Synthesis of new unnatural Nα-Fmoc pyrimidin-4-one amino acids: Use of the p-benzyloxybenzyloxy group as a pyrimidinone masking group

Article abstract of DOI:10.1039/c4ob02235a

The p-benzyloxybenzyloxy group is used to mask the oxo function of the 4(3H)-pyrimidinone ring in the synthesis of new unnatural amino acids. The synthetic approach is based on an aromatic nucleophilic substitution reaction between 4-[4-(benzyloxy)benzylo

Full text of DOI:10.1039/c4ob02235a

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Synthesis of new unnatural Nα-Fmoc pyrimidin-
4-one amino acids: use of the p-benzyloxybenzyloxy
group as a pyrimidinone masking group†
Cite this: DOI: 10.1039/c4ob02235a
Abdellatif ElMarrouni and Montserrat Heras*
The p-benzyloxybenzyloxy group is used to mask the oxo function of the 4(3H)-pyrimidinone ring in the
synthesis of new unnatural amino acids. The synthetic approach is based on an aromatic nucleophilic
substitution reaction between 4-[4-(benzyloxy)benzyloxy]-2-(benzylsulfonyl)pyrimidine and the nucleo-
philic side chain of several Nα-Boc amino esters, as the key step, followed by a series of standard protect-
ing group transformations. p-Benzyloxybenzyloxy is efficiently removed under mild acid conditions to
recover the 4(3H)-pyrimidinone system.
Received 21st October 2014,
Accepted 6th November 2014
DOI: 10.1039/c4ob02235a
www.rsc.org/obc
diine10 and L-lathyrine,11 two naturally occurring α-amino
acids carrying a pyrimidine ring on their side chain, which
Introduction
Unnatural amino acids, the non-genetically-coded amino exhibit a diverse range of bioactivities. In peptides incorporat-
acids, including α-, β- and γ-amino acids,1 represent a nearly ing heterocycle-containing α-amino acids, the nature of the
infinite array of compounds useful in different fields such as heterocycle plays an essential role in improving their biological
drug discovery, medicinal chemistry and protein engineering.2 properties and preventing their enzymatic degradation. The
They can be either naturally3 occurring or chemically syn- pyrimidine ring and its various oxo derivatives, pyrimidinones,
thesized. Amongst many other applications, synthetic α-amino are valuable scaffolds present in many natural and synthetic
acids4 are particularly suitable for the replacement of proteino- therapeutics, including anticancer, antiviral and antibacterial
genic α-amino acids into bioactive peptide sequences.5 It is agents.12 Thus, we focused our attention on the development
well known that the introduction of non-coded amino acids, of synthetic methods14 to access unusual pyrimidine α-amino
which generates modifications in the secondary and tertiary acids13 with the objective of preparing new antimicrobial pep-
structures of a peptide, is a helpful approach to increase its tides. In particular, we recently reported the synthesis of a set
proteolytic stability, and enhance its biological selectivity and of Nα-Fmoc-pyrimidinyl α-amino acids, types 1 and 2, contain-
activity.6 In addition, the recent advances in genetic encoding ing a pyrimidine ring in their side chain.15 These compounds
of unnatural amino acids into proteins and peptides has pro- are useful building blocks for solid-phase peptide synthesis
vided a potent tool to design and synthesize new customized following the Fmoc/tert-butyl strategy. As an extension of this
peptides.7 Therefore, the development of efficient synthetic work the preparation of Nα-Fmoc-pyrimidin-4-one α-amino
routes towards new unnatural α-amino acids is of a great acids type 3 is described in this manuscript (Fig. 1).
importance to date.4 Among them, heterocyclic α-amino acids
are particularly interesting, due to their diverse range of
chemical and biomedicinal applications, not only as com-
ponents of peptides, but also as a result of their intrinsic bio-
logical properties.3 Several examples include L-azatyrosine,8
a
pyridine-containing α-amino acid, which displays anticancer
and antibacterial activities, synthetic tryptophan derivatives,9
as key precursors of biologically active compounds, or willar-
Department of Chemistry, Faculty of Science, University of Girona, Campus de
Montilivi, E-17071 Girona, Spain. E-mail: montserrat.heras@udg.edu
†Electronic supplementary information (ESI) available: Experimental details and
copies of NMR spectra (1H, 13C, dept) for all new compounds. See DOI: 10.1039/
c4ob02235a
Fig. 1 New unnatural pyrimidinyl α-amino acids.
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Results and discussion
The synthesis of Nα-Fmoc-pyrimidin-2-yl amino acids 1 was
achieved via an aromatic nucleophilic substitution reaction
(SNAr) between sulfone 4 and the nucleophilic side chain of
several natural α-amino acids 5, properly protected, followed
by a series of standard protecting group transformations: (i)
ester saponification, (ii) acid treatment to remove the Boc
group, and (iii) Fmoc protection of the free amino acid
(Scheme 1).15b The preparation of pyrimidin-4-one amino
acids 3 was initially envisioned by a simple cleavage of the iso-
propoxy group of the pyrimidin-2-yl amino esters 6 under
acidic conditions (H2SO4) as previously described.14c Under
these conditions, simultaneous cleavage of both the isopropoxy
and Boc groups should take place. Disappointingly, the required
strong acidic media to remove the isopropoxy moiety caused the
decomposition of the starting substrates (Scheme 1). These
results revealed the need for a more labile group to mask the
pyrimidinone function during the nucleophilic substitution
step. It is mandatory to mask the pyrimidinone function
Scheme 2 Synthetic approach to Nα-Fmoc-pyrimidin-4-one amino
acids 3.
