Synthesis of novel 5'-uridine-head amphiphiles as model for DNA molecular recognition

Article abstract of DOI:10.1080/15257770903306625

Here we describe uridine functionalization in the 5' position, which provides new classes of cationic and nonionic amphiphiles specifically designed as DNA transfection agents. The synthetic procedures developed to obtain the cationic uridine-head surfactants prevented intramolecular cyclization that occurs when uridine is functionalized in this position without using protecting groups in the uracil.

Full text of DOI:10.1080/15257770903306625

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Synthesis of Novel 5′-Uridine-Head
Amphiphiles as Model for DNA Molecular
Recognition
Matteo Tiecco a , Pietro Di Profio a , Raimondo Germani a
Gianfranco Savelli a
&
a CEMIN–Excellence Centre on Innovative Nanostructured Materials,
Department of Chemistry , University of Perugia , Perugia, Italy
Published online: 20 Oct 2009.
To cite this article: Matteo Tiecco , Pietro Di Profio , Raimondo Germani & Gianfranco Savelli (2009)
Synthesis of Novel 5′-Uridine-Head Amphiphiles as Model for DNA Molecular Recognition, Nucleosides,
Nucleotides and Nucleic Acids, 28:10, 911-923, DOI: 10.1080/15257770903306625
To link to this article: http://dx.doi.org/10.1080/15257770903306625
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Nucleosides, Nucleotides and Nucleic Acids, 28:911–923, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903306625
SYNTHESIS OF NOVEL 5ꢀ-URIDINE-HEAD AMPHIPHILES AS MODEL
FOR DNA MOLECULAR RECOGNITION
Matteo Tiecco, Pietro Di Profio, Raimondo Germani, and Gianfranco Savelli
CEMIN–Excellence Centre on Innovative Nanostructured Materials, Department of
Chemistry, University of Perugia, Perugia, Italy
Here we describe uridine functionalization in the 5ꢁ position, which provides new classes of
2
cationic and nonionic amphiphiles specifically designed as DNA transfection agents. The synthetic
procedures developed to obtain the cationic uridine-head surfactants prevented intramolecular
cyclization that occurs when uridine is functionalized in this position without using protecting
groups in the uracil.
Keywords DNA transfection agents; uridine-head amphiphiles; uracil
Surfactants play important roles in studies of interaction with biomacro-
molecules: e.g. stabilization and super-activation of enzymes[1] and DNA
interaction and super-coiling effects.[2] Synthetic approaches are fo-
cused on preparing novel surfactant structures functionalized for specific
roles.
Recent publications[3] have focused on the synthesis of amphiphilic
nucleosides and nucleotides that are functionalized primarily in the base
and in 2ꢁ and 3ꢁ hydroxyl groups of the ribose. These amphiphiles have
interesting supramolecular properties in water, organic, and mixed solvents
because they form vesicles, helices, wormlike aggregates, fibers, and gels.
However, nucleosidic or nucleotidic amphiphiles specifically designed to
interact with DNA for transfection are still lacking considering the efficiency
of amphiphiles in this process.[4] The few available reports focus on
amphiphiles with only the base involved in interactions because the ribose
is functionalized in a way that strongly reduces the capability of recognition
of the headgroup.[5] Unfortunately, when functionalized in the 5ꢁ position,
Received 1 December 2008; accepted 18 May 2008.
Support of this work by Ministero dell’Universita` e Ricerca (PRIN 2006).
Address correspondence to Gianfranco Savelli, CEMIN–Excellence Centre on Innovative Nanos-
tructured Materials, Department of Chemistry, Via Elce di Sotto n◦8-I 06100 Perugia, University of
Perugia, Italy. E-mail: savelli@unipg.it
911

912
M. Tiecco et al.
SCHEME 1 a) p-toluenesulphonic acid, 2,2-dimethoxypropane, room temperature, 1.5 hours; b)
CH3SO2Cl, TEA, 0◦C, 16 hours; c) R = C12H25, reflux, 24 hours.
uridine can easily undergo intramolecular cyclization process that is some-
times the main reaction path.[6] For this reason we developed synthetic
routes to novel compounds functionalized in 5ꢁ position that are more effec-
tive. Here we describe alternative approaches that prevent intramolecular
cyclization and may prove useful not only for uridine amphiphiles synthesis,
but also for generic functionalization in the 5ꢁ position of uridine.
All our synthetic procedures start from 2ꢁ,3ꢁ-O-isopropylidene uri-
dine (2) that is prepared from commercially available uridine (1).
Our method improves on reported procedures[7] by decreasing reaction
times and increasing yields (Scheme 1). To synthesize the first class of
cationic compounds (which contains an ammonium group bound to
the 5ꢁ position) we functionalized 2 with mesyl group in the 5ꢁ posi-
tion. Reaction with dimethyldodecylamine provided the ammonium salt
(Scheme 1).
Nucleophilic substitution of the mesyl group in the 5ꢁ position of 3 was
very hard to reproduce and yields were low, ranging from 0 to about 30%
under different experimental conditions. The dominant process is uridine
intramolecular cyclization, as illustrated in Figure 1. That is triggered by
deprotonation by the amine. In fact, methyl didodecylamine, which is more
basic, gives only the cyclization product in this reaction. This well-known
intramolecular process[6] is an obstacle in oligo-nucleoside synthesis, that
is favoured by polar solvents and high temperatures. It occurs with a wide
range of leaving groups and bases, even in the 2ꢁ and 3ꢁ positions. Uridine
conformation is another major factor in this process. By rotation of the
FIGURE 1 Uridine intramolecular cyclization process.

Synthesis of Novel 5ꢁ-Uridine-Head Amphiphiles
913
FIGURE 2 syn and anti conformations of uridine.
C-N (1-1ꢁ) bond, uridine assumes two limit conformations: syn and anti
(Figure 2). Although uridine exists in both conformations, NOESY 1H-NMR
data[8] show that uridine 2ꢁ,3ꢁ-O-isopropylidene exists mainly in the syn
conformation (99:1) due to steric hindrance, the cyclization occurs from the
syn conformation. This unwanted product competing reaction was reduced
by removing the protecting group before mesyl substitution to increase the
population of the reactive anti conformation (Scheme 2).
When the same reaction was conducted on deprotected uridine, yields
were much higher and the process was reproducible. At reflux, only ∼10%
of cyclization product was obtained. At a lower temperature (65◦C) with
a longer reaction time (7 days) no cyclization product was observed. To
separate the ammonium salt from the cyclization product, the product
mixture was treated with NaClO4 and then passed over a Cl− ion exchange
to give the chloride salt. To obtain further evidence that removing the
protecting group to prevent cyclization is an efficient process, we repeated
the same reaction with methyl didodecylamine. On the deprotected mesyl
substrate (6) no product, neither the cyclization nor the substitution, was
observed even when the temperature was raised to 160◦C and DMF was
used instead of CH3CN, probably because of lower amine nucleophilicity.
Because the same reaction on the protected 5ꢁ-mesyl uridine (3) led only
to the cyclization product, these results shows that removal of 2ꢁ,3ꢁ-O-
isopropylidene to prevent cyclization is efficacious.
Another approach to obtain an ammonium salt via nucleophilic substi-
tution and preventing unwanted cyclization reactions is to functionalize the
uridine with a group that can be substituted by a tertiary amine at lower
temperatures. With this approach we synthesized a second class of cationic
SCHEME 2 a) HCl (10%), room temperature, 12 hours; b) 65◦C, 1 week.

914
M. Tiecco et al.
SCHEME 3 a) Chloroacetyl chloride, Py, -10◦C, 2 hours; b) dimethyldodecylamine (R = C12H25), 40◦C,
24 hours; c) DOWEX monosphere 650L resin, room temperature, 12 hours.
products with the ammonium bound in the 5ꢁ position with an ester spacer
(10) (Scheme 3).
2ꢁ,3ꢁ-O-isopropylidene uridine (2) was functionalized with chloroacetyl
chloride to obtain 5ꢁ-O–chloroacetyl-2ꢁ,3ꢁ-O-isopropylidene uridine (8).[9]
Reaction with dimethyldodecylamine gave an amphiphilic ammonium salt
(9). Because the reaction temperature was low (40◦C), no cyclization occurs
and the isopropylidene group does not need to be removed to prevent
cyclization. The final step is removal of the protecting group with an acid
resin such as DOWEX monosphere 650 L. At higher temperatures (reflux)
cyclization product (5) and a carboxy-betaine were also obtained (Figure 3).
This intramolecular process is somewhat different from that of the other
class of cationic compounds. In this case amine substitutes chloride and
then the amine excess that is needed to obtain the substitution product
promotes cyclization by acting as a base. This compound (10) is base-labile
and thermo-labile. We also observed degradation with mild sonication in
water or organic solvents.
The last class of uridine-head compounds are nonionic compounds.
Scheme 4 illustrates the procedures that provide these novel nonionic
amphiphilic uridine derivatives.
Functionalization in the 5ꢁ position was performed using lauroyl
chloride[10] and p-octyloxybenzoyl chloride. Isopropylidene was removed
FIGURE 3 The cyclization product and the carboxy-betaine obtained refluxing 5ꢁ-O-chloroacetyl-2ꢁ,3ꢁ-
isopropilidene uridine and dimethyldodecylamine.

Synthesis of Novel 5ꢁ-Uridine-Head Amphiphiles
915
SCHEME 4 a) Py, lauroyl chloride (11a), p-octyloxybenzoylchloride (11b), room temperature, 24 hours;
b) HCl (10%), room temperature, 12 hours (12a), 14 hours (12b).
with HCl to obtain very good yields of 5ꢁ-lauroyl uridine and 5ꢁ-p-
octyloxybenzoyl uridine (90% for 11a -80% for 11b and 87% for 12). Be-
cause uridine was functionalized with these substrates at room temperature,
no cyclization products were observed.
In summary, we functionalized uridine in the 5ꢁ position to give novel
uridine amphiphilic compounds. In the procedures developed to obtain
the two cationic surfactants, the methods prevented unwanted cyclization
products. Interestingly, the amide in uracil position 3 did not require
protection even though, given the role of this portion in the cyclization
process (Figure 1), this might have appeared to be the most efficient way
of preventing cyclization.
In the procedure for the first cationic amphiphile (7), removing the
protecting group in the uridine increased yields and ensured reproducibility
of nucleophilic substitution of the mesyl in 5ꢁ position even at high tem-
peratures that favour cyclization.[6] In the synthesis of the second cationic
product (10), we functionalized uridine as a quaternary ammonium, by us-
ing chloroacetyl chloride that yielded the nucleophilic substitution product
at temperatures of about 40◦C. The synthetic procedures for the nonionic
compounds (11 and 12) gave very good yields.
These novel compounds are currently being used on DNA interaction
studies to evaluate their capacity to super-coil DNA and to assess their
transfection efficiency.
EXPERIMENTAL
Unless otherwise specified, all reactions were carried out under anhy-
drous conditions with freshly distilled reagents and solvents or commercially
available anhydrous solvents and under nitrogen atmosphere. Melting
points were determined on Barloworld Scientific Stewart (Dunmow, UK)
SMP3 apparatusand are uncorrected. Gas-chromatography analyses were
performed on an Agilent 6850 Series II Network GC instrument (Agilent

916
M. Tiecco et al.
Technologies, Santa Clara, CA, USA). TLC separations were performed on
1
silica gel on TLC-cards on aluminium foils. H-NMR and 13C-NMR were
registered on a Bruker 200 MHz and on a Bruker 400 MHz instruments.
ESI-MS experiments were obtained on a QUATTRO triplequadrupole mass
spectrometer (Micromass, Manchester, UK), operating in the positive and
negative ion mode and equipped with a Z-spray electrospray source. Matrix-
assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectra
were measured on a Voyager DE-PRO MALDI-TOF mass spectrometer
(Applied Biosystems, Foster City, CA, USA) operating in the reflection
positive ion mode. MALDI mass spectra were calibrated through external
calibration using an Applied Biosystems standard calibration mixture.
2ꢁ,3ꢁ-O-isopropylidene-uridine (2)
Uridine (1) (3 g, 12.3 mmol), 2,2-dimethoxypropane (20 mL, 0.1626
mol), and p-toluenesulfonic acid (100 mg, 0.525 mmol) were dissolved in
dry acetone (100 mL) and stirred at room temperature for 1.5 hour. The
mixture was dried under vacuum at room temperature to give a spongy
yellow raw; the crude product was left in this condition for about 4 hours.
Amberlyst A-21 basic resin was then added with 20 mL of dry acetone. The
resin was filtered and the mixture dried under vacuum. The product was
crystallized from dichloromethane (DCM) at −20◦C to give white crystals.
Yield 90% (3.15 g); m.p. = 165–168◦C.
1H-NMR (200 MHz, CD3OD) δ = 1.3 (s, 3H, CH3 isop.), 1.5 (s, 3H, CH3
isop.), 3.8 (m, 2H, CH2 in 5ꢁ), 4.2 (m, 1H, H in 4ꢁ), 4.8 (m, 2H, H in 2ꢁ and
3ꢁ), 5.7 (d, 1H, A of AMX, JAX = 8 Hz, H in 5), 5.9 (m, 1H, H in 1ꢁ), 7.8 (d,
1H, M of AMX, JAX = 8 Hz, H in 6).
13C-NMR (200 MHz, CD3OD) δ = 25.7 (isop.), 27.7 (isop.), 63.2 (5ꢁ),
82.4 (2ꢁ), 86.0 (3ꢁ), 88.6 (4ꢁ), 94.3 (1ꢁ), 102.8 (5), 115.4 (ipso isop.), 144.1
(6), 152.4 (ipso 2), 166.4 (ipso 4).
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-mesyl-uridine (3)
2ꢁ,3ꢁ-O-isopropylidene-uridine (2) (1.314 g, 4.62 mmol) was dissolved in
anhydrous DCM (35 mL) with TEA (1 mL, 7.17 mmol). At the temperature
of 0◦C, CH3SO2Cl (0.4 mL, 5.17 mmol) was added dropwise. After 16 hours,
the mixture was washed with water and ice. The organic phases were dried
with anhydrous Na2SO4 and evaporated under vacuum: a pale yellow solid
was obtained. Yield 90% (1.50 g,); m.p. = 108–110◦C.
1H-NMR (200 MHz, CDCl3) δ = 1.4 (s, 3H, CH3 isop.), 1.6 (s, 3H, CH3
isop.), 3.0 (s, 3H, mesyl), 4.4 (m, 1H, H in 4ꢁ), 4.5 (m, 2H, CH2 in 5ꢁ), 4.9
(m, 1H, H in 2ꢁ), 5.0 (m, 1H, H in 3ꢁ), 5.6 (m, 1H, H in 1ꢁ), 5.7 (d, 1H, A of
AMX, JAX = 8 Hz, H in 5), 7.2 (d, 1H, M of AMX, JAX = 8 Hz, H in 6), 8.7
(sbr, 1H, NH in 3).

Synthesis of Novel 5ꢁ-Uridine-Head Amphiphiles
917
13C-NMR (200 MHz, CD3OD) δ = 24.6 (isop.), 26.6 (isop.), 37.6 (mesyl),
70.8 (5ꢁ), 82.7 (2ꢁ), 85.8 (3ꢁ), 86.7 (4ꢁ), 96.3 (1ꢁ), 103.1 (5), 115.7 (ipso isop.),
145.1 (6), 152.1 (ipso 2), 166.4 (ipso 4).
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-dimethyldodecyl Ammonium-uridine
Mesylate (4)
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-mesyl-uridine (3) (1.61 g, 4.44 mmol) was dis-
solved in anhydrous CH3CN (30 mL) with freshly distilled dimethyldodecy-
lamine (1.8 mL, 6.49 mmol), the mixture was refluxed for 24 hours. The
solvent was evaporated by vacuum and the resultant yellow oil washed many
times with anhydrous diethyl ether. The solid was crystallized in ethyl acetate
at -20◦C. Yield 28% (0.72 g,); m.p. = 184–186◦C.
1H-NMR (400 MHz, CD3OD) δ = 0.9 (t, 3H, CH3-R), 1.3 (m, 18H,
(CH2)9), 1.4 (s, 3H, CH3 isop.), 1.5 (s, 3H, CH3 isop.), 1.8 (m, 2H, CH2
in β at N+), 2.7 (s, 3H, CH3SO3−), 2.9 (ss, 6H, N+(CH3)2), 3.1 (m, 2H,
R-CH2-N+), 4.3 (m, 1H, H in 2ꢁ), 4.6 (m, 1H, H in 3ꢁ), 5.0 (m, 1H, H in 4ꢁ),
5.1 (dd, 2H, CH2 in 5ꢁ), 5.7 (m, 1H, H in 1ꢁ), 6.1 (d, 1H, A of AMX, JAX = 8
Hz, H in 5), 7.9 (d, 1H, M of AMX, JAX = 8 Hz, H in 6).
13C-NMR (400 MHz, CD3OD) δ = 14.6 (CH3 chain), 24.9 (isop.), 26.6
(isop.), 23.9, 25.9, 27.6, 30.4-30.9 (CH2- chain), 33.2 (mesyl), 39.7 (5ꢁ), 44.6
(α at N+), 59.3 (N+(CH3)2), 76.7 (3ꢁ), 83.2 (4ꢁ), 86.7 (2ꢁ), 100.6 (1ꢁ), 110.5
(5), 114.3 (ipso isop.), 145.8 (6), 160.0 (ipso 2), 175.0 (ipso 4).
Didodecylmethylamine
Didodecylamine (3.1 g, 8.76 mmol) was dissolved in 40 mL of ethanol at
40◦C and HCOOH (1.6 mL, 42.4 mmol) was added dropwise. After 30 min-
utes, HCOH (1.6 mL, 23.2 mmol) was added dropwise and the temperature
was raised to 82◦C for 2 hours. After cooling to room temperature aqueous
10% NaOH solution was added to quench the reaction. DCM was added
and the organic phase was separated and evaporated to obtain a pale yellow
liquid. This liquid was purified on a silica gel column, eluent EtOH. Yield
74% (2.38 g); GC > 99%.
1H-NMR (400 MHz, CDCl3) δ = 0.8 (t, 6H, 2CH3), 1.1 (m, 36H, -
(CH2)18-), 1.3 (m, 4H, 2CH2 in β to N), 2.1 (s, 3H, CH3), 2.2 (t, 4H, CH2-N).
Cyclonucleoside (5)
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-mesyl-uridine (3) (0.955 g, 2.64 mmol) was
dissolved in anhydrous CH3CN (40 mL) with didodecylmethylamine (1.4 g,
3.81 mmol), and refluxed for 6 days. The solvent was evaporated by vacuum
and the resultant yellow oil washed many times with diethyl ether. The crude

918
M. Tiecco et al.
product was recrystallized from diethyl ether/ethyl acetate (50/50) to give
white crystals. Yield 60% (0.45 g); m.p. = decomposes at T ≥ 230◦C.
1H-NMR (200 MHz, CDCl3) δ = 1.3 (s, 3H, CH3 isop.), 1.5 (s, 3H,
CH3isop.), 4.1 (m, 1H, H in 3ꢁ), 4.4 (m, 1H, H in 2ꢁ), 4.6 (m, 1H, H in
4ꢁ), 4.9 (m, 2H, CH2 in 5ꢁ), 5.3 (m, 1H, H in 1ꢁ), 6.0 (d, 1H, A of AMX, JAX
= 8 Hz, H in 5), 7.2 (d, 1H, M of AMX, JAX = 8 Hz, H in 6).
Anal. Calcd for C12H16N2O5: N 9.99, C 55.71, H 5.75; found N 10.12, C
56.00, H 5.98.
5ꢁ-Mesyl-uridine (6)
2ꢁ3ꢁ-O-isopropylidene-5-mesyl-uridine (3) (1.504 g, 4.15 mmol) was dis-
solved in MeOH (7 mL) at room temperature; a water solution of HCl
(10%) was added dropwise. After 12 hours the mixture was dried under
vacuum, extracted with MeOH, filtered, the filtrate was dried under vacuum,
and then treated with P2O5 under vacuum for 3 days to remove the residual
H2O. Yield 85% (1.137 g,); amorphous.
1H-NMR (200 MHz, CD3OD) δ = 3.0 (s, 3H, mesyl), 4.2 (m, 2H, CH2 in
5ꢁ), 4.3 (m, 1H, H in 4ꢁ), 4.5 (m, 1H, 2ꢁ), 4.6 (5.5 m, 1H, 3ꢁ), 5.9 (m, 1H, H
in 1ꢁ), 5.7 (d, 1H, A of AMX, JAX = 8 Hz, H in 5), 7.7 (d, 1H, M of AMX, JAX
= 8 Hz, H in 6).
13C-NMR (200 MHz, CD3OD) δ = 37.5(mesyl), 70.3 (5ꢁ), 70.4 (3ꢁ), 74.9
(2ꢁ), 83.0 (4ꢁ), 91.7 (1ꢁ), 103.1 (5), 142.6 (6), 152.4 (ipso 2), 166.2 (ipso 4).
Uridine-5ꢁ-dimethyldodecyl Ammonium Mesylate (7)
5-mesyl-uridine (6) (1.00 g, 3.10 mmol) was dissolved in anhydrous
CH3CN (40 mL) with dimethyldodecylamine (1.29 mL, 4.65 mmol), treated
at 65◦C for 1 week, and dried under vacuum to give a yellow oil. This crude
product was washed many times with Et2O giving a semi-solid material that
was crystallized from Et2O/MeOH (75/25) giving white, highly hygroscopic
crystals. Yield 75% (1.24 g); m.p. = 154–156◦C.
1H-NMR (200 MHz, CD3OD) δ = 0.8 (t, 3H, CH3-R), 1.3 (m, 18H,
(CH2)9), 1.8 (m, 2H, CH2 in β at N+), 2.7 (s, 3H, mesyl), 3.2 (ss, 6H,
N+(CH3)2), 3.4 (m, 2H, R-CH2-N+), 3.7 (m, 1H, H in 2ꢁ), 3.8 (m, 1H, H in
3ꢁ), 4.1 (m, 1H, H in 4ꢁ), 4.3 (m, 1H, H in 5ꢁ), 5.7 (d, 1H, A of AMX, JAX = 8
Hz, H in 5), 5.8 (m, 1H, H in 1ꢁ), 7.7 (d, 1H, M of AMX, JAX = 8 Hz, H in 6).
13C-NMR (400 MHz, CD3OD) δ = 14.7 (CH3 chain), 23.9-33.3 (-CH2-
chain), 39.7 (mesyl), 52.6 (5ꢁ), 52.7 (α at N+), 66.7 (N+(CH3)2), 66.9
(N+(CH3)2), 73.4 (2ꢁ), 73.6 (3ꢁ), 78.1 (4ꢁ), 95.7 (1ꢁ), 103.5 (5), 144.2 (6),
152.1 (ipso 2), 166.1 (ipso 4).
MALDI-TOF MS: m/z [M+H]+ calcd for C23H42N3O5 440.32 found
440.34.
ESI-MS: m/z [M+H]+ calcd for C23H42N3O5 440.32 found 440.50.

Synthesis of Novel 5ꢁ-Uridine-Head Amphiphiles
919
Uridine 2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-O-chloroacetyl (8)
Uridine 2ꢁ,3ꢁ-O-isopropylidene (2) (1.49 g, 5.24 mmol) was dissolved
in anhydrous DCM (30 mL) with pyridine (0.85 mL, 10.5 mmol), cooled
to −10◦C, and chloroacetyl chloride (0.83 mL, 10.4 mmol) was added
dropwise with magnetic stirring. After 2 hours, cold water saturated of NaCl
was added, the organic phase washed many times with water, dried with
anhydrous Na2SO4, and evaporated under vacuum. The crude product was
crystallized from DCM/Et2O (60/40) at -20◦C. Yield 72% (1.36 g); m.p. =
139–141◦C.
1H-NMR (200 MHz, CDCl3) δ = 1.4 (s, 3H, CH3 isop.), 1.6 (s, 3H, CH3
isop.), 4.1 (s, 2H, ClCH2CO), 4.4 (m, 1H, H in 4ꢁ), 4.5 (m, 2H, CH2 in 5ꢁ),
4.9 (m, 1H, H in 2ꢁ), 5.1 (m, 1H, H in 3ꢁ), 5.6 (m, 1H, H in 1ꢁ), 5.8 (d, 1H,
A of AMX, JAX = 8 Hz, H in 5), 7.3 (d, 1H, M of AMX, JAX = 8 Hz, H in 6),
9.7 (sbr, 1H, NH in 3).
13C-NMR (200 MHz, CDCl3) δ = 24.9 (isop.), 26.7 (isop.), 40.4 (Cl-
CH2), 65.3 (5ꢁ), 80.6 (4ꢁ), 84.2 (2ꢁ), 85.0 (3ꢁ), 95.0 (1ꢁ), 102.4 (5), 114.4
(ipso isop.), 142.4 (6), 150.4 (ipso 2), 163.3 (ipso 4), 166.6 (ipso acetyl).
Uridine 2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-O-Acetyl-dimethyl Dodecyl
Ammonium Chloride (9)
Uridine 2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-O-chloroacetyl (8) (3.18 g, 8.83 mmol)
was dissolved in anhydrous CH3CN (40 mL) with dimethyldodecylamine
(3 mL, 10.8 mmol) and warmed at 40◦C for 24 hours. Removal of the solvent
under vacuum to gave a pale yellow oil. The crude product was washed many
times with diethyl ether giving a noncrystalline sticky solid. Yield 70% (3.55
g); amorphous.
1H-NMR (200 MHz, CD3) δ = 0.9 (t, 3H, CH3-R), 1.3 (m, 20H, (CH2)10-
), 1.4 (s, 3H, CH3 isop.), 1.6 (s, 3H, CH3 isop.), 1.8 (m, 2H, CH2 in β at N+),
3.3 (m, 6H, CH3-N+), 3.5 (m, 2H, CH2 in α at N+), 3.6 (m, 1H, H in 3ꢁ), 3.8
(m, 2H, H in 2ꢁ), 3.9 (m, 2H, CO-CH2-N+), 4.3 (H in 4ꢁ), 4.4 (m, 2H, CH2
in 5ꢁ), 5.7 (d, 1H, A of AMX, JAX = 8 Hz, H in 5), 5.9 (m, 1H, H in 1ꢁ), 7.8
(d, 1H, M of AMX, JAX = 8 Hz, H in 6).
13C-NMR (200 MHz, CD3OD) δ = 12.5 (CH3- chain), 25.7 (CH3 isop),
28.3 (CH3 isop), 21.8, 22.7, 23.7, 25.4, 28.6, 28.8, 31.2 (-CH2- chain), 49.8
(-CH2-CH2-N+), 50.3 (R-CH2-N+), 60.4, 61.2 (CH3-N ), 63.7 (N+-CH2-CO),
+
65.1 (5ꢁ), 80.3 (3ꢁ), 83.9 (2ꢁ), 86.5 (4ꢁ), 92.2 (1ꢁ), 100.8 (5), 113.2 (ipso isop),
141.9 (6), 151.1 (ipso 2), 164.8 (ipso N+-CH2-CO), 165.0 (ipso 4).
Uridine-5ꢁ-O-Acetyl-dimethyl Dodecyl Ammonium Chloride (10)
Uridine 2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-O-acetyl-dimethyl dodecyl ammonium
chloride (9) (3.55 g, 6.18 mmol) was dissolved in MeOH at room tempera-
ture, and DOWEX monosphere 650L (H+) resin beads (∼8 g) were added,

920
M. Tiecco et al.
stirred at room temperature overnight, passed through a column containing
the same resin, and dried under vacuum to give a slightly yellow solid. Yield
88% (2.90 g); m.p. = 168–171◦C.
1H-NMR (200 MHz, CD3OD) δ = 0.9 (t, 3H, CH3-R), 1.3 (m, 20H,
(CH2)10-), 1.7 (m, 2H, CH2 in β at N+), 3.3 (s, 6H, CH3-N+), 3.6 (m, 2H,
CH2 in α at N+), 3.7 (m, 2H, H in 2ꢁ), 3.8 (s, 2H, N+CH2CO), 4.1 (m, 2H,
H in 3ꢁ), 4.2 (m, 2H, CH2 in 5ꢁ), 4.4 (m, 1H, H in 4ꢁ), 5.7 (d, 1H, A of AMX,
JAX = 8 Hz, H in 5), 5.9 (m, 1H, H in 1ꢁ), 8.0 (d, 1H, M of AMX, JAX = 8 Hz,
H in 6).
13C-NMR (200 MHz, CD3OD) δ = 14.7 (CH3 chain), 23.8, 27.4, 30.3,
30.9, 33.2, 52.6 (-CH2- chain), 54.0 (R-CH2-N+), 62.2, 62.4 (CH3-N ), 66.8
+
(N+-CH2-CO), 67.2 (5ꢁ), 71.4 (3ꢁ), 75.5 (2ꢁ), 86.5 (4ꢁ), 90.8 (1ꢁ), 103.0 (5),
143.0 (6), 152.7 (ipso 2), 166.5 (ipso N+-CH2-CO), 166.7 (ipso 4).
MALDI-TOF MS: m/z [M+H]+ calcd for C25H44N3O7 498.63 found
498.48.
ESI-MS: m/z [M+H]+ calcd for C25H44N3O7 498.63 found 498.52.
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-lauroyl-uridine (11a)
2ꢁ3ꢁ-O-isopropylidene-uridine (2) (2.87 g, 10.1 mmol) was dissolved with
anhydrous pyridine (1 mL, 12.4 mmol) in dry DCM/THF (70/30) at room
temperature. Lauroyl chloride (2.6 mL, 11.2 mmol) was added dropwise
with magnetic stirring for 24 hours. The solvent was removed under vacuum
to give a yellow oil that was dissolved in Et2O, the organic phase was
washed with water, dried with anhydrous Na2SO4, and dried under vacuum.
The crude product was purified by column chromatography (SiO2, Et2O),
yielding a sticky solid. Yield 90% (4.24 g); amorphous.
1H-NMR (200 MHz, CDCl3) δ = 0.6 (t, 3H, R-CH3), 1.0 (m, 18H, -CH2-),
1.1 (s, 3H, CH3 isop.), 1.3 (s, 3H, CH3 isop.), 2.0 (t, 2H, CH2CO), 4.0
(m, 1H, H in 4ꢁ), 4.1 (m, 2H, CH2 in 5ꢁ), 4.5 (m, 1H, H in 2ꢁ), 4.7 (m,
1H, H in 3ꢁ), 5.4 (m, 1H, H in 1ꢁ), 5.5 (d, 1H, A of AMX, JAX = 8 Hz,
H in 5), 7.0 (d, 1H, M of AMX, JAX = 8 Hz, H in 6), 8.7 (sbr, 1H, NH
in 3).
13C-NMR (200 MHz, CD3OD) δ = 14.6 (CH3- chain), 23.9 (CH3- isop),
27.6 (CH3- isop), 14.6-33.2 (-CH2- chain), 35.0 (R-CH2-CO), 65.3 (5ꢁ), 82.9
(3ꢁ), 86.0 (2ꢁ), 86.5 (4ꢁ), 95.8 (1ꢁ), 104.8 (5), 115.5 (ipso isop), 144.6 (6),
152.0 (ipso 2), 166.3 (ipso 4), 170.8 (ipso R-CO).
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-p-octyloxybenzoyl-uridine (11b)
2ꢁ3ꢁ-O-isopropylidene-uridine (2) (1.72 g, 6.16 mmol) was dissolved
in anhydrous pyridine (25 mL) at room temperature. p-Octyloxybenzoyl
chloride (2.6 mL, 11.2 mmol) was added dropwise and the solution was
stirred for 24 hours. The pyridine was removed under vacuum, the resultant

Synthesis of Novel 5ꢁ-Uridine-Head Amphiphiles
921
yellow raw was dissolved in Et2O and washed with aqueous HCl (10%). The
organic phase was dried with anhydrous Na2SO4 and Et2O removed under
vacuum. The product was purified by column chromatography (SiO2, Et2O)
giving a white sticky solid. Yield 80% (2.54 g); amorphous.
1H-NMR (200 MHz, CD3OD) δ = 0.9 (t, 3H, CH3-R), 1.3 (m, 12H, -
(CH2)6-), 1.4 (s, 3H, CH3 isop.), 1.6 (s, 3H, CH3 isop.), 1.8 (m, 2H, O-CH2-
CH2-), 4.0 (t, 2H, O-CH2), 4.5 (m, 1H, H in 4ꢁ), 4.5 (m, 2H, CH2 in 5ꢁ), 5.0
(m, 1H, H in 2ꢁ), 5.1 (m, 1H, H in 3ꢁ), 5.6 (d, 1H, A of AMX, JAX = 8 Hz, H
in 5), 5.8 (m, 1H, H in 1ꢁ), 7.0 (m, 2H, Ar), 7.6 (d, 1H, M of AMX, JAX = 8
Hz, H in 6), 7.9 (m, 2H, Ar).
13C-NMR (200 MHz, CD3OD) δ = 14.6 (CH3- chain), 25.6 (CH3- isop),
27.2 (CH3- isop), 23.8, 24.3, 27.6, 30.4, 30.5, 33.1 (-CH2- chain), 65.6 (5ꢁ),
69.5 (O-CH2-), 82.9 (3ꢁ), 86.2 (2ꢁ), 86.8 (4ꢁ), 95.9 (1ꢁ), 102.8 (5), 115.5 (ipso
isop), 115.5 (Ar), 123.0 (ipso Ar-CO), 132.8 (Ar), 144.2 (6), 152.1 (ipso 2),
165.0 (ipso Ar-O), 166.3 (ipso 4), 170.8 (ipso Ar-CO).
5ꢁ-lauroyl-uridine (12a)
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-lauroyl-uridine (11a) (4.2 g, 9.0 mmol) was
dissolved in acetone at room temperature (50 mL), aqueous HCl (10%) was
added until the reaction ended as shown by TLC. The reaction required 40
mL of HCl (10%) over about 12 hours. The product was insoluble in this
mixture, and collected by filtration giving white crystals. Yield 87% (3.34 g);
m.p. = 138–140◦C.
1H-NMR (200 MHz, DMSO-d6) δ = 0.9 (t, 3H, R-CH3), 1.2 (m, 16H,
-(CH2)9-), 1.5 (t, 2H, CH2-CH2-CO), 2.3 (t, 2H, CH2-CO), 3.6 (m, 1H, H in
2ꢁ), 3.9 (m, 2H, CH2 in 5ꢁ), 4.2 (m, 1H, H in 4ꢁ), 5.2 (m, 1H, H in 3ꢁ), 5.5
(m, 1H, H in 2ꢁ), 5.7 (d, 1H, A of AMX, JAX = 8 Hz, H in 5), 5.8 (m, 1H, H
in 1ꢁ), 7.6 (d, 1H, M of AMX, JAX = 8 Hz, H in 6), 11.3 (sbr, 1H, H in 3).
13C-NMR (200 MHz, DMSO-d6) δ = 12.6 (CH3- chain), 22.1, 24.4, 28.7,
29.0, 31.3, (-CH2- chain), 33.3 (R-CH2-CO), 63.6 (5ꢁ), 69.7 (3ꢁ), 72.7 (2ꢁ),
81.0 (4ꢁ), 88.6 (1ꢁ), 101.9 (5), 140.7 (6), 150.6 (ipso 2), 164.0 (ipso 4), 172.7
(ipso R-CO).
ESI-MS: calcd for C21H34N2O7 426.51, m/z [M+H]+ found 427.50; m/z
[M-H]− found 425.32.
5ꢁ-p-octyloxybenzoyl-uridine (12b)
2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-p-octyloxybenzoyl-uridine (11b) (0.78 g, 1.51
mmol) was dissolved in 5 mL of acetone; aqueous HCl (10%) added until
the reaction was complete as shown by TLC. The 25 mL of HCl 10% was
added over about 14 hours. The insoluble product was collected by filtration
giving white crystals. Yield 87% (0.63 g); m.p. = 156–159◦C.

922
M. Tiecco et al.
1H-NMR (200 MHz, DMSO-d6) δ = 0.9 (t, 3H, R-CH3), 1.3 (m, 10H,
(-CH2)5-), 1.7 (m, 2H, O-CH2-CH2-), 4.1 (t, 2H, O-CH2-CH2-), 4.2 (m, 1H,
H in 4ꢁ), 4.4 (m, 2H, CH2 in 5ꢁ), 5.3 (m, 1H, H in 2ꢁ), 5.6 (m, 1H, H in 3ꢁ),
5.7 (d, 1H, A of AMX, JAX = 8 Hz, H in 5), 5.8 (m, 1H, H in 1ꢁ), 7.1 (m, 2H,
Ar), 7.6 (d, 1H, M of AMX, JAX = 8 Hz, H in 6), 7.9 (m, 2H, Ar), 11.4 (sbr,
1H, H in 3).
13C-NMR (200 MHz, DMSO-d6) δ = 12.6 (CH3- chain), 22.1, 25.4, 28.7,
31.2 (-CH2- chain), 63.9 (O-CH2-), 67.9 (5ꢁ), 69.7 (3ꢁ), 72.8 (2ꢁ), 81.1 (4ꢁ),
89.0 (1ꢁ), 101.9 (5), 114.5 (Ar), 121.3 (ipso Ar-CO), 131.4 (Ar), 140.8 (6),
150.5 (ipso 2), 162.8 (ipso Ar-O), 163.0 (ipso 4), 165.2 (ipso Ar-CO).
ESI-MS: calcd for C24H34N2O7 476.52, m/z [M+H]+ found 477.53; m/z
[M-H]− found 475.36.
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