Synthesis of N-propynyl analogues of peptide nucleic acid (PNA) monomers and their use in the click reaction to prepare N-functionalized PNAs

Article abstract of DOI:10.1016/j.tet.2011.09.124

Application of the click reaction for coupling a 2-(2-aminoethoxy)ethoxy (AEE) function to thyminyl, cytosinyl and adeninyl peptide nucleic acid (PNA) monomer analogues bearing a N-propynyl group, in place of the original N-2-aminoethyl moiety, has been explored. The N-propynyl PNA analogues were prepared from glycine ethyl ester hydrochloride and the structure of the thyminyl derivative was confirmed by X-ray diffraction. These monomers were employed in click reactions with Boc-protected AEE azide to afford the corresponding triazolyl-linked PNA-AEE conjugates in yields ranging from 64 to 76%. [1,4]-Regiochemistry was verified from a NOESY correlation experiment.

Full text of DOI:10.1016/j.tet.2011.09.124

Tetrahedron 67 (2011) 9588e9594
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of N-propynyl analogues of peptide nucleic acid (PNA) monomers and
their use in the click reaction to prepare N-functionalized PNAs
*
ꢀ
Nicola M. Howarth , Jennyfer Ricci (nee Goujon)
Chemistry, School of Engineering & Physical Sciences, William H. Perkin Building, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Received in revised form 9 September 2011
Accepted 26 September 2011
Available online 6 October 2011
Application of the click reaction for coupling a 2-(2-aminoethoxy)ethoxy (AEE) function to thyminyl,
cytosinyl and adeninyl peptide nucleic acid (PNA) monomer analogues bearing a N-propynyl group, in
place of the original N-2-aminoethyl moiety, has been explored. The N-propynyl PNA analogues were
prepared from glycine ethyl ester hydrochloride and the structure of the thyminyl derivative was con-
firmed by X-ray diffraction. These monomers were employed in click reactions with Boc-protected AEE
azide to afford the corresponding triazolyl-linked PNAeAEE conjugates in yields ranging from 64 to 76%.
[1,4]-Regiochemistry was verified from a NOESY correlation experiment.
Keywords:
Click chemistry
Dipolar cycloadditions
Peptide nucleic acids
PEG analogues
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and a range of thyminyl PNA oligomers bearing either pentacosa-
10,12-diynoyl or stearoyl moieties at either the N-or C-termini (1
In 2002, Sharpless et al. and Meldal et al. independently re-
and 2, respectively, Fig. 1).9,10 PNAs are DNA mimics in which the
entire deoxyribose-phosphate backbone has been replaced by
a structurally homomorphous uncharged, achiral polyamide back-
bone composed of N-(2-aminoethyl)glycine subunits.11 An impor-
tant feature of PNAs is that they bind with higher affinity and
sequence specificity to both single-stranded (ss-DNA) and RNA
than their natural oligonucleotide counterparts.12 Monomers 1 and
oligomers 2 were subsequently investigated for their abilities to be
incorporated into polydiacetylene (PDA)-containing liposomes for
possible use as colorimetric nucleic acid biosensors. PDAs are
conjugated polymers, which exhibit interesting chromic effects;
blue to red transitions can be induced by heat,13 solvent varia-
tions,14 changes in pH15,16 and, most notably, upon exposing PDA
assemblies incorporating biologically active head groups to their
appropriate biomolecular targets.17
In order to enhance the aqueous solubilities of both the lipid-
functionalized PNA monomers and oligomers and their resulting
PDA liposomes, we have recently explored the use of the click re-
action to couple a 2-(2-aminoethoxy)ethoxy (AEE) hydrophilic
spacer to the N-terminus of PNA (3, Fig. 1). Despite the numerous
advantages offered by the click reaction, there are relatively few
reports in the literature to date involving the application of this
copper (I)-catalyzed cycloaddition to the N-functionalization of
PNAs. This alkyneeazide coupling has been successfully used to: (i)
attach ferrocene moieties to PNA oligomers;18e21 (ii) prepare pep-
tideePNA conjugates;22 (iii) immobilize PNA capture probes onto
microarrays;23 and, (iv) synthesize novel rhenium and
€
ported copper(I)-catalyzed Huisgen 1,3-dipolar [3þ2] cycloaddi-
tions involving organic azides and terminal alkynes (Scheme 1).1,2
In both cases, the corresponding [1,4]-disubstituted 1,2,3-triazole
products were formed chemo- and regioselectively in high yields.
It is this cycloaddition which has become associated with the term
‘click chemistry’.3 Copper(I)-catalyzed alkyneeazide couplings are
reliable, efficient, simple, and extremely versatile; they can be
performed in a variety of solvents, even water, and in the presence
of many functional groups.4,5 Given these advantages, it is therefore
not surprising that this reaction has found much use in recent years
for generating a vast array of compounds for many diverse appli-
cations,6 including those with biological potential.7,8
Cu(I)
N
N
N
+
R1
R2 N3
R1
H
R2
t-BuOH: H2O
H
[1,4]
€
Scheme 1. Copper(I)-catalyzed Huisgen [3þ2] cycloaddition.
Previously, we have reported the design and synthesis of ade-
ninyl, cytosinyl and thyminyl peptide nucleic acid (PNA) monomers
* Corresponding author. Tel.: þ44 131 451 8026; fax: þ44 131 451 3180; e-mail
address: N.M.Howarth@hw.ac.uk (N.M. Howarth).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.124

ꢀ
N.M. Howarth, J. Ricci (nee Goujon) / Tetrahedron 67 (2011) 9588e9594
9589
B
iii (98%)
iv
i (63%)
ii (76%)
Boc
N
O
HCl.H2N CO2Et
CO2Et
O
N
R1HN
R2
4
5
1
R5HN
B
B
O
O
N
O
N
O
N
N
CO2Et
R3(T)10R4
N
OR6
2
6a B = T (60%)
2
3
6b B = CCbz (51%)
6c B = ACbz (36%)
Fig. 1. B¼Adenin-9-yl; thymin-1-yl; N6-Cbz-adenin-9-yl; N6-Cbz-cytosin-1-yl. R1¼H;
CH3(CH2)11C4(CH2)8CO; CH3(CH2)16CO. R2¼OH; NH(CH2)2{O(CH2)2}2NHCO(CH2)8C4-
(CH2)11CH3; NH(CH2)9C4(CH2)11CH3. T¼Thyminyl PNA monomer. R3¼CH3(CH2)16CO;
CH3(CH2)11C4(CH2)8CO; AcAsp. R4¼LysNH2; AspNH2; Lys(CO(CH2)8C4(CH2)11CH3)NH2;
Lys(CO(CH2)16CH3)NH2. R5¼Boc; pentacosa-10,12-diynoyl; stearoyl. R6¼OEt; PNA
oligomer.
Scheme 2. Boc¼(CH3)3COCO; ACbz¼N6-Cbz-adenin-9-yl; CCbz¼N6-Cbz-cytosin-1-yl;
T¼thymin-1-yl. Reagents and conditions: (i) [(CH3)3COCO]2O, dioxane, H2O, NaOH (pH
w10.5), 0 ꢀC to rt; (ii) HChCCH2Br, NaH, DMF, toluene, 0 ꢀC to rt; (iii) TFA, CH2Cl2, rt;
(iv) BCH2CO2H,19,20 HBTU, DIPEA or TEA, DMF, rt.
