Carboxyalkyl peptoid PNAs: Synthesis and hybridization properties

Article abstract of DOI:10.1016/j.tet.2011.11.017

N^γ-Carboxyalkyl modified peptide nucleic acids (PNAs), containing the four canonical nucleobases, were prepared via solid-phase oligomerization. The inserted peptoid monomers 1 and 2 were constructed through simple synthetic procedures, utilizi

Full text of DOI:10.1016/j.tet.2011.11.017

Tetrahedron 68 (2012) 499e506
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Carboxyalkyl peptoid PNAs: synthesis and hybridization properties
,
a,
*
Chiara De Cola a, Alex Manicardi b, Roberto Corradini b, Irene Izzo a , Francesco De Riccardis
*
a
ꢀ
Dipartimento di Chimica e Biologia, Universita di Salerno, Via Ponte don Melillo, Fisciano (SA) 84084, Italy
b
ꢀ
Dipartimento di Chimica Organica e Industriale, Universita di Parma, Parco Area delle Scienze 17/A, Parma 43124, Italy
a r t i c l e i n f o
a b s t r a c t
Ng-Carboxyalkyl modified peptide nucleic acids (PNAs), containing the four canonical nucleobases, were
prepared via solid-phase oligomerization. The inserted peptoid monomers 1 and 2 were constructed
through simple synthetic procedures, utilizing appropriate glycidol and iodoalkyl electrophiles. Thermal
denaturation studies, performed with complementary antiparallel DNA strands, demonstrated that the
length of the Ng-side chain strongly influences the modified PNAs hybridization properties. Moreover,
Article history:
Received 2 August 2011
Received in revised form 21 October 2011
Accepted 7 November 2011
Available online 15 November 2011
multiple negative charges on the oligoamide backbone, when present on
proved to be beneficial for the oligomers’ water solubility and DNA hybridization specificity.
Ó 2011 Elsevier Ltd. All rights reserved.
g-nitrogen C6 side chains
Keywords:
Peptide nucleic acids
Peptoids
DNA recognition
1. Introduction
triazine-tagged oligoglycines systems,10 the oligomeric thymine-
functionalized peptoids,8d the achiral Ng-
u
-aminoalkyl nucleic
Nucleic acids encode structures and functions of all living sys-
tems. None of the multitudinous synthetically available homo-
morphous frameworks has ever displayed the phospho(deoxy)
ribosyl backbone properties.1 However, polyamides bearing ca-
nonical nucleobases, such as N-(2-aminoethyl)glycine PNA
(aegPNA),2 deeply interfere with the DNA/RNA functions and, in 20
years of biophysical and biological studies, have demonstrated
unmatched recognition and antisense properties.3
acids,8a constitute convincing examples of
g-nitrogen beneficial
modification. In particular, the Liu group contribution,8a revealed an
unexpected stereoelectronic effect played by the Ng-side chain
length. In their stringent analysis it was demonstrated that while
short
PNAs hybridization properties, longer
itively modulated nucleic acids binding. It was also found that sup-
pression of the positive -aminoalkyl charge (i.e., through
u
-amino Ng-side chains negatively influenced the modified
u
-amino Ng-side chains pos-
u
The considerable biological stability, the excellent nucleic acids
binding properties, and the appreciable chemical simplicity, make
PNA an invaluable tool in molecular biology.4 Unfortunately, despite
these remarkable properties, PNA has two serious limitations: low
water solubility5 and poor cellular uptake.6 Considerable efforts
have been made to circumvent these drawbacks, and a conspicuous
number of new analogs have been proposed,7 including those with
acetylation) caused no reduction in the hybridization affinity, sug-
gesting that factors different from mere electrostatic stabilizing in-
teractions were at play in the hybrid aminopeptoid-PNA/DNA (RNA)
duplexes.11
Considering the interesting results achieved in the case of N-(2-
alkylaminoethyl)-glycine units,8,9 and on the basis of poor hybrid-
ization properties showed by two fully peptoidic homopyrimidine
oligomers synthesized by our group,8b we decided to explore the
the
g
-nitrogen modified N-(2-aminoethyl)-glycine (aeg) units.8
In an elegant contribution by the Nielsen group,9 an accurate in-
effects of anionic residues at the g-nitrogen in a PNA framework on
the in vitro hybridization properties.
vestigation of the Ng-methylated PNA hybridization properties was
reported. Inthisstudy itwasfound that the formation ofPNA/DNA (or
RNA) duplexes was not altered in case of a 30% Ng-methyl nucleobase
substitution. However, the hybridization efficiency per N-methyl unit
in a PNA, decreased on increasing the N-methyl content.
The N-(carboxymethyl) and the N-(carboxypentamethylene)
Ng-residues, present in monomers 1 and 2 (Fig. 1) were chosen in
order to evaluate possible side chains length-dependent thermal
denaturations effects, and with the aim to respond to the water-
solubility issue, which is crucial for the specific subcellular
distribution.6a
The negative impact of the g-N alteration reported by Nielsen, did
not discourage further investigations. The potentially informative
The synthesis of a negatively charged N-(2-carboxyalkyla-
minoethyl)-glycine backbone (negative charged PNA are rarely
found in literature)12 was based on the idea of taking advantage of
the availability of a multitude of efficient methods for the gene
* Corresponding authors. Tel.: þ39 (0)89 969560; fax: þ39 (0)89 969603 (I.I.);
tel.: þ39 (0)89 969552; fax: þ39 (0)89 969603 (F.D.R.); e-mail addresses: iizzo@
unisa.it (I. Izzo), dericca@unisa.it (F. De Riccardis).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.11.017

500
C. De Cola et al. / Tetrahedron 68 (2012) 499e506
NH2
O
c
a
R
N
OH
+
t-BuO
OH
t-BuO
OH
O
O
b
9, R = H
7
8
10, R = Fmoc
O
H
Fmoc
Fig. 1. Structures of bis-protected thyminylated Ng-
u
-carboxyalkyl monomers 1 and 2.
Fmoc
O
N
e
d
N
N
O
t-BuO
t-BuO
O
O
cellular delivery based on the interaction of carriers with negatively
charged groups. Most of the nonviral gene delivery systems are, in
fact, based on cationic lipids13 or cationic polymers14 interacting
with negative charged genetic vectors. Furthermore, the neutral
backbone of PNA prevents them being recognized by proteins,
which interact with DNA, and PNA/DNA chimeras should be syn-
thesized for applications such as transcription factors scavenging
(decoy)15 or activation of RNA degradation by RNase-H (as in an-
tisense drugs).3d This lack of recognition is partly due to the lack of
negatively charged groups and of the corresponding electrostatic
interactions with the protein counterpart.16
12
11
O
NH
O
N
O
13, R = CH3
1, R = H
O
f
R
Fmoc
N
N
O
t-BuO
O
Scheme 1. Synthesis of the PNA monomer 1. Reagents and conditions: (a) DMF, DIPEA,
70 ꢀC, 3 days, 41%; (b) fluorenylmethoxycarbonyl chloride (FmoceCl), NaHCO3, 1,4-
dioxane/H2O, 18 h, 63%; (c) NaIO4, THF/H2O, 2 h, 97%; (d) H2NCH2COOCH3, NaH-
B(AcO)3, triethylamine in CH2Cl2, 18 h, 70%; (e) thymin-1-yl-acetic acid, Et3N, HATU in
DMF, 18 h, 49%; (f) LiOH$H2O, 1,4-dioxane/H2O, 0 ꢀC, 30 min, 69%.
In the present work we report the synthesis of the bis-protected
thyminylated Ng-
u-carboxyalkyl monomers 1 and 2 (Fig. 1), the
solid-phase oligomerization and the base-pairing behavior of four
oligomeric peptoid sequences 3e6 (Fig. 2) incorporating, to various
extents and at different positions, the monomers 1 and 2.
