Nucleo-β-amino acids: Synthesis and oligomerization to β-homoalanyl-PNA

Article abstract of DOI:10.1002/1522-2675(200211)85:11<3855::AID-HLCA3855>3.0.CO;2-A

The synthesis of thyminyl-, uracilyl-, cytosinyl-, and guaninyl-β³-amino acids and the oligomerization of the cytosinyl- and guaninyl-β³-amino acids to β-homoalanyl-PNA are presented. The pyrimidinyl nucleobases were connected to the

Full text of DOI:10.1002/1522-2675(200211)85:11<3855::AID-HLCA3855>3.0.CO;2-A

Helvetica Chimica Acta Vol. 85 (2002)
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Nucleo-b-Amino Acids: Synthesis and Oligomerization to
b-Homoalanyl-PNA
by Arndt M. Br¸ckner, Harald W. Schmitt, and Ulf Diederichsen*
Institut f¸r Organische Chemie, Georg-August-Universit‰t Gˆttingen, Tammannstrasse 2, D-37077 Gˆttingen
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
The synthesis of thyminyl-, uracilyl-, cytosinyl-, and guaninyl-b3-amino acids and the oligomerization of the
cytosinyl- and guaninyl-b3-amino acids to b-homoalanyl-PNA are presented. The pyrimidinyl nucleobases were
connected to the g-position of b-homoalanine by Mitsunobu reaction with a b-homoserine derivative or by
nucleophilic substitution of methanesulfonates. For the preparation of the guaninyl-b3-amino acid, a b-lactam
route was established that might be of interest also for the synthesis of other b3-amino acid derivatives. The
cytosinyl and guaninyl building blocks were oligomerized to hexamers. They form quite stable self-pairing
complexes in H2O as indicated by temperature dependent UV and CDspectroscopy.
1. Introduction. Peptide nucleic acids (PNAs) with a backbone composed of N-(2-
aminoethyl)glycine units have attracted considerable interest because of their potential
use as diagnostic tools and application in antigene or antisense therapy [1]. With alanyl-
PNA, a new kind of PNA oligomer was introduced that is based on a regular peptide
backbone: alanyl amino acids with alternating configuration carry nucleobases in the b-
position of the side chain [2]. These oligomers are able to form linear double strands
based on nucleobase recognition. Recently, oligomers have been reported that are
composed of nucleo-b3-amino acid building blocks1) in which adenine or 7-carbaade-
nine are connected to b-homoalanine at the g-position [4]. These b-homoalanyl-PNAs
are a special case of b-peptides with a b-sheet-like backbone conformation. They
organize in higher ordered structures by nucleobase pairing.
While b-peptides with proteinogenic and cyclic nonproteinogenic side chains
usually are known to fold into well-defined helical or sheet like secondary structures
[5], nucleo-b3-amino acids offer the potential to influence the b-peptide structure
through base pairing and aromatic p-interactions (stacking).
Here, we describe the syntheses of new N-Boc-nucleo-b3-amino acids with uracil,
thymine, cytosine, and guanine in the side chains. The cytosine- and guanine-containing
b-amino acids were oligomerized to b-homoalanyl-PNA by solid-phase techniques.
Furthermore, initial studies of the pairing properties of these oligomers are presented.
2. Synthesis of Pyrimidinyl-b3-Amino Acids. Stereoselective syntheses of b-amino
acids have already been reviewed extensively [5a][6]. Many methods known for the
preparation of b-amino acids with proteinogenic side chains are not applicable to the
The designation of b3-amino acids or b3-peptides is proposed by Seebach to specify the position of the side
chain [3a]. The preparation of a nucleo-b2-amino acid has recently been described [3b].
1
)

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Helvetica Chimica Acta Vol. 85 (2002)
synthesis of nucleo-b-amino acids. As a standard procedure, the ArndtÀEistert
homologation of a-amino acids was established by Seebach and co-workers [5a][7].
However, since ArndtÀEistert homologation of aromatic a-amino acids is expected to
proceed with racemization [7b], it should not be applied to the generation of nucleo-b3-
amino acids. Another obstacle is the poor solubility of nucleobases in most organic
solvents. In case of Boc-b-HalA-OH and Boc-b-Hal7CA-OH (H-b-HalA-OH ˆ (S)-g-
(adenin-9-yl)-b-homoalanine, H-b-Hal7CA-OH ˆ (S)-g-(7-carbaadenin-9-yl)-b-homo-
alanine) the Mitsunobu reaction was used to attach the nucleobase precursors 6-
chloropurine and 6-chloro-7-carbapurine to the g-position of a suitably protected b-
homoalanyl derivative [4].
The Mitsunobu reaction was chosen also as the key step in the syntheses of the
pyrimidinyl-nucleo-b-amino acids 1 and 2 (Scheme 1). Reduction of the commercially
available Boc-l-Asp(Bzl)-OH (3) led to the required b-homoserine derivative 4 [8].
For the syntheses of the uracil and thymine monomers, the N3-benzoyl-protected
nucleobases [9] were used. The protecting group not only prevents N3-alkylation but
Scheme 1. Synthesis of Pyrimidinyl-nucleo-b-amino Acids 1, 2, and 9 from b-Homoserine Derivative 4 as the
Key Intermediate

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3857
also improves the solubility. The way of bringing the reactants together seems to play a
crucial role to avoid the conversion of N-Boc-amino acids into N-Boc-aziridines under
Mitsunobu conditions [10]. Consequently, diethyl azodicarboxylate (DEAD), Ph3P,
and the N3-benzoyl-protected nucleobases were dissolved in THFat À308 and added to
a precooled solution of b-homoserine derivative 4. After stirring for 4 5 d at low
temperature, the coupling products 5 and 6 were isolated in yields of 70 and 65%,
respectively. Saponification of the benzyl ester and deprotection of the N3-atom were
achieved by catalytic hydrogenation on Pd/C. The analogous Mitsunobu reaction of the
N4-protected cytosine derivatives N4-(benzyloxycarbonyl)cytosine (cytosine(Z)), N4-
benzoylcytosine, and N4-[4-(tert-butyl)benzoyl]cytosine failed. However, the prepara-
tion of 7 by alkylation of cytosine(Z) was achieved with methanesulfonate 8. The latter
compound was prepared according to the literature [11]. The low yield of 23% for the
alkylation contrasts with the high yield obtained for the analogous reaction with 3-
benzoylthymine in preliminary studies. Not only the extremely poor solubility of
cytosine(Z) even in polar aprotic solvents accounts for the difficulties in the
preparation of 7. Methanesulfonate 8 showed a tendency to decompose under the
reaction conditions. Nevertheless, the cytosinyl-b3-amino acid 9 was prepared by
saponification of the benzyl ester 7 on gram-scale.
HMBC-NMR Experiments were performed to establish the N1-connectivity of 9.
However, no correlation between HÀC(g) and C(6), or HÀC(6) and C(g) in the
spectra was detectable. Therefore, the unprotected cytosinyl-b3-amino acid 10 was
prepared from the uracilyl-b3-amino acid 1 with known N1-connectivity as depicted in
Scheme 2. The manipulation of the C(4)-substituent in 1 was carried out by the 1,2,4-
triazole method [12]. The b3-amino acid 1 was first converted into the benzyl ester 11.
After activation of the C(4)-position with 4-chlorophenyl dichlorophosphate, the
Scheme 2. Conversion of the Uracilyl-b-amino Acid 1 into the Unprotected Cytosinyl-b-amino Acid 10 to
Establish the Connectivity of the Protected Cytosinyl-b-amino Acid 9

