Synthesis of amino acids with unnatural nucleobases or chromophores suitable for use in model electron-transfer studies

Article abstract of DOI:10.1002/ejoc.200300242

We describe the syntheses of Boc-protected amino acids bearing the chromophores pyromellitic diimide, naphthalene diimide, and pyrene in their side chains, as well as nucleo amino acids with the artificial purines 7-carbaguanine, 7-carbaadenine, isoadenine, and 7-carbaisoadenine covalently linked to the alanyl side chain. These amino acids incorporating chromophores are suitable building blocks for incorporation into alanyl-PNA for future investigations of the mechanism of nucleobase-mediated electron transfer. ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

Full text of DOI:10.1002/ejoc.200300242

FULL PAPER
Synthesis of Amino Acids with Unnatural Nucleobases or Chromophores
Suitable for Use in Model Electron-Transfer Studies
Thomas Wagner,[a] William B. Davis,[b] Katrin B. Lorenz,[a] Maria E. Michel-Beyerle,[b] and
Ulf Diederichsen*[a]
Keywords: Amino acids / Chromophores / Electron transfer / Peptide nucleic acids / Purines / Solid-phase synthesis
We describe the syntheses of Boc-protected amino acids
bearing the chromophores pyromellitic diimide, naphthalene
diimide, and pyrene in their side chains, as well as nucleo
amino acids with the artificial purines 7-carbaguanine, 7-car-
baadenine, isoadenine, and 7-carbaisoadenine covalently
ing chromophores are suitable building blocks for incorpora-
tion into alanyl-PNA for future investigations of the mech-
anism of nucleobase-mediated electron transfer.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
linked to the alanyl side chain. These amino acids incorporat- Germany, 2003)
Introduction
Electron transfer and hole transfer mediated by the nu-
cleobase stack of a DNA double strand has developed to a
lively research area during the last decade.[1] Mechanistic
details, however, regarding electron transfer, hole transfer,
and charge migration still need to be evaluated.[2] An exper-
imental drawback for electron transfer studies in DNA
double strands results from the helix topology. A DNA du-
plex may unwind or change its conformation when intercal-
ators are bound, or when constitutional changes are re-
quired to create an excess charge, electron or hole within
the base stack. Therefore, it is quite difficult to predict the
base-pair orientation within a perturbed DNA base stack.
In this context, we have introduced alanyl peptide nucleic
acids (alanyl-PNA) as model systems for DNA base stacks
(Scheme 1).[3] An alanyl-PNA single strand is based on a
regular peptide backbone having an alternating configura-
tion of alanyl amino acids. Nucleobases are covalently
bound to the alanyl side chains. These artificial oligomers
form linear double strands with high rigidity and very well
defined base-pair orientations. The linearity of the double
strands allows the ability to specifically generate unnatural
base pairs and different pairing modes.[4] Furthermore, the
orientation of the base-pair stacking can be varied by side-
chain homology, whereas the distance between the base
pairs can by influenced by the backbone homology.[5] The
base stack of alanyl-PNA can be addressed by intercalating
Scheme 1. Linear alanyl peptide nucleic acid double strand as a
model system for a DNA nucleobase stack
chromophores[6] or by chromophores covalently linked to
the amino acid side chains.[7] In both cases, a site lacking a
base pair is required, since alanyl-PNA is already linear and
cannot unwind any further. In contrast to DNA, contact
between a chromophore and the nucleobase stack in alanyl-
PNA proceeds regioselectively without conformational re-
organization. This feature makes alanyl-PNA especially
valuable as a DNA base-stack model for investigating elec-
tron transfer. Alanyl-PNA donor and acceptor chromoph-
ores having various oxidation potentials are needed to per-
form electron-transfer studies with this model system. Fur-
thermore, there is interest in incorporating modified purines
next to the canonical nucleobases to allow mediation of the
redox potential within the alanyl-PNA base stacks.
[a]
Institut für Organische Chemie, Universität Göttingen,
Tammannstr. 2, 37077 Göttingen, Germany
Fax: (internat.) ϩ49-(0)551-392944
E-mail: udieder@gwdg.de
Synthesis of Alanyl Amino Acids with Covalently Bound
Chromophores
For initial electron-transfer studies in alanyl-PNA, the
chromophores 9-amino-6-chloro-2-methoxyacridine and
[b]
Institut für Physikalische und Theoretische Chemie, Technische
Universität München,
Lichtenbergstr. 4, 85748 Garching, Germany
Eur. J. Org. Chem. 2003, 3673Ϫ3679
DOI: 10.1002/ejoc.200300242
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. Wagner, W. B. Davis, K. B. Lorenz, M. E. Michel-Beyerle, U. Diederichsen
FULL PAPER
Scheme 2. Synthesis of amino acids having naphthalene diimide and pyromellitic diimide chromophores starting from Boc-ornithine
ethidium were covalently linked to the alanyl side chain[7] both cytosine and thymine within a nucleobase stack. Initial
and incorporated into alanyl-PNA double strands. In the attempts to synthesize amino acid 6 by amide-bond forma-
acridine-modified alanyl-PNA oligomers, there is clear evi- tion between N-Boc-aminoalanine (7) and pyreneacetic acid
dence for electron transfer with a distance dependency simi- (10) in DMF were hampered because of the poor solubility
lar to that found in DNA double strands.[8]
and aggregation of the respective amino acid derivatives.[10]
The amino acid with a naphthalene diimide chromophore Therefore, we decided to link the amino acid to a trityl-
1 and amino acid 2 carrying a pyromellitic diimide dye chloro-polystyrene (TCP) solid support prior to the intro-
(Scheme 2) were synthesized to function as electron traps in duction of the pyrene residue.
