Chiral peptide nucleic acid monomers (PNAM) with modified backbones

Article abstract of DOI:10.1039/b806141f

Convenient high yielding syntheses of optically pure PNAMs comprising l- or d-serine, l-lysine and l-arginine units linked to thymine or Cbz-cytosine are described. Simple workup and inexpensive reagents are employed and free amino acids are used as coupl

Full text of DOI:10.1039/b806141f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Chiral peptide nucleic acid monomers (PNAM) with modified backbones†
Alan Roy Katritzky* and Tamari Narindoshvili
Received 11th April 2008, Accepted 4th June 2008
First published as an Advance Article on the web 9th July 2008
DOI: 10.1039/b806141f
Convenient high yielding syntheses of optically pure PNAMs comprising L- or D-serine, L-lysine and
L-arginine units linked to thymine or Cbz-cytosine are described. Simple workup and inexpensive
reagents are employed and free amino acids are used as coupling components.
Lysine, arginine and serine are of considerable interest among
the a-amino acids utilized for backbone modifications of
Peptide nucleic acids are synthetic analogs of nucleic acids in
PNA. Positively charged lysine-based monomers are considered to
Introduction
be efficient in creating stable PNA–DNA duplexes.20 Replacements
with D-lysine exhibited DNA hybridization properties equal to that
of the original PNA.20–22 The lysine-containing oligomers were
also readily soluble in aqueous systems.22 The lysine moiety allows
modification of backbones and the introduction of a broad range
of functional groups without interfering with the binding to DNA
or RNA.23 In many cases, PNAs were constructed with a lysine
residue at the C-terminus24,25 in order to improve the solubility
of the whole molecule or to avoid self-aggregation. Zhang et al.
designed novel chiral PNA molecules with lysine as the main
chain unit; however, monomer 4 was obtained utilizing a multistep
synthetic methodology.23c,d
Incorporation of the arginine side chain (guanidinium func-
tional group) into the PNA backbone facilitates PNA uptake into
mammalian cells.16 However, application of the recent method-
ology for the preparation of nucleobase–lysine and arginine
monomers 5 suffers from the use of DMF as the solvent, long
reaction times (23–25h), the need to deprotect esters, and moderate
yields of products.26
which the phosphate–sugar polynucleotide backbone is replaced
by a flexible pseudo-peptide polymer to which the nucleobases are
linked. Nielsen and co-workers have reported “classical” PNA
1 derived from PNA monomer (PNAM) units 2 comprising
N-protected (2-aminoethyl)glycine with an attached protected
nucleobase. The PNA 1 has the capacity to bind with high affinity
and specificity to a complementary sequence of DNA1 or RNA2
and 1 is also resistant to DNAses and proteinases.3
The unique physico-chemical characteristics of PNAs 1 have
led to their use in a wide range of biological assays,4,5 in vitro
antigen and antisense studies,6–9 and in situ studies of cancer and
aging.10–12 However, 1 lacks the formal charges found in DNA and
classical transfection agents are often unable to deliver a PNA into
cells. Other limitations include low aqueous solubility, ambiguity
in DNA binding orientation and poor membrane permeability.13
During the last decades modified PNAs aiming to improve the
characteristics of Nielsen’s PNA have been reported,13–15 however,
the search for new ligands with alternative modes of binding to
DNA duplexes remains of paramount importance. Ideal ligands
would: (i) bind DNA (and RNA) with both high affinity and high
selectivity, (ii) possess sufficient biostability and (iii) lack sequence
restrictions.
In addition to their utilization in the construction of PNA
oligomers, nucleobase–amino acid (lysine, arginine) building
blocks are appropriate for the design of new drugs with a neamine
scaffold such as 6. Neamine derivatives, bearing a nucleobase
with lysine and arginine as linkers, are active inhibitors of the
HIV (human immunodeficiency virus) TAR-Tat (trans-activator
responsive region-trans-activator protein) interaction.26
One important strategy for improving the properties of PNA 1 is
the utilization of monomeric PNA building blocks with modified
peptide backbones.13,15–18 The PNA backbone has been modified in
several ways: importantly, the replacement of glycine by a-amino
acids having hydrophobic, hydrophilic or charged a-substituents
leads to chiral PNA 3 with different orientational selectivity in
complementary DNA binding, as well as better solubility and cell
uptake rate.13–15,19
Utilization of serine-containing PNA monomers for the con-
struction of PNA oligomers leads to SNE 7 (serine-based
nucleobase-linked polyester), polyester analogs of nucleic acids.27
The replacement of the amide linkages by ester linkages should
improve the water solubility and backbone flexibility of a PNA;
however, the increased liability of esters to strong acids or bases
Center for Heterocyclic Compounds, Department of Chemistry, University
of Florida, Gainesville, FL 32611-7200, USA. E-mail: katritzky@chem.ufl.