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substitution of the O-atom by 3-nitro-1,2,4-triazole proceeded smoothly. When the
cytosine derivative 12 was treated with aqueous NH3/dioxane the cytosinyl-b3-amino
acid benzyl ester 13 was obtained. Deprotection of the N4-protected cytosinyl-b3-amino
acid 9, as well as saponification of the unprotected cytosinyl-b3-amino acid benzyl ester
13, gave the same cytosinyl-b3-amino acid 10 and hereby established the connectivity of
compound 9.
3. Synthesis of the Guaninyl-b3-Amino Acid. Guanine cannot be selectively
alkylated at N9. 2-Amino-6-chloropurine, however, gives almost exclusively the N9-
substituted product and is usually employed as a synthon for guanine [13]. Therefore,
its reaction with b-homoserine derivative 4 under Mitsunobu conditions was
investigated. Compound 14 was isolated in 34% yield after laborious chromatography
(Scheme 3). Unfortunately, the treatment of 14 with aqueous NaOH/dioxane not only
gave the desired guaninyl-b3-amino acid 15 in 59% yield but also the O6-benzylgua-
ninyl-b3-amino acid 16 in 19% yield.
Scheme 3. Synthesis of the Guaninyl-b-amino Acid 15 from the b-Homoserine Derivative 4 with the Mitsunobu
Reaction as the Key Step
In the light of these difficulties, preparation of 15 from a cheaper starting material
that is unable to undergo aziridine formation, and that does not contain an ester
functionality was desirable. Methanesulfonate 17 was the candidate of choice
(Scheme 4). It can easily be prepared from l-aspartic acid by a procedure of Salzmann
et al. [14]. The preformed Na salt of 2-amino-6-chloropurine was alkylated by this
compound in 39% yield. The nucleo-b-lactam 18 was then converted to the guaninyl-b3-

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amino acid 15 by treatment with refluxing HCl, followed by protection with Boc2O to
give 15 in high yield and excellent optical purity (> 99% ee as determined by Boc
deprotection, coupling with Boc-(S)-Ala-OSu and Boc-(R)-Ala-OSu resp., and HPLC
analysis of the diastereoisomers).
Scheme 4. Preparation of Guaninyl-nucleo-b-amino Acid 15 from Methanesulfonate 17
4. Guanine- and Cytosine-Containing b-Homoalanyl-PNAs. The synthesis and
structural characterization of b-HalA and b-Hal7CA containing b-homoalanyl-PNAs
has recently been reported [4]. The oligomers H-(b-HalA)n-Lys-NH2, where n ˆ 5 or 6,
form very stable higher aggregates in aqueous solution. This was explained by an
extended backbone conformation enabling simultaneous pairing of the WatsonÀCrick
and Hoogsteen pairing planes. As part of our ongoing studies of association processes in
b-homoalanyl-PNAs, the following oligomers were synthesized: H-b-HalG-b-HalG-b-
HalC-b-HalG-b-HalC-b-HalC-Lys-NH2 (19, sequence GGCGCC, b-HalG ˆ (S)-g-
(guanin-9-yl)-b-homoalanine, b-HalC ˆ (S)-g-(cytosin-9-yl)-b-homoalanine) and H-b-
HalG-b-HalC-b-HalG-b-HalC-b-HalG-b-HalC-Lys-NH2 (20, sequence GCGCGC).
Oligomerization of Boc-b-HalG-OH (15) and Boc-b-HalC(Z)-OH (9) was carried out
by solid-phase peptide synthesis on a MBHA-polystyrene (MBHA ˆ 4-methylbenzhy-
drylamine) resin loaded with N-a-Boc-w-Z-protected d-lysine. The lysine amide at the
C-terminal of the PNAs was incorporated to increase the solubility of the oligomer. The
amino acids were coupled in DMF with O-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole
(HOAt), and (ethyl)diisopropylamine (DIEA) for activation. To ensure chain
elongation yields ꢀ 97% each coupling step was performed twice first with 5 and
then 3 equiv. of nucleo-b3-amino acid. After deprotection and cleavage from the solid

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support with trifluoroacetic acid (TFA)/trifluoromethanesulfonic acid/dimethyl sul-
fide/m-cresol 5 :1:3 :1, the oligomers were precipitated with Et2O and purified by
HPLC on a RP-C18 column. They were characterized with electrospray ionization mass
spectrometry (ESI-MS).
The formation of pairing complexes and their stabilities in an aqueous solution were
examined by temperature dependent UV spectroscopy. Water was chosen as the
solvent, since the oligomers precipitated in phosphate buffer. Cooperative depairing of
double strands or higher aggregates leads to destacking of the nucleobases, which can
be recognized by the sigmoidal increase of the absorption (hyperchromicity, Arel) with
rising temperature [15]. The temperature Tm at the point of inflection characterizes the
double strand stability. The self-pairing complex of oligomer 19 (sequence GGCGCC)
showed a stability of Tm ˆ 468 (15 mm, 25% Arel), whereas for oligomer 20 (sequence
GCGCGC) a stability of Tm ˆ 518 (14 mm, 21% Arel) was found (Fig. 1).
Fig. 1. UV Melting curves (270 nm) of oligomers 19 (left, 15 mm in H2O) and 20 (right, 14 mm in H2O)
Similar stabilities and hyperchromicities found for both oligomers indicate the
formation of double strands with six G-C base pairs each. From simple model studies,
the antiparallel double strand formation in the WatsonÀCrick-pairing mode is
expected. In comparison to the alanyl-PNA hexamer with GGCGCC sequence
(Tm ˆ 588, 6 mm, 14% Arel) [2b], the b-homoalanyl-PNA double strands are slightly less
stable. This is probably due to higher conformational freedom of the homologated
oligomers. In case of the linear alanyl-PNA G-C hexamers, the GGCGCC sequence
clearly defines the formation of an antiparallel double strand, whereas higher
aggregation is preferred for the GCGCGC hexamer. In contrast, the b-homoalanyl-
PNA oligomer 20 does not show higher aggregation. Since linear double strand
formation is a prerequisite for band-like aggregates in alanyl-PNA, a deviation from
the linearity is likely for b-homoalanyl-PNA. Helicalization would be in agreement
with the need to lower the base pair distance of ca. 4.5 ä in b-homoalanyl-PNA double
strands with linear topology.
With circular dichroism (CD) spectroscopy, it is possible to distinguish between the
pairing complex in which the chromophore (nucleobase) is conformationally fixed in a
chiral environment (backbone) and the statistical distribution in a single strand at
higher temperatures. The CDspectra of oligomer 19 (sequence GGCGCC) and

