Synthesis of protected ω-mercapto amino acids: Precursors for incorporation of elongated cysteines into peptides

Article abstract of DOI:10.1016/S0957-4166(98)00261-4

A series of protected ω-mercapto amino acids with side-chain lengths ranging from 3-5 methylene units has been synthesized via nucleophilic substitution of ω-bromo-α-azido acids by 4-methoxy-α-toluenethiol followed by reduction of the azido functionality

Full text of DOI:10.1016/S0957-4166(98)00261-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2739–2743
Synthesis of protected ω-mercapto amino acids: precursors for
incorporation of elongated cysteines into peptides
Joseph T. Lundquist IV,a Erika E. Büllesbach b and Thomas A. Dix a,∗
aDepartment of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA
bDepartment of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29425, USA
Received 22 June 1998; accepted 23 June 1998
Abstract
A series of protected ω-mercapto amino acids with side-chain lengths ranging from 3–5 methylene units has
been synthesized via nucleophilic substitution of ω-bromo-α-azido acids by 4-methoxy-α-toluenethiol followed
by reduction of the azido functionality with SnCl2. These enantiomerically pure protected cysteine analogues can
be used to optimize the length of disulfide connections in cyclically constrained peptide pharmacophores. © 1998
Elsevier Science Ltd. All rights reserved.
1. Introduction
There is considerable interest in producing small peptides and peptidomimetics with the structural
conformation of the active binding pharmacophore of much larger peptides or proteins. These small active
moieties offer potential advantages of improved binding potency and selectivity, chemical and biological
stability, and pharmacokinetic characteristics when compared to their more complex molecules of origin.
A popular strategy has been to cyclize the essential functional elements via a disulfide linker which
can lock in the necessary three-dimensional structure for binding and biological activity as evidenced in
somatostatin mimetics.1 A pair of sulfhydryl containing amino acids placed in the proper position of the
peptide can stabilize a β-turn,1 one turn of an α-helix,2 or two turns of an α-helix.3 To establish these
motifs, the variety of natural thiol-containing amino acids, cysteine and homocysteine, is insufficient:
i.e. D-enantiomers and extended side-chains are required for optimization of the disulfide connection and
structure stabilization. Thus, we are interested in strategically protected ω-mercapto amino acids (Fig. 1)
for incorporation into relevant peptides.
D- and L-2-Amino-6-mercaptohexanoic acids have been prepared previously3 by converting the ω-
amino group of lysine to a pyridinium salt which is then displaced by a thiol, leaving S-protected D- or L-
∗
Corresponding author. Tel: (843)-953-6614; fax: (843)-953-6615; e-mail: dixta@musc.edu
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00261-4

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Fig. 1. Protected ω-mercapto amino acids
2-amino-6-mercaptohexanoic acids.3,4 Disadvantages of this chemistry are the use of a large chromogenic
leaving group, high excess of reagent, harsh reaction conditions, and subsequently low overall yields.
Although identical chemistry could be used to generate D- and L-2-amino-5-mercaptovaleric acids using
the corresponding ornithine analogue, other ω-mercapto-α-amino acids are not accessible via this route.
In this paper, we describe a straightforward scheme which may be used to produce ω-sulfur containing
α-amino acids of various side-chain lengths in good yields.
2. Results
The 2-(S)-azido-ω-bromo acids synthesized previously in this laboratory5 were chosen as start-
ing materials (Scheme 1) to produce ω-mercapto-containing amino acid analogues as follows: 2-
(S)-azido-5-bromovaleric acid (1a) for amino acids possessing three methylene groups in the side-
chain, 2-(S)-azido-6-bromohexanoic acid (1b) for analogues containing four methylene units, and
2-(S)-azido-7-bromoheptanoic acid (1c) for analogues containing five methylene units in the side-
chain. These chiral building blocks initially are substituted at the ω-carbon by equimolar amounts
of 4-methoxy-α-toluenethiol under basic conditions providing 2a–c. The reactions occur at room
temperature and are complete after 60 min. The azido group is reduced to the free amine with
stannous chloride in methanol following the method of Maiti et al.6 To provide monomers com-
patible with liquid- or automated solid-phase peptide synthesis, the amino group of each 2-amino-
ω-mercapto acid was either 9-fluorenylmethoxycarbonyl (Fmoc) protected7 yielding 3a–c or the D-
enantiomer of 3b was tert-butoxycarbonyl (t-Boc) protected providing D-Nα-(tert-butoxycarbonyl)-6-
(4-methoxybenzylmercapto)hexanoic acid 4 (depicted in Fig. 1).
3. Discussion
Since a general route to produce a variety of sulfur containing amino acids does not exist, we
designed and carried out the straightforward synthesis of a homologous series of protected 2-amino-ω-
mercapto acids with variable side-chain lengths. The products, L-Nα-(9-fluorenylmethoxycarbonyl)-
5-(4-methoxybenzylmercapto)valeric
acid
(3a),
L-Nα-(9-fluorenylmethoxycarbonyl)-6-(4-
methoxybenzylmercapto)hexanoic acid (3b), and L-Nα-(9-fluorenylmethoxycarbonyl)-7-(4-
methoxybenzylmercapto)heptanoic acid (3c), have been prepared from the appropriate 2-(S)-azido
ω-bromo acid in 44%, 55%, and 53% overall yields, respectively. The t-Boc-protected enantiomer
of 3b, D-Nα-(tert-butoxycarbonyl)-6-(4-methoxybenzylmercapto)hexanoic acid (4), was prepared
following the same general route in 79% overall yield. The versatility of our route is that any amino
protecting group can be incorporated at the end of the synthesis. Also advantageous is that the sulfur
is introduced with a trifluoroacetic acid labile protecting group (4-methoxybenzyl) attached. The

