α-Peptide/β-sulfonamidopeptide hybrids: Analogs of the chemotactic agent for-Met-Leu-Phe-OMe

Article abstract of DOI:10.1016/j.bmc.2005.11.043

In order to gain information on the activity shown by α-peptide/ β-sulfonamidopeptide hybrid analogs of the potent chemotactic agent fMLF-OMe, a structure-activity study is reported on N-Boc- and N-formyl tripeptide models containing an aminoalkanesulfoni

Full text of DOI:10.1016/j.bmc.2005.11.043

Bioorganic & Medicinal Chemistry 14 (2006) 2642–2652
a-Peptide/b-sulfonamidopeptide hybrids: Analogs
of the chemotactic agent for-Met-Leu-Phe-OMe
Cesare Giordano,a,* Gino Lucente,a Annalisa Masi,a Mario Paglialunga Paradisi,a
Anna Sansonea and Susanna Spisanib
a
`
Dipartimento di Studi farmaceutici, Universita di Roma ‘‘La Sapienza’’ and Istituto di Chimica Biomolecolare del CNR,
Sezione di Roma, P.le A. Moro 5, 00185 Roma, Italy
b
`
Dipartimento di Biochimica e Biologia Molecolare, Via Borsari 46, Universita di Ferrara, 44100, Ferrara, Italy
Received 21 July 2005; revised 23 November 2005; accepted 23 November 2005
Available online 13 December 2005
Abstract—In order to gain information on the activity shown by a-peptide/b-sulfonamidopeptide hybrid analogs of the potent che-
motactic agent fMLF-OMe, a structure–activity study is reported on N-Boc- and N-formyl tripeptide models containing an amino-
alkanesulfonic acid as central residue. Directed migration (chemotaxis), superoxide anion production, and lysozyme release have
been measured. The biochemical functions and the conformational properties of the new compounds are discussed and related
to previously studied models containing b-residues.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
are based on the synthesis of b-aminoethanesulfonic
acids as monomeric units and the study of the corre-
sponding oligomers.1,7 The high stability, the possibility
of mono- and disubstitution on the b-skeleton as well as
the physicochemical properties of the sulfonamide junc-
tion continue to stimulate interest on these peptidomi-
metics and on the related hybrid oligomers made up of
both a-aminocarboxylic- and b-aminosulfonic-acids.
Among the promising surrogates which can be consid-
ered for the replacement of the native amide bond of
peptides, a particular place should be reserved for
SO2-NH. This junction possesses chemical and physico-
chemical properties, some of them particularly useful to
overcome the well-known drawbacks which make native
peptides hardly useful as therapeutic agents. Sulfon-
amides are metabolically stable, can effectively interact
through hydrogen bond formation, adopt preferential
secondary structures, and possess a tetrahedral sulfur
atom mimicking the intermediate formed during the
process of amide-bond hydrolysis.1 Unfortunately, the
intrinsic chemical lability of the sulfonamido-a-peptides,
which are the closest sulfur analogs of natural peptides,
prevents the exploitation of the benefits associated with
this bioisosteric replacement.2,3
In this context, we reported previously8,9 studies on the
biochemical consequences of the introduction of a tau-
rine (Tau, b-aminoethanesulfonic acid) into the peptide
backbone of the potent chemotactic agent N-formyl-L-
methionyl-L-leucyl-L-phenylalanine methyl ester (for-
Met-Leu-Phe-OMe; fMLF-OMe)10–12 in place of the
native central L-leucine. This structural modification
was carried out as part of a research program, focused
on fMLF-OMe analogs containing x-amino acids at
the central position, in order to investigate the structural
and biological properties of such derivatives.13 A de-
tailed analysis of the role of the single residues on the
activity of formyl peptide chemoattractants has been pre-
viously reported by Freer et al.14 Data indicate that the
alteration at the central position with residues bearing
lipophilic side chains is well tolerated.12 In accordance
with these findings (see also Table V of Ref. 12) we found
that the introduction of a short spacer such as –HN-
CH2-CH2-CO– (i.e., b-Ala) generated the completely
As viable alternative to sulfonamido-a-peptides the
structurally related family of sulfonamido-b-peptides
has been developed. These are peptidomimetics structur-
ally related to the well-known class of b-peptides4–6 and
Keywords: Aminoalkanesulfonates; Chemotactic tripeptides; fMLF;
fMLP; Structure–activity relationships; Sulfonamidopeptides; Taurine.
*
Corresponding author. Tel.: +39 06 49913892; fax: +39 06 491491;
e-mail: cesare.giordano@uniroma1.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.043

C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
2643
inactive chemotactic peptide for-Met-bAla-Phe-OMe.9a
On the contrary and quite unexpectedly, the analogous
structural modification performed by introducing as
short spacer the sulfonyl analog of bAla (i.e.: Tau) gave
rise to the highly active chemotactic peptide for-Met-
Tau-Phe-OMe.9a Different sequences of the hybrid
pseudopeptides, in which the Tau was located at the
N-terminal position, did not generate active analogs.8
Tau, and the SO2–NH between the Tau and Phe have
been replaced by the corresponding CO-N(Me) and
SO2-N(Me) bonds. Finally, in compounds 4f–5f the
Tau residue is substituted by its higher homolog, the
3-aminopropanesulfonic acid (homotaurine; HTau).
2.1. Chemistry
The synthesis of the N-Boc pseudotripeptides 4a–f and
the corresponding N-formyl analogs 5a–f was per-
formed according to Scheme 1. The Cbz-amino-alka-
nesulfonyl chlorides 1a–c were obtained from taurine,
N-methyl-taurine, and homotaurine, respectively, which
were first N-protected by reaction with benzylchlorofor-
mate and then converted into the respective sulfonyl-
chlorides by treatment with phosphorus pentachloride.
This latter reaction, when performed on the homo-
taurine derivative Cbz-NH-(CH2)3-SO3H, afforded, in
addition to the desired sulfonyl chloride 1c, the corre-
sponding N-benzyloxycarbonyl-isothiazolidine-1,1-di-
oxide (N-Cbz-c-sultam)22 derived from the concurrent
intramolecular cyclization reaction.
By taking into account the relevance of the activation of
the formyl peptide receptor (FPR) in both antibacterial
host defense and tissue repair processes15,16 and in view
of the continuing interest on b-sulfonamidopep-
tides,1,17–21 we report here a study on a new group of
a-peptide/b-sulfonamidopeptide hybrids, specifically de-
signed to examine the role of the central sulfonamide
junction on the structural properties and biological
activities of chemotactic N-formyltripeptides.
2. Results and discussion
We have synthesized the Boc (4a–f) and formyl (5a–f)
pseudotripeptides (Table 1), which are sulfonamide ana-
logs of fMLF. In the models 4a–5a and 4b–5b, an L-al-
anine has been introduced in place of the N-terminal
Met and C-terminal Phe residues, respectively. The ana-
logs 4c and 5c contain palindromic sequence of fMLF,
where the positions of the terminal residues Met and
Phe are interchanged. In compounds 4d–5d and 4e–5e,
the modification has been focused on the alteration of
the chemical and physicochemical properties of the cen-
tral junctions; thus, the CO–NH between the Met and
Coupling of the sulfonyl chlorides 1a–c with the re-
quired amino acid methyl esters 2a–d, by adopting the
EDC/HOBT procedure, afforded the pseudodipeptides
3a–f. N-deprotection followed by coupling with the N-
Boc-protected amino acids gave the pseudotripeptides
4a–f. A direct transformation of the N-Boc derivatives
4a–f into the corresponding N-formyl analogs 5a–f
was performed by following the procedure of Lajoie
and Kraus.23
2.2. Solution conformation
Table 1.
In order to investigate the conformations of formyl
pseudotripeptides, the involvement of the NH groups
in intramolecular hydrogen bonding for analogs 5a–f
was probed using H NMR solvent-induced chemical
shifts. In Figure 1, the chemical shift dependence of
R-Ala-Tau-Phe-OMe (a)
R-Met-Tau-Ala-OMe (b)
R-Phe-Tau-Met-OMe (c)
R-Met-Tau-MePhe-OMe (d)
R-Met-MeTau-Phe-OMe (e)
R-Met-HTau-Phe-OMe (f)
1
4a–f: R = Boc; 5a–f: R = for
Scheme 1. Synthesis of pseudotripeptides 4a–f and 5a–f.

2644
C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
A
B
C
8
8
7.5
7.5
7
7.5
7
6.5
7
6.5
6
6.5
Ala NH
Met NH
Tau NH
Ala NH
Phe NH
Tau NH
Met NH
Tau NH
Phe NH
5.5
6
6
0
5
10
0
5
10
0
5
10
DMSO %
DMSO%
DMSO %
D
E
F
7.5
7.5
8
7
7
7.5
6.5
6.5
7
6
6.5
6
Met NH
Tau NH
5.5
Met NH
Phe NH
Met NH
HTau NH
Phe NH
6
5.5
5
0
5
10
0
5
10
0
5
10
DMSO %
DMSO%
DMSO %
Figure 1. Plots of NH proton chemical shifts in the 1H NMR spectra of the tripeptide derivatives 5a (A), 5b (B), 5c (C), 5d (D), 5e (E), and 5f (F) as a
function of increasing amounts of DMSO-d6 (v/v) added to CDCl3 solution (peptide concentration 10 mM).
Table 2. 1H NMR solvent accessibility of peptide NH groups: differences (Dd, ppm) between NH proton chemical shift values observed in a CDCl3
solution containing 10% DMSO-d6 and in neat CDCl3
Compound
N-terminal NH
Central NH
C-terminal NH
HCO-Ala-Tau-Phe-OMe (5a)
HCO-Met-Tau-Ala-OMe (5b)
HCO-Phe-Tau-Met-OMe (5c)
0.79
0.71
0.89
1.14
0.79
0.84
0.23
0.07
0.16
0.68
1.15
0.91
1.06
HCO-Met-Tau-(N-Me)Phe-OMe (5d)
HCO-Met-(N-Me)Tau-Phe-OMe (5e)
HCO-Met-NH(CH2)3-SO2-Phe-OMe (5f)
1.01
1.30
0.55
the NH resonances of 5a–f as a function of increasing
DMSO-d6 concentration in CDCl3 solution (10 mM) is
shown. The resulting solvent exposure of the NH groups
of 5a–f, expressed as the difference (Dd, ppm) between
the NH chemical shift values observed in a CDCl3 solu-
tion containing 10% DMSO and in neat CDCl3, is
reported in Table 2.
group of analogs interact efficiently with the solvent
especially when located at the C-terminal position (Dd
values ranging from 0.91 to 1.15 ppm). This behavior
suggests that, at variance with the NH groups of exter-
nal residues, the central amide Tau NH of 5a–c is in-
volved in an intramolecular hydrogen bond.
A
different behavior is observed in the case of the other
three peptides 5d–f (Figs. 1D–F and Table 2) where
the NH groups of all the residues show appreciable sol-
vent dependence of chemical shift with only a moderate
inaccessibility exhibited by the HTau NH group of 5f.
Although the NMR titration experiments only provide
an identification of potentially intramolecularly hydro-
gen bonded NH groups, without giving any information
on the nature of the acceptors, the present data indicate
The results of the solvent perturbation experiments
clearly show that in the three models 5a–c the central
CO-NH presents a pronounced solvent inaccessibility;
in particular, the Tau NH of the tripeptide 5b is practi-
cally unaffected by the increase of the DMSO-d6 concen-
tration (Dd = 0.07 ppm). On the contrary, the NH
groups of the external Ala, Met, and Phe of the same

C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
2645
that, at variance with the three analogs 5d–f, character-
ized by a large extent of unfolded conformations, the
analogs 5a–c appear to be in a folded conformation
involving the Tau NH as hydrogen bond donor. In this
context, it is interesting to note that an analogous result
more active than 5a and clearly more active than the
three N-Boc analogs 4a–c. However, all these com-
pounds are significantly less active than the previously
studied analogs Boc-Met-Tau-Phe-OMe and for-Met-
Tau-Phe-OMe which reach, at physiological concentra-
tion (10ꢀ9 M), C.I. values of 1.0 ca. When the activity
of the three N-formyl derivatives 5a–c is considered, it
can be seen that 5b, which maintains the Met at the first
position and contains the C-terminal Ala, exhibits the
highest activity, while 5a, where the Ala replaces the
Met, is the least active. This indicates that in these
Tau-containing pseudopeptides the lipophilic side chain
at the N-terminal position plays an important role sim-
ilar to that found in the usual a-peptidic fMLF-OMe
analogs. This is evident by the higher activity shown
by for-Phe-Tau-Met-OMe (5c) as compared with that
for-Ala-Tau-Phe-OMe (5a) as well as by the behavior
of Boc-Ala-Tau-Phe-OMe (4a) which has the lowest
activity among the peptides reported in Figure 2A. Fig-
ure 2B shows the chemotactic activity of the N-Boc
pseudotripeptides 4d–f and of the corresponding N-for-
myl derivatives 5d–f. For this group of peptides, as
against those shown in Figure 2A, the chemotactic activ-
ity exhibited by Boc derivatives is significantly different
from that exhibited by the respective formyl derivatives.
Compounds 4d–f demonstrate marginal activity (the
peaks of C.I. do not exceed 0.35 at a concentration of
10ꢀ9 M), while all the three N-formyl analogs are strong
agonists which, in the case of for-Met-(N-Me)Tau-Phe-
1
was obtained when the H NMR solvent perturbation
experiments were performed on small linear a/b-hybrid
peptides containing a b-Ala residue.9b Here the recur-
rent behavior was the solvent inaccessibility shown
by the b-Ala CO–NH group and this, on the basis of
literature data, was interpreted as indicative of the
occurrence of locally folded conformers centered at
such residue through
conformation).24
a
six-membered ring (C6
2.3. Biological activity
The biological activities, namely, the direct migration
(chemotaxis), superoxide anion production, and lyso-
zyme release have been determined on human neutro-
phils for all the synthesized compounds. The results of
the chemotactic activity, expressed as chemotactic index
(C.I.) (Section 4.7.2), are summarized in Figure 2. In Fig-
ure 2A, the activity of the tripeptides 4a–c and 5a–c is
reported.
The data suggest that these six compounds are able to
stimulate only moderate chemotactic activity (partial
agonists) with the two N-formyl derivatives 5b and 5c
1
0.5
0
1
0.5
0
-14 -12
Log [M]
-10
-8
-6
-4
-14 -12 -10
Log [M]
-8
-6
-4
4d: Boc-Met-Tau-(N-Me)Phe-OMe
5d: HCO-Met-Tau-(N-Me)Phe-OMe
4e: Boc-Met-(N-Me)Tau-Phe-OMe
4a: Boc-Ala-Tau-Phe-OMe
5a: HCO-Ala-Tau-Phe-OMe
4b: Boc-Met-Tau-Ala-OMe
5b: HCO-Met-Tau-Ala-OMe
5e: HCO-Met-(N-Me)Tau-Phe-OMe
4f: Boc-Met-NH(CH ) SO -Phe-OMe
4c : Boc-Phe-Tau-Met-OMe
5c : HCO-Phe-Tau-Met-OMe
fMLF-OMe
2 3
2
5f:: HCO-Met-NH(CH ) SO -Phe-OMe
2 3
2
fMLF-OMe
Figure 2. Chemotactic activity of tripeptide derivatives 4a–f and 5a–f expressed as chemotactic index (see Section 4.7.2).

2646
C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
OMe 5d, reach a maximum C.I. value of 0.85 at a con-
is found for the peptide for-Met-HTau-Phe-OMe (5f)
containing a c-aminopropanesulfonic acid (HTau) at
the central position of the backbone. A comparable val-
ue of activity, at the same concentration, is shown by the
previously studied sulfonamidopeptide for-Met-Tau-
Phe-OMe.9a In contrast, similar structure modifications,
involving the residues of b-Ala and c-Abu, cause inac-
tivity or significant loss of activity, respectively.9a,13
Therefore, both the Tau and HTau residues can be
incorporated at the central position of fMLF-OMe gen-
erating pseudopeptide analogs which show either high
or significant chemotactic activity in comparison to the
corresponding carboxamido models.
centration of 10ꢀ10 M.
Previous papers report the biological consequences of
the alteration or elimination of the NH groups at posi-
tion 2 and 3 of the parent peptide fMLF-OMe.25,26
The results, performed on analogs containing N-methyl
amide or ester bonds in place of the native CO-NH,
clearly demonstrate the importance for the activity of
the presence of the hydrogen bond donor NH groups
at the Met-Leu and Leu-Phe junctions. In the present
study, we have examined the two analogs 5d and 5e con-
taining (N-Me)Phe and (N-Me)Tau at position 2 and 3,
respectively. These derivatives exhibit good activity
(Fig. 2B) which is, however, lower than that shown by
the corresponding previously studied non-N-methylated
pseudopeptidic analog for-Met-Tau-Phe-OMe.9a Thus,
the consequences of the alkylation of NH-groups in sul-
fonamidopeptides are distinctly different from non-sul-
fonamide analogs, where the elimination of the protic
bonds at positions 2 and 3 leads to practically inactive
chemotactic peptides.25,26
Concerning the superoxide anion production (Fig. 3B)
all the tested derivatives, except the N-formyl derivatives
5d and 5e, resulted to be practically inactive. For the two
analogs 5d and 5e, both characterized by the presence of
a N-Me residue (Table 1), an agonistic activity was
detected only at high concentrations (10ꢀ4–10ꢀ5 M)
(Fig. 3B) and this indicates weak efficacy by the peptides
whꢀich can be considered partial agonists with regard to
O2 production. The corresponding N-Boc derivatives,
4d and 4e, also reported in Figure 3B, do not show
activity.
As reported in Figure 2B, significant chemotactic
activity (0.80 C.I. peak at a concentration of 10ꢀ10 M)
50
40
30
20
10
0
4a
5a
4b
5b
4c
5c
fMLF-OMe
-11 -10 -9
Log [M]
-8
-7
-6
-5
-4
80
60
40
20
0
60
50
40
30
20
10
0
B
C
-11
-9
-7
-5
-3
-12
-10
-8
-6
-4
Log [M]
Log [M]
4d
5d
4e
5e
4f
5f
fMLF-OMe
Figure 3. Release of neutrophil granule enzymes evaluated by determining lysozyme activity induced by 4a–c and 5a–c (A) and by 4d–f and 5d–f (C).
Superoxide anion (O2ꢀ) production triggered by 4d–f and 5d–f (B) (see Sections 4.7.3 and 4.7.4.).

C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
2647
As shown in Figure 3A, the activity as secretagogue
agents of the N-Boc and N-for peptides, characterized
by the presence of Ala at the external positions (4a–b
and 5a–b) or by the Met/Phe interchange (4c–5c) (see
Table 1), is very low. In Figure 3C, the enzyme release
activity is reported for the group of models 4d–f and
5d–f which present, as compared with peptides reported
in Figure 3A, only minor modifications of the Met-Tau-
Phe side-chain sequence (see Table 1). Here, similar to
the superoxide anion production, an activity compara-
ble to that of the fMLP-OMe is shown only at high con-
centration by the two analogs 5d and 5e characterized by
MePhe or MeTau replacements.
compounds a very useful tool for studying the signal
transduction mechanisms of the fMLF receptors and
their different subtypes.28
4. Experimental
4.1. General methods
Melting points were determined with a Buchi B 540
¨
apparatus and are uncorrected. Optical rotations were
taken at 20 ꢁC with a Schmidt-Haensch Polartronic D
polarimeter (1 dm cell, c 1.0 in CHCl3, unless otherwise
specified). IR spectra were recorded in 1% CHCl3 (un-
less otherwise specified) solution employing a Perkin-
It can be recalled that the consequences of the alteration
of the native sequence of amino acids on the lysozyme
release, here examined in the model for-Phe-Tau-Met-
OMe (5c), have been previously evaluated by Bonora
et al.27 while studying a group of retroisomers of fMLF.
The authors found that for-Phe-Leu-Met-NH2 was
approximately 100-fold less active than the parent for-
Met-Leu-Phe-NH2. The high fall in activity exhibited
by this retroisomer parallels the result here found for
the related pseudopeptide 5c (see Fig. 3A). Thus, the
maintenance of the native side chain sequence at the
N- and C-terminal positions is confirmed as an impor-
tant factor in the receptor recognition for usual peptides
as well as for the here studied sulfonamido analogs.
1
Elmer FT-IR Spectrum 1000 spectrometer. H NMR
spectra were determined in CDCl3 solution with a
Bruker AM 400 spectrometer and chemical shifts were
indirectly referred to TMS. Thin-layer and preparative
layer chromatographies were performed on silica gel
Merck 60 F254 plates. The drying agent was sodium sul-
fate. Elemental analyses were performed in the laborato-
ries of the Servizio Microanalisi del CNR, Area della
Ricerca di Roma, Montelibretti, Italy, and were within
0.4% of the theoretical values. The abbreviations used
are as follows: Boc, tert-butyloxycarbonyl; EEDQ, ethyl
2-ethoxy-1,2-dihydro-1-quinolinecarboxylate; EDC, N-
(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride; HOBt, 1-hydroxybenzotriazole; KRPG,
Krebs–Ringer-phosphate containing 0.1% w/v D-glu-
cose; NMM, N-methylmorpholine; DMF, dimethyl-
formamide; TEA, triethylamine.
3. Conclusion
In conclusion, we have synthesized and studied a series
of pseudopeptidic fMLF analogs characterized by the
presence of a central sulfonamide junction and different
alterations of both the backbone and the native side-
chain sequence. Results show that, similar to the litera-
ture findings on related classical peptide analogs,12,14 the
presence in sulfonamide models of the for-Met at the N-
terminal position is of primary importance for optimal
biological activity. On the contrary, the N-methylation
of the Tau and Phe NH groups, as well as the homolo-
gation of the central b-residue (see compounds 5d–f),
can be performed with only moderate decrease of the
chemotactic activity shown by for-Met-Tau-Phe-OMe.9a
Results on lysozyme release and superoxide anion pro-
duction (Fig. 3) show a clear negative effect of the cen-
tral sulfonamide bond on the two main biological
functions associated with chemotaxis. Analogs of fMLF
containing a central b-sulfonamido residue seem then
promising and flexible models for designing selective
agonists, with particular reference to chemoattractants
devoid of superoxide anion production.28
4.2. General procedure for the preparation of Cbz-amino-
alkanesulfonyl chlorides 1a–c
A solution of the amino-alkanesulfonic acid (1.0 mmol)
in H2O (10 mL) was adjusted to pH 8.5 by using an
aqueous solution of 1 M NaOH. Under vigorous stir-
ring, benzylchloroformate (1.1 mmol) was added in five
portions at room temperature, maintaining the pH at
the above value by addition of small amount of NaOH,
then the reaction mixture was stirred overnight. After
dilution with H2O (10 mL), the aqueous phase was
washed with Et2O (2· 10 mL) and evaporated. The
crude solid residue was coevaporated three times with
toluene and dried under high vacuum over P2O5 over-
night. To a suspension of the crude sodium sulfonate
salt in anhydrous Et2O (10 mL), PCl5 (1.5 mmol) was
added and the mixture was stirred at room temperature
overnight. The mixture was washed with water and the
organic phase dried and evaporated. Compounds 1a29
and 1b30 were obtained as colorless oil which solidified
by trituration with hexane.
Taken together the reported results indicate that the sul-
fonamide junction, due to its high polar character and
conformational properties, when located at the central
position of fMLF analogs to replace the native Leu (res-
idue), produces a profound alteration of the established
requirements for optimal ligand-receptor interaction.
The resulting properties, together with the observed
capability to discriminate among the biological activities
exerted by the fMLF, might render the here-described
4.3. General procedure for the preparation of pseudo-
dipeptides 3a–f
To an ice-cooled mixture containing the Cbz-amino-
alkanesulfonyl chloride 1a–c (1.0 mmol) and the amino
acid methyl ester hydrochlorides 2a–d (1.0 mmol) in
dry CH2Cl2 (8 mL), a solution of TEA (2.0 mmol) in
dry CH2Cl2 (8 mL) was added dropwise under nitrogen.

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C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
The resulting suspension was stirred overnight allowing
to warm to room temperature. After dilution with
CH2Cl2 (25 mL), the mixture was washed with 1 M
HCl (2 · 10 mL), saturated NaHCO3 (2 · 10 mL), and
brine (10 mL). The organic phase was dried and evapo-
rated under reduced pressure.
4.3.5. Cbz-homotauryl-L-phenylalanine methyl ester (3f).
From Cbz-homotauryl chloride 1c (0.660 g, 2.26 mmol)
and L-phenylalanine methyl ester hydrochloride 2a
(0.478 g). Silica gel flash chromatography (CHCl3) gave
the pure product as colorless oil. (0.501 g, 51%). [a]D
1
+7ꢁ. IR m: 3445, 2959, 1740, 1718 cmꢀ1. H NMR d:
1.94 (2H, m, HTau b-CH2), 2.82 (2H, m, HTau a-
CH2), 3.05 and 3.17 (4H, two m, Phe b-CH2 and HTau
c-CH2), 3.92 (3H, s, COOCH3), 4.43 (1H, m, Phe a-
CH), 4.95–5.06 (2H, m, Phe and HTau NH), 5.24 (2H,
s, CH2Ph), 7.23–7.58 (10H, m, aromatics). Anal. Calcd
for C21H26N2O6S: C, 58.05; H, 6.03; N, 6.45. Found:
C, 57.98; H, 6.22; N, 6.17.
4.3.1. Cbz-tauryl-L-alanine methyl ester (3b). From
Cbz-tauryl chloride 1a (0.800 g, 2.88 mmol) and L-ala-
nine methyl ester hydrochloride (2b) (0.400 g). Pale yel-
low oil, used in the next step without further
purification. (0.694 g, 70%). [a]D ꢀ45ꢁ. IR m: 3448,
3367, 2957, 1717 cmꢀ1 1H NMR d: 1.35 (3H, d,
.
J = 6.4 Hz, Ala CH3), 3.14 (2H, m, Tau a-CH2), 3.62
(2H, m, Tau b-CH2), 3.70 (3H, s, COOCH3), 4.13
(1H, m, Ala a-CH), 5.05 (2H, s, CH2Ph), 5.38 (1H,
d, J = 9.4 Hz, Ala NH), 5.35 (1H, poorly resolved t,
Tau NH), 7.28 (5H, s, aromatics). Anal. Calcd for
C14H20N2O6S: C, 48.83; H, 5.85; N, 8.13. Found: C,
48.58; H, 5.71; N, 8.15.
4.4. General procedure for the N-deprotection of pseudo-
dipeptides 3a–f
Method a (HBr/AcOH): the N-protected pseudodipep-
tide (1.0 mmol) was dissolved in a solution of 36%
HBr in acetic acid (3.0 mL). After 15 min at room tem-
perature, the reaction mixture was evaporated to dry-
ness without heating. The residue was repeatedly
coevaporated with anhydrous Et2O and then dried un-
der high vacuum overnight.
4.3.2. Cbz-tauryl-L-methionine methyl ester (3c). From
Cbz-tauryl chloride 1a (0.500 g, 1.80 mmol) and
L-methionine methyl ester hydrochloride 2c (0.360 g).
The crude product was triturated with hexane. Pale yel-
low solid. (0.60 g, 82%). [a]D ꢀ37ꢁ. IR m: 3448, 2957,
Method b (catalytic hydrogenation): a solution of the
N-protected pseudodipeptide (1.0 mmol) and trifluoro-
acetic acid (1.2 mmol) in MeOH (10 mL) was flushed
with an H2 stream in the presence of 10% palladium
on charcoal at room temperature until the reaction
was complete (TLC). The mixture was filtered on Celite
and the filtrate was evaporated to dryness under reduced
pressure. The oily residue was repeatedly coevaporated
with anhydrous diethyl ether to remove excess TFA
and then kept under high vacuum overnight.
1718 cmꢀ1
.
1H NMR d: 1.75–2.18 (2H, m, Met
b-CH2), 2.03 (3H, s, S-CH3), 2.51 (2H, m, Tau
a-CH2), 3.13 (2H, m, Tau b-CH2), 3.63 (2H, m, Met
c-CH2), 3.70 (3H, s, COOCH3), 4.28 (1H, m, Met
a-CH), 5.03 (2H, s, CH2Ph), 5.30 (1H, d, J = 9.6 Hz,
Met NH), 5.40 (1H, poorly resolved t, Tau NH),
7.14 (5H, s, aromatics). Anal. Calcd for C16H24N2O6S2:
C, 47.51; H, 5.98; N, 6.93. Found: C, 47.72; H, 6.04;
N, 6.81.
4.3.3. Cbz-tauryl-(N-methyl)-L-phenylalanine methyl
ester (3d). From Cbz-tauryl chloride 1a (0.233 g,
0.84 mmol) and (N-methyl)-L-phenylalanine methyl
ester hydrochloride 2d (0.163 g). Silica gel flash chroma-
tography (CHCl3) gave the pure product as a pale yel-
low oil. (0.298 g, 82%). [a]D ꢀ15ꢁ. IR m: 3688, 3446,
4.5. General procedure for the preparation of pseudotri-
peptides 4a–f. Carbodiimide coupling
To an ice-cooled mixture containing the required Boc-
protected amino acid (1.0 mmol), the pseudodipeptide
salt 3a–f (1.0 mmol), HOBt (1.2 mmol), and TEA
(1.2 mmol) in anhydrous ethyl acetate (6 mL), EDC
(1.0 mmol) was added and the reaction mixture was
allowed to warm to room temperature. After 12 h, the
reaction mixture was diluted with EtOAc (20 mL) and
washed with 1M KHSO4 (2 · 15 mL), saturated aqueous
NaHCO3 (2 · 15 mL), and brine (15 mL). The organic
phase was dried and evaporated under reduced pressure.
1
2955, 2406, 1718 cmꢀ1. H NMR d: 2.65 (2H, m, Tau
a-CH2), 2.74–3.00 (2H, m, Phe b-CH2), 2.68 (3H, s,
N-CH3), 3.24–3.45 (2H, m, Tau b-CH2), 3.76 (3H, s,
COOCH3), 4.86 (1H, m, Phe a-CH), 5.10 (2H, s,
CH2Ph), 5.35 (1H, br s, Tau NH), 7.14–7.40 (10H, m,
aromatics). Anal. Calcd for C21H26N2O6S: C, 58.05;
H, 6.03; N, 6.45. Found: C, 58.15; H, 5.92; N, 6.40.
4.3.4. Cbz-(N-methyl)tauryl-L-phenylalanine methyl ester
(3e). From Cbz-(N-methyl)tauryl chloride 1b (0.361 g,
1.24 mmol) and L-phenylalanine methyl ester hydro-
chloride 2a (0.267 g). Pale yellow oil used in the next
step without further purification. (0.390 g, 72%). [a]D
4.5.1. Boc-L-alanyl-tauryl-L-phenylalanine methyl ester
(4a). Cbz-tauryl-L-phenylalanine methyl ester31 3a
(0.441 g, 1.04 mmol) was N-deprotected according to
the above general procedure (method b). The dipeptide,
as trifluoroacetate salt (white solid), was acylated with
Boc-L-alanine (0.197 g, 1.04 mmol) according to the car-
bodiimide coupling general procedure. Silica gel flash
chromatography (CH2Cl2/MeOH 98:2) gave the pure
product as a colorless oil (0.414 g, 87%). [a]D ꢀ49ꢁ. IR
ꢀ10ꢁ. IR m: 3550, 3360, 2960, 1743, 1697 cmꢀ1
.
1H
NMR d: 2.70–3.12 (4H, m, Tau a-CH2 and Phe b-
CH2), 2.90 (3H, s, N-CH3), 3.42 (2H, m, Tau b-CH2),
3.62 (3H, s, COOCH3), 4.31 (1H, m, Phe a-CH), 5.02
(2H, s, CH2Ph), 5.41 (1H, d, J = 9.6 Hz, Phe NH),
7.01–7.48 (10H, m, aromatics). Anal. Calcd for
C21H26N2O6S: C, 58.05; H, 6.03; N, 6.45. Found: C,
58.02; H, 6.18; N, 6.77.
1
m: 3436, 3017, 1742, 1682 cmꢀ1. H NMR d: 1.26 (3H,
d, J = 7.2 Hz, Ala CH3), 1.36 [9H, s, C(CH3)3], 2.78
and 2.85 (2H, m, Tau a-CH2), 2.95 and 3.10 (2H, A
and B of on ABX, J = 5.1, 8.7 and 13.9 Hz, Phe

C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
2649
b-CH2), 3.42 and 3.51 (2H, two m, Tau b-CH2), 3.70
(3H, s, COOCH3), 4.02 (1H, m, Ala a-CH), 4.34 (1H,
m, Phe a-CH), 4.93 (1H, d, J = 7.5 Hz, Ala NH), 5.84
(1H, br s, Phe NH), 6.95 (1H, br s, Tau NH), 7.13–7.26
(5H, m, aromatics). Anal. Calcd for C20H31N3O7S: C,
52.50; H, 6.83; N, 9.18. Found: C, 52.37; H, 6.95; N, 9.08.
m, Met a-CH), 4.86 (1H, m, Phe a-CH), 5.12 (1H, br s,
Met NH), 6.68 (1H, br s, Tau NH), 7.14–7.26 (5H, m,
aromatics). Anal. Calcd for C23H37N3O7S2: C, 51.96;
H, 7.01; N, 7.90. Found: C, 51.68; H, 6.93; N, 7.78.
4.5.5. Boc-L-methionyl-(N-methyl)tauryl-L-phenylalanine
methyl ester (4e). Cbz-(N-methyl)tauryl-L-phenylalanine
methyl ester 3e (0.364 g, 0.84 mmol) was N-deprotected
according to the above general procedure (method b).
The dipeptide, as trifluoroacetate salt (white solid),
was acylated by Boc-L-methionine (0.210 g, 0.84 mmol)
according to the carbodiimide coupling general proce-
dure. Silica gel flash chromatography (CHCl3) gave
the pure product as colorless oil. (0.366 g, 82%). [a]D
4.5.2. