Stereoselective Synthesis of a Thymine Derivative of (S)-2-Hydroxy-4-(2-aminophenyl)butanoic Acid. A Novel Building Block for the Synthesis of Aromatic Peptidic Nucleic Acid Oligomers

Article abstract of DOI:10.1021/jo962326p

The synthesis of a thymine derivative of (S)-2-hydroxy-4-(2-aminophenyl)butanoic acid, compound 1, was achieved in high enantiomeric purity. The acyclic pyrimidine analog (S)-1 is a useful building block for the synthesis of a novel class of oligomers, the aromatic peptide nucleic acids (APNA, Scheme 1). The APNA tetramer 18 was prepared from the amino acid monomer (S)-1 using classical peptide synthesis. UV absorption spectra and ¹H NMR data of this tetramer suggested that base stacking interactions in the APNA oligomers may be favorable.

Full text of DOI:10.1021/jo962326p

J . Org. Chem. 1997, 62, 5451-5457
5451
Ster eoselective Syn th esis of a Th ym in e Der iva tive of
(S)-2-Hyd r oxy-4-(2-a m in op h en yl)bu ta n oic Acid . A Novel Bu ild in g
Block for th e Syn th esis of Ar om a tic P ep tid ic Nu cleic Acid
Oligom er s1
Youla S. Tsantrizos,* J acqueline F. Lunetta, Michael Boyd, Lee D. Fader,† and
Marie-Claire Wilson†
Department of Chemistry and Biochemistry, Concordia University, Montre´al, Que´bec H3G 1M8, Canada
Received December 11, 1996X
The synthesis of a thymine derivative of (S)-2-hydroxy-4-(2-aminophenyl)butanoic acid, compound
1, was achieved in high enantiomeric purity. The acyclic pyrimidine analog (S)-1 is a useful building
block for the synthesis of a novel class of oligomers, the aromatic peptide nucleic acids (APNA,
Scheme 1). The APNA tetramer 18 was prepared from the amino acid monomer (S)-1 using classical
1
peptide synthesis. UV absorption spectra and H NMR data of this tetramer suggested that base
stacking interactions in the APNA oligomers may be favorable.
In tr od u ction
binding affinity of a synthetic ODN analog, were ad-
dressed in the design and synthesis of the first peptide
nucleic acids by Nielson’s group (PNAs, Scheme 1).6
Although PNAs do not cross cell membranes readily,
their nuclear microinjection into cells has been shown
to result in the efficient and site-specific termination of
both transcription and translation.6c,7 Contrary to stud-
ies showing that incorporation of flexible acyclonucleo-
sides into synthetic ODNs decrease the stability of duplex
formation,8 the PNA oligomers exhibit a remarkable
affinity for both complementary DNA and RNA. Molec-
ular mechanics calculations,9 based on the solution NMR
data of a hexameric PNA-RNA duplex,10 suggested that
interresidue hydrogen-bonding interactions in the back-
bone of the PNAs provide conformational stability in the
duplexes and triplexes formed with these molecules.
A new class of peptide oligonucleotide analogs, the
aromatic peptide nucleic acids (APNA), having general
structure 2 (Scheme 1), is currently under investigation
in our laboratory. These molecules were designed in
order to investigate possible π-stacking interactions in
the backbone of the APNA oligomers and their imparting
stabilizing effect on a duplex or triplex structure.11 We
have speculated that the backbone N-H protons could
Numerous synthetic analogs of natural oligonucleotides
(ODN) have been described in the literature for their
promising applications in antisense chemotherapy.2 The
fidelity of the base-pairing interactions in the formation
of DNA-DNA or RNA-DNA duplexes (Watson-Crick
base pairing) is governed by an array of complementary
hydrogen bonds between the nucleotide bases. However,
the major stabilizing force for the formation of a duplex
is the electrostatic/hydrophobic attraction between the
stacked aromatic rings of the purine and pyrimidine
bases.3 Thus, ODN derivatives possessing structural
features which enhance π-stacking interactions exhibit
stronger hybridization affinity with a complementary
single strand of RNA or DNA (antisense),4 as well as with
double-stranded DNA (antigene, Hoogsteen or reversed
Hoogsteen base pairing),5 in the formation of a helix.
Two of the critical requirements for both antisense and
antigene applications, nuclease stability and enhanced
* Author to whom correspondence may be addressed at Department
of Chemistry and Biochemistry, Concordia University, 1455 de Mai-
sonneuve Blvd West, Montre´al, Que´bec H3G 1M8, Canada. Tel: (514)
848-3335. FAX: (514) 848-2868. E-mail: Youla@vax2.concordia.ca.
† Undergraduate research participants.
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
(1) The abbreviation APNA for aromatic peptide nucleic acids has
been assigned to this new class of analogs.
(5) (a) Perrouault, L.; Asseline, U.; Rivalle, C; Thuong, N. T.; Bisagni,
E.; Giovannangeli, C.; Doan, T. L.; He´le`ne, C. Nature 1990, 344, 358.
(b) Wilson, W. D.; Tanious, F. A.; Mizan, S.; Yao, S.; Kiselyov, A. S.;
Zon, G.; Strekowski, L. Biochemistry 1993, 32, 10614.
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Rev. 1990, 90, 543. (b) Crooke, S. T. Annu. Rev. Pharmacol. Toxicol.
1992, 32, 329. (c) Agrawal, S. Antisense oligonucleotides as antiviral
agents. Trends Biotechnol. 1992, 152. (d) Sanghvi, Y. S.; Cook, P. D.
In Nucleoside and Nucleotides as Antitumor and Antiviral Agents; Chu,
C. K., Baker, D. C., Eds.; Plenum: New York, 1993; p 311. (e)
Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123. (f) Milligan,
J . F.; Matteucci, M. D.; Martin, J . C. J . Med. Chem. 1993, 36, 1923.
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Res. 1995, 28, 366.
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Res. Commun. 1987, 148, 790 and references cited therein. (b) Delcourt,
S. G.; Blake, R. D. J . Biol. Chem. 1991, 266, 15160. (c) Zimmerman,
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L.; Schott, M. E. J . Am. Chem. Soc. 1981, 103, 7394. (c) Asseline, U.;
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J .; Terhorst, T. J .; Matteucci, M. D.; Froehler, B. C. J . Am. Chem. Soc.
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1991, 254, 1497. (b) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg,
R. H. J . Am. Chem. Soc. 1992, 114, 1895. (c) Hanvey, J . C.; Peffer, N.
J .; Bisi, J . E.; Thomson, S. A.; Cadila, R.; J osey, J . A.; Ricca, D. J .;
Hassman, C. F.; Bonham, M. A.; Au, K. G.; Carter, S. G.; Bruckenstein,
D. A.; Boyd, A. L.; Noble, S. A.; Babiss, L. E. Science 1992, 258, 1481.
(d) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Nucleic Acids
Res. 1993, 21, 197. (e) Dueholm, K. L.; Egholm, M.; Behrens, C.;
Christensen, L.; Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R.