The synthesis of compound 2 required a different strategy, due
to the low yields obtained in the glycidol opening induced by the
tert-butyl ester of the 6-aminocaproic acid 16 (see Experimental
section). A better electrophile was devised in the benzyl 2-
iodoethylcarbamate 1918 (Scheme 2). The nucleophilic displace-
ment gave the secondary amine 20, containing the Cbz-protected
ethylendiamine core. Compound 20, after a straightforward pro-
tective group adjustment and a subsequent reductive amination,
produced the fully protected bis-carboxyalkyl ethylenediamine key
intermediate 23. Compound 23 was reacted with thymine-1-acetic
acid and benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP), as condensing agent, and gave the
amide 24. Finally, after careful chemoselective hydrolysis of the
ethyl ester of 24, the required monomer 2 was obtained in ac-
ceptable yields.
The oligomers 3e6 were manually assembled in a stepwise
fashion on a Rink amide NOVA-PEG resin solid support. The un-
modified PNA monomers were coupled using 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU).
HATU was used for the coupling reactions involving the less re-
active secondary amino groups of the modified monomers 1 and 2.
The decamers were detached from the solid support and quanti-
tatively deprotected from the tert-butyl protecting groups, using
a 9:1 mixture of trifluoroacetic acid and m-cresol. The water-
soluble oligomers were purified by RP-HPLC, yielding the desired
3e6 as pure compounds. Their identity was confirmed by MALDI-
TOF mass spectrometry.
GTAGAT*1CACT–Gly–NH2 ,
G T*1AGAT*1CAC T*1–Gly–NH2 , 4
GTAGAT*2CACT–Gly–NH2 ,
G T*2AGAT*2CAC T*2–Gly–NH2 , 6
3
5
Fig. 2. Structures of target oligomers 3e6. T
*
represents the modified thyminylated Ng-
u
-carboxyalkyl monomers. T
*
incorporates monomer 1, T
*
incorporates monomer 2.
1
2
The carboxy termini of the modified mixed purine/pyrimidine
decamer PNA sequences were linked to a glycinamide unit. T 1 and
represent the insertion of the modified 1 and 2 Ng-
-carbox-
*
T
*
u
2
yalkyl monomer units, respectively.
The mixed-base sequence has been chosen since it has been
proposed by Nielsen and co-workers3e and subsequently used by
several groups as a benchmark for the evaluation of the effect of
modification of the PNA structure on PNA/DNA thermal stability.17
2. Results and discussion
2.1. Chemistry
The elaboration of monomers 1 and 2 (Fig. 1), suitable for the
Fmoc-based oligomerization, took advantage of the chemistry uti-
lized to construct the regular PNA monomers. In particular, the
synthesis of the N-protected monomer 1 started with the t-Bue-
glycine (7) glycidol amination,9 as shown in Scheme 1. N-Fluo-
renylmethoxycarbonyl protection of the adduct 9, and subsequent
diol oxidative cleavage, gave the labile aldehyde 11. Compound 11
was subjected to reductive amination in the presence of methyl-
glycine to obtain the triply protected bis-carboxyalkyl ethylenedi-
amine key intermediate 12. The 2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) pro-
moted condensation of 12 with thymin-1-yl-acetic acid gave the
expected tertiary amide 13. Careful LiOH-mediated hydrolysis
preserves the base-labile Fmoc group, affording the target mono-
mer unit 1.
2.2. Hybridization studies
In order to verify the ability of decamers 3e6 to bind to com-
plementary DNA, UV-monitored melting experiments were per-
formed mixing the water-soluble oligomers with the
complementary antiparallel deoxyribonucleic strands (5
centration each strand,
mM con-
l
¼260 nm). Table 1 presents the thermal
stability studies of the duplexes formed between the modified
PNAs and the DNA antiparallel strand, in comparison with the
unmodified PNA.

C. De Cola et al. / Tetrahedron 68 (2012) 499e506
501
cases the ability to discriminate closely related sequences is mag-
nified, with respect to the unmodified PNA.
NHCbz
5
NH2
5
NHCbz
5
a
b
d
For the binding of the Ng-caproic acid derivatives with the full-
matched antiparallel DNA, the table shows an evident increase of
the affinity (entries 4 and 5), when compared with the modified
sequences with shorter side chains (entries 2 and 3). Comparison
with the corresponding aegPNA shows, for the single insertion,
a 3.8 ꢀC Tm drop, while, for triple substitution, a Tm decrease of
1.5 ꢀC per Ng-alkylated monomer. It is also worth noting, in both 5
HO
HO
t-BuO
t-BuO
O
O
O
14
15
16
c
NHCbz
NH2
NHCbz
I
HO
and 6, the slight increase of the binding specificity (
D
Tm¼5.6 ꢀC and
0.8 ꢀC, entries 4 and 5) respect to unmodified PNA.
17
19
18
In previous studies, reporting the performances of backbone
modified PNA containing negatively charged monomers derived
from amino acids, the drop in melting temperature was found to be
R
NHR'
N
3.3 ꢀC in the case of the -Asp monomer and 2.3 ꢀC in the case of
L D-
e
h
Glu.3e The present results are in line with these data, with a de-
crease in melting temperatures, which still allows stronger binding
than natural DNA (entry 6). Thus it is possible to introduce nega-
tively charged groups via alkylation of the amide nitrogen in the
PNA backbone without significant loss of stability of the PNA/DNA
duplex, provided that a five methylene spacer is used.
t-BuO
5
+
16
19
O
20, R = H, R' = Cbz
21, R = Fmoc, R' = Cbz
22, R = Fmoc, R' = H
f
g
O
3. Conclusions
NH
O
In this contribution, we have constructed two orthogonally
O
H
N
protected Ng-
u-carboxy alkylated units. The successful insertion in
Fmoc
N
O
i
O
N
5
PNA-based decamers, through standard solid-phase synthesis
protocols, and the following hybridization studies, in the presence
of DNA antiparallel strand, demonstrate that the N-substitution
with negative charged groups is compatible with the formation of
a stable PNA/DNA duplex. The present study also extends the ob-
servation that correlates the efficacy of the nucleic acids hybrid-
ization with the length of the Ng alkyl substitution,8a expanding the
O
R
Fmoc
N
t-BuO
O
N
O
t-BuO
5
O
23
24, R = CH2CH3
2, R = H
l
validity also to Ng-
u-negative charged side chains.
The newly produced structures can create new possibilities for
PNA with functional groups enabling further improvement in their
ability to perform gene-regulation.
Scheme 2. Synthesis of the PNA monomer 2. Reagents and conditions: (a) tert-butanol,
DMAP, DCC, CH2Cl2, 18 h, 58%; (b) H2, Pd/C (10% w/w), acetic acid, methanol, 1 h and
30 min, quant.; (c) CbzeCl, CH2Cl2, 0 ꢀC (2 h)/rt, 18 h, quant.; (d) I2, imidazole, PPh3,
CH2Cl2, 3 h, 77%; (e) K2CO3, CH3CN, reflux, 18 h, 67%; (f) fluorenylmethoxycarbonyl
chloride (FmoceCl), NaHCO3, 1,4-dioxane/H2O, 18 h, 97%; (g) H2, Pd/C (10% w/w), acetic
acid, methanol, 1 h, quant.; (h) ethyl glyoxalate, NaHB(AcO)3, triethylamine in CH2Cl2,
18 h, 25%; (i) thymin-1-yl-acetic acid, Et3N, PyBOP in DMF, 18 h, 70%; (l) LiOH$H2O, 1,4-
dioxane/H2O, 30 min, 30%.
4. Experimental section
4.1. General methods
All reactions involving air or moisture sensitive reagents were
carried out under a dry argon or nitrogen atmosphere using freshly
distilled solvents. Tetrahydrofuran (THF) was distilled from LiAlH4
under argon. Toluene and CH2Cl2 were distilled from CaH2. Glass-
ware was flame-dried (0.05 Torr) prior to use. When necessary,
compounds were dried in vacuo over P2O5 or by azeotropic removal
of water with toluene under reduced pressure. Starting materials
and reagents purchased from commercial suppliers were generally
used without purification unless otherwise mentioned. Reaction
temperatures were measured externally; reactions were monitored
by TLC on Merck silica gel plates (0.25 mm) and visualized by UV
light, I2, or by spraying with H2SO4/Ce(SO4)2, phosphomolybdic
acid or ninhydrin solutions and drying. Flash chromatography was
performed on Merck silica gel 60 (particle size: 0.040e0.063 mm)
and the solvents employed were of analytical grade. Yields refer to
chromatographically and spectroscopically (1H and 13C NMR) pure
materials. The NMR spectra were recorded on Bruker DRX 400 (1H
at 400.13 MHz, 13C at 100.03 MHz), Bruker DRX 250 (1H at
250.13 MHz, 13C at 62.89 MHz), and Bruker DRX 300 (1H at
Table 1
Thermal stabilities (Tm
,
ꢀC) of modified PNA/DNA duplexes
Antiparallel
Entry PNA
DNA
DNA duplexa mis-matchedb
1
AceGTAGATCACT-GlyeNH2
48.6
36.4
(PNA sequence)8a
2
3
4
5
6
GTAGAT
GT 1AGAT
GTAGAT 2CACT-GlyeNH2 (5)
GT 2AGAT 2CACT 2-GlyeNH2 (6)
*
1CACT-GlyeNH2 (3)
43.2
40.7
44.8
44.1
33.5
34.4
30.8
35.6
26.5
*
*
1CACT 1-GlyeNH2 (4)
*
*
*
*
*
50-GTAGATCACT-30 (DNA sequence)9 33.5
a
50-AGTGATCTAC-30.