J. T. Lundquist IV et al. / Tetrahedron: Asymmetry 9 (1998) 2739–2743
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Scheme 1. Synthesis of protected L-2-amino-ω-mercapto acids
conditions for this nucleophilic substitution are mild enough so that even sterically hindered thiols
like triphenylmethanethiol might be considered for this reaction. The series of 2-amino-ω-mercapto
acids offer the possibility to optimize the active binding motifs in many settings including simple
one-turn cyclizations, two-turn α-helixes, and macromolecular disulfide optimizations. Additionally, the
availability of a variety of alkylated thiols offer the potential to produce many additional products, such
as non-natural methionine homologues and derivatives.
4. Experimental
1H and 13C NMR were obtained on a Varian Gemini 300 spectrometer. All solvents were reagent
grade obtained from commercial suppliers and used without distillation. Reagents were obtained as
follows: 4-methoxy-α-toluenethiol from Fluka, 9-fluorenylmethyl succinimidyl carbonate and di-tert-
butyl pyrocarbonate from Acros Organics, and stannous chloride from Aldrich. Flash chromatography
was performed using Whatman silica gel 60 (230–400 mesh) and monitored using Whatman UV254 thin
layer chromatography plates. Analytical HPLC was used to monitor nucleophilic substitution reactions
with the following system: the mobile phase consisted of 0.1% trifluoroacetic acid in water (solvent A)
and 0.1% trifluoroacetic acid in 80% acetonitrile (solvent B). Analysis was performed on a Waters dual
pump HPLC system in combination with a Bakerbond (C18, wide bore) column. Approximately 10 to
50 µg of azido acid was injected and separated at a flow rate of 1 mL/min. Linear gradients from 20% to
80% B over 30 min were used and the effluent detected by UV absorbance at 220 nm. Optical rotations
were measured in 1 and 0.5 dm cells of 10 and 0.1 mL capacity, respectively, and are quoted in units of
deg cm−3 g−1 dm−1
.
4.1. Representative synthesis of 2-(S)-azido-7-(4-methoxybenzylmercapto)heptanoic acid (2c)
To a solution of 1c (310 mg, 1.24 mmol) in EtOH (9 mL) was added 4-methoxy-α-toluenethiol
(purity 90%, 212 mg, 1.00 equiv.) followed by immediate addition of aqueous KOH (344 µL, 9.0 M
KOH solution, 2.5 equiv., diluted to 3 mL with distilled H2O). The reaction was allowed to stir at room