Helvetica Chimica Acta Vol. 85 (2002)
3861
oligomer 20 (sequence GCGCGC) distinctly show that the secondary structures at
lower temperatures are very similar (Fig. 2). Both oligomers seem to form double
strands with the same pairing mode and topology that differ from linearity because of
the huge Cotton effect. Furthermore, the decrease of the Cotton effect between 408 and
608 is in agreement with the results obtained with the temperature dependent UV
spectroscopy.
Fig. 2. CD Spectra of oligomers 19 (left, 10 mm in H2O) and 20 (right, 12 mm in H2O)
5. Conclusions. As the syntheses of the nucleo-b3-amino acids with adenine and 7-
carbaadenine were described in previous articles [4], now the preparation of the
remaining canonical nucleo-b3-amino acids has been presented. Next to the Mitsunobu
reaction for the attachment of the nucleobases to the g-position of b-homoalanine, the
nucleophilic substitution of a methanesulfonate was used in case of Z-protected
cytosine. For the preparation of the guaninyl-b3-amino acid, an efficient nucleo-b-
lactam route has been established that might also be applied for the syntheses of the
other nucleo-b3-amino acids. Finally, b-homoalanyl-PNA G-C hexamers were shown to
form quite stable WatsonÀCrick-pairing complexes with a double-strand topology that
is most likely helical.
This work was supported by the Deutsche Forschungsgemeinschaft. We are grateful for fellowships by the
Fonds der Chemischen Industrie (A. M. B.) and the Hermann-Schlosser-Stiftung (H. W. S.).
Experimental Part
General. All reagents were of anal. grade and used as supplied. Solvents were of the highest grade available.
HPLC: Pharmacia ækta basic with YMCJ×sphere ODS-H80, RP-C18 , 150 Â 10 mm, 4 mm, 80 ä for preparation
of the b-homoalanyl-PNAs, and 150 Â 4.6 mm, 4 mm, 80 ä for anal. samples of the b-homoalanyl-PNAs, and
Pharmacia Biotech Sephasil Peptide C18 ST, 250 Â 4.6 mm, 5 mm, 100 ä for ee determination. The oligomer
concentration was calculated by taking the extinction coefficient at 908 as being the sum of the extinction
coefficients of the nucleo-b3-amino acids. M.p.: Apparatus according to Dr. Tottoli, B¸chi 501. Optical rotation:
Perkin-Elmer 241 polarimeter. UV Melting curves: JASCO V-550 UV/VIS spectrophotometer with JASCO
ETC-505S/ETC-505T temp. controller. CDSpectra: JASCO J-500A spectropolarimeter with JASCO temp.
controller. IR: Perkin-Elmer 841. NMR Spectra: Bruker AC-250, Bruker AMX-500, Bruker DMX-500, Varian
Unity-300, or Varian INOVA-500 spectrometer. ESI-MS: LCQ Finnigan spectrometer.

