol-1-yl)-tetra-
hydro-furan-3,4-diol 2b. δH (500 MHz, D2O) δ 7.83 (1H, s, CH = C), 5.80
(1H, d, J 1–2 4.0, H-1), 4.34 (1H, appt J 1–2and 2–3 4.5, H-2), 4.09 (1H, t,
J 2–3 and 3–4 5.1, H-3), 3.92 (1H, dt, J 4–5 3.3, J 3–4 5.0, H-4), 3.53 (1H, dd,
J 4–5 3.2, J 5–5 12.7, H-5), 3.41 (1H, dd, J 4–5 5.0, J 5–5 12.7, H-5ꢁ), 3.21 (1H,
appd, J 12.6), 3.13 (1H, appd, J 12.7), 3.04–2.99 (1H, m), 2.83 (1H, t, J
12.1), 2.76–2.71 (1H, m), 1.89–1.86 (1H, m), 1.75–1.71 (1H, m), 1.60–1.43
(2H, m); δC (125 MHz, D2O) δ 162.9 (q, J 35.7 Hz, C = O), 147.9 (CH =
C), 122.3 (CH = C), 116. (q, J 291.3 Hz), 92.6 (C-1), 85.7 (C-3), 75.3 (C-2),
70.6 (C-4), 61.5 (C-5), 47.7, 44.4, 31.1, 28.0, 21.8; HRMS (ES) Calcd for
C12H21N4O4 (M+H+) 285.1563 found 285.1561.
ꢁ
ꢁ
ꢁ
(2R,5R)-2-Hydroxymethyl-5-(4-piperidin-4-yl-[1,2,3]triazol-1-yl)-tetra-
hydro-furan-3,4-diol 2c. δH (500 MHz, D2O) 8.01 (1H, s, CH = C), 6.01
(1H, d, J 1–2 3.8, H-1), 4.56 (1H, appt J 1–2and 2–3 4.3, H-2), 4.31 (1H, t,
ꢁ
J 2–3 and 3–4 4.9, H-3), 4.13 (1H, dd, J 3–4 4.3, J 4–5 7.4, H-4), 3.74 (1H, dd, J 4–5
2.5, J 5–5 12.7, H-5), 3.62 (1H, dd, J 4–5 4.8, J 5–5 12.7, H-5ꢁ), 3.40 (2H, appd,
J 12.9), 3.11–3.04 (3H, m), 2.17 (2H, appd, J 14.1), 1.86–1.79 (2H, m),
1.65–1.51 (2H, m, H-γ ꢁꢁ, H-δꢁꢁ); δC (125 MHz, D2O) δ 163.2 (q, J 36.4 Hz, C
= O), 150.4 (CH = C), 121.8 (CH = C), 116.8 (q, J 291.0 Hz), 92.6 (C-1),
85.7 (C-3), 75.3 (C-2), 70.6 (C-4), 61.5 (C-5), 43.9, 30.6, 28.1; HRMS (ES)
Calcd for C12H21N4O4 (M+H+) 285.1563 found 285.1562.
ꢁ
ꢁ
ꢁ
Benzoic acid (2R,3R,4R,5R)-3,4-dibenzoyl-5-(4-piperidin-3-yl-[1,2,3]
triazol-1-yl)-tetrahydro-furan-2-ylmethyl ester 12b. Pd/C (10% wt) was
added to a solution of benzoic acid (2R,3R,4R,5R)-3,4-dibenzoyl-5-(4-
pyridin-3-yl-[1,2,3]triazol-1-yl)-tetrahydro-furan-2-ylmethyl ester 6b diluted
in MeOH. The resulting suspension was stirred vigorously under an
atmosphere of H2 overnight. The crude reaction mixture was filtered
through a pad of celite, which was thoroughly rinsed with MeOH. The
combined filtrates were evaporated to dryness, coevaporated twice with
toluene to afford the crude piperidine derivative, quantitatively. Further
purification by flash chromatography (9/1, CHCl3/EtOH, v/v) afforded
the product as a clear oil (32.8 mg, 32%).
δH (500 MHz, CDCl3) δ 7.97–7.95 (2H,m, Ar), 7.90–7.86 (4H, m,
Ar), 7.66 (1H, s br, CH = C), 7.52–7.45 (3H, m, Ar), 7.39–7.36 (2H, m,
Ar), 7.31–7.27 (4H, m, Ar), 7.29–7.19 (1H, m, Ar), 6.37–6.35 (1H, m,

β-1,2,3-Triazolyl-Nucleosides
259
H-1), 6.17–6.16 (1H,m), 6.07 (1H, t, J 5.0), 4.80–4.74 (2H, m), 4.54–4.48
(1H, m), 3.61–3.56 (1H, m), 3.43–3.35 (2H, m), 2.97–2.92 (1H, m),
2.87–2.80 (1H, m), 2.01–1.97 (2H, m), 1.87–1.81 (1H, m); δC (125 MHz,
CDCl3) δ 166.0, 165.1, 165.0, 147.6, 133.8, 133.6 (2), 133.5, 129.9, 129.8,
129.3, 129.2, 128.6, 128.5 (2), 128.4, 120.3 (2), 90.3 (C-1), 81.2, 81.1, 75.1,
71.7, 71.6, 63.7, 63.6, 47.7 (2), 44.0, 30.5, 28.5, 21.7; HRMS (ES) calcd for
C33H33N4O7 (M+ H+) 597.2349 found 597.2353.
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