First, the introduction of the p-benzyloxybenzyloxy group at
because the nucleophilic substitution of a leaving group at posi- position 4 of the pyrimidine ring was attempted through a
selective O-alkylation of 2-benzylsulfanyl-4(3H)-pyrimidinone 7
with p-benzyloxybenzyl alcohol under Mitsunobu conditions
tion 2 of the 4(3H)-pyrimidinone ring is too labile and easily
decomposes to the corresponding uracil.14c
Our first alternative was the use of the more labile tert- as was performed in the synthesis of sulfone 4.14c In this case,
the Mitsunobu reaction was not completely selective and along
butoxy group to mask the pyrimidinone function instead of iso-
propoxy one.14c However, the synthesis of 4-tert-butoxy-pyrimi- with the desired O-alkylated product 10, a significant amount
dine derivatives afforded poor yields rendering this approach of N-alkylated product 11 was observed in a 4 : 1 ratio,
inefficient.14c Instead the p-benzyloxybenzyloxy group was
respectively.
chosen due to its structural similarity to Wang resin,16 which
Chromatography separation of these isomers was proble-
can be cleaved under mild acidic conditions (TFA at 25 °C) in matic rendering this reaction impractical. This byproduct
the solid-phase.17 For example, Lam et al. reported the synthesis
could be avoided using the phosphonium coupling reaction
of a library of purine analogues using solid-phase synthesis previously employed in the synthesis of pyrimidin-4yl α-amino
where the final cleavage from Wang resin released the pyrimidi- acids 2.15a While direct coupling of pyrimidinone 7 with p-ben-
none ring employing 30% TFA in CH2Cl2 at 25 °C.18
zyloxybenzyl alcohol mediated by (benzotriazol-1-yloxy)tris(di-
Hence, we planned the synthesis of target amino acids 3 via methylamino)phosphonium
a SNAr between the 2-benzylsulfonylpyrimidine 8 (R = p-benzy- failed, the use of a two-step sequence through the isolation of
the benzotriazolyloxy (OBt) intermediate 12 successfully
protected amino esters 5 followed by a series of protecting afforded compound 10. Thus, pyrimidinone 7 was reacted with
hexafluorophosphate
(BOP)
loxybenzyloxy group) and the nucleophilic side chain of the
group transformations (Scheme 2).
BOP and DBU in CH3CN to form the OBt-intermediate 12, fol-
Scheme 1 Synthesis of Nα-Fmoc-pyrimidinyl amino acids 1.
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Scheme 3 Synthesis of pyrimidinylsulfone 8.
lowed by treatment with p-benzyloxybenzyl alcohol in the pres- After 5 hours, the 4-alkoxy pyrimidine was fully converted to
ence of t-BuOK to provide the O-alkylated product 10 in 84% 2-morphonlino-4(3H)-pyrimidinone 14 in 85% yield (Scheme 3).
yield over two steps. The latter was then oxidized with m-CPBA
to afford the desired sulfone 8 in 79% yield (Scheme 3).
The base and reaction temperature are crucial parameters
to avoid racemization during the SNAr using Nα-Boc-amino
In our previous studies,15a we observed an unusual nucleo- esters 5 as nucleophiles.15 The SNAr between pyrimidinyl
philic substitution of several alkoxy groups at position 4 of the sulfone 8 and the suitably protected amino esters 5a–c – tyro-
pyrimidine ring. The stability of the p-benzyloxybenzyloxy sine, histidine and lysine – was carried out using potassium
group under SNAr conditions was tested by subjecting sulfone carbonate as a base in DMF under a controlled temperature
8 to an excess of morpholine at 60 °C. Compound 13 was ranging from 25 °C to 50 °C. Under these conditions, the
obtained in 83% yield as the sole product with no traces of any corresponding Nα-Boc pyrimidin-2-yl amino esters 9 were iso-
side product corresponding to the displacement of the alkoxy lated in excellent yields and without appreciable racemization
group. In addition, we also tested the deprotection conditions (Table 1). In the case of amino ester 5b, the nucleophilic
of this group using 30% TFA in CH2Cl2 at room temperature. attack of the imidazole ring took place through N(τ)nitrogen
Table 1 Synthesis of Nα-Boc-pyrimidinyl amino esters 9 and Nα-Fmoc-pyrimidin-4-one amino acids 3
Entry
1
Amino ester 5
Compound 9
Yield of 9 a (%)
Compound 3
Yield of 3 b (%)
5a
89
68
2
3
5b
5c
94
94
65
63
a Isolated yields. b Isolated yields over three steps.
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Scheme 4 Solid-phase synthesis of dipeptides 16a–c.
affording the compound 9b as a single regioisomer. Interest- dine ring proved to be stable under the basic conditions in the
ingly, the 1H-NMR spectrum of the lysine derivative 9c revealed presence of nucleophiles and could be easily removed under
a dynamic process confirmed by variable temperature NMR mild acid conditions. The features of this group allow the suc-
experiments. While at room temperature, the pyrimidine ring cessful synthesis of Nα-Fmoc-pyrimidin-4-one amino acids 3
protons and benzylic protons appeared as broad signals, these through an aromatic nucleophilic substitution reaction
signals became sharper with increasing temperature most between sulfone 8 and the nucleophilic side chain of several
probably due to the fast exchange of the protons on the guani- natural α-amino acids properly protected, followed by a series
dine function.19
of standard protecting group transformations. These Fmoc
The optical integrity of compounds 9a–c was determined derivatives 3a–c are useful building-blocks for the solid-phase
using a chromatography method established in our laboratory. peptide synthesis following a Fmoc/tert-butyl strategy. The bio-
Pyrimidinyl amino acids 9a–c were saponificated with LiOH logical effect of the incorporation of these unusual amino
and the resulting Boc-amino acids 15a–c were coupled with acids into antimicrobial peptides is currently underway.
both a resin-supported racemic phenylalanine and a resin sup-
ported S-phenylalanine using standard solid-phase peptide
synthesis. Final cleavage from the resin with TFA concomi-
tantly removed both the Boc and p-benzyloxybenzyloxy groups
leading to dipeptides 16a–c. In all cases, the HPLC analysis of
Experimental
General remarks
All commercially available chemicals were used as purchased
without further purification. Melting points (capillary tube)
were measured with an Electrothermal digital melting point
apparatus IA 91000 and are uncorrected. IR spectra were
recorded on a Mattson-Galaxy Satellite FT-IR using a single
reflection ATR system as a sampling accessory. NMR spectra
compounds (S,S)-16a–c resulted in only one peak corres-
ponding to a single diastereoisomer, while the analysis of
(S/R,S)-16a–c showed two peaks (Scheme 4).
