Synthesis and hybridization studies of a 5-aminopentanoic acid nucleobase (APN) dimer

Article abstract of DOI:10.1081/NCN-120003176

We have prepared a 5-aminopentanoic acid nucleobase (APN) dimer and investigated its hybridization capabilities to complementary DNA using both UV melting and NMR techniques.

Full text of DOI:10.1081/NCN-120003176

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SYNTHESIS AND HYBRIDIZATION STUDIES OF A 5-
AMINOPENTANOIC ACID NUCLEOBASE (APN) DIMER
Susan F. Donaldson a , Stephen C. Bergmeier b , Jennifer V. Hines b & Melinda S. Gerdeman a
a Division of Medicinal Chemistry , Ohio State University , Columbus, Ohio, 43210, U.S.A.
b Department of Chemistry & Biochemistry , Ohio University , Athens, Ohio, 45701, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Susan F. Donaldson , Stephen C. Bergmeier , Jennifer V. Hines & Melinda S. Gerdeman (2002) SYNTHESIS
AND HYBRIDIZATION STUDIES OF A 5-AMINOPENTANOIC ACID NUCLEOBASE (APN) DIMER, Nucleosides, Nucleotides and Nucleic
Acids, 21:2, 111-123, DOI: 10.1081/NCN-120003176
To link to this article: http://dx.doi.org/10.1081/NCN-120003176
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 21(2), 111–123 (2002)
SYNTHESIS AND HYBRIDIZATION STUDIES OF
A 5-AMINOPENTANOIC ACID NUCLEOBASE
(APN) DIMER
Susan F. Donaldson,1 Stephen C. Bergmeier,2,* Jennifer V. Hines,2
and Melinda S. Gerdeman1
1Division of Medicinal Chemistry, Ohio State University,
Columbus, Ohio 43210
2Department of Chemistry & Biochemistry, Ohio University,
Athens, Ohio 45701
ABSTRACT
We have prepared a 5-aminopentanoic acid nucleobase (APN) dimer and
investigated its hybridization capabilities to complementary DNA using
both UV melting and NMR techniques.
The principle of antisense=antigene inhibition of gene expression requires the
binding of a complementary oligonucleotide to either DNA (antigene)1 or
RNA (antisense)2. Binding of the oligonucleotide to target DNA or RNA
ultimately results in inhibition of transcription, translation, or RNA pro-
cessing through a variety of mechanisms3. Due to severe limitations in sta-
bility and utility of natural oligonucleotides as drugs, most antisense and
antigene agents are based on modified oligonucleotides.
Peptide nucleic acids (PNAs, 2) are a unique type of oligonucleotide
analog in which both the phosphodiester backbone and the sugar moiety
have been replaced with a peptide based structure4. PNAs have shown
excellent hybridization properties to both DNA and RNA targets5. Since
their introduction in 1991, many analogs have been synthesized and
*Corresponding author.
111
Copyright # 2002 by Marcel Dekker, Inc.
www.dekker.com

112
DONALDSON ET AL.
Figure 1.
evaluated for their ability to bind DNA and RNA6. Of particular note to our
studies has been the preparation of a variety of very flexible PNA type
molecules7. While some of these molecules have shown decreased hybridi-
zation ability, others have shown very similar hybridization properties to
PNAs. In order to further investigate the structural requirements of PNAs,
we reported8 the synthesis of a PNA of the general structure 3 in which we
oligomerized 5-aminopentanoic acid nucleobase (APN) monomers. We now
wish to report the results of UV melting and NMR hybridization studies with
Scheme 1.

5-AMINOPENTANOIC ACID NUCLEOBASE DIMER
113
an APN-T2 dimer as well as full experimental details for the synthesis of these
novel oligonucleotide analogues.
Synthesis of the dimer was carried out as previously described8. As
reported, to obtain APN-T2, it was necessary to synthesize two separate pie-
ces, the 50-end starter unit 6 and the 30-end monomer unit 11. Pieces 6 and 11
could be deprotected and coupled together to form the APN dimer. This dimer
was then used to study the hybridization capabilities of the APN molecule.
The synthesis of the 50-end of the oligomer is shown in scheme one.
Starting from diethanolamine (4), the nitrogen is protected as a t-butyl car-
bamate and one of the alcohols is protected as a benzyl carbonate to
produce 5. The remaining alcohol is coupled to N3-benzoylthymine9 via
Mitsunobu conditions10 to give 611.
The route to the 30-end monomer unit 11 starts from d-valerolactam (7)
with an N-allylation to provide 8. The lactam was hydrolyzed, the acid
esterified and the nitrogen protected as the t-butyl carbamate derivative to
give 9 in 62% yield from 8. The olefin was then cleaved with O3, and a
reductive workupwith NaBH gave alcohol 10. Finally the thymine was
4
introduced via a Mitsunobu reaction as before to give 11.
With monomer units 6 and 11 in hand, the amine of 6 was deprotected
to provide 12 using HCl12. The carboxyl of 11 was deprotected with hydrogen
and Pd/C to provide 13. For the coupling of 12 and 13 both 2-(1H-Benzo-
triazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU)13
and 2-(1H-Benzotriazol-1-yl)-1,1,3,3 tetramethyl uronium tetrafluoroborate
(TBTU)14 were examined, with TBTU providing the highest yield (67%)15.
In order to examine the hybridization capabilities of APN-T2 to target
DNA, it was necessary to deprotect the dimer. Using ammonium hydroxide
in dioxane, the benzoyl groups of the dimer were removed. Not suprisingly,
the Cbz groupwas also removed under these conditions thus, eliminating the
need for a catalytic hydrogenation step. Treatment of the crude material with
triflouroacetic acid to remove the remaining BOC group, gave salt 14 in 87%
yield over two steps.
Scheme 2.

114
DONALDSON ET AL.
With fully deprotected dimer in hand, hybridization studies using both
UV and NMR techniques were carried out. Hybridization studies are not
usually carried out on such a small oligonucleotides. The Tm of a hybrid
system decreases with sequence length because there are less hydrogen bonds
involved in the duplex structure17. Similarly the stacking of the bases, which
produces the change in hypochromicity upon melting is a function of oli-
gonucleotide length. Therefore, the Tm of a very short sequence occurs at a
low temperature and may be difficult to detect by UV. However, others have
used small synthetic oligonucleotide analogues and successfully studied their
annealing with UV melting.
UV melting studies18 were carried out in a buffer containing 1 M NaCl,
10 mM Tris and 10 mM MgCl2. Three samples were prepared with the fol-
lowing concentrations: (a) 4.30610À 7 M in polydA (control sample), (b)
4.30610À 7 M in polydA and 2.19610À 4 M in dT2, (c) 4.30610À 7 M in
polydA and 2.19610À 4 M in 15. Samples (b) and (c) were prepared so as to
result in a 1 base to 1 base ratio between the adenines in the polydA and the
thymines in either the native dT2 or APN-T2.
The melting experiment was conducted over a temperature range from
4ꢀC to 80ꢀC with a ramprate of 0.5 ꢀC=min. First derivatives of the melt data
(Fig. 2) were performed using the OD Deriv program19. As expected, melting
transitions for the dimers with polydA were observed at temperatures less
than 20ꢀC. While the data was noisy (due to the small hypochromicity
change) it appeared to indicate that our synthetic dimer APN-T2 might be
hybridizing to polydA with only a slightly reduced Tm relative to dT2
(polydA: T2, Tm ꢁ 16ꢀC, polydA: APN-T2, Tm ꢁ 11ꢀC). While inconclusive,
these promising results prompted further investigation into APN hybridiza-
tion utilizing NMR experiments.
Figure 2. First derivative of melt data for polydA:T2 and polydA:APN-T2.