50-AGTGGTCTAC-30.
b
The data obtained clearly demonstrates that the distance of the
negative charged carboxy group from the oligoamide backbone
strongly affects the PNA/DNA duplex stability. In particular, when
the
g-nitrogen brings an acetic acid substituent (with a single
methylene connecting the oligoamide backbone and the charged
group, entry 2), a drop of 5.4 ꢀC in Tm of the carboxypeptoid-PNA/
DNA (ap) duplex is observed, when compared with unmodified
PNA (entry 1). Triple insertion of monomer 1 (entry 3), results in
a decrease of 2.6 ꢀC per N-acetyl unit, showing no Ng-substitution
detrimental additive effects on the annealing properties. In both
300.10 MHz, 13C at 75.50 MHz) spectrometers. Chemical shifts (
d)
are reported in parts per million relatively to the residual solvent
peak (CHCl3,
d
¼7.26, 13CDCl3,
d
¼77.0; CD2HOD,
d
¼3.34, 13CD3OD,
d¼49.0) and the multiplicity of each signal is designated by the
following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;

502
C. De Cola et al. / Tetrahedron 68 (2012) 499e506
quint, quintuplet; m, multiplet; br, broad. Coupling costants (J) are
quoted in hertz. Homonuclear decoupling, COSY-45, and DEPT ex-
periments completed the full assignment of each signal. Elemental
analyses were performed on a CHNS-O FlashEA apparatus (Thermo
Electron Corporation) and are reported in percent abundance. High
resolution ESI-MS spectra were performed on a Q-Star Applied
Biosystem mass spectrometer. ESI-MS analysis in positive ion mode
was performed using a Finnigan LCQ Deca ion trap mass spec-
mixture of rotamers) 28.2, 47.4, 52.1, 52.3, 52.9, 53.2, 63.5, 64.0, 67.3,
68.1, 68.5, 70.1, 70.5, 83.1,120.1,120.2,124.9,125.2,127.3,127.9,128.0,
141.5, 143.8, 156.4, 157.3, 170.4, 171.3; m/z (ES) 428 (MHþ); (HRES)
MHþ, found 428.2070. C24H30NOþ6 requires 428.2068.
4.2.3. (9H-Fluoren-9-yl)methyl(tert-butoxycarbonyl)methyl-
formylmethylcarbamate (11). To a solution of 10 (0.80 g, 1.87 mmol)
in a 5:1 mixture of THF and water (5 mL), sodium periodate (0.44 g,
2.06 mmol) was added in one portion. The mixture was sonicated
for 15 min and stirred for another 2 h at room temperature. The
reaction mixture was filtered, the filtrate was washed with CH2Cl2,
and the solvent evaporated in vacuo. The crude product was dis-
solved in CH2Cl2/H2O, and the organic phase was dried over MgSO4,
filtered and the solvent evaporated in vacuo to give the labile al-
dehyde 11 (0.72 g, 97%), as white solid; Rf (92:8 CH2Cl2/CH3OH)
0.56; crude 11 was used immediately in the subsequent reductive
amination reaction; dH (300.10 MHz, CDCl3, mixture of rotamers)
1.43 (4.05H, s, (CH3)3C), 1.45 (4.95H, s, (CH3)3C), 3.81 (0.9H, br s,
CH2COOet-Bu), 3.99 (2H, br s, CH2CHO and CH2COOet-Bu, over-
lapped), 4.08 (1.1H, s, CH2CHO), 4.22e4.19 (1H, m, CHeFmoc), 4.42
(1.1H, d, J 6.0 Hz, CH2eFmoc), 4.50 (0.9H, d, J 6.0 Hz, CH2eFmoc),
7.29 (0.9H, br t, J 7.0 Hz, Ar. (Fmoc)), 7.38 (1.1H, t, J 7.0 Hz, Ar.
(Fmoc)), 7.49 (0.9H, d, J 9.0 Hz, Ar. (Fmoc)), 7.56 (1.1H, d, J 9.0 Hz, Ar.
(Fmoc)), 7.73 (2H, m, Ar. (Fmoc)), 9.35 (0.45H, br s, CHO), 9.64
(0.55H, br s, CHO); dC (75.50 MHz, CDCl3) 28.2, 47.3, 50.6, 50.8, 57.8,
58.4, 68.1, 68.6, 82.6, 82.7, 120.2, 124.9, 125.2, 127.3, 128.0, 141.5,
143.8, 143.9, 156.0, 156.4, 168.6, 168.7, 198.7; m/z (ES) 396 (MHþ);
(HRES) MHþ, found 396.1809. C23H26NOþ5 requires 396.1805.
ꢀ
trometer (ThermoFinnigan, San Jose, CA, USA) and the mass spectra
were acquired and processed using the Xcalibur software provided
by Thermo Finnigan. Samples were dissolved in 1:1 CH3OH/H2O,
0.1% formic acid, and infused in the ESI source by using a syringe
pump; the flow rate was 5 mL/min. The capillary voltage was set at
4.0 V, the spray voltage at 5 kV, and the tube lens offset at ꢁ40 V.
The capillary temperature was 220 ꢀC. MALDI-TOF mass spectro-
metric analyses were performed on a PerSeptive Biosystems
Voyager-De Pro MALDI mass spectrometer in the linear mode using
a
-cyano-4-hydroxycinnamic acid as the matrix. HPLC analyses
were performed on a Jasco BS 997-01 series, equipped with a qua-
ternary pumps Jasco PU-2089 Plus, and an UV detector Jasco MD-
2010 Plus. The resulting residues were purified by semi-
ꢁ
preparative reverse-phase C18 (Waters, Bondapak, 10
m
m, 125 A,
7.8ꢂ300 mm).