The Nα-Boc-pyrimidin-2-yl amino esters 9a–c were then con-
verted to the target Nα-Fmoc-pyrimidin-2-one amino acids
3a–c. Hydrolysis of the methyl ester followed by TFA treatment
that removed both the Boc and the p-benzyloxybenzyloxy
groups as expected. The resulting amino acids 17a–c were
immediately protected as Fmoc carbamate to achieve the
target Nα-Fmoc-pyrimidin-2-one amino acids 3a–c in good
yields over three steps (Table 1). Determination of the optical
integrity of products 3a–c was also realized through the chrom-
atography method described above, including the Fmoc
removal step before cleavage from the resin.19 These tests
showed that no detectable racemization occurred, in addition,
proved the stability of these substances by solid-phase peptide
synthesis following the Fmoc strategy.
1
were recorded on a Bruker DPX200 Advance spectrometer. H
NMR spectra were recorded at 300 or 400 MHz.13C NMR
spectra and DEPT experiments were performed at 75 or
100 MHz. Spectra recorded in CDCl3 were referenced to
residual CHCl3 at 7.26 ppm for 1H or 77.0 ppm for 13C. Spectra
recorded in DMSO-d6 were referenced to residual DMSO at
1
2.50 ppm for H or 39.5 ppm for 13C. Coupling constants (J)
are given in hertz (Hz). The following abbreviations were used
for spin multiplicity: s = singlet, d = doblet, t = triplet, q =
quartet, m = multiplet, dd = doublet of doublets, br = broad.
ESI mass spectra were recorded using a Navigator quadrupole
instrument. High resolution mass spectra (HRMS) were deter-
mined under conditions of ESI on a Bruker Micro Q-TOF
instrument using a hybrid quadrupole time-of-flight mass
spectrometer. Optical rotations were measured on a Perkin
Conclusions
In summary, we have demonstrated the use of the p-benzyloxy- Elmer polarimeter 343 Plus, using the sodium D line. Specific
benzyloxy group as a masking group of the pyrimidinone rotation [α]D is given in 10−1 cm2 g−1, and the concentration (c)
moiety during the synthesis of new unnatural α-amino acids 3. are expressed in g per 100 mL. Analytical thin layer chromato-
The p-benzyloxybenzyloxy group at position 4 of the pyrimi- graphy (TLC) was performed on precoated TLC plates, silica
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gel 60 F254 (Merck). The spots on the TLC plates were visual- and brine (1 × 20 mL). The organic layer was dried over
ized under a UV lamp (254 nm) and/or with an aqueous solu- MgSO4, filtered, concentrated under reduced pressure and the
tion of potassium permanganate (1.5% w/w). Flash resulting crude purified by flash chromatography (n-hexane–
chromatography purifications were performed on silica gel 60 EtOAc, 6 : 4) to afford sulfone 8 (2.89 g, 79%) as a colourless
(230–400 mesh, Merck).
oil. TLC: Rf (n-hexane–EtOAc, 1 : 1): 0.41; IR (neat): 3062, 3032,
Synthesis of 1-[2-(benzylsulfanyl)pyrimidin-4-yloxy]-1H-[b]- 1578, 1535, 1511, 1467, 1449, 1318, 1239, 1174, 1123 cm−1; H
[1,2,3]-benzotriazole (12). To a stirred solution of pyrimidi- NMR (300 MHz, CDCl3) δ 8.52 (d, J = 5.7 Hz, 1H, H(6)pyrim),
none 7 (2 g, 9.17 mmol) in CH3CN (27 mL), DBU (2 mL, 7.42–7.3 (m, 12H, Haryl), 6.99 (d, J = 8.7 Hz, 2H, Haryl), 6.85 (d,
13.75 mmol) and BOP (6 g, 13.75 mmol) were added succes- J = 5.7 Hz, 1H, H(5)pyrim), 5.42 (s, 2H, CH2O), 5.06 (s, 2H,
sively. The mixture was stirred at room temperature for 3 hours CH2O), 4.71 (s, 2H, CH2S); 13C NMR (75 MHz, CDCl3) δ 169.9
and the reaction completion was confirmed by TLC analysis. (s), 164.3 (s), 159.2 (s), 157.7 (d), 136.7 (s), 131.2 (d, 2C), 130.6
The solvent was evaporated under reduced pressure, and the (d, 2C), 128.8 (d), 128.7 (d, 2C), 128.6 (d, 2C), 128.0 (d), 127.4
resulting residue was purified by flash chromatography (d, 2C), 127.3 (s), 126.8 (s), 115.0 (d, 2C), 111.6 (d), 70.0 (t),
(n-hexane–EtOAc, 7 : 3) to afford OBt-adduct 12 (2.66 g, 87%) 69.6 (t), 57.6 (t); HRMS (ESI) m/z: calculated for C25H2N2NaO4S
as a white solid. mp 131–132 °C; TLC: Rf (n-hexane–EtOAc, [M + Na]+ 469.1192, found 469.1177.