PNA, since their reduction potentials are lower than any of
the native nucleobases. In addition, both 1 and 2 are ex-
Nα-Boc-aminoalanine (7) was Fmoc protected at the side
pected to undergo photoexcited hole-transfer reactions with chain to allow binding of the amino acid 8[11] as an ester to
guanine and adenine.[9] The synthesis of amino acid 1 was a TCP-Cl resin. After Fmoc deprotection, amide coupling
undertaken by simultaneous reaction of Nα-tert-butoxycar- of the resin-bound aminoalanine 9 to pyreneacetic acid 10
bonyl (Boc)-protected ornithine (3) and n-butylamine with was effected using O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetra-
naphthalenetetracarboxylic bisanhydride (4). Derivative 2 methyluronium tetrafluoroborate (TBTU) as the coupling
was obtained in an analogous manner by reaction of pyro- reagent, 1-hydroxybenzotriazole (HOBt) as the coupling
mellitic dianhydride (5) with Boc-Orn-OH and n-butylam- auxiliary, N,N-diisopropylethylamine (DIPEA) as the base,
ine.
and N-methyl-2-pyrrolidone (NMP) as the solvent. For
We are also interested in the pyrene derivative 6 purification, it was sufficient merely to wash the resin-
(Scheme 3) since it should serve as an electron donor for bound derivative 11. The desired amino acid 6 was obtained
Scheme 3. Solid-supported synthesis of a Boc-protected pyrene-appended amino acid
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Eur. J. Org. Chem. 2003, 3673Ϫ3679

Amino Acids with Unnatural Nucleobases or Chromophores
FULL PAPER
after acidic cleavage from the solid support, followed by ceeded only with modest yield (Scheme 4). The synthesis of
evaporation of the solvent.
the carbaguaninylalanine 12 was completed by acidic sub-
stitution of the purinyl chlorine atom in nucleo amino acid
17, followed by selective Boc-reprotection of the α-amino
group.
Synthesis of Alanyl Nucleo Amino Acids having Modified
Nucleobases
6-Chloro7-carbapurine (16) turned out to be a good nu-
cleophile for serine lactone 14 to yield the desired nucleo
amino acid 18 (Scheme 5). Purine modification by exchange
of the chlorine atom into an amino group was performed
in three steps — esterification, introduction of the azide,
and subsequent one-pot reduction and saponification — to
yield the carbaadeninyl nucleo amino acid 13. Without es-
terification, reduction of the azide was unsuccessful. Purinyl
azides are known to exist in a solvent-dependent equilib-
rium between the azide and the tetrazole forms.[16] In the
case of the carbapurinyl derivative having a free carboxylic
acid functionality, only the azide form was detected by
NMR spectroscopy in [D6]DMSO. Therefore, it seems likely
that only the tetrazole form 21 was reduced under the hy-
drogenolytic conditions. Esterification allowed the use of a
less polar solvent mixture, which shifts the equilibrium be-
tween azide 20 and tetrazole 21 (3:97 in CDCl3) and, conse-
quently, facilitates the reduction.
7-Carbaguanine and 7-carbaadenine are better electron
donors than the respective guanine and adenine nu-
cleobases.[12] Since regular hydrogen bonding in the
WatsonϪCrick mode is still possible with the 7-deaza de-
rivatives, both these nucleobases can be incorporated into
double strands without changing the overall pairing ge-
ometry. For incorporation in alanyl-PNA, the Boc-pro-
tected nucleo amino acids 12 (Scheme 4) and 13 (Scheme 5)
were required. Stereoselective generation of Boc-alanyl nu-
cleo amino acids usually is performed by nucleophilic ring
opening of Boc-serine lactone (14) with the respective nu-
cleobase.[13] Since both 7-carbaguanine and 7-carbaadenine
turned out to be unsuitable nucleophiles, the 6-chloropurine
derivatives 15 and 16 were used instead, with subsequent
steps needed to generate the desired carboxyl and amino
groups. 2-Amino-7-carba-6-chloropurine (15) and 7-carba-
6-chloropurine (16) were prepared by analogy to the pro-
cedures of Davoll[14] and Seela[15]
The ring opening of serine lactone 14 was quite difficult,
.
Finally, the isoadeninyl nucleo amino acid 22 was ob-
even with 2-amino-6-chloro-7-carbapurine (15), and suc- tained by catalytic hydrogenolysis of the 6-chloro-2-amino-
Scheme 4. Synthesis of the N7-carbaguaninyl nucleo amino acid 12 by ring opening of Boc-serine lactone
Scheme 5. Synthesis of N7-carbaadeninyl nucleo amino acid
Eur. J. Org. Chem. 2003, 3673Ϫ3679
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T. Wagner, W. B. Davis, K. B. Lorenz, M. E. Michel-Beyerle, U. Diederichsen
FULL PAPER
purinyl derivative 23[17] (Scheme 6), which is an intermedi-
ate in the guaninyl nucleo amino acid synthesis.[3b] In an
analogous procedure, the corresponding 7-carbapurinyl de-
rivative 17 was converted into the 7-carbaisoadeninyl nu-
cleo amino acid 24.[18] For this reaction to proceed well, it
was crucial to avoid further reduction, which happens read-
ily at increased temperatures or with higher amounts of
catalyst.
