Synthesis of a conformationally constrained δ-amino acid building block

Article abstract of DOI:10.1007/s00726-012-1362-3

Conformationally restricted amino acids are important components in peptidomimetics and drug design. Herein, we describe the synthesis of a novel, non-proteinogenic constrained delta amino acid containing a cyclobutane ring, cis-3(aminomethyl)cyclobutane

Full text of DOI:10.1007/s00726-012-1362-3

Amino Acids (2013) 44:511–518
DOI 10.1007/s00726-012-1362-3
ORIGINAL ARTICLE
Synthesis of a conformationally constrained d-amino acid
building block
•
Elaine O’Reilly Lara Pes Yannick Ortin
•
•
•
Helge Mu¨ller-Bunz Francesca Paradisi
Received: 11 May 2012 / Accepted: 6 July 2012 / Published online: 1 August 2012
Ó Springer-Verlag 2012
Abstract Conformationally restricted amino acids are
important components in peptidomimetics and drug design.
Herein, we describe the synthesis of a novel, non-protein-
ogenic constrained delta amino acid containing a cyclo-
butane ring, cis-3(aminomethyl)cyclobutane carboxylic
acid (ACCA). The synthesis of the target amino acid was
achieved in seven steps, with the key reaction being a base
induced intramolecular nucleophilic substitution. A small
library of dipeptides was prepared through the coupling of
ACCA with proteinogenic amino acids.
peptidases (Giannis and Kolter 1993). Another limitation
associated with the use of peptides as drugs is their high
hydrophilicity and high flexibility. Peptidomimetics have
been designed to mimic bioactive peptides and display
enhanced pharmacological properties, such as increased
bioavailability and metabolic stability (Gante 1994; Smith
et al. 2002; Giannis and Kolter 1993). Further to this, it is
proposed that appropriate conformational restriction of
lead compounds can increase the binding affinity of the
drug for its receptor (Boger et al. 2000). Conformationally
constrained amino acids are a useful way of tailoring the
rigidity of peptides. There have been a number of reports
on the synthesis of conformationally constrained amino
acids (Miller and Grubbs 1995) and in particular, great
interest has been shown for amino acids containing a
cyclobutane moiety (Burgess et al. 1997; Aguilera et al.
2008a; Andre’ et al. 2011). Some of these residues have
been incorporated into peptides (Aguilera et al. 2008b),
peptide dendrimers (Gutierrez-Abad et al. 2010) and even
into nucleotides (Fernandez et al. 1995). The presence of
these amino acids can lead to unusual conformations and
hydrogen-bonding interactions in the modified dipeptides
(Izquierdo et al. 2005; Gorrea et al. 2011) and peptides
(Fernandez-Tejada et al. 2009; Torres et al. 2009;
Keywords Non-proteinogenic amino acid ꢀ Dipeptides ꢀ
Conformational restriction ꢀ Cyclobutane ring
Introduction
Many neurotransmitters, neuromodulators and hormones
are peptides which interact with receptors and effect bio-
logical processes. Peptide agonists or antagonists have the
potential to be used as therapeutic agents by mimicking the
messenger molecule and disrupting the biological process.
However, studies have shown that bioactive peptides often
display poor bioavailability and can easily be degraded by
´
Gutierrez-Abad et al. 2011; Celis et al. 2012).
The incorporation of ACCA into peptides can result in
well-defined conformations and molecular rigidity.
Cyclobutane rings retain considerable flexibility due to
flipping between the two conformations through low
energy barriers and this may favour interactions between
peptidomimetics and receptors or active sites (Allan et al.
1990).
E. O’Reilly and L. Pes contributed equally to this paper.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1362-3) contains supplementary
material, which is available to authorized users.
E. O’Reilly ꢀ L. Pes ꢀ Y. Ortin ꢀ H. Mu
¨ller-Bunz ꢀ
F. Paradisi (&)
Centre for Synthesis and Chemical Biology,
UCD School of Chemistry and Chemical Biology,
Belfield, Dublin 4, Ireland
Further to their application in the field of peptidomi-
metics, cyclobutane derivatives have been widely exploited
in organic synthesis (Lee-Ruff and Mladenova 2003;
e-mail: francesca.paradisi@ucd.ie
123

512
E. O’Reilly et al.
Namyslo and Kaufmann 2003) and the strained motif
features in several natural products and synthetic com-
pounds which often display interesting biological proper-
ties (Dembitsky 2008).
that only the cis isomer was obtained upon hydrolysis due
to 8 being ‘‘conformationally locked’’. 1H NMR data
suggested that no epimerization had occurred during
hydrolysis and the X-ray crystal structure of the Boc-pro-
tected amino acid 13 supported this result (Fig. 3). In order
to investigate the possibility of epimerisation, amino acid 1
was also treated with a base [NaOH (2 M) and stirred
In this context, we describe a seven steps synthesis for the
preparation of a novel achiral, conformationally constrained
amino acid (Fig. 1). An enantioselective synthesis of the 2,2-
dimethyl derivative of 1 has been previously reported starting
from a-pinene (Fernandez et al. 1995). We have also syn-
thesised a small library of dipeptides by coupling amino acid
1 with the proteinogenic amino acids glycine, valine and
phenylalanine as well as self-coupling of 1.
1
overnight at 100 °C] with no changes noticed in this H-
NMR spectrum denoting a great stability of this compound.