99mtechnetium tricarbonyl-containing PNA bioconjugates.24,25 In
these examples, the click reactions were facilitated either by deri-
vatisation of the N-terminal PNA residue through coupling of
a suitable alkynoic or azido acid spacer or by replacement of the
nucleobase of the N-terminal PNA monomer with an alkyne-
containing side chain. However, for our studies, we wished to
maintain the PNA nucleobase for recognition purposes and we
wanted to avoid addition of an alkyl spacer, as it was envisaged that
this may further exacerbate the aqueous solubility difficulties al-
ready encountered. Therefore, taking these considerations on
board, PNA monomer analogues have been designed in which the
original N-2-aminoethyl moiety has been replaced with a N-pro-
pynyl group. Thus, here we report the synthesis of the N-propynyl
analogues of the adeninyl, cytosinyl and thyminyl PNA monomers,
preparation of Boc-protected AEE azide and, finally, their use in the
click reaction to afford triazolyl-linked PNAeAEE conjugates 3
(Fig. 1, where R5¼Boc and R6¼OEt).
is shown in Fig. 2. Compound 6a was found to crystallize with one
molecule in the asymmetric unit and in the same monoclinic space
group, P2/(1)n, as the original N-Boc-protected thyminyl PNA
monomer.30 Interestingly, although in solution 6a had been found
to exist in two rotameric forms, in the crystal structure only the cis
rotamer was present. Packing analysis revealed that the crystal of
6a had the expected centrosymmetric hydrogen-bonded dimeric
structure, which had been observed in the crystal structure of the
original thyminyl PNA monomer30 as well as other PNA ana-
logues.31 The N(3)eH/O(4) distance was 1.90 A. The dimers were
ꢁ
positioned vertically such that the thyminyl rings were stacked
ꢁ
above each other at a distance of 4.90 A. Further study of the in-
termolecular interactions showed several close contacts between
adjacent molecules of 6a including one involving alkynyl C(13)eH
ꢁ
and side chain carbonyl O(19), with an H/O distance of 2.30 A.
2. Results and discussion
2.1. N-Propynyl PNA monomers
The N-propynyl analogues of the thyminyl, cytosinyl and ade-
ninyl PNA monomers (6aec, respectively) were successfully pre-
pared from commercially available glycine ethyl ester
hydrochloride 4 as outlined in Scheme 2. Thus, the amino function
of 4 was first protected with a Boc group using a standard pro-
cedure to afford Boc-glycine ethyl ester.26 Subsequent treatment of
a solution of this product in anhydrous DMF with 3-bromoprop-1-
yne in the presence of sodium hydride gave the N-alkynyl de-
rivative 5. Finally, following Boc removal, the resulting amino
deprotected compound was coupled to thymin-1-ylacetic acid,27
N4-Cbz-cytosin-1-yl acetic acid28 and N6-Cbz-adenin-9-ylacetic
acid28 using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate (HBTU) as the activating agent in the pres-
ence of an organic base. After work-up and purification by flash
chromatography, the desired N-propynyl PNA analogues 6aec were
obtained in yields ranging from 35 to 59% over the two steps.
As had been found for the original PNA monomers,29 the 1H and
13C NMR spectra recorded for N-propynyl PNA analogues 6aec
showed the presence of two rotameric forms in solution. However,
unlike the original PNA monomers, which exhibited a 70:30 pref-
erence for the cis rotamer of the tertiary amide bond over the trans
rotamer (where the cis rotamer is defined as the conformer in
which the side chain methylene carbonyl is directed towards the C-
terminus of the PNA monomer), N-propynyl PNA analogues 6aec
showed no such preference with both rotamers present in equiv-
alent amounts.
Fig. 2. ORTEP diagram of thymin-1-yl derivative 6a.
2.2. Boc-protected AEE azide
The required Boc-protected AEE azide 10 was synthesized from
commercially available 2-(2-aminoethoxy)ethanol 7 as shown in
Scheme 3. The dibenzyl protection of 7 and subsequent alkylation
to afford 8 were accomplished following the procedures reported
by Koskinen et al.,32 except that ethyl bromoacetate was employed
in the latter reaction in place of methyl bromoacetate. The next step
involved reduction of 8 to give the corresponding alcohol. Although
this could be achieved by treatment with LiAlH4, the best yield of
the alcohol was obtained when using the NaBH4emethanol sys-
tem33 (82% cf. 72% with LiAlH4). The dibenzylamino protecting
group was subsequently removed by hydrogenation, using an H-
The structure of the thyminyl derivative 6a was further con-
firmed by an X-ray crystallographic study. The ORTEP diagram of 6a

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N.M. Howarth, J. Ricci (nee Goujon) / Tetrahedron 67 (2011) 9588e9594
9590
CubeÒ continuous-flow reactor, to give amino alcohol 9 in a near
quantitative yield. After re-protection of the free amino moiety of 9
with a Boc group, the hydroxyl function was successfully converted
into the corresponding mesylate by treatment with meth-
anesulfonyl chloride in the presence of triethylamine. Finally, the
desired Boc-protected AEE azide 10 was obtained, in a 60% yield
over the last three steps, by treatment of a solution of the mesylate
in anhydrous DMF with sodium azide at 60 ꢀC.
Fig. 3). If the corresponding [1,5]-regioisomer had been produced,
the triazolyl proton would have only shown an NOE correlation
with the C5-methylene protons. Compound 12 was examined here,
rather than click products 11aec, as it is devoid of aromatic protons
other than the triazolyl proton which facilitated identification of
NOE correlation peaks for this proton.
i (80%)
iii (82%)
iv (99%)
O
Bn2N
H2N
H2N
OH
OH
O
CO2Et
ii (40%)
2
7
8
v (84%)
BocHN
N3
O
O
vi (78%)
vii (92%)
2
2
9
10
Scheme 3. Reagents and conditions: (i) BnBr, K2CO3, H2O, rt;32 (ii) (a) NaH, THF,
BrCH2CO2Et, 0 ꢀC to rt; (iii) (a) NaBH4, THF,
(b) MeOH, ; (iv) 10% Pd/C, EtOH, H-
cubeÒ, 70 bar, 70 ꢀC; (v) [(CH3)3COCO]2O, Et3N, MeOH,
; (vi) MeSO2Cl, Et3N, CH2Cl2,
ꢀC to rt; (vii) NaN3, DMF, 60 ꢀC.
D (b)
D
D
D
0
2.3. Click reactions
Click reactions involving the thyminyl, cytosinyl and adeninyl N-
propynyl PNA analogues 6aec, respectively, and Boc-protected AEE
azide 10 have been explored (Scheme 4). The procedure reported by
Sharpless et al.1 had to be slightly modified as it was noticed that
the N-propynyl PNA derivatives 6aec displayed limited solubility in
the standard tert-butanol/water (1:1, v/v) solvent mixture. This
problem was overcome by adding 6aec to the reaction mixture as
a solution in dichloromethane. This modification also simplified
work-up as the organic products were easily separated from the
copper salts by mere separation of the organic and aqueous phases
upon completion of the reaction. Thus, a solution of the required N-
propynyl PNA analogue 6aec in dichloromethane was added to
a solution of azide 10 in tert-butanol/water followed by copper(II)
sulfate pentahydrate and a 1 M(aq) solution of sodium ascorbate at
rt. After work-up and purification by flash chromatography, the
desired click products 11aec were afforded in yields ranging from
64 to 76%. These reactions have not been optimized.
Fig. 3. 2D NOESY spectrum recorded for [1,4]-regioisomer of triazole 12 (CDCl3) and
key NOE contacts.
3. Conclusion
B
In summary, a viable synthetic route for the preparation of N-
propynyl analogues of the thyminyl, cytosinyl and adeninyl PNA
monomers 6aec, respectively, has been developed. These mono-
mers have been successfully employed in click reactions with Boc-
protected AEE azide 10 to afford the corresponding triazolyl-linked
conjugates. Studies are currently underway using these N-propynyl
PNA monomers for preparing lipid-functionalized PNA oligomers
bearing ‘clicked’ hydrophilic spacers for incorporation into PDA
liposomes.
O
N
i
N
10 + 6a-c
BocHN
O
N
N
CO2Et
O
11a B = T (76%)
11b B = CCbz (76%)
11c B = ACbz (64%)
Scheme 4. Reagents and conditions: CuSO4$5H2O, sodium ascorbate, t-BuOH/H2O/
CH2Cl2 (1:1:1, v/v/v), rt.
