One-pot synthesis of orthogonally protected enantiopure S-(aminoalkyl)-cysteine derivatives

Article abstract of DOI:10.1002/ejoc.200500464

The general synthesis of a new class of non-natural diamino acids, 2-amino-3-[(2′-aminoalkyl)thio]propanoic acids or S-(aminoalkyl)cysteines, is reported. Under the conditions devised, enantiopure N-Boc-protected β-iodoamines, readily generated from proteinogenic α-amino acids, are treated with L-cysteine ethyl ester hydrochloride, using Cs₂CO ₃ as a base. The S-alkylation products, obtained in high yields (96-98 %) and without any detectable traces of accompanying byproducts, are hydrolysed to yield the free carboxyl group. An orthogonal protection is then introduced on the free amino group by treatment with Fmoc-OSu under standard conditions. The inclusion of one of these orthogonally protected diamino acids in a solid-phase growing pentapeptide is also reported. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500464

FULL PAPER
DOI: 10.1002/ejoc.200500464
One-Pot Synthesis of Orthogonally Protected Enantiopure S-(Aminoalkyl)-
cysteine Derivatives[‡]
Adele Bolognese,[a] Olga Fierro,[b] Daniela Guarino,[b] Luigi Longobardo,*[a,b] and
Romualdo Caputo[a]
Dedicated to the memory of Arno F. Spatola[‡‡]
Keywords: Thia diamino acids / S-Alkylation / Diversity / Cysteine / β-Iodoamines
The general synthesis of a new class of non-natural diamino
acids, 2-amino-3-[(2Ј-aminoalkyl)thio]propanoic acids or S-
(aminoalkyl)cysteines, is reported. Under the conditions de-
vised, enantiopure N-Boc-protected β-iodoamines, readily
generated from proteinogenic α-amino acids, are treated
byproducts, are hydrolysed to yield the free carboxyl group.
An orthogonal protection is then introduced on the free
amino group by treatment with Fmoc-OSu under standard
conditions. The inclusion of one of these orthogonally pro-
tected diamino acids in a solid-phase growing pentapeptide
is also reported.
with L-cysteine ethyl ester hydrochloride, using Cs2CO3 as a
base. The S-alkylation products, obtained in high yields (96–
98%) and without any detectable traces of accompanying
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
tural elements such as helices, turns and small sheetlike
structures. When peptide bioactivity is contingent upon a
precise 3-D structure, the capacity of a biomimetic oligomer
to fold can be indispensable. Examples of simple peptidomi-
metics include azapeptides, oligocarbamates and oligoureas,
and common examples of foldamers include β-peptides, γ-
peptides, oligo(phenylene ethynylene)s, vinylogous sulfono-
peptides and poly-N-substituted glycines (peptoids).[2–5]
However, the main road to generate peptide diversity still
resides on the preparation of non-natural amino acids and
their incorporation in specific (bioactive) peptide sequences.
In this paper, we report the enantioselective synthesis of
new thia diamino acids 1 that represent a new class of non-
natural amino acids resembling natural lanthionine (2)[6]
one of whose carboxyl groups has been replaced by the side
chain (R in 1) of a proteinogenic α-amino acid.
Introduction
Nowadays it is thoroughly recognized that natural pep-
tides, despite the potential interest to exploit them as phar-
maceutical lead compounds, are indeed poor drug candi-
dates because of their low oral bioavailability, potential im-
munogenicity and insufficient metabolic stability in vivo.[1]
Recent efforts to ameliorate disadvantageous peptide
characteristics and thus generate viable pharmaceutical
therapies have focused on the creation of non-natural pep-
tide mimics. These “peptidomimetics” can be based on any
oligomer that mimics peptide primary structure through the
use of amide bond isosteres and/or modification of the
native peptide backbone, including chain extension or het-
eroatom incorporation.[2] Peptidomimetic oligomers are
often protease-resistant and may have reduced immunoge-
nicity and improved bioavailability relative to peptide ana-
logues.
In addition to primary structural mimicry, a select subset
of the sequence-specific peptidomimetic oligomers, the so-
called “foldamers”,[3] exhibits well-defined secondary struc-
To the best of our knowledge, the sole literature pre-
cedent for these compounds is the so-called “thialysine”
(“Tlys”) (R = H in 1), known for its interesting cytotoxic
properties towards human acute leukaemia Jurkat T cells.[7]
Thialysine, whose preparation dates back to nearly half a
century ago,[8,9] should formally represent the parent com-
pound of this class of thia diamino acids.
