Exploring the interest of 1,2-Dithiolane ring system in peptide chemistry. Synthesis of a chemotactic tripeptide and x-ray crystal structure of a 4-amino-1,2-dithiolane-4-carboxylic acid derivative

Article abstract of DOI:10.1016/S0968-0896(01)00256-5

Due to their relevant biological functions and specific chemical reactivity 1,2-dithiolanes (five-membered cyclic disulfides) represent an emerging class of heterocyclic compounds. However, despite the extensive research centered on lipoic acid and its analogues, only very few data are at the present available on peptides containing this ring system. We report here synthesis, conformation and bioactivity of a fMLF-OMe analogue, namely For-Met-Adt-Phe-OMe (7), in which the residue of the 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) (4) replaces the central L-leucine. The crystal conformation of the synthetic intermediate Boc-Adt-OMe (5) is also described and compared to that of lipoic acid (R-1,2-dithiolane-3-pentanoic acid) (3) and asparagusic acid (1,2-dithiolane-4-carboxylic acid) (2). Copyright

Full text of DOI:10.1016/S0968-0896(01)00256-5

Bioorganic & Medicinal Chemistry10 (2002) 147–157
Exploring the Interest of 1,2-Dithiolane Ring System in
Peptide Chemistry. Synthesis of a Chemotactic Tripeptide
and X-ray Crystal Structure of a 4-Amino-1,2-dithiolane-4-
carboxylic Acid Derivative
Enrico Morera,a Gino Lucente,a,* Giorgio Ortar,a Marianna Nalli,a Fernando Mazza,b
Enrico Gavuzzob and Susanna Spisanic
a
`
Dipartimento di Studi Farmaceutici and Centro di Studio per la Chimica del Farmaco del CNR, Universita degli Studi di Roma
‘‘La Sapienza’’, P.le A. Moro 5, 00185 Rome, Italy
bIstituto di Strutturistica Chimica, CNR, C.P. n. 10, 00016 Monterotondo Stazione, Rome, Italy
c
`
Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, 44100 Ferrara, Italy
Received 30 January2001; accepted 10 July2001
Abstract—Due to their relevant biological functions and specific chemical reactivity1,2-dithiolanes (five-membered cyclic disulfides)
represent an emerging class of heterocyclic compounds. However, despite the extensive research centered on lipoic acid and its
analogues, only very few data are at the present available on peptides containing this ring system. We report here synthesis, con-
formation and bioactivityof a fMLF-OMe analogue, namelyFor-Met-Adt-Phe-OMe ( 7), in which the residue of the 4-amino-1,2-
dithiolane-4-carboxylic acid (Adt) (4) replaces the central l-leucine. The crystal conformation of the synthetic intermediate Boc-
Adt-OMe (5) is also described and compared to that of lipoic acid (R-1,2-dithiolane-3-pentanoic acid) (3) and asparagusic acid (1,2-
dithiolane-4-carboxylic acid) (2). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
sents a well documented critical sequence in some pro-
teins where it acts, with its opened and closed forms, as
a molecular switch able to control receptor or enzyme
activation.1ꢀ4 The lipoic acid, on the other hand, is an
extensivelystudied natural product which plays a dual
role in living organisms: it is responsible for the transfer
of acetyl groups from pyruvic acid to coenzyme A in the
metabolic process which converts carbohydrates into
energyand is a powerful antioxidant capable to defend
The relevant role played by thiols and disulfides in bio-
logical systems is well known. The thiol–disulfide inter-
change reaction and the related formation and cleavage,
under physiological conditions, of strong S–S bonds
represent in fact one of the most versatile and efficient
mechanisms through which the tertiarystructure of
polypeptides and proteins is controlled. However, the
large majorityof these structures involves linear or large
ring disulfides; small ring systems, in which the S–S
bond makes part of a conformationallyconstrained
structure, are less frequentlyencountered. Two relevant,
albeit different examples, which can be found among
natural products, are represented bythe eight-mem-
bered ring of the oxidized form of the -Cys-Cys- dyad 1
and the five-membered 1,2-dithiolane system of the
lipoic acid (3). The cyclic form of the dipeptide 1 repre-
5ꢀ10
the bodyagainst the free radical damage.
During our studies on small ring disulfides and con-
formationallyconstrained analogues of csyteine we
reported recentlysynthesis and properties of glutathione
analogues incorporating the oxidized form of the -Cys-
Cys- dyad 1 or the 4-amino-1,2-dithiolane-4-carboxylic
acid (Adt) residue (4) in place of the native l-
cysteine.11,12 The interest for an a-aminoacid containing
the 1,2-dithiolane ring system stems from the unique
chemical properties shown bythis structural fragment
on which the biological functions of lipoic acid are
based. Of particular relevance appear the fast rate of
*Corresponding author. Tel.: +39-06-445-4218; fax: +39-06-491491;
e-mail: gino.lucente@uniromal.it
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00256-5

148
E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
ring opening bynucleophiles as compared with related
open-chain and cyclic disulfides,13 the trans-acylating
properties,14 and the efficiencyin the metal-ion coordi-
nation.15,16 Although all these properties, as well as
manyothers, have been the object of detailed studies,
the incorporation of the 1,2-dithiolane ring system into
the peptide backbone and the studyof the chemical,
conformational and biochemical consequences of this
modification are just emerging in recent literature. Spe-
cifically, the so far available data concern in fact the
glutathione analogue tripeptide g-Glu-Adt-Gly-OH (9)
in which the dithiolanic residue has been incorporated
into the backbone through an isopeptidic bond (Scheme
1).12
Leu replacement, have been studied. In particular, it has
been observed that the tripeptide For-Met-Ac5c-Phe-
OMe, containing the achiral 1-amino-1-cyclopentane-
carboxylic acid (Ac5c or cycloleucine) which is the
cycloaliphatic analogue of the Adt, maintains the
bioactivityof the parent while adopting a well defined
b-turn backbone conformation.21 On the basis of these
findings, the synthesis of the new analogue For-Met-
Adt-Phe-OMe (7) has been considered an interesting
opportunityin order to gain information on two main
points: (i) definition of the synthetic strategies suitable
to incorporate the Adt residue into a peptide backbone
and (ii) examination of the tendencyof the five-mem-
bered cyclic disulfide residue to maintain the biological
activityand the tpyical backbone folding which are
encountered when the cyclopentane ring system is pre-
sent at the central position of the bioactive tripeptide.
