4-Amino-1,2-dithiolane-4-carboxylic acid (Adt) as cysteine conformationally restricted analogue. Synthetic protocol for Adt containing peptides

Article abstract of DOI:10.1016/S0960-894X(00)00281-X

An efficient and versatile protocol to incorporate the achiral and C(α,α)-tetrasubstituted 4-amino-1,2-dithiolane-4-carboxylic acid Adt (1) residue into peptides is described. The 2,2-bis[(benzylthio)methyl]glycine N-carboxy anhydride (5) was found to be the key reactive intermediate from which both Boc-Adt-OMe (8) and the glutathione analogue H-Glu(-Adt-Gly-OH)-OH (12) can be obtained. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00281-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1585±1588
4-Amino-1,2-dithiolane-4-carboxylic Acid (Adt) as Cysteine
Conformationally Restricted Analogue. Synthetic Protocol for
Adt Containing Peptides
Enrico Morera,a Marianna Nalli,a Francesco Pinnen,b Domenico Rossia
and Gino Lucentea,*
a
Á
Centro di Studio per la Chimica del Farmaco del CNR c/o Dipartimento di Studi Farmaceutici, Universita degli Studi di Roma ``La
Sapienza'', P.le A. Moro 5, 00185 Roma, Italy
b
Á
Dipartimento di Scienze del Farmaco, Universita ``G. D'Annunzio'', 66100 Chieti, Italy
Received 10 March 2000; accepted 11 May 2000
AbstractÐAn ecient and versatile protocol to incorporate the achiral and Ca,a-tetrasubstituted 4-amino-1,2-dithiolane-4-car-
boxylic acid Adt (1) residue into peptides is described. The 2,2-bis[(benzylthio)methyl]glycine N-carboxy anhydride (5) was found to
be the key reactive intermediate from which both Boc-Adt-OMe (8) and the glutathione analogue H-Glu(-Adt-Gly-OH)-OH (12)
can be obtained. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
heterocyclic Ca,a-tetrasubstituted residue for replacing
cysteine in the glutathione as well as in other bioactive
peptides. This achiral a-amino acid contains in fact the
1,2-dithiolane ring, a ®ve-membered system which, with
varying substituent patterns, is found in a variety of
natural and synthetic compounds (Fig. 1).4,5 1,2-
Dithiolanes can be easily obtained by the corresponding
1,3-dithiols by oxidation with a number of reagents
under mild conditions. However, due to an interplay of
conformational and electronic e€ects, connected with
the size of the ring, the chemical reactivity of 1,2-
dithiolanes is well distinct from that of linear and related
cyclic disul®des,6 8 and represents the key structural
feature on which the activity of relevant biomolecules,
such as lipoic acid, is based.4,5,9 Thus, the incorporation
of this structural fragment into peptide backbones and
the study of the chemical and biochemical consequences
of this modi®cation should represent a new and inter-
esting ®eld to be explored.
Chiral and achiral aminoacids possessing a quaternary
a-carbon atom (Ca,a-tetrasubstituted aminoacids) are
the subject of considerable interest. Their incorporation
into peptides represents in fact an e€ective and versatile
strategy to restrict the conformational freedom, to sta-
bilize de®ned secondary structures and to enhance the
stability towards chemical and enzymatic hydrolysis.1
Among these amino acids particular interest has
recently been devoted to congeners in which the two Ca
substituents make part of a ring containing one or two
heteroatoms.2 These heterocyclic Ca,a-tetrasubstituted
models, when inserted into a peptide backbone, combine
the tendency to generate speci®c constraints with the
capacity to establish intra- or inter-molecular non covalent
interactions through the side chain heteroatoms. As a
consequence of this structural feature some members of
this group are actually conformationally restricted
mimetics of side chain functionalized proteinogenic
amino acids which play crucial roles in binding sites of
receptors and enzymes (Fig. 1).
A literature examination revealed that data referring to
Adt are very scarce and actually limited to the synthesis,
®nalized to the determination of the activity as anti-
in¯ammatory or antiviral agent,10 and enzymatic inhi-
bitor.11 In this paper we report a general and ecient
protocol to prepare Adt derivatives suitable to incorporate
this residue into peptides and their expeditious utilization
for the synthesis of a glutathione analogue containing
the Adt residue in place of the native R-cysteine.
