Synthesis of C-protected 2,2-dideutero β3-amino acids

Article abstract of DOI:10.1055/s-2006-950314

A simple three-step procedure for the preparation of C-protected 2,2- ²H-β³-amino acids has been developed starting from the natural α-amino acids. Our synthetic path is based on the homologation reaction of α-amino acids through the

Full text of DOI:10.1055/s-2006-950314

PAPER
4013
Synthesis of C-Protected 2,2-Dideutero b3-Amino Acids
2, 2-Dideutero
b
3-AnminoAcidsnalisa Guaragna,* Silvana Pedatella, Vittoria Pinto, Giovanni Palumbo
Università di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica, Via Cynthia 4, 80126 Napoli, Italy
Fax +39(081)674119; E-mail: guaragna@unina.it
Received 2 August 2006
So far, only little information is available about the phar-
macokinetic properties of bioactive b-peptides. Recently
Seebach7,8 and co-workers have performed an interesting
Abstract: A simple three-step procedure for the preparation of C-
protected 2,2-2H-b3-amino acids has been developed starting from
the natural a-amino acids. Our synthetic path is based on the ho-
mologation reaction of a-amino acids through the formation of di-
14
study using C-labelled peptides to follow their absorp-
deuterated alcohol intermediates obtained by heavy isotope tion, distribution, metabolism, and excretion (ADME).
reduction (NaBD4) of the carboxylic function.
Key words: b3-amino acids, deuterated amino acids, isotopically
labeled compounds
Pharmacokinetic studies traditionally used radiolabelled
target compounds as a means for ADME studies; new
analysis technologies now make it possible to use targets
enriched with stable isotopes, such as carbon-13 and deu-
terium, as alternatives to radioisotopes.9 The use of stable
isotopes allows, ADME studies to be directly replicated in
humans, providing unequivocal validation of animal mod-
els. Recently, deuterium labeling of proteins and peptides
have also been used in quantitative proteomics analysis.10
The discovery of nonproteinogenic amino acids among
natural products1 has increased the level of interest in this
family of molecules. b-Amino acids show interesting
pharmacological properties either in free form, for in-
stance emeriamine2 (1), or as key components of a variety
of bioactive molecules such as taxol3 (2), one of the most
active antitumor agents which contains phenylisoserine as
its side chain (Figure 1). Furthermore, b-amino acids, al-
though not as abundant as their a-analogues, are also seg-
ments in peptidic natural products with various biological
activities, such as (R)-b-dopa (3,4-dihydroxy-b-phenyla-
lanine, 3) contained in mushroom Cortinarius violaceus.4
As a part of our ongoing project on the preparation of a
new ICAT (Isotope-Coded Affinity Tag) reagent11 as a
powerful tool for quantitative proteome analysis, contain-
ing a deuterated b-amino acidic linker moiety, we have
applied a simple methodology to obtain C-protected 2,2-
2H-b3-amino acids12 starting from the natural a-amino ac-
ids. The current path consists of a homologation reaction
by reduction of carboxylic function13 and then substitu-
tion of the hydroxyl group of the corresponding primary
hydroxyl function with a cyano group (Scheme 1). We
have tested our methodology using various a-amino acids
and the obtained results are reported in Tables 1– 3.
AcO
OH
O
BzHN
O
_
+
COO
O
Me3N
O
HO
i
NH2
BocHN
COOH
BocHN
CD2OH
AcO
OBz
HO
see Table 1
1
2
R
R
NH2
4a–e
HO
HO
COOH
see Table 2 ii
3
iii
HCl·H2N
CD2COOMe
BocHN
R
CD2CN
see Table 3
Figure 1 Naturally occurring compounds containing b-amino acid
R
moiety
6a–e
5a–e
The incorporation of b-amino acids5 has been successful
in creating peptidomimetics6 that not only have potent bi-
ological activity, but are also resistant to proteolysis. Nev-
ertheless, metabolism and pharmacokinetics are other
important aspects in addition to biological activity for the
potential use of these compounds as drugs.
