Solid-Phase Synthesis of Dipeptide-Conjugated Nucleosides and Their Interaction with RNA

Article abstract of DOI:10.1002/hlca.200390295

Dipeptide-conjugated nucleosides were efficiently synthesized from the intermediates of 3′-amino-3′-deoxy-nucleosides by using the solid-phase synthetic strategy with HOBt/HBTU (1-hydroxy-1H-benzotriazole/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate) as the coupling reagents (Schemes 1-3). CD Spectra and thermal melting studies showed that the synthesized hydrophobic dipeptide-thymidine and -uridine derivatives 8a-8d, 13a-d, and 18 had a mild affinity with the polyA · polyU duplex and could induce the change of RNA conformation. The results also implied that the interaction of conjugates with RNA might be related to the sugar pucker conformation of the nucleoside.

Full text of DOI:10.1002/hlca.200390295

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Helvetica Chimica Acta Vol. 86 (2003)
Solid-Phase Synthesis of Dipeptide-Conjugated Nucleosides and Their
Interaction with RNA
by Guimin Dong, Liangren Zhang*, and Lihe Zhang
National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China
(tel: ‡86-10-82801570; fax: ‡86-10-82802724; e-mail: liangren@bjmu.edu.cn)
Dipeptide-conjugated nucleosides were efficiently synthesized from the intermediates of 3'-amino-3'-
deoxy-nucleosides by using the solid-phase synthetic strategy with HOBt/HBTU (1-hydroxy-1H-benzotriazole/
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate) as the coupling reagents (Schemes 1
3). CD Spectra and thermal melting studies showed that the synthesized hydrophobic dipeptideÀthymidine and
Àuridine derivatives 8a 8d, 13a d, and 18 had a mild affinity with the polyA ¥ polyU duplex and could induce
the change of RNA conformation. The results also implied that the interaction of conjugates with RNA might be
related to the sugar pucker conformation of the nucleoside.
Introduction. The functional and structural diversities of RNA provide numerous
opportunities for academic researchers and pharmaceutical industry to develop small
molecules to target specific RNA for treating a variety of diseases, such as bacteria or
virus infections [1]. The RNA secondary structure of base pairing is more conservative
than its primary sequence, so the potential for slower development of drug resistance
against small molecules is one of the advantages of targeting RNA over traditional
protein targets. Aminoglycosides, a class of structurally diverse aminocyclitols with
potent antibiotic and antiviral activities are intensively and well-studied RNA binders.
They can selectively and stoichiometrically bind with functional RNA motifs and
disrupt the proteinÀRNA, RNPÀRNA, or RNAÀRNA interaction [2]. On the basis
of the study of RNA in the presence of aminoglycosides and other small molecules, the
interactions of RNA with small molecules are affected by the distribution of charged,
aromatic, and H-bonding groups of a relatively rigid scaffold [1]. Considering the
interaction of proteinÀRNA, the a-helix conformation of the protein provides a
scaffold for H-bonding with RNA bases, the b-sheet form is suitable for binding
aromatic groups with unstacked bases, and the negative phosphodiester moiety of RNA
supplies a function for the electrostatic interaction. Furthermore, the structure of RNA
is dynamic, and the conformational change can be induced by a small molecule [2]. The
structural and functional features of RNA stimulated our enthusiasm to find more-
selective and potent small molecules, and the adoption of virtual screening, surface
plasmon resonance (SPR), and other technologies also speeded up the process [3].
Our previous findings indicated that aminoglycosyl-nucleosides could bind to RNA
with high affinity [4]. Besides that the configuration of the glycosyl moiety could affect
their interaction, the heterocyclic-base moiety also showed an effect on the base
stacking of the RNA duplex. Peptides have a relatively flexible conformation as
compared to a glycosyl moiety. The diverse functional groups of a peptide, such as
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Helvetica Chimica Acta Vol. 86 (2003)
3517
hydrophobic, basic, or acidic groups, could provide multiform H-bonding or electro-
static interactions with RNA. Therefore, a small-peptide-conjugated nucleoside could
be expected to exhibit affinity with an RNA duplex and interrupt RNA function, and
selectivity could be achieved by choosing an appropriate nucleoside and peptide.
