Enantioselective Synthesis of D -α-(Uracil-5-yl)glycine Derivatives and Their Racemization-Free Incorporation into a Model Peptide

Article abstract of DOI:10.1002/ejoc.201500914

An efficient asymmetric synthesis of D-α-(uracil-5-yl)glycine derivatives using an enzymatic dynamic kinetic resolution of D,L-hydantoin intermediates with an immobilized D-hydantoinase (D-Hyd-1) is described. In addition, mild conditions for a racemization-free solid-phase peptide synthesis (SPPS) using the Fmoc strategy have been found.

Full text of DOI:10.1002/ejoc.201500914

FULL PAPER
DOI: 10.1002/ejoc.201500914
Enantioselective Synthesis of -α-(Uracil-5-yl)glycine Derivatives and Their
D
Racemization-Free Incorporation into a Model Peptide
Nicole M. Weckenmann[a] and Boris J. Nachtsheim*[a]
Keywords: Nucleobases / Amino acids / Peptides / Kinetic resolution / Enzyme catalysis / Chiral pool
An efficient asymmetric synthesis of
derivatives using an enzymatic dynamic kinetic resolution of
-hydantoin intermediates with an immobilized -hydan- using the Fmoc strategy have been found.
D-α-(uracil-5-yl)glycine
toinase (
D-Hyd-1) is described. In addition, mild conditions
for a racemization-free solid-phase peptide synthesis (SPPS)
D,L
D
Introduction
In recent decades, the synthesis of enantiomerically pure
non-canonical amino acids has been given considerable at-
tention. This is an important field in bioorganic chemistry,
as the genetic incorporation of such structures allows de-
tailed studies of the structure and function of proteins.[1–8]
Nucleobase amino acids (NBAs) are of particular interest,
since they can be found in a variety of natural products.
Examples include the secondary metabolites (S)-willardiine
and (S)-isowillardiine, which show interesting pharmaco-
logical properties and can act as potent antagonists of
AMPA receptors (AMPA = α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid) or GluR5-containing kainate re-
ceptors (Figure 1, a).[9–12]
Furthermore, it has been demonstrated that peptides
containing NBAs show a good intrinsic cell permeability,
and form stable complexes with endogenous nucleobases
and polynucleotides.[13] In addition, with complementary
base pairing at appropriate positions, they can adopt a β-
hairpin fold with a unique stability.[14] They are able to in-
teract with complementary RNA, enhance peptide–peptide
recognition, and inhibit the activity of MMLV (Moloney
murine leukemia virus) reverse transcriptase by the forma-
tion of complexes with polyA at the primer site.[15–17] To
date, most studies have used NBAs derived from alanine
or homoalanine. The corresponding glycine-derived NBAs
have been investigated to a much lesser extent, since they
have not been synthetically accessible in enantiomerically
pure form. Torrence and coworkers developed a synthesis
of glycine derivatives of deoxyuridine using an Ugi reaction,
but a mixture of diastereomers was obtained.[18] In this arti-
Figure 1. Structures of selected known nucleobase-containing
amino acids.
cle, we want to fill this gap and present the first enantio-
selective synthesis of d-α-(uracil-5-yl)glycine (1a) and its N-
protected derivatives (Figure 1, b). A variety of d-phenyl
glycine derivatives are easily accessible in enantiomerically
pure form through the so-called “hydantoinase process”
(Scheme 1).
In this procedure, a 5-aryl-substituted hydantoin is
formed through a Bucherer–Bergs reaction between an aro-
matic carbaldehyde, ammonium carbonate, and a cyanide
source. Subsequently, the d,l-hydantoin is treated with an
enzyme of the hydantoinase class; this selectively converts
the d-(5-aryl)-hydantoin into the corresponding d-N-carb-
amoyl amino acid. Final deprotection provides the desired
d-5-aryl glycine with a high enantiomeric excess.[19–22] Since
the hydantoin is prone to racemization under moderately
basic conditions (pHϾ 8), this dynamic kinetic resolution
could theoretically give the desired nonproteinogenic (R)-
amino acid in a very high yield. Due to the structural simi-
larity of phenyl glycine to our target structure 1a, we
planned to use the hydantoinase process for its synthesis.
[a] Eberhard Karls University, Institut of Organic Chemistry,
Auf der Morgenstelle 18A, 72076 Tübingen, Germany
E-mail: boris.nachtsheim@uni-tuebingen.de
http://www.uni-tuebingen.de/fakultaeten/
mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/
chemie/institute/organische-chemie/institut/willkommen.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500914.
6624
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6624–6630

Enantioselective Synthesis of d-α-(Uracil-5-yl)glycine Derivatives
the free amino acid at this stage (14–18%). However, treat-
ment of N-carbamoyl derivatives 6a–6c with NaNO2 in HCl
provided the desired racemic α-(uracil-5-yl)glycine deriva-
tives (i.e., 1a–1c) in 60–74% isolated yield.[25]
Enantioselective Synthesis
Once we had established a synthetic route to compounds
1a–1c, we were interested in an enantioselective synthesis
of the corresponding amino acids, using the hydantoinase
process as the method of choice for the dynamic kinetic
resolution of hydantoin intermediates 5a–5c. To the best
of our knowledge, no enzymatic synthesis using such polar
molecules as substrates for hydantoinases has been pub-
lished to date. Thus, we were very pleased to quickly find
optimized reaction conditions for this transformation (Tris-
buffer at pH 8.5, immobilized d-hydantoinase-1 from Bacil-
lus thermoglucosidasius). This two-step process allowed us
to convert the racemic hydantoins (i.e., 5a–5c) into the tar-
get compounds [i.e., (R)-1a–(R)-1c] with excellent enantio-
meric excesses of 95–99% (Scheme 3).