5-AMINOPENTANOIC ACID NUCLEOBASE DIMER
115
NMR experiments were conducted to observe the imino protons of the
APN dimer in the presence of dA4 (note: polydA was too large to use in these
NMR experiments). Observation of an imino proton indicates base pair
formation as H-bonding protects the imino proton from exchange. This type
of experiment has been carried out previously to provide information
regarding base pair stability of an oligonucleotide containing 5-fluorour-
acil20. In our experiments, water supression was achieved using a WATER-
GATE pulse sequence21. The aqueous buffer used was 1 M NaCl, 10 mM
NaH2PO4, 1 mM EDTA. Magnesium was not included in the experiments in
order to minimize degradation of the dA4. Samples were prepared as 90%
buffer and 10% D2O with DSS as an internal standard. Five samples were
prepared: (a) 1.0610À 3 dA4 (control sample), (b) 2.0610À 3 APN-T2
(control sample) (c) 2.0610À 3 T2 (control sample) (d) 1.0610À 3 dA4 and
2.0610À 3 APN-T2 (e) 1.0610À 3 dA4 and 2.0610À 3 T2. Samples of (d) and
(e) were prepared so as to result in a 1 base to 1 base ratio. Experiments were
conducted at 1ꢀ increments from 1ꢀC to 12ꢀC. As expected, no imino proton
signals were observed with control samples (a), (b), or (c). Figs. 3 and 4 show
the results obtained with samples (d) and (e) containing the two sets of
complementary strands.
Figure 3 shows the imino region of the dA4:APN-T2 experiment. At 1ꢀC
an imino peak is clearly visible. As the temperature is slowly increased from
1ꢀ to 8ꢀC the imino peak gradually disappears. At 5ꢀC the imino peak has
virtually disappeared. Figure 4 shows the imino peak of the dA4:T2 sample.
Again at low temperature the imino peak is clearly visible. Upon warming the
imino peak gradually disappears. In the dA4:T2 sample the imino peak is
visible until 6–7ꢀC. This again indicates that the dT2 forms dimers that are
only slightly more stable than our synthetic analogue, APN-T2. The overall
lower Tm values in the NMR experiments vs. the UV experiments is most
likely due to differences in the buffers.
Based on the UV melting experiments and the NMR observations of the
imino protons we can conclude that our APN analogues of oligonucleotides
should have similar hybridization properties as native DNA-DNA hybridi-
zation. Further experiments with longer, solid-phase synthesized oligomers
will investigate this possibility.
EXPERIMENTAL
1H and 13C spectra were recorded on a Bruker AF 250, Bruker AF 270
or a Bruker DRX 400 model spectrometer. Chemical shifts are reported in
ppm relative to tetramethylsilane unless otherwise noted. Melting points
were taken using a Thomas Hoover capillary melting point apparatus and
are uncorrected. Thin layer chromatography (TLC) was performed on
Whatman pre-coated silica gel F254 aluminum foils. Purification of the

116
DONALDSON ET AL.
Figure 3. Imino region of dA4:APN-T2.
reaction products was carried out by flash column chromatography using a
glass column dry packed with silica gel (230–400 mesh ASTM) according to
the method of Still21. Visualization was accomplished with UV light and/or
ninhydrin solution followed by heating. Exact mass measurements recorded
in the electron impact (EI) mode were determined at The Ohio State Uni-
versity Chemical Instrument Center with a Kratos MS-30 mass spectro-
meter. UV spectra were recorded on a Beckman DU-40 spectrometer.
Combustion analyses were performed at Quantitative Technologies, Inc.,
Whitehouse, New Jersey. Infrared spectra were obtained on salt plates in
CCl4 or as a KBr pellet, using a Nicolet Protege 460 model spectrometer.
Peaks are reported in cmÀ 1. UV melting studies were carried out using a
Perkin Elmer Lambda 10 UV/Vis Spectrometer equipped with a Peltier
Temperature Programmer. NMR hybridization studies were carried out on a
Bruker DRX 400 model spectrometer. THF was distilled from sodium and
benzophenone. CH3CN was distilled from phosphorus pentoxide before use.
CH2Cl2 was distilled from CaH2. Et3N, iPr2NEt, and pyridine were distilled
from CaH2 and stored over KOH pellets. Benzoyl chloride was distilled