H.; Nielsen, P. E.; Buchardt, O J . Org. Chem. 1994, 59, 5767. (f) Lenzi,
A.; Reginato, G.; Taddei, M. Tetrahedron Lett. 1995, 36, 1713. (g)
Review: Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. 1996, 4, 5.
(7) Wittung, P.; Kajanus, J .; Edwards, K.; Nielsen, P.; Norde´n, B.;
Malmstrom, B. G. FEBS Lett. 1995, 365, 27.
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453.
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93, 649.
(10) Brown, S. C.; Thomson, S. A.; Veal, J . M.; Davis, D. G. Science
1994, 265, 777.
(11) During preparation of this manuscript, a study on modified
oligonucleotides having a N-phenylamide backbone linkage was re-
ported, which showed that the phenyl ring has a negative influence
on the stability of a duplex: Waldner, A.; De Mesmaeker, A.; Wende-
born, S. Bioorg. Med. Chem. Lett. 1996, 6, 2363.
S0022-3263(96)02326-2 CCC: $14.00 © 1997 American Chemical Society

5452 J . Org. Chem., Vol. 62, No. 16, 1997
Tsantrizos et al.
Sch em e 2. Syn th esis of th e Th ym id in e
Sch em e 1. Str u ctu r a l Com p a r ison of AP NA a n d
P NA An a logs
Acyclon u cleosid e An a log (S)-1
potentially form stabilizing intramolecular interactions
with the center of the adjacent benzene ring (dipole/cation
π interactions12 or hydrogen bonds13) thus contributing
to the precise conformation and stabilty of the APNA
macromolecules in a hybridized duplex or triplex, analo-
gous to that proposed for the PNA oligopeptides.9
The APNA analogs were designed to have a distance
of three bonds between the nucleobases and the back-
bone, as in the case of natural oligonucleotides. Since
three of the bonds in the aniline moieties of the APNA
oligomers are coplanar, the spatial distance between the
bases would greately depend on the conformation and
presence, or absence, of π-stacking interactions along the
backbone. Therefore, the most favorable number of
bonds between the bases in these new analogs remains
to be determined. This report outlines the stereocon-
trolled synthesis of a thymine derivative of (S)-2-hydroxy-
4-(2-aminophenyl)butanoic acid (1), the first monomer in
the series of analogs which will be used as building blocks
in the synthesis of APNA oligomers (2a ). The dimer and
tetramer of 1 were also prepared using classical peptide
synthesis, and their conformation in solution was exam-
ined by 1H NMR and UV spectroscopy. The distance
between nucleobases in analog 2a is seven bonds along
the backbone; thus it is homomorphous to the acetami-
date and carboxymethyl-linked ODN analogs; the latter
are known to form stable duplex structures with comple-
mentary RNA.14
R-hydroxy acid 4 in high chemical yield (98%) and
enantiomeric purity (>98% ee).17 Compound (S)-4 was
subsequently converted to the methyl ester with diazo-
methane and reduced to (S)-5 by catalytic hydrogena-
tion,18 and the resulting aniline was protected to give the
Fmoc derivative (S)-6 in good overall yield (>70% from
4).19
Initial efforts to extend the R-hydroxy moiety of 6 to
the (methylthio)methyl ether using methyl sulfide and
benzoyl peroxide,20 or DMSO in a Pummerer type rear-
rangement,21 gave primarily the R-keto ester and only
15-20% yield of the desired product. However, the
synthesis of the analogous intermediate (ethylthio)methyl
ether (S)-8 was easier to achieve in reasonable yields
(55-60%) by converting the MOM ether (S)-7 first to an
(16) Casy, G.; Lee, T. V.; Lovell, H. Tetrahedron Lett. 1992, 33, 817.
(17) The enantiomers of 4 could not be separated by chiral HPLC.
Thus, compound 4 was first converted to 5 and then the optical purity
of 5 was determined from its 1H NMR in the presence of (R)-(-)-2,2,2-
trifluoro-1-(9-anthryl)ethanol. (a) Pirkle, W. H.; Rinaldi, P. L J . Org.
Chem. 1978, 43, 4475. (b) Tsantrizos, Y. S.; Ogilvie, K. K. Can. J . Chem.
1991, 69, 772.
(18) Reduction of both the R-ketone and the â,γ-olefin of 3 with a
L-proline/NaBH4 complex gave (R)-2-hydroxy-4-(2-nitrophenyl)butanoic
acid, with ∼50% ee. The â,γ-olefin reduction has been shown to involve
conjugation with the ortho nitro group in the phenyl ring: Kitajima,
H.; Ikebe, T.; Murakami, S. Unpublished results. After reaction with
CH2N2 and catalytic hydrogenation, the (R)-2-hydroxy-4-(2-nitrophen-
yl)butanoic acid was then converted to 5. This sample of (R)-5 (∼50%
ee) was subsequently used to produce the R enantiomers of all key
intermediates in Scheme 2; these compounds were used as reference
samples in measurments of optical purity.
Resu lts a n d Discu ssion
The novel acyclic thymidine analog (S)-1 was obtained
using the R-keto acid 3 as the key starting material
(Scheme 2).15 Enzymatic reduction of 3 using Bacillus
stearothermophilus lactate dehydrogenase16 gave the (S)-
(12) Dougherty, D. A. Science 1996, 271, 163.
(13) (a) Levitt, M; Perutz, M. F. J . Mol. Biol. 1988, 201, 751. (b)
Adams, H.; Harris, K. D. M.; Hembury, G. A.; Hunter, C. A.;
Livingstone, D.; McCabe, J . F. Chem. Commun. 1996, 2531. (c) Adams,
H.; Carver, F. J .; Hunter, C. A.; Osborne, N. J . Chem. Commun. 1996,
2529.
(19) Carpino, L. A.; Han, G. Y. J . Org. Chem. 1972, 37, 3404.
(20) Medina, J . C.; Salomon, M.; Kyler, K. S. Tetrahedron Lett. 1988,
29, 3773.
(14) J ones, A. S.; MacCoss, M.; Walker, R. T Biochim. Biophys. Acta
1973, 365, 365.
(15) Stecher, E. D.; Gelblum, E. J . Org. Chem. 1961, 26, 2693.
(21) Pojer, P. M.; Angyal, S. J . Aust. J . Chem. 1978, 31, 1031.

Thymine Derivative of (S)-2-Hydroxy-4-(2-aminophenyl)butanoic Acid
J . Org. Chem., Vol. 62, No. 16, 1997 5453
Sch em e 3. Syn th esis of th e Th ym id in e AP NA Oligom er s
R-bromo ether with dimethylboron bromide and subse-
quently displacing the bromide with ethanethiol.22 Sev-
eral attempts to prepare 9 directly from 7 (one-pot
reaction), by reacting the unstable R-bromo ether inter-
mediate with bis(trimethylsilyl)thymine in the presence
of n-Bu4NI, led to a very low yield of the desired
compound 9 (∼5% yield), with alcohol 6 being the only
other detectable product.23 In contrast, coupling of (S)-8
with bis(trimethylsilyl)thymine24 in the presence of iodine
gave the acyclonucleoside thymidine analog (S)-9 in 58%
yield, and the unreacted (S)-8 was recovered (based on
the recovery of 8, compound 9 was formed with 98%
efficiency). The enantiomeric purity of compound (S)-9
was analyzed by chiral HPLC and found to be greater
than 98% ee.25
The Fmoc protecting group was partly unstable to
conditions required for ester hydrolysis;26 thus it was
replaced with the base stable Boc group using standard
literature procedures (Scheme 3). Peptide bond forma-
tion between the aniline monomer (S)-10 and the Boc-
protected free acid (S)-12 was easily achieved using O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HATU) as the coupling reagent in the
presence of diisopropylethylamine (DIPEA).27 The ad-
dition and removal of the Boc group as well as the
hydrolysis of the methyl ester were almost quantitative,
whereas the peptide coupling reaction gave typically a
70% yield. The preparation of dimer (S,S)-13 and tet-
ramer (S,S,S,S)-17 were achieved using the same reac-
tion conditions (Scheme 3).28
The structural identity of all new compounds was
1
confirmed by their MS and NMR data. The H and 13C
chemical shifts of all new compounds were assigned after
extensive analysis of their high-field 2D NMR spectra.
The chemical shift assignments of tetramer 17 were
based on its 1H, COSY, NOESY, HMQC, HMBC (500
MHz), and 13C (150 MHz) NMR spectra and are sum-
marized in Table 1. In addition, the high-resolution FAB
MS data of the fully deprotected APNA monomer, dimer,
and tetramer (1, 16, 18) confirmed the elemental com-
position of these new molecules.
The fully deprotected compounds, monomer 1, dimer
16, and tetramer 18 were found to be completely water
soluble. Thus, the 1H resonances of the thymine moieties
in D2O were compared, in the hope of gaining some
insight into the preorganization of these molecules in an
aqueous solution.29 Small upfield shifts of both the H6
and methyl resonances in the tetramer, as compared to
those of the dimer were observed (Table 2). This phe-
nomenon is generally attributed to the ring-current
magnetic anisotropy effect of the purine and pyrimidine
bases and is usually observed in conformations involving
intramolecular base-stacking interactions. Unfortu-
nately, the monomer did not show the expected chemical
shifts, downfield from those of the dimer. We have
speculated that this discrepancy may be due to the
presence of two chromophores in the APNA monomer
which can π-stack with each other. We have further
(28) The diastereomeric mixture of 13 was prepared from racemic
monomers 10 and 12, and examined by 1H NMR (300 MHz, CDCl3).
Two sets of diastereomeric protons were clearly observed (so that
integrals could be obtained with a high degree of accuracy) for the
following resonances: backbone-NHCO (δ 8.24 and 8.53), -CO2CH3 (δ
3.64 and 3.69), Th-CH3 (δ 1.91 and 1.95), and the Boc group (δ 1.50
and 1.53). However, the 1H NMR of compound (S,S)-13 (300 MHz,
CDCl3) showed only one set of signals [δ 8.53 (NHCO), 3.69 (CO2CH3),
1.91 (Th-CH3), and 1.50 (Boc)]. Since it is highly unlikely that coupling
of the enantiomerically pure S monomers would lead to the formation
of the (R,R) dimer 13, we feel fairly confident that the peptide coupling
reaction does not suffer from racemisation to any significant extent
under the reaction conditions used.
(22) Morton, H. E.; Guindon, Y. J . Org. Chem. 1985, 50, 5379.
(23) When menthol was used as a model secondary alcohol to test
the reaction conditions, the desired thymine derivative was produced
in 90% yield.
(24) Nishimura, T.; Iwai, I. Chem. Pharm. Bull. 1964, 12, 352.
(25) The two enantiomers of racemic 9 were clearly separated on a
chiralcel OD HPLC column using 10% ethanol in hexane at a flow rate
of 1 mL/min.
(26) Hydrolysis of the methyl ester did not lead to any racemization
under the reaction conditions used; deuterium exchange of the R-proton
was not observed when the reaction was carried out using 3 equiv of
LiOH in dioxane-D2O (1:1) at rt.
(29) The chemical shifts were obtained from COSY NMR spectra
using sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as the
reference standard.
(27) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397.

5454 J . Org. Chem., Vol. 62, No. 16, 1997
Ta ble 1. 1H (500 MHz) a n d 13C (150 MHz) NMR Da ta of AP NA Tetr a m er 17 in CDCl3
Tsantrizos et al.
assignment
1′ (CO2CH3)
(CO2CH3)
13C (δ)
1H (δ)
3.64
int, mult, J (Hz)
assignment
4
13C (δ)
1H (δ)
int, mult, J (Hz)
172.51
52.90
77.94
78.22
78.76
79.88
32.46
33.07
33.43
33.75
26.65
26.78
26.90
27.11
76.40a
76.71a
77.03a
77.03a
164.04
164.22
164.43
164.73
112.03
112.17c
112.24
12.50c
12.50c
139.64
139.81
140.02c
131.63
131.64
132.97
133.43
134.66
134.83
135.03
136.34
123.42
124.50
124.55
124.87
127.24
127.32
127.33c
125.11
126.09
126.27
126.48
129.35
129.61
129.78
129.87
3H, s
4H, m
2′
3′
4′
4.17-4.23b
1.90-2.24b
2.62-2.75b
5
8H, m
8H, m
Th-CH3
6
1.86
9H, bs
3H, bs
1.88
7.05-7.18b
1′′
2′′
3′′
O-CH2-Th
Ph-NH-CO
5.12b
5.06b
5.18b
5.26b
8.62
8.72
8.84
8.71
2H, bs
2H, AB quartet
2H, AB quartet
2H, AB quartet
1H, bs
1H, bs
1H, bs
7.66
7.74
7.61
1H, bs
Ph-NH-Boc
Ph-NH-CO
1H, bs
1H, d, J ) 8.1
1H, d, J ) 7.3
1H, d, J ) 7.3
170.33
170.72
170.80
151.94
80.76
7.54
4′′
5′′
7.15-7.20b
Ph-NH-CO2C(CH3)3
Ph-NH-CO2C(CH3)3
Ph-NH-CO2C(CH3)3
2
28.57
1.47
9H, s
6.98-7.01b
7.09-7.19b
151.94
152.00
152.16
152.23
6′′
Th-NH
9.95
10.02
10.16
10.27
1H, bs
1H, bs
1H, bs
1H, bs
a
b
Due to overlapping 13C resonances, these chemical shifts assignments were based on the HMQC and HMBC NMR data. Due to
overlapping resonances in the 1H NMR spectrum, the exact chemical shifts and coupling constants could not be determined; the values
given were obtained from the HMQC and HMBC data. c The strong intensity of the signal suggested overlapping 13C resonances.