Concerning the crystallographic analysis of Boc-Adt-
OMe (5), an examination of the literature on 1,2-
dithiolanes puts in clear evidence the importance of the
steric arrangement of the bonds around the S–S group
and the deriving sulfur lone-pair–lone-pair interaction
for a complete understanding of the unusual reactivity
of this class of small ring disulfides.22ꢀ25 In this context,
the here reported molecular structure can provide the
first geometrical and conformational details of an
orthogonallyprotected a-aminoacid residue whose Ca
carbon atom makes part of the 1,2-dithiolane ring sys-
tem; these structural parameters are compared with
those concerning asparagusic acid (2),26 lipoic acid (3),27
and related literature available 1,2-dithiolanes.
As a development of our interest in this subject, we
report here the crystal and molecular structure of the
Boc-Adt-OMe (5) and the synthesis of 7, an analogue of
the prototypical chemotactic tripeptide N-formyl-l-
methionyl-l-leucyl-l-phenylalanine methyl ester (For-
Met-Leu-Phe-OMe; fMLF-OMe) in which the central
native Leu has been replaced bythe Adt residue. The
importance of the central aminoacidic residue in deter-
mining the activityof chemotactic fMLF-OMe analo-
gues is well known and several position-2 modified
analogues, incorporating Ca,a-dialkylglycines17ꢀ20 as the
Results and Discussion
Chemistry
The products described in this paper have been synthe-
sized according to Scheme 2; the here adopted strategy
is different from that referring to the synthesis of the
previouslyreported Adt containing glutathione analo-
gue where the dithiolanic ring was formed onlyin the
last step of the reaction sequence bydeprotecting and
oxidizing the residue of 2,2-bis-(benzylthiomethyl)-gly-
cine (6) initiallyincorporated with the intermediacyof
the corresponding N-carboxyanhydride 8 (Scheme 1).12
In the case of the synthesis of the tripeptide under study
For-Met-Adt-Leu-OMe (7), the above reported strategy
cannot be followed due to the sensitivityof the methio-
nine side-chain thioether function to the Na/liquid-NH3
conditions requested to debenzylate the sulphydrylic
groups of 6.28 Thus, as illustrated in Scheme 2, the
methionine containing tripeptide 7 was obtained byfol-
lowing two new routes, both employing the orthogon-
allyprotected Boc-Adt-OMe derivative ( 5) as the
starting product. However, the N-carboxyanhydride 8
plays a key role also in these two procedures, being
the starting material from which 5 is obtained
through N-carbamoylation followed by methanolysis.
Byfollowing the first route (see Scheme 2), the dithio-
lanic derivative Boc-Adt-OMe (5) is C-deprotected by
mild aqueous alkaline hydrolysis and the resulting acid

E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
149
Scheme 1. Reagents and conditions: (i) Na-liq NH3, ꢀ78 ꢁC; (ii) I2, EtOH, 90% (two steps).
Scheme 2. Reagents and conditions: (i) COCl2–toluene, THF, 88%; (ii) (Boc)2O, Py, THF; (iii) MeOH, NMM, 93% (overall from 8); (iv) Na-liq
NH3, ꢀ78 ꢁC; (v) I2, MeOH, 89% (overall from 10); (vi) NaOH 2 N, MeOH; (vii) HOBt, EDC, H-Phe-OMe HCl, TEA, DMF, 84% (from 5) and
82% (from 12); (viii) SOCl2, MeOH; (ix) HOBt, EDC, N-Boc-Met-OH, TEA, DMF, 60% (from 5) and 59% (from 11); (x) HCO2H; (xi) EEDQ,
DMF, 79% (from 13).

150
E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
is used for the coupling reaction with phenylalanine
methyl ester to give the dipeptide 11. In the alternative
sequence the Boc-Adt-OMe derivative is first depro-
tected at the amino group bytreatment with thionyl
It is clearlyseen that the Met and the Adt NH groups
are significantlymore sensitive to changes in solvent
composition as compared with the Phe NH group which
appears appreciablyshielded. This result suggests that a
significant population of conformers of the two tripep-
tides adopts, in CDCl3 solution, a folded conformation
characterized byan H-bond interaction between the Phe
NH and a carbonylic group. An analogous situation has
been evidenced in a series of conformationallyrestricted
formyl-methionyl tripeptides of general formula HCO-
Met-Xxx-Phe-OMe incorporating at the central posi-
tion Ca,a-symmetrically disubstituted glycyl residues.21
All these models show, together with their N-Boc pre-
cursors, a marked tendencyto fold into b-turn con-
formations involving H-bond interaction between the
Phe NH and the carbonyl group bound at the Met NH.
In particular the DMSO-d6 titration curve obtained by
us with For-Met-Adt-Leu-OMe (7) (see Fig. 1A) shows
practicallythe same values exhibited bythe N-formyl-
tripeptide For-Met-Ac5c-Leu-OMe.21
chloride in drymethanol and the resulting H N-Adt-
2
OMe is N-acylated with Boc-Met-OH, by using EDC as
the coupling reagent, to give the dipeptide methyl ester
12. This was neatlyhydrolyzed with methanolic NaOH
and then coupled to H-Phe-OMe, byusing the same
condensing agent of the previous step, to afford the
N-Boc-tripeptide 13. This latter derivative could also be
obtained byusing, as the starting material, the dipeptide
11 and adopting analogous deprotecting and coupling
procedures. In the last common step, the N-Boc tri-
peptide 13 is directlytransformed into the correspond-
ing N-Formyl analogue 7 byfollowing the procedure of
Lajoie and Kraus.29 Despite its reactivityand in accor-
dance with literature data on the reduced tendencyof
4,4-disubstituted 1,2-dithiolanes to polymerize,30 the
dithiolanic residue is found stable under the adopted
deprotection and coupling conditions and the yields,
although not optimized, are in both cases satisfactory
and nearlyidentical.