Scheme 1. Hydantoinase process for the enantioselective synthesis
of optically pure (hetero)aryl glycines.
Results and Discussion
Racemic Synthesis
First, we investigated the racemic synthesis of 1a–1c by
a fast four-step procedure starting from 5-(formyl)uracils
4a–4c.[23] Strecker-type Bucherer–Bergs reaction of 4a–4c
yielded the key d,l-hydantoins (i.e., 5a–5c) in good overall
yields ranging from 60 to 84% (Scheme 2).[24]
Scheme 3. Dynamic kinetic resolution of hydantoins 5a–5c using
d-hydantoinase-1 from Bacillus thermoglucosidasius. Enantio-
selectivities were measured by chiral HPLC analysis after Fmoc
protection. [a] Reaction was stopped without full conversion of the
starting material.
Due to the low solubility of 5a in the reaction medium,
(R)-1a was obtained in only 30% yield with 95% ee after a
reaction time of 7 d. In addition, more than half of the
starting material was recovered. Hydantoins 5b and 5c were
completely soluble and gave better results. While (R)-1b
could be isolated in 55% yield with 98% ee after 7 d, the
conversion of 5c was very fast, yielding (R)-1c in 77% yield
with an excellent 99% ee after only 5 h.
Scheme 2. (a) (NH4)2CO3, NaCN, 55 °C, 2 h. (b) NaOH (1 m aq.),
95 °C, 2 h. (c) NaNO2, HCl (3.5 m aq.), 0 °C, 1 h.
It proved necessary to carry out the reaction in a closed
vial to keep the CO2 concentration at an adequate level.
Hydrolysis of 5a–5c with NaOH (1 m aq.) yielded N-carb-
Solid-Phase Peptide Synthesis (SPPS)
To incorporate these amino acids into peptides and use
amoyl-protected derivatives 6a–6c in 54–76% yield. For the them in SPPS, protection with standard protecting groups
hydrolysis of 5a, it is worth mentioning that significant was examined. Fmoc (fluorenylmethyloxycarbonyl) protec-
amounts of 1a (44%) could already be isolated after this tion was achieved for all the α-(uracil-5-yl)glycines in up to
step; the other two derivatives gave only trace amounts of 85% yield (7a–7c) using Fmoc-OSu (Su = succinimide) in
Eur. J. Org. Chem. 2015, 6624–6630
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6625

N. M. Weckenmann, B. J. Nachtsheim
FULL PAPER
MeCN/H2O (Scheme 4). Boc (tert-butoxycarbonyl) protec- (Table 1, entry 1). The use of DEPBT, an alternative cou-
tion and N-acetylation were demonstrated for 1a, yielding pling reagent commonly used with epimerization-sensitive
Boc-protected amino acid 8 in 86% yield, the N-acetyl-α- aryl glycines, together with diisopropylethylamine (DIPEA)
(uracil-5-yl)glycine 9 in 75% yield, respectively. Faced with as a base,[27–29] resulted in a higher diastereomeric excess of
the potential base lability of 1a, we decided to investigate the model tripeptide, but racemization was still significant
its compatibility with standard solid-phase peptide synthe- (Table 1, entry 2).
sis and determine the extent of racemization at the C-α po-
sition during this process. A model tripeptide, already used
Table 1. Optimization studies towards the incorporation of (R)-7a
into model tripeptide 10.
by Barany and coworkers to investigate cysteine racemiza-
tion, was prepared.[26] Incorporation of racemic 7a into
Incorporation of
Entry[a] Fmoc cleavage
Activator
Base
correct enantiomer
model tripeptide 10 (Figure 2) under standard Fmoc SPPS
conditions using HBTU and NMM gave the diastereomeric
product with an expected de of 0% (Figure 2, a). Incorpora-
tion of enantiopure (R)-7a (95% ee) into the model peptide
gave the tripeptide with a de of only 8%, which proved our
hypothesis on the potential epimerization of the C-α posi-
tion under this standard peptide-coupling protocol
[%] (de of 10 [%])[b]
1
2
3
4
5
6
7
piperidine
piperidine
DBU (1%)
DBU (1%)
DBU (1%)
DBU (1%)
DBU (1%)
HBTU
DEPBT
DEPBT
HATU
COMU
COMU
COMU
NMM
DIPEA
DIPEA
DIPEA
DIPEA
TMP
55 (8)
65 (24)
72 (40)
83 (62)
91 (78)
99 (93)
99 (93)
DMP
[a] DMF was used as solvent; see Exp. Sect. for detailed procedures.
[b] de was determined using a Nucleodur C-18 column with a linear
gradient of MeCN (80% aq. with 0.1% TFA) and water (with 0.1%
TFA) from 5:95 to 35:65 over 10 min. HBTU = O-(benzotriazol-1-
yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate; NMM =
N-methylmorpholine; DMF = N,N-dimethylformamide; DEPBT =
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one; DIPEA
= N,N-diisopropylethylamine; DBU = 1,8-diazabicyclo[5.4.0]undec-
7-ene; HATU = N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide;
COMU = (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-
morpholino-carbenium hexafluorophosphate; TMP = 2,4,6-trimeth-
ylpyridine; DMP = 2,6-dimethylpyridine.
To address this base lability of the amino acid, only
1 equiv. of the base (relative to the activator) was used. The
stronger and non-nucleophilic amidine base 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) gave better results than the
commonly used piperidine for Fmoc cleavage, and the time
for deprotection was decreased to 30 s (Table 1, en-
try 3).[30,31] Cryle and coworkers investigated other coupling
reagents to access epimerization-prone biosynthetic inter-
mediates, and found HATU[32] and COMU[33] to be advan-
tageous.[34,35]
Scheme 4. (a) Fmoc-OSu, Et3N, room temp., 16 h. (b) Boc2O,
NaHCO3, r.t., 19 h. (c) Ac2O, Et3N, r.t., 24 h.