As a prosecution of our interest in the synthesis of glu-
tathione analogues,3 we were attracted by 4-amino-1,2-
dithiolane-4-carboxylic acid (Adt, 1) as an interesting
*Corresponding author. Tel.: +39-06-445-4218; fax: +39-06-491-491;
e-mail: glucente@axrma.uniromal.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00281-X

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E. Morera et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1585±1588
Chemistry
As could be expected, steric hindrance at the Ca-carbon
atom of the aminoacid 4 renders slow and low yielding
the usual amino-protection and carboxy-activation meth-
ods. However, a very ecient and ¯exible procedure was
recognized when the sterically hindered aminoacid 4
was converted to the corresponding N-carboxyanhydride
(NCA, 5).16 This intermediate is a stable and crystalline
compound which can be cleanly and quantitatively
transformed into the N-Boc derivative. To achieve this
result the NCA 5 was treated with (Boc)2O/pyridine,17
to give the corresponding urethane protected N-carboxy-
anhydride (UNCA, 6); this compound is highly reactive
and can be quantitatively converted to the methyl ester
7 by treatment at room temperature with methanol in
the presence of N-methylmorpholine. Deprotection of
the two mercapto groups with sodium in liquid ammo-
nia followed by oxidation with iodine gave the ortho-
gonally protected Adt derivative 8 in 69% yield from 5.
The strategy followed to obtain Adt derivatives is
reported in Scheme 1.12 The 5,5-bis[(benzylthio)methyl]
hydantoin 2 was prepared starting from 1,3-dibromo-
acetone.10 The aminoacid 4 was obtained by re¯uxing 2
with aqueous Ba(OH)2 for eight days.13 In order to ®nd
more rapid and less drastic hydrolysis conditions, the
procedure recently reported by Rebek,14 was explored.
This is based on the activation of the hydantoin, towards
the nucleophilic ring opening, by N-carbamoylation with
tert-butyloxycarbonyl (Boc) groups. Thus, the bis-Boc
derivative 3 was prepared and subjected to milder
hydrolytic conditions.14 In the present case, however,
the method failed to give satisfactory results leading in
any case to mixtures and partial hydrolysis.15
The reactivity evidenced for NCA 5 was then exploited
to synthesize an Adt containing glutathione analogue.
In Scheme 218 two di€erent strategies are reported. In
the ®rst route the NCA 5 is used as carboxy activated
intermediate of the amino acid 4 and allowed to react
Figure 1. Some representative Ca,a-tetrasubstituted heterocyclic ami-
noacids and 1,2-dithiolane derivatives. Abbreviations used: Pip: 4-ami-
nopiperidin-4-carboxylic acid (Ref. 2b); Thp: 4-aminotetrahydrothio-
pyran-4-carboxylic acid (Ref. 2a); TOAC: 2,2,6,6-tetramethylpyperidin-
1-oxyl-4-amino-4-carboxylic acid (Ref. 2e); HmS(Ipr): O,O-isopropyli-
dene-a-hydroxymethylserine (Ref. 2c).
Scheme 2. (a) H-Gly-OBzl.TsOH, TEA, NMM, THF, rt, 24 h, 95%;
(b) Cbz-Glu-OBzl, DCC, Py, THF, rt, 90 h, 60%; (c) Cbz-Glu-OBzl,
Scheme 1. (a) (Boc)2O, DMAP, THF, rt, 2h; (b) Ref. 13; (c) COCl2-
toluene, THF, rt, 1 h, 86%; (d) (Boc)2O, Py, THF, rt, 22h; (e) MeOH,
NMM, rt, 24 h, 86% (overall from 5); (f) Na±NH3 liq., 78 ꢀC; (g) I2,
EtOH, 80% (overall from 7).
.
DCC, Py, THF, rt, 24 h; (d) H-Gly-OBzl TsOH, TEA, NMM, THF,
rt, 3 h, 40% (overall from 5); (e) Na±NH3 liq., 78 ꢀC; (f) I2, EtOH,
90% (overall from 11).

E. Morera et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1585±1588
1587
with equimolar quantity of glycine benzyl ester (H-Gly-
OBzl) to give the dipeptide benzylester 9 in almost
quantitative yield. Contrary to our expectation, the
dicyclohexylcarbodiimide (DCC) mediated acylation of
the sterically hindered amino group of 9 with N-benzyl-
oxycarbonyl-(S)-glutamic acid 1-benzyl ester (Cbz-Glu-
OBzl) was found to be an ecient step and a€orded,
although slowly (see Scheme 2), the fully protected glu-
tathione analogue 11 in 60% yield.
3. (a) Lucente, G.; Luisi, G.; Pinnen, F. Il Farmaco 1998, 53,
721 and references therein. (b) Calcagni, A.; Lucente, G.;
Luisi, G.; Pinnen, F.; Rossi, D. Amino Acids 1999, 17, 257.
4. Breslow, D. S.; Skolnik, H. In The Chemistry of Hetero-
cyclic Compounds. Multi-sulfur and Sulfur and Oxygen 5- and
6-Membered Heterocycles; Weissberger, A., Ed.; Interscience
Publishers: New York, 1966; Part One, p 313.
5. Teuber, L. Sulfur Reports 1990, 9, 257.
6. Singh, R.; Whitesides, G. M. J. Am. Chem. Soc. 1990, 112,
1190.
7. Houk, J.; Whitesides, G. M. J. Am. Chem. Soc. 1987, 109,
6825.
8. Takagi, M.; Goto, S.; Ishihara, R.; Matsuda, T. Chem.
Commun. 1976, 993.
It was also possible to obtain the above reported ana-
logue 11 by adopting a di€erent route based on the
procedure devised by Shin during the synthesis of oligo-
peptides containing didehydroaminoacid residues.19 We
found in fact that, in analogy with NCAs of unsaturated
amino acids, NCA 5 is eciently N-acylated by N-pro-
tected amino acids by using DCC in the presence of
pyridine; the so-obtained N-acyl intermediates can be
used in situ for the subsequent nucleophilic ring opening
by carboxy-protected aminoacid residues. This procedure
was adopted for the synthesis of 11 and is reported in
Scheme 2. Here the NCA 5 is acylated with Cbz-Glu-
OBzl and the obtained g-glutamyl-NCA 10 is con-
secutively used to acylate the amino group of H-Gly-
OBzl to a€ord the fully protected tripeptide derivative
11. Deprotection and oxidation under the conditions
adopted to obtain 8, a€orded the Adt containing glu-
tathione analogue 12.
9. For a discussion on the signi®cance of the evolutionary
selection of the 1,2-dithiolane ring system as enzymatic cofac-
tor of 2-oxo acid dehydrogenase see: Singh, R.; Whitesides, G.
M. J. Am. Chem. Soc. 1990, 112, 6304.
10. (a) Shen, T.-Y.; Walford, G. L. U.S. Patent 3,547,948,
1970; Chem. Abstr. 1971, 75, 6336j. (b) Shen, T.-Y.; Walford, G.
L. U. S. Patent 3,655,692, 1972; Chem. Abstr. 1972, 77, 5331h. (c)
Rice, W. G.; Schultz, R. R.; Baker, D. C.; Henderson, L. E. PCT
Int. Appl. WO 98 01,440, 1998; Chem. Abstr. 1998, 128,
123799m.
11. Coulter, A. W.; Lombardini, J. B.; Sufrin, J. R.; Talalay,
P. Mol. Pharmacol. 1974, 10, 319.
12. All new compounds were fully characterized. Selected
1
data: 3: H NMR (300 MHz, CDCl3) d 1.51 (s, 9H, t-butyl),
1.61 (s, 9H, t-butyl), 3.06 (ABq, 4H, J=14.0 Hz, 2ÂCCH2S),
3.67 (ABq, 4H, J=13.2 Hz, 2ÂCH2C6H5), 7.23±7.35 (m, 10H,
aromatics); 13C NMR (75.43 MHz, CDCl3) d 27.8, 28.0
[2ÂC(CH3)3], 35.5, 37.1, (4ÂCH2S), 70.5 [C(CH2)2], 85.1, 86.7
[2ÂC(CH3)3], 127.3, 128.6, 129.0, 137.3 (aromatics), 145.0,
147.9, 148.6, 169.3 (4ÂCO). 5: mp 94±95 ꢀC; IR (KBr) 3251,
Properties of Adt containing peptides together with
extension of the studies to other 1,2-dithiolane ring systems,
will be reported in due course.
