Novel thiol- and thioether-containing amino acids: Cystathionine and homocysteine families

Article abstract of DOI:10.1007/s00726-012-1352-5

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation react

Full text of DOI:10.1007/s00726-012-1352-5

Amino Acids
DOI 10.1007/s00726-012-1352-5
ORIGINAL ARTICLE
Novel thiol- and thioether-containing amino acids: cystathionine
and homocysteine families
•
Luigi Longobardo Nunzia Cecere
•
•
Marina DellaGreca Ivan de Paola
Received: 11 May 2012 / Accepted: 22 June 2012
Ó Springer-Verlag 2012
Abstract Natural L-homocysteine and L,L-cystathionine,
along with a series of unnatural analogues, have been
prepared from L-aspartic and L-glutamic acid. Manipulation
of the protected derivatives provided x-iodoamino acids,
which were used in thioalkylation reactions of sulfur
nucleophiles, such as the ester of L-cysteine and potassium
thioacetate.
Nva
Pg
Norvaline
Protecting group
SPPS Solid-phase peptide synthesis
THF
TPP
Tetrahydrofurane
Triphenylphosphine
Introduction
Keywords Cystathionine ꢀ Homocysteine ꢀ Cysteine
thioalkylation ꢀ Bis-amino acids ꢀ Unnatural amino acids
The metabolism of sulfur-containing amino acids has been
associated with several aspects of human health and cel-
lular functions. Sulfur-containing amino acids, and small
peptides containing them, contribute significantly to the
protection and integrity of cellular systems because of their
ability to influence the cellular redox state and their
capacity to detoxify toxic compounds, free radicals and
reactive oxygen species (Finkelstein et al. 1984; Mason
2003; Townsend et al. 2004). Methionine, an amino acid
acquired through the diet, is our primary source of sulfur.
Sulfur is distributed in various amino acid forms such as
cysteine, homocysteine (Hcy), cystathionine (Cth), lanthi-
onine (Lan), and taurine. L,L-Cth (1, Fig. 1), also known as
carbacystine, is a metabolic intermediate in the pathway to
make cysteine from homocysteine. It has been isolated
from the urine of humans with alteration in sulfur metab-
olism (Frimpter 1965), the leaves of Astragalus pectinari,
(Nigam et al. 1972), and the deproteinized hemolymph of
Bombix mori (Kondo 1962). It is also a component of
ezomycin, the Streptomyces derived antifungal that belongs
to a family of complex peptidyl nucleoside super-antibi-
otics (Knapp et al. 2000; Khalaf et al. 2008).
Abbreviations
Allyl
All
Bn
Benzyl
Boc
Cth
Hcy
DCC
Fm
tert-Butoxycarbonyl
Cystathionine
Homocysteine
N,N0-Dicyclohexylcarbodiimide
9-Fluorenylmethyl
Fmoc 9-Fluorenylmethoxycarbonyl
HOBt 1-Hydroxybenzotriazole
ImH
Imidazole
NMM N-Methylmorpholine
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1352-5) contains supplementary
material, which is available to authorized users.
L. Longobardo (&) ꢀ N. Cecere ꢀ M. DellaGreca ꢀ I. de Paola
`
Dipartimento di Scienze Chimiche, Universita degli Studi di
Napoli Federico II, Via Cinthia 4, 80126 Naples, Italy
e-mail: luilongo@unina.it
Present Address:
I. de Paola
`
Dipartimento delle Scienze Biologiche, Universita degli Studi di
Napoli Federico II, Via Mezzocannone 16, 80134 Naples, Italy
From a chemical perspective, the interest in Hcy and
especially in Cth amino acids are due to their extraordinary
structural resemblance to cystine. The substitution of a
123

L. Longobardo et al.
NH2
CO2H
Materials and methods
NH2
NH2
CO2H
HO2C
HO2C
S
HO2C
S
NH2
NH2
2
4
α-CyD
General methods
1 L,L-Cth = β-CyD
NH2
NH2
CO2H
NH2
S
The melting points were measured with a Kofler apparatus,
and they are reported uncorrected. The optical rotations
S
HO2C
CO2H
3
γ-CyE
α-CyE
were measured with
a
Jasco1010 polarimeter
1
(k = 589 nm). H- and 13C-NMR spectra were recorded
with Varian Inova 500 and Bruker DRX-400 spectrome-
ters: chemical shifts are in ppm (d), and J coupling con-
stants are in Hz. RP-HPLC were carried out with a
Shimadzu SCL-10 Avp system with a photodiode array
detector, using a binary solvent system consisting of
0.01 % TFA in H2O and 0.01 % TFA in CH3CN; an ana-
lytical Vydac C18 column was used (4.6 mm diameter with
a flow rate of 1 mL/min). Low-resolution mass spectra
were recorded on a Thermo Finnigan LXQ linear trap.