PerkinϪElmer 241 MC polarimeter was used for optical rotation
measurements. A Büchi SMP-20 apparatus was used for melting
point determination. HPLC was performed using either a Beckman
System Gold or Pharmacia Äkta Basic using YMC-Pack ODS,
˚
RP-C18 columns for preparative runs (250 ϫ 20 mm, 5 µm, 120 A)
˚
and for analytical samples (250 ϫ 4.6 mm, 5 µm, 120 A). The op-
tical purity was determined by HPLC analysis of the amino acid
dimers obtained with (S)-Boc-Ala-OSu.
1,8:4,5-Naphthalenetetracarboxydiimide Derivative 1: n-Butylamine
(213 µL, 2.15 mmol) was added dropwise under argon to a solution
of naphthalene-1,4,5,8-tetracarboxylic dianhydride (4; 500 mg,
2.15 mmol) in dry DMF (25 mL). The reaction mixture was heated
to 130 °C and stirred for 12 h. After evaporation of the solvent
under reduced pressure, the crude material was purified by flash
chromatography on silica gel with dichloromethane/acetone (95:5)
as eluent. After evaporation of the solvent, the residue was dis-
solved in dry DMF (10 mL) and treated under argon with (S)-N-
Boc-ornithine (3; 470 mg, 2.15 mmol). The reaction mixture was
heated to 120 °C and stirred for 18 h. The solvent was evaporated
under reduced pressure. Purification of the residue was performed
by flash chromatography on silica gel with chloroform/methanol
(9:1) as eluent to give 1 (500 mg, 41%) as a pale-green solid. M.p.
252 °C (decomposition). RF (chloroform/methanol, 9:1) ϭ 0.40.
[α]2D5 ϭ ϩ37.9 (DMSO, c ϭ 0.31). IR (KBr): ν˜ ϭ 3394 br, 2961 s,
1707 s, 1666 s, 1581 s, 1517 m, 1456 s, 1375 s, 1338 s, 1245 s, 1167
s, 1079 m, 1059 m, 880 w, 771 s, 583 w cmϪ1. 1H NMR (250 MHz,
[D6]DMSO, room. temp.): δ ϭ 0.89Ϫ0.95 [t, 3 H, 4-H (nBu)], 1.31
(s, 9 H, tBu), 1.25Ϫ1.45 [m, 2 H, 3-H (nBu)], 1.55Ϫ1.75 [m, 6 H,
β-H, γ-H, 2-H (nBu)], 3.62Ϫ3.77 (m, 1 H, α-H), 3.92Ϫ4.08 [m, 4
H, δ-H, 1-H (nBu)], 6.41 (br. s, 1 H, NH), 8.60 (s, 4 H, Ar-H) ppm.
13C NMR (62.5 MHz, [D6]DMSO, room. temp.): δ ϭ 14.0, 20.1,
28.2, 28.5, 28.8, 29.9, 53.8, 78.3, 79.6, 126.5, 126.6, 130.0, 154.3,
162.9 ppm. ESI-MS: m/z (%) ϭ 560.0 (100) [M ϩ Na]ϩ. HRMS
(ESI) C28H31N3O8 (537.6): calcd. 538.2184; found 538.2186 [M ϩ
H]ϩ.
Scheme 6. Synthesis of isoadeninyl and N7-carbaisoadeninyl nu-
cleo amino acids by dechlorination of 2-amino-6-chloropurinyl nu-
cleo amino acids
Conclusions
The synthesis of Boc-protected amino acids carrying the
chromophores naphthalene diimide, pyromellitic diimide,
and pyrene, or the modified nucleobases 7-carbaguanine, 7-
carbaadenine, isoadenine, and 7-carbaisoadenine, was per-
formed to allow their incorporation into alanyl-PNA by
solid-phase peptide synthesis. Since this set of chromoph-
ores provides a wide spectrum of redox potentials, it should
be possible for oxidative or reductive interactions with the
nucleobase stack of alanyl-PNA double strands to be in-
itiated by the chromophores’ excited states. In the special
case of alanyl-PNA duplexes, charge-transfer experiments
profit from this well-defined and rigid DNA model system.
In particular, the conformational reorganization in DNA
that results from stacking of a chromophore with the nu-
cleobases can be avoided in alanyl-PNA. The corresponding
electron-transfer studies using the new derivatives are in
progress.
Pyromellitdiimide Derivative 2: Pyromellitic dianhydride (5; 470 mg,
2.15 mmol) and (S)-N-Boc-ornithine (3; 500 mg, 2.15 mmol) were
dissolved in dry DMF (25 mL) under argon. n-Butylamine
(157 mg, 2.15 mmol, 213 µL) was added dropwise to the suspension
with vigorous stirring. The mixture was heated at 130 °C for 20 h.
The solvent was evaporated under reduced pressure. Purification
was performed by flash chromatography on silica gel with chloro-
form, followed by a methanol gradient (2% per 250 mL), as the
eluent. The white solid obtained after evaporation of the solvents
was precipitated from a mixture of hexane and chloroform (1:1).