[‡] Chiral Aminoalkyl Cation Equivalents, 1
[a] Dipartimento di Chimica Organica e Biochimica, Università di
Napoli Federico II
4, via Cynthia, 80126 Napoli, Italy
Fax: +39-081-674102
E-mail: luilongo@unina.it
[b] CNR-Istituto di Scienze dell’Alimentazione 52,
Via Roma, 83100 Avellino, Italy
[‡‡]A pioneer of pseudopeptide chemistry
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
In view of the biological properties that could be ex-
pected for such compounds and also their usefulness in in-
Eur. J. Org. Chem. 2006, 169–173
© 2006 Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim
169

A. Bolognese, O. Fierro, D. Guarino, L. Longobardo, R. Caputo
FULL PAPER
troducing elements of diversity if incorporated in synthetic
peptide molecules, we have realized a simple and efficient
synthesis that leads to orthogonally protected thia diamino
acids 1, by S-alkylation of C-protected l-cysteine with dif-
ferent N-Boc-β-iodoamines 5, in their turn prepared as de-
picted in Scheme 1.
Scheme 2. Synthesis of orthogonally protected thia diamino acids.
i) HCl·l-Cys-OEt, Cs2CO3, DMF, room temp., 3 h; ii) aq. LiOH,
MeOH, room temp., 2 h; iii) Fmoc-OSu, THF, Na2CO3, 0 °C, 1 h.
Scheme 1. Conversion of N-Boc-α-amino acids into N-Boc-β-
iodoamines. i) THF, NMM, MeOCOCl, then NaBH4 in H2O; ii)
TPP-I2, ImH (imidazole), DMC, reflux, 1 h.
The S-alkylation procedure may appear as a slight modi-
fication of a method already reported for the synthesis of
lanthionine derivatives.[17–19] Indeed, the resemblance is
only formal, since in our case the S-alkylation occurs on an
iodide β to a carbamate function (N-Boc-β-iodoamines 5)
whereas the cysteine alkylation leading to lanthionines oc-
curs on the β-iodide of N-trityl-protected serine alkyl ester
that is naturally prone to undergo either hydrogen iodide
elimination or aziridine ring closure.
Following alkylation, the products 6a–c were hydrolysed
by LiOH in MeOH to the corresponding monoprotected
thia diamino acids 7a–c that, without being isolated, were
treated with Fmoc-OSu under standard conditions. The fi-
nal orthogonally protected thia diamino acids 8a–c were
obtained in excellent (79–82%) overall yields (based on the
starting N-Boc-β-iodoamines) after purification by flash
chromatography. Their regio- and stereochemical integrity
In fact, the reaction is known, and several reports of S-
alkylation of N-Boc-β-iodoamines by miscellaneous mer-
captans can be found in the current literature.[10–14] The ra-
tionale of their use in the synthesis of thia diamino acids 1
lies in the opportunity to access a series of synthetic build-
ing blocks, chiral aminoalkyl cation equivalents that carry
through the chirality of their parent proteinogenic α-amino
acids.
Results and Discussion
The enantiopure N-Boc-β-iodoamines 5a–c were readily was ascertained by TLC, RP-HPLC, 1H and 13C NMR
prepared in excellent yields from the corresponding natural spectroscopy, and MS analyses. DEPT and COSY 1H
α-amino acids 3a–c, as depicted in Scheme 1. The N-Boc- NMR experiments also showed the absence of traces of
protected β-amino alcohols 4a–c, coming from the re- other isomers.
duction of the α-amino acids, were converted into the corre-
In order to avoid complicated systematic names in cur-
sponding iodoamines by using a polymer-bound triarylpho- rent laboratory practice, we propose for these new thia di-
sphane/I2 complex in dry dichloromethane (DCM) in the amino acids a nomenclature system in which each com-
presence of imidazole.[15] In consideration of the somewhat pound is given an acronym composed of “Cy” (from “Cys”)
high cost of such a triarylphosphane, we also carried out and the one-letter code denoting the α-amino acid whose
the conversion by using the inexpensive triphenylphos- side chain (R in formula 1) is present in it. A referee sug-
phane, with quite comparable results (see Exp. Sect.).