High-resolution ES mass spectra were obtained with a
Micromass Q-TOF UltimaTM API. The microwave reac-
tions were performed on a Mars system from CEM. TLC
was carried out on silica gel Merck 60 F254 plates (0.2 mm
layer thickness) and developed with ninhydrin (0.25 % in
MeOH) or visualised by UV. Column chromatography was
performed on a Merck Kieselgel 60 (70–230 mesh). Inor-
ganic, protected a-amino acids and other reagents, includ-
ing NaBD4 (98 % D) and D2O (99.9 % D), were purchased
from Aldrich at the highest purity grade and used without
further purification. Dry solvents were distilled immedi-
ately before use. All compounds for which analytical and
spectroscopic data are reported were homogeneous by TLC
and HPLC, and the solids were crystallised.
NH2
HO2C
SH
HO2C
SH
NH2
L-Hcy
6
β3-L-Cy s
5
NH2
NH2
SH
SH
HO2C
HO2C
γ3-L-Cys
7
L-5-mercapto-Nva
8
NH2
NH2
Y = S, CyX amino acids
Y = Se, SeX amin o acids
Y
R
CO2H
R= proteinogenic side chains
Fig. 1 Cystathionine and homocysteine families and the definition of
CyX and SeX amino acids
disulfide bond with a reduction-resistant thioether bridge is
an attractive means of improving stability while imposing
minimal structural perturbation to a natural bioactive
peptide. Cth-containing bioactive peptides have been pre-
pared by SPPS methods based on in situ Cth formation via
cysteine thioalkylation (Mayer et al. 1995), by the incor-
poration of pre-formed Cth-containing segments (Jones
et al. 1998), or through the direct desulfurization of cys-
tine-containing peptides (Galande et al. 2003).
Two recent examples of thioether-containing peptides
(Tabor 2011), the analogues of the therapeutic peptide
Compstatin obtained by SPPS methods (Knerr et al. 2011)
and the a-conotoxin ImI (Dekan et al. 2011), demonstrated
that the thioether analogues maintained a similar biological
activity, but increased its metabolic stability to reduction,
making this new class of analogues a promising alternative
for the therapeutic applications.
Preparation of N,C-protected x-iodoamino acids
Methyl chloroformate (1.8 mL, 23.3 mmol) was added
dropwise to a solution of NMM (2.6 mL, 23.3 mmol) and
Boc-amino acids (21.2 mmol) in THF (70 mL) at 0 °C
with magnetic stirring. After 15 min, the solution was fil-
tered, and the salt was washed with THF (3 9 20 mL). A
suspension of NaBH4 (1.1 g, 26.5 mmol) in H2O (8 mL)
was then added dropwise to the filtrate in an ice bath and
under magnetic stirring. The ice bath was removed, and the
temperature of the mixture was allowed to increase to room
temperature. After 10 min, the solvent was evaporated
under reduced pressure. The remaining residue was dis-
solved in EtOAc (100 mL), and the solution was washed
with brine until neutral. The organic layer was separated
and dried over Na2SO4, and the solvent was evaporated
under reduced pressure to give the crude alcohol
(95–98 %). To a solution of triphenylphosphine (2.8 g,
10.6 mmol) and dry imidazole (1.4 g, 21.2 mmol) in
anhydrous THF (70 mL), iodine (2.7 g, 10.8 mmol) was
added at room temperature under an argon atmosphere and
Although L,L-Cth 1 is a key intermediate for studies on
sulfur metabolism and an excellent candidate for pepti-
domimetic chemistry, few synthetic methods for this
compound have been reported. One example involves a
procedure starting from methionine (Shiraiwa et al. 2002).
Another method, which was first developed for lanthionine
synthesis (Zhu et al. 2003), involves the phase-transfer
condensation of Fmoc- and tert-butyl ester L-cysteine with
protected c-bromo-L-aminobutyrate that is derived from
L-homoserine (Knerr et al. 2011). Additionally, some
biotechnological procedures employ the bacterial CGL
(Kanzaki et al. 1987) or the aminohexyl-Sepharose immobi-
lised enzyme (Yamagata et al. 2003), but these procedures
are limited to the preparation of natural L,L-Cth 1.