edu; Fax: +1-352-392-9199; Tel: +1-352-392-0554
† Electronic supplementary information (ESI) available: Copies of 1H
and 13C NMR spectra of 20–23, 22ꢀ, 23ꢀ, 27–29, 32, 33. See DOI:
10.1039/b806141f
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as compared with amide linkages requires the development of
new synthetic strategies for SNE oligomers. Lacking an efficient
synthetic procedure, SNEs have received little attention. Recently
Murata and Wada first reported27 the multistep synthesis of the op-
tically active unprotected thymine–serine monomer 13 (Scheme 1).
(Thymin-1-yl)acetic acid 8 was converted to the pentafluorophenyl
ester 9 (37%), and coupled with TMS-protected L-serine 11 to
obtain protected SNE monomer 12, which undergoes subsequent
deprotection of the TMS groups to give 13.27
Scheme 2 Synthesis of nucleobase (thymine and Cbz-protected cyto-
sine)-1-acetic acid derivatives.
Scheme 1 Reagents and conditions: (i) pentafluorophenol, DCC, DMF,
rt, 17 h; (ii) TMS-Cl, CH2Cl2, 60 ◦C, 1.5 h; (iii) MeOH, CH2Cl2, rt; (iv) 9,
CH2Cl2, rt, 12 h; (v) TFA, CH2Cl2, MeOH, rt, 1 h.
Scheme 3
room temperature (Scheme 3) took 12 h to complete, but pure
20 (82%) and 21 (75%) were isolated directly from the reaction
media. Compounds 20 and 21 are stable indefinitely at 20 ◦C.
We consider the published synthetic protocols23c,d,26,27 challeng-
ing for the preparation of nucleobase–lysine, arginine and serine
monomers as they include complex procedures, low yields and
difficulties with product purification.
We have previously reported the extensive use of N-
acylbenzotriazoles for N-,28 C-,29 and O-acylation30 reactions.
We now report a simple and efficient synthesis, utilizing N-
acylbenzotriazoles, of backbone modified chiral PNA and SNE
monomers comprising L- and D-serine, L-lysine and L-arginine
amino acids.
2. Preparation of nucleobase–serine SNE monomers 22, 22ꢀ, 23,
23ꢀ
N-Acylbenzotriazoles 20, 21 we◦re treated with L- and D-serine 10,
10ꢀ in aqueous acetonitrile at 20 C for 30 min (Scheme 4, Table 1).
After acidifying, and evaporation of the solvent, the residue was
washed with water to achieve complete removal of side product
BtH and afford optically pure nucleobase (thymine and Cbz-
cytosine)–serine monomers 22, 22ꢀ and 23, 23ꢀ in 78–85% yields.
Advantageously, no protection of the side chain functionality is
required to selectively form an amide bond to serine.
Results and Discussion
Our strategy was to utilize benzotriazole mediated solution phase
methodology, which enables the incorporation of nucleobases and
important amino acids in two simple steps, affording backbone
modified, chiral PNA and SNE monomers and building blocks
appropriate for new drug design strategies.
The synthesis of our desired monomers starts with the prepara-
tion of the nucleobase (thymine, cytosine) acetic acid derivatives
8, 19. (Thymin-1-yl)acetic acid31 8 was prepared in 80% yield
from thymine 14 (Scheme 2). The Cbz-cytosine derivative32 19
was prepared (43% overall) from cytosine 16 by (i) alkylation with
methyl bromoacetate, (ii) Cbz-protection of the exocyclic amine
17 using Cbz-Cl–DMAP reagents and (iii) saponification of the
corresponding methyl ester 18 (Scheme 2).