Further information on the solution conformations and
the type of b-turn involved can be obtained byexamin-
ing the results of nuclear Overhauser effect (NOE)
experiments summarized in Table 1. It should be recal-
led that in standard b-turns the interproton distances
which differentiate type-I from type-II structures are
Ci+1H–Ni+2H (3.4 and 2.2 A in type I and II, respec-
tively) and Ni+1H–Ni+2H (2.6 and 4.5 A in type I and
II, respectively); furthermore, a short distance of 2.4 A
between Ni+2H–Ni+3H is observed in both the types of
turns.31 As shown in Table 1 the two peptides 7 and 13
show strong sequential NOEs Met CaH Adt NH; this,
together with the Phe NH Adt NH NOEs and the
absence of connections between Adt NH and Met NH,
clearlysupports the presence of type-II b-turns with the
Adt residue located at i+2 position. In this type of b-
turns, contrarilyto type I, the central N–H amide bond
(residue i+2) is cis to the CaH of the preceding residue
(i+1) and this conformational feature is reflected bythe
strong NOE observed between the Met CaH and the
Adt NH of both 7 and 13. Fig. 2 illustrates the proposed
folding byputting also in evidence the intramolecular
hydrogen bond (i+3!i) between H–CO (or ButOCO)
and Phe NH closing a pseudocyclic 10-membered ring.
Solution conformation
To gain information on the conformational preferences
of the Adt containing tripeptides 7 and 13, the chemical
shift dependence of the NH resonances as a function of
DMSO-d6 concentration in CDCl3 solution (10 mM)
was studied. The results are reported in Figure 1 where
the resonances of the three NH groups of the tripeptides
are shown.
Table 1. Observed nuclear Overhauser effects (NOEs) in the NOESY
spectra of HCO-Met-Adt-Phe-OMe (7) and Boc-Met-Adt-Phe-OMe
(13)a
7
13
Met NH. . .H–CO
s
—
s
s
s
m
m
m
w
s
—
—
m
m
s
m
w
m
w
s
Met NH. . .Adt NH
Met CaH. . .Met CgH2
Met CaH. . .Met CbH2
Met CaH. . .Adt NH
Adt NH. . .Adt CH2
Phe NH. . .Adt NH
Phe NH. . .Adt CH2
Phe NH. . .Phe CbH2
Phe NH. . .Phe CaH
Phe CaH. . .Phe CbH2
Phe CbH2. . .aromatic–H
Figure 1. Plots of the NH chemical shifts (1H NMR) as a function of
the increasing DMSO-d6 concentration (% v/v) in CDCl3 solution
(10 mM) for (A) the tripeptide For-Met-Adt-Phe-OMe (7) and (B)
Boc-Met-Adt-Phe-OMe (13).
s
s
s
s
as, strong; m, medium; w, weak.

E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
151
A final consideration concerns the NOE between Phe
NH and Adt NH which, in the case of the N-Boc tri-
peptide 13, is found to be weaker than that observed in
the N-For tripeptide 7 (see Table 1). This finding could
indicate that a H-bond interaction, involving the Met
CO instead of the ButOCO as acceptor could be opera-
tive in 13. An examination of the models shows in fact
that this latter H-bond interaction, closing a seven-
membered pseudocyclic ring (g-turn), is associated with
a larger interproton Phe NH–Adt NH distance as com-
pared with the b-turn.32
intraring valence angles at the sulfur atoms which
show significantlydifferent values, being 91.4 and
g1
97.2ꢁ, at S1 and S1 g2, respectively. The S–S bond
length (2.056 A) corresponds to that found in the case
of lipoic acid (2.053 A) and is longer than that found in
strain-free open-chain disulfides (ca. 2.044 A).
Further relevant features in the structure of 5 are the
non planarityof the ring and the torsion angle around
the S–S bond. An examination of Figure 3 reveals that
the dithiolane ring of 5 can be described as an envelope
with four atoms, including the sulfur bridge, on a plane
and one carbon atom (the C1b1) at the envelope flap; this
latter atom is oriented on the opposite side of the Boc–
NH substituent and bound at the sulfur atom S1g1 which
exhibits the smaller intraring valence angle. The degree
of deviation from planarityof the ring can be evaluated
Conformation in the crystal of Boc-Adt-OMe (5)
Figure 3 reports a perspective view of the conformation
in the crystal of Boc-Adt-OMe (5) together with the
numbering scheme usuallyadopted for aminoacid deri-
vatives and peptides. As can be deduced bythe figure
and bythe data in Table 2, the Boc–NH–chain adopts a
trans–trans planar conformation and shows aꢁC04–C01–
O01 valence angle (102.3ꢁ) smaller byabout 7 than the
tetrahedral value; both these findings are in accordance
with the geometrical and conformational peculiarities
derived from crystallographic investigations on peptides
containing the tert-butyloxycarbonylamino group.33
Considering the geometrical dimension of the five-
membered ring it can be seen that the two C–S bond
lengths are nearlyidentical and the same is true for the
two C–C bonds. A quite different situation concerns the
b1
from the distance between the C1 and the mean plane
of the other four ring atoms which was found to be 0.76
A. On the basis of these features a pseudomirror plane,
g2
bisecting the C1b2-S1 bond and passing through the
carbon at the flap, can be individuated. A final obser-
a
0
vation concerns the valence angle N1–C1 –C1 (t angle;
see Table 2) whose value, in peptides containing Ca,a
-
symmetrically substituted glycines, is highly sensible to
the type of adopted local conformation and is always
smaller than the tetrahedral value in the fully-extended
conformations (typical values: 102–107ꢁ).34,35 The value
here found for the Adt derivative 5 is 110.1ꢁ and is in
perfect accordance with the value observed in Ac5c
containing peptides and related folded structures.34
In order to compare the above reported data with those
of alreadyknown 1,2-dithiolanes, an examination of the
literature has been performed bytaking into considera-
tion saturated rings not fused with other cyclic systems
and containing disulfide groups not involved in
metal ion complexes. Whereas several X-raystruc-
tures of 3-monosubstituted-1,2-dithiolanes related to
lipoic acid are available, onlyasparagusic acid structure
has been found as representative of 4-monosubstituted
derivatives.26 Five 1,2-dithiolane structures concern 3,4-
Figure 2. Relevant interproton correlations as deduced byNOESY
experiments for peptides 7 and 13.
Table 2. Relevant geometrical and conformational data of Boc-Adt-
OMe (5)a
Bond lengths (A)
Torsion angles (ꢁ)b
Main chain
S1g1-S1gb21
2.056(3)
1.806(6)
1.809(6)
1.534(8)
1.555(8)
1.539(8)
S1g1-C1b2
S1g2-C1
a
O01-C00-N1-C1
177.1(5)
50.1(6)
45.3(6)
179.7(5)
a
C00-N1-C1a-C1
0
0
0
C1 -C1b2
C1a-C1b1
C1a-C1
a
0
N1-C1 -C1 -O1
C1a-C1 -O1 -C2
0
0
Valence angles (ꢁ)
1,2 Dithiolane ring
g2
C1b1-S1g1-S1g1
91.4(2)
97.2(2)
C1a-C1b1-S1g1-S1g2
C1a-C1b2-S1g2-S1gb12
C1b1-S1g1-S1g2-C1
47.8(4)
10.5(4)
C1b2-S1g2-S1b2
C1b1-C1a-C1
109.3(5)
110.1(4)
110.6(5)
107.3(4)
102.3(5)
ꢀ31.5(3)
N1-C1a-C1
C1b1-C1a-C1b2-S1g1
20.6(6)
ꢀ47.8(5)
g2
0
a
S1g2-C1b2-C1a
C1b2-C1a-C1b1-S1
S1g1-C1b1-C1
C04-C01-O01
aAtom numbering is that reported in Figure 3.
bTorsion angles have been computed according to ref 44.
Figure 3. A perspective view of the crystal conformation of Boc-Adt-
OMe (5).

152
E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
disubstituted derivatives and have been determined in
connection with the assignment of the stereochemistry
to gerrardine36 and guinesine37,38 alkaloids. No struc-
tures referring to 4,4-disubstituted rings have been
found. The analysis of the data shows that the S–S bond
length and the torsion angle of 3-monosubstituted deri-
vatives fall in the range of 2.03–2.06 A and ꢀ29.5 to
ꢀ35.3ꢁ, respectively.
the implication of this finding should be evaluated in the
light of the literature data which register the different
bioactivityshown bythe oxidized forms of glutathione
GSSG (a constraint-free linear disulfide) and oxidized
lipoic acid (a five-membered cyclic disulfide).8,39 The
appropriate example in this context is the abilityto
scavenge hypochlorous acid, a dangerous oxidant of
several biological systems: whereas the reduced forms of
both GSH and lipoic acid are potent HClO scavengers,
onlythe oxidized (1,2-dithiolane form) of lipoic acid is
able to quench the oxidant activityof HClO, the GSSG
being practicallyinactive. This quite surprising finding
can be explained bytaking into account the different
nature of the S–S bond in the two molecules.8,39
Examination of these values, together with those found
for asparagusic acid and reported in Table 3, shows
that S–S bond length and torsion angle of the Boc-
Adt-OMe (5) (2.056 A and 31.5ꢁ) are more consistent
with 3-monosubstituted derivative structures, including
lipoic acid, than with the 4-monosubstituted model
represented bythe asparagusic acid. This latter com-
pound presents in fact a lower S–S torsion angle (26.6ꢁ)
and a longer S–S bond length (2.10 A). A different pic-
ture is presented bythe 3,4-disubstituted models as
esemplified bythe two diastereoisomeric forms of a gui-
nesine derivative containing the 3,4-disubstituted-1,2-
dithiolane ring. Here the cis 3,4-isomer exhibits usual
values of S–S bond length and torsion angle (2.04 A and
36.4ꢁ) whereas the corresponding values of the trans 3,4-
isomer are 2.09 A and ꢀ2.6ꢁ, respectively. Thus, this
compound shows the smallest value of S–S torsion angle
observed in X-raycrystal structures of 1,2-dithiolanes
and underlines, at the same time, the strong influence of
the substitution pattern on the ring conformation.
Biological activity
The biological activityof the Adt containing tripeptide
7 was determined on human neutrophils and compared
with that of the prototypical bioactive agent fMLF-
OMe. Directed migration (chemotaxis), lysozyme
release, and superoxide anion production have been
measured. As shown in Figure 4A, compound 7 is active
in inducing neutrophil chemotaxis, even though with
lower potencythan the reference model. The activity
as secretagogue agent on the lysozyme release (Fig.
4B) and in terms of superoxide anion production
(Fig. 4C) remains verylow at concentrations ranging
from 10ꢀ9 to 10ꢀ7 M but increases rapidly, reaching the
value shown bythe reference tripeptide, starting from
10ꢀ5 M.
Structure–reactivity relationship
An interesting correlation between the decreasing of the
torsion angle around the disulfide bridge with the
increasing of the S–S bond length has been evidenced by
Bock et al.22 It is now well established that the major
contribution to the enhanced reactivityof 1,2-dithio-
lanes is given bythe small dihedral angle around the S–
S bond associated with the reduced size of the ring and
bythe consequent unfavourable interaction between the
adjacent sulfur lone-pairs.25 Accordingly, the S–S bond
in 1,2-dithiolanes is longer and weaker than that found
in open chain disulfides whose dihedral angle is ca. 90ꢁ
and the S–S bond length about 2.02 A. The interest and
Conclusions
In the present paper, we reported synthetic strategies
which allow the direct incorporation of a 1,2-dithiolanic
aminoacid residue into peptide backbones. The results
show that the achiral and conformationallyconstrained
Adt, possessing a quaternarycarbon atom at position 4
of the dithiolanic ring, is stable under the conventional
reaction conditions adopted during peptide synthesis.