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J. T. Lundquist IV et al. / Tetrahedron: Asymmetry 9 (1998) 2739–2743
temperature and monitored by analytical HPLC. The reaction was complete after 1 h and concentrated
under reduced pressure, diluted with 40 mL H2O, and acidified to pH 2 by dropwise addition of 37% HCl.
The crude product was extracted with ethyl acetate (3×), dried (MgSO4), and concentrated. Purification
on silica gel (elution with 5% MeOH in CH2Cl2, Rf=0.54) provided 353 mg of 2c as a clear oil (88%
yield). [α]D22=−15.6 (c=1.0, methanol, l=1 dm). 1H NMR (300 MHz, CDCl3) δ 7.23 (d, J=8.6 Hz, 2H),
6.86 (d, J=8.7 Hz, 2H), 3.89 (dd, J=5.2, 8.5 Hz, 1H), 3.80 (s, 3H), 3.67 (s, 2H), 2.41 (t, J=6.9 Hz, 2H),
1.90–1.70 (m, 2H), 1.65–1.50 (m, 2H), 1.50–1.35 (m, 4H); 13C NMR (75.5 MHz, CDCl3) δ 176.4, 158.6,
130.6, 129.9, 113.9, 61.7, 55.3, 35.7, 31.2, 31.1, 28.9, 28.2, 25.3.
4.2. 2-(S)-Azido-5-(4-methoxybenzylmercapto)valeric acid (2a)
[α]D22=−26.0 (c=1.0, methanol, l=1 dm). H NMR (300 MHz, CDCl3) δ 7.23 (d, J=8.3 Hz, 2H),
1
6.86 (d, J=8.2 Hz, 2H), 3.89 (dd, J=5.0, 8.2 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 2H), 2.44 (t, J=6.9 Hz, 2H),
2.00–1.80 (m, 2H), 1.80–1.60 (m, 2H); 13C NMR (75.5 MHz, CDCl3) δ 175.9, 158.6, 130.3, 130.0,
114.0, 61.4, 55.4, 35.5, 30.4, 30.3, 25.2.
4.3. 2-(S)-Azido-6-(4-methoxybenzylmercapto)hexanoic acid (2b)
[α]D22=−16.7 (c=1.0, methanol, l=1 dm). H NMR (300 MHz, CDCl3) δ 7.23 (d, J=8.5 Hz, 2H),
1
6.85 (d, J=8.7 Hz, 2H), 3.89 (dd, J=5.2, 8.3 Hz, 1H), 3.80 (s, 3H), 3.67 (s, 2H), 2.41 (t, J=6.9 Hz, 2H),
1.90–1.70 (m, 2H), 1.65–1.45 (m, 4H); 13C NMR (75.5 MHz, CDCl3) δ 176.1, 158.6, 130.4, 129.9,
114.0, 61.9, 55.3, 35.7, 30.9, 31.0, 28.6, 25.0.
4.4. Representative synthesis of L-Nα-(9-fluorenylmethoxycarbonyl)-7-(4-methoxybenzylmercapto)-
heptanoic acid (3c)
The SnCl2 reduction follows the method of Maiti et al.6 for aliphatic amine production,
except that the workup is modified to accommodate the amino acid product. 2-(S)-Azido-7-(4-
methoxybenzylmercapto)heptanoic acid (2c) (353 mg, 1.09 mmol), dissolved in 1 mL MeOH, is added
dropwise to SnCl2 (414 mg, 2 equiv.) suspended in MeOH (1 mL). The reaction was followed by
monitoring the disappearance of 2c using the TLC system described in Section 4.1 and was complete
in 1 h at which time MeOH was removed in vacuo. The solid was diluted with a 50:50 mixture of
H2O:dioxane and brought to pH 11 by addition of KOH solution. The inorganic precipitate was filtered
(Whatman 0.45 µm nylon membrane filter) and washed once with the pH 11 mixture. If necessary,
the amino acid can be purified by reverse phase HPLC at this point, to further remove the inorganic
material. The solution containing the amino acid was concentrated under reduced pressure to 2–3 mL.
The pH was adjusted to 10–11 with HCl or triethylamine (TEA) as needed to facilitate protection of the
amino group following a general procedure,7 utilizing the dropwise addition of dioxane to aid solubility.
9-Fluorenylmethyl succinimidyl carbonate (441 mg, 1.31 mmol) was solubilized in acetonitrile (2 mL)
and added in one portion. The pH was maintained between 9 and 9.5 by dropwise TEA addition. After
1 h, the reaction was concentrated in vacuo, diluted with H2O (75 ml), acidified to pH 2 with HCl, and
extracted with ethyl acetate (3×), which was dried (MgSO4) and concentrated. Purification on silica
gel (elution with 5% MeOH in CH2Cl2, Rf=0.38) provided 340 mg of 3c as a clear oil (60% yield).
[α]D22=−3.3 (c=2.5, methanol, l=0.5 dm). 1H NMR (300 MHz, CDCl3) δ 7.80–7.25 (m, 8H), 7.20 (d,
J=8.7 Hz, 2H), 6.82 (d, J=8.7 Hz, 2H), 5.27 (d, J=8.4 Hz, 1H, N–H), 4.25–4.35 (m, 3H), 4.22 (t, J=6.9
Hz, 1H), 3.77 (s, 3H), 3.64 (s, 2H), 2.37 (t, J=6.7 Hz, 2H), 1.90–1.25 (m, 8H); 13C NMR (75.5 MHz,

J. T. Lundquist IV et al. / Tetrahedron: Asymmetry 9 (1998) 2739–2743
2743
CDCl3) δ 177.1, 158.6, 156.2, 143.7, 141.4, 130.6, 129.9, 127.8, 127.1, 125.1, 120.1, 114.0, 67.1, 55.3,
53.7, 47.2, 35.7, 32.2, 31.1, 28.9, 28.3, 24.9.
4.5. L-Nα-(9-Fluorenylmethoxycarbonyl)-7-(4-methoxybenzylmercapto)valeric acid (3a)
[α]D22=−5.8 (c=2.5, methanol, l=0.5 dm). H NMR (300 MHz, CDCl3) δ 7.80–7.25 (m, 8H), 7.22
1
(d, J=8.5 Hz, 2H), 6.84 (d, J=8.5 Hz, 2H), 5.40 (d, J=8.2 Hz, 1H, N–H), 4.55–4.40 (m, 3H), 4.23 (t,
J=6.8 Hz, 1H), 3.78 (s, 3H), 3.65 (s, 2H), 2.50–2.30 (m, 2H), 2.10–1.50 (m, 4H); 13C NMR (75.5 MHz,
CDCl3) δ 176.6, 158.6, 156.1, 143.7, 141.4, 130.3, 129.9, 127.8, 127.2, 125.1, 120.1, 114.0, 67.2, 55.3,
53.4, 47.2, 35.7, 31.4, 30.7, 24.9.
Acknowledgements
This work was supported by a Faculty Research Award (to T.A.D.) from the Medical University of
South Carolina. J.T.L. is an American Foundation for Pharmaceutical Education predoctoral scholar.
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