As expected, the 1H and 13C NMR spectra recorded for 11aec
revealed the presence of two rotamers. The formation of the tri-
azolyl moiety of 11aec was verified from the 1H NMR spectra by the
disappearance of two triplet peaks between 2.21e2.30 ppm and
2.32e2.48 ppm, corresponding to the terminal alkyne proton in the
two rotameric forms of 6aec, and the appearance of two singlet
peaks between 7.71e7.73 ppm and 7.94e8.12 ppm due to the tri-
azolyl proton in the two rotameric forms of 11aec. The 1,4-
regiochemistry of this click reaction was confirmed by a two di-
mensional NOESY correlation experiment performed on the model
triazole compound 12 (Fig. 3), prepared from propynyl in-
termediate 5 (Scheme 2) and Boc-protected AEE azide 10. This
study clearly showed NOE correlations between the triazolyl proton
and both the N1- and C4-methylene protons (a and b, respectively,
4. Experimental section
4.1. General
All starting materials were purchased from either Alfa Aesar,
Fisher Scientific, Novabiochem or SigmaeAldrich Chemical
companies and were generally used as supplied without further
purification. Anhydrous solvents were prepared following stan-
dard procedures.34 Analytical thin layer chromatography (TLC)
was performed on aluminium plates pre-coated with Merck
Kieselgel 60 GF254 (Art. 05554). Products were visualised by UV
light and/or by staining the plate with an alkaline KMnO4 solu-
tion [KMnO4 (3 g), K2CO3 (20 g), 5% (w/v) (aq) NaOH (5 mL) and

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N.M. Howarth, J. Ricci (nee Goujon) / Tetrahedron 67 (2011) 9588e9594
9591
H2O (300 mL)] followed by heating. Column chromatography
(CI) 242 ([MþH]þ, 15%), 203 (75), 142 (100), 68 (26); HRMS (ESI) m/
z: [MþH]þ, found 242.1387. C12H20NO4 requires 242.1387.
refers to the method of Still et al.35 and was performed using
DAVISILÒ silica (60 A; 35e70
m
m) purchased from Fisher (cat. S/
ꢁ
0693/60). Melting points were determined using a Stuart Melting
Point SMP10 apparatus and are uncorrected. IR spectra were
4.1.2. N-(Thymin-1-ylacetyl)-N-(propyn-3-yl)glycine ethyl ester
(6a). TFA (8 mL) was added dropwise to a stirred solution of 5
(1.00 g, 4.14 mmol) in CH2Cl2 (8 mL) at rt and the mixture was left
to stir for 1 h. Subsequently, the solvent was removed under re-
duced pressure and the residue afforded was co-evaporated with
toluene (2ꢁ10 mL) followed by Et2O (2ꢁ10 mL) before being dried
in a vacuum desiccator over P2O5. The trifluoroacetic acid salt of N-
(propyn-3-yl)glycine ethyl ester (1.04 g, 98%) was obtained as
a brown oil and was used in the next stage without further
purification.
HBTU (1.34 g, 3.53 mmol) followed by DIPEA (2.00 mL,
12.0 mmol) were added to a stirred solution of thymin-1-ylacetic
acid27 (0.68 g, 3.70 mmol) in DMF (10 mL) at rt and the mixture
was left to stir for 15 min. Subsequently, a solution of the tri-
fluoroacetic acid salt of N-(propyn-3-yl)glycine ethyl ester (8.86 g,
3.36 mmol) in DMF (10 mL) was slowly added to the reaction
recorded on a PerkineElmer 1600 FTeIR spectrometer. 1H and 13
C
NMR spectra were recorded on Bruker AC 200 or DPX 400
spectrometers at 200 MHz (1H) and 50 MHz (13C) or at 400 MHz
(1H) and 101 MHz (13C), respectively. 1H and 13C chemical shifts
(d) are reported in parts per million (ppm) relative to SiMe4, using
the 13C signals or residual proton signals of deuterated solvents
as internal standards. Coupling constants (J) are reported in hertz
(Hz). Low resolution (LRMS) and high resolution (HRMS) mass
spectra were recorded at the EPSRC National Mass Spectrometry
Service Centre, Swansea, UK. Elemental analyses and X-ray
crystallography were carried out by analytical services in
Chemistry, School of Engineering & Physical Sciences at Heriot-
Watt University.
4.1.1. N-(tert-Butoxycarbonyl)-N-(propyn-3-yl)glycine ethyl ester
(5). A solution of di-tert-butyl dicarbonate (5.14 g, 23.5 mmol) in
dioxane (7 mL) was added dropwise to a stirred solution of glycine
ethyl ester hydrochloride 4 (3.02 g, 21.6 mmol) in water (60 mL) at
0 ꢀC. After being left to stir for a further 15 min at this temperature,
the reaction was allowed to warm slowly to rt whilst the pH was
maintained at 10.5 by the addition of 2 M(aq) NaOH. After 2 h, the
reaction mixture was concentrated in vacuo and the residual paste
was dissolved in CH2Cl2 (50 mL). The resulting organic solution was
washed with water (20 mL), the two phases were separated and the
aqueous layer was re-extracted with CH2Cl2 (30 mL). The combined
organic layers were dried (MgSO4) and the solvent was evaporated
under reduced pressure. The crude product obtained was purified
by column chromatography (EtOAc/petroleum ether (40e60 ꢀC),
6:4) to afford N-Boc-glycine ethyl ester (2.77 g, 63%) as a yellow
liquid; Rf 0.66 (EtOAc/petroleum ether (40e60 ꢀC), 6:4); dH
(200 MHz; CDCl3) 1.22 (2H, t, J 7.2, OCH2CH3), 1.39 (9H, s, C(CH3)3),
3.83 (2H, d, J 5.6, NHCH2CO), 4.10 (2H, q, J 7.2, OCH2CH3), 5.04 (1H,
br s, NH); dC (50 MHz; CDCl3) 14.0, 28.2, 42.3, 61.1, 79.6, 155.7, 170.3;
m/z (CI) 204 ([MþH]þ, 20%),165 (90), 104 (100), 58 (10), 44 (16);
HRMS (ESI) m/z: [MþH]þ, found 204.1230. C9H18NO4 requires
204.1230.
mixture followed by a further quantity of DIPEA (3.00 mL,
28.0 mmol). The mixture was stirred at rt overnight and then the
solvent was removed in vacuo. The residue was dissolved in CH2Cl2
(30 mL) and the resulting organic solution was washed successively
with 1 M(aq) NaHCO3 (3ꢁ15 mL), 1 M(aq) KHSO4 (2ꢁ15 mL), water
(15 mL) and, finally, brine (15 mL). The organic layer was dried
(MgSO4) and the solvent was evaporated under reduced pressure.
The crude orange foam afforded was purified by column chroma-
tography (EtOAc/MeOH, 95:5) to yield the title compound 6a (0.62 g,
60%) as a cream coloured foam; mp 162e163 ꢀC; [Found: C, 54.77;
H, 5.53; N, 13.38. C14H17N3O5 requires C, 54.72; H, 5.58; N, 13.67%];
Rf 0.32 (EtOAc/MeOH, 95:5); nmax(film) 3238, 3165, 2999, 2832,
1726, 1724, 1682, 1664 cmꢂ1
; dH (400 MHz; CDCl3) (two rotational
isomers observed) 1.26 and 1.31 (2H, t, J 7.1, OCH2CH3), 1.34 (3H, s,
thyminyl C(5)-CH3), 2.30 and 2.48 (1H, t, J 2.4, CH2C^CH), 4.18 and
4.26 (2H, q, J 7.1, OCH2CH3), 4.27e4.29 (4H, m, COCH2NCH2C^CH),
4.50 and 4.70 (2H, s, NCOCH2N), 7.01 (1H, s, thyminyl C(6)-H), 9.50
(1H, s, thyminyl N(3)-H); dC (101 MHz; CDCl3) (two rotational iso-
mers observed) 12.2,14.0 and 14.1, 36.3 and 38.0, 47.7, 48.0 and 48.1,
61.7 and 62.3, 73.8 and 74.6, 76.6 and 77.1, 110.9, 141.1, 151.3, 164.5,
167.1, 168.7 and 168.9; m/z (ESI) 308 ([MþH]þ, 100%), 325 (70), 142
(25); HRMS (ESI) m/z: [MþH]þ, found 308.1240. C14H18N3O5 re-
quires 308.1241.