The product was collected and dried to provide the pure amino
acid 2 (470 mg, 45%) as a slightly yellow solid. M.p. 230 °C (de-
composition). RF (chloroform/methanol, 4:1) ϭ 0.50. [α]2D5 ϭ Ϫ19.4
(DMSO, c ϭ 0.34). IR (KBr): ν˜ ϭ 3387 br, 2964 s, 2874 s, 1698 s,
1516 m, 1396 s, 1252 m, 1163 s, 1093 m, 1045 s, 932 m, 895 w, 853
w, 821 w, 800 w, 780 w, 727 s, 609 w, 560 w, 462 w cmϪ1. 1H NMR
(250 MHz, [D6]DMSO, room. temp.): δ ϭ 0.86Ϫ0.92 [t, 3 H, 4-H
(nBu)], 1.32 (s, 9 H, tBu), 1.37Ϫ1.39 [m, 2 H, 3-H (nBu)],
1.53Ϫ1.76 [m, 6 H, β-H, γ-H, 2-H (nBu)], 3.58Ϫ3.64 [m, 4 H, δ-
3
H, 1-H (nBu)], 3.79Ϫ3.83 (m, 1 H, α-H), 7.00 (d, J ϭ 8 Hz, 1 H,
NH), 8.17 (s, 2 H, Ph). 13C NMR (62.5 MHz, [D6]DMSO, room.
temp.): δ ϭ 13.8, 19.8, 25.3, 28.3, 28.5, 30.2, 53.6, 78.3, 79.8, 117.5,
137.4, 166.7, 170.4, 174.3 ppm. ESI-MS: m/z (rel. %) ϭ 510.0 (100)
[M ϩ Na]ϩ. HRMS (ESI) of C24H28N3O8 (486.5): calcd. 510.1847;
found 510.1844 [M ϩ Na]ϩ.
Experimental Section
General: All reagents were of analytical grade and used as supplied.
Solvents were of the highest grade available. NMR spectra were
recorded with a Bruker AC 250, Bruker AMX 500 or Bruker DMX
500 spectrometer. ESI mass spectra were measured with a TSQ 700 2-Chlorotrityl Resin-Bound (S)-N-tert-Butoxycarbonyl-β-N-(9-fluor-
Finnigan or Bruker APEX-QIII, 7T spectrometer. enylmethoxycarbonyl)aminoalanine (9): 2-Chlorotrityl resin (3.00 g,
A
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Eur. J. Org. Chem. 2003, 3673Ϫ3679

Amino Acids with Unnatural Nucleobases or Chromophores
FULL PAPER
940 µmol Cl/g) was weighed in a fritted reaction chamber. The resin
was swelled for 20 min in dichloromethane. (S)-N-tert-Butoxycar-
bonyl-β-N-(9-fluorenylmethoxycarbonyl)aminoalanine (8; 1.50 g, (m, 1 H, β-H), 4.16 (m, 1 H, α-H), 4.55 (dd, J ϭ 14, J ϭ 4 Hz,
3.50 mmol) was suspended in dry dichloromethane (30 mL) and
then N,N-diisopropylethylamine (400 µL, 2.30 mmol) was added.
The solution was stirred until the solid had dissolved. This solution
acid, 70:30:3:0.35). 1H NMR (250 MHz, [D6]DMSO, room. temp.):
δ ϭ 0.99 (br. s, 1.5 H, tBu rotamer), 1.25 (br. s, 7.5 H, tBu), 3.99
3
4
1 H, β-H), 6.20 (d, 3J ϭ 4 Hz, 1 H, 7-H), 6.60Ϫ6.69 (m, 3 H,
BocNH, NH2), 7.04 (d, 3J ϭ 4 Hz, 1 H, 8-H) ppm. 13C NMR
(62.5 MHz, [D6]DMSO, room. temp.): δ ϭ 28.3 [CCH(Boc)], 45.7
was added to the swelled resin and the mixture was shaken for (CHβ), 54.5 (CHα), 78.1 [CC(Boc)], 98.1 (C-7), 108.0 (C-5), 127.4 (C-
5 min. Another portion of N,N-diisopropylethylamine (800 µL,
4.60 mmol) was then added and the mixture shaken for another
2 h. Methanol (3 mL) was then added and mixed for 20 min. The
resin was washed two times with dichloromethane and three times
with DMF. After drying for 48 h under vacuum, the modified resin
9 was collected. The loading of the resin was calculated by weight
difference to be 587 µmol 8/g resin 9.
8), 151.0 (C-4), 153.9 (C-6), 155.1 (CBocCO), 159.4 (C-2), 172.3
(CCOOH) ppm. ESI-MS: m/z (%) ϭ 356.2 (100) [M ϩ H]ϩ. HRMS
(EI) of C14H18ClN5O4 (355.8): calcd. 355.1047; found 355.1047
[Mϩ] .
(S)-N-tert-Butoxycarbonyl-β-(7-carbaguanin-9-yl)alanine (12): (S)-
N-tert-Butoxycarbonyl-β-(2-amino-7-carba-6-chloro-9-purinyl)-
alanine (17; 100 mg, 281 µmol) was dissolved in 1  hydrochloric
acid (10 mL) and heated for 18 h at 80 °C. After removal of the
solvent, the brown residue was dissolved in a mixture of sodium
hydroxide, water, and dioxane (1:1:2) and then cooled to 0 °C. Di-
tert-butylhydrogencarbonate was added and the reaction mixture
was stirred for 45 min at 0 °C. The dark-brown solution was
warmed slowly to room temperature and then stirred for another
72 h. After evaporation of the solvent, the crude product was puri-
fied by flash chromatography on silica gel with ethyl acetate/meth-
anol/acetic acid (80:20:5) as eluent to give 12 (37 mg, 39%, ee ϭ
99.4%) as a white solid. Rf ϭ 0.53 (chloroform/methanol/water/
acetic acid, 70:30:3:0.35). [α]2D5 ϭ ϩ55.2 (MeOH, c ϭ 0.19). M.p.