4.2. Chemistry
4.2.1. tert-Butyl 2-(2,3-dihydroxypropylamino)acetate (9). To a solu-
tion of glycidol (8, 436 L, 6.56 mmol) in DMF (5 mL), glycine tert-
butyl ester (7, 1.00 g, 5.96 mmol) in DMF (10 mL), and DIPEA
(1600 L, 8.94 mmol) were added. The reaction mixture was refluxed
m
m
4.2.4. (9H-Fluoren-9-yl)methyl(tert-butoxycarbonyl)methyl 2-((me-
thoxycarbonyl)methylamino)ethylcarbamate (12). To a solution of
crude aldehyde 11 (0.72 g, 1.83 mmol) in dry CH2Cl2 (12 mL), a so-
lution of glycine methyl ester hydrochloride (0.30 g, 2.39 mmol)
and Et3N (0.41 mL, 2.93 mmol) was added. The reaction mixture
was stirred for 1 h. Sodium triacetoxyborohydride (0.78 g,
3.66 mmol) was then added and the reaction mixture was stirred
for 18 h at room temperature. The resulting mixture was washed
with an aqueous saturated solution of NaHCO3 and the aqueous
phase extracted with CH2Cl2 (3ꢂ20 mL). The organic phase was
dried over MgSO4, filtered, and the solvent evaporated in vacuo to
give the crude product, which was purified by flash chromatogra-
phy (AcOEt/petroleum ether/NH3 2.0 M solution in ethyl alcohol
from 40:60:0.1 to 90:10:0.1) to give 12 (0.60 g, 70%) as a colorless
oil. Found: C, 66.7; H, 6.9. C26H32N2O6 requires C, 66.65; H, 6.88%; Rf
(98:2:0.1 CH2Cl2/CH3OH/NH3 2.0 M solution in ethyl alcohol) 0.63;
dH (400.13 MHz, CDC13, mixture of rotamers) 1.45 (9H, s, (CH3)3C),
2.56 (0.9H, t, J 6.0 Hz, N(Fmoc)CH2CH2NH), 2.83 (1.1H, t, J 6.0,
N(Fmoc)CH2CH2NH), 3.27 (0.9H, t, J 6.0 Hz, N(Fmoc)CH2CH2NH),
3.29 (0.9H, s, CH2COOMe), 3.44 (1.1H, s, CH2COOMe), 3.49 (1.1H, t, J
6.0 Hz, N(Fmoc)CH2CH2NH), 3.72 (3H, s, CH3), 3.91 (0.9H, s,
CH2COOt-Bu), 3.96 (1.1H, s, CH2COOt-Bu), 4.21 (0.45H, t, J 6.0 Hz,
CH2CHFmoc), 4.26 (0.55H, t, J 6.0 Hz, CH2CHFmoc), 4.37 (1.1H, d, J
6.0 Hz, CH2CHFmoc), 4.51 (0.9H, d, J 6.0 Hz, CH2CHFmoc), 7.29 (2H,
t, J 7.0 Hz, Ar. (Fmoc)), 7.39 (2H, t, J 7.0 Hz, Ar. (Fmoc)), 7.58 (2H, m,
Ar. (Fmoc)), 7.75 (2H, d, J 7.0 Hz, Ar. (Fmoc)); dC (100.03 MHz, CDCl3)
27.8, 47.0, 47.2, 47.4, 48.2, 48.6, 50.1, 50.3, 50.5, 53.3, 67.2, 67.6, 81.5,
81.7, 119.7, 124.7, 124.9, 126.8, 127.5, 141.0, 143.7, 156.0, 156.2, 168.7,
169.4, 171.9, 172.1; m/z (ES) 469 (MHþ); (HRES) MHþ, found
469.2341. C26H33N2Oþ6 requires 469.2333.
for 3 days. NaHCO3 (0.50 g, 5.96 mmol) was added and the solvent
was concentrated in vacuo to give the crude product, which was
purified by flash chromatography (CH2Cl2/CH3OH/NH3 2.0 M solu-
tion in ethyl alcohol from 100:0:0.1 to 88:12:0.1) to give 9 (0.50 g,
41%) as a pale yellow oil. Found: C, 52.7; H, 9.4. C9H19NO4 requires C,
52.67; H, 9.33%; Rf (97:3:0.1 CH2Cl2/CH3OH/NH3 2.0 M solution in
ethyl alcohol) 0.36; dH (400.13 MHz, CDC13) 1.42 (9H, s, (CH3)3C), 2.62
(1H, dd, J 12.0, 7.7 Hz, CHHCH(OH)CH2OH), 2.71 (1H, dd, J 12.0, 2.9 Hz,
CHHCH(OH)CH2OH), 3.28 (2H, br s, CH2COOt-Bu), 3.51 (1H, dd, J 11.0,
5.4 Hz, CH2CH(OH)CHHOH), 3.62 (1H, dd, J 11.0, 1.2 Hz, CH2CH(OH)
CHHOH), 3.72 (1H, m, CH2CH(OH)CH2OH); dC (100.03 MHz, CDCl3)
29.2, 52.6, 53.1, 66.4, 71.6, 82.7, 172.8; m/z (ES) 206 (MHþ); (HRES)
MHþ, found 206.1390. C9H20NOþ4 requires 206.1387.
4.2.2. (9H-Fluoren-9-yl)methyl(tert-butoxycarbonyl)methyl 2,3-
dihydroxypropylcarbamate (10). To
a solution of 9 (0.681 g,
3.33 mmol) in a 1:1 mixture of 1,4-dioxane/water (46 mL), NaHCO3
(0.559 g, 6.66 mmol) was added. The mixture was sonicated until
complete dissolution and FmoceCl (1.03 g, 3.99 mmol) was added.
The reaction mixture was stirred for 18 h, then, through addition of
a saturated solution of NaHSO4, the pH was adjusted to 3 and the
solvent was concentrated in vacuo to remove the excess of 1,4-
dioxane. The water layer was extracted with CH2Cl2 (3ꢂ50 mL),
the organic phase was dried over MgSO4, filtered, and the solvent
evaporated in vacuo to give the crude product, which was purified by
flash chromatography (CH2Cl2/CH3OH from 100:0 to 90:10) to give
10 (0.90 g, 63%) as a pale yellow oil. Found: C, 67.4; H, 6.9. C24H29NO6
requires C, 67.43; H, 6.84%; Rf (95:5:0.1 CH2Cl2/CH3OH/NH3 2.0 M
solution in ethyl alcohol) 0.44; dH (300.10 MHz, CDC13, mixture of
rotamers) 1.45 (9H, s, (CH3)3C), 3.04e3.25 (1.7H, m, CH2CH(OH)
CH2OH), 3.40 (0.3H, m, CH2CH(OH)CH2OH), 3.43e3.92 (3H, m,
CH2CH(OH)CH2OH, CH2CH(OH)CH2OH), 3.93 (2H, brs, CH2COOt-Bu),
4.22 (0.9H, m, CHeFmoc and CH2eFmoc), 4.42 (1.4H, br d, J 9.0 Hz,
CH2eFmoc), 4.61 (0.7H, m, J 9.0 Hz, CHeFmoc), 7.29 (2H, br t, J 7.0 Hz,
Ar. (Fmoc)), 7.38 (2H, br t, J 7.0 Hz, Ar. (Fmoc)), 7.57 (2H, br d, J 9.0 Hz,
Ar. (Fmoc)), 7.76 (2H, br d, J 9.0 Hz, Ar. (Fmoc)); dC (75.50 MHz, CDCl3,
4.2.5. Compound 13. To a solution of 12 (0.60 g, 1.28 mmol) in DMF
(30 mL), thymine-1-acetic acid (0.35 g, 1.90 mmol), HATU (0.73 g,
1.90 mmol), and triethylamine (0.54 mL, 3.84 mmol) were added.
The reaction mixture was stirred for 18 h, concentrated in vacuo,
dissolved in CH2Cl2 (20 mL), and washed with 1 M HCl solution. The
aqueous layer was extracted with CH2Cl2 (3ꢂ20 mL). The combined

C. De Cola et al. / Tetrahedron 68 (2012) 499e506
503
organic phases were dried over MgSO4, filtered, and the solvent
evaporated in vacuo to give a crude material, which was purified by
flash chromatography (AcOEt/petroleum ether from 30:70 to
100:0) to give 13 (0.40 g, 49%) as yellow oil. Found: C, 62.5; H, 6.1.
C33H38N4O9 requires C, 62.45; H, 6.03%; Rf (8:2 AcOEt/petroleum
ether) 0.38; dH (300.10 MHz, CDC13, mixture of rotamers) 1.39e1.44
(9H, m, (CH3)3C), 1.88 (3H, br s, CH3ethymine), 2.99 (0.3H, m,
CH2CH2N(Fmoc)), 3.08 (0.3H, m, CH2CH2N(Fmoc)), 3.52 (2.8H, m,
CH2CH2N(Fmoc) and CH2CH2N(Fmoc)), 3.77e3.89 (3.6H, m,
CH2CH2N(Fmoc) and CH3OOC), 3.96e4.38 (6H, m, CH2ethymine,
CH2COOCH3, CH2COO-t-Bu), 4.45e4.80 (3H, m, CH(Fmoc) and
CH2CH(Fmoc)), 6.90e7.06 (1H, complex signal, CHethymine), 7.28
(2H, m, Ar. (Fmoc)), 7.41 (2H, t, J 7.0 Hz, Ar (Fmoc)), 7.55 (2H, d, J
7.0 Hz, Ar. (Fmoc)), 7.75 (2H, t, J 7.0 Hz, Ar. (Fmoc)), 8.86 (1H, br s,
NHethymine); dC (75.5 MHz, CDCl3) 12.5, 28.2, 31.6, 36.6, 47.2, 47.4,
47.5, 47.8, 48.2, 49.1, 50.6, 51.8, 52.1, 52.5, 53.0, 66.5, 68.2, 82.3, 82.5,
110.5, 120.2, 124.5, 124.8, 125.14, 125.3, 127.3, 127.4, 127.5, 128.0,
128.1, 141.3, 141.4, 143.8, 144.0, 151.1, 156.4, 162.7, 164.4, 169.1, 169.2;
m/z (ES) 635 (MHþ); (HRES) MHþ, found 635.2741. C33H39N4O9þ
requires 635.2712.
and palladium on charcoal (10% w/w, 0.19 g) were added. The re-
action mixture was stirred under a hydrogen atmosphere at room
temperature for 1 h and filtered through Celite. The solvent was
evaporated in vacuo to give crude 16 (1.13 g, 100%, colorless oil),
which was used in the next step without purification; Rf (95:5
CH2Cl2/CH3OH) 0.46; dH (400.13 MHz, CDC13) 1.33 (2H, q, J 6.5 Hz,
CH2CH2CH2COOt-Bu), 1.39 (9H, s, COOC(CH3)3), 1.55 (2H, q, J 6.5 Hz,
CH2CH2CH2CH2CH2NH), 1.62 (2H, q, J 6.5 Hz, CH2CH2CH2COOt-Bu),
2.17 (2H, t, J 6.5 Hz, CH2CH2CH2COOt-Bu), 2.84 (2H, t, J 6.5 Hz,
CH2CH2CH2NH); dC (75.50 MHz, CDCl3) 24.3, 25.8, 27.2, 28.0, 35.1,
39.4, 80.2, 177.2; m/z (ES) 188 (MHþ); (HRES) MHþ, found 188.1653.
C10H22NOþ2 requires 188.1645.
4.2.9. Benzyl 2-hydroxyethylcarbamate (18). To a solution of etha-
nolamine (17, 2.00 g, 32.8 mmol) in dry CH2Cl2 (30 mL) at 0 ꢀC,
a solution CbzeCl (3.73 mL, 26.2 mmol) in dry CH2Cl2 (20 mL) was
slowly added. The reaction mixture was stirred for 2 h at 0 ꢀC and
at room temperature for 18 h. The resulting mixture was washed
with an aqueous saturated solution of NaHCO3 and the aqueous
phase extracted with CH2Cl2 (3ꢂ50 mL). The organic phase was
dried over MgSO4, filtered, and the solvent evaporated in vacuo to
give crude 18 (5.11 g, 100%, pale yellow oil), which was used in the
next step without purification; Rf (92:8 CH2Cl2/CH3OH) 0.47; dH
(250.13 MHz, CDC13) 3.36 (2H, br t, J 6.5 Hz, CH2OH), 3.71 (2H, q, J
6.5 Hz, CH2NH), 5.11 (2H, s, CH2Bn), 7.35 (5H, m, Ar.); dC
(62.89 MHz, CDCl3) 43.3, 61.7, 66.7, 127.9, 128.0, 128.4, 136.2, 157.0;
m/z (ES) 196 (MHþ); (HRES) MHþ, found 196.0977. C10H14NOþ3
requires 196.0968.
4.2.6. Compound 1. To a solution of 13 (0.40 g, 0.63 mmol) in a 1:1
mixture of 1,4-dioxane/water (8 mL) at 0 ꢀC, LiOH monohydrate
(58 mg,1.39 mmol) was added. The reaction mixture was stirred for
30 min and a saturated solution of NaHSO4 was added until pH w3.
The aqueous layer was extracted with CH2Cl2 (3ꢂ15 mL) and once
with AcOEt. The combined organic phases were dried over MgSO4,
filtered, and the solvent evaporated in vacuo to give a crude ma-
terial, which was purified by flash chromatography (CH2Cl2/
CH3OH/AcOH from 95:5:0.1 to 80:20:0.1) to give 1 (0.27 g, 69%) as
a white solid. Found: C, 62.0; H, 6.0. C32H36N4O9 requires C, 61.93;
H, 5.85%; Rf (9:1:0.1 CH2Cl2/CH3OH/NH3 2.0 M solution in ethyl
alcohol) 0.12; dH (250.13 MHz, CDC13, mixture of rotamers)
1.39e1.43 (9H, m, (CH3)3C), 1.83 (3H, br s, CH3 (thymine)), 3.03
(0.3H, m, CH2CH2N(Fmoc)), 3.17 (0.3H, m, CH2CH2N(Fmoc)),
3.55e3.72 (2.8H, m, CH2CH2N(Fmoc) and CH2CH2N(Fmoc)),
3.95e4.06 (6.6H m, CH2CH2N(Fmoc), CH2ethymine, CH2COOH,
CH2COOet-Bu), 4.14e4.77 (3H, m, CH(Fmoc) and CH2CH(Fmoc)),
6.97e7.11 (1H, complex signal, CHethymine), 7.23e7.40 (4H, m, Ar.
(Fmoc)), 7.55 (2H, d, J 7.0 Hz, Ar. (Fmoc)), 7.75 (2H, m, Ar. (Fmoc)),
10.00 (1H, br s, NHethymine); dC (75.50 MHz, CDCl3) 12.3, 28.2,
29.9, 46.4, 47.3, 48.7, 50.2, 50.9, 53.6, 68.3, 82.3, 82.6, 110.8, 120.2,
124.9, 125.2, 125.3,127.3, 127.5, 128.0, 141.4,142.1,143.8, 144.0,151.7,
156.6, 165.0, 165.2, 168.2, 169.0, 169.2, 172.3; m/z (ES) 621 (MHþ);
(HRES) MHþ, found 621.2541. C32H37N4Oþ9 requires 621.2555.
4.2.10. Benzyl 2-iodoethylcarbamate (19). To a solution of PPh3
(2.66 g, 10.2 mmol) in CH2Cl2 (10 mL), I2 (2.59 g, 10.2 mmol) in
CH2Cl2 (10 mL) was slowly added. The reaction mixture was stirred
for 30 min. Imidazole (1.39 g, 20.4 mmol) in CH2Cl2 (10 mL) was
then added and the reaction mixture was stirred for further 30 min.
Finally, 18 (1.00 g, 5.13 mmol) was added and the reaction mixture
stirred for 3 h. The resulting mixture was washed with an aqueous
saturated solution of NaHCO3 and 10% w/w of Na2S2O3 and the
aqueous phase extracted with CH2Cl2 (3ꢂ25 mL). The organic phase
was dried over MgSO4, filtered, and the solvent evaporated in vacuo
to give a crude material, which was purified by flash chromatog-
raphy (AcOEt/petroleum ether from 0:100 to 100:0) to give 19
(1.20 g, 77%) as white amorphous solid. Found: C, 39.4; H, 4.0.
C10H12INO2 requires C, 39.36; H, 3.96%; Rf (6:4 AcOEt/petroleum
ether) 0.88; dH (250.13 MHz, CDC13) 3.25 (2H, t, J 6.5 Hz, CH2I), 3.55
(2H, q, J 6.5 Hz, CH2NH), 5.11 (2H, s, CH2Bn), 7.36 (5H, m, Ar.); dC
(62.89 MHz, CDCl3) 5.1, 43.0, 66.5, 127.8, 128.1, 128.2, 136.0, 155.8;
m/z (ES) 306 (MHþ); (HRES) MHþ, found 305.9991. C10H13INOþ2
requires 305.9985.