i) NMM, MeOCOCl in THF, then NaBD4 in D2O; ii) –PPh2, I2,
HIm in CH2Cl2, then Et4N+CN– in CH2Cl2; iii) HCl in MeOH
Scheme 1
We began the synthesis of our deuterium labelled com-
pounds by treating N-protected a-amino acids with meth-
yl chloroformate. The corresponding carbomethoxy
anhydrides formed in situ were next reduced and labelled
by means of NaBD4 (98% atom D) in D2O (99.9%), af-
fording the related 1,1-2H-b-amino alcohols in high yield
SYNTHESIS 2006, No. 23, pp 4013–4016
x
x.
x
x
.
2
0
0
6
Advanced online publication: 12.10.2006
DOI: 10.1055/s-2006-950314; Art ID: Z16406SS
© Georg Thieme Verlag Stuttgart · New York

4014
A. Guaragna et al.
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Table 1 1,1-Dideutero b-Amino Alcohols 4a–e Prepared
25
Compound
Yield (%)
Mp (°C)
[a]D
1H NMR (300 MHz, CDCl3/TMS) d, J (Hz)
13C NMR (75 MHz,
CDCl3/TMS) d
4a
85
oil
–
1.43 (s, 9 H), 3.22 (d, J = 5.5, 2 H), 3.86 (br s, 28.2, 42.8, 61.8 (CD2),
R = H
1 H, exchangeable with D2O), 5.10 (br t, J =
79.6, 156.8
5.5, 1 H)
4b
R = Me
94
89
58.2–59.1
(hexane)
–9.8
1.15 (d, J = 6.8, 3 H), 1.43 (s, 9 H), 2.95 (br s, 17.1, 28.2, 48.3, 66.5
(c = 1.3, CHCl3) 1 H, exchangeable with D2O), 3.71 (br q, J = (CD2), 79.5, 156.2
6.8, 1 H), 4.75 (br d, J = 6.6, 1 H)
4c
64.9–65.7
(hexane)
+14.8
1.45 (s, 9 H), 2.60 (br s, 1 H, exchangeable
28.8, 51.7, 63.1 (CD2),
70.6, 73.4, 79.5, 127.0,
127.2, 127.8, 136.9, 155.1
R = CH2OBn
(c = 1.7, CHCl3) with D2O), 3.67 (dd, J = 4.3, 9.3, 1 H), 3.71
(dd, J = 3.8, 9.3, 1 H), 3.80–3.90 (m, 1 H),
4.38 (d, J = 12.0, 1 H), 4.62 (d, J = 12.0, 1 H),
5.22 (br s, 1 H), 7.27–7.42 (m, 5 H)
4d
90
85
97.3–98.9
(hexane)
–23.1
1.43 (s, 9 H), 1.92 (br s, 1 H, exchangeable
28.2, 37.3, 49.1, 53.4
(CD2), 79.6, 126.4, 128.4,
R = CH2Ph
(c = 1.2, CHCl3) with D2O), 2.82 (d, J = 7.1, 2 H), 3.78–3.92
(m, 1 H), 4.72 (br s, 1 H), 7.18–7.28 (m, 5 H) 129.1, 137.6, 157.7
4e
oil
–10.1
0.93–1.32 (m, 6 H), 1.43 (s, 18 H), 2.14 (br s, 23.0, 28.7, 30.2, 30.9,
R =
(CH2)4NHBoc
(c = 1.9, MeOH) 1 H, exchangeable with D2O), 2.78–2.98 (m, 2 40.0, 52.6, 65.8 (CD2),
H), 3.43–3.58 (m, 1 H), 4.13 (br s, 1 H), 4.51 79.4, 79.7, 156.6, 156.7
(br s, 1 H)a
a Recorded in C6D6 at 75 °C.