In fact, the use of HATU provided tripeptide 10 with
62% de, and COMU was able to couple the amino acid to
the resin to give a good 78% de with 91% of the correct
diastereomer (Table 1, entries 4 and 5). An examination of
other bases, such as 2,4,6-trimethylpyridine (TMP, sym-
collidine; pKa = 7.43) and 2,6-dimethylpyridine (DMP,
lutidine; pKa = 6.60), with weakly nucleophilic and less ba-
sic properties, was undertaken.[36] With both of these bases,
minimal racemization was observed, giving 99% of the cor-
rect diastereomer of the model tripeptide 10 (Table 1, en-
tries 6 and 7; Figure 2, b). The coupling efficiency of every
activator for the tripeptide was above 85%.
Figure 2. (A) HPLC trace of a mixture of H-Gly-d-Uracil-Phe-
NH2 (tR = 7.89 min) and H-Gly-l-Uracil-Phe-NH2 (tR = 8.55 min).
(B) Structure of model tripeptide 10, and HPLC trace of a reaction
mixture run under the optimized reaction conditions given in
Table 1, entry 7. Baseline separation was achieved using a Nucleo-
dur C-18 column with a linear gradient of MeCN [80% aq. with
0.1% TFA (trifluoroacetic acid)] and water (with 0.1% TFA) from
5:95 to 35:65 over 10 min.
Conclusions
In conclusion, we have developed an efficient enantio-
selective synthesis of three d-α-(uracil-5-yl)glycine deriva-
tives based on the hydantoinase process. Based on a model
6626
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6624–6630

Enantioselective Synthesis of d-α-(Uracil-5-yl)glycine Derivatives
resealed, and the temperature was raised to 55 °C. After 4 h, the
reaction mixture was slowly cooled to room temperature and
stirred overnight. The product was collected by filtration and
recrystallized from EtOH/H2O (2:1) to give 5a (600 mg, 2.86 mmol,
tripeptide, we have also developed a racemization-free pro-
tocol for their utilization in SPPS using the activator
COMU with equal amounts of DMP as a base. This im-
proved protocol makes these amino acids useful building
blocks for solid-phase peptide synthesis using the Fmoc
strategy. The biological effects of the incorporation of these
new amino acids into antimicrobial peptides are currently
under investigation in our laboratory.
1
84%) as a beige solid. H NMR (400 MHz, [D6]DMSO): δ = 9.92
(br. s, 3 H), 7.74 (s, 1 H), 7.55 (s, 1 H), 4.71 (s, 1 H) ppm. 13C
NMR (101 MHz, [D6]DMSO): δ = 174.3, 162.7, 157.7, 151.1,
142.6, 107.1, 56.4 ppm. IR ν = 1734, 1684, 1635, 1623, 1539, 1158,
˜
752, 733, 713, 706 cm–1. HRMS (ESI): calcd. for C7H5N4O4 [M –
H]– 209.0305; found 209.0307. Rf (A) = 0.56.
5-(2,5-Dioxoimidazolidin-4-yl)-1-methylpyrimidine-2,4(1H,3H)-di-
one (5b): N-1-Methyl-5-(formyl)uracil (4b; 56.0 mg, 0.36 mmol,
1.00 equiv.) was dissolved in EtOH (1 mL) and deionized water
(1 mL). Ammonium carbonate (121 mg, 1.26 mmol, 3.50 equiv.)
was added, the reaction flask was sealed with a septum, and the
reaction mixture was stirred for 30 min at room temperature. Sub-
sequently, sodium cyanide (21.7 mg, 0.43 mmol, 1.20 equiv.) was
added, the reaction flask was resealed, and the temperature was
raised to 55 °C. After 2 h, the reaction mixture was slowly cooled
to room temperature, and adjusted to pH = 3 with HCl (3 m aq.)
(caution: HCN liberation!). The solvent was removed under re-
duced pressure, and the crude product was purified by flash
chromatography (EtOAc/MeOH, 8:1) to give 5b (49 mg,
Experimental Section
1
General Remarks: H NMR spectra were recorded with an Avance
400 MHz instrument in [D6]DMSO or D2O. Chemical shifts (δ) are
given in parts per million (ppm). 1H NMR spectra were referenced
to the residual proton signal of [D6]DMSO at δ = 2.50 ppm (quint)
or of D2O at δ = 4.79 ppm. 13C NMR spectra were fully 1H-de-
coupled by broadband decoupling, and were referenced to the [D6]-
DMSO signal at δ = 39.43 ppm (sept). The following abbreviations
are used to describe splitting patterns: br = broad, s = singlet, d =
doublet, t = triplet, q = quartet, quint = quintet, sept = septet, m
= multiplet. Coupling constants J are given in Hz. High-resolution
mass spectra were recorded with an APEX II Bruker Daltonic in-
strument using the ESI method in negative-ion mode. Thin-layer
chromatography was carried out on precoated silica gel 60 plates
1
0.22 mmol, 60%) as a pale yellow solid. H NMR (400 MHz, [D6]
DMSO): δ = 11.46 (s, 1 H), 10.64 (s, 1 H), 7.81 (br. s, 2 H), 4.71
marked with
a
fluorescence indicator (Macherey–Nagel,
(s, 1 H), 3.25 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ =
ALUGRAM Xtra SIL G/UV254), and visualization was achieved
using UV light (254 nm) or by spraying with ninhydrin. Flash col-
umn chromatography was carried out on silica gel (0.040–
0.063 mm). Unless otherwise stated, all reactions were carried out
under air. IR spectra were measured neat, and were recorded with
a JASCO FTIR-4100 instrument. A selection of the most intense
signals is given in wavenumbers (cm–1). Ion-exchange chromatog-
raphy was carried out on either strongly acidic Amberlyst 15(H)
resin or strongly basic Amberlyst A-26(OH) resin (Alfa Aesar).