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(S)-N-[(tert-Butoxy)carbonyl]-g-(3-benzoyluracil-1-yl)-b-homoalanine Benzyl Ester (5). DEAD (1.64 ml,
10.4 mmol) was added to a suspension of 3-benzoyluracil (1.80 g, 8.33 mmol) and Ph3P (2.73 g, 10.4 mmol) in
dry THF (21 ml) at À308 under Ar. The mixture was allowed to warm to À158 and added dropwise to a soln. of
(S)-N-Boc-b-homoserine benzyl ester (4, 1.25 g, 4.04 mmol) in dry THF, which had been precooled to À158.
After 4 d, the reaction was quenched with H2O (570 ml), and the mixture was concentrated. Purification by
repeated chromatography (silica gel; hexane/AcOEt 2 :3 and CHCl3/MeCN 9 :1) yielded 5 (1.44 g, 70%).
Colorless solid. M.p. 80 818. Rf (CHCl3/MeCN 9 :1) 0.30. [a]2D0 ˆ ‡86.0 (c ˆ 1.1, CHCl3). IR: 3401, 3093, 3069,
3040, 2983, 2936, 1752, 1710, 1665, 1599, 1513, 1445, 1390, 1365, 1345, 1239, 1169, 1115, 1090, 1053, 1029, 982, 904,
t
860, 802, 784, 763, 699, 629. 1H-NMR (CDCl3): 1.41 (br. s, Bu), 2.60 2.75 (m, 2 HÀC(a)); 3.50 3.66 (m,
HÀC(g)); 4.00 4.12 (m, HÀC(g)); 4.25 4.36 (m, HÀC(b)); 5.11 (s, PhCH2); 5.43 (d, 3J ˆ 8, BocNH); 5.78 (d,
3J ˆ 8, HÀC(5)); 7.20 (d, 3J ˆ 8, HÀC(6)); 7.25 7.40 (m, 5 arom. H); 7.46 (m, 2 arom. H); 7.61 (m, 1 arom. H);
8.08 (m, 2 arom. H). 13C-NMR (CDCl3): 28.4 (tBu), 36.0 (C(a)); 45.9 (C(b)); 52.4 (C(g)); 66.9 (PhCH2); 80.0
(tBu); 101.7 (C(5)); 128.3 (Ph); 128.5 (Ph); 128.7 (Ph); 129.0 (Ph); 130.8 (Ph); 131.6 (Ph); 134.9 (Ph); 135.2
(Ph); 144.4 (C(6)); 150.1 (C(2)); 155.5 (CˆO (Boc)); 162.4 (C(4)); 168.8 (PhCO); 171.0 (COOBzl). ESI-MS:
‡
‡
‡
1036.9 (89, [2 M ‡ Na] ), 1014.8 (100, [2 M ‡ H] ), 530.1 (89, [M ‡ Na] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-(uracil-1-yl)-b-homoalanine (1). Pd/C (751 mg, 30% Pd on charcoal
containing 53.1% H2O) was added to a soln. of 5 (753 mg, 1.48 mmol) in a mixture of dioxane (40 ml), H2O
(20 ml), and AcOH (2 ml). The mixture was stirred overnight under an atmosphere of H2 (1 bar). The catalyst
was filtered off and washed several times with MeOH. The filtrate and the washing solns. were pooled. After
removal of the solvents, the crude product was recrystallized from AcOEt/MeOH 4 :1 (10 ml) and as much H2O
as necessary to obtain a clear soln. under reflux. Additional material was obtained from the mother liquor.
Compound 1 (399 mg, 86%) was isolated as a colorless solid. M.p. 2038 (dec.). Rf (AcOEt/MeOH/H2O 80 :14 :6,
2% AcOH) 0.48. [a]2D0 ˆ ‡117.0 (c ˆ 1.0, DMSO). IR: 3466, 3449, 3194, 2984, 2609, 1721, 1694, 1651, 1493, 1474,
1410, 1395, 1369, 1350, 1298, 1284, 1248, 1212, 1168, 1117, 1060, 993, 890, 842, 815, 771, 717, 637. 1H-NMR
t
((D6)DMSO): 1.10 1.52 (br. s, Bu); 2.19 2.45 (m, 2 HÀC(a)); 3.11 3.50 (m, HÀC(g)); 3.78 4.00 (m,
3
3
3
HÀC(g)); 4.01 4.22 (m, HÀC(b)); 5.48 (d, J ˆ 7, H ÀC(5)); 6.39 (d, J ˆ 9, 0.13 H, BocNH); 6.72 (d, J ˆ 9,
0.87 H, BocNH); 7.32 (d, 3J ˆ 8, HÀC(6)); 11.11 (s, 0.8 H, HÀC(3)); 11.26 (s, 0.2 H, HÀC(3)); 12.20 (br. s,
COOH). 13C-NMR ((D6)DMSO): 27.7; 27.8; 28.1; 36.8; 37.2; 46.0; 51.5; 51.9; 77.9; 78.1; 100.2; 100.4; 145.8;
‡
‡
‡
146.1; 151.0; 155.0; 163.8; 171.8. ESI-MS: 627.1 (100, [2 M ‡ H] ), 649.1 (87, [2 M ‡ Na] ), 314.0 (28, [M ‡ H] ).
Anal. calc. for C13H19N3O6 (313.31): C 49.84, H 6.11, N 13.41; found: C 49.88, H 6.10, N 13.36.
(S)-N-[(tert-Butoxy)carbonyl]-g-(3-benzoylthymin-1-yl)-b-homoalanine Benzyl Ester (6) from 4.
3-Benzoylthymine (1.92 g, 8.33 mmol), Ph3P (2.73 g, 10.4 mmol), and DEAD (1.64 ml, 10.4 mmol) were added
to dry THF (20 ml) at À308 under Ar. After 10 min, the now clear soln. was added dropwise to a soln. of 4
(1.25 g, 4.04 mmol) in dry THF (10 ml) at À308 within 10 min. The mixture was stirred for 5 d at À108. After
removal of the solvent, purification was achieved by repeated chromatography (silica gel; CHCl3/MeOH 9 :1
and hexane/acetone 7:3) to give 6 (1.37 g, 65%). Colorless solid. M.p. 73 758. Rf (CHCl3/MeCN 9 :1) 0.45.
[a]2D0 ˆ ‡77.0 (c ˆ 0.9, CHCl3). IR: 3365, 3071, 3038, 2982, 2938, 2343, 1749, 1706, 1658, 1599, 1513, 1439, 1391,
1367, 1349, 1245, 1169, 1063, 1031, 984, 903, 874, 850, 820, 781, 764, 699, 685, 631. 1H-NMR (CDCl3): 1.39 (s, tBu);
1.89 (s, Me); 2.55 2.75 (m, 2 HÀC(a)), 3.46 3.70 (m, HÀC(g)), 3.96 4.13 (m, HÀC(g)); 4.18 4.40 (m,
HÀC(b)); 5.11 (s, PhCH2); 5.45 (d, 3J ˆ 9, BocNH); 7.04 (d, J ˆ 1, HÀC(6)); 7.26 7.39 (m, 5 arom. H); 7.40
7.55 (m, 2 arom. H); 7.56 7.69 (m, 1 arom. H); 7.98 8.13 (m, 2 arom. H). 13C-NMR (CDCl3): 12.3; 28.3; 30.9;
36.0; 46.0; 51.9; 66.9; 80.0; 110.2; 128.3; 128.5; 128.6; 129.0; 130.7; 131.6; 134.8; 135.2; 140.5; 150.1; 155.3; 163.1;
‡
169.1; 171.0. ESI-MS: 544.0 (100, [M ‡ Na] ).
Benzyl Ester 6 from (S)-N-[(tert-Butoxy)carbonyl]-g-(methanesulfonyl)-b-homoserine Benzyl Ester (8).
A
soln. of 8 (500 mg, 1.29 mmol), Bu4NI (143 mg, 387 mmol), K2CO3 (267 mg, 1.93 mmol), and 3-
benzoylthymine (327 mg, 1.42 mmol) in dry DMF (50 ml) was stirred under Ar at r.t. for 3 d. After quenching
the reaction with AcOH (221 ml, 3.86 mmol), the solvent was removed and the residue was subjected to
chromatography (silica gel; CHCl3/MeCN 9 :1). Compound 6 (442 mg, 66%) was isolated as a colorless solid.
The anal. data correspond to those obtained from 6 prepared via the Mitsunobu reaction.
(S)-N-[(tert-Butoxy)carbonyl]-g-(thymin-1-yl)-b-homoalanine (2). Pd/C (1.80 g, 30% Pd/C containing
53.1% H2O) was added to a soln. of 6 (1.18 g, 2.27 mmol) in a mixture of dioxane (60 ml), H2O (15 ml), and
AcOH (1.5 ml). The mixture was stirred overnight under an atmosphere of H2 (1 bar). The catalyst was filtered
off and washed several times with MeOH. The filtrate and the washing solns. were pooled. After removal of the
solvents and repeated co-evaporation with toluene, the crude product was purified by recrystallization from
AcOEt/MeOH 4 :1 and as much H2O as necessary to obtain a clear soln. under reflux. Compound 2 (638 mg,
86%) was isolated as a colorless solid. M.p. 2128 (dec.). Rf (AcOEt/MeOH/H2O 80 :14 :6, 2% AcOH) 0.54.