A small library of dipeptides containing ACCA 1 was
then prepared (Schemes 2, 3). The carboxylic acid moiety
was first protected through esterification. Lewis acids such
as TiCl4 and ZrCl4 have previously been employed for
highly successful esterifications (Singh et al. 2008; Shang
et al. 2007). A catalytic quantity of zirconium chloride was
found to be an effective catalyst for the esterification of
amino acid 1. The reaction proceeded with high efficiency
in n-propanol yielding amino ester 9 in 92 % yield.
Results and discussion
Our approach to the synthesis of amino acid 1 involved the
construction of nitrogen heterocycle 3 (Scheme 1), whose
synthesis in two steps has previously been reported (Cook
et al. 1994). The reaction involved the formation of an
enamine via the conjugate addition of benzylamine to
methyl propiolate 2, followed by the aza-annulation reac-
tion with acryloyl chloride, giving ester 3 in 53 % yield
over 2 steps (Cook et al. 1994). Hydrogenation of 3 gave
racemic ester 4 in 97 % yield. Treatment of 4 with lithium
borohydride afforded racemic alcohol 5, which was con-
verted to the iodo-derivative 6 passing through a mesylated
intermediate with 93 % yield over the 2 steps. Direct
iodination of 5 was also achieved, albeit with lower yield
(Garegg and Samuelsson 1980). It is noteworthy that a
kinetic resolution of racemic ester 4 has previously been
reported, providing access to the enantio-enriched com-
pounds in high yields (Gray and Gallagher 2006). In the
same study, the direct bromination of 5 has also been
reported. Treatment of 6 with LHMDS resulted in a rapid
intramolecular nucleophilic substitution, yielding 7 in
93 % yield. This type of intramolecular cyclisation has
been previously reported. Galeazzi et al. employed this
strategy for the synthesis of both enantiomers of cis-2-
aminomethylcyclobutane (Galeazzi et al. 1999). Com-
pound 7 was deprotected under Birch conditions affording
bicyclic derivative 8 in 94 % yield. The formation of 8 was
confirmed by X-ray crystallography (Fig. 2).
O
O
O
CO2Me
BnN
O
BnN
O
BnN
a)
b)
c)
OMe
OMe
OH
2
3
4
5
O
O
O
d)
BnN
e)
f)
BnN
HN
I
7
8
6
O
OH
Cl
g)
H
H3N
H
1
Scheme 1 Synthesis of conformationally constrained amino acid salt
1ꢀHCl. Reagents and conditions: a) (i) BnNH2, Et2O, -10 °C rt, 1 h;
(ii) acryloyl chloride, THF, rt, 12 h 53 %; b) H2, Pd/C, Na2CO3,
EtOH, rt, 12 h, 97 %; c) LiBH4, THF, 0 °C rt, 12 h, 77 %; d)
(i) CH3SO2Cl, TEA, CH2Cl2, -10 °C rt, 16 h; (ii) NaI, acetone,
reflux, 24 h, 93 %; or PPh3, imidazole, I2, toluene, 3 h, 68 %; e)
LHMDS, THF, -20 °C, 1 h, 93 %; f) NH3(l), Li(s), THF, tBuOH,
-78 °C, 94 %; g) HCl 2 M, reflux, 12 h, 99 %
Acidic hydrolysis of 8 afforded amino acid salt 1ꢀHCl in
quantitative yield. The nature of the cyclic precursor means
O
OH
H
H3N
H
1
Fig. 1 d-Amino acid 1
Fig. 2 X-ray crystal structure of bicyclic amide 8
123

Synthesis of a conformationally constrained d-amino acid building block
513
O
could not be isolated with a sufficient degree of purity.
Triethylamine yielded the best results, and compound 12c
was obtained after an overnight reflux and subsequent
purification with ion exchange chromatography in excel-
lent yields. The ion exchange resin purification step was
sufficient to catalyse also the hydrolysis of the ester pro-
tecting group, affording the free carboxylic acid. The
efficiency of this hydrolysis technique was already dem-
onstrated in the literature (Basu et al. 1989; Qureshi et al.
1968).
O
OH
O
N
H
H
H
13
Experimental
Fig. 3 X-ray crystal structure of Boc-protected ACCA 13
General methods
Amino acids 10 a–c were protected at the N-terminus with
tert-Butyloxycarbonyl (t-Boc) or 9-fluorenylmethylox-
ycarbonyl(Fmoc)andthencoupledwithester9 toachievethe
protected dipeptides 11 a–c. Best results were achieved using
O-(7-Azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU) as the coupling agent. Base-
catalysed hydrolysis was employed for deprotection of the
C-terminus ester. The deprotection of the N-terminus Fmoc
took place under the same conditions while the deprotection
of the N-terminus t-Boc in 11a was achieved under standard
acidic conditions using HCl/Et2O. In this case, the non-
aqueous conditions aid isolation of the deprotected amino
acid, which precipitates as a solid.
All chemicals were purchased from Sigma Aldrich. Dry
solvents were obtained from a Puresol Grubbs Speciality
Chemicals and Stream. When necessary, all reactions were
performed under an atmosphere of nitrogen using oven
dried glassware. Oxygen-free nitrogen was obtained from
BOC gases and used without further drying.