gested using the one-letter code in lower case, to be more in
Under our experimental conditions, the N-Boc-β- connection with the three-letter symbolism of amino acids.
iodoamines 5a–c, generated from Phe, Leu and Pro,[16] Accordingly, the three compounds we have synthesised and
respectively, were treated with l-cysteine ethyl ester hydro- reported in this paper should be indicated as Fmoc-l-
chloride and Cs2CO3, in dry DMF under argon (Scheme 2). Cyf(Boc)-OH (8a), Fmoc-l-Cyl(Boc)-OH (8b) and Fmoc-
The S-alkylations proceeded in high yields (96–98%); nei- l-Cyp(Boc)-OH (8c).
ther alkylation of the cysteinyl amino group nor traces of
As an illustrative example of the versatility of our or-
elimination products and/or aziridines from 5a–c could be thogonally protected thia diamino acids, compound 8a was
detected by TLC or 1H NMR of the crude alkylation prod- included in a growing pentapeptide on a PAC-PEG-PS (4-
ucts 6a–c.
hydroxymethylphenoxyacetic acid polyethyleneglycol poly-
170
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 169–173

Orthogonally Protected Enantiopure S-(Aminoalkyl)cysteine Derivatives
FULL PAPER
1010 polarimeter. RP-HPLC was carried out with a Shimadzu
SCL-10 Avp system with a photodiode-array detector, using a bi-
nary solvent system consisting of 0.01% TFA in H2O (solvent A)
and 0.01% TFA in MeCN (solvent B); an analytical Vydac C18
column was used (4.6 mm diameter with a flow rate of 1 mL/ min).
1H and 13C NMR spectra were recorded with Varian Inova 500
and Bruker DRX-400 spectrometers: chemical shifts are in ppm (δ)
and J coupling constants in Hz. Low-resolution MALDI-TOF
mass spectra were obtained with a Voyager DE-PRO (PE-Biosys-
tem), using 2,3-dihydroxybenzoic acid (DHB) as matrix. High-reso-
lution ES mass spectra were obtained with a Micromass Q-TOF
UltimaTM API using Leu-Enk as standard and NaI for calibration.
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 visualized by UV. Column chromatography was performed on
Merck Kieselgel 60 (70–230 mesh). Dry solvents were distilled im-
mediately before use.
styrene) support with a classical peptide coupling in DMF.
HATU [2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetrameth-
yluronium hexafluorophosphate] and NMM (N-methyl-
morpholine) were used as activating agents (Scheme 3).
Preparation of N-Boc-β-Iodoamines 5a–c. General Procedure: To a
stirred suspension of polystyryl-diphenylphosphane (ca. 3 mmol/g,
4 g, ca. 12 mmol) in dry DMC (70 mL), under argon, I2 (3.62 g,
12.7 mmol) and imidazole (1.70 g, 25 mmol) were added in se-
quence. After 15 min, one of the N-Boc-β-aminols 4a–c (5 mmol),
dissolved in the same solvent (10 mL), was added in one portion
to the heterogeneous reaction mixture, that was then refluxed for
1 h (TLC monitoring, EtOAc/hexane, 15:85). After cooling, it was
diluted with DMC (100 mL) and filtered through a glass septum
funnel to remove the polymeric material. The filtrate was washed
with aq. 10% Na2S2O3 (2×50 mL) and finally with pure water,
then concentrated in vacuo. The resulting N-Boc-β-iodoamines 5a–
c, obtained in high yields (95–98%), were used directly in the next
step. When, under the same experimental conditions, polystyryl-
diphenylphosphane was replaced by soluble TPP (triphenylphos-
phane) (1.64 g, 6.25 mmol), the final N-Boc-β-iodoamines needed
further purification by flash chromatography (EtOAc/hexane, 7:3)
and were obtained in somewhat lower yields (82–86%).
Scheme 3. Solid-phase synthesis of pentapeptide foldamer 9. i) 2%
DBU in DMF, Fmoc-Cyf(Boc)-OH, HATU, NMM, 1 h; ii) 2%
DBU in DMF, Boc-Phe-OH, HATU, NMM, 30 min; iii) TFA,
TES, TA, room temp., 2 h.