4.2.7. Benzyl 5-(tert-butoxycarbonyl)pentylcarbamate (15). To a so-
lution of 14 (3.00 g, 11.3 mmol), DMAP (0.14 g, 1.13 mmol), and t-
BuOH (1.30 mL, 13.9 mmol) in dry CH2Cl2 (5.0 mL), a solution of
DCC (13.6 mL, 13.6 mmol, 1.0 M in CH2Cl2) was added. The reaction
mixture was filtered, the filtrate washed with CH2Cl2, and the sol-
vent evaporated in vacuo to give the crude product, which was
purified by flash chromatography (AcOEt/petroleum ether from
10:90 to 100:0) to give 15 (2.10 g, 58%) as white solid. Found: C,
67.2; H, 8.4. C18H27NO4 requires C, 67.26; H, 8.47%; Rf (97:3 CH2Cl2/
CH3OH) 0.84; dH (400.13 MHz, CDC13) 1.31 (2H, q, J 6.5 Hz,
CH2CH2CH2COOt-Bu), 1.41 (9H, s, COOC(CH3)3), 1.48 (2H, q, J 6.5 Hz,
CH2CH2CH2NH),1.56 (2H, q, J 6.5 Hz, CH2CH2CH2COOt-Bu), 2.18 (2H,
4.2.11. Benzyl 2-(5-(tert-butoxycarbonyl)pentylamino)ethylcarbamate
(20). To a solution of 16 (0.35 g, 1.87 mmol) in dry acetonitrile
(10 mL), at reflux, K2CO3 (0.88 g, 6.38 mmol) was added. The reaction
mixture was stirred for 10 min. After that, a solution of 19 (0.40 g,
1.31 mmol) in dry acetonitrile (5 mL) was added and the reaction
mixture was stirred at reflux 18 h. The product was filtered and the
crude was purified by flash column chromatography (CH2Cl2/CH3OH
from 100:0 to 90:10) to give 20 (0.32 g, 67%) as yellow light oil.
Found: C, 65.9; H, 8.6. C20H32N2O4 requires C, 65.91; H, 8.85%; Rf (93:7
CH2Cl2/CH3OH) 0.71; dH (300.10 MHz, CDC13) 1.35 (2H, q, J 6.5 Hz,
CH2CH2CH2CH2CH2NH), 1.43 (9H, s, COOC(CH3)3), 1.57 (2H, q, J 6.0 Hz,
CH2CH2CH2CH2CH2NH), 1.67 (2H, q, J 6.0 Hz, CH2CH2CH2CH2CH2NH),
t,
J
6.5 Hz, CH2CH2CH2COOt-Bu), 3.15 (2H, q,
J
6.5 Hz,
CH2CH2CH2NH), 4.91 (1H, br s, NH), 5.07 (2H, br s, CH2Bn), 7.32 (5H,
m, Ar.); dC (75.50 MHz, CDCl3) 24.5, 26.0, 28.0, 29.5, 35.3, 40.8, 66.4,
79.9, 127.9, 128.0, 128.3, 136.6, 156.3, 172.8; m/z (ES) 322 (MHþ);
(HRES) MHþ, found 322.2009. C18H28NOþ4 requires 322.2013.
2.21 (2H, t,
J 6.0 Hz, OOCCH2CH2), 2.82 (2H, t, J 6.0 Hz,
CH2CH2CH2CH2CH2NH), 2.99 (2H, t, J 6.0 Hz, CONHCH2CH2NH), 3.46
(2H, q, J 6.0 Hz, CONHCH2CH2NH), 5.09 (2H, s, CH2Ar), 5.73 (1H, br s,
NHCOO), 7.34 (5H, m, Ar.); dC (75.50 MHz, CDCl3) 24.4, 26.2, 27.9, 35.0,
39.3, 48.4, 48.6, 66.6, 79.9, 127.9, 128.0, 128.3, 136.1, 156.6, 172.8; m/z
4.2.8. tert-Butyl 6-aminohexanoate (16). To a solution of 15 (1.95 g,
6.07 mmol) in dry MeOH (150 mL), acetic acid (1.39 mL, 24.0 mmol)

504
C. De Cola et al. / Tetrahedron 68 (2012) 499e506
(ES) 365 (MHþ); (HRES) MHþ, found 365.2426. C20H33N2Oþ4 requires
365.2435.
7 Hz, Ar. (Fmoc)), 7.39 (2H, t, J 7 Hz, Ar. (Fmoc)), 7.57 (2H, d, J 7 Hz,
Ar. (Fmoc)), 7.75 (2H, t, J 7 Hz, Ar. (Fmoc)); dC (100.03 MHz, CDCl3,
mixture of rotamers) 14.1, 24.7, 26.1, 28.0, 35.3, 46.8, 47.3, 47.8, 50.6,
60.7, 66.5, 79.9, 119.8, 124.6, 126.9, 127.5, 141.3, 144.0, 155.9, 156.2,
172.2, 172.9; m/z (ES) 539 (MHþ); (HRES) MHþ, found 539.3108.
C31H43N2Oþ6 requires 539.3116.
4.2.12. Compound (21). To a solution of 20 (0.32 g, 0.88 mmol) in
a 1:1 mixture of 1,4-dioxane/water (20 mL), NaHCO3 (148 mg,
1.76 mmol) was added. The mixture was sonicated until complete
dissolution and FmoceCl (0.29 g, 1.12 mmol) was added. The re-
action mixture was stirred 18 h, then, through addition of a satu-
rated of NaHSO4, the pH was adjusted to 3 and the solvent was
concentrated in vacuo to remove the excess of dioxane. The water
layer was extracted with CH2Cl2 (3ꢂ25 mL), the organic phase was
dried over MgSO4, filtered, and the solvent evaporated in vacuo to
give the crude product, which was purified by flash chromatogra-
phy (CH2Cl2/CH3OH from 100:0 to 98:2) to give 21 (0.50 g, 97%) as
a yellow light oil. Found: C, 71.7; H, 7.3. C35H42N2O6 requires C,
71.65; H, 7.22%; Rf (95:5 CH2Cl2/CH3OH) 0.61; dH (400.13 MHz,
CDC13, mixture of rotamers) 1.08e1.60 (15H, m, CH2CH2CH2CH2
CH2N, COOC(CH3)3), 2.16 (2H, t, J 6.0 Hz, CH2COO), 2.85 (0.8H, br s,
4.2.15. Compound (24). To a solution of 23 (100 mg, 0.186 mmol) in
DMF (6 mL), thymine-1-acetic acid (55 mg, 0.30 mmol), PyBOP
(154 mg, 0.30 mmol), and triethylamine (83 mL, 0.60 mmol) were
added. The reaction mixture was stirred for 18 h, concentrated in
vacuo, dissolved in CH2Cl2 (20 mL), and washed 1 M HCl solution.
The aqueous layer was extracted with CH2Cl2 (3ꢂ20 mL). The
combined organic phases were dried over MgSO4, filtered, and the
solvent evaporated in vacuo to give a crude material, which was
purified by flash chromatography (AcOEt/petroleum ether from
30:70 to 100:0) to give 24 (92 mg, 70%) as amorphous white solid.
Found: C, 64.7; H, 6.9. C38H48N4O9 requires C, 64.76; H, 6.86%; Rf
(6:40 AcOEt/petroleum ether) 0.11; dH (250.13 MHz, CDC13, mixture
of rotamers) 1.00e1.60 (18H, m, CH2CH2CH2CH2CH2N, COOC(CH3)3
and COOCH2CH3), 1.89 (3H, s, CH3ethymine), 2.18 (2H, m,
CH2COOC(CH3)3), 2.95e3.65 (6H, m, NHCH2CH2NCH2), 4.00e4.80
(9H, m, CH2OOCCH2NHCH2, CH2CHFmoc and CH2ethymine),
6.94e7.00 (1H, complex signal, CHethymine), 7.39 (2H, t, J 7.0 Hz,
Ar. (Fmoc)), 7.42 (2H, t, J 7.0 Hz, Ar. (Fmoc)), 7.56 (2H, d, J 7.0 Hz, Ar.
(Fmoc)), 7.76 (2H, t, J 7.0 Hz, Ar. (Fmoc)), 8.43 (1H, br s, NHethy-
mine); dC (75.50 MHz, CDCl3, mixture of rotamers) 12.1, 13.9, 24.6,
26.0, 28.0, 35.3, 46.4, 46.7, 47.2, 47.8, 48.1, 50.7, 61.7, 66.9, 80.1, 110.6,
111.0, 119.9, 124.6, 127.0, 127.6, 141.3, 143.8, 143.9, 151.2, 156.4, 164.3,
167.7, 168.9, 173.0; m/z (ES) 705 (MHþ); (HRES) MHþ, found
705.3506. C38H49N4Oþ9 requires 705.3494.
CH2CH2CH2CH2CH2N),
2.96
(2.4H,
CONHCH2CH2N
and
CH2CH2CH2CH2CH2N), 3.13 (0.8H, br s, CONHCH2CH2N), 3.30 (2H, br
s, CONHCH2CH2N), 4.19 (1H, br s, CH2CHFmoc), 4.53e4.57 (2H, br s,
CH2CHFmoc), 5.05 (2H, br s, CH2Ar), 7.29 (7H, m, Ar. (Cbz) and Ar.