1
1 : 1): 0.59; IR (neat): 3062, 3028, 1581, 1542, 1424, 1336, 1258,
Synthesis of 4-[4-(benzyloxy)benzyloxy]-2-morpholinopyrimi-
1236, 1213, 1081 cm−1; H NMR (300 MHz, CDCl3) δ 8.53 (d, dine (13). To a stirred solution of pyrimidinyl sulfone 8
J = 5.6 Hz, 1H, H(6)pyrim), 8.09 (dd, J = 8.1, 1.5 Hz, 1H, HOBt), (132 mg, 0.29 mmol) in 1,4-dioxane (1 mL), morpholine
7.58–7.52 (m, 1H, HOBt), 7.47–7.41 (m, 2H, HOBt), 7.15–7.12 (m, (75 mL, 0.87 mmol) was added. The mixture was stirred at
3H, Haryl), 6.94–6.91 (m, 2H, Haryl), 6.79 (d, J = 5.6 Hz, 1H, 60 °C for 12 hours. The solvent was evaporated under reduced
H(5)pyrim), 3.79 (s, 2H, CH2S); 13C NMR (75 MHz, CDCl3) pressure, and the resulting residue was purified by flash
δ 173.1 (s), 168.8 (s), 160.1 (d), 143.4 (s), 136.4 (s), 128.9 (d), chromatography (n-hexane–EtOAc, 7 : 3) to afford compound
128.8 (s), 128.6 (d, 2C), 128.4 (d, 2C), 127.2 (d), 125.0 (d), 120.6 13 (91 mg, 83%) as a white solid. Mp 101–104 °C; TLC: Rf (n-
(d), 108.7 (d), 100.4 (d), 35.1 (t); HRMS (ESI) m/z: calculated for hexane–EtOAc, 1 : 1): 0.65; IR (neat): 3060, 3030, 1610, 1553,
1
C17H14N5OS [M + H]+ 336.0914, found 336.0900; calculated for 1511, 1437, 1307, 1225, 1173, 983, 819, 696 cm−1
;
1H NMR
C17H13N5NaOS [M + Na]+ 358.0733, found 358.0717.
(400 MHz, CDCl3) δ 8.07 (d, J = 5.6 Hz, 1H, H(6)pyrim),
Synthesis of 4-[4-(benzyloxy)benzyloxy]-2-(benzylsulfanyl)pyr- 7.45–7.33 (m, 7H, Haryl), 6.80 (d, J = 8.5 Hz, 2H, Haryl), 6.04 (d,
imidine (10). To a stirred solution of OBt-adduct 12 (3.0 g, J = 5.6 Hz, 1H, H(5)pyrim), 5.28 (s, 2H, CH2O), 5.08 (s, CH2O),
9.0 mmol) in CH3CN (30 mL), 4-benzyloxybenzyl alcohol 3.80–3.76 (m, 8H, CH2morph); 13C NMR (100 MHz, CDCl3)
(2.9 g, 13.5 mmol) and t-BuOK (2.0 g, 18 mmol) were added δ 169.3 (s), 161.6 (s), 158.6 (s), 157.9 (d), 136.8 (s), 129.7 (d,
successively. The mixture was stirred at 25 °C for 90 minutes. 2C), 129.0 (s), 128.5 (d, 2C), 127.9 (d), 127.4 (d, 2C), 114.8 (d,
The solvent was evaporated under reduced pressure, and the 2C), 97.3 (d), 70.0 (t), 67.1 (t), 66.7 (t, 2C), 44.3 (t, 2C); MS (ESI)
resulting residue was purified by flash chromatography m/z: 378.1 [M + H]+.
(n-hexane–EtOAc, 9 : 1) to afford compound 10 (3.62 g, 97%) as
a white solid. mp 65–66 °C; TLC: Rf (n-hexane–EtOAc, 1 : 1):
General procedure for the synthesis of Nα-Boc pyrimidin-2-yl
amino esters (9)
0.72; IR (neat): 3060, 3030, 1610, 1553, 1511, 1437, 1307, 1225,
1173 cm−1 1H NMR (300 MHz, CDCl3) δ 8.24 (d, J = 5.7 Hz, To a stirred solution of the corresponding Nα-Boc-amino ester
;
1H, H(6)pyrim), 7.45–7.26 (m, 12H, Haryl), 6.98 (d, J = 8.7 Hz, 5 (1.1 equiv.) in dry DMF (0.4 M), K2CO3 (1.2–2.4 equiv.) was
2H, Haryl), 6.42 (d, J = 5.7 Hz, 1H, H(5)pyrim), 5.32 (s, 2H, added and the resulting mixture was stirred at room tempera-
CH2O), 5.08 (s, 2H, CH2O), 4.44 (s, 2H, CH2S); 13C NMR ture for 15 min. Then, pyrimidinyl sulfone 8 (1.0 equiv.) was
(75 MHz, CDCl3) δ 171.2 (s,), 168.5 (s), 158.9 (s), 157.4 (d), added dissolved in DMF (1 M). The resulting mixture was
137.6 (s), 136.9 (s), 130.1 (d, 2C), 128.9 (d, 2C), 128.7 (d, 2C), stirred at the temperature specified for each compound. The
128.6 (d, 2C), 128.4 (s), 128.0 (d), 127.5 (d, 2C), 127.2 (d), 115.0 solvent was removed under reduced pressure, and the resulting
(d, 2C), 104.2 (d), 70.1 (t), 68.1 (t), 35.4 (t); HRMS (ESI) m/z: cal- residue was purified by flash chromatography (n-hexane–
culated for C25H23N2O2S [M + H]+ 415.1475, found 415.1478; EtOAc, from 15 : 1 to 1 : 1) to afford Nα-Boc-pyrimidin-2-yl
calculated for C25H22N2NaO2S [M + Na]+ 437.1294, found amino esters 9.
437.1301.