Helvetica Chimica Acta Vol. 85 (2002)
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[a]2D0 ˆ ‡97.0 (c ˆ 1.1, DMSO). IR: 3442, 3186, 3068, 3000, 2953, 1723, 1709, 1682, 1660, 1504, 1465, 1445, 1416,
1
1401, 1373, 1348, 1272, 1225, 1173, 1133, 1063, 1052, 1035, 924, 894, 853, 818, 788, 779, 761, 716, 618. H-NMR
((D6)DMSO): 1.05 1.39 (br. s, tBu); 1.70 (s, Me); 2.20 2.50 (m, 2 HÀC(a)); 3.10 3.51 (m, HÀC(g)); 3.73
3.97 (m, HÀC(g)); 3.99 4.21 (m, HÀC(b)); 6.32 (d, 3J ˆ 9, 0.15 H, BocNH); 6.74 (d, 3J ˆ 9, 0.85 H, BocNH);
7.23 (s, HÀC(6)); 11.11 (s, 0.87 H, HÀC(3)); 11.25 (s, 0.13 H, HÀC(3)); 12.00 12.55 (br. s, COOH). 13C-NMR
((D6)DMSO): 12.0; 27.8; 28.0; 36.7; 37.1; 46.0; 51.3; 77.8; 107.6; 141.9; 150.9; 155.0; 164.3; 171.9. ESI-MS: 350.0
‡
‡
(100, [M ‡ Na] ), 676.9 (48, [2 M ‡ Na] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-[N4-(benzyloxycarbonyl)cytosin-1-yl]-b-homoalanine Benzyl Ester (7).
A suspension of 8 (5.91 g, 15.3 mmol), N4-(benzyloxycarbonyl)cytosine (4.88 g, 19.9 mmol), and K2CO3 (3.39 g,
24.5 mmol) in dry DMF (100 ml) was stirred at r.t. for 14 d under N2. The solvent was removed, and the residue
was extracted with AcOEt and acetone. After evaporation of the solvents and purification by chromatography
(silica gel: AcOEt/hexane 3 :1 to AcOEt), 7 (1.85 g, 23%) was isolated as a colorless solid. M.p. 1628. Rf
(AcOEt/hexane 3 :1) 0.29. [a]2D0 ˆ ‡74.4 (c ˆ 0.25, MeOH). IR: 3367, 2984, 2361, 1743, 1729, 1684, 1627, 1558,
t
1516, 1369, 1344, 1223, 1167, 1055, 787. 1H-NMR (CDCl3): 1.36 (s, Bu), 2.58 (br. m, HÀC(a)); 2.86 (br. m,
HÀC(a)); 4.07 (br. m, 2 HÀC(g)); 4.23 (br. m, HÀC(b)); 5.13 (s, PhCH2); 5.21 (s, PhCH2); 5.51 (br. m,
HÀC(5)); 7.25 7.45 (m, 10 arom. H), 7.49 7.62 (br. m, HÀC(6)). 13C-NMR (CDCl3): 28.2; 36.2; 47.3; 52.4;
66.7; 67.7; 80.5; 95.0; 127.9; 128.1; 128.3; 128.4; 128.5; 128.6; 135.0; 149.2; 152.5; 155.3; 162.6; 170.9. ESI-MS:
‡
‡
559.5 (75, [M ‡ Na] ), 1095.4 (100, [2 M ‡ Na] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-[N4-(benzyloxycarbonyl)cytosin-1-yl]-b-homoalanine (9). A soln. of 7
(1.68 g, 3.13 mmol) in a mixture of dioxane (15 ml), H2O (10 ml), and 1m aq. NaOH (5 ml) was stirred at r.t. for
9 h. The mixture was then neutralized with 1m aq. HCl, concentrated, and freeze-dried. The residue was
subjected to chromatography (RP-C18 silica gel; H2O to H2O/MeOH 4 :6) to yield 9 (1.15 g, 82%). Colorless
solid. M.p. 172 1828 (dec.). Rf (AcOEt/MeOH/H2O 80 :14 :6, 3% AcOH) 0.55. [a]2D0 ˆ ‡94.0 (c ˆ 0.25,
MeOH). IR: 3410, 2977, 1750, 1653, 1574, 1504, 1370, 1213, 1053, 789, 746, 697. 1H-NMR ((D6)DMSO): 1.25 (s,
tBu); 1.97 2.13 (m, 2 HÀC(a)); 3.56 3.66 (m, HÀC(g)); 3.85 4.00 (m, 2 HÀC(b), HÀC(g)); 5.16 (s,
3
3
PhCH2); 6.86 (d, J ˆ 7, H ÀC(5)); 7.26 7.40 (m, 5 arom. H); 7.81 (d, J ˆ 7, H ÀC(6)); 10.57 (br. s, COOH).
13C-NMR ((D6)DMSO): 28.1 (tBu); C(a) signal probably overlaps with the solvent signal; 46.5 (C(b)); 53.6
(C(g)); 66.3 (PhCH2); 77.5 (tBu); 93.7 (C(5)); 127.8 128.4 (Ph); 136.0 (Ph); 150.0 (C(6)); 153.3; 154.8; 162.8;