123

Novel thiol- and thioether-containing amino acids
magnetic stirring. After 15 min, the crude alcohol
(8.5 mmol), which was dissolved in the same solvent
(20 mL), was added; the reaction mixture was allowed to
reflux until the consumption of the starting material (*1 h,
TLC). The mixture was then cooled, diluted with EtOAc
(100 mL), and washed with 10 % aq. Na2S2O4 (40 mL)
and brine (2 9 50 mL). The organic layer was dried, and
the solvent was evaporated under reduced pressure to give
the crude iodide moiety in mixture with Ph3PO as a white
solid. Rapid flash chromatography purification (20 % of
EtOAc in Hexane) yielded (77–90 %) the pure iodides.
Using these conditions, iodides 9–15 were prepared. For
their characterization, see the electronic supplementary
material.
and 16 Hz), 3.62 (dd, 1H, J = 5 and 7 Hz), 4.05–4.14 (m,
1H), 4.19 (q, 2H, J = 7 Hz), 5.12 (brs, 2H), 5.35 (brd, 1H,
J = 9 Hz), 7.31–7.38 (m, 5 H). 13C-NMR (126 MHz,
CDCl3) d: 14.2, 28.4, 36.9, 37.7, 47.4, 54.4, 61.3, 66.6,
79.6, 128.3, 128.4, 128.6, 135.6, 155.1, 171.1, 173.6.
HRMS (ESI), calcd for [C21H33N2O6S, (M?H)?]
441.2054, found 441.2047. For the characterization of
compounds 19, 23, 20, 24 and 25, see the electronic sup-
plementary material.
Hydrolysis and peptide coupling of protected CyD
amino acids
(S)-2-amino 4-[(R)-2-amino-2-carboxyethylsulfanyl] buta-
noic acid dihydrochloride, H-b-CyD-OH 2HCl (26), L,L-
Cth: H-b-CyD(Boc,OtBu)-OEt 23 (100 mg) was mixed in
a microreactor with 10 mL of 3 M aq. HCl and placed in
the microwave apparatus. The temperature was fixed to
120 °C, and the mixture was stirred magnetically for
15 min. After this time, a direct LC–MS aliquot control
showed the presence of only molecular species with a mass
of MH? 223.08. The mixture was then cooled to room
temperature, diluted with 10 mL of H2O, and frozen and
lyophilised twice to give the product as a foam (70 mg,
97 %), [a]2D5 = 22.8 (c 0.7, 1 M HCl), in agreement with
the literature value ([a]D25 = 23.4) (Shiraiwa et al. 2002);
1H-NMR (500 MHz, D2O) d: 2.05 (m, 1H); 2.17 (m, 1H);
2.60–2.70 (m, 2H); 3.01 (dd, 1H, J = 6 and 15 Hz); 3.10
(dd, 1H, J = 5 and 15 Hz); 4.04 (dd, 1H, J = 5 and 7 Hz);
4.09 (dd, 1H, J = 5 and 7 Hz). 13C-NMR, (125 MHz,
D2O) d: 24.3, 26.7, 28.3, 48.9 (minor), 49.0 (major), 49.6
(minor), 49.7 (major). HRMS (ESI), calcd for
[C7H15N2O4S, (M?H)?] 223.0747, found 223.0749,
246.0646 [(M?Na)?].
S-Alkylation of potassium thioacetate with protected x-
iodoamino acids
Under an argon atmosphere, potassium thioacetate
(228 mg, 2 mmol) was added in one portion to a stirred
solution of N,C-protected x-iodoamino acids (1 mmol) in
dry acetonitrile (5 mL). Stirring was continued in the dark
at room temperature for 1 h. The mixture was evaporated
under vacuum, and the residue was suspended in EtOAc
(50 mL) and shaken with H2O (3 9 25 mL). The organic
layer was separated and dried, and the solvent was evap-
orated under reduced pressure. The residue was purified by
flash chromatography (Hex–EtOAc 80/20). Using these
conditions, the fully protected thiol amino acids 16, 17, 20,
21 and 22 were obtained (91–96 %). For their character-
ization, see the electronic supplementary material.