5-AMINOPENTANOIC ACID NUCLEOBASE DIMER
117
Figure 4. Imino region of dA4:T2.
under vacuum just before use. Acetyl chloride and EtOAc were distilled
under N2 before use. All reactions were carried out under an N2 atmosphere
unless otherwise specified.
O-Benzyloxycarbonyl-N-tert-butoxycarbonyl diethanolamine (5). A solu-
tion of diethanolamine (4.8 g, 45.4 mmol) in THF (45.4 mL) was cooled to 0ꢀC.
To this solution, di-tert-butyl dicarbonate (9.9 g, 45.4 mmol) was added. The
reaction was stirred at room temperature for 24 h. Concentration followed by
column chromatography (EtOAc) gave 9.3 g of N-BOC diethanolamine (100 )
as an oil: 1H NMR (CDCl3, 250 MHz) d 1.46 (s, 9H), 3.43 (t, 4H), 3.76 (t, 4H).
13C NMR (CDCl3, 62.5 MHz) d 28.02, 51.58, 61.04, 79.72, 155.93. IR (CCl4)
1669, 2977, 3385 cmÀ 1. HRMS calculated for C9H19NO4 was 205.1315, found
205.1319. Next, N-BOC diethanolamine (5.0 g, 24.4 mmol), DMAP (300 mg,
2.43 mmol), and Et3N (3.4 mL, 24.36 mmol) were dissolved in CH2Cl2 (49 mL).
The mixture was cooled to 0ꢀC and benzylchloroformate (3.5 mL, 24.4 mmol)
was slowly added. The reaction was stirred at room temperature for 18 h. The
reaction was washed with brine, extracted with CH2Cl2, dried over MgSO4,
filtered and concentrated. Chromatography (25% EtOAc/hexanes) gave 4.44 g
of 5 (54%) as an oil: 1H NMR (CDCl3, 250 MHz) d 1.43 (s, 9H), 3.39 (m, 2H),
3.51 (m, 2H), 3.66 (m, 2H), 4.25 (m, 2H), 5.17 (s, 2H), 7.34 (m, 5H). 13C NMR
(CDCl3, 62.5 MHz) d 27.85, 46.98, 50.46, 60.79, 65.81, 69.12, 79.71, 127.74,
129.10, 134.88, 154.58, 155.51. IR (CCl4) 1693, 1747, 2975, 3450 cmÀ 1. Anal.
Calcd. for C17H25NO6: C, 60.16; H, 7.42; N, 4.13. Found: C, 59.82; H, 7.44;
N, 4.03.

118
DONALDSON ET AL.
1-(5-O-Benzyloxycarbonyl-3-aza-(N-tert-butoxycarbonyl)-pentyl)-N3-ben-
zoyl thymine (6). A solution of 5 (4.3 g, 12.6 mmol), N3-benzoyl thymine9
(2.9 g, 12.6 mmol), and Ph3P (4.3 g, 16.4 mmol) in THF (79 mL) was cooled to
0ꢀC and diethylazodicarboxylate (2.6 mL, 16.4 mmol) was slowly added. The
reaction was stirred at room temperature for 2.5 h. Concentration followed by
chromatography (first in 30% iPrOH=hexanes then 1% MeOH/CHCl3) gave
5.62 g of 6 (65%) as an oil: 1H NMR (CDCl3) d 1.39 (s, 9 H), 1.77 (s, 3 H), 3.45
(m, 4H), 3.74 (m, 2H), 4.22 (m, 2H), 5.13 (s, 2H), 7.03 (s, 1H), 7.32 (m, 5H),
7.42 (t, 2H), 7.57 (t, 1H), 7.85 (m, 1H), 8.08 (d, 1H). 13C NMR (CDCl3) d 11.94,
28.08, 46.25, 47.29, 65.86, 69.64, 80.54, 109.73, 128.14, 128.42, 128.82, 130.36,
131.74, 134.58, 134.92, 140.53, 149.71, 154.68, 155.14, 163.06, 169.05. IR (CCl4)
1651, 1694, 1747, 2977, 3482 cmÀ 1. UV (EtOH) lmax ¼ 251.0 FABMS (MþH)
calculated for C29H33N3O8 was 551.2377, found 552.2360.
N-Allyl d-valerolactam (8). KH (9.85 g of a 30% suspension in oil,
150 mmol) was suspended in THF (181 mL). In a second flask, d-valerolactam
(4.95 g, 50 mmol) was dissolved in THF (61 mL). This solution was slowly
added to the KH suspension and stirred for 2 h. The reaction was cooled to 0ꢀC
and freshly distilled allyl bromide (5.25 mL, 60 mmol) was slowly added. The
reaction was stirred for an additional 20 h after which time it was diluted with a
6:1 solution of EtOAc:MeOH. The solution was further diluted with H2O and
extracted with EtOAc. Column chromatography (10% EtOAc/hexanes!5%
1
MeOH=CHCl3) gave 6.21 g (89%) of 8 as a yellow oil. H NMR (CDCl3,
250 MHz) d 1.70 (m, 4H), 2.31 (t, 2H), 3.15 (t, 2H), 3.88 (d, 2H), 5.14 (m, 2H),
5.65 (m, 1H). 13C NMR (CDCl3, 62.5 MHz) d 21.0, 22.8, 31.7, 46.8, 48.8, 116.5,
132.6, 166.8. IR (CCl4) 1637.3 cmÀ 1. HRMS calculated for C8H13NO was
139.0998 found 139.0995.
Benzyl N-allyl-N-tert-butoxycarbonyl-5-aminopentanoate (9). Lactam 8
(5.3 g, 38 mmol) was refluxed in 7M HCl (38 mL) for 24 h. The crude material
was concentrated and suspended in benzene (38 mL). To this suspension, p-
toluenesulfonic acid (9.0 g, 47.5 mmol) and benzyl alcohol (25.0 mL,
241.7 mmol) were added. The solution was refluxed for 24 h, after which time it
was extracted with ether, and the aqueous phase concentrated. The crude
concentrate was resuspended in CH2Cl2 (38 mL) and di-tert-butyl dicarbonate
(12.4 g, 57 mmol) was added. The solution was cooled to 0ꢀC for addition of
Et3N (21.2 mL, 152.1 mmol). The reaction was stirred at room temperature for
24 h after which time additional di-tert-butyl dicarbonate (4.1 g, 19 mmol) was
added. The reaction was stirred for another 24 h and washed with H2O. Con-
centration of the organic phase followed by column chromatography (10%
1
EtOAc/hexanes) gave 8.2 g (62%) of 9 as a colorless oil. H NMR (CDCl3,
250 MHz) d 1.45 (s, 9H), 1.59 (m, 4H), 2.36 (t, 2H), 3.16 (t, 2H), 3.76 (t, 2H),
5.05 (m, 4H), 5.71 (m, 1H), 7.31 (m, 5H). 13C NMR (CDCl3, 62.5 MHz) d 21.9,
27.5, 28.1, 33.6, 45.8, 49.2, 65.7, 115.8, 127.8, 128.2, 134.1, 135.9, 155.1, 172.6.

5-AMINOPENTANOIC ACID NUCLEOBASE DIMER
119
IR (CCl4) 1162, 1247, 1693, 1736, 2975 cmÀ 1. Anal Calcd. for C20H29NO4: C,
69.13; H, 8.41; N, 4.03. Found C, 69.23; H, 8.52; N, 4.00.
Benzyl-N-tert-butoxycarbonyl-N-(2-hydroxyethyl)-5-aminopentanoate (10).
Olefin 9 (7.0 g, 20.2 mmol) was dissolved in CH2Cl2 (20 mL) and THF (80 mL)
and cooled to À 78ꢀC. O3 was bubbled through the solution for 45 min fol-
lowed by N2 purging for an additional 45 minutes. Next, NaBH4 (0.76 g,
20.2 mmol) was added and the reaction was stirred 1 h after which time an
additional portion of NaBH4 (1.53 g, 40.3 mmol) was added. The reaction was
warmed to room temperature, stirred for 1.5 h, and partitioned between 1 M
HCl and EtOAc. Column chromatography (50% EtOAc/hexanes) gave 5.7 g
(79%) of 10 as a colorless oil. 1H NMR (CDCl3, 250 MHz) d 1.45 (s, 9H), 1.59
(m, 4H), 2.36 (t, 2H), 3.25 (m, 4H), 3.66 (m, 2H), 5.05 (s, 2H), 7.29 (m, 5H). 13C
NMR (CDCl3, 62.5 MHz) d 22.1, 27.9, 28.3. 29.7, 33.8, 48.0, 50.0, 62.0, 66.1,
128.1, 128.5, 136.1, 156.7, 173.2. IR (CCl4) 1167, 1366, 1693, 1736, 2974,
3445 cmÀ 1. Anal Calcd. for C19H29NO5: C, 64.94; H, 8.32; N, 3.99. Found C,
65.32; H, 8.53; N, 3.83.
Benzyl-N-(tert-butoxycarbonyl)-N-(ethyl-2-(N3-benzoyl thymine)-5-amino-
pentanoate (11). A solution of 10 (1.3 g, 3.8 mmol), N3-benzoyl thymine9
(0.86 g, 3.8 mmol), and Ph3P (1.30 g, 4.9 mmol) in THF (79 mL) was cooled to
0ꢀC and diethylazodicarboxylate (0.77 mL, 4.9 mmol) was slowly added. The
reaction was stirred at room temperature for 2.5 h. Concentration followed by
1
chromatography (50% EtOAc/hexanes) gave 1.81 g of 11 (85%) as an oil: H
NMR (CDCl3, 250 MHz) d 1.53 (s,m 13H), 1.88 (s, 3H), 2.35 (t, 2H), 3.18 (t,
2H), 3.43 (t, 2H), 5.09 (s, 5H), 7.01 (s, 1H), 7.31 (m, 5H), 7.46 (t, 2H), 7.61 (t,
1H), 8.02 (d, 1H). 13C NMR (CDCl3, 62.5 MHz) d 11.9, 21.8, 27.7, 28.1, 33.5,
44.8, 47.0, 65.8, 109.6, 127.9, 128.3, 128.8, 130.2, 131.9, 134.5, 135.9, 140.6,
149.6, 155.3, 163.0, 168.9, 172.6. IR (CCl4) 1155, 11,249, 1656, 1697, 1739,
2958 cmÀ 1. UV (EtOH) lmax ¼ 251.5 Anal Calcd. for C31H37N3O7: C, 66.06;
H, 6.62; N, 7.45. Found C, 65.79; H, 6.45; N, 7.40.
1-(5-O-Benzyloxycarbonyl-3-aza-pentyl)-N3-benzoyl thymine hydrochloride
(12). A solution of 6 (0.55 g, 1 mmol) in EtOAc (10 mL) was added to an-
hydrous HCl (10 mmol, prepared by adding acetyl chloride (0.71 mL, 10 mmol)
to anhydrous MeOH (0.41 mL, 10 mmol) in EtOAc (2 mL)) at 0ꢀC. The ice bath
was removed and the mixture was stirred at room temperature for 18 h. Con-
1
centration gave 0.38 g (78%) of crude 12 as a yellow solid: H NMR (DMSO-
d6, 250 MHz) d 1.83 (s, 3H), 3.29 (m, 7H), 4.07 (m, 2H), 4.40 (m, 2H), 5.16 (s,
2H), 7.38 (m, 5H), 7.55 (m, 2H), 7.76 (m, 2H), 8.03 (d, 2H), 9.35 (br s, 1H).
N-(tert-butoxycarbonyl)-N-(ethyl-2-(N3-benzoyl thymine)-5-aminopenta-
noic acid (13). To benzyl ester 11 (0.65 g, 1.15 mmol) was added 5% Pd(OH)2/
C (0.03 g) and THF (4 mL). The reaction stirred under an atmosphere of H2