Methyl (2S)-N-Boc-2-amino-3-{4-[4-(4-(benzyloxy)-benzyloxy)-
Synthesis of 4-[4-(benzyloxy)benzyloxy]-2-(benzylsulfonyl)pyr- pyrimidin-2-yloxy]phenyl}propanoate (9a). Synthesized accord-
imidine (8). To an ice-cooled solution of benzylsulfanylpyrimi- ing to the general procedure from pyrimidinyl sulfone 8
dine 10 (3.5 g, 8.4 mmol) in CH2Cl2 (42 mL), m-CPBA (3.6 g, (250 mg, 0.56 mmol), Nα-Boc-(S)-tyrosine methyl ester 5a
21.1 mmol) was added in small portions. The resulting (182 mg, 0.61 mmol) and K2CO3 (93 mg, 0.67 mmol) at 50 °C
mixture was allowed to warm up to room temperature and for 24 h, compound 9a (291 mg, 89%) was obtained as a col-
stirred for 1 h. The reaction completion was confirmed by TLC ourless oil. TLC: Rf (n-hexane–EtOAc, 3 : 2): 0.25; [α]2D0 +22.9
analysis. Next, the solvent was removed under reduced (c 1.36, CHCl3); IR (neat): 3433, 1741, 1709, 1568, 1508, 1380,
1
pressure and the residue was dissolved in EtOAc (100 mL), 1334, 1271, 1240, 1213, 1166 cm−1; H NMR (400 MHz, CDCl3)
washed with saturated aqueous NaHCO3 solution (2 × 20 mL) δ 8.19 (d, J = 5.7 Hz, 1H, H(6)pyrim), 7.44–7.25 (m, 7H, Haryl),
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7.19 (d, J = 8.5 Hz, 2H, Haryl), 7.13 (d, J = 8.5 Hz, 2H, Haryl), General procedure for the synthesis of Nα-Fmoc pyrimidin-2-
6.95 (d, J = 8.6 Hz, 2H, Haryl), 6.45 (d, J = 5.7 Hz, 1H, H(5)pyrim), one amino acids 3
5.26 (s, 2H, CH2O), 5.08 (s, 2H, CH2O), 5.05 (br s, 1H, NH),
4.61 (m, 1H, CHα), 3.71 (s, 3H, OCH3), 3.15 (dd, J = 13.9, 5.8 To a stirred solution of appropriate Nα-Boc-amino methyl
Hz, 1H, CH2β), 3.07 (dd, J = 13.9, 6.1 Hz, 1H, CH2β), 1.42 (s, 9H, esters 9 (1 equiv.) in THF–MeOH–H2O (1 : 2 : 2; 0.12 M), LiOH
C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 172.3 (s), 171.2 (s), 164.9 monohydrate (2.5 equiv.) was added. The mixture was stirred
(s), 159.0 (s), 158.9 (d), 152.0 (s), 136.8 (s), 133.1 (s, 2C), 130.4 at room temperature for 3–4 h. The organic solvents were
(d, 2C), 130.3 (d, 2C), 128.6 (d, 2C), 128.1 (s), 128.0 (d), 127.5 removed under reduced pressure and the pH of the resulting
(d, 2C), 122.0 (d, 2C), 114.9 (d, 2C), 103.6 (d), 80.2 (s), 70.1 (t), aqueous solution was then adjusted to 4 with glacial acetic
68.2 (t), 54.5 (d), 52.3 (q), 37.8 (t), 28.4 (q, 3C); HRMS (ESI) m/z: acid. The solution was extracted with CH2Cl2 (3 × 5 mL) and
calculated for C33H36N3O7 [M + H]+ 586.2548, found 586.2555; the combined organic layers were dried over MgSO4, filtered,
calculated for C33H35N3NaO7 [M + Na]+ 608.2367, found and concentrated under reduced pressure. The resulting
608.2384.
residue was used without further purification in the next step.
Methyl (2S)-N-Boc-2-amino-3-{1-[4-(4-(benzyloxy)benzyloxy)- The free Nα-Boc-amino acid was dissolved in CH2Cl2 (0.7 M)
pyrimidin-2-yl]-1H-imidazol-4-yl}propanoate (9b). Synthesized and the solution was cooled in an ice bath. TFA (0.3 M) was
according to the general procedure from pyrimidinyl sulfone 8 added dropwise and the resulting mixture was stirred at 0 °C
(250 mg, 0.56 mmol), Nα-Boc-(S)-histidine methyl ester 5b for 5 h. The solvent was removed under reduced pressure and
(164 mg, 0.61 mmol) and K2CO3 (93 mg, 0.67 mmol) at 50 °C the crude material was dissolved in 1,4-dioxane (0.3 M). The
for 24 h, compound 9b (293 mg, 94%) was obtained as a col- resulting solution was adjusted with 5% aqueous NaHCO3
ourless oil. TLC: Rf (n-hexane–EtOAc, 3 : 2): 0.25; [α]2D0 +17.2 solution to pH 7. Fmoc-Osu (1.02–1.05 equiv.) was then added
(c 1.5, CHCl3); IR (neat): 2961, 2919, 1745, 1710, 1590, 1565, slowly. During this addition, pH was readjusted with 5%
1
1512, 1486, 1446, 1361, 1298, 1247, 1170, 1027 cm−1; H NMR aqueous NaHCO3 solution to pH 7. The resulting mixture was
(400 MHz, CDCl3) δ 8.51 (s, 1H, H(2)imid), 8.33 (d, J = 5.7 Hz, stirred at rt for 8–12 h and the reaction completion was con-
1H, H(6)pyrim), 7.64 (s, 1H, H(5)imid), 7.44–7.31 (m, 7H, Haryl), firmed by TLC analysis. After this time, the solvent was
7.00 (d, J = 8.7 Hz, 2H, Haryl), 6.61 (d, J = 5.7 Hz, 1H, H(5)pyrim), removed under reduced pressure and the resulting residue was
5.83 (d, J = 8.2 Hz, 1H, NH), 5.41 (s, 2H, CH2O), 5.08 (s, 2H, diluted in water (10 mL) and extracted with EtOAc (3 × 5 mL).
CH2O), 4.62 (m, 1H, CHα), 3.73 (s, 3H, H17), 3.17 (dd, J = 14.7, The combined organic layers were back extracted with satu-
5.5 Hz, 1H, CH2β), 3.07 (dd, J = 14.7, 4.8 Hz, 1H, CH2β), 1.44 (s, rated NaHCO3 solution (3 × 5 mL). The combined basic
9H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 172.5 (s), 170.3 (s), aqueous layers were then acidified to pH 1–2 with 1% aqueous
159.2 (s), 158.5 (d), 155.6 (s), 136.9 (s), 135.9 (s), 132.2 (d), HCl solution, and extracted with EtOAc (3 × 5 mL). These
130.2 (d, 2C), 128.7 (d, 2C), 128.1 (d), 127.9 (s, 2C), 127.5 (d, organic layers were combined and dried over MgSO4, filtered,
2C), 115.2 (d, 2C), 114.3 (d), 106.4 (d), 79.8 (s), 70.2 (t), 68.7 (t), and concentrated under reduced pressure. The resulting
53.5 (d), 52.3 (q), 29.8 (t), 28.4 (q, 3C); HRMS (ESI) m/z: calcu- residue was triturated with diethyl ether, filtered, washed with
lated for C30H34N5O6 [M + H]+ 560.2504, found 560.2511; cal- diethyl ether and n-pentane, and dried to afford Nα-Fmoc pyri-
culated for C30H33N5NaO6 [M + Na]+ 582.2323, found 582.2294. midin-2-one amino acids 3.