S-Alkylation of L-cysteine ethyl ester with protected x-
iodoamino acids
(S)-allyl 4-[(R)-2-(S)-2-{[(9H-fluoren-9-yl)methoxycar-
bonyl]amino}propanamido-3-ethoxy-3-oxopropylsulfanyl]-
2-[(tert-butoxycarbonyl) amino] butanoate, Fmoc-Ala-b-
CyD(Boc,OAll)-OEt, (27): H-b-CyD(Boc,OAll)-OEt 22,
(177 mg, 0.45 mmol), Fmoc-L-Ala-OH (140 mg, 0.45
mmol), and dry HOBt (61 mg, 0.45 mmol) were dissolved
in dry THF (5 mL). The solution was cooled to 0 °C, and
DCC (93 mg, 0.45 mmol) was added in one portion. After
stirring for 1 h at 0 °C and 2 h at room temperature, the
white precipitate was filtered, and the filtrate was diluted
with 50 ml of EtOAc and washed with H2O (3 9 25 mL).
The organic phase was separated, dried over Na2SO4 and
concentrated under vacuum. The residue, which was puri-
fied by flash chromatography using CHCl3, furnished
To a stirred solution of N,C-protected x-iodoamino acids
(1 mmol) and L-cysteine ethyl ester hydrochloride
(185 mg, 1 mmol) in dry DMF (5 mL) under argon, solid
Cs2CO3 (652 mg, 2 mmol) was added in one portion.
Stirring was continued in the dark at room temperature for
3 h. The mixture was suspended in EtOAc (50 mL) and
shaken with H2O (3 9 25 mL). The organic layer was then
dried, and the solvents were evaporated under reduced
pressure. The residue was purified by flash chromatography
(CH2Cl2–MeOH 98/2). Using these conditions, the tri
protected thioether containing amino acids 18, 19, 23, 24,
and 25 was obtained (82–88 %).
(S)-benzyl-4-[(R)-2-amino-3-ethoxy-3-oxopropylsulfa-
nyl]-3-[(tert-butoxycarbonyl)amino] butanoate, H-a-
CyD(Boc,OBn)-OEt, (18), viscous oil, [a]2D5 = 12.4 (c 1.1,
CHCl3); 1H-NMR (500 MHz, CDCl3) d: 1.28 (t, 3H,
J = 7 Hz), 1.43 (s, 9H), 1.73 (brs, 2H), 2.69 (dd, 1H,
J = 6 and 16 Hz), 2.71–2.84 (m, 2H), 2.92 (dd, 1H, J = 6
292 mg (95 %) of pure dipeptide 27 as a white solid: m.p.
25
(CH2Cl2/Hex) 61–62 °C; [a] = ?18.7 (c 0.9, CHCl3);
D
1H-NMR (500 MHz, CD3OD) d: 1.25 (t, 3H, J = 7 Hz);
1.36 (d, 3H, J = 7 Hz); 1.40 (s, 9H); 1.60 (m, 1H);
1.65–1.75 (m, 2H); 1.80–1.88 (m, 2H); 1.93 (m, 1H); 2.02
123

L. Longobardo et al.
I
(m, 1H); 2.56 (m, 1H); 2.68 (m, 1H); 2.90 (dd, 1H, J = 7
and 14 Hz); 3.00 (dd, 1H, J = 6 and 16 Hz); 3.45 (m, 1H);
4.20 (dd, 2H, J = 7 and 16 Hz); 4.36 (m, 1H); 4.50–4.70
(m, 2H); 5.18 (d, 1H, J = 10 Hz); 5.30 (d, 1H,
J = 16 Hz); 5.90 (m, 1H); 7.31 (t, 2H, J = 8 Hz); 7.39 (t.
2H, J = 8 Hz); 7.68 (t, 2H, J = 8 Hz); 7.78 (t, 2H,
J = 8 Hz). 13C-NMR (126 MHz, CDCl3) d: 14.1; 18.3;
28.3; 29.7; 32.6; 33.9; 34.1; 47.1; 50.4; 52.3; 52.6; 61.9;
66.0; 67.2; 80.1; 119.9; 125.1; 127.1; 127.7; 131.4; 141.2;
143.8; 155.5; 156.1; 170.2; 171.8; 172.2. HPLC method:
gradient start from 50 % CH3CN–H2O 10 min to 100 %
CH3CN over 15 min and 15 min at 100 % CH3CN, with
monitoring at k 280 nm. A single peak was collected at
17.55 min. HRMS (ESI), calcd for [C35H46N3O9S, (M?H)
?] 684.1529, found 684.1525.