Proton and carbon nuclear magnetic resonance spectra (1H
and 13C-NMR, respectively) were recorded on 400 MHz
(operating frequencies: 1H, 399.75 MHz; 13C, 101.00),
500 MHz (operating frequencies: 1H, 499.72 MHz; 13C,
125.65) and 600 MHz (operating frequencies: 1H,
599.78 MHz; 13C, 150.82) FT spectrometers. Tetramethyl-
silane was used as an internal reference in the deuterated
The same strategy was followed for the synthesis of 15.
NMR spectra of the dipeptides indicated the formation of a
single product, again with no epimerization observed.
In the case of the Gly-ACCA dipeptide (12c), unusual
1
chloroform (CDCl3) (d = 0.00 ppm) for H NMR spectra.
The middle CDCl3 solvent peak was referenced to 77.16 ppm
for 13C NMR spectra. The residual solvent peak in deuterated
methanol (CD3OD) was referenced to 3.31 and 49.00 ppm for
1H NMR and 13C NMR spectra, respectively. The residual
solvent peak in deuterated water (D2O) was referenced to
4.79 ppm for 1H NMR. The coupling constants (J) are in Hz
and the chemical shifts (d) are given in parts per million. High
resolutionmassspectrawereobtainedonaWaters/Micromass
instrument. Melting points are uncorrected. Evaporation in
vacuo refers to the removal of solvent on a Bu¨chi rotary
1
behaviour was observed. Initially, H-NMR analysis indi-
cated a splitting of the signals, which was attributed to
epimerization of the compound. However, 2D NMR stud-
ies and variable temperature NMR experiments indicated
that neither diasteromers nor rotamers were involved in the
phenomenon. To investigate this further, an alternative
stepwise strategy was adopted for the deprotection of
compound 11c. Piperidine was initially employed for the
selective cleavage of the Fmoc group, however, the product
O
O
OnPr
O
OH
OnPr
H
O
O
H
(a)
(b)
H
N
COOH
Cl
H2N
N
H
H
GP
N
N
H
+
GP
H3N
H
H
H
R
H
R
H
H
9
11 a-c
12 a-c
10 a-c
PG = Fmoc or Boc
R = CH2Ph (Phe)
CH(CH3)2 (Val)
H (Gly)
Scheme 2 Synthesis of dipeptides 12 a–c. Reagents and conditions:
i
a) HATU, Pr2EtN, CH2Cl2, 18 h, 38–92 %; b) for 12a: (i) NaOH
2 M, CH3CN, 45 °C, 3 h; (ii) HCl/Et2O, CH2Cl2, 54 %; for 12b:
NaOH 2 M, dioxane/water 7:3, 45 °C, 3 h, 90 %; for 12c: CH2Cl2/
TEA 1:1, 45 °C, 24 h, 90 %
123

514
E. O’Reilly et al.
Cl
O
OnPr
O
O
O
OnPr
OH
BocHN
H3N
Cl
OH
O
O
(a)
(b)
+
H
H3N
BocHN
H
H
H
H
H
N
N
H
H
H
H
H
H
H
H
9
13
14
15
i
Scheme 3 Synthesis of self-coupling dipeptide 15. Reagents and conditions: a) HATU, Pr2EtN, CH2Cl2, 18 h, 58 %; b) (i) NaOH 2 M,
CH3CN, 45 °C, 3 h; (ii) HCl/Et2O, CH2Cl2 57 %
evaporator with an integrated vacuum pump. Thin-layer
chromatography (TLC) was carried out on aluminium backed
60 F254 silica gel.
stirred overnight. The organic layer was concentrated in
vacuo and dissolved in acetone (50 mL) and NaI (3.98 g,
26.58 mmol) was added to the stirring solution. The mix-
ture was stirred under reflux for 24 h. The organic solvent
was removed in vacuo and H2O (40 mL) was added along
with EtOAc (50 mL), the organic layer was separated and
the aqueous layer was extracted with EtOAc (5 9 20 mL).
The organic layers were combined, washed with brine,
dried over MgSO4 and concentrated in vacuo. The crude oil
was purified by silica gel column chromatography (cyclo-
hexane/EtOAc, 70:30) to give 6 (3.36 g, 96 % yield) as a
yellow/orange oil. (Rf = 0.12, cyclohexane/EtOAc,
50:50). dH (500 MHz; CDCl3) 7.3–7.22 (5H, m, PhH),
4.63 (1H, d, J = 14.7, PhCH2), 4.54 (1H, d, J = 14.7,
PhCH2), 3.34 (1H, m, NCH2), 3.08 (2H, m, CH2I), 2.97
(1H, dd, J = 12.0 and 9.5, NCH2), 2.56 (1H, ddd,
J = 17.7, 6.2 and 3.6, C = OCH2), 2.45 (1H, ddd,
J = 17.7, 11.2 and 6.2, C = OCH2), 2.06–1.95 (2H, m,
C = OCH2CH2), 1.66–1.54 (1H, m, CHCH2); dC
(126 MHz; CDCl3) 169.0, 136.8, 128.5, 128.0, 127.4, 52.3,
50.1, 35.9, 30.7, 27.7, 7.9; m/z (EI) 329.0273 (M ? H?.
C13H16INO requires 329.0277).
Compounds 3 and 4 were prepared as described in the
work of Cook and colleagues (1994). Methods and char-
acterization are reported in the supporting information.