Methyl (2S)-N-Boc-2-amino-6-{N-[4-(4-(benzyloxy)benzyloxy)-
(2S)-N-Fmoc-2-amino-3-[4-(6-oxo-1,6-dihydropyrimidin-2-
pyrimidin-2-yl]amino}hexanoate (9c). Synthesized according yloxy)phenyl]propanoic acid (3a). Synthesized according to the
to the general procedure from pyrimidinyl sulfone 8 (250 mg, general procedure from Nα-Boc-pyrimidin-2-yl amino ester 9a
0.56 mmol), Nα-Boc-(S)-lysine methyl ester 5c (200 mg, (140 mg, 0.25 mmol), compound 3a (84 mg, 68%) was obtained
0.61 mmol) and K2CO3 (155 mg, 1.22 mmol) at 45 °C for 24 h, as a colourless solid. Mp: 117–118 °C; TLC: Rf (EtOAc–MeOH–
compound 9c (280 mg, 84%) was obtained as a colourless oil. AcOH, 10 : 2 : 0.1): 0.45; [α]2D0 −1.3 (c 0.4, DMF); IR (neat): 3318,
TLC: Rf (n-hexane–EtOAc, 3 : 7): 0.51; [α]2D0 +7.8 (c 1.0, CHCl3); 1663, 1592, 1551, 1502, 1306, 1193, 1137 cm−1 1H NMR
;
IR (neat): 3365, 3255, 2935, 1739, 1685, 1584, 1528, 1511, 1453, (400 MHz, d6-DMSO) δ 12.81 (br, 1H, OH), 7.88 (d, J = 7.4 Hz,
1
1427, 1282, 1243, 1225, 1171, 1159 cm−1; H NMR (300 MHz, 2H, HFmoc), 7.68–7.64 (m, 3H, H(6)pyrim, HFmoc), 7.42–7.38 (m,
CDCl3) δ 7.95 (d, J = 5.7 Hz, 1H, H(6)pyrim), 7.44–7.31 (m, 7H, 2H, HFmoc), 7.34–7.28 (m, 4H, Haryl), 7.12 (d, J = 8.4 Hz, 2H,
Haryl), 6.98 (d, J = 8.7 Hz, 2H, Haryl), 6.01 (d, J = 5.7 Hz, 1H, HFmoc), 6.07 (d, J = 6.3 Hz, 1H, H(5)pyrim), 4.24–4.12 (m, 4H,
H(5)pyrim), 5.27 (s, 2H, CH2O), 5.13 (br s, 1H, NH), 5.07 (s, 2H, CHCH2O, CHα), 3.10 (dd, J = 13.9, 4.1 Hz, 1H, CH2β), 2.89 (dd,
CH2O), 4.30 (br s, 1H, CHα), 3.71 (s, 3H, OCH3), 3.40 (q, J = 6.7 J = 13.9, 10.4 Hz, 1H, CH2β); 13C NMR (75 MHz, d6-DMSO) δ
Hz, 2H, NCH2), 1.83 (m, 1H, CH2β), 1.73–1.56 (m, 3H, CH2, 173.5 (s), 165.2 (s), 159.3 (s), 155.9 (s), 153.9 (d), 150.1 (s), 143.8
CH2β), 1.43 (s, 9H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 173.5 (s, 2C), 140.6 (s, 2C), 135.7 (s), 130.2 (d, 2C), 127.6 (d, 2C), 127.0
(s), 170.1 (s), 161.9 (s), 159.0 (s), 156.8 (d), 155.6 (s), 137.1 (s), (d, 2C), 125.2 (d), 125.1 (d), 121.3 (d, 2C), 120.0 (d, 2C), 108.4
130.1 (d, 2C), 129.0 (s), 128.8 (d, 2C), 128.2 (d), 127.6 (d, 2C), (d), 65.5 (t), 55.7 (d), 46.6 (d), 35.9 (t); HRMS (ESI) m/z: calcu-
115.1 (d, 2C), 97.7 (d), 80.1 (s), 70.3 (t), 67.6 (t), 53.6 (d), lated for C28H24N3O6 [M + H]+ 498.1660, found 498.1683; calcu-
52.4 (q), 41.3 (t), 32.7 (t), 29.4 (t), 28.5 (q), 23.0 (t); HRMS (ESI) lated for C28H23N3NaO6 [M + Na]+ 520.1479, found 520.1493.
m/z: calculated for C30H39N4O6 [M + H]+ 551.2864, found
551.2884.
(2S)-N-Fmoc-2-amino-3-[1-(6-oxo-1,6-dihydropyrimidin-2-yl)-
1H-imidazol-4-yl]propanoic acid (3b). Synthesized according
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to the general procedure from Nα-Boc-pyrimidin-2-yl amino
ester 9b (150 mg, 0.25 mmol), compound 3b (76 mg, 65%) was
obtained as a colourless solid. Mp: 163–164 °C; TLC: Rf
(EtOAc–MeOH–AcOH, 10 : 2 : 0.1): 0.25; [α]2D0 +17.4 (c 0.3,
DMF); IR (neat): 3135, 1685, 1598, 1526, 1494, 1445, 1389,
D. J. Scholten, M. J. Smit, T. P. Sakmar, R. Leurs and C. de
Graaf, Drug Discovery Today, 2012, 9, e301–e313;
(c) P. Conti, L. Tamborini, A. Pinto, A. Blondel,
P. Minoprio, A. Mozzarelli and C. De Micheli, Chem. Rev.,
2011, 111, 6919–6946; (d) W. L. Scott, J. Alsina, C. O. Audu,
E. Babaev, L. Cook, J. L. Dage, L. A. Goodwin,
J. G. Martynow, D. Matosiuk, M. Royo, J. G. Smith,
A. T. Strong, K. Wickizer, E. M. Woerly, Z. Zhou and
M. J. O’Donnell, J. Comb. Chem., 2009, 11, 14–23;
(e) J. S. Ma, Chim. Oggi, 2003, 65–68; (f) G. Beck, Synlett,
2002, 837–850.