A solution of 3-bromopropyne in toluene (80% w/v, 2.53 mL,
22.7 mmol) was added dropwise to a stirred solution of N-Boc-
glycine ethyl ester (1.54 g, 7.57 mmol) in anhydrous DMF (5 mL) at
rt under N2 atmosphere. The resulting mixture was cooled to 0 ꢀC
and NaH (60% disp. in mineral oil, 0.61 g, 15.2 mmol) was added
portionwise. Subsequently, the reaction was allowed to warm
slowly to rt whereupon it was left to stir for a further 18 h. The
reaction was quenched by the careful addition of water (15 mL) and
the mixture was concentrated in vacuo. The residual paste was
dissolved in EtOAc (30 mL) and the resulting organic solution was
washed with water (15 mL). The two phases were separated and
the aqueous layer was re-extracted with EtOAc (6ꢁ10 mL). The
combined organic layers were washed with water (2ꢁ10 mL) fol-
lowed by brine (10 mL) and dried (MgSO4). The solvent was evap-
orated under reduced pressure and the crude residue obtained was
purified by column chromatography (EtOAc/petroleum ether
(40e60 ꢀC), 1:5) to yield the title compound 5 (1.39 g, 76%) as a deep
yellow oil; Rf 0.53 (EtOAc/petroleum ether (40e60 ꢀC), 2:8);
4.1.3. N-[N4-(Benzyoxycarbonyl)cytosin-1-ylacetyl]-N-(propyn-3-yl)
glycine ethyl ester (6b). The trifluoroacetic acid salt of N-(propyn-3-
yl)glycine ethyl ester was prepared as described for 6a. HBTU
(0.36 g, 1.50 mmol) followed by TEA (0.28 mL, 2.00 mmol) were
added to a stirred solution of N4-benzyloxycarbonyl cytosin-1-
ylacetic acid28 (0.32 g, 1.10 mmol) in DMF (6 mL) at rt and the
mixture was left to stir for 15 min. Subsequently, a solution of the
trifluoroacetic acid salt of N-(propyn-3-yl)glycine ethyl ester
(0.23 g, 1.02 mmol) in DMF (6 mL) was slowly added to the reaction
mixture followed by a further quantity of TEA (0.85 mL, 6.10 mmol).
The mixture was stirred at rt overnight and then the solvent was
removed in vacuo. The residue was dissolved in CH2Cl2 (10 mL) and
the resulting organic solution was washed successively with
1 M(aq) NaHCO3 (3ꢁ15 mL), 1 M(aq) KHSO4 (2ꢁ15 mL), water
(15 mL) and, finally, brine (15 mL). The organic layer was dried
(MgSO4) and the solvent was evaporated under reduced pressure.
The crude orange foam afforded was purified by column chroma-
tography (EtOAc/MeOH, 95:5) to yield the title compound 6b (0.22 g,
51%) as a white foam; mp 130e132 ꢀC; [Found: C, 59.02; H, 5.07; N,
13.19. C21H22N4O6 requires C, 59.15; H, 5.20; N, 13.14%]; Rf 0.24
nmax(film) 3270, 2980, 2934, 1748, 1706 cmꢂ1
; dH (400 MHz; CDCl3)
(two rotational isomers observed) 1.26e1.30 (3H, m, OCH2CH3),
1.39 and 1.49 (9H, s, C(CH3)3), 2.22 (1H, t, J 2.5, CH2C^CH), 3.89 and
4.09 (2H, s, NCH2CO), 4.13 and 4.19 (2H, s, NCH2C^CH), 4.18e4.22
(2H, m, OCH2CH3); dC (101 MHz; CDCl3) (two rotational isomers
observed) 14.1 and 14.2, 28.1 and 28.2, 36.6 and 37.1, 46.9 and 47.5,
61.0, 72.3 and 72.6, 78.7, 80.9 and 81.0, 154.6 and 154.7, 169.7; m/z
(EtOAc); nmax (KBr) 3476, 3280, 2984, 1744, 1668, 1629, 1558 cmꢂ1
;
dH (400 MHz; CDCl3) (two rotational isomers observed) 1.16 and
1.22 (2H, t, J 7.1, OCH2CH3), 2.21 and 2.32 (1H, t, J 2.4, CH2C^CH),

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4.07 and 4.20 (2H, q, J 7.1, OCH2CH3), 4.11 and 4.23 (2H, s, NCH2CO),
4.17 and 4.18 (2H, s, NCH2C^CH), 4.52 and 4.78 (2H, s, NCOCH2N),
5.62 (2H, s, PhCH2O), 6.96e7.02 (1H, m, cytosinyl C(5)eH),
7.26e7.32 (5H, m, Ph), 7.38e7.43 (1H, m, cytosinyl C(6)eH), 9.24
(1H, br s, NH); dC (CDCl3) (two rotational isomers were observed)
13.8, 36.3 and 38.2, 47.8 and 48.1, 50.8 and 50.9, 62.0 and 62.5, 67.9,
73.9 and 74.7, 76.4 and 76.8, 96.3, 128.4, 128.6, 128.7, 134.9, 149.8,
152.2, 157.3, 163.3, 167.5 and 167.6, 169.4 and 169.5; m/z (ESI) 427
([MþH]þ, 61%), 319 (3), 85 (100); HRMS (ESI) m/z: [MþH]þ, found
427.1612. C21H23N4O6 requires 427.1612.
2.61 (2H, t, J 6.2, NCH2CH2O), 3.46e3.52 (6H, m, CH2O(CH2)2O), 3.57
(4H, s, 2ꢁ NCH2Ph), 4.03 (2H, s, OCH2CO), 4.10 (2H, q, J 7.1,
OCH2CH3), 7.12e7.32 (10H, m, 2ꢁ Ph); dC (50 MHz; CDCl3) 14.4,
52.8, 59.1, 60.9, 68.9, 70.1, 70.5, 71.1, 126.9, 128.3, 128.9, 139.8, 170.6;
m/z (ESI) 372 ([MþH]þ, 100%), 153 (50); HRMS (ESI) m/z: [MþH]þ,
found 372.2170. C22H30NO4 requires 372.2169.
4.1.6. 8-Amino-3,6-dioxaoctan-1-ol (9). Finely powdered NaBH4
(3.86 g, 102 mmol) was added portionwise, over a period of 15 min,
to a stirred solution of 8 (5.83 g, 15.7 mmol) in THF (80 mL) at
reflux. Subsequently, methanol (80 mL) was carefully added drop-
wise to the stirred reaction mixture at reflux over another 15 min.
The resulting mixture was left to stir at reflux for a further 60 min.
After this time, the mixture was allowed to cool to rt before the
reaction was quenched by the careful addition of a saturated
aqueous solution of NH4Cl (80 mL). The organic layer was separated
and the aqueous phase was extracted with EtOAc (6ꢁ80 mL). The
combined organic layers were dried (MgSO4) and the solvent was
removed under reduced pressure. The crude oil afforded was pu-
rified by column chromatography (EtOAc/petroleum ether
(40e60 ꢀC), 2:1) to yield the intermediate, 8-dibenzylamino-3,6-
dioxaoctan-1-ol (4.22 g, 82%), as a colourless oil; Rf 0.33 (EtOAc/
petroleum ether (40e60 ꢀC), 7:3); nmax (film) 3436, 3027, 2871,
4.1.4. N-[N6-(Benzyloxycarbonyl)adenin-9-ylacetyl]-N-(propyn-3-yl)
glycine ethyl ester (6c). The trifluoroacetic acid salt of N-(propyn-3-
yl)glycine ethyl ester was prepared as described for 6a. HBTU
(0.40 g, 1.06 mmol) followed by DIPEA (0.19 mL, 1.10 mmol) were
added to a stirred solution of N6-(benzyloxycarbonyl)adenin-9-
ylacetic acid28 (0.33 g, 1.01 mmol) in DMF (30 mL) at rt and the
mixture was left to stir for 15 min. Subsequently, a solution of the
trifluoroacetic acid salt of N-(propyn-3-yl)glycine ethyl ester
(0.30 g, 1.17 mmol) in DMF (20 mL) was slowly added to the re-
action mixture followed by a further quantity of DIPEA (0.34 mL,
1.93 mmol). The mixture was stirred at rt overnight and then the
solvent was removed in vacuo. The residue was dissolved in CH2Cl2
(10 mL) and the resulting organic solution was washed successively
with 1 M(aq) NaHCO3 (3ꢁ15 mL), 1 M(aq) KHSO4 (2ꢁ15 mL), water
(15 mL) and, finally, brine (15 mL). The organic layer was dried
(MgSO4) and the solvent was evaporated under reduced pressure.