Scheme 4
Table 1 Yields of nucleobase–serine monomers 22, 22ꢀ, 23, 23ꢀ
Thymine–serine
Cbz-cytosine–serine
monomers (Yielda)
monomers (Yielda)
Amino acid
1. Preparation of N-(nucleobase-1-yl-acetyl)-1H-benzotriazoles
20, 21
Thymine–L-serine 22
(85%)
L-Serine
10
Cbz-cytosine–L-serine 23
(82%)
Thymine–D-serine 22ꢀ
(83%)
D-Serine
Cbz-cytosine–D-serine 23ꢀ
(78%)
Conversion of
8
and 19 into the corresponding N-
10ꢀ
acylbenzotriazoles 20, 21 was performed by modified literature
procedures.28–30,33 Reaction of nucleobase-1-yl-acetic acids with
1H-benzotriazole and thionyl chloride in CH2Cl2 or THF at
a Isolated yield.
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In comparison with the recent literature procedure27 for the
preparation of SNE monomers, our methodology offers simple
preparative and workup procedures, short times for completion,
uses inexpensive reagents, gives high yields, and allows the use of
free amino acids as coupling components.
Scheme 7
3. Preparation of nucleobase–lysine/–arginine PNE monomers
27–29, 32, 33
Conclusion
Thymine was chosen for initial further study because it presented
the fewest synthetic challenges; however, the methodology should
be extendable to other natural and unnatural nucleobases. Opti-
cally active monomers 27, 28 containing thymine linked to L-lysine
were obtained similarly, by coupling (in aqueous acetonitrile)
benzotriazole activated (thymin-1-yl)acetic acid 20 with Ne-Fmoc
and -Cbz protected L-lysines 24, 25, respectively, in the presence
of Et3N. Simple work up procedures afforded pure monomers
27, 28 (80–92%) without chromatography. The preparation of the
free L-arginine PNA building block 29 (92%) did not require the
utilization of Et3N or any protecting group (Scheme 5).
In conclusion we have developed novel approaches to the conve-
nient and efficient synthesis of backbone modified serine-, lysine-
and arginine-containing chiral PNA monomers, utilizing a simple
two-step synthetic route involving: (i) activation of nucleobase-1-
yl acetic acids as stable benzotriazole derivatives and (ii) coupling
with amino acids in aqueous media requiring neither anhydrous
reaction conditions nor the use of expensive coupling reagents.
Experimental
General Methods
Melting points were determined on Fisher melting apparatus.
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded on a 300 MHz NMR spectrometer in CDCl3 or DMSO-
d6. N-Cbz-, N-Fmoc-, and free amino acids were purchased from
Fluka and Acros and used without further purification. Elemental
analyses were performed on a Carlo Erba-1106 instrument.
MALDI analyses were performed on a Bruker Reflex II TOF mass
spectrometer retrofilled with delayed extraction. Optical rotation
values were measured with the use of the sodium D line.
(Thymine-1-yl)acetic acid (8). Thymine (3.78 g, 30 mmol) was
Scheme 5
dissolved in a solution of KOH (6.458, 115 mmol) in 20 ml of
◦
water. While this solution was warmed in a 40 C water bath, a
The synthesis of optically active monomer 31, derived from
thymine and Nx-NO2-L-arginine 30, was accompanied by unex-
pected esterification, when crude 31 was crystallized from MeOH–
DCM–ether–HCl (Scheme 6) to give 32 (89%).
solution of bromoacetic acid (6.25 g, 45 mmol) in 10 ml of water
was added over 30 minutes. The reaction was stirred for another
30 minutes at this temperature. It was allowed to cool to room
temperature and the pH was adjusted to 5.5 with conc. HCl. The
solution was then cooled in a refrigerator for 2 h. Any precipitate
formed was removed by filtration. The solution was then adjusted
to pH 2 with conc. HCl and put in a freezer for 2 h. The resultant
white precipitate was isolated by filtration, washed with◦water and
dried, affording 8 (4.4 g, 80%). White solid; mp 252–253 C, (lit.31);
1H NMR (300 MHz, DMSO-d6, Me4Si): d 1.76 (s, 3H), 4.37 (s,
2H), 7.50 (s, 1H), 11.35 (s, 1H), 13.20 (s, 1H). 13C NMR (DMSO-
d6, Me4Si): d 11.9, 48.4, 108.3, 141.8, 151.0, 164.4, 169.6. Anal.
calcd for C7H8N2O4: C, 45.66; H, 4.38; N, 15.21. Found: C, 45.30;
H, 4.24; N, 15.19.