234 °C. IR (KBr): ν˜ ϭ 3368 br, 2925 s, 2846 s, 2362 m, 1654 s, 1560
s, 1543 s, 1528 s, 1508 s, 1458 m, 1396 s, 1253 m, 1165 m, 1051 m,
854 w, 785 w, 727 w, 681 w cmϪ1. 1H NMR (400 MHz, [D6]DMSO,
room. temp.): δ ϭ 1.07 (br. s, 1.5 H, tBu rotamer), 1.27 (br. s, 7.5
H, tBu), 3.85 (m, 1 H, α-H or β-H), 4.02 (m, 1 H, α-H or β-H),
(S)-N-tert-Butoxycarbonyl-β-(aminoalanyl)
1-Pyrenylacetoamide
(6): The amino acid-loaded resin 9 (501 mg, 587 µmol/g resin) was
placed in a 10-mL fritted syringe. The resin was swelled for 2 h in
dry DMF (9 mL). After removing the solvent, 20% piperidine in
DMF (5 mL) was added and reacted with the resin for 5 min. An-
other portion of 20% piperidine in DMF (5 mL) was then added
and the reaction proceeded for another 20 min. The resin was then
washed four times with DMF (8 mL) over 2 min. Pyreneacetic acid
(193 mg, 741 µmol), TBTU (238 mg, 741 µmol), and 1-hydroxy-
1H-benzotriazole (100 mg, 741 µmol) were dissolved in N-methyl-
2-pyrrolidone (7 mL) and then added to the resin. N,N-Diisopro-
pylethylamine (151 µL, 1.76 mmol) was added to initiate coupling.
The syringe was shielded from light and the reaction mixture was
shaken for 3 h. The resin was then washed four times, for 2 min
each time, with DMF (10 mL). To complete the reaction, the resin
was reacted a second time with 1-pyreneacetic acid, TBTU, HOBT,
and DIPEA, followed by two washes with DMF. The solid was
then washed three times with dichloromethane (10 mL). The final
product was obtained after cleavage from the resin using a mixture
of acetic acid, trifluoroacetic acid, and dichloromethane (3:1:6).
After evaporation under reduced pressure and drying under HV,
product 6 was collected (130 mg, 8% from 8). M.p. 137 °C. RF
3
4
3
4.45 (dd, J ϭ 13, J ϭ 3 Hz, 1 H, β-H), 6.10 (d, J ϭ 3 Hz, 1 H,
3
7-H), 6.25 (d, J ϭ 8 Hz, 1 H, BocNH), 6.52 (br. s, NH2), 6.63 (d,
3J ϭ 3 Hz, 1 H, 8-H), 10.89 (br. s, 1 H, 1-H) ppm. 13C NMR
(100 MHz, [D6]DMSO, room. temp.): δ ϭ 28.0 [CCH(Boc)], 28.4
[CCH(Boc)], 46.2 (CHβ), 55.9 (CHα), 77.7 [CC(Boc)], 99.9 (C-5), 100.6
(C-7), 121.0 (C-8), 150.8 (C-4), 152.7 (C-2), 155.0 (CBocCO), 159.2
(C-6), 173.7 (CCOOH) ppm. ESI-MS: m/z (%) ϭ 338.1 (100) [M
ϩ H]ϩ.
(chloroform/methanol/water/acetic acid, 70:30:3:0.3)
ϭ 0.60.
[α]2D5 ϭ Ϫ20.0 (acetone, c ϭ 0.31). IR (KBr): ν˜ ϭ 3335 br, 3042 m,
2976 s, 2929 s, 1927 w, 1712 br, 1652 s, 1525 s, 1433 m, 1391 m,
1366 s, 1345 m, 1249 s, 1162 s, 1061 m, 1026 w, 966 w, 845 s, 779
(S)-N-tert-Butoxycarbonyl-β-(6-chloro-7-carbapurin-9-yl)alanine
(18): N-Boc--serine lactone (14, 1.00 g, 5.70 mmol) was added to a
solution of 6-chloro-7-carbapurine (1.75 g, 11.4 mmol) in absolute
DMSO (15 mL). After adding DBU (868 mg, 5.70 mmol, 852 µL),
the reaction mixture was stirred for 1 h at room temperature. After
evaporation of the solvent, purification of the crude product was
performed by flash chromatography on silica gel (ethyl acetate/
methanol/acetic acid, 80:20:5) to give 18 (1.03 g, 53%) as a slightly
yellow solid. Rf ϭ 0.56 (chloroform/methanol/water/acetic acid,
w, 756 w, 710 m, 598 w cmϪ1 1H NMR (250 MHz, [D6]DMSO,
.
room. temp.): δ ϭ 1.35 (s, 9 H, tBu), 3.45Ϫ3.60 (m, 2 H, β-H),
3
4.06 (m, 1 H, α-H), 4.20 (s, 2 H, CH2-Ar), 6.96 (d, J ϭ 8 Hz, 1
H, NH), 7.96Ϫ8.35 (m, 9 H, Ar-H) ppm. 13C NMR (62.5 MHz,
[D6]DMSO, room. temp.): δ ϭ 28.5, 41.7, 53.8, 78.6, 80.3, 124.3,
124.4, 124.5, 125.2, 125.3, 125.4, 126.5, 127.2, 127.6, 127.7, 129.0,
130.1, 130.7, 131.0, 131.2, 131.7, 155.7, 163.4, 171.1 ppm. ESI-MS:
m/z (%) ϭ 347.0 (82) [M Ϫ Boc ϩ 2H]ϩ, 390.8 (44) [M Ϫ tBu ϩ
2H]ϩ, 469.0 (35) [M ϩ Na]ϩ, 892.8 (100) [2M ϩ H]ϩ, 914.9 (51)
[2M ϩ Na]ϩ, 1338.5 (7) [3M ϩ H]ϩ, 1360.7 (10) [3M ϩ Na]ϩ.