Ta ble 2. 1H Ch em ica l Sh ifts of Mon om er 1, Dim er 16,
a n d Tetr a m er 18 in D2Oa
Ta ble 3. UV Absor ba n ce a t 264 n m in CH3OH
compound
molar concentration
absorbance
1.525
assignment
1 (δ)
16 (δ)
18 (δ)
thymine
2.0 × 10-4
2.0 × 10-4
2.0 × 10-4
1.0 × 10-4
0.5 × 10-4
H6
Th-CH3
7.34
1.80
7.54, 7.41
1.87, 1.83
7.45, 7.41, 7.38, 7.30b
1.81, 1.80 (2Me), and 1.76
compound 6
monomer 11
dimer 15
∼0 (UVmax ) 232 nm)
1.222
1.789
1.418
a
b
DSS was used as the reference standard. Due to overlapping
resonances in the 1H NMR spectrum, the exact chemical shifts
were obtained from the 1D COSY spectra.
tetramer 17
tetramer 18 and a complementary DNA tetramer [(dA)4]
could not be detected at temperatures as low as 5 °C.
However, duplex formation is not usually observed
between two complementary tetramers, even with natu-
ral oligonucleotides; an octameric (dT)-(dA) complex
would be the minimum length required in order to
properly measure a Tm value.
explored this possibility by comparing the UV absorption
spectra of pure thymine to the APNA molecules at 264
nm (Table 3). Although the absorbance value of the
monomer was lower than that of the free thymine, it was
once again inconsistent with respect to the dimer and
tetramer. The APNA tetramer showed significant hy-
pochromism compared to the dimer, indicative of a
stacked structure. Duplex formation between the APNA
In conclusion, the chiral APNA tetramer 18 constitutes
a new class of synthetic nucleic acid analogs prepared

Thymine Derivative of (S)-2-Hydroxy-4-(2-aminophenyl)butanoic Acid
J . Org. Chem., Vol. 62, No. 16, 1997 5455
(3 atm) in the presence of 10% Pd/C (0.33g) in EtOH for 15 h.
The solution was filtered through Celite and concentrated to
give a 92% yield of the desired product as a light yellow oil.
The enantiomeric purity of the (S)-enantiomer was measured
from the novel amino acid monomer (S)-1 using classical
peptide synthesis. It is expected that the APNA analogs
will be stable to nuclease and protease degradation and
the lipophilic character of their novel backbone may have
a positive effects on the cell permeability of these com-
pounds. It is also encouraging that these analogs are
fairly soluble in aqueous media. A thorough evaluation
of their influence on the hybridization stability of a
duplex or a triplex requires the synthesis of longer APNA
homopolymers, as well as DNA-APNA chemical chime-
ras; this studies are currently in progress in our labora-
tory.
1
to be >98% ee from its H NMR observed in the presence of 1
equiv of (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol; (R)-5 hav-
ing ∼50% ee was also synthesized and used for reference in
the NMR analysis of the optical purity: [R]D +28 (c 0.40,
CHCl3). TLC (1:2 Hex/EtOAc): Rf ) 0.25. 1H NMR (270 MHz,
CDCl3): δ 1.85-2.14 (2m, 2H3′), 2.55-2.75 (m, 2H4′), 3.72 (s,
OCH3), 3.78 (bs, OH), 4.21 (dd, J ) 8.2, 3.7 Hz, H2′), 6.65-
6.78 (m, 2H, Ar), 7.00-7.05 (m, 2H, Ar). 13C NMR (67.5 MHz,
CDCl3): δ 25.99, 33.54, 52.34, 69.48, 115.87, 118.83, 125.34,
127.17, 129.70, 144.14, 175.30. MS (NH3)CI m/z (assign-
ment): 210 (base, MH+), 209 (M+), 178 (M - OCH3+), 177 (M
- CH3OH+), 149 (177 - CO).
Exp er im en ta l Section
Syn th esis of F m oc-P r otected (S)-6. Compound 5 (3.06
g, 14.66 mmol) was dissolved in a mixture of 10% aqueous Na2-
CO3 (17.6 mL, 1 equiv) and dioxane (10 mL) and cooled to 0
°C. Fluorenylmethyl chloroformate (3.79 g, 1 equiv, 14.66
mmol) was dissolved in dioxane (20 mL) and added dropwise
via a syringe, giving a white milky reaction mixture. The
mixture was stirred at 0 °C for 1.5 h and allowed to warm up
to rt for an additional hour. The reaction was then quenched
with H2O (30 mL), acidified to pH 3, and extracted with EtOAc
(4 × 30 mL); the combined organic layers were washed with
saturated NaCl (120 mL) and dried over anhydrous MgSO4.
Flash column chromatography using 3:2 Hex/EtOAc as the
solvent system led to the isolation of the desired product 6 as
a white solid in 84% yield. TLC (1:1 Hex/EtOAc): Rf ) 0.35.
Mp: 119-120 °C. 1H NMR (270 MHz, CDCl3): δ 1.89-2.15
(m, 2H3′), 2.65-2.89 (m, 2H4′), 3.09 (bs, OH), 3.70 (s, OCH3),
4.06 (dd, J ) 8.6, 3.7 Hz, H2′), 4.27, (t, J ) 6.9 Hz,
NHCOOCH2CH), 4.48 (d, J ) 6.9 Hz, NHCOOCH2CH)), 7.04-
7.78 (12H, Ar and Fmoc-Ar). 13C NMR (67.5 MHz, CDCl3): δ
25.87, 34.26, 47.09, 52.57, 66.85, 68.75, 119.88, 124.51, 125.05,
127.00, 127.14, 127.63, 129.82, 135.88, 141.24, 143.81, 154.27,
175.04.
Syn th esis of Meth oxym eth yl Eth er (S)-7. Compound
(S)-6 (1.1 g, 2.3 mmol) was dissolved in freshly distilled THF
(20 mL) at 0 °C under N2. Chloromethyl methyl ether (4.0
mL, 20 equiv, 53.2 mmol) was added, followed by the dropwise
addition of dry diisopropylethylamine (1.4 mL, 3 equiv, 0.8
mmol). The reaction mixture was allowed to warm up to rt
and to stir for 18 h. The reaction was quenched with the
addition of water (50 mL), followed by extraction with EtOAc
(3 × 50 mL); the organic layer was dried with anhydrous
MgSO4 and concentrated to give a light amber oil. Purification
by flash column chromatography using 2:1 Hex/EtOAc as the
solvent system gave product 7 as a light yellow oil in 90% yield:
[R]D -19.6 (c 0.30, CHCl3). TLC (2:1 Hex/EtOAc): Rf ) 0.24.