1206, 1142 cm−1 1H NMR (400 MHz, d6-DMSO) δ 12.81 (br,
;
1H, OH), 8.37 (s, 1H, H(2)imid), 8.24 (d, J = 5.6 Hz, 1H,
H(6)pyrim), 7.86 (d, J = 7.2 Hz, 2H, HFmoc), 7.66–7.63 (m, 4H,
H(5)imid, NH, HFmoc), 7.37 (t, J = 7.4 Hz, 2H, HFmoc), 7.29–7.24
(m, 2H, HFmoc), 6.47 (d, J = 5.6 Hz, 1H, H(5)pyrim), 4.31–4.17 (m,
4H, CHCH2O, CHα), 3.00 (dd, J = 14.7, 4.4 Hz, 1H, CH2β), 2.90
(dd, J = 14.7, 9.4 Hz, 1H, CH2β); 13C NMR (75 MHz, d6-DMSO)
δ 173.4 (s), 171.2 (s), 157.8 (d), 155.9 (s), 153.8 (s), 143.7 (s, 2C),
140.6 (s, 2C), 139.0 (s), 134.9 (d), 127.5 (d, 2C), 127.0 (d, 2C),
125.2 (d), 125.1 (d), 120.0 (d, 2C), 113.8 (d), 106.4 (d), 65.6 (t),
53.7 (d), 46.5 (d), 29.8 (t); HRMS (ESI) m/z: calculated for
C25H22N5O5 [M + H]+ 472.1615, found 472.1615.
3 (a) C. T. Walsh, R. V. O’Brien and C. Khosla, Angew. Chem.,
Int. Ed., 2013, 52, 7098–7124; (b) I. Wagner and H. Musso,
Angew. Chem., Int. Ed. Engl., 1983, 22, 816–828.
4 For recent reviews on the synthesis of unnatural α-amino
acids, see: (a) S. Kotha, D. Goyal and A. S. Chavan, J. Org.
Chem., 2013, 78, 12288–12313; (b) S. M. So, H. Kim, L. Mui
and J. Chim, Eur. J. Org. Chem., 2012, 229–241;
(c) R. Saladino, G. Botta and M. Crucianelli, Mini-Rev. Med.
Chem., 2012, 12, 277–300; (d) A. Perdih and M. S. Dolenc,
Curr. Org. Chem., 2011, 15, 3750–3799; (e) P. Jana, S. Maity
and D. Haldar, Curr. Org. Synth., 2010, 7, 224–234;
(f) C. Cativiela and M. Ordoñez, Tetrahedron: Asymmetry,
2009, 20, 1–63; (g) D. Haldar, Curr. Org. Synth., 2008, 5, 61–
80; (h) A. Perdih and M. D. Sollner, Curr. Org. Chem., 2007,
11, 801–832; (i) C. Najera and J. M. Sansano, Chem. Rev.,
2007, 107, 4584–4671.
5 (a) I. Ng-Choi, M. Soler, I. Güell, E. Badosa, J. Cabrefiga,
E. Bardají, E. Montesinos, M. Planas and L. Feliu, Protein
Pept. Lett., 2014, 21, 357–367; (b) R. P. Hicks,
J. J. Abercromide, R. K. Wong and K. P. Leung, Bioorg. Med.
Chem., 2013, 21, 205–214.
6 (a) J. R. Mallareddy, A. Borics, A. Keresztes, K. E. Kövér,
D. Tourwé and G. Tóth, J. Med. Chem., 2011, 54, 1462–1472;
(b) L. Gentilucci, R. De Marco and L. Cerisoli, Curr. Pharm.
Des., 2010, 16, 3185–3203; (c) R. Karstad, G. Isaksen,
B.-O. Brandsdal, J. S. Svendsen and J. Svenson, J. Med.
Chem., 2010, 53, 5558–5566; (d) C. Adessi and C. Soto, Curr.
Med. Chem., 2002, 9, 963–978.
(2S)-N-Fmoc-2-amino-6-[N-(4-oxo-3H-pyrimidin-2-yl) amino]-
hexanoic acid (3c). Synthesized according to the general pro-
cedure from Nα-Boc-pyrimidin-2-yl amino ester 9c (150 mg,
0.27 mmol), compound 3c (78 mg, 63%) was obtained as a col-
ourless solid. Mp: 121–122 °C; Rf (EtOAc–MeOH–AcOH,
10 : 2 : 0.1): 0.69; [α]2D0 +6.6 (c 1.0, DMF); IR (neat): 2944, 1686,
1645, 1534, 1446, 1193, 1132 cm−1 1H NMR (400 MHz,
;
d6-DMSO) δ 12.59 (br s, 1H, OH), 7.89 (d, J = 7.5 Hz, 2H,
HFmoc), 7.72 (d, J = 7.1 Hz, 2H, HFmoc), 7.65–7.61 (m, 2H,
H(6)pyrim, NH), 7.42 (t, J = 7.4 Hz, 2H, HFmoc), 7.33 (t, J = 7.4 Hz,
2H, HFmoc), 5.67 (d, J = 6.6 Hz, 1H, H(5)pyrim,), 4.30–4.20 (m,
3H, CHCH2O), 3.93 (m, 1H, CHα), 3.25 (q, 2H, CH2N),
1.75–1.68 (m, 2H, CH2CH2N), 1.66–1.54 (m, 2H,
CH2CH2CH2CH2N); 13C NMR (100 MHz, d6-DMSO) δ 173.9 (s),
162.5 (s), 162.3 (s), 156.2 (s), 153.8 (d), 143.8 (s, 2C), 140.7 (s,
2C), 127.6 (d, 2C), 127.1 (d, 2C), 125.3 (d, 2C), 120.1 (d, 2C),
102.9 (d), 65.6 (t), 53.8 (d), 46.7 (d,), 40.4 (t), 30.4 (t), 28.1 (t),
22.9 (t); HRMS (ESI) m/z: calculated for C25H27N4O5 [M + H]+
463.1976, found 463.1984.