HRMS (ESI) of C26H26N2O5 (446.5): calcd. 447.1915; found
447.1912 [M ϩ H]ϩ.
1
70:30:3:0.35). H NMR (250 MHz, [D6]DMSO, room. temp.): δ ϭ
0.94 (br. s, 1.5 H, tBu rotamer), 1.17 (br. s, 7.5 H, tBu), 4.17Ϫ4.35
3
(m, 2 H, α-H, β-H), 4.75 (m, 1 H, β-H), 6.54 (d, J ϭ 4 Hz, 1 H,
7-H), 6.58 (m, 0.2 H, BocNH), 6.67 (d, 3J ϭ 6 Hz, 0.8 H, BocNH),
3
7.60 (d, J ϭ 4 Hz, 1 H, 8-H), 8.56 (s, 1 H, 2-H) ppm. 13C NMR
(62.5 MHz, [D6]DMSO, room. temp.): δ ϭ 26.5 [CCH(Boc)], 28.4
[CCH(Boc)], 46.2 (CHβ), 54.4 (CHα), 78.2 [CC(Boc)], 98.3 (C-7), 117.0
(C-5), 128.4 (C-8), 150.2 (C-2), 150.6 (C-4, C-6), 151.2 (C-4, C-6),
155.0 (CBocCO), 172.2 (CCOOH) ppm. ESI-MS: m/z (%): 341.1 (100)
[M ϩ H]ϩ. HRMS (ESI) [M ϩ H]ϩ (C14H17ClN4O4): calcd.
341.1011; found 341.1009.
(S)-β-(2-Amino-6-chloro-7-carbapurin-9-yl)-N-(tert-butoxycarbon-
yl)alanine (17): 2-Amino-7-carba-6-chloropurine (15, 1.80 g,
10.7 mmol) was dissolved in absolute DMSO (15 mL) and treated
with N-Boc--serine lactone (14, 2.00 g, 10.7 mmol) in a darkened
apparatus. After adding DBU (1.63 g, 10.7 mmol, 1.60 mL), the
reaction mixture was stirred at room temperature for 2 h. After
removal of the solvent, purification of the product was achieved by
flash chromatography on silica gel with ethyl acetate/methanol/
acetic acid (80:20:5) as the eluent to give 17 (584 mg, 15%) as a
(S)-N-tert-Butoxycarbonyl-β-(6-chloro-7-carbapurin-9-yl)alanine
Benzyl Ester (19): A solution of (S)-N-tert-butoxycarbonyl-β-(-6-
chloro-7-carbapurin-9-yl)alanine (18, 990 mg, 2.91 mmol) in anhy-
slightly yellow solid. Rf ϭ 0.45 (chloroform/methanol/water/acetic drous DMF (5 mL) was diluted with benzyl alcohol (5 mL). After
Eur. J. Org. Chem. 2003, 3673Ϫ3679
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T. Wagner, W. B. Davis, K. B. Lorenz, M. E. Michel-Beyerle, U. Diederichsen
FULL PAPER
3
cooling to 0 °C, DMAP (354 mg, 2.91 mmol) and EDCI·HCl NH2), 7.03 (s, J ϭ 3 Hz, 1 H, 8-H), 7.98 (s, 1 H, 2-H) ppm. 13C
(612 mg, 3.20 mmol) were added and the reaction mixture was NMR (100 MHz, [D6]DMSO, room. temp.): δ ϭ 28.5 [CCH(Boc)],
warmed to room temperature and stirred for 18 h. After evapor-
ation of the solvents, purification of the residue was achieved by
48.9 (CHβ), 56.3 (CHα), 77.6 [CC(Boc)], 98.1 (C-7), 102.6 (C-1), 125.1
(C-8), 150.5 (C-4), 151.5 (C-2), 154.9 (CBocCO), 157.6 (C-6), 175.1
flash chromatography on silica gel (ethyl acetate/hexane, 1:1) to (CCOOH) ppm. ESI-MS: m/z (%) ϭ 322.1 (100) [M ϩ H]ϩ. HRMS
give 19 (1.14 g, 91%) as a white solid. Rf ϭ 0.68 (ethyl acetate/
(ESI) of C14H19N5O4 (321.3): calcd. 322.1510; found 322.1507 [M
1
hexane, 1:1). H NMR (400 MHz, CDCl3, room. temp.): δ ϭ 1.11 ϩ H]ϩ.