1H NMR (300 MHz, CDCl3): δ 2.06-2.13 (m, 2H3′), 2.73 (t, J
) 7.2 Hz, 2H4′), 3.35 (s, CH2OCH3), 3.68 (s, COOCH3), 4.19
(t, J ) 6 Hz, H2′), 4.29 (t, J ) 6.9 Hz, Fmoc-CHCH2), 4.48-
4.60 (m, Fmoc-CHCH2), 4.64 (d, J ) 6.9 Hz, A part of AB, 1H,
OCH2O), 4.71 (d, J ) 6.9 Hz, B part of AB, 1H, OCH2O), 6.90-
7.64 (13H, Ar, Fmoc-Ar, NH). 13C NMR (75 MHz, CDCl3): δ
26.37, 33.13, 47.41, 52.24, 56.30, 66.99, 74.40, 96.34, 120.15,
122.87, 124.72, 125.25, 127.26, 127.39, 127.90, 129.76, 131.70,
135.79, 141.49, 143.97, 144,03, 154.38, 172.67. EIMS m/z
(assignment): 476 (MH)+, 475 (M+), 297 [(M - Fmoc +
COOH)+], 279 [(297 - H2O)+].
In str u m en ta tion a n d Gen er a l Meth od s. NMR spectra
were obtained at 20-22 °C. 1H and 13C NMR chemical shifts
are quoted in ppm and are referenced to the internal deuter-
ated solvent unless otherwise indicated. Mass spectral data
were obtained at McGill University, Biomedical Mass Spec-
trometry Unit, and the Department of Chemistry. All reac-
tions were run under a nitrogen atmosphere using oven-dried
syringes and glassware when appropriate. THF was distilled
from Na/benzophenone, CH2Cl2 was distilled from P2O5, MeOH
were distilled from Mg turnings, and DMF was distilled from
CaO. Reagents and solvents were purchased from Aldrich
Chemical Co. and VWR Scientific of Canada, respectively. The
enzymes BSLDH and FDH were purchased from Genzyme
(Cambridge, MA) and Boeringer Mannheim (Montreal, Que-
bec), respectively. Reversed phase flash column chromatog-
raphy was carried out on silica gel reacted with n-octadecyl-
trichlorosilane, following previously reported procedures.30
Syn th esis of r-Hyd r oxy Acid (S)-4. An aqueous suspen-
sion of compound 2 was titrated to pH 7 with 1 N NaOH and
freeze-dried to obtain the sodium salt as a yellow powder. The
salt (2.42 g, 10 mmol) was dissolved in TRIS‚HCl buffer (5
mM, pH 6, 500 mL) containing sodium formate (2.3 equiv, 23
mmol, 1.56 g), NADH (0.02 equiv, 0.2 mmol, 150 mg), and
dithiothrietol (0.005 equiv, 0.05 mmol, 7.8 mg), and the
solution was degassed under vacuum for at least 30 min.
Lyophilized powders of the two enzymes, BSLDH (600 units)
and FDH (50 units), were added, and the mixture was gently
stirred at rt under N2 for 24 h with a periodic addition of acid
(0.2 N HCl) to maintain the pH at 6.0-6.2. The solution was
then acidified to pH 3 with 1 N HCl and extracted with EtOAc
(4 × 300 mL). The organic layer was concentrated to give the
desired compound as a light brown solid which was found to
1
be fairly pure by H NMR (98% yield): mp 123-125 °C. [R]D
+43.2 (c 0.10, MeOH), >98% ee. The enantiomers of 4 could
not be separated by chiral HPLC; however, the enantiomeric
purity was estimated from the % ee of compound 5. TLC on
normal silica gel (1:1 MeOH/EtOAc): Rf ) 0.56; on C18
reversed phase silica gel (1:1 MeOH/H2O), Rf ) 0.32. 1H NMR
(270 MHz, acetone-d6): δ 4.94 (dd, J ) 2.0, 4.9 Hz, H2′), 6.53
(dd, J ) 4.9, 15.8 Hz, H3′), 7.21 (dd, J ) 2.0, 15.8 Hz, H4′),
7.53 (dt, J ) 7.9, 1.5, H4′′), 7.70-7.73 (tm, J ) 8.0 Hz, H5′′),
7.81 (dd, J ) 1.5, 8.0 Hz, H6′′), 7.93 (dd, J ) 1.0, 8.0 Hz, H3′′).
13C NMR (67.5 MHz, CDCl3): δ 72.24, 125.30, 127.25, 129.62,
129.65, 132.84, 133.36, 134.18, 149.61, 175.00.
Syn th esis of An ilin e Meth yl Ester (S)-5. The R-hydroxy
acid (S)-4 was dissolved in methanol and reacted with excess
diazomethane at rt until the evolution of gas had ceased. Pure
methyl ester (S)-5 was obtained after flash column chroma-
tography in 90% yield: [R]D +55 (c 0.40, CHCl3). TLC (3:2 Hex/
EtOAc): Rf ) 0.23. 1H NMR (270 MHz, CDCl3): δ 3.85 (s,
OCH3), 4.89 (dd, J ) 1.7, 5.2 Hz, H2′), 6.22 (dd, J ) 5.2, 15.8
Hz, H3′), 7.29 (dd, J ) 1.7, 15.8 Hz, H4′′), 7.37-7.45 and 7.55-
7.59 (2m, 3H, Ar), 7.93 (d, J ) 7.9 Hz, H3′′).
Syn t h esis of (Met h ylt h io)et h yl E t h er In t er m ed ia t e
(S)-8. Compound (S)-7 (1.52 g, 3.2 mmol) was dissolved in
freshly distilled CH2Cl2 (40 mL) and cooled to -78 °C under
N2. A solution of dimethylboron bromide in CH2Cl2 (1.5 M,
7.6 mL, 11.2 mmol) was added along with diisopropylethyl-
amine (0.04 mL, 0.1 equiv, 0.32 mmol). After 20 min, more
diisopropylethylamine (1.2 mL, 6.4 mmol) was added followed
by ethanethiol (0.8 mL, 9.6 mmol), and the resulting mixture
was stirred for 2 h at -78 °C. The reaction was quenched
with saturated aqueous NaHCO3 (50 mL), and the mixture
was allowed to warm up to rt. The aqueous layer was
extracted with EtOAc (3 × 100 mL), and the combined organic
layers were washed with saturated NaCl (150 mL). The
organic layer was then dried with anhydrous MgSO4 and
Hydrogenation of both the nitro group and the double bond
was achieved by reacting the methyl ester (3.3 g) with H2 gas
(30) (a) Tsantrizos, Y. S.; Ogilvie, K. K.; Watson, A. K. Can. J . Chem.
1992, 70, 2276. (b) Blunt, J . W.; Calder, V. L.; Fenwick, G. D.; Lake,
R. J .; McCombs, J . D.; Munro, M. H. G.; Perry, N. B. J . Nat. Prod.
1987, 50, 290.