Furthermore, the incorporation of such residue at the
central position of a bioactive tripeptide, leads to an
analogue which maintains the bioactivityand induces a
backbone folding analogous to that imposed bythe
Ac5c, the Caa-tetrasubstituted aminoacid which is the
dicarba analogue of the Adt. This latter finding is in
accordance with literature data concerning isosterism
and bioisosterism of disulfides, a topic relevant to the
development of cystine isosteres; it is well known in fact
that the most frequentlyexploited cystine isostere is its
dicarba analogue (2S,7S)-2,7-diaminosuberic acid.40
Biological active peptides containing this residue have
been developed in order to improve the stabilityand
obtain analogues devoid of the chemical and metabolic
labilitytypical of the disulfide bonds. The here reported
results suggest an inverse isosteric relationship concern-
ing the couple Adt/Ac5c whose interest lies on the
possibilityto exploit the chemical versatilityoffered by
the constrained disulfide system including the hydro-
Table 3. Structural parameters characterizing lipoic acid (3), aspara-
gusic acid (2), and N-Boc-Adt-OMe (5)a
Lipoic acidb
Asparagusic
acidc
Boc-Adt-OMe
Bond length (A)
C–S
C–S*
S–S
1.83
1.79
2.053
1.83
1.85
2.10
1.806
1.809
2.056
Intraring valence angle (ꢁ)
S
S*
92.8
95.5
92.6
96.6
91.4
97.2
Torsion angle (ꢁ)
C–S–S*–C
35.0
26.6
31.5
aThe asterisk indicates the sulfur atom with the major intraring
valence angle.
bSee ref 27.
cSee ref 26.
genolytic transformation into
cysteine analogues.12
C
a,a-disubstituted

E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
153
Finally, a comparison of the structural parameters of
the molecule of Boc-Adt-OMe (5) with those of aspar-
agusic acid shows that the presence of two substituents
on C-4 carbon atom of the 1,2-dithiolane ring, as com-
pared with the monosubstitution on the same atom
present in the asparagusic acid, favours the S–S bonding
interaction, thus leading to a stereoelectronic arrange-
ment which closelyresembles that found in lipoic acid
(3). This finding, together with the stabilityduring the
synthetical steps and the properties shown by the tri-
peptide 7, stimulates further studies on peptides con-
taining 1,2-dithiolane ring systems and in particular the
4,4-disubstituted and achiral Adt residue; the avail-
abilityand the possible exploitation of peptide models
possessing lipoic acid related properties, seem in fact a
rewarding objective.
64 transients were collected for two-dimensional experi-
ments. The data sets were linearlypredicted to
512ꢂ1024 data points. A Gaussian window was applied
in both dimensions. Zero filling was used for a final
spectrum size of 1024ꢂ1024 data points. Analytical
results were within ꢃ0.4% of the theoretical values.
Column chromatographies were carried out using
Merck silica gel 60 (230–400 mesh). Thin layer chroma-
tographies were performed on silica gel Merck 60 F254
plates. The drying agent was sodium sulfate. Following
abbreviations are used for reagents and solvents: Boc2O
(di-t-butyldicarbonate), DMF (N,N-dimethylformamide),
EDC (N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride), EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-
dihydroquinoline),
HOBt
(1-hydroxybenzotriazole),
NMM (4-methylmorpholine), TEA (triethylamine). All
reagents and solvents were purchased at highest com-
mercial qualityand used without further purification,
except for dry ethyl ether and tetrahydrofuran (THF)
which were distilled from LiAlH4.
Studies on further aminoacids and peptides, character-
ized bythe presence of the 1,2-dithiolane ring, are at
present in progress in our laboratories.
N-Carboxy-2,2-bis(benzylthiomethyl)glycine anhydride
(NCA) (8). To a stirred suspension of 2,2-bis(ben-
zylthiomethyl)glycine (6)41 (0.500 g, 1.44 mmol) in dry
THF (20 mL) 4.5 mL of a 1.93 M phosgene solution in
toluene (Fluka 79380) were added dropwise at 0 ꢁC.
After 2 h at room temperature, the solvent was removed
under reduced pressure below 40 ꢁC and the resulting
residue (0.505 g), dissolved in dryethyl ether, was fil-
tered on a short pad of silica gel to give pure NCA 8
(0.472 g, 88%).12
Experimental
Melting points were determined with a Kofler hot stage
apparatus and are uncorrected. Optical rotations were
taken at 20 ꢁC with a Schmidt-Haensch Polartronic D
polarimeter in a 1 dm cell (CHCl3). Infrared spectra
were recorded with a Perkin-Elmer 983 spectro-
photometer. H and 13C NMR spectra were measured
1
with a Varian Mercury300 spectrometer using tetra-
methylsilane as internal standard. NOESY spectra were
recorded in CDCl3 containing 5.0% of DMSO-d6 on an
Avance 2000 Bruker console with a 1200-ms mixing
time and displayed in the phase-sensitive mode. Usually
256ꢂ1024 data points were collected and for each block
N-Boc-2,2-bis(benzylthiomethyl)-Gly-OMe (10). The
NCA 8 (0.485 g, 1.3 mmol) was dissolved in dryTHF
(3 mL) and the solution was then cooled to ꢀ15 ꢁC with
stirring. Boc2O (0.340 g, 1.56 mmol) in dryTHF (2 mL)
Figure 4. Biological activityfor For-Met-Adt-Phe-OMe ( 7) towards human neutrophils: (A) chemotactic activity; (B) release of neutrophil granule
enzymes evaluated by determining lysozyme activity; (C) superoxide anion production.

154
E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
and drypyridine (0.21 mL, 2.6 mmol) were added suc-
cessivelyand the resulting mixture was stirred 10 min at
ꢀ15 ꢁC and 20 h at room temperature, at which time the
a-C), 81.3 [(C(CH3)3], 127.2, 128.6, 129.2, 135.7 (aro-
matics), 154.5, 169.4, 171.6 (3ꢂCO). Anal.