In the final step, the product was cleaved from the poly-
meric support, and the expected pentapeptide 9 was ob-
tained. One single compound with the expected molecular
weight could be detected by analytical RP-HPLC and
MALDI-TOF MS analysis of the crude product.
tert-Butyl [(1S)-1-Benzyl-2-iodoethyl)carbamate (5a): 96%; m.p.
110–111 °C (from Et2O/hexane). [α]2D0 = +18.3 (c = 1.3, CHCl3)
{ref.[22] m.p. 118 °C; [α]2D0 = +18.9 (c = 2.9)}; Rf (EtOAc/hexane,
85:15) = 0.51. 1H NMR (CDCl3, 500 MHz): δ = 1.42 (s, 9 H), 2.76
(dd, J = 6.7 and 13.4 Hz, 1 H), 2.90 (dd, J = 5.0 and 13.4 Hz, 1
H), 3.16 (dd, J = 3.3 and 10.0 Hz, 1 H), 3.38 (br. dd, J = 4.0 and
10.0 Hz, 1 H), 3.45–3.70 (m, 1 H), 4.57–4.72 (m, 1 H), 7.20–7.40
(m, 5 H) ppm.
Conclusions
We have proposed a synthesis of orthogonally protected
thia diamino acids that are ready to be inserted into solid-
phase growing peptides to obtain foldamers.
It is noteworthy that the same synthetic approach can
even be applied to the modification of native proteins (bio-
conjugation),[20,21] considering that the most widely used
tert-Butyl [(1S)-1-(Iodomethyl)-3-methylbutyl]carbamate (5b): 98%;
m.p. 58 °C (from Et2O/hexane). [α]2D0 = –31.6 (c = 1.5, CHCl3)
{refs.[15,23] m.p. 55–57 °C; [α]2D0 = –29.9 (c = 1.3)}; Rf (EtOAc/hex-
1
ane, 85:15) = 0.56. H NMR (CDCl3, 500 MHz): δ = 0.92 (d, J =
bioconjugation strategy exploits the latent nucleophilicity 6.7, 3 H), 0.93 (d, J = 6.7, 3 H), 1.30–1.37 (m, 2 H), 1.45 (s, 9 H),
1.53–1.68 (m, 1 H), 3.28 (dd, J = 2.7 and 9.2 Hz, 1 H), 3.35–3.45
(m, 1 H), 3.47 (dd, J = 3.7 and 9.2 Hz, 1 H), 4.52 (br. d, J = 6.0,
1 H) ppm.
of the thiol side chain of cysteine. It might be interesting to
use chiral β-iodoamines as aminoalkyl cation equivalents
for the S-alkylation of cysteines and the introduction of
proteinogenic α-amino acid moieties in native proteins.
However, successful work is already in progress in our
laboratory in preparing new orthogonally protected non-
natural diamino acids such as 8a–c with various hetero-
atoms other than sulfur.
tert-Butyl [(1S)-2-Iodo-1-(pyrrolidin-1-yl)ethyl]carbamate (5c): 95%;
m.p. 38–40 °C (from EtOAc/hexane). [α]2D0 = –34.7 (c = 1.3, CHCl3)
{ref.[24] m.p. 102–104 °C; [α]2D0 = –32.8 (c = 1.5)}; Rf (EtOAc/hex-
1
ane, 85:15) = 0.48. H NMR (CDCl3, 500 MHz): δ = 1.46 (s, 9 H),
1.81–1.83 (m, 1 H), 1.86–1.89 (m, 2 H), 2.03–2.05 (m, 1 H), 3.11–
3.15 (t, J = 9.5, 1 H), 3.34–3.37 (m, 2 H), 3.43–3.48 (t, 1 H), 3.82–
3.85 (m, 1 H) ppm.