1
1839, 1787, 1490, 1451 cm 1; H NMR (300 MHz, CDCl3) d
2.75 (ABq, 4H, J=14.4, 2ÂCCH2S), 3.70 (s, 4H, 2ÂCH2C6
H5), 5.49 (br s, 1H, NH), 7.25±7.38 (m, 10H, aromatics); 13C
NMR (75.43 MHz, CDCl3) d 36.7 and 37.6 (4ÂCH2S), 68.9
Acknowledgements
[C(CH2)2], 127.7, 128.9, 129.0, 137.0 (aromatics), 150.8, 170.3
1
(2ÂCO). 7: IR (CHCl3) 3415, 1741, 1708, 1492 cm
;
1H
The authors thank Professor Bruno Danieli (Dip. Chi-
mica Organica e IndustrialeÐUniversita degli Studi di
Milano, Italy) for FABMS spectral analysis.
NMR (300 MHz, CDCl3) d 1.46 (s, 9H, t-butyl), 2.83 and 3.50
(dd, 4H, J=13.5 and J=13.6 Hz, 2ÂCCH2S), 3.67 (ABq, 4H,
J=13.1 Hz, 2ÂCH2C6H5), 3.70 (s, 3H, OCH3), 5.91 (br s, 1H,
NH), 7.25±7.33 (m, 10H, aromatics); 13C NMR (75.43 MHz,
CDCl3) d 28.4 [C(CH3)3], 36.5, 37.3 (4ÂCH2S), 53.0 (OCH3),
65.5 [C(CH2)2], 80.0 [C(CH3)3], 127.1, 128.5, 128.9, 138.2
(aromatics), 154.1, 171.6 (2ÂCO) 8: IR (KBr) 3354, 1736,
1707, 1485; 1H NMR (300 MHz, CDCl3) d 1.43 (s, 9H, t-
butyl), 3.37 and 3.63 (dd, 4H, J=13.0 and J=12.9 Hz,
2ÂCH2), 3.82 (s, 3H, OCH3), 5.2 (br s, 1H, NH); 13C NMR
(75.43 MHz, CDCl3) d 28.4 [C(CH3)3], 47.8 (2ÂCH2S), 53.5
(OCH3), 71.6 [C(CH2)2], 81.1 [C(CH3)3], 155.1, 171.5 (2ÂCO);
FAB±MS m/e 279 (M+).
References and Notes
1. (a) Toniolo, C.; Benedetti, E. Macromolecules 1991, 24,
4004. (b) Balaram, P. Curr. Opin. Struct. Biol. 1992, 2, 845. (c)
Toniolo, C.; Crisma, M.; Fabiano, N.; Melchiorri, P.; Negri,
L.; Krause, J. A.; Eggleston, D. S. Int. J. Peptide Protein Res.
1994, 44, 85. (d) Goodman, M.; Shao, H. Pure Appl. Chem.
1996, 68, 1303. (e) Horikawa, M.; Nakajama, T.; Ohfune, Y.
Synlett 1997, 253. (f) Torrini, I.; Paglialunga Paradisi, M.;
Pagani Zecchini, G.; Lucente, G.; Gavuzzo, E.; Mazza, F.;
Pochetti, G.; Traniello, S.; Spisani, S. Biopolymers 1997, 42,
415.
13. Comber, R. N.; Reynolds, R. C.; Friedrich, J. D.;
Manguikian, R. A.; Buckheit, R. W. Jr.; Truss, J. W.; Shan-
non, W. M.; Secrist, J. A. III J. Med. Chem. 1992, 35, 3567.
14. Kubik, S.; Meissner, R. S.; Rebek, J. Jr. Tetrahedron Lett.
1994, 35, 6635.
2. (a) Torrini, I.; Pagani Zecchini, G.; Paglialunga Paradisi,
M.; Lucente, G.; Gavuzzo, E.; Mazza, F.; Pochetti, G.;
Spisani, S.; Giuliani A. L. Int. J. Pept. Protein Res. 1991, 38,
495. (b) Wysong, C. L.; Yokum, T. S.; Morales, G. A.; Gundry,
R. L.; McLaughin, M. L.; Hammer, R. P. J. Org. Chem. 1996,
61, 7650. (c) Wolf, W. M.; Stasiak, M.; Leplawy, M. T.;
Bianco, A.; Formaggio, F.; Crisma, M.; Toniolo, C. J. Am.
Chem. Soc. 1998, 120, 11588. (d) Stasiak, M.; Wolf, W. M.;
Leplawy, M. T. J. Pept. Sci. 1998, 4, 46. (e) Monaco, V.;
Formaggio, F.; Crisma, M.; Toniolo, C.; Hanson, P.; Mill-
hauser, G. L. Biopolymers 1999, 50, 239 and references therein.
15. For analogous results see: (a) Ornstein, P. L.; Bleisch, T.
J.; Arnold, M. B.; Kennedy, J. H.; Wright, R. A.; Johnson, B.
G.; Tizzano, J. P.; Helton, D. R.; Kallman, M. J.; Schoepp, D.
D. J. Med. Chem. 1998, 41, 358. (b) Horwell, D. C.; McKiernan,
M. J.; Osborne, S. Tetrahedron Lett. 1998, 39, 8729.
16. Katakai, R. G. J. Org. Chem. 1975, 40, 2697 and refer-
ences cited therein.
17. (a) Fuller, W. D.; Cohen, M. P.; Shabankareh, M.; Blair,
R. K. J. Am. Chem. Soc. 1990, 112, 7414. (b) Fuller, W. D.;
Goodman, M.; Naider, F. R.; Zhu, Y.-F. Biopolymers 1996,
40, 183 and references cited therein.

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E. Morera et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1585±1588
18. All compounds were fully characterized. Selected data: 11:
[a]d= 7.0ꢀ (c 1.0, CHCl3); IR (CHCl3) 3375, 1735, 1712,
OCH2), 127.3, 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 129.0,
135.2, 136.1, 138.3 (aromatics), 156.4, 169.4, 171.3, 171.9,
172.0 (5ÂCO). 12: [a]d=+92.1ꢀ (c 0.5, H2O/MeOH 2:1 vol.);
1
1673, 1494, 1452 cm 1; H NMR (300 MHz, CDCl3) d 1.83
1
(m, 1H, Glu b-CHA), 2.09 (m, 2H, Glu g-CH2), 2.25 (m, 1H,
Glu b-CHB), 3.12 (ABq, 2H, J=13.8, CCH2S), 3.23 (ABq,
2H, J=13.7, CCH2S), 3.64 (ABq, 4H, J=13.3 Hz,
2ÂCH2C6H5), 3.95 (AB part of ABX system, 2H, Jgem=19.0,
Jvic=5.0 Hz, Gly CH2), 4.48 (m, 1H, Glu a-CH), 5.12 (m, 6H,
3ÂOCH2), 5.73 (d, 1H, J=8.3 Hz, Glu NH), 6.12 (1H, s, NH),
7.22±7.34 (m, 25H, aromatics), 7.45 (X part of ABX system,
1H, Gly NH); 13C NMR (75.43 MHz, CDCl3) d 27.9 (Glu b-
CH2), 32.2 (Glu g-CH2), 37.0 and 37.8 (4ÂCH2S), 41.7 (Gly
CH2), 53.3 (Glu a-CH), 63.0 [C(CH2)2], 67.1, 67.2, 67.4 (3 x,
IR (KBr) 3371, 1746, 1659, 1547, 1372 cm 1; H NMR (300
MHz, D2O) d 1.92 (m, 2H, Glu bCH2), 2.31 (m, 2H, Glu g-
CH2), 3.32 (d, 2H, J=12.0 Hz, Adt CH2), 3.47±3.56 (m, 5H,
Adt CH2, Glu a-CH, and Gly CH2); 13C NMR (75.43 MHz,
D2O) d 26.2 (Glu b-CH2), 31.3 (Glu g-CH2), 43.9 (Gly CH2),
47.8, 47.9 (2ÂCH2S), 54.2 (Glu a-CH), 71.8 [C(CH2)2], 171.9,
174.8, 176.4. (4ÂCO); FAB±MS m/e 374 (M+Na+)
19. Shin, C.; Yonezawa, Y.; Ikeda, M. Bull. Chem. Soc. Jpn.
1986, 59, 3573.