C19H26N2O5S2 (426.54), C, H, N, S.
reaction was deemed complete byTLC (CH Cl2). Dry
2
MeOH (1.5 mL) and NMM (0.286 mL, 2.6 mmol) were
added to the mixture and the stirring was continued at
room temperature for further 24 h. After removing all
volatile components under reduced pressure, the resi-
dual syrup (0.675 g) was chromatographed on silica gel
(20 g) using CH2Cl2/hexane=6/4 as eluent to give pure
N-Boc-Met-Adt-OMe (12). To a solution of 5 (0.200 g,
0.72 mmol) in dryMeOH (5.0 mL) cooled at 0 ꢁC thio-
nyl chloride (0.052 mL, 0.72 mmol) was added and the
mixture was stirred at 50 ꢁC for 3 h. Evaporation under
reduced pressure of the solution afforded H-Adt-
.
OMe HCl (0.160 g). To the stirred suspension of the
hydrochloride in dry DMF (2 mL) cooled at 0 ꢁC
N-Boc-Met-OH (0.197 g, 0.79 mmol), HOBt (0.122 g,
0.79 mmol), EDC (0.151 g, 0.79 mmol), and, after 15 min
at 0 ꢁC and 20 min at room temperature, TEA
(0.100 mL, 0.72 mmol) were added. The mixture was
stirred at room temperature for 18 h and then worked
up as described for 11 to give a residue (0.238 g) which,
chromatographed on silica gel (10 g) using hexane/
EtOAc=65:35 as eluent, afforded 0.178 g of 12 (60%).
Mp 132–134 ꢁC (from CH2Cl2/hexane); [a]D ꢀ21ꢁ
(c 1.0); IR (CHCl3) 3341, 1744, 1710, 1497, 1368,
1292, 1163 cmꢀ1; 1H NMR (300 MHz, CDCl3) d 1.46 (s,
9H, t-butyl), 1.96 (m, 1H, Met b-CHA), 2.07 (m, 1H,
Met b-CHB), 2.12 (s, 3H, S-CH3), 2.60 (m, 2H, Met
g-CH2), 3.40 (m, 2H, Adt 2ꢂCHA), 3.65 (apparent d,
2H, Adt 2ꢂCHB), 3.78 (s, 3H, OCH3), 4.30 (m, 1H, Met
a-CH), 5.20 (d, 1H, J=7.5 Hz, Met NH), 7.17 (s, 1H,
Adt NH); 13C NMR (75.43 MHz, CDCl3) d 15.3 (Met
CH3), 28.3 [(C(CH3)3], 30.0 (Met g-CH2), 30.8 (Met b-
CH2), 47.2, 47.4 (2ꢂAdt b-CH2), 52.9 (Met a-CH), 53.3
(OCH3), 70.8 (Adt a-C), 80.5 [(C(CH3)3], 155.7, 170.0,
171.4 (3ꢂCO). Anal. C15H26N2O5S3 (410.56), C, H, N,
S.
12
10 as a waxyoil (0.560 g, 93%).
N-Boc-Adt-OMe (5). Compound 10 (1.491 g,
3.23 mmol) was dissolved at ꢀ78 ꢁC in approximately
140 mL of freshlydistilled liquid NH , carefullyexclud-
3
ing moisture. Then Na was slowlyadded (ca. 0.35 g) in
small portions until the blue colour of the solution
persisted for 10 min. Ammonium chloride (0.814 g,
15.2 mmol) was added and the solvent allowed to eva-
porate under a stream of drynitrogen. The residue was
dilute with MeOH (100 mL), neutralized with 2 N HCl,
and additioned dropwise with an aqueous solution of
iodine 0.1 M until a persistent pale yellow colour
appeared. The solution was decolourized with 1 M
Na2S2O3, concentrated under reduced pressure, diluted
with water, and extracted with EtOAc. The organic
phase was washed with water, dried, and evaporated to
give crude 5 (0.889 g) which was further purified by
crystallization from MeOH (0.801 g, 89%). Mp 104–
105 ꢁC.12
N-Boc-Adt-Phe-OMe (11). To a solution of 5 (0.213 g,
0.76 mmol) in MeOH (3.5 mL) 2N NaOH (0.76 mL) was
added under stirring. After 6 h at room temperature, the
solution was concentrated under vacuum, acidified with
0.5 N HCl, and extracted with EtOAc; the organic phase
was washed with water, dried, and evaporated. The
resulting residue (0.206 g) was used in the next step
without further purification.
N-Boc-Met-Adt-Phe-OMe (13). From 11: to a solution
of 11 (0.187 g, 0.44 mmol) in dryMeOH (3.0 mL) cooled
at 0 ꢁC thionyl chloride (0.032 mL, 0.44 mmol) was
added and the mixture was stirred at 50 ꢁC for 5 h.
Evaporation under reduced pressure of the solution
.
afforded H-Adt-Phe-OMe HCl as a foam. To the stirred
To the previous residue, dissolved in DMF (2.0 mL) and
cooled to 0 ꢁC, HOBt (0.130 g, 0.84 mmol) and EDC
(0.161 g, 0.84 mmol) were added. After stirring for
15 min at 0 ꢁC and for 20 min at room temperature,
suspension of the hydrochloride in dry DMF (1.5 mL)
cooled at 0 ꢁC N-Boc-Met-OH (0.120 g, 0.48 mmol),
HOBt (0.074 g, 0.48 mmol), EDC (0.092 g, 0.48 mmol),
and, after 15 min at 0 ꢁC and 20 min at room tempera-
ture, TEA (0.062 mL, 0.44 mmol) were added. The mix-
ture was stirred at room temperature for 20 h and then
worked-up as above. Chromatographyof the residue
(0.221 g) on silica gel (7 g) using CHCl3/EtOAc=9:1 as
eluent afforded 144 mg of 13 (59%). Mp 164–165 ꢁC
(from acetone); [a]D ꢀ13ꢁ (c 1.0); IR (CHCl3) 3340,
.
H-Phe-OMe HCl (0.181 g, 0.84 mmol) and TEA
(0.118 mL, 0.84 mmol) were added. The reaction mix-
ture was stirred at room temperature for 18 h and then
diluted with water and extracted with EtOAc. The
organic phase was washed with water, 10% citric acid
solution, saturated NaHCO3, and water, dried, and
evaporated under reduced pressure. The residue
(0.310 g) was chromatographed on silica gel (10 g) using
CHCl3/EtOAc=95:5 as eluent to give 0.271 g of 11
(84%). Mp 193–195 ꢁC (from acetone); [a]D+39ꢁ
(c 1.0); IR (CHCl3) 3411, 1720, 1682, 1505, 1369,
1740, 1692, 1492, 1437, 1367, 1162 cmꢀ1 1H NMR
;
(300 MHz, CDCl3) d 1.43 (s, 9H, t-butyl), 1.94 (m, 1H,
Met b-CHA), 2.09 (m, 1H, Met b-CHB), 2.11 (s, 3H,
SCH3), 2.59 (m, 2H, Met g-CH2), 3.06 (dd, 1H, J=7.2
and 14.1 Hz, Phe b-CHA), 3.19 (dd, 1H, J=6.0 and
14.1 Hz, Phe b-CHB), 3.49 (m, 4H, 2ꢂAdt b-CH2), 3.71
(s, 3H, OCH3), 4.22 (m, 1H, Met a-CH), 4.83 (m, 1H,
Phe a-CH), 5.22 (d, 1H, J=7.2 Hz, Met NH), 6.98 (s,
1H, Adt NH), 7.12–7.31 (m, 6H, aromatics and Phe
NH); 13C NMR (75.43 MHz, CDCl3) d 15.3 (Met CH3),
28.3 [(C(CH3)3], 30.2 (Met b and g-CH2), 37.7 (Phe b-
CH2), 45.9, 46.8 (2ꢂAdt b-CH2), 52.4 (Met and Phe a-
CH), 53.9 (OCH3), 71.7 (Adt a-C), 80.8 [(C(CH3)3],
1
1158 cmꢀ1; H NMR (300 MHz, CDCl3) d 1.43 (s, 9H,
t-butyl), 3.09 (dd, 1H, J=6.5 and 14.1 Hz, Phe b-CHA),
3.18 (dd, 1H, J=5.7 and 14.1 Hz, Phe b-CHB), 3.43 (m,
4H, 2ꢂAdt b-CH2), 3.72 (s, 3H, OCH3), 4.89 (m, 1H,
Phe a-CH), 5.29 (s, 1H, Adt NH), 7.10–7.29 (m, 6H,
aromatics and Phe NH); 13C NMR (75.43 MHz,
CDCl3) d 28.1 [(C(CH3)3], 37.9 (Phe b-CH2), 46.4
(2ꢂCH2S), 52.4 (Phe a-CH), 53.5 (OCH3), 71.7 (Adt

E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
155
127.1, 128.6, 129.2, 136.0 (aromatics), 155.9, 169.0, 171.6,
171.9 (4ꢂCO). Anal. C24H35N3O6S3 (557.74), C, H, N, S.
counting statistics. An empirical absorption correction,
based on azimuthal scans of several reflections, was
applied. The data were finallycorrected for Lorentz and
polarization effects. The structure was solved bydirect
methods. The non-H atoms were refined anisotropically
byfull-matrix least-squares method. The H atoms were
located at the expected positions and included in the last
structure factors calculations with isotropic thermal
parameters deduced from the carrier atoms. All the cal-
culations were performed using TEXSAN crystallo-
graphic software package.42
From 12: to a solution of 12 (0.150 g, 0.37 mmol) in
MeOH (2.0 mL), 2 N NaOH (0.37 mL) was added under
stirring. After 6 h at room temperature, the solution was
concentrated under vacuum below 30 ꢁC, acidified with
0.5 N HCl, and extracted with EtOAc; the organic phase
was washed with water, dried, and evaporated. The
resulting residue (0.153 g) was used in the next step
without further purification.
To the previous residue dissolved in DMF (2.0 mL) and
cooled to 0 ꢁC, HOBt (0.063 g, 0.41 mmol) and EDC
(0.078 g, 0.41 mmol) were added. After stirring 15 min at
Biological assay
Cells. Human periferal blood neutrophiles were purified
employing the standard techniques of dextran (Phar-
macia, Uppsala, Sweden) sedimentation, centrifugation
on Ficoll-Paque (Pharmacia), and hypotonic lysis of red
cells. The cells were washed twice and resuspended in
KRPG (Krebs–Ringer phosphate containing 0.1% w/v
glucose, pH 7.4) at a concentration of 50ꢂ106 cells/mL.
Neutrophils were 98–100% pure.
ꢁ
.
0 C and 20 min at room temperature, H-Phe-OMe HCl
(0.088 g, 0.41 mmol) and TEA (0.057 mL, 0.41 mmol)
were added. The reaction mixture was stirred at room
temperature for 18 h and then worked up as described
for 11 to give a residue (0.170 g) which, chromato-
graphed on silica gel (7 g) using CHCl3/EtOAc=95:5 as
eluent, afforded 0.154 g of 13 (82%) identical to the
sample obtained from 11.