CO2R1
i
( )
n
ii
R = H
PgHN
9-10
RO2C
PgHN
CO2R1
( )
n
RO2C
PgHN
I
ii
( )
= H
R1
n
i
11-15
i: NMM, MeOCOCl, THF, 0°C, 15 min, then NaBH4 (or NaBD4), H2O
ii: TPP-I2, ImH, THF, reflux, 1 h
Scheme 1 Preparation of N,C-protected x-iodoamino acids
acid 10 and the deuterated analogue 10-d2, while Boc-L-
Glu(OH)-OBn and Fmoc-L-Glu(OH)-OAll generated the
protected d-iodo a-amino acids 14 and 15, respectively.
Cysteine esters have traditionally been S-alkylated by alkyl
halides, using NaOEt in refluxing EtOH (Perrey et al. 2001) or
with the reagent system Cs2CO3/TBAI in DMF (Salvatore
et al. 2005); however, products were generally obtained in
modest yields. The best results were recently obtained using
free amino acid in the presence of 1,1,3,3-tetramethylguani-
dine (Włostowski et al. 2010). The S-alkylation of C-protected
cysteine with a x-iodoamino acid proceeds very efficiently in
DMF and using commercial L-cysteine ethyl ester hydro-
chloride and Cs2CO3 as the base. The substitution products
were obtained within 3 h in high yields (Table 2), except for
the iodides 12 and 15. Iodides 9 and 10 gave protected
a-CyD 18 and a-CyE 19, respectively, while iodide 11 and 13
provided the two protected Cth, (b-CyD) 23 and 24, respec-
tively. Finally, the iodide 14 yielded the protected c-CyE 25.
These S-alkylations were all chemoselective using two eq. of
Cs2CO3. Neither alkylation of the cysteine amino group nor
traces of elimination and/or cyclization products were detected
by TLC or 1H-NMR analysis of the crude alkylation products.
These bis-amino acids were obtained with three orthogonal
protections and one free amino group. However, these amino
esters are not suitable for long-term storage, and there con-
version into hydrochloride salt is preferred.
Results and discussion
In an extension of our efforts to create new classes of amino
acids containing sulfur and selenium atoms, we report the
preparation of new members belonging to the CyX series. The
CyX and SeX series (Fig. 1), where theX denotestheone-letter
symbol of a proteinogenic residue attached to the sulfur or
selenium atoms of cysteine or selenocysteine through its
reduced carboxylic group, are efficiently obtained by the thi-
oalkylation of a cysteine ethyl ester and/or a free selenocys-
teine with N-(Boc)-b-iodoamines (Bolognese et al. 2006;
Caputo et al. 2007). These series include (Fig. 1) the com-
pounds a-CyD 2 and b-CyD 1 (L,L-cystathionine, (2S, 20R)-
Cth), which were acquired from Asp and Cys, and the couple
a-CyE 4 and c-CyE 3, which were acquired from Glu and Cys,
respectively.
The synthesis of Cth and its analogues starts from
L-aspartic and L-glutamic acids that bear various orthogonal
protecting groups, such as N-Boc, N-Fmoc and allyl, flu-
orenyl, benzyl, and t-butyl esters. The free carboxylic acid
functionality of these derivatives is first converted to a
mixed anhydride using MeOCOCl in the presence of NMM
as the base. The reductions to the alcohols were obtained
with NaBH4 in H2O (Kokotos 1990).
Treatment of the resultant N,C-protect L-hydroxyl amino
acids with the TPP-I2 complex in the presence of imidazole
(Rajagopal et al. 1994; Caputo et al. 1995a, b; Koseki et al.
2011) produced the N,C-protected L-x-iodoamino acids in
high yields (Scheme 1; Table 1). Using NaBD4 in H2O in
the reduction step (Caputo and Longobardo 2007), the
related deuterated halides were obtained.
In the reaction of iodides 12 and 15, extensive depro-
tection of the Fm and Fmoc protecting groups was
observed. This S-alkylation procedure has been recently
used to fix a Cys and/or a Sec residue at position 4 of a
protected proline (Caputo et al. 2010). S-Alkylations with
two eq. of CH3COSK in dry CH3CN at room temperature
released the expected substitution products in high yields
(Scheme 2; Table 2). Iodide 9 yielded the protected b3-Cys
16, while iodide 10 and 10-d2 generated c3-Cys 17 and 17-
d2, respectively. The halides 12 and 13 provided protected
L-Hcy 20 and 21, respectively, while iodide 14 afforded the
protected L-5-mercaptonorvaline 22.