Methyl (cytosine-1-yl)acetate (17). To a suspension of cytosine
(5.0 g, 45.0 mmol) in DMF (100mL) was added NaH (1.08 g,
45.0 mmol) under N2 at 0 ◦C. The mixture was stirred for 2 h at rt,
then methyl bromoacetate (4.3 mL, 45.0 mmol) was added. The
mixture was stirred for 48 h at rt. The solvent was evaporated in
vacuo. The crude residue was triturated with water (100mL) and
the precipitate was filtered off. Recrystallization from MeOH–H2O
gave 17 (5.27◦g, 64%) as white microcrystals. Mp 225–226 ◦C, (lit.32
mp 225–227 C); 1H NMR (300 MHz, DMSO-d6, Me4Si): d 3.68
(s, 3H), 4.54 (s, 2H), 5.87 (d, J = 7.3 Hz, 1H), 7.60–7.75 (m, 2H),
Scheme 6
Esterification was also observed under the isolation conditions
used (MeOH–H+) in the synthesis of the Cbz-cytosine–L-serine
monomer 23 (Scheme 7) to give 33 (75%). Such esterification could
be useful for the synthesis of methyl esters of thymine and Cbz-
cytosine monomers derived from Nx-NO2-arginine and serine.
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8.14 (br s, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d 49.7,
52.2, 93.7, 147.6, 153.0, 164.0, 168.8.
5H), 7.68 (t, J = 7.8 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 8.20 (t, J =
8.0 Hz, 2H), 8.33 (d, J = 8.2 Hz, 1H), 10.96 (s, 1H). 13C NMR
(75 MHz, DMSO-d6, Me4Si): d 52.4, 66.6, 94.6, 113.7, 120.4, 127.0,
128.0, 128.2, 128.5, 130.5, 131.4, 135.9, 145.4, 150.6, 153.1, 155.1,
163.8, 166.8. m/z (TOF MS) 405.1306 [M+ + H] 405.1298 (100%).
Methyl (N4-benzyloxycarbonyl)cytosin-1-ylacetate (18). Cbz-
Cl (3.12 mL, 21.8 mmol) and DMAP (2.67 g, 21.8 mmol) were
dissolved at −15 ◦C in CH2Cl2 (20 mL). The mixture was stirred
for 15 min then 17 (2.0 g, 10.9 mmol) was gradually added.
After stirring for 15 min at −15 ◦C then 5 h at rt, the mixture
was evaporated in vacuo and the crude residue was taken up in
CHCl3. The organic layer was washed with 1M HCl and with
water, then dried over Na2SO4 and lastly evaporated under reduced
pressure. Trituration of the crude residue in ether gave a white
precipitate which was then filtered◦off, affording 18 (2.65 g, 77%) as
white microcrystals. Mp 161–162 C, (lit.32); 1H NMR (300 MHz,
DMSO-d6, Me4Si): d 3.68 (s, 3H), 4.63 (s, 2H), 5.20 (s, 2H), 7.05
(d, J = 7.1 Hz, 1H), 7.30–7.450 (m, 5H), 8.06 (d, J = 7.1 Hz, 1H),
10.89 (bs, 1H). 13C NMR (75 Mz, DMSO-d6, Me4Si): d 50.5, 52.3,
66.5, 94.2, 128.0, 128.2, 128.5, 135.9, 150.3, 151.5, 154.9, 163.5,
168.5. Anal. calcd for C15H15N3O5: C, 56.78; H, 4.76; N, 13.24.
Found: C, 56.92; H, 4.67; N, 12.95.