Actually, many naturally occurring or synthesized peptide-conjugated nucleosides have
shown antibacterial or antiviral activities [5]. In the early years, Robins and co-workers
indicated that some natural nucleosideÀpeptides were inhibitors of protein synthesis,
and peptides bound to DNA may play a role in protein synthesis by acting as
−derepressors× of structural genes [6]. Puromycin, an amino acid conjugated nucleoside,
resembles the structure of the 3'-end of aminoacyl-tRNA which can bind to the
ribosomal A-site [7]. On the other hand, many nucleoside or nucleoside-mimetic drugs
have been conjugated to the structural and functional peptides to improve their
selectivity or efficiency and to overcome the problems of side effects [8]. In this paper,
we present a concise method for the synthesis of peptide-conjugated nucleosides. This
solid-phase peptide-synthesis strategy can be modified and used for constructing a
compound library. Our results also show that the dipeptide-conjugated thymidines and
uridines could bind to RNA and change the RNA conformation slightly.
Results and Discussion. Chemical Syntheses of the Dipeptide-Conjugated Nucleo-
sides. As the starting compound for the synthesis of thymidine derivatives, 3'-amino-3'-
deoxythymidines were used. The 5'-OH of the nucleoside was used for the attachment
to the solid support. To improve the efficiency of the synthesis, a succinyl moiety was
used as a spacer connecting the solid support and the nucleoside (Scheme 1). Thus 3'-
amino-3'-deoxythymidine (2) was synthesized in six steps in 37.2% overall yield from
thymidine (1) [9]. The amino group of 2 was protected by the (tert-butoxy)carbonyl
(Boc) group [10] ( ! 3) and then converted to ester 4 by treatment with succinic
anhydride in the presence of N,N-dimethylpyridin-4-amine (DMAP) under Ar [11].
The free COOH group of 4 was activated as 4-nitrophenyl ester ( ! 5) for the efficient
coupling with the aminomethyl (AM) resin [12] ( ! 6). After removal of the Boc group
with 33% CF3COOH in CH2Cl2, an amount of 170 mmol/g of the nucleoside-loaded
resin 7 was obtained, which was treated with amino acids by the solid-phase peptide-
synthesis strategy to give the dipeptide-conjugated thymidines 8a d. The solid-phase
synthesis of these dipeptide-conjugated nucleosides was carried out by means of
standard Boc strategy, using HOBt (1-hydroxy-1H-benzotriazole) and HBTU (2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) as the coupling
reagents in the presence of diisopropylethylamine (iPr2EtN) [13]. Several kinds of
nonpolar amino acids were chosen to compose the dipeptides. The dipeptide-
conjugated thymidines 8a d were released from the resin with ammonia, purified by
1
column chromatography (silica gel), and identified by ESI-TOF-MS and H-NMR.
Dipeptide-conjugated xylofuranosyl-thymine was synthesized in the same way as
described for the synthesis of dipeptide-conjugated thymidines (Scheme 2). The
intermediate (3'-amino-2',3'-dideoxy-b-d-xylofuranosyl)thymine 12 was synthesized
starting from thymidine (1). The 5'-O-tritylthymidine 3'-(methanesulfonate) (9) was
obtained in an overall yield of 68.2% after thymidine had been successively treated
with trityl chloride and methanesulfonyl chloride [9]. Treatment of 9 with sodium azide
under Ar gave 10 in 71.0% yield. After deprotection of 10 by treatment with 80%

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 1
i) Di(tert-butyl) dicarbonate, EtOH. ii) Succinic anhydride, pyridine, DMAP. iii) 4-Nitrophenol, pyridine, DCC.
iv) AM resin, DMF. v) 33% CF3COOH in CH2Cl2. vi) Standard peptide synthesis.