5456 J . Org. Chem., Vol. 62, No. 16, 1997
Tsantrizos et al.
concentrated to dryness. Pure compound (S)-8 was isolated
as a light yellow oil (450 mg) after flash column chromatog-
raphy using 4:1 Hex/EtOAc as the solvent system. The
average yield of this reaction was 55-60%, based on the
amount of recovered alcohol 6: [R]D -54 (c 1.80, CHCl3). TLC
(4:1 Hex/EtOAc): Rf ) 0.19. 1H NMR (300 MHz, CDCl3): δ
1.24 (t, J ) 7.5 Hz, SCH2CH3), 2.07-2.14 (m, 2H3′), 2.60 (q, J
) 7.4 Hz, SCH2CH3), 2.73 (t, J ) 6.6 Hz, 2H4′), 3.67 (s, OCH3),
4.31 (t, OCH2CH-FMoc), 4.40 (t, J ) 5.5 Hz, H2′), 4.45-4.61
(m, OCH2CH-Fmoc), 4.67 (d, J ) 11 Hz, A part of AB, 1H,
OCH2S), 4.87 (d, J ) 11 Hz, B part of AB, 1H, OCH2S), 7.06-
7.81 (13H, ArH, NH). 13C NMR (75 MHz, CDCl3): δ 15.01,
25.29, 26.55, 32.99, 47.48, 52.37, 67.19, 73.04, 73.90, 120.26,
124.78, 125.36, 127.36, 127.51, 128.00, 129.85, 135.87, 141.58,
144.04, 144.12, 154.43, 172.64. FAB MS m/z (assignment):
506 (MH+), 505 (M+), 444 [(M - SEt)+].
both enantiomers;14 the retention times for the S- and R-
enantiomer are 23.8 and 27.7 min, respectively: [R]D -24 (c
0.40, MeOH). TLC (3:1 EtOAc/Hex): Rf ) 0.30. 1H NMR (300
MHz, CD3OD): δ 1.58 (9H, tBoc), 1.95 (Th-CH3), 1.94-2.16
(m, 2H3′), 2.67-2.74 (m, 2H4′), 3.75 (s, OCH3), 4.30 (dd, J )
6 Hz, H2′), 5.18 (d, A part of AB, J ) 10.5 Hz, 1H, OCH2Th),
5.39 (d, B part of AB, J ) 10.5 Hz, 1H, OCH2Th), 7.04-7.27
(m, 5H, ArH, H6), 9.35 (s, NHBoc). 13C NMR (75 MHz, CD3-
OD): δ 12.51, 26.44, 28.59, 32.66, 52.52, 76.44, 77.44, 80.68,
112.02, 122.97, 124.41, 127.38, 129.59, 131.09, 136.14, 139.52,
151.52, 153.69, 164.20, 172.12.
P r ep a r a tion of th e F r ee Acid An a log (S)-12. To a
solution of compound 11 (105 mg, 0.234 mmol) in 3:1 THF/
MeOH (5 mL) was added aqueous LiOH (250 µL, 1.4 M), and
the reaction mixture was stirred at rt for 3 h. The mixture
was subsequently evaporated to dryness, and the resulting
residue was dissolved in H2O (10 mL) at pH 3 and extracted
with EtOAc (3 × 20 mL). The organic layer was dried with
anhydrous MgSO4 and concentrated to give a fairly pure
Syn th esis of Th ym in e An a log (S)-9. Methoxymethyl
ethyl thioether (S)-8 (218 mg, 0.43 mmol) was dissolved in dry
THF (1 mL) in the presence of activated molecular sieves (3
Å). A solution of bis(trimethylsilyl)thymine (1.4 mL, 1.5 M in
dry THF) was added, followed by the addition of I2 (109 mg,
0.43 mmol), and the reaction mixture was stirred at rt under
N2 for 48 h. The mixture was then poured into a 5% aqueous
sodium sulfite solution (10 mL) and extracted with EtOAc (3
× 15 mL), and the organic layer was washed with H2O (45
mL) and saturated NaCl (45 mL). The organic layer was then
dried with anhydrous MgSO4 and concentrated. Purification
by flash column chromatography, using 1:2 Hex/EtOAc as the
eluting solvent, led to the isolation of the desired product in
58% yield (98% based on recovery of starting material). TLC
(1:2 Hex/EtOAc): Rf ) 0.25. 1H NMR (270 MHz, CDCl3): δ
1.84 (s, CH3), 2.00-2.18 (m, 2H3′), 2.61-2.74 (m, 2H4′), 3.65
(s, OCH3), 4.20 (dd, J ) 5.2 Hz, H2′), 4.29 (t, J ) 9 Hz,
-CHCH2- of Fmoc), 4.58-4.68 (m, -CHCH2- of Fmoc), 4.90 (d,
J ) 12 Hz, A part of AB, 1H, OCH2Th), 5.13 (d, J ) 12 Hz, B
part of AB, 1H, OCH2Th), 6.9-8.3 (14H, Ar, NH, H6). 13C
NMR (75 MHz, CDCl3): δ 12.46, 26.31, 32.81, 47.50, 52.55,
66.83, 76.38, 77.42, 111.87, 120.26, 125.21, 125.23, 127.31,
127.39, 127.57, 128.03, 129.81, 135.66, 139.58, 141.56, 143.89,
144.03. MALDI MS m/z (assignment): 592 [(M + Na)+].
P r ep a r a tion of An ilin e An a log (S)-10. Compound (S)-9
(460 mg, 0.81mmol) was treated with 5% piperidine in DMF
(13 mL) at rt for 20 min. The DMF was then removed by
evaporation, and the crude mixture was partitioned between
H2O (10 mL) and EtOAc (20 mL). The aqueous layer was
extracted with more EtOAc (3 × 20 mL), the combined organic
layers were dried with anhydrous MgSO4 and concentrated
to give a light yellow oil. Purification by flash column
chromatography using 5:1 EtOAc/Hex afforded compound 10
in 98% yield: [R]D -37 (c 0.2, MeOH). TLC (5:1 Hex/EtOAc):
Rf ) 0.20. 1H NMR (300 MHz, CD3OD): δ 1.87 (d, J ) 1.2
Hz, Th-CH3), 1.83-2.13 (m, 2H3′), 2.59 (t, J ) 7.5 Hz, 2H4′),
3.68 (s, OCH3), 4.17 (dd, J ) 3.6, 8.4 Hz, H2′), 5.14 (d, A part
of AB, J ) 11.1 Hz, OCH2Th), 5.29 (d, B part of AB, J ) 11.1
Hz, 1H, OCH2Th), 6.59 (dt, J ) 7.5, 1.2 1H, Ar), 6.68 (dd, J )
8.1, 1.2 Hz, 1H, Ar), 6.89 (dd, J ) 7.2, 1.2 Hz, 1H, Ar), 6.94
(dt, J ) 8.1, 1.2 Hz, 1H, Ar), 7.48 (q, J ) 1.2 H6). 13C NMR
(75 MHz, CD3OD): δ 12.36, 27.55, 32.96, 52.73, 77.80, 77.86,
112.05, 117.29, 119.51, 126.27, 128.36, 130.63, 142.31, 146.40,
153.38, 166.85, 174.48. MS EI m/z (assignment): 347 (M+),
348 (MH+).
sample of the free acid analog (S)-12 in 94% yield: [R]D
-15 (c
0.37, MeOH). TLC on C18-silica (2:1 MeOH/H2O): Rf ) 0.53.