The crude orange foam afforded was purified by column chroma-
tography (EtOAc/MeOH, 9:1) to yield the title compound 6c (0.16 g,
36%) as a white solid; mp 120e122 ꢀC; [Found C, 58.49; H, 4.62; N,
18.86. C22H22N6O5 requires C, 58.66; H, 4.92; N, 18.66%]; Rf 0.39
(EtOAc/MeOH, 95:5); nmax (KBr) 3237, 3190, 3125, 3029, 2957, 1763,
2871, 1494, 1453 cmꢂ1
; dH (200 MHz; CDCl3) 2.73 (2H, t, J 6.2,
NCH2CH2O), 2.90 (1H, br s, OH), 3.57e3.69 (m, 10H, CH2O(-
CH2)2O(CH2)2OH), 3.72 (4H, s, 2ꢁ NCH2Ph), 7.24e7.48 (10H, m, 2ꢁ
NCH2Ph); dC (50 MHz; CDCl3) 52.8, 59.1, 61.8, 70.1, 70.5, 72.7, 127.0,
128.3, 128.9, 139.8; m/z (CI) 330 ([MþH]þ, 100%), 268 (3), 254 (6),
240 (30), 238 (23), 210 (30); HRMS (ESI) m/z: [MþH]þ, found
330.2068. C20H28NO3 requires 330.2064.
Using a 10% Pd/C cartridge, a 0.05 M solution of 8-dibenzylamino-
3,6-dioxaoctan-1-ol (1.00 g, 3.04 mmol) in absolute ethanol (60 mL)
was cycled twice through an H-CubeÒ continuous-flow reactor at
a flow rate of 1 mL/min. The applied pressure was set to 70 bar and
the temperature to 70 ꢀC. At the end of the second run, the entire
reaction mixture was collected and concentrated in vacuo to give the
title compound 9 (0.45 g, 99%) as a colourless oil; nmax (film) 3365,
1749, 1646, 1610 cmꢂ1
; dH (400 MHz; CDCl3) (two rotational iso-
mers were observed) 1.24 and 1.32 (2H, t, J 7.1, OCH2CH3), 2.30 and
2.48 (1H, t, J 2.4, CH2C^CH), 4.18 and 4.28 (2H, q, J 7.1, OCH2CH3),
4.25 and 4.38 (2H, s, NCH2CO), 4.31 and 4.32 (2H, s, NCH2C^CH),
4.98 and 5.17 (2H, s, NCOCH2N), 5.28 (2H, s, PhCH2O), 7.32e7.42
(5H, m, Ph), 8.02 and 8.05 (1H, s, adeninyl C(8)eH), 8.71 and 8.74
(1H, s, adeninyl C(2)eH), 8.86 (1H, br s, NH); dC (101 MHz; CDCl3)
(two rotational isomers were observed) 14.0 and 14.1, 36.4 and 38.1,
43.7 and 43.8, 47.5 and 47.9, 61.6 and 62.3, 67.1, 73.9 and 74.7, 76.7
and 77.2, 121.4, 128.4, 128.6, 128.7, 135.4, 143.1 and 143.8, 149.4 and
149.5, 150.9 and 151.0, 151.4 and 151.5, 152.9 and 153.1, 165.8 and
165.9, 167.2; m/z (ESI) 451 ([MþH]þ, 100%), 342 (36); HRMS (ESI) m/
z: [MþH]þ, found 451.1725. C22H23N6O5 requires 451.1724.
3312, 3302, 2873, 2854, 1599 cmꢂ1
; dH (200 MHz; D2O) 2.88 (2H, t, J
5.3, NH2CH2CH2O), 3.54e3.72 (10H, m, CH2O(CH2)2O(CH2)2OH); dC
(50 MHz; D2O) 39.1, 60.0, 68.6, 69.2, 69.3, 71.4.
4.1.7. 8-tert-Butoxycarbonylamino-3,6-dioxaoct-1-yl azide (10). Di-
tert-butyl dicarbonate (1.43 g, 6.57 mmol) was added to a stirred
solution of 9 (0.48 g, 3.19 mmol) in 10% (v/v) TEA/MeOH (20 mL) at
rt. The reaction mixture was heated at reflux for 3 h and then
allowed to cool to rt. The solvent was removed in vacuo and the
residual oil was purified by column chromatography (EtOAc/MeOH,
95:5) to yield the intermediate, 8-tert-butoxycarbonylamino-3,6-
dioxaoctan-1-ol (0.67 g, 84%), as a colourless oil; Rf 0.40 (EtOAc/
4.1.5. 8-Dibenzylamino-3,6-dioxaoctanoic acid ethyl ester (8). A so-
lution of N,N-dibenzyl-5-amino-3-oxapentan-1-ol32 (2.86 g,
10.00 mmol) in anhydrous THF (8 mL) was added to a stirred slurry
of NaH (60% disp. in mineral oil, 0.48 g, 12.0 mmol) in anhydrous
THF (15 mL) at rt. The mixture was heated at reflux for 2 h. After
being cooled to 0 ꢀC, ethyl bromoacetate (1.22 mL, 11.0 mmol) was
slowly added dropwise and the mixture was allowed to warm to rt
before being left to stir for 17 h. Subsequently, the reaction was
quenched by the careful addition of water (5 mL) and THF was
removed under reduced pressure. The aqueous solution was
extracted with EtOAc (5ꢁ20 mL) and the combined organic layers
were washed with water (2ꢁ10 mL) followed by brine (10 mL). The
combined aqueous phases were re-extracted with a further quan-
tity of EtOAc (20 mL). The combined organic layers were dried
(MgSO4) and the solvent was removed under reduced pressure to
yield a brown liquid. The crude product was purified by column
chromatography (EtOAc/petroleum ether (40e60 ꢀC), 4:6) to give
the title compound 8 (2.16 g, 40%) as a pale yellow oil; Rf 0.38
(EtOAc/petroleum ether (40e60 ꢀC), 4:6); nmax (film) 3027, 2872,
MeOH, 95:5); nmax (film) 3356, 2976, 1694, 1526 cmꢂ1
; dH
(200 MHz; CDCl3) 1.38 (9H, s, C(CH3)3), 2.98 (1H, br s, OH), 3.25 (2H,
br dt, NHCH2CH2O), 3.49 (2H, t, J 5.3, NHCH2CH2O), 3.54e3.61 (6H,
m, O(CH2)2OCH2), 3.68 (2H, br dt, CH2CH2OH), 5.27 (1H, br s, NH); dC
(50 MHz; CDCl3) 28.7, 39.9, 61.3, 69.9, 70.5, 70.6, 72.3, 79.5, 156.3;
m/z (CI) 250 ([MþH]þ, 15%), 150 (100); HRMS (ESI) m/z: [MþH]þ,
found 250.1649. C11H24NO5 requires 250.1649.