AcOH ( ! 11), catalyzed hydrogenation gave (3'-amino-2',3'-dideoxy-b-d-xylofurano-
syl)thymine (12). Compound 12 was bound to the AM resin via a succinyl linker in the
same way as described for the synthesis of 7; an amount of 110 mmol/g of the
nucleoside-loaded resin was obtained. Subsequent standard solide-phase synthesis as
described for 8a d yielded the dipeptide-conjugated nucleosides 13a d, which were
similarly identified (see Exper. Part).
Regarding the synthesis of resin-bound 2'-amino-2'-deoxyuridine 17, a similar
sequence starting from 14 was performed (Scheme 3). Since both the 3'-OH and 5'-OH
groups were active towards esterification, two compounds 16a and 16b were obtained,
when 15 was treated with succinic anhydride. The FAB-MS showed only a monoester
‡
peak at 444.1 ([M ‡ 1] ); no diester was observed. The 1H-NMR spectrum established
that a ratio of ca. 1:1 of the 5'- and 3'-monoester 16a and 16b, respectively, was
obtained. These monoesters could not be easily separated by column chromatography
(silica gel); thus, they were coupled with the AM resin as a mixture ( ! 17a/17b). A
total amount of 87 mmol/g of nucleoside was loaded on the resin. The subsequent solid-

Helvetica Chimica Acta Vol. 86 (2003)
3519
Scheme 2
i) NaN3, DMF, Ar, 1008. ii) 80% AcOH, r.t. iii) Pd/C, H2. iv) Standard peptide synthesis.
Scheme 3
i) Di(tert-butyl) dicarbonate, MeCN, Et3N. ii) Succinic anhydride, pyridine, DMAP. iii) 1. 4-nitrophenol,
pyridine, DCC; 2. AM resin, DMF; 3. 33% CF3COOH in CH2Cl2. iv) Standard peptide synthesis.
phase peptide synthesis (!18) indicated that the solid-support-linkage mode had
almost no influence on the synthesis efficiency.
Interaction of Dipeptide-Conjugated Nucleosides with polyA ¥ polyU. Thermal
melting analysis and circular dichroism (CD) were used to investigate the interaction
of the dipeptide-conjugated nucleosides with the polyA ¥ polyU duplex. The thermal
melting curve showed that, in most cases, a slight decrease of Tm was observed, the
maximum being 3.88 (Fig. 1, Table). This means that the existence of dipeptide-

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Helvetica Chimica Acta Vol. 86 (2003)
conjugated nucleosides may affect the RNA-duplex stability to some extent. CD
Spectra showed that the RNA-typical A-form duplex was maintained in the presence of
dipeptide-conjugated nucleosides (Fig. 2). The change in peak shape implied a slight
change of RNA conformation. Comparing to other cases, a more obvious change was
observed in the presence of compound 8a, which indicated that a stronger interaction
existed. The structural difference between 8a and 13a resides in the sugar moiety; it is
certain that the interactions between the dipeptide-conjugated nucleoside and the
RNA duplex are related to the sugar-pucker conformation of the nucleoside, which
further affects the base stacking and the nonbonding interactions.
Fig. 1. Thermal melting curves of polyA ¥ polyU in the presence or absence of the dipeptide-conjugated
nucleosides a) 8a d and b) 13a d and 18

Helvetica Chimica Acta Vol. 86 (2003)
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Fig. 2. CD Spectra of polyA ¥ polyU in the presence or absence of the dipeptide-conjugated nucleosides a) 8a
and b) 13a d and 18
d
Table. Melting Temperatures Tm of PolyA ¥ PolyU Duplex in the Presence or Absence of NucleosideÀPeptide
Conjugates
Tm [8]
DTm [8]
PolyA ¥ polyU/8a
PolyA ¥ polyU/8b
PolyA ¥ polyU/8c
PolyA ¥ polyU/8d
PolyA ¥ polyU/13a
PolyA ¥ polyU/13b
PolyA ¥ polyU/13c
PolyA ¥ polyU/13d
PolyA ¥ polyU/18
PolyA ¥ polyU
59.4
56.3
60.1
58.0
58.0
59.5
58.2
57.2
59.1
60.1
À 0.7
À 3.8
0.0
À 2.1
À 2.1
À 0.6
À 1.9
À 2.9
À 1.0
This work was supported by the National Natural Science Foundation of China (SFCBIC 20320130046).