General procedure for the preparation of nucleobase–serine mono-
mers: thymine–L-serine (22), thymine–D-serine (22ꢀ), Cbz-cytosine–
L-serine (23), Cbz-cytosine–D-serine (23ꢀ). Nucleobase–N-
acylbenzotriazole 20 [for 22, 22ꢀ] or 21 [for 23, 23ꢀ] (1 mmol) was
added at 20 ◦C to a solution of L-serine (or D-serine) (1 mmol)
in MeCN–H2O (10 : 5 mL), in the presence of Et3N (1.2 mmol).
The reaction mixture was stirred at room temperature for 30 min.
After adding 4 N HCl (1 mL) the solvent was removed under
reduced pressure until fully dry. Water (3 mL) was added to the
dry residue and the mixture was stirred for 2 min to dissolve
BtH·HCl. The suspension was filtered off and the solid washed
with water until the pH was neutral, giving the pure product.
(2S)-3-Hydroxy-2-({2-[5-methyl-2,4-dioxo-3,4-dihydro-1(2H)-
pyrimidinyl]acetyl}amino)propanoic acid (22). (230 mg, 85%);
◦
(N4-Benzyloxycarbonyl)cytosin-1-ylacetic acid (19). 18 (0.2 g,
6.3 mmol) was dissolved in dioxane (50 mL) and aqueous 1M
NaOH (8.82 mL) was added. The mixture was stirred for 5 h at
rt and then concentrated in vacuo. The residue was taken up in
aqueous 1M KHSO4. The resultant white precipitate was isolated
by filtration, washed with water and dried,◦affording 19 (1.66 g,
white microcrystals; mp 218–219 C, (lit.27); [a]D23 −16.4 (c 1.68
in DMF); 1H NMR (300 MHz, DMSO-d6, Me4Si): d 1.74 (s, 3H),
3.61 (dd, J = 10.7, 4.1 Hz, 1H), 3.70 (dd, J = 10.7, 4.9 Hz, 1H),
4.25–4.32 (m, 1H), 4.37 (s, 2H), 5.06 (br s, 1H), 7.42 (s, 1H), 8.46
(d, J = 8.0 Hz, 1H), 11.27 (s, 1H), 12.69 (br s, 1H). 13C NMR
(75 MHz, DMSO-d6, Me4Si): d 12.0, 49.0, 54.7, 61.4, 107.9, 142.5,
151.0, 164.5, 167.1, 171.8. Anal. calcd for C10H13N3O6: C, 44.28;
H, 4.83; N, 15.49. Found: C, 43.91; H, 4.69; N, 15.22
1
87%) as white microcrystals. Mp 264–266 C, (lit.32); H NMR
(300 MHz, DMSO-d6, Me4Si): d 4.53 (s, 2H), 5.19 (s, 2H), 7.03
(d, J = 7.2 Hz, 1H), 7.33–7.43 (m, 5H), 8.04 (d, J = 7.3 Hz, 1H),
10.81 (br s, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d 50.5,
66.5, 94.0, 128.0, 128.2, 128.5, 136.0, 150.5, 153.2, 155.0, 163.3,
169.4. Anal. calcd for C14H13N3O5: C, 55.45; H, 4.32; N, 13.86.
Found: C, 55.50; H, 4.35; N, 13.72.
(2R)-3-Hydroxy-2-({2-[5-methyl-2,4-dioxo-3,4-dihydro-1(2H)-
pyrimidinyl]acetyl}amino)propanoic acid (22ꢀ). (225 mg, 83%);
◦
white microcrystals; mp 234–235 C, (lit.27); [a]D23 +19.2 (c 1.78
in DMF); 1H NMR (300 MHz, DMSO-d6, Me4Si): d 1.74 (s, 3H),
3.61 (dd, J = 10.7, 3.9, Hz, 1H), 3.70 (dd, J = 10.7, 4.1, Hz, 1H),
4.25–4.33 (m, 1H), 4.37 (s, 2H), 7.42 (s, 1H), 8.46 (d, J = 7.7 Hz,
1H), 11.28 (s, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d 12.0,
49.0, 54.7, 61.4, 107.9, 142.5, 151.0, 164.5, 167.1, 171.8. Anal. calcd
for C10H13N3O6: C, 44.28; H, 4.83; N, 15.49. Found: C, 44.42; H,
4.85; N, 15.21.
General procedure for the preparation of nucleobase–N-
acylbenzotriazoles: thymine–Bt (20), Cbz-cytosine–Bt (21). To a
solution of 1H-benzotriazole (0.476 g, 4 mmol) in 20 mL of dry
CH2Cl2 [for 20] [THF for 21], was added thionyl chloride (0.1 mL,
1.2 mmol) and the reaction mixture was stirred for 20 min at
20 ◦C. Nucleobase acetic acid 8 or 13 (1 mmol) was added to the
reaction mixture and stirred overnight at room temperature. The
precipitate obtained was filtered off and dried. Water (7 ml) was
added to the dry solid and stirred for 2 min to dissolve BtH·HCl.
The suspension was filtered off then the solid was washed with
water until the pH was neutral and dried to obtain pure product.
(S)-2-[2-(4-Benzyloxycarbonylamino-2-oxo-2H -pyrimidin-1-
yl)-acetylamino]-3-hydroxypropanoic acid (23). (320 mg, 82%).
White microcrystals; mp 192–194 ◦C; [a]D23 −11.5 (c 1.63 in DMF);
1H NMR (300 MHz, DMSO-d6, Me4Si): d 3.62 (dd, J = 10.7,
4.0 Hz, 1H), 3.72 (dd, J = 10.7, 4.8, Hz, 1H), 4.28–4.34 (m, 1H),
4.52–5.63 (m, 3H), 5.20 (s, 2H), 6.97–7.06 (m, 1H), 7.30–7.49 (m,
5H), 8.00 (d, J = 7.3, 0.8H), 8.06 (d, J = 7.3 Hz, 0.2H), 8.56
(d, J = 7.4 Hz, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d
51.1, 54.8, 61.5, 66.6, 93.7, 128.0, 128.2, 128.5, 136.0, 151.5, 153.2,
154.7, 162.9, 166.7, 171.8. m/z (TOF MS) 413.1068 [M+ + Na]
413.1053 (100%).
1-(2-Benzotriazol-1-yl-2-oxo-ethyl)-5-methyl-1H-pyrimidine-
2,4-dione (20). (234 mg, 82%). White microcrystals; mp 242–
◦
1
243 C; H NMR (300 MHz, DMSO-d6, Me4Si): d 1.81 (s, 3H),
5.60 (s, 2H), 7.60–7.72 (m, 2H), 7.84 (t, J = 7.7 Hz, 1H), 8.22 (d,
J = 8.3 Hz, 1H), 8.33 (d, J = 8.3 Hz, 1H), 11.56 (s, 1H). 13C NMR
(75 MHz, DMSO-d6, Me4Si): d 12.1, 50.3, 108.9, 113.6, 120.4,
127.0, 130.5, 131.4, 141.6, 145.3, 151.2, 164.4, 167.1. Anal. calcd
for C13H11N5O3: C, 54.74; H, 3.89; N, 24.55. Found: C, 54.45; H,
3.94; N, 24.31.
(R)-2-[2-(4-Benzyloxycarbonylamino-2-oxo-2H -pyrimidin-1-
yl)-acetylamino]-3-hydroxypropanoic acid (23ꢀ). (304 mg, 78%);
white microcrystals; mp 183–185 ◦C; [a]D23 +7.4 (c 0.68 in DMF);
1H NMR (300 MHz, DMSO-d6, Me4Si): d 3.62 (dd, J = 10.3,
4.0 Hz, 1H), 3.72 (dd, J = 10.7, 4.7, Hz, 1H), 4.26–4.34 (m, 1H),
4.52–5.62 (m, 3H), 5.19 (s, 2H), 6.95–7.04 (m, 1H), 7.30–7.52 (m,
5H), 8.00 (d, J = 7.4, 0.8H), 8.05 (d, J = 7.4 Hz, 0.2H), 8.55
(d, J = 7.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d
[1-(2-Benzotriazol-1-yl-2-oxo-ethyl)-2-oxo-1,2-dihydro-pyrimi-
din-4-yl]-benzyl carba◦mate (21). (303 mg, 75%). Yellowish micro-
crystals; mp 128–130 C; 1H NMR (300 MHz, DMSO-d6, Me4Si):
d 5.22 (s, 2H), 5.73 (s, 2H), 7.17 (d, J = 7.1 Hz, 1H), 7.33–7.45 (m,
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51.1, 54.8, 61.4, 66.7, 93.7, 128.0, 128.3, 128.6, 135.9, 151.6, 153.1,
154.5, 162.8, 166.7, 171.8. m/z (TOF MS) 413.1068 [M+ + Na]
413.1063 (100%).