Ph
(see Table 1) with a 98% deuterium incorporation (deter-
mined by H NMR spectroscopy and MS analyses). On
I
H+
Ph Ph
P
+
1
BocHN
CD2OH
Ph
P
I–
BocHN
CD2
I–
+
O
the basis of previously acquired knowledge,14 the dideu-
terated amino alcohols 4a–e were then converted into
their corresponding amino iodides using a suspension of
triphenylphosphine polymer-bound/iodine complex
(polystyryl diphenyliodophosphonium iodide) in anhy-
drous dichloromethane.
I
R
R
I–
BocHN
CD2I
Ph
Ph
O
+
P
R
The triphenylphosphine polymer-bound/halogen complex
is a Lewis acid and a dehydrating agent widely employed
in miscellaneous reactions15 with low environmental im-
pact. In fact it avoids contamination from by-products and
use of non-environmentally friendly solvents in the puri-
fication processes. The phosphine oxide, which under our
conditions is the only byproduct of the reaction, is linked
to the polymeric matrix and can thus be easily separated
by filtration (Scheme 2). Amino iodides so obtained16
were directly engaged, after a simple filtration, in the next
step in which the iodine atom was easily replaced with a
cyano group (Table 2) by means of a suitable cyanide ion
source (Et4N+CN–). Finally, hydrolysis of nitriles 5a–e us-
ing acid-catalyzed alcoholysis led to the corresponding
2,2-dideuterated amino esters 6a–e (Table 3). The deute-
rium content percentage was maintained unchanged dur-
ing the whole synthetic route, at a rate shown by MS
Scheme 2
tions were monitored by TLC (precoated silica gel plate F254,
Merck). Column chromatography: Merck Kieselgel 60 (70–230
mesh). All moisture-sensitive reactions were conducted under dry
N2 using oven-dried glassware. THF was distilled from sodium/
benzophenone immediately prior to use. Ph3P polymer-bound was
purchased from Fluka Chemical Co. D2O minimum isotopic purity
99.9 atom% D and NaBD4 98 atom% D were used.
2,2-2H-b3-Amino Esters; Typical Procedures
1,1-2H-N-Boc-b-amino Alcohol 4b
To a magnetically stirred solution of N-Boc-Ala (0.50 g, 2.6 mmol)
in anhyd THF (20 mL) at 0 °C, was added N-methylmorpholine
(0.35 mL, 3.2 mmol), followed by ethyl chloroformate (0.24 mL,
3.2 mmol). After 40 min, the mixture was filtered through a glass
sinter funnel on a Celite pad and washed with THF. To the filtrate
so obtained, at 0 °C and under magnetic stirring, was added a sus-
pension of NaBD4 (0.11 g, 2.6 mmol) in D2O (2 mL) in one portion.
The mixture was kept at r.t. for 10 min, then the solvent was re-
moved under reduced pressure and the obtained residue dissolved
with Et2O and washed with H2O until neutral. The organic layer was
dried (Na2SO4) and evaporated under reduced pressure. Chroma-
tography of the crude product on silica gel (CHCl3–MeOH, 8:2)
1
spectrometry and H NMR spectroscopy to be not less
than 97%.
1H and 13C NMR spectra: Varian Gemini 300 MHz and Varian Ino-
va 500 MHz spectrometers. HRMS-EI: Micromass Q-TOF micro.