‡
‡
174.5 (COOH). ESI-MS: 469.6 (100, [M ‡ Na] ), 915.4 (56, [2 M ‡ Na] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-(uracil-1-yl)-b-homoalanine Benzyl Ester (11). Compound 1 (1.00 g,
3.19 mmol) was dissolved in a mixture of DMF (5 ml) and BzlOH (5 ml). DMAP (195 mg, 1.60 mmol) and N-
[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride (EDCI ¥ HCl, 672 mg, 3.51 mmol) were added
at 08, and the mixture was stirred at r.t. for 16 h. After removal of the solvents, the residue was purified by
repeated chromatography (silica gel; CHCl3/EtOH 95 :5) to give 11 (1.11 g, 86%). Colorless solid. M.p. 148
1498. Rf (CHCl3/EtOH 95 :5) 0.24. [a]2D0 ˆ ‡113.0 (c ˆ 0.9, DMSO). IR: 3370, 3058, 2375, 2001, 1679, 1522, 1453,
1418, 1390, 1368, 1345, 1275, 1244, 1167, 1112, 1056, 982, 848, 804, 765, 697, 622. 1H-NMR (CDCl3): 1.36 (br. s,
tBu); 2.55 2.82 (m, 2 HÀC(a)); 3.70 3.87 (m, HÀC(g)); 3.88 4.00 (m, HÀC(g)); 4.13 4.30 (m, HÀC(b));
5.12 (s, PhCH2); 5.38 (d, 3J ˆ 8, BocNH); 5.61 (d, 3J ˆ 8, HÀC(5)); 7.11 (d, 3J ˆ 8, HÀC(6)): 7.27 7.41 (m,
5 arom. H); 9.16 (br. s, HÀC(3)). 13C-NMR (CDCl3): 28.2 (tBu), 36.1 (C(a)); 46.9 (C(b)); 51.0 (C(g)); 66.8
(PhCH2); 80.1 (tBu); 102.1 (C(5)); 128.3 (Ph); 128.5 (Ph); 128.6 (Ph); 135.4 (Ph); 144.7 (C(6)); 151.3 (C(2));
‡
155.3 (BocCO); 163.5 (C(4)); 170.9 (COOBzl). ESI-MS: 426.1 (100, [M ‡ Na] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-[4-(3'-nitro-1',2',4'-triazol-1'-yl)-2-oxo-1H-pyrimidin-1-yl]-b-homoala-
nine Benzyl Ester (12). A soln. of 11 (894 mg, 2.22 mmol) in dry pyridine (14 ml) was cooled to 08. 4-
Chlorophenyl dichlorophosphate (434 ml, 2.66 mmol) was added dropwise, followed by a soln. of 3-nitro-1,2,4-
1H-triazole (760 mg, 5.84 mmol) in dry pyridine (11 ml). After stirring for 16 h at r.t., the solvent was removed
by co-evaporation with toluene, and the residue was purified by chromatography (silica gel; CHCl3/EtOH 95 :5)
to give 12 (1.03 g, 93%). Yellow solid. M.p. 176 1778. Rf (CHCl3/EtOH 95 :5) 0.40. [a]2D0 ˆ ‡163.0 (c ˆ 1.0,
DMSO). IR: 3567, 3374, 3096, 2986, 1736, 1680, 1634, 1561, 1514, 1470, 1426, 1403, 1367, 1348, 1302, 1246, 1163,
t
1116, 1059, 1001, 939, 835, 786, 751, 697, 655. 1H-NMR (CDCl3): 1.31 (br. s, Bu), 2.70 (dd, 1J ˆ 17, 3J ˆ 5,
HÀC(a)); 2.84 (dd, 1J ˆ 17, 3J ˆ 5, HÀC(a)); 3.91 4.10 (m, HÀC(g)); 4.22 4.42 (m, HÀC(b), HÀC(g)); 5.14
(s, PhCH2); 5.40 (d, 3J ˆ 8, BocNH); 6.99 (d, 3J ˆ 7, H ÀC(5)); 7.26 7.42 (m, 5 arom. H); 7.93 (d, 3J ˆ 7,
HÀC(6)); 9.29 (s, HÀC(5')). 13C-NMR (CDCl3): 28.1; 36.2; 46.6; 54.0; 67.0; 80.4; 93.9; 95.8; 128.4; 128.6; 128.7;
‡
‡
135.2; 144.6; 152.6; 154.7; 155.3; 158.5; 170.8. ESI-MS: 998.7 (100, [2 M ‡ H] ), 522.0 (21, [M ‡ Na] ), 499.7 (16,
‡
[M ‡ H] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-(cytosin-1-yl)-b-homoalanine Benzyl Ester (13). Aq. NH3 (39 ml) was
added to a soln. of 12 (984 mg, 1.96 mmol) in dioxane (78 ml). After stirring for 1 h at r.t., the mixture was