1H NMR (300 MHz, CD3OD): δ 1.50 (s, C(CH3)3), 1.88 (d, J )
1.2 Hz, Th-CH3
), 1.86-2.14 (m, 2H3′), 2.62-2.81 (m, 2H4′),
4.10-4.15 (m, H2′), 5.14 (d, A part of AB, J ) 10.5 Hz, 1H,
OCH2Th), 5.39 (d, B part of AB, J ) 10.5 Hz, 1H, OCH2Th),
7.05-7.32 (m, 4H, ArH), 7.48 (d, J ) 1.2 Hz, H6), 8.21 (s, NH).
13C NMR (75 MHz, CD3OD): δ 12.40, 28.28, 28.87, 34.11,
77.69, 77.86, 81.11, 111.93, 126.94, 127.49, 127.96, 130.91,
136.92, 137.30, 142.33, 153.29, 156.83, 166.84, 175.60.
P r ep a r a tion of th e F u lly Dep r otected Mon om er (S)-
1. Boc-protected monomer 12 (∼10 mg) was dissolved in an
anhydrous solution of 15% trifluoroacetic acid in CH2Cl2 (2 mL)
and stirred at rt under N2 for 15 min. The fully deprotected
monomer 1 was obtained in high purity after evaporation of
the reaction mixture to dryness. C18-TLC (3:1 MeOH/H2O):
Rf ) 0.77. 1H NMR (300 MHz, D2O): δ 1.80 (d, J ) 1.2 Hz,
Th-CH3), 1.77-1.93 and 2.05-2.15 (2m, 2H3′), 2.57 (t, J ) 7
Hz, 2H4′), 3.85-3.89 (dd, J ) 9.3, 3.6, H2′), 5.01 (d, A part of
AB, J ) 11 Hz, 1H, OCH2Th), 5.22 (d, B part of AB, J ) 11
Hz, 1H, OCH2Th), 6.72-7.10 (m, 4H, ArH), 7.34 (d, J ) 1.2
Hz, H6). HRFAB MS m/z: 320.12457 (M + H)+, calcd mass
for C15H17N3O5 + H+ ) 320.124646.
Syn th esis of AP NA Dim er (S,S)-13. Free acid monomer
12 (96 mg in 2 mL of dry DMF, 0.22 mmol) was added to a
solution of the HATU coupling reagent (101 mg in dry DMF,
0.27 mmol) at 0 °C under N2. Diisopropylethylamine (80 µL,
0.44 mmol) was added, and the reaction was allowed to stir
for 10 min. The free aniline monomer 10 (100 mg in 1 mL of
dry DMF, 0.29 mmol) was then added via a syringe, and the
resulting solution was stirred at rt for 24 h. The reaction was
quenched by dilution with H2O (10 mL), and the product was
extracted with EtOAc (3 × 20 mL). Purification via C18
reversed phase chromatography using a solvent gradient from
100% H2O to 100% MeOH led to the isolation of the desired
product in 70% yield as a pale yellow solid (eluted from column
in ∼55% aqueous MeOH): [R]D -24 (c 0.40, MeOH). TLC on
C18-silica (1:1 MeOH/H2O): Rf ) 0.35. 1H NMR (300 MHz,
CD3OD): δ 1.47 (s, C(CH3)3), 1.84 (d, J ) 1.5 Hz, Th-CH3),
1.88 (d, J ) 1.5 Hz, Th-CH3), 1.88-2.18 (2m, 4H3′), 2.6-2.8
(2m, 4H4′), 3.60 (s, OCH3), 4.16-4.28 (m, 2H2′), 5.08 (d, A part
of AB, J ) 10.5 Hz, 1H, OCH2Th), 5.17 (d, B part of AB, J )
10.5 Hz, 1H, OCH2Th), 5.27 (d, A part of AB, J ) 10.2 Hz,
1H, OCH2Th), 5.35 (d, B part of AB, J ) 10.2 Hz, 1H, OCH2-
Th), 7.0-7.35 (8H, m, ArH), 7.38 (q, J ) 1.5 Hz, H6), 7.57 (q,
J ) 1.5 Hz, H6), 7.9 (s, NH). 13C NMR (300 MHz, CD3OD): δ
12.37, 12.24, 28.17, 28.26, 28.90, 34.38, 34.81, 52.81, 78.04,
78.18, 78.30, 79.71, 81.18, 111.95, 112.17, 127.06, 127.54,
127.93, 128.03, 128.14, 128.18, 131.03, 131.08, 142.31, 153.32,
156.89, 166.78, 173.52, 174.16, MALDI MS m/z (assignment):
786 [(M + Na)+].
P r ep a r a tion of Boc-P r otected An a log (S)-11. Com-
pound (S)-10 (52 mg, 0.15 mmol) was dissolved in 10%
triethylamine in MeOH (1 mL) and mixed with di-tert-butyl
dicarbonate (66 mg, 0.30 mmol). The mixture was stirred at
rt for 17 h. The solvent was then evaporated, and the residue
was partitioned between H2O (10 mL) and EtOAc (20 mL).
The aqueous layer was extracted with more EtOAc (3 × 20
mL), and the combined organic layers were dried with anhy-
drous MgSO4 and concentrated to give the Boc-protected
analog (11) as a light yellow foam with a 92% yield and 98-
99% ee. The enantiomeric purity of 11 was determined by
HPLC using a Chiralcel OD column with 10% ethanol in
hexane as the eluting solvent and a flow rate of 1 mL/min.
Under these conditions, base line separation of the two
enantiomers was observed with a reference sample containing
P r ep a r a tion of F r ee An ilin e Dim er (S,S)-14. Boc meth-
yl ester dimer 13 (53 mg, 0.07 mmol) was dissolved in an
anhydrous solution of 15% trifluoroacetic acid in CH2Cl2 (2 mL)
and stirred at rt under N2 for 15 min. The reaction mixture
was subsequently evaporated to dryness and partitioned by
chromatography on a dianion HP20 column using a solvent

Thymine Derivative of (S)-2-Hydroxy-4-(2-aminophenyl)butanoic Acid
J . Org. Chem., Vol. 62, No. 16, 1997 5457
gradient from 100% H2O to 100% MeOH. The aniline dimer
14 eluted from the column with 50-60% aqueous MeOH; 40%
yield of pure 14 was isolated. However, some unreacted
starting material was detected by TLC: [R]D -43 (c 0.17,
MeOH). TLC on C18-silica (2:1 MeOH/H2O): Rf ) 0.30. 1H
NMR (300 MHz, CD3OD): δ 1.82 (d, J ) 1.2 Hz, Th-CH3), 1.87
(d, J ) 1.2 Hz, Th-CH3), 1.9-2.21 (m, 4H3′), 2.56-2.80 (m,
4H4′), 3.58 (s, OCH3), 4.20 (dd, J ) 3.9, 8.1 Hz, H2′), 4.24 (dd,
J ) 3.9, 8.1 Hz, H2′), 5.07 (d, A part of AB, J ) 10.8 Hz, 1H,
OCH2Th), 5.17 (d, B part of AB, J ) 10.8 Hz, 1H, OCH2Th),
5.27 (d, A part of AB, J ) 10.2 Hz, 1H, OCH2Th), 5.34 (d, B
part of AB, J ) 10.2 Hz, 1H, OCH2Th), 6.62 (dt, J ) 7.5, 1.2
Hz, 1H, Ar), 6.70 (dd, J ) 7.2, 1.2 Hz, 1H, Ar), 6.91-7.00 (m,
2H, Ar), 7.17-7.23 (m, 3H, Ar), 7.29-7.30 (m, 1H, Ar), 7.32
(q, J ) 1.2 Hz, H6), 7.50 (q, J ) 1.2 Hz, H6). 13C NMR (75
MHz, CD3OD): δ 12.38, 12.42, 27.92, 28.34, 33.77, 34.44,
52.81,78.04, 78.27, 78.38, 79.80, 111.96, 112.19, 117.29, 119.60,
126.67, 128.05, 128.17, 128.26, 128.42, 130.75, 131.10, 136.01,
137.67, 142.31, 146.49, 153.32, 153.35, 166.81, 173.78, 174.15.