S-Alkylations of Cysteine Ethyl Ester. General Procedure: To a
stirred solution of one of the β-iodoamines 5a–c (0.33 mmol) and
l-cysteine ethyl ester hydrochloride (62 mg, 0.33 mmol) in dry
DMF (5 mL) and under argon, solid Cs2CO3 (216 mg, 0. 66 mmol)
Experimental Section
General: Melting points were measured with a Kofler apparatus
and are uncorrected. Optical rotations were measured with a Jasco
Eur. J. Org. Chem. 2006, 169–173
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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171

A. Bolognese, O. Fierro, D. Guarino, L. Longobardo, R. Caputo
FULL PAPER
was added in one portion. Stirring was continued in the dark at
room temperature for 3 h. Most of the solvent was carefully (Ͻ
45 °C) evaporated in vacuo and the residue was suspended in
EtOAc (50 mL) and shaken with H2O (3×20 mL). The organic
layer was then dried (Na2SO4) and the solvents were evaporated in
vacuo.
125 MHz): δ = 29.9, 36.8, 39.5, 42.1, 54.6, 56.6, 59.5, 69.3, 81.2,
122.0, 127.5, 128.4, 129.3, 129.9, 130.4, 131.5, 140.9, 143.7, 146.4,
170.2 ppm. MS-MALDI-TOF (C32H36N2O6S): m/z = 599.14 [M +
Na+], 615.18 [M + K+], 477.30 [M – Boc]. HR ES-MS (EI): calcd.
577.2765 [MH+], found 577.2789.
(2R)-3-[(2ЈS)-2Ј-(tert-Butyloxycarbonyl)amino-4Ј-methylpentylthio]-
2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-Cyl-
(Boc)-OH (8b): 82% (overall yield from 5b); Rf (DCM/MeOH, 9:1)
= 0.37. Analytical sample, crystallized from hot absolute EtOH:
M.p. 130–133 °C. [α]2D0 = –29.2 (c = 0.6, MeOH). 1H NMR
(CD3OD, 500 MHz): δ = 0.88 (d, J = 5.8 Hz, 6 H), 1.28–1.36 (m,
2 H), 1.42 (s, 9 H), 1.62–1.65 (m, 1 H), 2.61–2.64 (m, 2 H), 2.91–
2.94 (m, 1 H), 3.05–3.08 (m, 1 H), 3.66–3.70 (m, 1 H), 4.23–4.33
(m, 4 H), 7.29–7.32 (t, J = 7.5 Hz, 2 H), 7.36–7.40 (t, 2 H), 7.69–
7.70 (d, J = 7.4 Hz, 2 H), 7.78–7.80 (d, 2 H) ppm. 13C NMR
(CD3OD, 125 MHz): δ = 19.5, 29.9, 36.8, 39.5, 42.1, 54.6, 56.6,
59.5, 69.3, 81.1, 122.0, 127.5, 129.3, 129.9, 130.4, 131.5 140.9,
143.7, 146.4, 159.0, 159.6, 160.5, 171.4 ppm. MS-MALDI-TOF
(C29H38N2O6S): m/z = 565.32 [M + Na+], 581.33 [M + K+], 443.25
[M – Boc]. HR ES-MS (EI): calcd. 543.2940 [MH+], found
543.2965.
Ethyl (2R)-2-Amino-3-{(2ЈS)-2Ј-[(tert-butyloxycarbonyl)amino]-3Ј-
phenylpropylthio}propanoate, H-Cyf(Boc)-OEt (6a): Oil, ca. 97%;
Rf (DCM/MeOH, 95:5) = 0.23. Chromatographically pure sample:
1H NMR (CDCl3, 400 MHz): δ = 1.24 (t, 3 H), 1.39 (s, 9 H), 2.62
(d, J = 5.5, 2 H), 2.76–2.93 (m, 3 H), 2.9 (dd, J = 4.6 and 13.4 Hz,
1 H), 3.60 (dd, J = 4.6 and 7.0 Hz, 1 H), 3.92–4.01 (m, 1 H), 4.15
(q, 2 H), 5.03 (d, J = 8.4, 1 H), 7.16–7.28 (m, 5 H) ppm. 13C NMR
(CDCl3, 125 MHz): δ = 13.9, 28.1, 36.6, 37.7, 39.3, 51.2, 54.1, 61.0,
79.0, 126.3, 128.2, 129.2, 137.4, 155.1, 173.7 ppm. C19H30N2O4S
(382.5): calcd. C 59.66, H 7.91; found C 59.61, H 7.87 (specially
prepared analytical sample).