3864
Helvetica Chimica Acta Vol. 85 (2002)
concentrated, and the residue was purified by repeated chromatography (silica gel; CHCl3/MeOH 4 :1).
Compound 13 (761 mg, 96%) was isolated as a colorless solid. M.p. 94 978. Rf (CHCl3/MeOH 4 :1) 0.88. [a]D20
ˆ
‡95.0 (c ˆ 0.9, DMSO). IR: 3346, 2982, 2001, 1699, 1649, 1522, 1494, 1455, 1438, 1392, 1367, 1282, 1170, 1053,
1027, 979, 912, 855, 789, 751, 697, 676, 619. 1H-NMR (CDCl3): 1.36 (s, tBu), 2.48 2.60 (m, HÀC(a)); 2.78 2.92
(m, HÀC(a)); 3.89 4.07 (m, 2 HÀC(g)); 4.12 4.27 (m, HÀC(b)); 5.11 (s, PhCH2); 5.30 6.60 (br. s, NH2);
5.63 (d, 3J ˆ 7, H ÀC(5)); 5.67 (d, 3J ˆ 7, BocNH); 7.21 (d, 3J ˆ 7, H ÀC(6)), 7.28 7.41 (m, 5 arom. H). 13C-NMR
(CDCl3): 28.3; 36.4; 48.3; 51.8; 66.7; 79.7; 94.0; 105.3; 128.4; 128.6; 135.6; 146.4; 155.5; 157.0; 165.6; 171.0. ESI-
‡
‡
MS: 805.0 (100, [2 M ‡ H] ), 403.0 (37, [M ‡ H] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-(cytosin-1-yl)-b-homoalanine (10) from 13. A soln. of NaOH (100 mg,
2.50 mmol) in H2O (5.5 ml) was added to a soln. of 13 (586 mg, 1.46 mmol) in dioxane (5 ml). After being
stirred overnight at 308, the mixture was acidified with 1m aq. HCl (pH 2 3), and the precipitating crystalline 10
was separated. The mother liquor was concentrated, and additional product was isolated after chromatography
(RP-C18 silica gel, first H2O then H2O/MeOH with increasing amounts of MeOH). A total of 385 mg of 10
(85%) was obtained as a colorless solid. M.p. 211 2138 (dec.). Rf (AcOEt/MeOH/H2O 80 :14 :6, 3% AcOH)
0.39. ee 96% (determined by HPLC of the dimer prepared with Boc-(S)-Leu-OSu on RP-C18; 10 30% B (B ˆ
MeCN ‡ 0.1% TFA) in 30 min, tR(like) 22.8 min; tR(unlike) 21.4 min). [a]2D0 ˆ ‡120.3 (c ˆ 0.8, DMSO). IR:
3414, 3079, 2986, 2924, 2749, 1959, 1692, 1650, 1559, 1486, 1447, 1419, 1397, 1352, 1308, 1286, 1274, 1220, 1168,
1108, 1059, 1018, 1002, 900, 850, 806, 768, 715, 611. 1H-NMR ((D6)DMSO): 1.19 1.40 (br. s, tBu); 2.23 2.41 (m,
2 HÀC(a)); 3.35 3.51 (m, HÀC(g)); 3.82 3.95 (m, HÀC(g)); 3.98 4.15 (m, HÀC(b)); 5.60 (d, 3J ˆ 7,
HÀC(5)); 6.12 6.31 (br. s, 0.2 H, BocNH); 6.65 (d, 3J ˆ 7, 0.8 H, BocNH); 6.72 7.12 (br. s, NH2); 7.33 (d, 3J ˆ 7,
HÀC(6)). 13C-NMR ((D6)DMSO): 28.1; 37.0; 46.8; 51.7; 77.7; 92.8; 146.0; 155.7; 165.8; 171.8. ESI-MS: 313.0 (98,
‡
‡
[M ‡ H] ), 625.0 (100, [2 M ‡ H] ).
Compound 10 from 9. A soln. of NaOH (145 mg, 3.63 mmol) in H2O (4 ml) was added to a soln. of 9
(50.0 mg, 112 mmol) in dioxane (3 ml). After being stirred for 2 d at 508, the mixture was acidified with 1m aq.
HCl (pH 2 3) and concentrated. The product 10 (32.2 mg, 92%) was isolated after chromatography (RP-C18
silica gel; first H2O then H2O/MeOH with increasing amounts of MeOH) as a colorless solid. The anal. data
correspond to those of 10 obtained from 13 as described above.
(S)-N-[(tert-Butoxy)carbonyl]-g-(2-amino-6-chloropurin-9-yl)-b-homoalanine Benzyl Ester (14). Ph3P
(2.11 g, 8.04 mmol) was dissolved in dry MeCN (30 ml) under N2, and the soln. was cooled to À458. DEAD
(1.47 ml, 9.34 mmol) was added within 20 min, and the soln. was allowed to warm to À308. A À208 cold
suspension of 4 (1.38 g, 4.46 mmol) and 2-amino-6-chloropurine (1.14 g, 6.72 mmol) in a mixture of dry THF
(30 ml) and dry MeCN (10 ml) was added within 30 min. After stirring for 2 h at À208, the soln. was warmed to
08, and stirring was continued for 11 d. The solvents were removed, and the residue was taken up in CHCl3 and
centrifuged. After concentration of the supernatent suspension and precipitation of Ph3PO in cold Et2O,
purification by repeated chromatography (silica gel; CHCl3/MeOH 95 :5 and CHCl3/MeCN 3 :1) afforded 14
(707 mg, 34%). Yellowish solid. M.p. 79 808. Rf (CHCl3/EtOH 95 :5) 0.32. [a]2D0 ˆ ‡23.0 (c ˆ 1.0, CHCl3). IR:
3392, 2983, 1716, 1616, 1565, 1518, 1464, 1409, 1394, 1368, 1311, 1283, 1253, 1163, 1055, 1001, 913, 786, 752, 698,
642. 1H-NMR (CDCl3): 1.18 1.55 (br. s, tBu); 2.58 (dd, J ˆ 17, 6, HÀC(a)); 2.67 (dd, J ˆ 15, 4, HÀC(a)); 4.10
4.42 (br. m, HÀC(b), HÀC(g)); 4.97 5.10 (br. s, NH2); 5.10 5.16 (m, PhCH2); 5.34 5.51 (m, BocNH); 7.30
7.41 (m, 5 arom. H); 7.65 (s, HÀC(8)). 13C-NMR (CDCl3): 28.2; 36.1; 46.0; 47.7; 66.9; 80.2; 125.2; 128.4; 128.6;
‡
128.7; 135.3; 142.8; 151.5; 154.1; 155.0; 159.1; 170.7. ESI-MS: 461.0 (100, [M ‡ H] ).
(S)-N-[(tert-Butoxy)carbonyl]-g-(guanin-9-yl)-b-homoalanine (15) from 14. A soln. of 14 (1.00 g,
2.17 mmol) in a mixture of dioxane (11 ml) and 1m aq. NaOH (22 ml) was stirred at 508 for 20 h. The mixture
was then neutralized with 1m aq. HCl and concentrated. The residue was subjected to chromatography (silica
gel; AcOEt/MeOH/H2O 80 :14 :6, 3% AcOH; after removal of (S)-N-[tert-butoxy)carbonyl]-g-(O6-benzyl-
guanin-9-yl)-b-homoalanine (16): THF/MeOH/H2O 80 :14 :6, 3% AcOH). Desalting was achieved by
chromatography (RP-C18 silica gel; first H2O then H2O/MeOH 9 :1). Compounds 16 (184 mg, 19%) and 15
(453 mg, 59%) were isolated as colorless solids.
Data of 16: M.p. 187 1908. Rf (AcOEt/MeOH/H2O 80 :14 :6, 3% AcOH) 0.70. [a]2D0 ˆ ‡54.0 (c ˆ 0.8,
DMSO). IR: 3368, 2985, 2001, 1687, 1620, 1588, 1525, 1455, 1414, 1361, 1336, 1251, 1162, 1070, 1028, 911, 848,
789, 753, 700, 639. 1H-NMR ((D6)DMSO): 0.90 1.10 (br. s, 1.4 H, tBu); 1.12 1.55 (br. s, 7.6 H, tBu); 2.26 2.45
(m, 2 HÀC(a)); 3.90 4.31 (m, HÀC(b), 2 HÀC(g)); 5.39 5.62 (m, PhCH2); 6.40 6.52 (br. s, NH2); 6.87 (d,
3J ˆ 8, BocNH); 7.28 7.43 (m, 3 arom. H); 7.44 7.58 (m, 2 arom. H); 7.63 (s, HÀC(8)). 13C-NMR
((D6)DMSO): 27.5; 28.0; 37.0; 46.0; 47.3; 66.7; 77.8; 90.6; 113.6; 128.0; 128.3; 136.7; 140.0; 154.6; 154.7; 159.6;
‡
159.9; 171.9. ESI-MS: 443.1 (85, [M ‡ H] ).