(S)-5-Nitroguanidino-2-[2-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-acetylamino]pentanoic acid methyl ester (32). 20
◦
(0.15 g, 0.5 mmol) was added at 20 C to a solution of Nx-
NO2-L-arginine (0.115 g, 0.5 mmol) in MeCN–H2O (10 : 5 mL),
in the presence of Et3N (2 mmol). The reaction mixture was
stirred for 1 h. 4 N HCl (1 mL) was then added, the acetonitrile
removed under reduced pressure and the water evaporated by a
current of air. The residue was dissolved with a minimum amount
of methanol (2–3 mL), then dichloromethane–ether (CH3OH :
CH2Cl2 : ether; 1 : 1 : 0.5) were added and the clear solution
was allowed to stand to give pure 32 (355 mg, 89%). White
(S)-6-(9H-Fluoren-9-ylmethoxycarbonylamino)-2-[2-(5-methyl-
2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-acetylamino]hexanoic
acid (27). 20 (0.285 g, 1 mmol) was added at 20 ◦C to a solution
of Ne-Fmoc–L-Lys-OH (0.384 g, 1 mmol) in MeCN–H2O (10 :
5 mL) in the presence of Et3N (2 mmol). The reaction mixture was
stirred for 1 h. After adding 4 N HCl (1 mL) the acetonitrile was
removed under reduced pressure. The water layer was extracted
with EtOAc (50 mL) and washed with 4 N HCl (10 mL). White
crystals precipitated from the organic solution and were filtered
to give 27 (0.427 g, 80%) which was reprecipitated from CH2Cl2–
hexanes. White microcrystals, mp 223–224 ◦C; [a]D23 −2.46 (c 1.83
◦
microcrystals, mp 215–217 C; [a]2D3 −5.03 (c 1.45 in DMF); H
NMR (300 MHz, DMSO-d6, Me4Si): d 1.43–1.72 (m, 4H), 1.75
(s, 3H), 3.05–3.23 (m, 2H), 3.64 (s, 2H), 4.20–4.35 (m, 1H), 4.35
(s, 2H), 7.43 (s, 1H), 7.52–8.60 (m, 3H), 8.65 (d, J = 7.3 Hz, 1H),
11.29 (s, 1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d 11.9,
24.7, 28.3, 49.1, 51.8, 52.0, 108.0, 142.4, 151.0, 159.3, 164.5, 167.3,
172.2. Anal. calcd for C14H21N7O7 : C, 42.10; H, 5.30; N, 24.55.
Found: C, 42.33; H, 5.40; N, 24.20.
1
1
in DMF); H NMR (300 MHz, DMSO-d6, Me4Si): d 1.22–1.70
(m, 6H), 1.77 (s, 3H), 2.88–3.02 (m, 2H), 4.12–4.26 (m, 2H), 4.27–
4.32 (m, 2H), 4.37 (s, 2H), 7.27–7.48 (m, 6H), 7.67 (d, J = 7.1 Hz,
2H), 7.88 (d, J = 7.4 Hz, 2H), 8.48 (d, J = 7.8 Hz, 1H), 11.29 (s,
1H). 13C NMR (75 MHz, DMSO-d6, Me4Si): d 12.1, 22.7, 29.1,
31.0, 46.9, 49.2, 52.1, 65.4, 108.1, 120.3, 125.3, 127.3, 127.8, 140.9,
142.6, 144.1, 151.2, 156.3, 164.7, 167.2, 173.6. m/z (TOF MS)
557.2007 [M+ + Na] 557.2028 (100%).