Experimental Part
1. General. All solvents were dried and distilled prior to use. Evaporations were carried out under reduced
pressure below 458. (Aminomethyl)polystyrene (AM) resin was from Advanced ChemTech. TLC: GF254 silica

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Helvetica Chimica Acta Vol. 86 (2003)
gel (Qingdao Haiyang Chemical Factory, China). CC ˆ Column chromatography. Optical rotations: Perkin-
Elmer-243-B polarimeter. NMR Spectra: Jeol-AL-300 or Varian-VXR-500 instrument, d in ppm rel. to SiMe4 as
an internal standard, J in Hz. ESI-TOF-MS: ABI Qstar; in m/z.
2. Dipeptide-Conjugated 3'-Amino-3'-deoxythymidines 8a d. 3'-{[(tert-Butoxy)carbonyl]amino}-3'-deoxy-
thymidine (3). Di(tert-butyl) dicarbonate (2.2 ml, 9.58mmol) was slowly added to a soln. of 2 (2.0 g, 8.33 mmol)
in EtOH (260 ml). The mixture was allowed to react for 1 h at r.t. After evaporation, the residue was purified by
CC (silica gel, AcOEt/hexanes 3 :2): 2.38 g (83.8%) of 3. M.p. 115 1188. 1H-NMR ((D6)DMSO): 11.27
(s, NH); 7.74 (s, HÀC(6)); 6.11 (t, HÀC(1')); 5.04 (s, OHÀC(5')); 4.05 (m, HÀC(4')); 3.64 (m, HÀC(3'),
‡
2 HÀC(5')); 2.17 (m, 2 HÀC(2')); 1.76 (s, MeÀC(5)); 1.37 (s, tBu). FAB-MS: 342.1 ([M ‡ 1] ).
3'-{[(tert-Butoxy)carbonyl]amino}-3'-deoxythymidine 5'-(Hydrogen Butanedioate) (4). DMAP (788 mg,
6.45 mmol) was added to a soln. of 3 (2.2 g, 6.45 mmol) in pyridine (65 ml). After the solid was dissolved,
succinic anhydride (775 mg, 7.74 mmol) was added under Ar, and the mixture was stirred for 30 h at r.t. Then the
solvent was evaporated, the residue dissolved in CH2Cl2 (80 ml), the soln. washed with 15% aq. citric acid
(10 ml) and H2O (2 Â 30 ml) and evaporated, and the residue was purified by CC (silica gel, AcOEt/hexanes
3 :2): 2.07 g (73.0%) of 4. M.p. 107 1098. 1H-NMR ((D6)DMSO): 7.45 (s, HÀC(6)); 6.07 (t, HÀC(1')); 4.26
(m, HÀC(3')); 4.08( m, HÀC(4')); 3.63 (m, 2 HÀC(5')); 2.55 (m, CH2CH2); 2.23 (m, 2 HÀC(2')); 1.91
‡
(s, MeÀC(5)); 1.34 (s, tBu). FAB-MS: 441.8([ M ‡ 1] ).
3'-{[(tert-Butoxy)carbonyl]amino}-3'-deoxythymidine 5'-(Resin-Bound Butanedioate) (6). To a soln. of 4-
nitrophenol (286 mg, 2.06 mmol) and pyridine (0.4 ml) in dioxane (10 ml), 4 (793 mg, 1.80 mmol) and DCC
(dicyclohexyl carbodiimide) (793 mg, 3.84 mmol) were added. The mixture was allowed to react for 2 h at r.t.,
and then the precipitate was filtered off. Aminomethyl (AM) resin (700 mg, 0.91 mmol; 1.3 mmol/g), DMF
(6 ml), and DMAP (22 mg; 0.18mmol) were added to the filtrate. The mixture was stirred for 2 d at r.t., then it
was filtered and washed with DMF, MeOH, and Et2O. After it was dried, Ac2O (8ml) and pyridine (20 ml) were
added, and the mixture was stirred at r.t. for 1 h to cap the unreacted amino group. Then the mixture was filtered
and washed with DMF, MeOH, and Et2O, and dried.