Random locomotion. Random locomotion was per-
formed with 48-well microchemotaxis chamber (Bio
HCO-Met-Adt-Phe-OMe (7). A solution of 13 (0.097 g,
0.174 mmol) in 0.7 mL of 98% formic acid was stirred at
room temperature for 18 h. After removal of the excess
of formic acid under vacuum, the residue was dissolved
in dryDMF (0.7 mL) and EEDQ (0.052 g, 0.209 mmol)
was added. The solution was stirred at room tempera-
ture for 18 h. Evaporation under reduced pressure
afforded a residue (0.168 g) which was chromato-
graphed on silica gel (5 g) using CHCl3/EtOAc=6:4 as
eluent to give pure 7 (0.067 g, 79%). Mp 112–116 ꢁC
(from acetone); [a]D ꢀ20ꢁ (c 1.0); IR (CHCl3) 3319,
1742, 1673, 1494, 1436, 1360 cmꢀ1; 1H NMR (300 MHz,
CDCl3) d 1.98 (m, 1H, Met b-CHA), 2.10 (m, 1H, Met
b-CHB), 2.12 (s, 3H, SCH3), 2.62 (m, 2H, Met g-CH2),
3.08 (dd, 1H, J=7.2 and 14.1 Hz, Phe b-CHA), 3.19 (dd,
1H, J=5.8 and 14.1 Hz, Phe b-CHB), 3.38, 3.50 (ABq,
2H, J=12.4 Hz, Adt CAH2), 3.60 (s, 2H, Adt CBH2),
3.72 (s, 3H, OCH3), 4.61 (m, 1H, Met a-CH), 4.84 (m,
1H, Phe a-CH), 6.21 (d, 1H, J=6.3 Hz, Met NH), 6.95
(s, 1H, Adt NH), 7.11–7.32 (m, 6H, aromatics and Phe
NH), 8.10 (s, 1H, HCO); 13C NMR (75.43 MHz,
CDCl3) d 15.3 (Met CH3), 30.0 (Met g-CH2), 30.9 (Met
b-CH2), 37.6 (Phe b-CH2), 45.9, 47.3 (2ꢂAdt b-CH2),
51.4 (Met a-CH), 52.4 (Phe a-CH), 53.9 (OCH3), 71.9
(Adt a-C), 127.1, 128.5, 129.3, 136.0 (aromatics), 161.8,
169.2, 171.2, 171.9 (4ꢂCO). Anal. C20H27N3O5S3
(485.63), C, H, N, S.
Probe, Milan, Italy) and the migration into the filter
43
was evaluated bythe method of leading-front.
The
actual control random movement is 32 mmꢃ3 SE of 10
separate experiments performed in duplicate.
Chemotaxis. In order to studythe potential chemotactic
activity, each peptide was added to the lower compart-
ment of the chemotaxis chamber. Peptides were diluted
from a stock solution (DMSO; 10ꢀ2 M) with KRPG
containing 1 mg/mL of bovine serum albumin (Orha
Behringwerke, Germany) and used at concentrations
ranging from 10ꢀ12 to 10ꢀ5 M. Data were expressed in
terms of chemotactic index, which is the ratio: (migration
toward test attractant minus migration toward the buf-
fer)/migration toward the buffer; the values are the mean
of six separate experiments performed in duplicate. Stan-
dard errors are in the 0.02–0.09 chemotactic index range.
Table 4. Crystal data of N-Boc-Adt-OMe (5)
Empirical formula
Formula weight
C10H17NO4S2
279.4
Crystal system
a
b
c
b
V
Space group
dc
Z
F(000)
Monoclinic
6.081(2) A
19.783(5) A
11.358(2) A
91.49(2)ꢁ
1365.8(6) A3
P21/n
1.36 g cmꢀ3
4
592
X-ray single crystal analysis
l(Cu-Ka)
ꢀ(Cu-Ka)
Crystal size
1.5418 A
3.46 mmꢀ1
0.75ꢂ0.10ꢂ0.05 mm
124ꢁ
Crystals were obtained by slow evaporation of a
methanol solution of 5. X-raydata (see Table 4) were
collected at room temperature on a Rigaku AFC5R
2ꢁmax
diffractometer equipped with
a
graphite mono-
Reflections with I>3s(I)
No. variables
R, Rw
Full-matrix least-squares weight
S
Max and min residual electron density0.3
1310
154
0.057, 0.066
sin #/l
0.9
toꢀ0.2 e/A3
chromated Cu-Ka radiation (l=1.5418 A) and 12 KW
rotating anode source.
Reflections with I<15s(I) were rescanned up to four
times with an accumulation of counts to improve

156
E.Morera et al./ Bioorg.Med.Chem.10 (2002) 147–157
Enzyme assay. The release of neutrophil granule
enzymes was evaluated by determination of lysozyme
activity, modified for microplate-based assays. Cells,
3ꢂ106/well, were first incubated in triplicate wells of
microplates with 5 mg/mL cytochalasin B at 37 ꢁC for
15 min and then in the presence of each peptide in a final
concentration of 10ꢀ9ꢀ2ꢂ10ꢀ5 M for further 15 min.
The plates were then centrifuged at 400g for 5 min and
the lysozyme was quantified nephelometrically by the
rate of lysis of cell wall suspension of Micrococcus lyso-
deikticus. The reaction rate was measured using a
microplate reader at 465 nm. Enzyme release was
expressed as a net percentage of total enzyme content
released by0.1% Triton X-100. Total enzyme activity
was 85ꢃ1 mg/1ꢂ107 cells/min. The values are the mean
of five separate experiments done in duplicate. Standard
errors are in 1–6% range.
W. W.; Zhang, Y. F.; Davidson, V. L. Biochemistry 1993, 32,
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Louis, MO, USA) modified for microplate-based
assays. Tests were carried out in a final volume of
200 mL containing 4ꢂ105 neutrophils, 100 nmol cyto-
chrome c (Sigma) and KRPG. At zero time, different
amounts (10ꢀ9ꢀ2ꢂ10ꢀ5 M) of each peptide were added
and the plates were incubated into a microplate reader
(Ceres 900, Bio-Tek Instruments, Inc.) with the com-
partment temperature set at 37 ꢁC. Absorbance was
recorded at wavelengths of 550 and 465 nm. Differences
in absorbance at the two wavelengths were used to cal-
culate nmol of Oꢀ2 produced using an absorptivityfor
cytochrome c of 18.5 mMꢀ1/cm. Neutrophils were incu-
bated with 5 mg/ml cytochalasin B (Sigma) for 5 min
prior to activation bypeptides. The points are the mean
of five separate experiments done in duplicate. Standard
errors are in the 0.1–4 nmols Oꢀ2 range.
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Acknowledgements
This work was supported in part bythe Ministero
dell’Universita e della Ricerca Scientifica e Tecnologica.
We are grateful to ‘Banca del Sangue’ of Ferrara (Italy) for
providing fresh blood. Thanks are due to Prof. Giancarlo
Fabrizi (Dipartimento di Studi di Chimica e Tecnologia
delle Sostanze Biologicamente Attive–Universita di
Roma ‘La Sapienza’) for NOESY experiments.
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