120
DONALDSON ET AL.
(balloon) for 18 h. The reaction was filtered through celite with EtOAc and
extracted using sat. aq. NaHCO3. The aqueous layer was acidified with 1 M
HCl to pH 3 and then extracted with EtOAc. Concentration of the organic
1
phase gave 0.49 g (90%) of 13 as a white solid. H NMR (CDCl3, 250 MHz) d
1.48 (s,m 13H), 1.82 (s, 3H), 2.39 (m, 2H), 3.12 (m, 2H), 3.39 (m, 2H), 3.75 (m,
2H), 7.06( s, 1H), 7.42 (m, 2H), 7.86 (m, 1H), 7.99 (m, 1H). 13C NMR (CDCl3,
62.5 MHz) d 11.9, 21.6, 27.7, 28.1, 33.2, 45.0, 47.0, 80.2, 109.7, 128.8, 130.3,
131.6, 134.7, 140.8, 149.7, 155.5, 163.1, 168.9, 177.6. IR (CCl4) 1161, 1249,
1436, 1654, 1697, 1747, 2976 cmÀ 1. UV (EtOH) lmax ¼ 254.5. HRMS (MþNa)
ꢂ
calculated for C24H31N3O7 Na was 496.2060 found 496.2027.
APN Dimer (14). To a cold (0ꢀC) suspension of amine hydrochloride 12
(0.38 g, 0.77 mmol) in CH3CN (2 mL), was added iPr2EtN (0.27 mL,
1.54 mmol). This solution was added to a solution of acid 13 (0.37 g, 0.77 mmol)
and TBTU (0.25 g, 0.77 mmol) in CH3CN (2 mL). The mixture was stirred at
room temperature for 24 h. Concentration followed by chromatography (1%
MeOH/CHCl3) gave 0.47 g (67%) of fully protected 15 as a white solid: mp91–
93ꢀC 1H NMR (CDCl3, 250 MHz) d 1.42 (m, 13H), 1.73 (m, 2H), 1.89 (m, 5H),
2.29 (m, 2H), 3.15 (m, 2H), 3.48 (m, m, 6H), 3.83 (m, 4H), 4.25 (m, m, 2H), 5.12
(m, 2H), 6.97 (m, 1H), 7.11 (s, 1H), 7.34 (m, 5H), 7.44 (m, 4H), 7.60 (m, 2H),
7.91 (m, 3H). 13C NMR (CDCl3, 62.5 MHz) d 0.4, 12.5, 12.7, 22.4, 23.1, 28.8,
32.8, 45.5, 45.9, 46.8, 47.1, 47.6, 65.5, 66.8, 70.5, 80.6, 111.2, 111.6, 128.8, 128.9,
129.07, 129.14, 129.2, 129.5, 129.6, 130.8, 130.9, 132.0, 135.3, 135.4, 135.6,
140.6, 140.9, 150.3, 150.5, 155.2, 163.6, 169.4, 173.9 IR (CCl4) 1162, 1257, 1437,
1653, 1698, 1746, 2955, 3067 cmÀ 1. UV (EtOH) lmax ¼ 252.5. FABMS (MþNa)
calculated for C48H54N6O12ꢂNa was 929.3699, found 929.3693. The fully pro-
tected APN dimer (0.34 g, 0.37 mmol) was dissolved in dioxane (14.8 mL), and
conc. aq. NH4OH (37 mL, 28–30%) was slowly added. The reaction was stirred
at room temperature for 45 min and concentrated. Chromatography (5%
MeOH/CHCl3!10% MeOH/CHCl3) resulted in 0.21 g. The crude material was
dissolved in CH2Cl2 (1 mL) and TFA (0.34 mL, 4.4 mmol) was added at 0ꢀC.
The reaction was stirred at room temperature for 3 h. Concentration gave 0.19 g
1
ꢀ
(82%) of 14 as a yellow solid: mp91–110 C (decomposes) H NMR (D2O,
400 MHz, 85ꢀC) d 1.59 (m, 5H), 1.82 (m, 6H), 2.37 (m, 2H), 3.01 (m, 2H), 3.40
(m, 4H), 3.64 (m, 4H), 4.02 (m, 1H), 7.37 (m, 2H). 13C NMR (CDCl3,
62.5 MHz) d 11.6, 11.7, 22.0, 25.5, 32.3, 43.9, 45.4, 46.6, 46.8, 47.3, 48.0, 49.3,
59.3, 62.7, 110.7, 112.0, 142.6, 143.6, 152.7, 153.2, 167.3, 176.6. FABMS
ꢂ
(MþNa) calculated for C21H32N6O6 Na was 487.2283, found 487.2299.
ACKNOWLEDGMENTS
We would like to acknowledge support of this work by the American Cancer
Society, Ohio Division, Inc.

5-AMINOPENTANOIC ACID NUCLEOBASE DIMER
121
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Received June 12, 2001
Accepted November 20, 2001