Dipeptide-Conjugates 8a d from 3'-Amino-3'-deoxythymidine 5'-(Resin-Bound Butanedioate) (7). Resin-
bound nucleoside 6 was added to 33% CF3COOH in CH2Cl2 and was stirred for 30 min at r.t., then filtered, and
washed successively with DMF, MeOH, and Et2O. The loading amount of nucleoside on resin was determined
following the standard procedure. After the resin was dried, standard solid-phase peptide synthesis (Boc
strategy) yielded the dipeptide-conjugated nucleosides 8a d. The final products were purified by CC (silica
gel).
l-Alanyl-N1-[(2S,3S,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimidin-
1-yl)furan-3-yl]-l-phenylalaninamide (8a): Yield 75.6%. M.p. 162 1658. [a]1D8 ˆ ‡21.5 (c ˆ 0.28, MeOH).
1H-NMR ((D6)DMSO)1): 7.78( s, HÀC(6)); 7.25 7.30 (m, 5 arom. H, (Phe)); 6.19 (t, HÀC(1')); 3.98 4.32
(m, CH2(b)(Phe), HÀC(a)(Ala)); 3.48 3.75 ( m, HÀC(4'), 2 HÀC(5')); 2.99 3.10 (m, HÀC(3')); 2.84
(m, HÀC(a)(Phe)); 2.04 2.30 (m, 2HÀC(2')); 1.79 (s, MeÀC(5)); 1.23 (d, Me(b)(Ala)). ESI-TOF-MS:
‡
460.3002 ([M ‡ 1] ; calc. 460.5034).
l-Alanyl-N1-[(2S,3S,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimidin-
1-yl)furan-3-yl]-l-alaninamide (8b): Yield 80.4%. M.p. 115.7 116.08. [a]1D8 ˆ ‡63.75 (c ˆ 0.8, MeOH).
1H-NMR (CD3OD)1): 7.78( s, HÀC(6)); 6.12 (t, HÀC(1')); 4.34 (q, HÀC(a)(Ala1)); 4.22 (q, HÀC(a)(Ala2));
3.58 3.84 ( m, HÀC(3'), HÀC(4'), 2 HÀC(5')); 2.18 2.31 ( m, 2 HÀC(2')); 1.79 (s, MeÀC(5)); 1.38( d, Me-
‡
(b)(Ala1)); 1.27 (d, Me(b)(Ala2)). ESI-TOF-MS: 384.1197 ([M ‡ 1] ; calc. 384.4074).
l-Alanyl-N1-[(2S,3S,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimidin-
1
1-yl)furan-3-yl]-l-leucinamide (8c): Yield 73.5%. M.p. 156 1588. [a]1D8 ˆ ‡10.17 (c ˆ 0.59, MeOH). H-NMR
(CD3OD)1): 7.78( s, HÀC(6)); 6.12 (t, HÀC(1')); 4.22 4.40 (m, 2 HÀC(2')); 3.58 3.78 ( m, HÀC(3'),
HÀC(4'), 2 HÀC(5')); 2.17 2.28( m, CH2(b)(Leu)); 1.79 (s, MeÀC(5)); 1.49 1.63 (m, HÀC(a)(Ala),
HÀC(a)(Leu), HÀC(g)(Leu)); 1.26 (d, Me(b)(Ala)); 0.89 (m, 2 Me(d)(Leu)). ESI-TOF-MS: 426.1686
‡
([M ‡ 1] ; calc. 426.4871).
l-Valyl-N1-[(2S,3S,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimidin-
1-yl)furan-3-yl]-l-phenylalaninamide (8d): Yield 84.3%. M.p. 179 1828. [a]1D8 ˆ ‡6.23 (c ˆ 1.3, MeOH).