Preparation of ( )1-benzyl-5-(hydroxymethyl)piperidin-2-
one (5)
( )4 (5.23 g, 23.85 mmol) was dissolved in anhydrous
THF (35 mL) and cooled to 0 °C. LiBH4 (1.04 g,
47.70 mmol) was added and the solution was allowed to
warm to room temperature and stirred overnight. The
reaction was quenched at 0 °C with water (20 mL) and
then with 10 % HCl. The organic layer was separated and
the aqueous layer was extracted with EtOAc (4 9 20 mL).
The organic layers were combined, dried over MgSO4 and
concentrated in vacuo. The crude oil was purified by silica
gel column chromatography (cyclohexane/EtOAc, 60:40)
to give ( )7 (4.03 g, 77 % yield) as a clear oil. (Rf = 0.11,
cyclohexane/EtOAc, 50:50). dH (600 MHz; CDCl3)
7.33–7.18 (5H, m, PhH), 4.59 (1H, d, J = 14.6, PhCH2),
4.53 (1H, d, J = 14.6, PhCH2), 3.53 (1H, dd, J = 10.7 and
5.6, CH2OH), 3.44 (1H, dd, J = 10.7 and 7.2, CH2OH),
3.30 (1H, ddd, J = 12.2, 5.2 and 1.5, NCH2), 2.99 (1H, dd,
J = 12.2 and 10.1, NCH2), 2.92 (1H, br s, OH), 2.51 (1H,
ddd, J = 17.8, 5.8 and 3.4, C = OCH2), 2.39 (ddd,
J = 17.8, 11.3 and 6.5, C = OCH2), 2.04–1.95 (1H, m,
OHCH2CH), 1.89–1.82 (1H, m, C = OCH2CH2), 1.54–
1.46 (1H, m, C = OCH2CH2); dC (151 MHz; CDCl3)
170.0, 137.0, 128.5, 127.9, 127.3, 64.3, 50.3, 49.8, 36.4,
31.1, 23.8; m/z (ES) 220.1329 (M ? H? C13H18NO2
requires 220.1338).
Preparation of 3-benzyl-3-aza-bicyclo[3.1.1]heptan-2-one
(7)
( )6 (3.2 g, 9.72 mmol) was dissolved in anhydrous THF
(5 mL) and LHMDS (1 M soln./THF, 9.72 mL) was added at
-20 °C. The solution was stirred for 1 h before being
quenched with H2O (15 mL). EtOAc (20 mL) was added and
the organic layer was separated. The aqueous layer was
extracted with EtOAc (3 9 15 mL). The organic layers were
combined, dried over MgSO4 and concentrated in vacuo. The
crude oil was filtered through a short silica column (cyclo-
hexane/EtOAc, 2:1) to give 7 (1.82 g, 93 % yield) as a yel-
low/orange oil. (Rf = 0.24, cyclohexane/EtOAc, 50:50). dH
(500 MHz; CDCl3) 7.36–7.22 (5H, m, PhH), 4.61 (2H, s,
PhCH2), 3.26 (2H, d, J = 1.5, NCH2), 2.84 (1H, q,
J = 5.7 Hz, C = OCH), 2.72–2.65 (1H, m, NCH2CH),
2.38–2.28 (2H, m, C = OCH(CHH)2, 1.73–1.63 (2H, m,
C = OCH(CHH)2; dC (126 MHz; CDCl3) 175.7, 137.4,
128.5, 128.0, 127.2, 50.0, 48.0, 41.1, 33.2, 30.9; m/z (ES)
202.1222 (M ? H? C13H16NO requires 202.1232).
Preparation of ( ) 1-benzyl-5-(iodomethyl)piperidin-2-one
(6)
( )5 (3.92 g, 13.18 mmol) was dissolved in anhydrous
CH2Cl2 (25 mL). The solution was cooled to -10 °C and
TEA (2.0 g, 19.77 mmol) was added, followed by the slow
addition of CH3SO2Cl (1.81 g, 15.19 mmol). The solution
was allowed to warm slowly to room temperature and
123

Synthesis of a conformationally constrained d-amino acid building block
515
Preparation of 3-aza-bicyclo[3.1.1]heptan-2-one (8)
(101 MHz; CD3OD) 176.7, 64.7, 45.4, 34.8, 30.1, 29.9,
26.6, 10.5; m/z (ES?) 172.1342 (M ? H? C9H18NO2
requires 172.1338).
7 (1.5 g, 7.45 mmol) was dissolved in anhydrous THF/
tBuOH, 10:1 (10 mL) and the mixture was added to a
stirring liquid ammonia at -78 °C. Metallic Li pellets were
added slowly to the stirring solution until a constant blue/
black colour was observed. The reaction was then quen-
ched by the addition of solid ammonium chloride. After
removal of the NH3, EtOAc (30 mL) and H2O (8 ml) were
added. The organic layer was separated and the aqueous
layer was extracted with EtOAc (3 9 15 mL). The organic
layers were combined, dried over MgSO4 and concentrated
in vacuo. The crude oil was filtered through a short silica
column (cyclohexane/EtOAc, 1:1) to give 8 (800 mg, 94 %
yield) as a yellow oil. (Rf = 0.1, cyclohexane/EtOAc,
50:50). dH (500 MHz; CD3OD) 7.46–7.13 (1H, m, NH)
3.43 (2H, d, J = 2.0, NCH2), 2.81–2.71 (1H, m,
NCH2CH), 2.60 (1H, q, J = 5.6 Hz, C = OCH), 2.48–2.37
(2H, m, C = OCH(CHH)2, 1.70–1.62 (2H, m,
C = OCH(CHH)2). dC (126 MHz; CD3OD) 181.5, 46.3,
42.0, 34.3, 31.4; m/z (EI?) 111.0684 (M ? H? C6H9NO
requires 111.0684).