(S)-2-[2-(4-Benzyloxycarbonylamino-2-oxo-2H -pyrimidin-1-
yl)-acetylamino]-3-hydroxypropionic acid methyl ester (33). 21
(0.2 g, 0.5 mmol) was added at 20 ◦C to a solution of L-serine
(0.05 g, 0.5 mmol) in MeCN–H2O (10 : 5 mL), in the presence of
Et3N (2 mmol). The reaction mixture was stirred for 1 h. 4 N HCl
(1 mL) was then added, the acetonitrile removed under reduced
pressure and the water evaporated by a current of air. The residue
was dissolved with a minimum amount of methanol (2–3 mL) and
dichloromethane–ether (CH3OH : CH2Cl2 : ether; 1 : 1 : 0.5) were
added. The clear solution was allowed to stand to give pure 33
(S)-6-Benzyloxycarbonylamino-2-[2-(5-methyl-2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-acetylamino]hexanoic acid (28). 20
◦
(0.285 g, 1 mmol) was added at 20 C to a solution of Ne-Cbz–
L-Lys-OH (0.280 g, 1 mmol) in MeCN–H2O (10 : 5 mL), in the
presence of Et3N (2 mmol). The reaction mixture was stirred for
1 h. After adding 4 N HCl (1 mL) the acetonitrile was removed
under reduced pressure. The water layer was extracted with EtOAc
(50 mL), washed with 4 N HCl soln (3 × 30 mL) and sat. NaCl
soln (20 mL), and dried over MgSO4. Evaporation of the solvent
gave 28 (392 mg, 92%) in pure form, which was reprecipitated from
CH2Cl2–hexanes. White microcrystals, mp 204–206 ◦C, (lit.26); [a]2D3
◦
1
(303 mg, 75%). White microcrystals, mp 204–205 C; H NMR
(300 MHz, DMSO-d6, Me4Si): d 3.01–3.09 (m, 1H), 3.64 (s, 3H),
3.59–3.76 (m, 1H), 4.32–4.42 (m, 1H), 4.57 (s, 2H), 5.20 (s, 2H),
7.00 (d, J = 7.3 Hz, 1H), 7.30–7.46 (m, 5H), 8.00 (d, J = 7.1 Hz,
1H), 8.73 (d, J = 7.3 Hz, 1H). 13C NMR (75 MHz, DMSO-d6,
Me4Si): d 51.1, 52.0, 54.9, 61.3, 66.8, 93.7, 128.1, 128.3, 128.6,
135.8, 152.0, 153.0, 153.9, 162.5, 166.8, 170.9. m/z (TOF MS)
427.1224 [M+ + Na] 427.1278 (100%).
1
−5.9 (c 1.70 in DMF); H NMR (300 MHz, DMSO-d6, Me4Si):
d 1.22–1.48 (m, 4H), 1.50–1.74 (m, 2H), 1.74 (s, 3H), 2.91–3.04
(m, 2H), 4.12–4.24 (m, 1H), 4.33 (s, 2H), 5.00 (s, 2H), 7.23–7.29
(m, 1H), 7.29–7.39 (m, 5H), 7.41 (s, 1H), 8.47 (d, J = 7.6 Hz,
1H), 11.3 (s, 1H), 12.66 (br s, 1H). 13C NMR (75 MHz, DMSO-
d6, Me4Si): d 12.0, 22.6, 29.0, 30.9, 49.0, 52.0, 65.2, 107.9, 127.8,
128.4, 137.3, 142.5, 151.0, 156.2, 164.5, 167.0, 173.4. Anal. calcd
for C21H26N4O7 : C, 56.50; H, 5.87; N, 12.55. Found: C, 56.30; H,
5.94; N, 12.45.
Acknowledgements
We thank Dr C. D. Hall for helpful suggestions.
References
(S)-5-Guanidino-2-[2-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-acetylamino]pentanoic acid, (29). 20 (0.1 g, 0.35 mmol)
was added at 20 ◦C to a solution of L-arginine (0.061 g, 0.35 mmol)
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The acetonitrile was removed under reduced pressure and most
of the water was evaporated by a current of air. The obtained
suspension was filtered off to give pure 29 (312 mg, 92%), which
was ◦then recrystallized from water. White microcrystals, mp 290–
292 C; 1H NMR (300 MHz, TFA, Me4Si): d 1.76–1.97 (m, 3H),
1.99 (s, 3H), 2.08–2.24 (m, 1H), 3.28–3.42 (m, 2H), 4.55–4.88
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179.2. m/z (TOF MS) 341.1568 [M+ + H] 341.1575 (100%).
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