1H-NMR (CD3OD)1): 7.89 (s, HÀC(6)); 7.22 7.32 (m, 5 arom. H(Phe)); 6.21 (t, HÀC(1')); 4.46 4.51
(m, HÀC(4')); 4.10 (d, HÀC(a)(Val)); 3.96 4.00 (t, HÀC(a)(Phe)); 3.82 3.89 (m, 2 HÀC(5')); 3.68 3.74
1
)
The nucleoside numbering is retained.

Helvetica Chimica Acta Vol. 86 (2003)
3523
(m, HÀC(3')); 2.89 3.19 (m, CH2(b)(Phe)); 2.25 2.40 (m, 2 HÀC(2')); 1.96 2.08( m, HÀC(b)(Val)); 1.88
‡
(s, MeÀC(5)); 0.94 0.97 (m, 2 Me(g)(Val)). ESI-TOF-MS: 488.1780 ([M ‡ 1] ; calc. 488.5565).
3. Dipeptide-Conjugated 1-(3'-Amino-2',3'-dideoxy-b-d-xylofuranosyl)thymines 13a d. 1-(3'-Azido-2',3'-
dideoxy-5'-O-trityl-b-d-xylofuranosyl)thymine (ˆ1-[3'-Azido-2',3'-dideoxy-5'-(triphenylmethyl)-b-d-threo-pen-
tofuranosyl]-5-methylpyrimidine-2,4(1H,3H)-dione; 10). To a soln. of 5'-O-tritylthymidine 3'-(methanesulfo-
nate) (9; 3.10 g, 5.52 mmol) in DMF (45 ml), NaN3 (1.15 g, 17.7 mmol) was added. The mixture was kept at 1008
under Ar for 4 h, then it was poured into ice-water (1000 ml). After filtration, the filtrate was purified by CC
(silica gel, hexanes/AcOEt 4 :1): 1.99 g (71%) of 10. M.p. 149 1518. 1H-NMR ((D6)DMSO): 11.35 (s, NH); 7.44
(s, HÀC(6)); 7.35 (m, 15 arom. H); 6.07 (m, HÀC(1')); 4.56 (m, HÀC(3')); 4.27 (m, HÀC(4')); 3.18
‡
(m, 2 HÀC(5')); 2.73 (m, 1 HÀC(2')); 2.06 (m, 1 HÀC(2')); 1.66 (s, MeÀC(5)). FAB-MS: 509.7 ([M ‡ 1] ).
1-(3'-Azido-2',3'-dideoxy-b-d-xylofuranosyl)thymine (ˆ1-(3'-Azido-2',3'-dideoxy-b-d-threo-pentofurano-
syl)-5-methylpyrimidine-2,4(1H,3H)-dione; 11). A soln. of 10 (2.85 g; 5.60 mmol) in 80% AcOH/H2O (40 ml)
was heated at 1208 for 20 min. After evaporation, the residue was purified by CC (silica gel, hexanes/AcOEt
5 :4): 1.23 g (83.0%) of 11. M.p. 154 1578. 1H-NMR ((D6)DMSO): 11.32 (s, NH); 7.48( s, HÀC(6)); 6.02
(m, HÀC(1')); 5.02 (m, OHÀC(5')); 4.45 (m, HÀC(4')); 3.98( m, HÀC(3')); 3.68( m, HÀC(5')); 2.72
(m, 1 HÀC(2')); 2.07 (m, 1 HÀC(2')); 1.78( s, MeÀC(5)).
1-(3'-Amino-2',3'-dideoxy-b-d-xylofuranosyl)thymine (ˆ1-(1'-Amino-2',3'-dideoxy-b-d-threo-pentofurano-
syl)-5-methylpyrimidine-2,4-(1H,3H)-dione; 12). To a soln. of 11 (2.07 g, 7.78mmol) in MeOH (80 ml), 10% Pd/
C (225 mg) was added. The mixture was hydrogenated under 50 p.s.i. at r.t. for 6.5 h. After filtration, the filtrate
was evaporated and the residue extracted repeatedly with AcOEt: 1.61 g (86.2%) of 12. M.p. 129 1318.