Helvetica Chimica Acta Vol. 85 (2002)
3865
Data of 15: M.p. 2348 (dec.). Rf (AcOEt/MeOH/H2O 80 :14 :6, 3% AcOH) 0.32. [a]2D0 ˆ ‡45.0 (c ˆ 1.2,
DMSO). IR: 3422, 3129, 2980, 1688, 1575, 1536, 1484, 1410, 1366, 1251, 1165, 1054, 1027, 850, 784, 691, 638.
t
t
1H-NMR ((D6)DMSO): 0.99-1.15 (br. s, 1.5 H, Bu); 1.16 1.32 (br. s, 7.5 H, Bu); 2.12 2.32 (m, 2 HÀC(a));
3.89 4.10 (m, HÀC(b), 2 HÀC(g)); 6.50 (s, 0.15 H, BocNH); 6.65 6.79 (br. s, NH2); 6.80 6.90 (m, 0.85 H,
BocNH); 7.50 (s, HÀC(8)). 13C-NMR ((D6)DMSO): 27.5; 28.0; 46.1; 47.8; 77.5; 116.3; 137.6; 151.4; 153.7; 154.6;
‡
‡
157.1; 175.6. ESI-MS: 353.1 (100, [M ‡ H] ), 705.0 (39, [2 M ‡ H] ).
(S)-4-[(2'-Amino-6'-chloropurin-9'-yl)methyl]azetidin-2-one (18). NaH (1.74 g suspension in mineral oil,
43.5 mmol) was added to a suspension of 2-amino-6-chloropurine (6.82 g, 40.2 mmol) in dry DMF (100 ml)
under Ar. After H2 evolution had ceased, the mixture was heated to 508, and a soln. of 17 (6.00 g, 33.5 mmol) in
dry DMF (50 ml) was added. After stirring the suspension at 858 for 4 h, the solvent was removed, and the
residue was thoroughly washed with THF/MeOH 9 :1 (filtration through Celite). The soluble material was
absorbed on silica gel and subjected to chromatography (silica gel; THF/CH2Cl2 4 :1). Evaporation of the
solvents yielded 18 (3.31 g, 39%). Cream-colored solid. Rf (THF/CH2Cl2 4 :1) 0.24. [a]2D0 ˆ À6.1 (c ˆ 0.25,
DMSO). IR: 3319, 3200, 1752, 1650, 1615, 1560, 1525, 1477, 1386, 1346, 1310, 1166, 997, 907, 782, 733. 1H-NMR
((D6)DMSO): 2.69 (m, HÀC(3)); 2.97 (m, HÀC(3)); 3.93 (m, HÀC(4)); 4.22 (m, CH2); 6.92 (s, NH2); 8.07 (s,
NH); 8.11 (s, HÀC(8')). 13C-NMR ((D6)DMSO): 41.4 (C(3)); 45.4 (C(4)); 47.3 (CH2); 123.4 (C(5')); 143.4
‡
(C(8')); 149.6 (C(6')); 154.4 (C(4')); 160.0 (C(2')); 166.6 (CˆO). ESI-MS: 294 (100, [M ‡ H ‡ MeCN] ), 253
‡
(38, [M ‡ H] ).
Compound 15 from 18. Compound 18 (3.10 g, 12.3 mmol) was dissolved in 1m aq. HCl (600 ml). The soln.
was refluxed for 9 h and then evaporated to dryness. A stirred suspension of the residue in H2O/1m aq. NaOH/
dioxane 1 :1:2 (80 ml) was treated portionwise with Boc2O (7.12 g, 32.6 mmol) within 5 d. pH 9 was maintained
by repeated addition of 1m aq. NaOH. The resultant mixture was washed with Et2O (2 Â 50 ml) and
concentrated in vacuo. Purification of the residue by chromatography (RP-C18 silica gel, first H2O then H2O/
MeOH 9 :1) provided, after freeze drying, 15 (3.00 g, 69%) as a colorless solid. The anal. data correspond to the
those of 15 obtained from 14 as described above except for [a]2D0 ˆ ‡54.9 (c ˆ 0.25, DMSO). ee > 99%
(determined by HPLC of the dimers prepared with Boc-(S)-Ala-OSu and Boc-(R)-Ala-OSu on RP-C18; 7.8
14.4% B (B ˆ MeCN/H2O 9 :1 ‡ 0.1% TFA) in 30 min, tR(like) 21.8 min; tR(unlike) 19.2 min).
General Procedure for SPPS of b-Homoalanyl-PNA. Oligomerization was performed as a manual solid-
phase peptide synthesis on a 4-methylbenzhydrylamine(MBHA)-polystyrene resin loaded with d-lysine(Z)-
OH (39.4 mg, 22.4 mmol lysine amide). For the first step of the double coupling, an excess of 5 equiv. N-Boc-
protected nucleo-b3-amino acid (112 mmol) was used and activated by O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU, 38.4 mg, 101 mmol), 1-hydroxy-7-aza-1H-benzotriazole
(HOAt, 15.2 mg, 112 mmol), and (ethyl)diisopropylamine (DIEA, 39.1 ml, 224 mmol) in DMF (500 ml). For the
second step of the double coupling, an excess of 3 equiv. N-Boc-protected nucleo-b3-amino acid (67.2 mmol) was
used and activated by HATU (23.0 mg, 60.5 mmol), HOAt (9.2 mg, 68 mmol), and DIEA (23.5 ml, 134 mmol) in
DMF (500 ml). After swelling the loaded resin for 1 h in 2 ml CH2Cl2, the following procedure was repeated for
every nucleo-b3-amino acid unit: 1) deprotection twice, for 3 min with TFA/m-cresol 95 :5 (2 ml); 2) washing
first 5Â with CH2Cl2/DMF 1 :1 (2 ml) and then 5Â with pyridine (2 ml); 3) double coupling steps, each 1 h
gentle moving in a small column; 4) washing with CH2Cl2/DMF 1:1 (3 Â 2 ml), DMF/piperidine 95 :5 (3 Â
2 ml), and CH2Cl2/DMF 1:1 (3 Â 2 ml). The resin was washed with TFA (3 Â 2 ml) and CH2Cl2 (5 Â 2 ml),
dried overnight in vacuo, suspended in dimethyl sulfide (600 ml)/m-cresol (200 ml), and cooled to À208. TFA
(1 ml) was added, followed by trifluoromethanesulfonic acid (200 ml) after 10 min. The mixture was warmed to
r.t. within 1.5 h, and stirring was continued for 1.5 h. The filtrate was concentrated by freeze drying, and the b-
homoalanyl-PNA precipitated with Et2O (30 ml at À108) as a white solid and was purified by HPLC (RP-C18).
The yield of each coupling step was estimated from HPLC to be higher than 97%.
H-b-HalG-b-HalG-b-HalC-b-HalG-b-HalC-b-HalC-Lys-NH2 (19): Anal. HPLC: 15.1 min, RP-C18, gra-
dient: 4 12% B (B ˆ MeCN/H2O 9 :1 ‡ 0.1% TFA) in 16 min. ESI-MS: 716.4 (92%, [M ‡ 2 H]2‡), 728.2
‡
‡
(100%, [M ‡ H ‡ Na]2‡), 738.9 (86%, [M ‡ 2 Na]2‡), 1430.7 (16%, [M ‡ H] ), 1452.7 (20%, [M ‡ Na] ).
H-b-HalG-b-HalC-b-HalG-b-HalC-b-HalG-b-HalC-Lys-NH2 (20): Anal. HPLC: 18.6 min, RP-C18, gra-
dient: 4 15% B (B ˆ MeCN/H2O 9 :1 ‡ 0.1% TFA) in 33 min. ESI-MS: 716.2 (100%, [M ‡ 2 H]2‡), 727.1
(31%, [M ‡ H ‡ Na]2‡), 738.2 (20%, [M ‡ 2 Na]2‡), 1430.5 (6%, [M ‡ H] ).
‡

3866
Helvetica Chimica Acta Vol. 85 (2002)
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Received June 10, 2002