FAB MS m/z (assignment): 663 (M+).
P r ep a r a tion of F r ee Acid Dim er (S,S)-15. Hydrolysis
of methyl ester dimer 13 to the corresponding free acid 15
(∼99% yield) was carried out using the same reaction condi-
tions as in the preparation of the free acid analog (S)-12. 15:
[R]D -25 (c 0.25, MeOH). TLC on C18-silica (2:1 MeOH/H2O):
Rf ) 0.38. 1H NMR (300 MHz, CD3OD): δ 1.47 (s, C(CH3)3),
1.84 (bs, Th-CH3), 1.88 (bs, Th-CH3), 2.00-2.15 (m, 4H3′),
2.60-2.85 (m, 4H4′), 4.16-4.25 (m, 2H2′), 5.08 (d, A part of
AB, J ) 11.1 Hz, 1H, OCH2Th), 5.20 (d, B part of AB, J )
11.1 Hz, 1H, OCH2Th), 5.26 (d, A part of AB, J ) 10.2 Hz,
1H, OCH2Th), 5.33 (d, B part of AB, J ) 10.2 Hz, 1H, OCH2-
Th), 7.05-7.35 (8H, m, Ar), 7.38 (bs, H6). 7.55 (bs, H6), 8.25
(s, NH). 13C NMR (75 MHz, CD3OD): δ 12.42, 12.45, 24.37,
28.10, 28.49, 28.90, 30.83, 34.54, 34.74, 77.92, 78.17, 79.66,
81.18, 111.86, 112.16, 127.06, 127.52, 127.85, 128.00, 128.08,
128.17, 131.00, 131.20, 136.00, 137.09, 137.33, 137.70, 142.37,
153.29, 156.90, 166.70, 173.54, 175.73, 210.21. FAB MS m/z
(assignment): 749 (M+).
MS m/z: 621.23111 (M + H)+, calcd mass for C30H32N6O9
+
H+ ) 621.230902.
Syth esis of AP NA Tetr a m er s (S,S,S,S)-17 a n d -18. The
peptide coupling reaction between dimers 14 and 15 was
achieved using the same reaction procedure as in the coupling
of their respective monomers. The crude product was purified
by C18 reversed phase flash column chromatography using a
gradient from 100% H2O to 100% MeOH. The desired APNA
tetramer 17 eluted with 70-80% aqueous MeOH: [R]D -31 (c
0.08, MeOH). TLC on C18-silica (2:1 MeOH/H2O): Rf ) 0.13.
NMR data in Table 1. ES MS m/z (assignment): 1392.6 (M+),
1393.6 (MH+). Hydrolysis of the methyl ester followed by
removal of the Boc group (using standard conditions in both
cases) led to the isolation of tetramer 18 which was purified
by preparative C18-TLC (3:1 MeOH/H2O): Rf ) 0.50. 1H NMR
(500 MHz, D2O) showed resonances in the expected chemical
1
shifts regions (based on the H NMR spectra of compound 17).
However, due to the great extend of overlapping resonances,
the exact chemical shifts of all resonances were not assigned;
only those resonances associated with the methyls and H6
protons of the thymine moieties were assigned from the 1D
COSY NMR spectra. HRFAB MS m/z: 1355.3408 (M + Cs)+,
calcd mass for C60H62N19O17 + Cs+ ) 1355.341022.
Ack n ow led gm en t. This work was supported by
grants to Y.S.T. from the Banting Research Foundation,
the Canadian Foundation for AIDS Research, Natural
Sciences and Engineering Research Council of Canada,
and Fonds pour la Formation de Chercheurs et l’Aide a`
la Recherche du Que´bec. We are grateful to Dr. Tsuguo
Ikebe, Yoshitomi Pharmaceutical Industries, LTD.,
Fukuoka, J apan, for his generous gift of compound 2.
We thank Dr. Daniel Boismenu and Dr. Orval Mamer
of the Biomedical Mass Spectrometry Unit for assistance
with the ES MS and HRFAB MS data. We also thank
Dr. Phan Viet Minh Tan (Regional Center for high field
NMR at the Universite´ de Montre´al) for recording the
13C NMR (150 MHz) of 16 and Dr. Franc¸oise Sauriol
(Chemistry Department of McGill University) for re-
cording the 1H, COSY, HMQC, and HMBC NMR spectra
of 16.
P r ep a r a tion of F u lly Dep r otected Dim er (S,S)-16. Boc-
protected dimer 15 (∼15 mg) was dissolved in an anhydrous
solution of 15% trifluoroacetic acid in CH2Cl2 (2 mL) and
stirred at rt under N2 for 15 min. The fully deprotected dimer
16 was obtained after evaporation of the reaction mixture to
dryness. C18-TLC (2:1 MeOH/H2O): Rf ) 0.71. 1H NMR (300
MHz, D2O): δ 1.83 (bs, Th-CH3), 1.87 (bs, Th-CH3), 1.70-2.33
(4m, 4H3′), 2.63-2.71 and 2.84-2.91 (2m, 4H4′), 4.00-4.04
and 4.23-4.27 (2m, 2H2′), 5.08 (s, 2H OCH2Th), 5.26 (d, A
part of AB, J ) 11.1 Hz, 1H, OCH2Th), 5.32 (d, B part of AB,
J ) 11.1 Hz, 1H, OCH2Th), 7.05-7.43 (8H, m, Ar), 7.41 (bs,
H6). 7.54 (bs, H6). HRFAB MS m/z: 1355.3408 (M + Cs)+,
calcd mass for C60H62N19O17 + Cs+ ) 1355.341022]. HRFAB
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of all new compounds (24 pages). This material is
contained in libraries on microfiche, immediately following this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O962326P