Chiral HPLC analysis was carried out using a Reprosil Chiral-AM
column (4.6ϫ250 mm, Dr. Maisch) at a flow rate of 1.0–
1.5 mLmin–1, using mixtures of hexane (with 0.1% TFA) and iso-
propanol (with 0.1% TFA) as the solvent system. HPLC analysis
of peptides was achieved using a Nucleodur C-18 column at a flow
rate of 1.5 mLmin–1, with MeCN (80% aq. with 0.1% TFA) and
water (with 0.1% TFA) as the solvent system. LC–MS analysis of
peptides was carried out with an LC–MS-2020 Shimadzu Single
Quadrupol MS instrument using the ESI method. The column used
for LC–MS analysis was a Kinetex 2.6u c18 column (2.1ϫ100 mm,
Phenomenex), used at a flow rate of 0.3 mLmin–1, with a linear
gradient of MeCN (80% aq. with 0.1% formic acid) and water
(with 0.1% formic acid) from 5:95 to 95:5 over 13 min. Polarimetric
analysis was carried out using a JASCO P-1020 spectropolarimeter
at the sodium d-line with a 1 dm path-length cell; concentrations
are given in g100 mL–1. Immobilized d-Hydantoinase-1 from
Bacillus thermoglucosidasius (195 Ug–1, using phenylhydantoin as
substrate) was purchased from Iris Biotech GmbH. TentaGel R
RAM resin (0.20 mmolg–1 capacity, 90 μm particle size) was pur-
chased from RAPP Polymere. Solvent systems for TLC: (A)
nBuOH/MeOH/H2O/Et3N (60:20:20:1, v/v).
174.1, 162.3, 157.7, 150.9, 146.5, 107.4, 56.3, 35.4 ppm. IR ν =
˜
1733, 1717, 1683, 1675, 1645, 1635, 639, 636, 603 cm–1. HRMS
(ESI): calcd. for C8H7N4O4 [M – H]– 223.0473; found 223.0475. Rf
(EtOAc/MeOH, 3:1) = 0.57.
5-(2,5-Dioxoimidazolidin-4-yl)-1,3-dimethylpyrimidine-2,4(1H,3H)-
dione (5c): N,N-1,3-Dimethyl-5-(formyl)uracil (4c; 100 mg,
0.59 mmol, 1.00 equiv.) was dissolved in EtOH (1 mL) and deion-
ized water (1 mL). Ammonium carbonate (200 mg, 2.08 mmol,
3.50 equiv.) was added, the reaction flask was sealed with a septum,
and the reaction mixture was stirred for 30 min at room tempera-
ture. Subsequently sodium cyanide (34.7 mg, 0.71 mmol,
1.20 equiv.) was added, the reaction flask was resealed, and the
temperature was raised to 55 °C. After 2 h, the reaction mixture
was slowly cooled to room temperature, and adjusted to pH = 3
with HCl (3 m aq.) (caution: HCN liberation!). The solvent was
removed under reduced pressure, and the crude product was puri-
fied by flash chromatography (CHCl3/MeOH, 12:1) to give 5c
(98 mg, 0.41 mmol, 71 %) as a pale yellow solid. 1H NMR
(400 MHz, [D6]DMSO): δ = 10.67 (s, 1 H), 7.89 (s, 1 H), 7.82 (s, 1
H), 4.75 (s, 1 H), 3.33 (s, 3 H), 3.15 (s, 3 H) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 174.1, 161.2, 157.7, 151.0, 144.9,
106.4, 56.7, 36.6, 27.4 ppm. IR ν = 3837, 3752, 3734, 3670, 1743,
˜
1729, 1706, 1704, 1675, 1635 cm–1. HRMS (ESI): calcd. for
C9H9N4O4 [M – H]– 237.0629; found 237.0629. Rf (CHCl3/MeOH,
9:1) = 0.21.
General Procedure for the Basic Hydrolysis of Hydantoins to N-
Carbamoyl-Protected Amino Acids (6): Hydantoin 5 (0.5 mmol,
1.00 equiv.) was dissolved in NaOH (1 m aq.; 2 mL), and the tem-
perature was raised to 95 °C. After the starting material had been
consumed (typically 2 h), the solution was adjusted to pH = 3–4
with HCl (3 m aq.) and applied to an Amberlyst 15(H) cation-ex-
change column. The column was washed with water to neutral,
eluting the N-carbamyl amino acid 6. Subsequently, the pure amino
acid 1 was eluted with NH4OH (1 m aq.).
5-(2,5-Dioxoimidazolidin-4-yl)pyrimidine-2,4(1H,3H)-dione (5a): 5-
(Formyl)uracil (4a; 475 mg, 3.40 mmol, 1.00 equiv.) was dissolved
in EtOH (5 mL) and deionized water (5 mL). Ammonium carb-
onate (1.14 g, 11.9 mmol, 3.50 equiv.) was added, the reaction flask
was sealed with a septum, and the reaction mixture was stirred for
30 min at room temperature. Subsequently sodium cyanide
(182 mg, 3.72 mmol, 1.10 equiv.) was added, the reaction flask was
N-Carbamoyl-(uracil-5-yl)glycine (6a): Yield 54%, white solid. 1H
NMR (400 MHz, D2O + 1 equiv. AcOH): δ = 7.65 (s, 1 H), 4.97
Eur. J. Org. Chem. 2015, 6624–6630
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6627

N. M. Weckenmann, B. J. Nachtsheim
(s, 1 H) ppm. 13C NMR (101 MHz, D2O + 1 equiv. AcOH): δ = 1 (d-HYD-1; 0.5 g) was added to a solution of hydantoin 5
FULL PAPER
179.1, 168.2, 159.0, 158.1, 147.7, 108.5, 50.9 ppm. IR ν = 3854, (0.21 mmol, 1.00 equiv.) in Tris-HCl buffer (0.1 m; pH = 8.5 at
˜
3744, 3469, 3308, 3267, 3244, 659, 639, 616 cm–1. HRMS (ESI):
calcd. for C7H4N4O5 [M – H]– 227.0422; found 227.0419. Rf (A) =
0.26.