Methanesulfonyl chloride (0.18 mL, 2.29 mmol) was added
slowly dropwise to
a
stirred solution of 8-tert-butox-
ycarbonylamino-3,6-dioxaoctan-1-ol (0.52 g, 2.08 mmol) and TEA
(0.44 mL, 3.12 mmol) in anhydrous CH2Cl2 (10 mL) at 0 ꢀC under N2
atmosphere. The reaction mixture was allowed to slowly warm to rt
before being left to stir overnight. After this time, the reaction was
quenched by the addition of ice cool water (10 mL) and the two
phases were separated. The organic layer was washed with a satu-
rated aqueous solution of NaHCO3 (10 mL) followed by brine
(10 mL) and dried (MgSO4). The solvent was removed under
1754, 1494 cmꢂ1
; dH (200 MHz; CDCl3) 1.16 (3H, t, J 7.1, OCH2CH3),

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9593
reduced pressure and the crude residue obtained was purified by
column chromatography (EtOAc/petroleum ether (40e60 ꢀC), 4:6)
to give the intermediate, 8-tert-butoxycarbonylamino-3,6-
dioxaoct-1-yl methanesulfonate (0.53 g, 78%), as a colourless oil:
Rf 0.46 (EtOAc/MeOH, 95:5); nmax (film) 2976, 2935, 2874, 1708,
glycine ethyl ester (11b). A solution of the cytosinyl monomer 6b
(0.29 g, 0.69 mmol) in CH2Cl2 (5 mL) was added to a stirred solution
of the azide 10 (0.18 g, 0.68 mmol) in H2O/t-BuOH (1:1) (10 mL) at
rt. Subsequently, CuSO4$5H2O (0.04 g, 0.17 mmol) followed by
1 M(aq) sodium ascorbate solution (0.34 mL, 0.34 mmol) were
added and the resulting yellow solution was left to stir for 48 h.
After this time, the two layers were separated and the organic
phase was dried (MgSO4) before being concentrated in vacuo. The
crude residue obtained was purified by column chromatography
(EtOAc/MeOH, 93:7) to give the title compound 11b (0.32 g, 76%) as
an off-white foam; Rf 0.26 (EtOAc/MeOH, 93:7); nmax (film) 3262,
1521, 1352, 1175 cmꢂ1
; dH (200 MHz; CDCl3) 1.38 (9H, s, C(CH3)3),
3.01 (3H, s, OSO2CH3), 3.24 (2H, br dt, NHCH2CH2O), 3.47 (2H, t, J
5.3, NHCH2CH2O), 3.58e3.61 (4H, m, OCH2CH2O), 3.71 (2H, t, J 4.6,
OCH2CH2OSO2), 4.33 (2H, t, J 4.6, OCH2CH2OSO2), 4.93 (1H, br s,
NH); dC (50 MHz; CDCl3) 28.6, 37.9, 40.5, 69.2, 69.4, 70.4, 70.5, 70.8,
79.5, 155.8; m/z (CI) 345 ([MþNH3]þ, 17%), 328 ([MþH]þ, 20), 289
(70), 271 (100), 228 (35); HRMS (ESI) m/z: [MþH]þ, found 328.1426.
C12H26NO7S requires 328.1424.
3143, 2979, 2931, 1745, 1692, 1668, 1625 cmꢂ1
; dH (400 MHz; CDCl3)
(two rotational isomers were observed) 1.22 and 1.27 (3H, t, J 7.1,
OCH2CH3), 1.42 (9H, s, C(CH3)3), 3.22e3.31 (2H, m, NHCH2CH2O),
3.47e3.67 (6H, m, CH2O(CH2)2O), 3.79e3.88 (2H, m, triazolyl N(1)e
CH2CH2O), 4.09 (2H, br s, NCH2CO), 4.13 and 4.18 (2H, q, J 7.1,
OCH2CH3), 4.39 and 4.88 (2H, s, NCOCH2N), 4.48 and 4.55 (2H, br t, J
4.9, triazolyl N(1)eCH2CH2O), 4.67 and 4.76 (2H, s, triazolyl C(4)e
CH2N), 5.19 (2H, s, PhCH2O), 6.67 and 6.99 (1H, br t, NH), 7.20e7.65
(7H, m, Ph and cytosinyl C(5)eH and C(6)eH), 7.71 and 8.12 (1H, s,
triazolyl C(5)eH); dC (101 MHz; CDCl3) (two rotational isomers
were observed) 14.1, 28.3, 40.3, 42.8 and 43.8, 47.5, 49.1, 49.7 and
50.3, 61.4, 67.9, 69.9, 70.2, 70.4, 79.3, 95.2, 123.1, 128.3, 128.4, 128.7,
134.4, 143.2, 149.9, 152.1, 153.0, 155.6, 163.2, 167.1, 168.6; m/z (ESI)
701 ([MþH]þ, 100%), 601(13); HRMS (ESI) m/z: [MþH]þ, found
701.3243. C32H45N8O10 requires 701.3253.
Sodium azide (0.31 g, 4.76 mmol) was added to a stirred solu-
tion of 8-tert-butoxycarbonylamino-3,6-dioxaoct-1-yl meth-
anesulfonate (0.59 g, 1.81 mmol) in anhydrous DMF (20 mL) at rt
under N2 atmosphere. The reaction mixture was gently heated at
60 ꢀC for 4 h. After this time, the reaction was poured onto a mix-
ture of ice/water (1:1) (ca. 20 mL) and the resulting aqueous solu-
tion was extracted with CH2Cl2 (3ꢁ20 mL). The organic phase was
separated, dried (MgSO4) and the solvent was carefully removed
under reduced pressure to give the title compound 10 (0.45 g, 92%)
as a yellow oil. This was used directly, without further purification,
in the click reactions to prepare 11aec and 12; nmax (film) 3360,
2977, 2930, 2870, 2109, 1713 cmꢂ1
; dH (400 MHz; CDCl3) 1.42 (9H, s,
C(CH3)3), 3.27e3.30 (2H, m, NHCH2CH2O), 3.38 (2H, t, J 5.3,
OCH2CH2N3), 3.52 (2H, t, J 5.3, NHCH2CH2O), 3.59e3.64 (4H, m,
OCH2CH2O), 3.65 (2H, t, J 5.3, OCH2CH2N3), 5.00 (1H, br s, NH); dC
(101 MHz; CDCl3) 28.4, 40.3, 50.6, 70.0, 70.2, 70.3, 70.5, 79.1, 155.9;
m/z (CI) 292 ([MþNH4]þ, 7%), 275 ([MþH]þ, 12), 236 (50), 175 (95),
44 (100); HRMS (ESI) m/z: [MþH]þ, found 275.1715. C11H23N4O4
requires 275.1714.
4.1.10. N-[N1-(8-tert-Butoxycarbonylamino-3,6-dioxaoct-1-yl)-1,2,3-
triazol-4-yl]methyl-N-[N6-(benzyloxycarbonyl)adenin-9-ylacetyl]gly-
cine ethyl ester (11c). A solution of the adeninyl monomer 6c
(0.47 g, 1.05 mmol) in CH2Cl2 (5 mL) was added to a stirred solution
of the azide 10 (0.24 g, 0.87 mmol) in H2O/t-BuOH (1:1) (10 mL) at
rt. Subsequently, CuSO4$5H2O (0.05 g, 0.22 mmol) followed by
1 M(aq) sodium ascorbate solution (0.44 mL, 0.44 mmol) were
added and the resulting yellow solution was left to stir for 48 h.