1H-NMR ((D6)DMSO): 8.17 (s, HÀC(6)); 6.00 (q, HÀC(1')); 3.79 (m, HÀC(3'), HÀC(4')); 3.67
‡
(m, 2 HÀC(5'), NH2ÀC(3')); 2.44 (m, HÀC(2')); 1.74 (s, MeÀC(5)). FAB-MS: 241.0 ([M ‡ 1] ).
Dipeptide Conjugates 13a d from 12. As described for 8a d.
l-Alanyl-N1-[(2S,3R,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimi-
din-1-yl)furan-3-yl]-l-phenylalaninamide (13a): Yield 82.9%. M.p. 150 1538. [a]1D8 ˆ ‡51.67 (c ˆ 0.31, MeOH).
1H-NMR ((D6)DMSO)1): 7.78( s, HÀC(6)); 7.24 7.33 (m, 5 arom. H (Phe)); 5.96 (t, HÀC(1')); 4.28 4.52
(m, 2 HÀC(2')); 3.96 4.01 (m, HÀC(4')); 3.86 3.91 (m, HÀC(3')); 3.49 3.59 (m, 2 HÀC(5')); 2.79 3.09
(m, CH2(b)(Phe)); 2.44 2.53 (m, HÀC(a)(Phe)); 1.86 1.92 (q, HÀC(a)(Ala)); 1.86 (s, MeÀC(5)); 1.20
‡
(d, Me(b)(Ala)). ESI-TOF-MS: 460.3002 ([M ‡ 1] ; calc. 460.5034).
l-Phenylalanyl-N1-[(2S,3R,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxo-
pyrimidin-1-yl)furan-3-yl]-l-phenylalaninamide (13b): Yield 93.2%. M.p. 149 1508. [a]1D8 ˆ ‡46.75 (c ˆ 0.40,
MeOH). 1H-NMR (CD3OD)1): 7.70 (s, HÀC(6)); 7.19 7.37 (m, 10 arom. H (Phe1,Phe2)); 5.84 (t, HÀC(1'));
4.46 4.56 (m, HÀC(2')); 4.03 4.10 (m, HÀC(3'), HÀC(4')); 3.71 (m, 2 HÀC(5')); 4.10 (d, HÀC(a)(Phe2));
2.95 3.31 (m, 4 H, CH2(b)(Phe1,Phe2)); 2.43 2.52 (m, HÀC(a)(Phe1)); 1.88 (s, MeÀC(5)); 1.21 1.28
‡
(m, HÀC(a)(Phe2)). ESI-TOF-MS: 536.1493 ([M ‡ 1] ; calc. 536.5993).
l-Alanyl-N1-[(2S,3R,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimi-
din-1-yl)furan-3-yl]-l-valinamide (13c): Yield 79.8%. M.p. 167 1688. [a]1D8 ˆ ‡39.0 (c ˆ 0.30, MeOH).
1H-NMR (CD3OD)1): 7.76 (s, HÀC(6)); 5.89 (t, HÀC(1')); 4.17 4.53 (m, 2 HÀC(2')); 3.96 4.03
(m, HÀC(3'), HÀC(4')); 3.60 3.74 (m, 2 HÀC(5')); 3.51 (d, HÀC(a)(Val)); 1.99 2.14 (m, HÀC(a)(Ala),
HÀC(b)(Val)); 1.80 (s, MeÀC(5)); 1.26 (d, Me(b)(Ala)); 0.92 0.99 (m, 2 Me(g)(Val)). ESI-TOF-MS:
‡
412.1451 ([M ‡ 1] ; calc. 412.4606).
l-Phenylalanyl-N1-[(2S,3R,5R)-tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxo-
pyrimidin-1-yl)furan-3-yl]-l-leucinamide (13d): Yield 81.8%. M.p. 147 1498. [a]1D8 ˆ ‡24.47 (c ˆ 0.38, MeOH).