Optical rotations: Jasco P-1010 (1.0 dm cell). Melting points are un-
corrected and were determined with a capillary apparatus. Reac-
Synthesis 2006, No. 23, 4013–4016 © Thieme Stuttgart · New York

PAPER
2,2-Dideutero b3-Amino Acids
4015
Table 2 2,2-Dideutero b-Amino Nitriles 5a–e Prepared
25
Compound
Yield (%) Mp (°C)
[a]D
1H NMR
(300 MHz, CDCl3/TMS)
13C NMR
(75 MHz, CDCl3/TMS)
d, J (Hz)
d
5a
R = H
88
90
42.4–45.0
(hexane)
–
1.45 (s, 9 H), 3.38 (d, J = 6.3,
2H), 4.90 (br s, 1 H)
28.1, 29.5 (CD2), 36.5, 80.1,
118.0, 155.0
5b
R = CH3
68.4–70.0
(hexane)
–120
1.33 (d, J = 6.8, 3H), 1.43 (s, 9 19.4, 25.0 (CD2), 28.3, 43.0,
(c = 1.4, CHCl3) H), 3.95–4.15 (m, 1 H), 4.69 (br 80.1, 117.4, 154.8b
d, J = 7.05, 1 H)a
5c
70
80
75
oil
–9.7
1.50 (s, 9 H), 3.60 (dd, J = 4.9, 28.1, 29.6 (CD2), 46.8, 69.5,
R = CH2OBn
(c = 1.6, CHCl3) 9.6, 1 H), 3.70 (dd, J = 3.8, 9.6, 73.4, 80.1, 117.1, 127.6, 127.9,
1 H), 4.05–4.18 (m, 1 H), 4.58 128.4, 137.1, 154.8
(s, 2 H), 5.10 (br d, J = 6.4, 1
H), 7.31–7.40 (m, 5 H)
5d
123.8–125.0
(hexane)
–18.2
1.43 (s, 9 H), 2.86 (dd, J = 7.8, 28.2, 29.9 (CD2), 39.3, 48.3,
79.6, 117.3, 126.4, 128.4,
13.6, 1 H), 4.00–4.17 (m, 1 H), 129.1, 137.6, 154.8
4.72 (br s, 1 H), 7.18–7.28 (m,
5 H)
R = CH2Ph
(c = 1.2, CHCl3) 13.6, 1 H), 2.99 (dd, J = 5.8,
5e
62.7–64.0
(hexane)
–45.2
0.72–1.18 (m, 6 H), 1.38 (s, 9
23.0, 28.5, 28.6, 29.9 (CD2),
R = (CH2)4NHBoc
(c = 0.6, CHCl3) H), 1.44 (s, 9 H), 2.64–2.81 (m, 30.0, 33.1, 40.0, 47.3, 79.5,
2 H), 3.29–3.42 (m, 1 H), 3.92 80.3, 117.5, 155.4, 156.4
(br s, 1 H), 4.21 (br s, 1 H)c
a Recorded at 500 MHz.
b Recorded at 125 MHz.
c Recorded in C6D6 at 75 °C.
Table 3 2,2-Dideutero b3-Amino Ester Hydrochlorides 6a–e Prepared
13C NMR
25
Compound
Yield (%)
Mp (°C)
[a]D
1H NMR
(300 MHz, CD3OD/TMS)
d, J (Hz)
(75 MHz, CD3OD/TMS)
d
6a
R = H
93
90
85
104.4–106.0
(CHCl3)
–
3.18 (br s, 2 H), 3.73 (s, 3 H)
32.1, 36.4 (CD2), 52.7, 172.6
6b
R = Me
250.0 (dec.)
(CHCl3)
+0.19
1.47 (d, J = 6.8, 3 H), 3.48 (q, 18.3, 38.2 (CD2), 44.7, 52.2,
(c = 2.5, MeOH) J = 6.8, 1 H), 3.72 (s, 3 H)
170.8
6c
oil
+0.68 3.63 – 3.78 (m, 4 H), 4.48 (d,
37.0 (CD2), 48.0, 51.8, 72.4,
R = CH2OBn
(c = 0.7, MeOH) J = 12.0, 1 H), 4.54 (d, J = 12.0, 73.1, 127.2, 127.5, 127.8,
1 H), 4.78 (m, 1 H), 7.31–7.40 138.2, 171.1
(m, 5 H)
6d
87
83
255.0 (dec.)