Preparation of 3-(((tert-butoxycarbonyl)amino)
methyl)cyclobutanecarboxylic acid (13)
1 HCl (150 mg, 0.9 mmol) was dissolved in dioxane and
water 2:1 (12 mL) and cooled to 0 °C. 1 M NaOH (5 mL)
was added and the solution was stirred for 10 min before
adding 2 eq of bis-tert-butyl dicarbonate (390 mg,
1.8 mmol). The reaction was left under stirring for 3 h
before being concentrated in vacuo. The aqueous layer was
extracted with Et2O (15 mL 9 2) and adjusted to pH 3 by
the addition of 1 M HCl. The acidified aqueous layer was
extracted with CH2Cl2 (15 mL 9 5) and the combined
CH2Cl2 layers were dried over MgSO4 and concentrated in
vacuo to give 13 (227 mg, 95 % yield) as a brown solid.
No further purification was required. (Rf = 0.28, EtOAc/
CH3OH 90:10). Mp: 63–65 °C. dH (400 MHz; CDCl3)
5.99 (1H, br s, COOH), 4.61 (1H, br s, NH), 3.13 (2H, s,
NCH2), 3.07–2.98 (1H, quin, J 8.9 Hz, C = OCH),
2.51–2.36 (1H, m, NCH2CH), 2.37–2.25 (2H, m,
C = OCH(CHH)2, 2.00–2.09 (2H, m, C = OCH(CHH)2,
1.44 (9H, s, C(CH3)3); dC (101 MHz; CDCl3) 180.8, 156.5,
79.5, 45.7, 34.5, 31.4, 28.8, 28.4; m/z (ES?) 252.1212
(M ? Na? C11H19NO4Na requires 252.1212).
Preparation of cis-3-(aminomethyl)cyclobutanecarboxylic
acid (1 HCl)
8 (220 mg, 1.98 mmol) was refluxed in 2 M HCl (10 mL)
overnight. The H2O was removed in vacuo to give 1
(328 mg, quantitative yield) as a beige solid with no further
purification required. Mp: 169–170 °C. dH (500 MHz;
CD3OD) 3.18–3.08 (1H, m, C = OCH), 2.96 (2H, d,
J = 7.3, NCH2), 2.63–2.51 (1H, m, NCH2CH), 2.46–2.37
General method: coupling of protected dipeptides
9, the N-protected amino acid (10a–c and 13), and
O-(7-Azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU, 1 eq) were dissolved in
anhydrous THF (20 mL). The solution was cooled to 0 °C
and N,N-diisopropylethyl amine (3 eq) was added. The
reaction was stirred at room temperature overnight. The
solvent was evaporated in vacuo and the crude oil dis-
solved in CH2Cl2 (15 mL) and washed with NaHCO3
(15 mL), acetic acid 10 % (15 mL 9 2) and brine
(15 mL). The combined organic layers were dried over
MgSO4 and concentrated in vacuo.
(2H,
m,
C = OCH(CHH)2,
2.02
(2H,
m,
C = OCH(CHH)2; dC (126 MHz; CD3OD) 176.8, 45.5,
34.9, 30.2, 29.9; m/z (ES?) 130.0863 (M ? H? C6H12NO2
requires 130.0868).
Preparation of (3-(Propoxycarbonyl)cyclobutyl)
methanaminium chloride (9)
1ꢀHCl (370 mg, 2.86 mmol) was dissolved in n-propanol
(10 mL) and ZrCl4 (133 mg, 0.572 mmol, 20 mol %) was
added. The solution was refluxed for 24 h before being
concentrated in vacuo. The crude oil was dissolved in
CHCl3 (2 mL) and left overnight. The precipitate was fil-
tered off and the solution concentrated in vacuo to give 11
(570 mg, 92 % yield) as an off-white solid. Mp:
159–160.5 °C. dH (400 MHz; CD3OD) 3.46 (2H, t,
J = 6.7, CH2CH2CH3), 3.08 (1H, m, C = OCH), 2.93 (2H,
d, J = 7.4, NCH2), 2.60–2.49 (1H, m, NCH2CH),
2.43–2.33 (2H, m, C = OCH(CHH)2, 1.98 (2H, ddd,
J = 18.7, 9.3 and 2.4, C = OCH(CHH)2, 1.56–1.43 (2H,
m, CH2CH3), 0.88 (3H, t, J = 7.4, CH2CH3); dC
Preparation of propyl (S)-3-((2-((tert-
butoxycarbonyl)amino)-3-
phenylpropanamido)methyl)cyclobutanecarboxylate (11a)
11a was prepared following the General Procedure, by
reacting 9 (300 mg, 1.45 mmol) with N-Boc-L-Phenylala-
nine 10a (500 mg, 1.89 mmol). The crude oil was purified
by silica gel column chromatography (cyclohexane/EtOAc,
100:0 then 70:30) to give 11a as a white solid (557 mg,
92 % yield). (Rf = 0.65, cyclohexane/EtOAc, 50:50).