1H-NMR (CD3OD)1): 7.62 (s, HÀC(6)); 5.74 (t, HÀC(1')); 4.37 4.45 (m, 2 HÀC(2')); 3.93 3.98
(m, HÀC(4')); 3.52 3.67 (m, HÀC(3'), 2 HÀC(5')); 2.87 2.99 (m, CH2(b)(Phe)); 2.34 2.44
(m, HÀC(a)(Phe)); 1.78( s, MeÀC(5)); 1.43 1.70 (m, HÀC(a)(Leu), HÀC(g)(Leu), CH2(b)(Leu)); 0.76
‡
0.89 (m, 2 Me(d)(Leu)). ESI-TOF-MS: 502.1980 ([M ‡ 1] ; calc. 502.5831).
4. Dipeptide Conjugate 18. 2'-{[(tert-Butoxy)carbonyl]amino}-2'-deoxyuridine (15). To a suspension of 14
(778mg, 3.20 mmol) in MeCN (200 ml), ( tert-butyl) dicarbonate (0.8ml, 3.48mmol) and Et 3N (0.7 ml,
5.02 mmol) were added. The mixture was stirred overnight at r.t. and then evaporated. The residue was purified
1
by CC (silica gel, AcOEt/hexanes 5 :2): 760 mg (69.2%) of 15. M.p. 224 2258. H-NMR ((D6)DMSO): 11.44
(s, HÀN(3)); 7.85 (s, HÀC(6)); 5.85 (d, HÀC(5)); 5.56 (d, HÀC(1')); 5.16 (m, 1 HÀC(2')); 4.18 3.99
‡
(m, HÀC(3'), HÀC(4')); 3.57 (m, 2 HÀC(5')); 1.33 (s, tBu). FAB-MS: 343.9 ([M ‡ 1] ).
l-Phenylalanyl-N1-[(2R,3R,4S,5R)-tetrahydro-4-hydroxy-5-(hydroxymethyl)-2-(1,2,3,4-tetrahydro-2,4-di-
oxopyrimidin-1-yl)furan-3-yl]-l-phenylalaninamide (18). As described for 8a d, except that the intermediates
16a/16b and 17a/17b were used directly without separation. After the conjugate was released from the resin, it

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Helvetica Chimica Acta Vol. 86 (2003)
was purified by CC (silica gel): 18 (91.6%). [a]1D8 ˆ ‡21.3 (c ˆ 0.27, MeOH). M.p. 156 1588. 1H-NMR
(CD3OD)1); 8.02 (d, J(5,6) ˆ 8.4, HÀC(6)); 7.36 7.17 (m, 10 arom. H (Phe1,Phe2)); 6.02 (t, HÀC(1')); 4.68
(m, HÀC(a)(Phe)); 4.55 (m, HÀC(a)(Phe)); 4.21 (m, HÀC(2')); 4.07 (m, HÀC(3')); 3.91 (m, HÀC(4')); 3.75
‡
(m, HÀC(5')); 3.17 (m, CH2(b)(Phe1)); 2.86 (m, CH2(b)(Phe2)). ESI-TOF-MS: 538.1515 ([M ‡ 1] ; calc.
538.5721).
5. Circular-Dichroism and Thermal-Melting Measurements. CD Spectra: Jasco J-715 spectropolarimeter; at
158 in thermostatically controlled 1-cm cuvette. Thermal denaturation studies of polyA/polyU: Varian Cary 300.
CD Spectra and Tm values in the absence and presence of synthetic dipeptide-conjugated nucleosides were
determined in buffer soln. containing 10 mm Na2HPO4, 0.14m NaCl, and 1.0 mm EDTA (pH 7.2). The soln.
containing dipeptide-conjugated nucleoside was mixed with equimolar amounts of polyA and polyU per
nucleoside. The concentration of dipeptide-conjugated nucleosides was 40 and 60 mm in the case of thermal-
melting measurements and CD spectra, respectively.
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Received June 2, 2003