Ethyl
(2R)-2-Amino-3-{(2ЈS)-2Ј-[(tert-butoxycarbonyl)amino]-4Ј-
methylpentylthio}propanoate, H-Cyl(Boc)-OEt (6b): Oil, ca. 98%;
Rf (DCM/MeOH, 95:5) = 0.21. Chromatographically pure sample:
1H NMR (CDCl3, 400 MHz): δ = 0.82 (d, 6 H), 1.21 (t, 3 H), 1.31
(t, 2 H), 1.41 (s, 9 H), 1.6 (m, 1 H), 2.61 (d, J = 5.5, 2 H), 2.75
(dd, J = 7.0 and 13.4 Hz, 1 H), 2.79 (dd, J = 4.6 and 13.4 Hz, 1
H), 3.51 (dd, J = 4.6 and 7.0 Hz, 1 H), 3.9 (m, 1 H), 4.18 (q, 2 H),
4.9 (br. d, 1 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 14.3, 22.3,
23.2, 25.1, 28.6, 38.4, 39.1, 43.2, 48.6, 54.5, 61.3, 79.7, 155.6,
174.1 ppm. C16H32N2O4S (348.5): calcd. C 55.14, H 9.26; found C
55.20, H 9.27 (specially prepared analytical sample).
(2R)-3-{[(2ЈS)-1-(tert-Butyloxycarbonyl)pyrrolidin-2Ј-ylmethyl]-
thio}-2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-
Cyp(Boc)-OH (8c): 79% (overall yield from 5c); Rf (DCM/MeOH,
9:1) = 0.45. Analytical sample, crystallized from hot absolute
EtOH: M.p. 130–133 °C. [α]2D0 = –25.2 (c = 0.6, MeOH). H NMR
1
(CD3OD, 500 MHz): δ = 1.36 (s, 9 H), 1.67–1.69 (m, 1 H), 1.80–
1.85 (m, 3 H), 2.49–2.57 (m, 1 H), 2.76–2.81 (m, 3 H), 2.99–3.01
(m, 2 H), 3.07–3.10 (m, 1 H), 3.80–3.82 (m, 2 H), 4.13–4.16 (t, J =
7.7 Hz, 1 H), 4.20–4.26 (m, 3 H), 7.20–7.22 (t, J = 7.5 Hz, 2 H),
7.26–7.28 (t, 2 H), 7.57–7.59 (d, J = 7.4 Hz, 2 H), 7.68–7.70 (d, J
= 7.4 Hz, 2 H) ppm. 13C NMR (CD3OD, 125 MHz): δ = 22.1, 28.1,
29.1, 33.6, 35.6, 46.3, 52.4, 57.1, 65.7, 79.8, 120.1 125.3, 127.1,
127.6, 140.7, 143.8, 153.9, 157.2, 172.3 ppm. MS-MALDI-TOF
(C28H34N2O6S): m/z = 549.6 [M + Na+], 565.7 [M + K+], 426.5
[M – Boc]. HR ES-MS (EI): calcd. 527.2731 [MH+], found
527.2704.
Ethyl (2R)-2-Amino-3-{[(2ЈS)-1-(tert-butoxycarbonyl)pyrrolidin-2Ј-
yl]methylthio}propanoate, H-Cyp(Boc)-OEt (6c): Oil, ca. 96%; Rf
(DCM/MeOH, 95:5) = 0.29. Chromatographically pure sample: 1H
NMR (CD3OD, 400 MHz): δ = 1.28 (t, 3 H), 1.45 (s, 9 H), 1.85–
1.97 (m, 4 H), 2.55–2.58 (m, 1 H), 2.84–2.88 (m, 3 H), 3.36–3.38
(m, 2 H), 3.60–3.63 (m, 1 H), 3.87–3.89 (m, 1 H), 4.16–4.21 (q, 2
H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 14.4, 22.9, 28.7, 30.2,
36.5, 37.8, 47.2, 54.5, 57.1, 61.3, 79.8, 154.4, 174.1 ppm.
C15H28N2O4S (332.5), calcd. C 54.19, H 8.49; found C 54.24, H
8.43 (specially prepared analytical sample).
Alkaline Hydrolysis of Crude 6a–c and Fmoc Protection of the Crude
Diamino Acids 7a–c. General Procedure: To a stirred solution of
one of the S-alkylated compounds 6a–c (0.3 mmol) in MeOH
(5 mL) aq. 1 m LiOH (1 mL) was added in one portion. After 2 h
of stirring at room temperature, MeOH was evaporated in vacuo.