(br. s, 1.5 H, tBu rotamer), 1.26 (br. s, 7.5 H, tBu), 4.45Ϫ4.58 (m,
(S)-β-(2-Aminopurin-9-yl)-N-(tert-butoxycarbonyl)alanine (22): (S)-
N-tert-Butoxycarbonyl-β-(2-amino-6-chloropurin-9-yl)alanine (23,
830 mg, 2.33 mmol) was dissolved in a mixture of methanol
(100 mL) and glacial acetic acid (3 mL). Palladium on charcoal
(111 mg, 5% Pd on charcoal containing 50.5% water) was added,
the mixture was saturated with hydrogen gas, and then left to react
for 38 h. The charcoal was separated from the reaction product by
filtration and then washed with methanol. The solvent was evapo-
rated and the product isolated by flash chromatography on silica
gel (ethyl acetate/methanol/acetic acid, 80:20:5) to give 22 (437 mg,
58%; ee ϭ 98.2%) as a white solid. Rf ϭ 0.60 (2-propanol/water/
acetic acid, 5:2:1, saturated with sodium chloride). [α]2D5 ϭ Ϫ33.9
(MeOH, c ϭ 0.2). M.p. 211 °C (decomp.). IR (KBr): ν˜ ϭ 3350 br,
3216 s, 2879 m, 1618 s, 1583 s, 1524 s, 1477 s, 1429 s, 1394 s, 1295
m, 1254 m, 1165 s, 1055 m, 1025 w, 965 w, 901 w, 854 w, 796 m,
3
4
2 H, α-H, β-H), 4.74 (dd, J ϭ 13, J ϭ 5 Hz, 1 H, β-H), 5.08 (dt,
3
2 H, PhCH2), 6.60 (d, J ϭ 4 Hz, 1 H, 7-H), 7.23Ϫ7.38 (m, 5 H,
3
3
Ph), 7.46 (d, J ϭ 8 Hz, 1 H, BocNH), 7.67 (d, J ϭ 4 Hz, 1 H, 8-
H), 8.61 (s, 1 H, 2-H) ppm. 13C NMR (100 MHz, CDCl3, room.
temp.): δ ϭ 27.7 [CCH(Boc)], 28.3 [CCH(Boc)], 45.2 (CHβ), 53.7 (CHα),
66.7 (CCHPh), 79.0 [CC(Boc)], 98.9 (C-7), 117.2 (C-5), 128.2 (CPhenyl
,
C-8), 128.4 (CPhenyl, C-8), 128.7 (CPhenyl, C-8), 132.3 (CPhenyl, C-8),
135.8 (CPhenyl C-1), 150.6 (C-2), 150.9 (C-4, C-6), 151.3 (C-4, C-6),
155.5 (CBocCO), 170.2 (CCOOBzl) ppm. ESI-MS: m/z (%) ϭ 431.2
(100) [M ϩ H]ϩ. HRMS (ESI) C21H23ClN4O4 (430.9): calcd.
431.1481; found 431.1478 [M ϩ H]ϩ.
(S)-β-(6-Azido-7-carbapurin-9-yl)-N-(tert-butoxycarbonyl)alanine
Benzyl Ester (20) and (S)-N-tert-Butoxycarbonyl-β-(tetrazolo[6,1-
i]purin-9-yl)alanine Benzyl Ester (21): Sodium azide (2.46 g,
38.0 mmol) was added to a solution of (S)-N-tert-butoxycarbonyl-
1
638 m cmϪ1. H NMR (400 MHz, [D6]DMSO, room. temp.): δ ϭ
0.97 (br. s, 1.5 H, tBu rotamer), 1.24 (br. s, 7.5 H, tBu), 4.00Ϫ4.23
(m, 2 H, α-H, β-H), 4.45Ϫ4.55 (m, 1 H, β-H), 6.49 (br. s, 2 H,
β-(6-chloro-7-carbapurin-9-yl)alanine
benzyl
ester
(1.09 g,
2.53 mmol) in anhydrous DMF (25 mL). The reaction mixture was
stirred at 60 °C for 18 h. The solvent was evaporated and the crude
product was purified by flash chromatography on silica gel (ethyl
acetate/hexane, 2:3) to give a mixture of 20 and 21 (251 mg, 23%;
ratio 3:97) as a white solid. Rf ϭ 0.47 (ethyl acetate/hexane, 2:3).
1H NMR (250 MHz, CDCl3, room. temp.): δ ϭ 1.29 (br. s, 9 H,
tBu), 4.28Ϫ4.37 (m, 1 H, β-H), 4.55Ϫ4.84 (m, 2 H, α-H, β-H),
4.94Ϫ5.10 (m, 2 H, PhCH2), 5.25 (d, 3J ϭ 5 Hz, 1 H, BocNH),
3
NH2), 6.68 (d, J ϭ 7 Hz, 1 H, BocNH), 7.88 (s, 1 H, 8-H), 8.51
(s, 1 H, 6-H) ppm. 13C NMR (100 MHz, [D6]DMSO, room. temp.):
δ ϭ 28.2 [CCH(Boc)], 44.3 (CHβ), 54.3 (CHα), 78.1 [CC(Boc)], 127.0 (C-
5), 143.3 (C-6), 148.8 (C-8), 153.4 (C-4), 155.2 (CBocCO), 160.6 (C-
2), 172.2 (CCOOH) ppm. ESI-MS: m/z (%) ϭ 323.1 (100) [M ϩ
H]ϩ. HRMS (ESI) of C14H18N6O4 (334.3): calcd. 322.1462; found
322.1460 [M ϩ H]ϩ.