(CHCl3)
+4.28
2.94 (dd, J =8 .3, 14.2, 1 H),
37.0, 39.5 (CD2), 48.3, 52.8,
128.7, 129.4, 130.1, 136.7,
R = CH2Ph
(c = 1.4, MeOH) 3.79 (dd, J = 6.3, 14.2, 1 H),
3.70 (s, 3 H), 3.82 (t, J = 7.3, 1 171.2b
H), 7.05–7.38 (m, 5 H)a
6e
R =
oil
+8.49
1.48–1.58 (m, 2 H), 1.68–1.72 23.0, 28.5, 30.0, 33.1 (CD2),
(c = 0.6, MeOH) (m, 4 H), 2.93 (t, J = 7.8, 2 H), 40.0, 50.3, 52.8, 170.2b
(CH2)4NH2·HCl
3.57 (t, J = 6.8, 1 H), 3.75 (s, 3
H)a
a Recorded at 500 MHz.
b Recorded at 125 MHz.
Synthesis 2006, No. 23, 4013–4016 © Thieme Stuttgart · New York

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A. Guaragna et al.
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gave the pure dideuterated N-Boc-amino alcohol 4b; yield: 0.43 g
(94%).
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2,2-2H-N-Boc-b-amino Nitrile 5b
To a magnetically stirred suspension of anhyd polystyryl diphe-
nylphosphine (0.56 g, ~1.67 phosphine units) in anhyd CH2Cl2 (10
mL) at r.t., was added dropwise a solution of I2 (0.42 g, 1.67 mmol)
in the same solvent (10 mL) in the dark and under dry N2. After 15
min, solid dideuterated N-Boc-amino alcohol 4b (0.27 g, 1.52
mmol) was added in one portion to the suspension. The reaction was
kept at 40 °C for 2 h (TLC monitoring: CHCl3–MeOH, 8:2) until all
the starting amino alcohol was completely consumed. The mixture
was then filtered through a glass sinter funnel and washed with
CH2Cl2. To the filtrate, under magnetical stirring, Et4N+CN– was
added in one portion and the reaction kept at reflux for 3 h until
complete consumption of the starting N-Boc-b-amino iodide. The
cooled mixture was poured on a silica gel column (1:5) and eluted
with CH2Cl2. The organic solvent evaporated under reduced pres-
sure afforded the pure dideuterated N-Boc-b-amino nitrile 5b; yield:
0.25 g (90%).
HRMS-EI: m/z calcd for C9H14D2N2O2: 186.1335; found: 186.1346.
2,2-2H-b3-Amino Ester Hydrochloride 6b
To a magnetically stirred solution of 5b (0.24 g, 1.3 mmol) in anhyd
Et2O (8 mL) at 0 °C, was added dropwise cold 12 M HCl in MeOH
(3 mL). The reaction was kept at r.t. overnight. To the solution were
added few drops of H2O (28 mL, 1.56 mmol) and the solvent was co-
evaporated with Et2O (3 × 10 mL) affording the desired compound
6b as a crystalline white solid; yield: 0.18 g (90%).
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Acknowledgment
We thank Guido Smiraglia for collecting valuable results while per-
forming his master thesis work. The authors would also like to
thank Dr. Emiliano Manzo from Instituto di Chimica Biomolecola-
re, CNR Napoli for HRMS analysis. 1H and 13C NMR spectra were
recorded at Centro Interdipartimentale di Metodologie Chimico-Fi-
siche, Università di Napoli Federico II. Varian Inova 500 MHz in-
strument is a property of Consorzio Interuniversitario Nazionale La
Chimica per l’Ambiente (INCA) and was used in the frame of a pro-
ject by INCA and M.I.U.R. (L. 488/92, Cluster 11-A).
(11) Unpublished results.
(12) b-Amino acids can be subdivided into b3, b2, and b2,3 amino
acids, depending upon the position of the side chain(s) on the
aminoalkanoic acid skeleton.
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polymer bound/iodine complex towards all the tested amino
acids.
Synthesis 2006, No. 23, 4013–4016 © Thieme Stuttgart · New York