40 °C; 4.2 mL) containing MnCl2 (1 mm), and the mixture was in-
cubated at 40 °C with a constant bubbling of nitrogen though the
solution. After 7 d, or after the starting material had been con-
sumed, the immobilized enzyme was separated by centrifugation
and washed three times with water. The combined aqueous frac-
tions were concentrated under reduced pressure, keeping the bath
temperature below 40 °C. The residue was applied to an Amberlyst
A-26(OH) anion-exchange column, which was then washed with
water to neutral, and subsequently eluted with HCl (0.5 m aq.).
Fractions containing the N-carbamoyl amino acid were concen-
trated under reduced pressure. The pure d-amino acids (R)-1 were
isolated as described above in the general procedure for the synthe-
sis of d,l-α-(uracil-5-yl)glycines. Recovered immobilized d-HYD-1
was washed with Tris-HCl buffer (0.1 m; pH = 8 at 4 °C) containing
MnCl2 (5 mm), the solution was decanted off, and the enzyme was
stored, slightly wet, at 2–8 °C under nitrogen.
N-Carbamoyl-(N-1-methyluracil-5-yl)glycine (6b): Yield 65%, white
solid. 1H NMR (400 MHz, [D6]DMSO): δ = 11.43 (s, 1 H), 7.74
(s, 1 H), 6.75 (br. s, 3 H), 4.98 (s, 1 H), 3.23 (s, 3 H) ppm. 13C
NMR (101 MHz, [D6]DMSO): δ = 172.0, 163.2, 158.0, 150.9,
144.8, 111.3, 50.4, 35.3 ppm. In the 1H NMR spectrum, trace
amounts of pyridine derived from the Amberlyst 15(H) ion-ex-
change resin are visible, and this was taken into account when cal-
culating the product yield (see Supporting Information). IR ν =
˜
3187, 2362, 1652, 1558, 1540, 663, 648, 624, 612, 602 cm–1. HRMS
(ESI): calcd. for C8H9N4O5 [M – H]– 241.0578; found 241.0581. Rf
(A) = 0.28.
N-Carbamoyl-(N,N-1,3-dimethyluracil-5-yl)glycine (6c): Yield 76%,
white solid. 1H NMR (400 MHz, [D6]DMSO): δ = 7.81 (s, 1 H),
6.51 (d, J = 8.5 Hz, 1 H), 5.79 (s, 2 H), 5.01 (d, J = 8.5 Hz, 1 H),
3.31 (s, 3 H), 3.16 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO):
δ = 172.1, 162.1, 157.9, 151.0, 142.9, 110.6, 50.9, 36.4, 27.4 ppm.
D
-α-(Uracil-5-yl)glycine [(R)-1a]: Yield 30%, white solid. [α]2D2
=
–51.4 (c = 0.1, DMF). Chiral HPLC [Reprosil Chiral-AM column
using hexane (with 0.1% TFA) and isopropanol (with 0.1% TFA),
70:30, at a flow rate of 1 mLmin–1]: 95% ee (as its Fmoc-protected
derivative).
IR ν = 3332, 3194, 2355, 1751, 1688, 1543, 1158, 880, 734,
˜
633 cm–1. HRMS (ESI): calcd. for C9H11N4O5 [M – H]– 255.0735;
found 255.0734. Rf (A) = 0.29.
D
-α-(N-1-Methyluracil-5-yl)glycine [(R)-1b]: Yield 55%, white solid.
[α]2D2 = –53.7 (c = 0.1, DMF). Chiral HPLC [Reprosil Chiral-AM
column using hexane (with 0.1% TFA) and isopropanol (with 0.1%
TFA), 70:30, at a flow rate of 1 mLmin–1]: 98% ee (as its Fmoc-
protected derivative).
General Procedure for the Synthesis of
D,L-α-(Uracil-5-yl)glycines
(1): A solution of NaNO2 (0.12 mmol, 1.20 equiv.) in H2O (0.5 mL)
was added dropwise over a period of 5 min to a solution of N-
carbamoyl amino acid 6 (0.10 mmol, 1.00 equiv.) in HCl (3.5 m aq.;
1 mL) at 0 °C. After complete conversion of the starting material
(typically 1 h), the mixture was applied to an Amberlyst 15(H) cat-
ion-exchange column. The column was washed with water to neu-
tral, and subsequently the pure amino acid 1 was eluted with
NH4OH (1 m aq.).
D
-α-(N,N-1,3-Dimethyluracil-5-yl)glycine [(R)-1c]: Yield 77%, white
solid. [α]2D2 = –31.6 (c = 0.1, DMF). Chiral HPLC [Reprosil Chiral-
AM column using hexane (with 0.1% TFA) and isopropanol (with
0.1% TFA), 90:10, at a flow rate of 1.5 mLmin–1]: 99% ee (as its
Fmoc-protected derivative).