(Fmoc)), 7.38 (2H, t, J 7.0 Hz, Ar. (Fmoc)), 7.55 (2H, d, J 7.0 Hz, Ar.
(Fmoc)), 7.76 (2H, t, J 7.0 Hz, Ar. (Fmoc)); dC (62.89 MHz, CDCl3) 24.5,
25.9, 27.8, 27.9, 35.2, 39.2, 39.6, 46.2, 46.8, 47.1, 47.6, 66.3, 79.7,
119.6, 124.4, 126.8, 127.4, 127.8, 128.2, 136.4, 141.1, 143.7, 155.6,
156.3, 172.7; m/z (ES) 587 (MHþ); (HRES) MHþ, found 587.3129.
C35H43N2Oþ6 requires 587.3116.
4.2.13. Compound (22). To a solution of 21 (150 mg, 0.26 mmol) in
dry MeOH (9 mL), acetic acid (29 mL, 0.512 mmol) and palladium on
charcoal (10% w/w, 15 mg) were added. The reaction mixture was
stirred under a hydrogen atmosphere at room temperature for 1 h
and filtered through Celite. The solvent was evaporated in vacuo to
give crude 22 (118 mg, 100%, colorless oil), which was used in the
next step without purification; Rf (95:5 CH2Cl2/CH3OH) 0.13; dH
(300.10 MHz, CDC13, mixture of rotamers) 1.05e1.60 (15H, m,
CH2CH2CH2CH2CH2N, COOC(CH3)3), 2.15 (2H, t, J 6.0 Hz, CH2COO),
2.60 (0.6H, br s, CH2CH2CH2CH2CH2N), 2.90e3.20 (4H, CONH
CH2CH2N, CH2CH2CH2CH2CH2N, CONHCH2CH2N and CONHCH2
CH2N), 3.38 (1.4H, br s, CONHCH2CH2N), 4.19 (1H, br s, CH2CHF
moc), 4.52 (2H, m, CH2CHFmoc), 7.38 (4H, m, Ar. (Fmoc)), 7.54 (2H,
d, J 7.0 Hz, Ar. (Fmoc)), 7.74 (2H, t, J 7.0 Hz, Ar. (Fmoc)); dC
(100.03 MHz, CDCl3) 22.6, 24.4, 24.7, 25.9, 26.1, 28.1, 35.1, 25.4, 38.8,
42.4, 46.5, 47.3, 47.9, 53.4, 67.0, 80.1, 119.8, 119.9, 124.0, 124.6, 126.9,
127.1, 127.7, 140.5, 141.3, 143.8, 149.0, 155.8, 157.1, 172.7, 172.9; m/z
(ES) 453 (MHþ); (HRES) MHþ, found 453.2740. C27H37N2Oþ4 requires
453.2748.
4.2.16. Compound (2). To a solution of 24 (175 mg, 0.25 mmol) in
a 1:1 mixture of 1,4-dioxane/water (6 mL) at 0 ꢀC, LiOH mono-
hydrate (23 mg, 0.55 mmol) was added. The reaction mixture was
stirred for 30 min and saturated solution of NaHSO4 added until pH
w3. The aqueous layer was extracted with CH2Cl2 (3ꢂ10 mL) and
once with AcOEt. The combined organic phases were dried over
MgSO4, filtered, and the solvent evaporated in vacuo to give a crude
material, which was purified by flash chromatography (CH2Cl2/
CH3OH from 95:5 to 80:20) to give 2 (50 mg, 30%) as amorphous
white solid. Found: C, 64.0; H, 6.6. C36H44N4O9 requires C, 63.89; H,
6.55%; Rf (9:1:0.1 CH2Cl2/CH3OH/NH3 2.0 M solution in ethyl alco-
hol) 0.22; dH (400.13 MHz, CDC13, mixture of rotamers) 1.00e1.50
(15H, m, CH2CH2CH2CH2CH2N and COOC(CH3)3), 1.86 (3H, s,
CH3ethymine), 2.17 (2H, m, J 6.0 Hz, CH2COOC(CH3)3), 2.90e3.70
(6H, m, NHCH2CH2NCH2), 3.90e4.85 (7H, m, OCCH2NHCH2,
CH2CHFmoc and CH2ethymine), 7.02 (1H, br s, CHethymine),
7.10e7.45 (4H, m, Ar. (Fmoc)), 7.57 (2H, d, J 7.0 Hz, Ar. (Fmoc)), 7.77
(2H, t, J 7.0 Hz, Ar. (Fmoc)); 10.00 (1H, br s, NHethymine); dC
(100.03 MHz, CDCl3, mixture of rotamers) 13.5, 22.7, 25.9, 26.0, 27.3,
28.8, 29.4, 29.7, 36.6, 36.7, 45.8, 47.8, 48.5, 48.8, 49.6, 54.7, 68.3, 81.4,
81.6, 111.8, 111.9, 121.2, 125.9, 126.5, 128.3, 129.0, 129.5, 130.3, 139.1,
142.6, 142.9, 145.0, 145.1, 152.6, 157.7, 166.2, 169.0, 172.7, 174.4; m/z
(ES) 677 (MHþ); (HRES) MHþ, found 677.3182. C36H45N4Oþ9 requires
677.3181.
4.2.14. Compound (23). To a solution of 22 (115 mg, 0.26 mmol) in
CH2Cl2 (5 mL), ethyl glyoxalate (33 mL, 0.33 mmol), Et3N (54 mL,
0.38 mmol), and NaHB(OAc)3 (109 mg, 0.52 mmol) were added. The
reaction mixture was stirred for 18 h. The resulting mixture was
washed with an aqueous saturated solution of NaHCO3 and the
aqueous phase extracted with CH2Cl2 (3ꢂ10 mL). The organic phase
was dried over MgSO4, filtered, and the solvent evaporated in vacuo
to give the crude product, which was purified by flash chroma-
tography (AcOEt/petroleum ether from 60:40 to 100:0) to give 23
(35 mg, 25%) as white light oil. Found: C, 69.2; H, 7.9. C31H42N2O6
requires C, 69.12; H, 7.86%; Rf (95:5:0.1 CH2Cl2/CH3OH/NH3 2.0 M
solution in ethyl alcohol) 0.46; dH (300.10 MHz, CDC13, mixture of
rotamers) 1.00e1.80 (18H, m, CH2CH2CH2CH2CH2N, COOC(CH3)3
and COOCH2CH3), 2.18 (2H, t, J 6.0 Hz, CH2COOC(CH3)3), 2.46 (0.8H,
br s, CH2CH2CH2CH2CH2N), 2.74 (1.2H, br s, CH2CH2CH2CH2CH2N),
2.90e3.45 (6H, m, COCH2NHCH2CH2N), 4.18e4.23 (3H, m,
COOCH2CH3, CH2CHFmoc), 4.52 (2H, m, CH2CHFmoc), 7.31 (2H, t, J
4.2.17. Low yield synthesis of tert-butyl 6-(2,3-dihydroxypropylamino)
hexanoate (25) and unwanted tert-butyl 6-(bis(2,3-dihydroxypropyl)
amino)hexanoate (26). To
a solution of glycidol (8, 134 mL,
2.02 mmol) in DMF (3 mL), tert-butyl 6-aminohexanoate (16,
456 mg, 2.44 mmol) in DMF (3 mL), and DIPEA (0.55 mL,
3.17 mmol) were added (Scheme 3). The reaction mixture was
refluxed for 3 days. NaHCO3 (320 mg, 1.79 mmol) was added and
the solvent was concentrated in vacuo to give the crude product,
which was purified by flash chromatography (CH2Cl2/CH3OH/NH3

C. De Cola et al. / Tetrahedron 68 (2012) 499e506
505
2.0 M solution in ethyl alcohol from 100:0:0.1 to 88:12:0.1) to give
25 (60 mg, 11%) and 26 (320 mg, 47%). Compound 25: pale yellow
oil. Found: C, 59.8; H,10.5. C13H27NO4 requires C, 59.74; H,10.41%; Rf
(90:10:0.1 CH2Cl2/CH3OH/NH3 2.0 M solution in ethyl alcohol) 0.31;
dH (300.10 MHz, CDC13) 1.31 (2H, q, J 6.5 Hz, CH2CH2CH2COOt-Bu),
1.43 (9H, s, COOC(CH3)3), 1.52 (2H, quint, J 6.5 Hz, CH2CH2CH2NH),
1.57 (2H, q, J 6.5 Hz, CH2CH2CH2COOt-Bu), 2.20 (2H, t, J 6.5 Hz,
CH2CH2CH2COOt-Bu), 2.58 (2H, m, CH2NHCH2CH(OH)CH2(OH)),
2.69 (1H, dd, J 15.0, 6.0 Hz, NHCHHCH(OH)CH2(OH)), 2.80 (1H, dd, J
15.0, 3.0 Hz, CHHCH(OH)CH2(OH)), 3.59 (1H, dd, J 9.0, 3.0 Hz,
CH2CH(OH)CHH(OH)), 3.70 (1H, dd, J 9.0, 3.0 Hz, CH2CH(OH)