After this time, the two layers were separated and the organic
phase was dried (MgSO4) before being concentrated in vacuo. The
crude residue obtained was purified by column chromatography
(EtOAc/MeOH, 9:1) to give the title compound 11c (0.40 g, 64%) as
a white foam; Rf 0.20 (EtOAc/MeOH, 9:1); nmax (film) 3362, 3248,
4.1.8. N-[N1-(8-tert-Butoxycarbonylamino-3,6-dioxaoct-1-yl)-1,2,3-
triazol-4-yl]methyl-N-(thymin-1-ylacetyl)glycine ethyl ester (11a). A
solution of the thyminyl monomer 6a (0.11 g, 0.34 mmol) in CH2Cl2
(3 mL) was added to a stirred solution of the azide 10 (0.09 g,
0.33 mmol) in H2O/t-BuOH (1:1) (6 mL) at rt. Subsequently,
CuSO4$5H2O (0.01 g, 0.03 mmol) followed by 1 M(aq) sodium
ascorbate solution (0.07 mL, 0.07 mmol) were added and the
resulting yellow solution was left to stir for 48 h. After this time, the
two layers were separated and the organic phase was dried
(MgSO4) before being concentrated in vacuo. The crude residue
obtained was purified by column chromatography (EtOAc/MeOH,
95:5) to give the title compound 11a (0.14 g, 76%) as a yellow foam;
Rf¼0.12 (EtOAc/MeOH, 95:5); nmax (film) 3357, 2978, 1743, 1679,
3134, 2988, 2936, 2884, 1747, 1706, 1674, 1612 cmꢂ1
; dH (400 MHz;
CDCl3) (two rotational isomers were observed) 1.19 and 1.22 (3H, t, J
7.1, OCH2CH3),1.41 (9H, s, C(CH3)3), 3.24e3.32 (2H, m, NHCH2CH2O),
3.45 (2H, t, J 4.9, NHCH2CH2O), 3.51e3.63 (4H, m, OCH2CH2O), 3.79
and 3.86 (2H, t, J 4.9, triazolyl N(1)eCH2CH2O), 4.13 and 4.24 (2H, q,
J 7.1, OCH2CH3), 4.18 and 4.42 (2H, s, NCH2CO), 4.50 and 4.60 (2H, t, J
4.9, triazolyl N(1)eCH2CH2O), 4.71 and 4.84 (2H, s, NCOCH2N), 5.01
and 5.42 (2H, s, triazolyl C(4)eCH2N), 5.10 and 5.22 (1H, br s, NH),
5.30 (2H, s, PhCH2O), 7.33e7.45 (5H, m, Ph), 7.73 and 7.97 (1H, s,
triazolyl C(5)eH), 8.12 (1H, br s, adeninyl C(8)eH), 8.72 and 8.76
(1H, s, adeninyl C(2)eH), 8.89 (1H, br s, NH); dC (101 MHz; CDCl3)
(two rotational isomers were observed) 14.1 and 14.2, 28.4, 40.3,
42.8 and 43.8, 43.8 and 44.2, 47.8 and 49.2, 50.2 and 50.5, 61.5 and
62.2, 67.7, 69.1 and 69.2, 70.1, 70.4, 70.6, 79.5, 121.4, 123.6 and 124.5,
128.5, 128.6, 128.7, 135.5, 141.8 and 142.3, 144.1, 149.3, 151.1, 151.4,
152.8, 156.0, 166.3, 168.6 and 168.7; m/z (ESI) 725 ([MþH]þ, 100%),
649 (15); HRMS (ESI) m/z: [MþH]þ, found 725.3358. C33H45N10O9
requires 725.3365.
1678, 1514 cmꢂ1
; dH (400 MHz; CDCl3) (two rotational isomers
were observed) 1.19 and 1.22 (3H, t, J 7.1, OCH2CH3), 1.42 (9H, s,
C(CH3)3), 1.89 and 1.91 (3H, s, thyminyl C(5)eH), 3.28e3.30 (2H, m,
NHCH2CH2O), 3.50e3.54 (2H, m, NHCH2CH2O), 3.56e3.61 (4H, m,
OCH2CH2O), 3.78e3.85 (2H, m, triazolyl N(1)eCH2CH2O), 4.14 and
4.28 (2H, s, NCH2CO), 4.16 and 4.18 (2H, q, J 7.1, OCH2CH3), 4.42 and
4.80 (2H, s, NCOCH2N), 4.50 and 4.56 (2H, t, J 4.9, triazolyl N(1)e
CH2CH2O), 4.68 and 4.73 (2H, s, triazolyl C(4)eCH2N), 5.17 and 5.23
(1H, br t, NH), 7.02 and 7.05 (1H, s, thyminyl C(6)eH), 7.73 and 7.94
(1H, s, triazolyl C(5)eH), 9.06 and 9.20 (1H, br s, thyminyl N(3)eH);
dC (101 MHz; CDCl3) (two rotational isomers were observed) 12.3,
15.2, 28.4, 40.1, 42.6 and 43.4, 47.6 and 48.1, 47.8 and 48.9, 50.2 and
50.4, 62.1, 69.3, 69.9, 70.2, 70.4, 79.2 and 79.3, 110.8 and 110.8, 123.7
and 124.3, 140.8 and 140.9, 144.2 and 144.3, 151.1, 156.0, 164.1, 167.1
and 167.2, 168.6 and 168.9; m/z (ESI) 582 ([MþH]þ, 100%), 482 (65);
HRMS (ESI) m/z: [MþH]þ, found 582.2881. C25H40N7O9 requires
582.2882.
4.1.11. N-[N1-(8-tert-Butoxycarbonylamino-3,6-dioxaoct-1-yl)-1,2,3-
triazol-4-yl]methyl-N-[tert-butoxycarbonylamino]glycine ethyl ester
(12). A solution of the propynyl derivative 5 (0.09 g, 0.36 mmol) in
CH2Cl2 (3 mL) was added to a stirred solution of the azide 10
(0.09 g, 0.33 mmol) in H2O/t-BuOH (1:1) (6 mL) at rt. Subsequently,
CuSO4$5H2O (0.01 g, 0.03 mmol) followed by 1 M(aq) sodium
ascorbate solution (0.07 mL, 0.07 mmol) were added and the
4.1.9. N-[N1-(8-tert-Butoxycarbonylamino-3,6-dioxaoct-1-yl)-1,2,3-
triazol-4-yl]methyl-N-[N4-(benzyloxycarbonyl)cytosin-1-ylacetyl]

ꢀ
N.M. Howarth, J. Ricci (nee Goujon) / Tetrahedron 67 (2011) 9588e9594
9594
resulting deep yellow solution was left to stir for 48 h. After this
time, the two layers were separated and the organic phase was
dried (MgSO4) before being concentrated in vacuo. The crude res-
idue obtained was purified by column chromatography (EtOAc/
MeOH, 9:1) to give the title compound 12 (0.13 g, 76%) as an off-
white solid: Rf 0.40 (EtOAc/MeOH, 9:1); nmax (film) 3362, 2977,
829606. Copies of the data can be obtained, free of charge, on ap-
plication to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44
(0)1223 336033 or email: deposit@ccdc.cam.ac.uk).
Acknowledgements
2933, 1748, 1704, 1514 cmꢂ1
; dH (400 MHz; CDCl3) (two rotational
The authors thank ScotChem and Heriot-Watt University for
financial support (to J.R.), Dr. A.S.F. Boyd for recording NMR spectra,
Dr. G.M. Rosair for X-ray crystallography, Mrs. C. Graham for ele-
mental analysis and the EPSRC National Mass Spectrometry Service
Centre, Swansea, U.K. for LRMS and HRMS data.
isomers were observed) 1.22 (3H, t, J 7.2, OCH2CH3), 1.38 (9H, s,
C(CH3)3), 1.40 (9H, s, C(CH3)3), 3.28 (2H, br dt, NHCH2CH2O), 3.52
(2H, m, NHCH2CH2O), 3.56 (4H, m, OCH2CH2O), 3.82 (2H, m, tri-
azolyl N(1)eCH2CH2O), 3.95 and 3.99 (2H, s, NCH2CO), 4.13 (2H, q, J
7.2, OCH2CH3), 4.44 (2H, m, triazolyl N(1)eCH2CH2O), 4.55 and 4.57
(2H, s, triazolyl C(4)eCH2N), 4.92 and 5.20 (1H, br t, NH), 7.56 and
7.75 (1H, s, triazolyl C(5)eH); dC (101 MHz; CDCl3) (two rotational
isomers were observed) 14.0 and 14.1, 28.2, 28.3, 40.3, 42.8 and
43.4, 48.1 and 48.8, 50.1 and 50.2, 60.8 and 60.9, 69.4 and 69.5, 70.0,
70.2, 70., 80.5, 80.6, 123.1 and 123.9, 144.2 and 144.6, 155.2, 155.9,
169.8; m/z (EI) 515 ([M]þ, 20%).