Boc-L-Asp(OBn)-OH furnished c-iodo-b-amino acid 9
(Scheme 1), while from Boc-L-Asp(OH)-OAll, Boc-L-
Asp(OH)-OFm and Boc-L-Asp(OH)-OtBu, the corre-
sponding c-iodo a-amino acids 11, 12 and 13 were
obtained. Boc-L-Glu(OBn)-OH furnished d-iodo c-amino
123

Novel thiol- and thioether-containing amino acids
NH3+Cl-
CO2H
Table 1 N,C-Protected x-Iodoamino Acids (Scheme 1)
HO2C
S
Iodide
n
R
R1
Pg
Yield
(%)a
NH3+Cl-
R = t-Bu
26
i
NH2
CO2Et
9
1
2
2
1
1
1
2
2
H
Bn
Bn
Bn
H
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Fmoc
80
77
84
79
76
81
83
90
RO2C
S
10
10-d2
11
12
13
14
15
a
H
NHBoc
O
ii
H
CO2Allyl
NHFmoc
R = Allyl
NH
Allyl
Fm
t-Bu
Bn
BocHN
S
CO2Et
H
27
H
i): aq HCl 3 M, MW, 120°C, 15 min; ii): Fmoc-L-Ala-OH, DCC, HOBt, THF, 3 h
H
Scheme 3 Hydrolysis and peptide coupling of protected b-CyD
Allyl
H
Isolated yield
of the ethyl ester and Na-Fmoc protection (Bolognese et al.
2006).
Table 2 Protected Cystathionine and Homocysteine Families
CyX residues can be used in direct coupling with
N-protected a-amino acids in solution synthesis or hydro-
lysed to the free amino acids. To this end, b-CyD 24 was
fully deprotected in a single step with aq. 3 M HCl in a
microwave oven at 120 °C for 15 min (Scheme 3).
Free L,L-Cth dihydrochloride salt 26 was obtained in
high yields after freeze-drying the crude reaction products
two times. b-CyD 23 was used in a standard peptide cou-
pling with Fmoc-L-Ala-OH to give the expected dipeptide
27 in high yield.
Amino
acids
n
Three-letter symbols
R
Yield
(%)a
16
17
17-d2
18
19
20
21
22
23
24
25
a
1
2
2
1
2
1
1
2
1
1
2
Boc-b3-Cys(Ac)-OBn
Boc-c3-Cys(Ac)-OBn
Boc-c3-Cys(Ac)-OBn-d2
H-a-CyD(Boc,OBn)-OEt
H-a-CyE(Boc,OBn)-OEt
Boc-Hcy(Ac)-OFm
Bn
95
93
96
85
88
92
94
91
82
87
84
Bn
Bn
Bn
Bn
Fm
t-Bu
Bn
Boc-Hcy(Ac)-OtBu
Boc-Nva(5-SAc)-OBn
H-b-CyD(Boc,OAll)-OEt
H-b-CyD(Boc,OtBu)-OEt
H-c-CyE(Boc,OBn)-OEt
Allyl
t-Bu
Bn
Conclusions
Protected cystathionine and homocysteine amino acid
analogues, including deuterium-labelled analogues, were
easily obtained from commercially available reagents.
These residues can be incorporated into growing peptide
chains and used as redox stable isosteres of natural disul-
fide bonds. Preparing small, cyclic unnatural peptides may
be useful in the search for bioactive and metabolically
stable peptidomimetics.
Isolated yields
As previously reported for the CyL, CyF, and
CyP amino acids, these sulfur-containing residues may be
further converted into related, orthogonally protected acid
derivatives, which are ready for use in SPPS, by hydrolysis
SAc
Conflict of interest The authors have declared no conflict of
interest.
CO2R
i
( )
n
I
BocHN
16-17
CO2R
( )
n
( )
CO2Et
NH2
RO2C
n
S
BocHN
ii
NHBoc
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SAc
i
( )
n
BocHN
20-22
RO2C
I
NH2
CO2Et
( )
n
RO2C
( )
S
BocHN
n
ii
NHBoc
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