Acknowledgements
7 (a) N. Budisa, Curr. Opin. Biotechnol., 2013, 24, 591–598;
(b) W. H. Zhang, G. Otting and C. J. Jackson, Curr. Opin.
Struct. Biol., 2013, 23, 581–587; (c) H. Xia, A. Chatterjee,
S.-H. Choi, K. M. Bajjuri, S. C. Sinha and P. G. Schultz,
Angew. Chem., Int. Ed., 2013, 52, 14080–14083; (d) C. C. Liu
and P. G. Schultz, Annu. Rev. Biochem., 2010, 79, 413–444.
8 (a) A. Moussa, P. Meffre, J. Martinez and U. Rolland, Amino
Acids, 2012, 42, 1339–1348; (b) H. P. Wang, T. L. Hwang,
O. Lee, Y. J. Tseng, C. Y. Shu and S. J. Lee, Bioorg. Med.
Chem. Lett., 2005, 15, 4272–4274; (c) M. Adamczyk,
S. R. Akireddy and R. E. Reddy, Org. Lett., 2001, 3, 3157–
3159; (d) B. Ye and T. R. Burke, J. Org. Chem., 1995, 60,
2640–2641.
Abdellatif ElMarrouni is a recipient of a predoctoral fellowship
from the Generalitat de Catalunya. This work was supported
by grants from the Spanish Ministerio de Economía y Competi-
tividad (MINECO) (AGL2009-13255-C02-02/AGR and AGL2012-
39880-C02-02). The authors also acknowledge the Serveis
Tècnics de Recerca of the University of Girona for the NMR
and mass spectrometry analysis.
Notes and references
1 Amino Acids, Peptides and Proteins in Organic Chemistry,
vol. 1 Origins and Synthesis of Amino Acids, ed. A. B. Hughes,
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009.
2 (a) W. H. Zhang, G. Otting and C. J. Jackson, Curr. Opin.
Struct. Biol., 2013, 23, 581–587; (b) M. Siderius, M. Wal,
9 (a) S. Preciado, L. Mendive-Tapia, F. Albericio and
R. Lavilla, J. Org. Chem., 2013, 78, 8129–8135;
(b) M. E. Kieffer, L. M. Repka and S. E. Reisman, J. Am.
Chem. Soc., 2012, 134, 5131–5137.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
10 (a) M. Martinez, A. H. Ahmed, A. P. Loh and R. E. Oswald,
Biochemistry, 2014, 53, 3790–3795; (b) N. P. Dolman,
E. Montesinos, M. Planas and L. Feliu, Eur. J. Org. Chem.,
2012, 4321–4332.
J. C. A. More, A. Alt, J. L. Knauss, T. O. Pentikäinen, 14 (a) D. Font, M. Heras and J. M. Villalgordo, Tetrahedron,
R. C. Glasser, D. Bleakman, M. L. Mayer, G. L. Collingridge
and D. E. Jane, J. Med. Chem., 2007, 50, 1558–1570.
11 (a) E. G. Brown and Y. Turan, Phytochemistry, 1996, 43,
1029–1031; (b) R. M. Adlington, J. E. Baldwin,
2008, 64, 5226–5235; (b) D. Font, M. Heras and
J. M. Villalgordo, J. Comb. Chem., 2003, 5, 311–321;
(c) D. Font, M. Heras and J. M. Villalgordo, Synthesis, 2002,
1833–1842.
D. Catterick and G. J. Pritchard, Chem. Commun., 1997, 15 (a) A. ElMarrouni, J. M. Fabrellas and M. Heras, Org.
1757–1758.
Biomol. Chem., 2011, 9, 5967–5977; (b) A. ElMarrouni,
M. Güell, C. Collell and M. Heras, Tetrahedron, 2010, 66,
612–623.
12 (a) G. W. Rewcastle, in Comprehensive Heterocyclic Chem-
istry III: Pyrimidines and their Benzo Derivatives, Elsevier,
2008, vol. 8, ch. 2, pp. 117–272; (b) K. S. Jain, 16 (a) J. W. James, Tetrahedron, 1999, 55, 4855–4946;
T. S. Chitre, P. B. Miniyar, M. K. Kathiravan, (b) S.-S. Wang, J. Am. Chem. Soc., 1973, 95, 1328–1333.
V. S. Bendre, V. S. Veer, S. R. Shahance and 17 (a) R. Salives, G. Dupas, N. Plé, G. Quéguiner and A. Turk,
C. J. Shishoo, Curr. Sci., 2006, 90, 793–803;
(c) C. Laimberth, Heterocycles, 2006, 68, 561–603;
(d) I. M. Lagoja, Chem. Biodiversity, 2005, 2, 1–50.
J. Comb. Chem., 2005, 7, 414–420; (b) J. L. Falcó,
J. L. Matallana, J. Barberena, J. Teixidor and J. I. Borrell,
Mol. Diversity, 2003, 6, 3–11.
13 (a) S. Vilà, C. Camó, E. Figueras, E. Badosa, E. Montesinos, 18 T. M. Chin-Tan, F. Yang, H. Fu, M. S. Raghavendra and
M. Planas and L. Feliu, Eur. J. Org. Chem., 2014, 4785–4794; Y. Lam, J. Comb. Chem., 2007, 9, 210–218.
(b) I. Ng-Choi, M. Soler, V. Cerezo, E. Badosa, 19 See ESI.†
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2014