[a]D20: ?1.6° (c 13ꢀ10-3 CHCl3). Mp: 106–108 °C. dH
123

516
E. O’Reilly et al.
(500 MHz; CDCl3) 7.33–7.17 (5H, m, PhH), 5.85 (1H, s,
NHC = OCH), 5.08 (1H, s, NHBoc), 4.27 (1H, dd,
J = 14.6, 7.3 Hz, CHCH2Ph), 4.02 (2H, t, J = 6.7 Hz,
CH2CH2CH3), 3.26–3.12 (2H, dd, J = 12.8 and 6.5 Hz,
NCH2), 3.11–3.04 (1H, dd, J = 6.5 and 13.6 Hz, CH2Ph),
3.04–2.98 (1H, dd, J = 7.7 and 13.6 Hz, CH2Ph),
2.97–2.88 (1H, m, C = OCH), 2.34–2.27 (1H, m, NCH2
CH), 2.25–2.12 (2H, m, C = OCH(CHH)2, 1.93–1.79 (2H,
m, C = OCH(CHH)2), 1.68–1.61 (2H, m, CH2CH3), 1.41
(9H, s, C(CH3)3), 0.93 (3H, t, J = 7.4 Hz, CH2CH3). dC
(125.65 MHz; CDCl3) 175.2, 171.6, 155.7, 137.3, 129.7,
128.9, 127.1, 80.2, 66.2, 56.4, 45.0, 39.4, 34.4, 30.8, 29.1,
28.8, 22.3, 10.6; m/z (ES?) 417.2370 (M-H?. C23H33N2O5
requires 417.2389).
0.97 mmol, 84 % yield). (Rf = 0.75, EtOAc/CH3OH,
90:10). Mp: 111–113 °C. dH (500 MHz; CDCl3) 7.79–7.75
(2H, d, J = 7.6 Hz, Ar), 7.61–7.6 (2H, d, J = 7.4 Hz, Ar),
7.41–7.38 (2H, t, J = 7.5 Hz, Ar), 7.34–7.29 (2H, m, Ar),
6.15 (1H, s, NHC = OCH2), 5.49 (1H, s, NHFmoc), 4.44
(1H, d, J = 6.9 Hz, CH2Fmoc), 4.23 (1H, t, J = 6.9 Hz,
CHFmoc), 4.02 (1H, t, J = 6.7 Hz, CH2CH2CH3), 3.86
(2H, d, J = 4.6 Hz, NHCH2C = O), 3.30 (2H, t,
J = 6.1 Hz, NCH2), 3.03–2.96 (1H, quin, J = 8.8 Hz,
C = OCH), 2.53–2.44 (1H, m, NCH2CH), 2.35–2.26 (2H,
m, C = OCH(CHH)2), 2.02–1.92 (2H, m, C = OCH
(CHH)2), 1.67–1.60 (1H, m, CH2CH2CH3), 0.92 (3H, t,
J = 7.4 Hz, CH2CH3); dC (125.65 MHz; CDCl3) 175.5,
169.0, 143.6, 141.6, 127.7, 127.2, 125.5, 120.0, 67.3, 66.2,
47.4, 44.3, 44.0, 34.2, 30.7, 28.4, 21.8, 10.3; m/z (ES?)
473.2033 (M ? Na?. C26H30N2O5Na requires 473.2052).
Preparation of propyl (S)-3-((2-((((9H-fluoren-9-
yl)methoxy)carbonyl)amino)-3-methylbutanamido)methyl)
cyclobutanecarboxylate (11b)
Preparation of propyl 3-((3-(((tert-butoxycarbonyl)
amino)methyl)cyclobutanecarboxamido)methyl)
cyclobutanecarboxylate (14)
11b was prepared according to General Procedure, by
reacting 9 (180 mg, 0.86 mmol) with N-Fmoc-L-Valine
10b (580 mg, 1.7 mmol). The crude oil was purified
through a silica gel column chromatography (cyclohexane/
EtOAc from 100:0 to 40:60) giving 11b as a yellow solid
(155 mg, 38 % yield). (Rf = 0.86, EtOAc). [a]2D0: -13.0°
(c 10.2 9 10-3 CHCl3). Mp: 152–154 °C. dH (500 MHz;
CD3OD) 7.75 (2H, d, J = 7.5 Hz, Ar), 7.58 (2H, d,
J = 7.4 Hz, Ar), 7.39 (2H, t, J = 7.4 Hz, Ar) 7.29 (2H, m,
Ar), 6.29 (1H, s, NHC = OCH), 5.57 (1H, d, J = 8.2 Hz,
NHFmoc), 4.43–4.39 (1H, m, CH2Fmoc), 4.35–4.32 (1H,
m, CH2Fmoc), 4.19 (1H, t, J = 7.0 Hz, FmocCH), 4.01
(2H, t, J = 6.7 Hz, CH2CH2CH3), 3.96–3.94 (1H, m,
CHCH(CH3)2), 3.36–3.27 (1H, m, NCH2), 3.27–3.18 (1H,
m, NCH2), 3.05–2.93 (1H, m, C = OCH), 2.53–2.38 (1H,
m, NCH2CH), 2.29–2.24 (2H, m, C = OCH(CHH)2),
2.16–2.07 (1H, m, CH(CH3)2), 2.01–1.90 (2H, m,
C = OCH(CHH)2), 1.68–1.56 (2H, m, CH2CH3), 0.92 (6H,
m, CH(CH3)2 and 3H, t, J = 7.4 Hz, CH2CH3); dC
(125.65 MHz; CDCl3) 175.5, 171.4, 156.6, 144.0, 141.6,
128.2, 127.4, 125.3, 120.1, 67.3, 66.3, 60.8, 47.3, 44.2,
34.0, 31.1, 30.7, 28.8, 28.7, 22.2, 19.4, 18.1, 10.6 m/z
(ES?) 515.2521 (M ? Na?. C29H36N2O5Na requires
515.2522).