3
6.40 (d, 3J ϭ 3 Hz, 0.03 H, 7-H, azide), 6.91 (d, J ϭ 3 Hz, 0.97
(S)-β-(2-Amino-7-carbapurin-9-yl)-N-(tert-butoxycarbonyl)alanine
(24): (S)-N-tert-Butoxycarbonyl-β-(2-amino-6-chloro-7-carbapu-
rin-9-yl)alanine (17; 58.0 mg, 163 µmol) was dissolved in a mixture
of methanol (20 mL) and glacial acetic acid (600 µL). Palladium
on charcoal (8.0 mg, 5% Pd on charcoal containing 50.5% water)
was added and then the mixture was saturated with hydrogen gas
and left to react for 90 min. The charcoal was separated from the
reaction product by filtration and subsequent washing with meth-
anol. The solvent was evaporated and the product was isolated by
flash chromatography on silica gel (ethyl acetate/methanol/acetic
acid, 80:20:5) to give 24 (21 mg, 40%; ee ϭ 85.1%) as a slightly
yellow solid. Rf ϭ 0.56 (chloroform/methanol/water/acetic acid,
H, 7-H, tetrazole), 7.02Ϫ7.30 (m, 6 H, 8-H, Ph), 8.46 (s, 0.03 H,
2-H, azide), 9.02 (s, 0.97 H, 2-H, tetrazole) ppm. 13C NMR
(62.5 MHz, CDCl3, room. temp.): δ ϭ 28.7 [CCH(Boc)], 47.4 (CHβ),
54.7 (CHα) 60.9, 65.8, 67.9, 78.0 [CC(Boc)], 102.7 (C-7), 103.8 (C-5),
128.1, 128.7, 129.0, 129.3, 131.6, 134.8, 147.1, 171.2 (CCOOBzl) ppm.
ESI-MS: m/z (%) ϭ 438.2 (100) [M ϩ H]ϩ, 460.2 (21) [M ϩ Na]ϩ.
HRMS (ESI) of C21H23N7O4 (437.5): calcd. 438.1884; found
438.1883 [M ϩ H]ϩ.
(S)-N-tert-Butoxycarbonyl-β-(7-carbaadenin-9-yl)alanine (13):
A
mixture of (S)-N-tert-butoxycarbonyl-β-(6-chloro-7-carbapurin-9-
yl)alanine benzyl ester (20) and (S)-N-tert-butoxycarbonyl-β-(tetra-
zolo[6,1-i]purin-9-yl)alanine benzyl ester (21) (250 mg, 571 µmol)
were dissolved in a mixture of dioxane (10 mL), water (6.25 mL)
and glacial acetic acid (500 µL). Palladium on charcoal (60.0 mg,
5% Pd containing 50.5% water) was added and the mixture was
kept for 24 h under an atmosphere of hydrogen gas. The reaction
product was separated from the charcoal by filtration and sub-
sequent washing with methanol. The solvent was evaporated and
the product isolated by flash chromatography on silica gel (ethyl
acetate/methanol/acetic acid, 80:20:5) to give 13 (61 mg, 33%; ee ϭ
88.5%) as a white solid. Rf ϭ 0.26 (chloroform/methanol/water/
acetic acid, 70:30:3:0.35). [α]2D5 ϭ ϩ43.4 (MeOH, c ϭ 0.14). M.p.
164Ϫ168 °C (dec.). IR (KBr): ν˜ ϭ 3676 s, 3448 s, 2925 m, 2362 m,
2545 m, 1943 w, 1918 w, 1717 s, 2654 s, 1560 s, 1522 s, 1508 s, 1490
s, 1419 s, 1363 s, 1340 m, 1168 m, 1052 m, 1025 m, 669 m, 518 w,
1
70:30:3:0.35). H NMR (400 MHz, [D6]DMSO, room. temp.): δ ϭ
0.96 (br. s, 1.5 H, tBu rotamer), 1.24 (br. s, 7.5 H, tBu), 3.88Ϫ4.12
3
4
(m, 2 H, α-H, β-H), 4.58 (dd, J ϭ 13, J ϭ 4 Hz, 1 H, β-H), 6.07
3
3
(br. s, 2 H, NH2), 6.14 (d, J ϭ 7 Hz, 1 H, BocNH), 6.18 (d, J ϭ
3 Hz, 1 H, 7-H), 6.99 (d, J ϭ 3 Hz, 1 H, 8-H), 8.38 (s, 1 H, 6-H)
3
ppm. 13C NMR (100 MHz, [D6]DMSO, room. temp.): δ ϭ 27.5
[CCH(Boc)], 28.3 [CCH(Boc)], 45.8 (CHβ), 55.6 (CHα), 77.5 [CC(Boc)],
98.5 (C-7), 111.2 (C-5), 126.6 (C-8), 150.0 (C-6), 153.0 (C-4), 154.9
(CBocCO), 159.9 (C-2), 172.8 (CCOOH) ppm. ESI-MS: m/z (%) ϭ
322.2 (100) [M ϩ H]ϩ. HRMS (ESI) of C14H19N5O4 (321.3): calcd.
322.1510; found 322.1506 [M ϩ H]ϩ.
Acknowledgments
1
472 w cmϪ1. H NMR (400 MHz, [D6]DMSO, room. temp.): δ ϭ
1.01 (br. s, 1.5 H, tBu rotamer), 1.25 (br. s, 7.5 H, tBu), 3.88Ϫ4.12
This work was supported by the Volkswagenstiftung. W. D. is grate-
3
4
(m, 2 H, α-H, β-H), 4.66 (dd, J ϭ 13, J ϭ 3 Hz, 1 H, β-H), 5.93 ful for a Fellowship from the Alexander von Humboldt Foun-
3
(m, 1 H, BocNH), 6.40 (d, J ϭ 3 Hz, 1 H, 7-H), 6.78 (br. s, 2 H, dation.
3678
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 3673Ϫ3679

Amino Acids with Unnatural Nucleobases or Chromophores
FULL PAPER
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Received April 17, 2003
Eur. J. Org. Chem. 2003, 3673Ϫ3679
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3679