CHH(OH)), 3.77 (1H, m, CH2CH(OH)CH2(OH)); dC (62.89 MHz,
CDCl3) 24.6, 26.4, 27.9, 28.7, 35.2, 49.2, 51.8, 65.1, 69.7, 80.0,173.0; m/
z (ES) 262 (MHþ); (HRES) MHþ, found 262.2017. C13H28NOþ4 requires
262.2013. Compound 26: yellow oil. Found: C, 57.3; H, 9.8.
C16H33NO6 requires C, 57.29; H, 9.92%; Rf (90:10:0.1 CH2Cl2/CH3OH/
NH3 2.0 M solution in ethyl alcohol) 0.44; dH (400.13 MHz, CDC13,
mixture of diastereoisomers) 1.27 (2H, q, J 6.5 Hz, CH2CH2CH2COOt-
Bu), 1.43 (11H, m, COOC(CH3)3 and CH2CH2CH2NH), 1.57 (2H, q,
J 6.5 Hz, CH2CH2CH2COOt-Bu), 2.19 (2H, t, J 6.5 Hz, CH2CH2CH2
COOt-Bu), 2.40e2.60 (6H, m, CH2NH[CH2CH(OH)CH2(OH)]2), 3.50
(2H, m, NH[CH2CH(OH)CHH(OH)]2), 3.63 (2H, m, NH[CH2CH
(OH)CHH(OH)]2), 3.77 (2H, m, NH[CH2CH(OH)CH2(OH)]2); dC
(62.89 MHz, CDCl3, mixture of diastereoisomers) 26.1, 27.5, 28.0,
29.3, 29.4, 36.7, 56.8, 56.9, 58.3, 59.1, 66.0, 66.2, 70.5, 71.0, 81.5,
174.6; m/z (ES) 336 (MHþ); (HRES) MHþ, found 336.2395. C16H34
NOþ6 requires 336.2381.
coupling the unreacted sites were capped by adding 5% acetic an-
hydride and 6% DIPEA in DMF and the reaction vessel was shaken
for 1 min (twice), and subsequently washed with a solution of 5% of
DIPEA in DMF. The resin was always washed thoroughly with DMF/
CH2Cl2. The cleavage from the resin was achieved by addition of
a 10% solution of m-cresol in TFA. The PNA was then precipitated
adding 10 volumes of diethyl ether, cooled at ꢁ18 ꢀC for at least 2 h
and finally collected through centrifugation (5 min@5000 rpm).
The resulting residue was redissolved in H2O and purified by
semipreparative reverse-phase C18 (Waters, Bondapak, 10 mm,
ꢁ
125 A, 7.8ꢂ300 mm) gradient elution from 100% H2O (0.1% TFA,
eluent A) to 60% CH3CN (0.1%TFA, eluent B) in 30 min. The product
was then lyophilized to get a white solid. MALDI-TOF MS analysis
confirmed the expected structures for the four oligomers (3e6),
with peaks at the following m/z values: compound 3 m/z (MALDIꢁ-
TOF MSdnegative mode) 2839 (MꢁH)ꢁ, calcd for C112H140N59O33
2839.11; compound 4 m/z (MALDI-TOF MSdnegative mode) 2955
(MꢁH)ꢁ, calcd for C116H144N59O3ꢁ7 2955.12; compound 5 m/z
(MALDI-TOF ꢁMSdnegative mode) 2895 (MꢁH)ꢁ, calcd for
C116H148N59O33 2895.17; compound 6 m/z (MALDꢁI-TOF MSdneg-
ative mode) 3123 (MꢁH)ꢁ, calcd for C128H168N59O37 3123.31.
4.4. Thermal denaturation studies
DNA oligonucleotides were purchased from CEINGE Bio-
tecnologie avanzate s.c. a r.l.
The concentrations of PNAs were quantified by measuring the
absorbance (A260) of the PNA solution at 260 nm. The values for the
molar extinction coefficients (
(A)¼13.7 mL/( molꢂcm),
11.7 mL/(
molꢂcm),
3
260) of the individual bases are:
3
260
NH2
5
R
O
m
3
260
(C)¼6.6 mL/(
mmolꢂcm),
3
260
(G)¼
N
OH
OH
+
OH
t-BuO
m
3
260
(T)¼8.6 mL/( molꢂcm) and molar ex-
m
OH
t-BuO
tinction coefficient of PNA was calculated as the sum of these values
according to sequence.
5
O
O
16
8
The PNA oligomers and DNA were hybridized in a buffer
100 mM NaCl, 10 mM sodium phosphate, and 0.1 mM EDTA, pH 7.0.
The concentrations of DNA and modified PNA oligomers were 5 mM
25, R = H
26, R =
OH
each for duplex formation. The samples were first heated to 90 ꢀC
for 5 min, followed by gradually cooling to room temperature.
Thermal denaturation profiles (abs vs T) of the hybrids were mea-
sured at 260 nm with an UV/Vis Lambda Bio 20 Spectrophotometer
equipped with a Peltier Temperature Programmer PTP6 interfaced
to a personal computer. UV-absorption was monitored at 260 nm
from in an 18e90 ꢀC range at the rate of 1 ꢀC per minute. A melting
curve was recorded for each duplex. The melting temperature (Tm)
was determined from the maximum of the first derivative of the
melting curves.
Scheme 3. Attempted synthesis of compound 25.
4.3. General procedure for manual solid-phase
oligomerization
PNA oligomers were assembled on a Rink amide PEGA resin
using the above obtained Fmoc-protected PNA modified monomers
as well as normal PNA monomers.
Rink amide PEGA resin (50e100 mg) was first swelled in CH2Cl2
for 30 min. Normal PNA monomers (from Link Technologies) were
provided by Advance Biosystem Italia srl. The Fmoc group was then
deprotected by 20% piperidine in DMF (8 minꢂ2). The resin was
washed with DMF and CH2Cl2 and tested to be positive by the
Kaiser test. The resin was preloaded with an Fmoceglycine and
subsequently, the coupling of the monomers (PNA or PNA modi-
fied) was conducted with either one of the following two methods
(A and B). Method A: monomer (5 equiv), HBTU (4.9 equiv), and
DIPEA (10 equiv); method B: modified monomer (2 equiv), HATU
(2 equiv), and DIPEA (10 equiv). When the monomer was coupled
to a primary amine, i.e., to a classic PNA monomer, method A was
used; when the coupling was to a secondary amine, i.e., to a mod-
ified PNA monomer, method B was used. The coupling mixture was
added directly to the Fmoc-deprotected resin. The coupling re-
action required 30 min at room temperature for the introduction of
both normal and modified monomers, in case of method B the
coupling time was raised to 6 h, and the resin was then washed by
DMF/CH2Cl2. The Fmoc deprotection and monomer coupling cycles
were continued until the coupling of the last residue. After every
Acknowledgements
ꢀ
This work has been supported by the Universita degli Studi di
Salerno and by the Universita degli Studi di Parma. We thank Dr.
Patrizia Iannece (University of Salerno) for mass spectra.
ꢀ
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€
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