14 was prepared according to General Procedure, by
reacting 9 (181 mg, 0.87 mmol) with 13 (195 mg,
0.85 mmol). The crude oil was purified through a silica gel
column chromatography (cyclohexane/EtOAc from 100:0
to 0:100) giving 14 as a clear yellow solid (194 mg, 58 %
yield). (Rf = 0.13, cyclohenane/EtOAc 50:50). Mp:
62–64 °C. dH (400 MHz; CDCl3) 5.60 (1H, s,
NHC = OCH), 4.72 (1H, s, NHBoc), 4.02 (2H, t,
J = 6.5 Hz, CH2CH2CH3), 3.25 (2H, t, J = 5.9, NCH2),
3.13–3.11 (2H, m, BocNCH2), 3.04–2.95 (1H, m,
CHC = OOnPr), 2.92–2.79 (1H, m, CHC = ON),
2.55–2.37 (2H, m, NCH2CH and BocNCH2CH), 2.33–2.21
(4H, m, OC = OCH(CHH)2 and NC = OCH(CHH)2),
2.01–1.92 (4H, m, OC = OCH(CHH)2 and NC = OCH
(CHH)2), 1.69–1.60 (2H, m, CH2CH3), 1.44 (9H, s,
C(CH3)3), 0.92 (3H, t, J = 7.4 Hz, CH2CH3). dC
(101 MHz; CDCl3) 175.7, 175.4, 156.4, 79.6, 66.5, 45.9,
44.6, 36.6, 34.4, 31.2, 28.9, 28.8, 22.4, 10.7; m/z (ES-)
381.2408 (M - H- C20H33N2O5 requires 381.2389).
Preparation of (S)-1-(((3-carboxycyclobutyl)methyl)
amino)-1-oxo-3-phenylpropan-2-aminium chloride (12a)
Preparation of propyl 3-((2-((((9H-fluoren-9-yl)methoxy)
carbonyl)amino)acetamido)methyl)
cyclobutanecarboxylate (11c)
11a (550 mg, 1.31 mmol) was dissolved in 20 mL of
CH3CN and NaOH 2 M (8 mL) was added. The reaction
was left under stirring for 2 h at 45 °C before bringing the
solution to pH 3 with HCl 1 M and extracting it with
CH2CH2 (15 mL 9 5). The organic layers were combined,
dried over MgSO4 and concentrated in vacuo. The white
solid obtained was dissolved in CH2CH2 (20 mL) and HCl
in Et2O 3 M (0.4 mL) was added at 0 °C. The reaction was
stirred for 4 h. The white precipitate was filtered off and
11c was prepared according to General Procedure, by
reacting 9 (240 mg, 1.15 mmol) with N-Fmoc-Glycine 10c
(350 mg, 1.18 mmol). The crude oil was purified through a
silica gel column chromatography (cyclohexane/EtOAc
from 100:0 to 20:80) giving 11c as a white solid (437 mg,
123

Synthesis of a conformationally constrained d-amino acid building block
517
washed with Et2O (190 mg, 0.69 mmol) with an overall
yield (over the 2 steps) of 53 %. (Rf = 0.08, EtOAc/
CH3OH, 90:10). [a]D20: ?26.0° (c 7.6 9 10-3 MeOH). Mp:
170–172 °C. dH (400 MHz; CD3OD) 7.46–7.18 (5H, m,
PhH), 4.08 (1H, t, J = 7.4 Hz, CHCH2Ph), 3.25–3.05 (4H,
m, NCH2 and CH2Ph), 3.05–2.93 (1H, m, C = OCH),
2.45–2.26 (1H, m, NCH2CH), 2.28–2.15 (2H, m,
C = OCH(CHH)2), 1.90–1.81 (2H, m, C = OCH(CHH)2);
dC (101 MHz; CD3OD) 177.1, 169.6, 135.7, 130.6, 129.9,
128.7, 55.8, 45.4, 38.6, 34.7, 31.8, 29.9; m/z (ES?)
277.1552 (M ? H? C15H21N2O3 requires 277.1552).
29.5, 29.3; m/z (ES?) 187.1082 (M ? H? C8H15N2O3
requires 187.1083).
Preparation of (3-(((3-carboxycyclobutyl)methyl)
carbamoyl)cyclobutyl)methanaminium chloride (15)
14 (180 mg, 0.47 mmol) was dissolved in CH3CN (15 mL)
and NaOH 2 M (5 mL) was added. The reaction was left
under stirring for 2 h at 45 °C before bringing the solution to
pH 3 with HCl 1 M and extracting it with CH2CH2
(15 mL 9 5). The organic layers were combined, dried over
MgSO4 and concentrated in vacuo giving 15 as a dark yellow
oil, 84 mg (0.25 mmol) of which was dissolved in CH2CH2
(10 mL). HCl in Et2O 5 M (0.1 mL) was added and the
reaction was stirred for 4 h. The clear yellow precipitate was
filtered off and washed with Et2O (38 mg, 0.14 mmol)
affording 15 in an overall yield (over the 2 steps) of 56 %.