References and notes
1. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596e2599.
2. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057e3064.
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2004e2021.
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6. Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249e1262.
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Med. Res. Rev. 2008, 28, 278e308.
4.2. X-ray crystallographic diffraction study of N-(thymin-1-
ylacetyl)-N-(propyn-3-yl)glycine ethyl ester (6a)
8. New, K.; Brechbiel, M. W. Cancer Biother. Radiopharm. 2009, 24, 289e302.
9. Howarth, N. M.; Lindsell, W. E.; Murray, E.; Preston, P. N. Tetrahedron Lett. 2003,
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8875e8887.
11. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 54, 1497e1500.
12. Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998,
37, 2796e2823.
The crystals of 6a were grown at rt using the vapour diffusion
method in which the solvent was methanol and the precipitant was
diethyl ether. Single crystal X-ray diffraction data were collected at
100 K on a Bruker X8 APEX2 CCD diffractometer36 using the APEX2
suite of programs.37 The positions of hydrogen atoms were calculated
and constrained to idealised geometry, except for the thyminyl N(3)e
H hydrogen atom, which was located from the difference Fourier map
and its isotropic displacement parameter was constrained to
13. Chance, R. R.; Patel, G. N.; Witt, J. D. J. Chem. Phys. 1979, 71, 206e211.
14. (a) Chance, R. R. Macromolecules 1980, 13, 396e398; (b) Rawiso, M.; Aime, J. P.;
€
Fave, J. L.; Schott, M.; Muller, M. A.; Schmidt, M.; Baumgartl, H.; Wegner, G. J.
Phys. 1988, 49, 861e880.
ꢁ
1.2ꢁU(eq) of N(3) and the NeH distance restrained to 0.90(2) A. The
15. (a) Preziosi, A. F.; Bhattacharjee, H. R.; Patel, G. N. Polym. Prepr. (Am. Chem. Soc.,
Div. Polym. Chem.) 1980, 21, 166e167; (b) Bhattacharjee, H. R.; Preziosi, A. F.;
Patel, G. N. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1980, 21, 168e169;
(c) Bhattacharjee, H. R.; Preziosi, A. F.; Patel, G. N. J. Chem. Phys. 1980, 73,
1478e1480; (d) Patel, G. N.; Preziosi, A. F.; Bhattacharjee, H. R. J. Polym. Sci. Pol.
Sym. 1984, 71, 247e257.
crystal was not single and was treated as having three components.
The cell was found using CELL_NOW and the intensity data was in-
tegrated using SAINT_PLUS followed by scaling and correction by
TWINABS. The crystal parameters and structure refinement details
for 6a are given in Table 1. Crystallographic data (excluding structure
factors) for this structure have been deposited in the Cambridge
Crystallographic Data Centre as supplementary publication no. CCDC
16. Agh-Atabay, N. M.; Lindsell, W. E.; Preston, P. N.; Tomb, P. J. Polym. Int. 1993, 31,
367e374.
17. Yoon, B.; Lee, S.; Kim, J.-M. Chem. Soc. Rev. 2009, 38, 1958e1968.
€
€
18. Gasser, G.; Husken, N.; Koster, S. D.; Metzler-Nolte, N. Chem. Commun. 2008,
3675e3677.
Table 1
19. Sosniak, A. M.; Gasser, G.; Metzler-Nolte, N. Org. Biomol. Chem. 2009, 7,
4992e5000.
Crystallographic data and structure refinement details for compound 6a
€
€
20. Husken, N.; Gasser, G.; Koster, S. D.; Metzler-Nolte, N. Bioconjugate Chem. 2009,
20, 1578e1586.
Parameter
6a
€
21. Husken, N.; Ge˛bala, M.; Schuhmann, W.; Metzler-Nolte, N. ChemBioChem 2010,
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C
14H17N3O5
11, 1754e1761.
307.31
100(2) K
0.71073 A
Monoclinic
P2(1)/n
22. Gogoi, K.; Mane, M. V.; Kunte, S. S.; Kumar, V. A. Nucleic Acids Res. 2007, 35
e139.
ꢁ
23. Lim, S. Y.; Chung, W.-Y.; Lee, H. K.; Park, M. S.; Park, H. G. Biochem. Biophys. Res.
Commun. 2008, 376, 633e636.
€
24. Gasser, G.; Jager, K.; Zenker, M.; Bergmann, R.; Steinbach, J.; Stephan, H.;
¼90ꢀ
ꢁ
a
Unit cell dimensions
a¼4.8969(15) A
Metzler-Nolte, N. J. Inorg. Biochem. 2010, 104, 1133e1140.
25. Gasser, G.; Sosniak, A. M.; Leonidova, A.; Braband, H.; Metzler-Nolte, N. Aust. J.
Chem. 2011, 64, 265e272.
¼96.444(13)ꢀ
ꢁ
b¼15.069(5) A
b
¼90ꢀ
ꢁ
c¼20.118(5) A
g
3
ꢁ
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1475.2(8) A
4
26. Bodansky, M.; Bodansky, A. The Practice of Peptide Synthesis, 2nd ed.; Springer:
Berlin, 1994.
1.384 Mg/m3
0.107 mmꢂ1
648
27. Kosynkina, L.; Wang, W.; Liang, T. C. Tetrahedron Lett. 1994, 35, 5173e5176.
28. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen, H. F.; Vulpius,
T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59,
5767e5773.
Crystal size
0.44ꢁ0.20ꢁ0.12 mm3
29. Chen, S.-M.; Mohan, V.; Kiely, J. S.; Griffith, M. C.; Griffey, R. H. Tetrahedron Lett.
q
Range for data collection
1.69e33.12ꢀ
1994, 35, 5105e5108.
Index ranges
Reflections collected
Independent reflections
ꢂ6ꢃhꢃ6, 0ꢃkꢃ21, 0ꢃlꢃ28
31,957
30. Goujon, J. Towards the Development of a Novel Colourimetric Nucleic Acid
Biosensor Based on Peptide Nucleic Acid-Functionalised Polydiacetylene Lipo-
somes. Ph.D. Thesis, Heriot-Watt University, U.K, 2009.
31. Sanjayan, G. J.; Pedireddi, V. R.; Ganesh, K. N. Org. Lett. 2000, 2, 2825e2828.
32. Koskinen, A. M. P.; Valo, T.; Vihavainen, S.; Hakala, J. M. L. Bioorg. Med. Chem.
Lett. 1995, 5, 573e578.
33. da Costa, J. C. S.; Pais, K. C.; Fernandes, E. L.; de Oliveira, P. S. M.; Mendonc¸ a, J. S.;
de Souza, M. V. N.; Peralta, M. A.; Vasconcelos, T. R. A. Arkivoc 2006, i, 128e133.
34. Procter, G.; Leonard, J.; Lygo, B. Advanced Practical Organic Chemistry, 2nd ed.;
CRC: Cheltenham, UK, 1994.
35. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923e2925.
36. Bruker. APEX2 Version 2.1; Bruker AXS: Madison, Wisconsin, USA, 2004.
37. Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112e122.
6683 [Rint¼0.0763]
Completeness to
q
¼25.00ꢀ
89.5%
Absorption correction
Max. and min transmission
Refinement method
Semi-empirical from equivalents
0.9873 and 0.6355
Full-matrix least-squares on F2
6683/19/206
Data/restraints/parameters
Goodness-of-fit on F2
0.996
Final R indices [I>2
R indices (all data)
Largest diff. peak and hole
s
(I)]
R1¼0.0922, wR2¼0.2518
R1¼0.1223, wR2¼0.2700
ꢁꢂ3
0.395 and ꢂ0.518 e A