The residue was redissolved in THF (10 mL) and the solution co-
oled to 0 °C. Fmoc-OSu (112 mg, 0.33 mmol) was then added por-
tionwise over 1 h, maintaining an alkaline pH (ca. 10) by dropwise
addition of 10% aq. Na2CO3, then by addition of excess solid
Na2CO3. THF was evaporated in vacuo and the crude reaction
product was dissolved in EtOAc (75 mL) and worked up as usual,
under acidic conditions (aq. 0.1 m HCl). The final N-Fmoc,NЈ-Boc
thia diamino acids 8a–c were purified by flash chromatography,
using a 0–5% MeOH gradient in DCM.
Solid-Phase Synthesis of H-Phe-Cyf-Asp-Lys-Gln-OH (9): In a
manual peptide synthesizer Fmoc-Asp(OtBu)-Lys(Boc)-Gln(Trt)-
PAC-PEG-PS was grown up starting from Fmoc-Gln(Trt)-PAC-
PEG-PS (118 mg, 0.16 mmol/g, 0.019 mmol). The Fmoc group was
removed using 2% DBU (1,8-diazobicyclo[5.4.0]undec-7-ene) in
DMF (3 mL) for 30 min. The orthogonally protected thia diamino
acid 8a (43 mg, 0.076 mmol) was then added together with HATU
(29 mg, 0.076 mmol) and NMM (8 μL) in DMF (3 mL). The coup-
ling proceeded for 1 h. The resin was washed with DMF (3×5 mL)
and DCM (3×5 mL) and the protecting Fmoc group was again
removed with 2% DBU in DMF (3 mL) for 30 min. The last coup-
ling was performed by using Boc-Phe-OH (20 mg, 0.076 mmol),
HATU (29 mg, 0.076 mmol) and NMM (8 μL) in DMF (3 mL).
The product was eventually washed with DMF, DCM, and Et2O,
dried and cleaved from the polymeric support using a mixture of
TFA (1.9 mL), TES (triethylsilane) (60 μL) and TA (thioanisole)
(2R)-3-[(2ЈS)-2Ј-(tert-Butyloxycarbonyl)amino-3Ј-phenylpropylthio]-
2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-
Cyf(Boc)-OH (8a): 80% (overall yield from 5a); Rf (DCM/MeOH, (60 μL) at room temperature for 2 h. After precipitation in dry
9:1) = 0.47. Analytical sample, crystallized from hot absolute
Et2O, it was washed 3 times with the same solvent. Under analyti-
cal RP-HPLC conditions (solvents A/B, 95:5 over 5 min, then from
95:5 to 8:2 over 10 min, finally from 8:2 to 1:1 over 10 min) a single
1
EtOH: M.p. 135–136 °C. [α]2D0 = –20.0 (c = 1.0, MeOH). H NMR
(CD3OD, 500 MHz): δ = 1.40 (s, 9 H), 2.62–2.67 (m, 3 H), 2.84–
2.90 (m, 2 H), 3.04–3.06 (dd, J = 4.5 Hz, 1 H), 3.86–3.87 (m, 1 H), peak could be detected at t = 12.6 min (220 nm). MS-MALDI-
4.22–4.27 (t, J = 7.7, 1 H), 4.31–4.37 (m, 3 H), 7.13–7.20 (m, 5 H), TOF (C36H52N8O9S): m/z (%) = 773.24 (100) [M], 795.21 (50) [M
7.27–7.31 (t, J = 7.5 Hz, 2 H), 7.35–7.39 (t, 2 H), 7.67–7.69 (d, J + Na+], 811.21 (10) [M + K+], 755.23 (30) [M – H2O]. HR ES-MS
= 7.4 Hz, 2 H), 7.77–7.79 (d, 2 H) ppm. 13C NMR (CD3OD,
(EI): calcd. 773.3746 [MH+], found 773.3759.
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Eur. J. Org. Chem. 2006, 169–173

Orthogonally Protected Enantiopure S-(Aminoalkyl)cysteine Derivatives
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Received: June 24, 2005
Published Online: November 7, 2005
Eur. J. Org. Chem. 2006, 169–173
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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