(Rf = 0.025, EtOAc/CH3OH 90:10). Mp: 156–158 °C. dH
(500 MHz; CD3OD) 3.17 (2H, d, J = 6.5 Hz,
C = ONHCH2), 3.06–2.94 (4H, m, C = OCH, NC = OCH,
CH2N), 2.60–2.50 (1H, m, C = ONCH2CH), 2.48–2.39 (1H,
m, NCH2CH), 2.37–2.32 (2H, m, OC = OCH(CHH)2),
2.29–2.23 (2H, m, NC = OCH(CHH)2), 2.03–1.92 (4H, m,
Preparation of (S)-3-((2-amino-3-methylbutanamido)
methyl)cyclobutanecarboxylic acid (12b)
11b (48 mg, 0.09 mmol) was dissolved in dioxane/meth-
anol 7:3 (7 mL) and NaOH 2 M (2 mL) was added. The
reaction was left under stirring for 4 h at 45 °C. Dioxane
was evaporated in vacuo and the water left was extracted
with Et2O (10 mL) and CH2CH2 (10 mL 9 2). The aque-
ous layer was neutralised with HCl 1 M and purified with
DOWEX 50WX8-200 ion exchange column affording
12b as a yellow solid (18 mg, 0.078 mmol, 90 % yield).
(Rf = 0.55, CH3OH). Mp could not be measured because
the compound is hydroscopic. [a]2D0: ?118.0° (c
0.5 9 10-3 MeOH). dH (500 MHz, CD3OD) 3.50 (1H,
brs, CHCH(CH3)2), 3.23 (2H, d, J = 6.2 Hz, NCH2), 2.90
(1H, quint, J = 8.9 Hz, C = OCH(CH2)2), 2.50–2.39 (1H,
m, NCH2CH), 2.35–2.20 (2H, m, C = OCH(CHH)2),
2.08–2.16 (1H, m, CH(CH3)2), 1.99–1.92 (2H, m,
C = OCH(CHH)2), 1.06–0.98 (3H, d, J = 6.8, Hz,
CH(CH3)2 and 3H, d, J = 6.8, Hz, CH(CH3)2). dC
(125.65 MHz; CD3OD) 180.1, 169.4, 60.4, 45.3, 37.1,
31.8, 30.4, 30.2, 19.2, 18.2; m/z (ES?) 229.1546 (M ? H?
C11H21N2O3 requires 229.1552).
OC = OCH(CHH)2
and
NC = OCH(CHH)2).
dC
(101 MHz; CD3OD) 177.2, 176.9, 45.3, 45.0, 36.3, 34.9,
32.2, 29.9, 29.7, 29.6; m/z (ES?) 241.1564 (M ? H?
C12H21N2O3 requires 241.1552).
Conclusions
We have developed an efficient route forthe preparation of an
achiral d-amino acid bearing a cyclobutane core. The syn-
thetic strategy employed means that only the cis isomer is
formed upon hydrolysis, providing cis-3(amino-
methyl)cyclobutane carboxylic acid in an overall yield of
34 %. NMR and X-ray crystallography data confirm that no
epimerization of the amino acid occurs under the reaction
conditions employed. This conformationally constrained d-
amino acid has applications in the field of peptidomimetics as
well as being a useful synthetic building block.
Preparation of cis-3-((2-aminoacetamido)methyl)
cyclobutanecarboxylic acid (12c)
11c (185 mg, 0.41 mmol) was dissolved in CH2Cl2/TEA
1:1 (20 mL). The reaction was left under stirring for 24 h at
45 °C. The white precipitate was filtered off and washed
with acetonitrile. The filtrate solution was concentrated in
vacuo and the crude yellow solid was purified with
DOWEX 50WX8-200 ion exchange column, affording 12c
as white solid (69 mg, 0.37 mmol, 90 % yield). (Rf = 0.3,
CH3OH). Decomposition temperature: 200 °C. dH
(500 MHz; D2O) 3.87 (2H, br s, NHCH2C = O), 3.30 (2H,
d, J = 6.4 Hz, NCH2), 3.00–2.92 (2H, m, C = OCH),
2.52–2.42 (1H, m, NCH2CH), 2.33–2.27 (2H, m,
C = OCH(CHH)2), 1.88–1.81 (2H, m, C = OCH(CHH)2).
dC (125.65 MHz; D2O) 184.9, 166.8, 44.2, 40.5, 36,5,
We have prepared a small library of dipeptides by
coupling amino acid 1 to a small panel of proteinogenic
amino acids. The dipeptide of amino acid 1 was also
synthesised.
Acknowledgments We would like to thank the Irish Research
Council for Science, Engineering and Technology (IRCSET) for
funding this research. We would also like to acknowledge the facil-
ities of the Centre for Synthesis and Chemical Biology (CSCB),
funded by the Higher Education Authority’s Programme for Research
in Third-Level Institutions (PRTLIs). We are grateful to Dr. Jimmy
Muldoon for his help with the NMR experiments.
123

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