D,L
-α-(Uracil-5-yl)glycine (1a): Yield 74%, white solid. 1H NMR
General Procedure for the Synthesis of Nα-Fmoc-(uracil-5-yl)glycines
(7): A solution of Fmoc-OSu (0.11 mmol, 1.10 equiv.) in MeCN
(500 μL) was added over a period of 5 min to a solution of amino
acid 1 (0.10 mmol, 1.00 equiv.) and Et3N (0.23 mmol, 2.30 equiv.)
in MeCN (500 μL) and H2O (500 μL), and the mixture was stirred
at room temp. overnight. The mixture was then washed with EtOAc
(2ϫ 1 mL), the aqueous phase was adjusted to pH = 3.5 with citric
acid (10% aq.), and then it was extracted with EtOAc (3ϫ 1 mL).
The combined organic fractions were dried with anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica.
(400 MHz, [D6]DMSO + 1 equiv. TFA): δ = 11.52 (s, 1 H), 11.30
(d, J = 5.9 Hz, 1 H), 8.36 (s, 3 H), 7.73 (d, J = 5.9 Hz, 1 H), 4.84
(s, 1 H) ppm. 13C NMR (101 MHz, [D6]DMSO + 1 equiv. TFA):
δ = 169.1, 163.1, 150.8, 143.9, 105.5, 49.6 ppm. IR ν = 3744, 1714,
˜
1706, 1695, 1671, 1508, 1380, 1224, 634, 613 cm–1. HRMS (ESI):
calcd. for C6H6N3O4 [M – H]– 184.0364; found 184.0364. Rf (A) =
0.15, yellow stain with ninhydrin.
D,L-α-(N-1-Methyluracil-5-yl)glycine (1b): Yield 70%, white solid.
1H NMR (400 MHz, [D6]DMSO + 1 equiv. TFA): δ = 11.52 (br. s,
1 H), 7.67 (s, 1 H), 5.42 (br. s, 3 H), 4.06 (s, 1 H), 3.26 (s, 3 H)
ppm. 13C NMR (101 MHz, [D6]DMSO + 1 equiv. TFA): δ = 166.8,
163.4, 150.8, 145.3, 109.5, 50.7, 35.4 ppm. In the 1H NMR spec-
trum, trace amounts of pyridine derived from the Amberlyst 15(H)
ion-exchange resin are visible, and this was taken into account
when calculating the product yield (see Supporting Information).
2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-2-(uracil-5-yl)acetic
Acid (7a): Yield 80 %, white solid, eluent: CHCl3/MeOH, 9:1 +
0.5% AcOH. 1H NMR (400 MHz, [D6]DMSO): δ = 11.10 (s, 1 H),
10.95 (br. s, 1 H), 7.88 (d, J = 7.7 Hz, 2 H), 7.69–7.68 (m, 2 H),
7.40 (t, J = 7.0 Hz, 2 H), 7.35–7.26 (m, 3 H), 7.22–7.06 (m, 1 H),
4.74 (d, J = 7.0 Hz, 1 H), 4.23–4.18 (m, 3 H) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 171.4, 163.7, 155.2, 151.2, 143.9,
143.8, 140.7, 127.6, 127.1, 125.3, 120.1, 119.2, 65.6, 51.9, 46.7 ppm.
IR ν = 3854, 3744, 3152, 1714, 1635, 1015, 848, 765, 671, 601 cm–1.
˜
HRMS (ESI): calcd. for C7H8N3O4 [M – H]– 198.0520; found
198.0520. Rf (A) = 0.16, yellow stain with ninhydrin.
D,L-α-(N,N-1,3-Dimethyluracil-5-yl)glycine (1c): Yield 60%, white
IR ν = 3854, 3744, 1706, 1683, 1635, 758, 737, 658, 614 cm–1
.
1
˜
solid. H NMR (400 MHz, [D6]DMSO + 1 equiv. TFA): δ = 8.44
(br. s, 3 H), 8.04 (s, 1 H), 4.82 (s, 1 H), 3.36 (s, 3 H), 3.20 (s, 3 H)
ppm. 13C NMR (101 MHz, [D6]DMSO + 1 equiv. TFA): δ = 168.8,
HRMS (ESI): calcd. for C21H16N3O6 [M – H]– 406.103362; found
406.103421. Rf (A) = 0.63. Chiral HPLC [Reprosil Chiral-AM col-
umn using hexane (with 0.1% TFA) and isopropanol (with 0.1%
TFA), 70:30, at a flow rate of 1 mLmin–1]: 49.2% (tR = 8.82 min)
and 50.8% (tR = 11.25 min).
161.7, 150.8, 146.3, 104.9, 50.2, 36.9, 27.6 ppm. IR ν = 1712, 1653,
˜
1473, 1456, 1340, 1185, 1123, 786, 665, 658 cm–1. HRMS (ESI):
calcd. for C8H10N3O4 [M – H]– 212.0677; found 212.0676. Rf (A)
= 0.16, yellow stain with ninhydrin.
2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-2-(1-methyluracil-
5-yl)acetic Acid (7b): Yield 72 %, white solid, eluent: CHCl3/
MeOH, 10:1 + 0.5% AcOH. 1H NMR (400 MHz, [D6]DMSO +
General Procedure for the Enzymatic Dynamic Kinetic Resolution to
give
D-α-(Uracil-5-yl)glycines [(R)-1]: Immobilized d-Hydantoinase-
6628
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6624–6630

Enantioselective Synthesis of d-α-(Uracil-5-yl)glycine Derivatives
1 equiv. AcOH): δ = 12.39 (br. s, 1 H), 11.49 (s, 1 H), 7.89 (d, J =
7.5 Hz, 2 H), 7.73 (d, J = 7.5 Hz, 2 H), 7.70 (s, 1 H), 7.56 (d, J =
8.1 Hz, 1 H), 7.41 (t, J = 7.5 Hz, 2 H), 7.34–7.30 (m, 2 H), 4.99 (d,
J = 8.1 Hz, 1 H), 4.32–4.19 (m, 3 H), 3.25 (s, 3 H) ppm. 13C NMR
(101 MHz, [D6]DMSO + 1 equiv. AcOH): δ = 172.8, 162.9, 155.7,
150.8, 145.0, 143.8, 140.7, 127.7, 127.1, 125.3, 120.1, 109.4, 65.9,
163.2, 151.0, 140.4, 109.6, 48.8, 22.3 ppm. IR ν = 1739, 1718, 1662,
˜
1224, 1117, 1010, 980, 882, 707, 603 cm–1. HRMS (ESI): calcd. for
C8H8N3O5 [M – H]– 226.0469; found 226.0470. Rf (A) = 0.33.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental details, spectroscopic data, HPLC/HPLC–MS
chromatograms, and copies of 1H and 13C NMR spectra for all
new compounds.
50.5, 46.6, 35.5 ppm. IR ν = 3744, 1751, 1739, 1717, 1695, 1635,
˜
1365, 1362, 1225, 1207 cm–1. HRMS (ESI): calcd. for C22H18N3O6
[M – H]– 420.1201; found 420.1203. Rf (CHCl3/MeOH, 9:1 + 0.5%
AcOH) = 0.27. Chiral HPLC [Reprosil Chiral-AM column using
hexane (with 0.1% TFA) and isopropanol (with 0.1% TFA), 70:30,
at a flow rate of 1 mLmin–1]: 49.5% (tR = 18.8 min) and 50.5% (tR
= 22.5 min).
Acknowledgments
This work was financially supported by the German Baden-
Württemberg Stiftung (Juniorprofessorenprogramm) and the
Fonds der Chemischen Industrie (general financial support and
Ph. D. stipend to N. M. W.). Prof. Dirk Schwarzer is thanked for
SPPS and HPLC support, and for helpful discussions.
2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-2-(1,3-dimethylura-
cil-5-yl)acetic Acid (7c): Yield 85 %, white solid, eluent: CHCl3/
MeOH, 20:1 + 0.5% AcOH. 1H NMR (400 MHz, [D6]DMSO +
1 equiv. AcOH): δ = 7.89–7.87 (m, 2 H), 7.77–7.74 (m, 3 H), 7.54
(d, J = 8.2 Hz, 1 H), 7.43–7.39 (m, 2 H), 7.33–7.29 (m, 2 H), 5.05
(d, J = 8.2 Hz, 1 H), 4.37–4.18 (m, 3 H), 3.32 (s, 3 H), 3.18 (s, 3
H) ppm. 13C NMR (101 MHz, [D6]DMSO + 1 equiv. AcOH): δ =
172.8, 161.9, 155.7, 150.9, 143.8, 143.4, 140.7, 127.6, 127.1, 125.3,
[1] K. Lang, J. W. Chin, Chem. Rev. 2014, 114, 4764–4806.
[2] S. A. Pless, C. A. Ahern, Annu. Rev. Pharmacol. Toxicol. 2013,
53, 211–229.
[3] L. Wang, P. G. Schultz, Chem. Commun. 2002, 1–11.
[4] J. Xie, P. G. Schultz, Nat. Rev. Mol. Cell Biol. 2006, 7, 775–
782.
120.1, 108.6, 65.9, 51.1, 46.6, 36.6, 27.6 ppm. IR ν = 1751, 1684,
˜
1635, 1557, 1375, 1362, 1225, 1205, 639, 617 cm–1. HRMS (ESI):
calcd. for C23H20N3O6 [M – H]– 434.1358; found 434.1358. Rf
(CHCl3/MeOH, 9:1 + 0.5% AcOH) = 0.48. Chiral HPLC [Reprosil
Chiral-AM column using hexane (with 0.1% TFA) and isoprop-
anol (with 0.1 % TFA), 90:10, at a flow rate of 1.5 mL min–1]:
49.7% (tR = 115.5 min) and 50.3% (tR = 126.4 min).
[5] I. Kwon, S. I. Lim, Macromol. Chem. Phys. 2013, 214, 1295–
1301.
[6] N. Budisa, Curr. Opin. Biotechnol. 2013, 24, 591–598.
[7] K. Wang, W. H. Schmied, J. W. Chin, Angew. Chem. Int. Ed.
2012, 51, 2288–2297; Angew. Chem. 2012, 124, 2334–2344.
[8] A. El Marrouni, M. Heras, Org. Biomol. Chem. 2015, 13, 851–
858.
2-[(tert-Butyloxycarbonyl)amino]-2-(2,4-dioxo-1,2,3,4-tetrahydro-
pyrimidin-5-yl)acetic Acid (8): A solution of d,l-α-(uracil-5-yl)-
glycine (1a; 0.20 mmol, 1.00 equiv.) in saturated aq. NaHCO3 solu-
tion (1 mL) was cooled in an ice bath, and a solution of Boc2O
(0.22 mmol, 1.10 equiv.) in THF (1 mL) was added dropwise. The
mixture was stirred for 19 h at room temp. The volatile components
were removed from the mixture under reduced pressure, then the
residue was cooled to 0 °C and acidified to pH = 2 by the addition
of HCl (3 m aq.). The solution was extracted with EtOAc (3ϫ
4 mL). The combined organic fractions were dried with anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography (EtOAc/MeOH,
6:1 + 0.5% AcOH) to give 8 (49 mg, 0.17 mmol, 86%) as a white
solid. 1H NMR (600 MHz, [D6]DMSO): δ = 10.98 (br. s, 2 H), 7.27
(s, 1 H), 6.42 (s, 1 H), 4.56 (s, 1 H), 1.35 (s, 9 H) ppm. 13C NMR
(150 MHz, [D6]DMSO): δ = 172.9, 164.0, 154.5, 151.3, 139.3,
[9] R. Gmelin, Hoppe-Seyler’s Z. Physiol. Chem. 1959, 316, 164.
[10] F. Lambein, R. Van Parijs, Biochem. Biophys. Res. Commun.
1968, 32, 474–479.
[11] J. C. A. More, H. M. Troop, N. P. Dolman, D. E. Jane, Br. J.
Pharmacol. 2003, 138, 1093–1100.
[12] P. B. Hitchcock, K. Papadopoulos, D. W. Young, Org. Biomol.
Chem. 2003, 1, 2670–2681.
[13] G. N. Roviello, E. Benedetti, C. Pedone, E. M. Bucci, Amino
Acids 2010, 39, 45–57.
[14] R. Uozumi, T. Takahashi, T. Yamazaki, V. Granholm, H. Mih-
ara, J. Pept. Sci. 2010, 94, 830–842.
[15] S. Matsumura, T. Takahashi, A. Ueno, H. Mihara, Chem. Eur.
J. 2003, 9, 4829–4837.
[16] H. Miyanishi, T. Takahashi, H. Mihara, Bioconjugate Chem.
2004, 15, 694–698.
[17] G. N. Roviello, C. Crescenzo, D. Capasso, S. Di Gaetano, S.
Franco, E. M. Bucci, C. Pedone, Amino Acids 2010, 39, 795–
800.
[18] X. Fan, X. Zhang, C. Bories, P. M. Loiseau, P. F. Torrence,
Bioorg. Chem. 2007, 35, 121–136.
[19] C. Syldatk, A. Läufer, R. Müller, H. Höke, in: Microbial Bio-
products, vol. 41, Springer, Berlin/Heidelberg, Germany, 1990,
p. 29–75.
[20] J. Ogawa, N. Horinouchi, S. Shimizu, in: Enzyme Catalysis in
Organic Synthesis Wiley-VCH, Weinheim, Germany, 2012, p.
651–674.
[21] R. Sharma, R. M. Vohra, Curr. Sci. 1999, 77, 127–136.
[22] O. Keil, M. P. Schneider, J. P. Rasor, Tetrahedron: Asymmetry
1995, 6, 1257–1260.
112.9, 77.7, 52.3, 28.3 ppm. IR ν = 1733, 1706, 1684, 1635, 1365,
˜
1010, 999, 938, 656, 601 cm–1. HRMS (ESI): calcd. for C11H14N3O6
[M – H]– 284.0888; found 284.0889. Rf (A) = 0.44.
2-Acetamido-2-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)acetic
Acid (9): A suspension of d,l-α-(uracil-5-yl)glycine (1a; 0.20 mmol,
1.00 equiv.) in H2O/DMF (1:1; 1 mL) was cooled in an ice bath.
Ac2O (0.22 mmol, 1.10 equiv.) and Et3N (0.46 mmol, 2.30 equiv.)
were added dropwise, and the mixture was stirred for 24 h at room
temperature. The reaction mixture was cooled to 0 °C, and acidified
to pH = 2 with HCl (3 m aq.). The mixture was extracted with
EtOAc (3ϫ 4 mL). The combined organic fractions were dried with
anhydrous Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography (EtOAc/
MeOH, 4:1 + 0.5% AcOH) to give 9 (34 mg, 0.15 mmol, 75%) as
a white solid. 1H NMR (400 MHz, [D6]DMSO): δ = 12.60 (s, 1 H),
11.24 (s, 1 H), 10.92 (d, J = 5.5 Hz, 1 H), 8.22 (d, J = 7.9 Hz, 1
H), 7.38 (d, J = 5.5 Hz, 1 H), 5.18 (d, J = 7.9 Hz, 1 H), 1.85 (s, 3
H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ = 171.4, 169.0,
[23] For a detailed description of the synthesis of 5-(formyl)uracils
4a–4c starting from uracil, see Supporting Information.
[24] H. T. Bucherer, W. Steiner, J. Prakt. Chem. 1934, 140, 291–316.
[25] H. Collet, L. Boiteau, J. Taillades, A. Commeyras, Tetrahedron
Lett. 1999, 40, 3355–3358.
[26] Y. Han, F. Albericio, G. Barany, J. Org. Chem. 1997, 62, 4307–
4312.
[27] D. B. Li, J. A. Robinson, Org. Biomol. Chem. 2005, 3, 1233–
1239.
Eur. J. Org. Chem. 2015, 6624–6630
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6629

N. M. Weckenmann, B. J. Nachtsheim
FULL PAPER
[28] H. Li, X. Jiang, Y.-h. Ye, C. Fan, T. Romoff, M. Goodman,
Org. Lett. 1999, 1, 91–94.
[29] F. Dettner, A. Hänchen, D. Schols, L. Toti, A. Nußer, R. D.
Süssmuth, Angew. Chem. Int. Ed. 2009, 48, 1856–1861; Angew.
Chem. 2009, 121, 1888–1893.
[30] J. D. Wade, J. Bedford, R. C. Sheppard, G. W. Tregear, Pept.
Res. 1991, 4, 194.
[31] M. Dettin, S. Pegoraro, P. Rovero, S. Bicciato, A. Bagno, C.
Di Bello, J. Pept. Res. 1997, 49, 103–111.
[32] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398.
[33]
[34]
A. El-Faham, R. S. Funosas, R. Prohens, F. Albericio, Chem.
Eur. J. 2009, 15, 9404–9416.
C. Brieke, M. J. Cryle, Org. Lett. 2014, 16, 2454–2457.
[35] C. Brieke, V. Kratzig, K. Haslinger, A. Winkler, M. J. Cryle,
Org. Biomol. Chem. 2015, 13, 2012–2021.
[36] L. A. Carpino, D. Ionescu, A. El-Faham, J. Org. Chem. 1996,
61, 2460–2465.
Received: July 9, 2015
Published Online: September 9, 2015
6630
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6624–6630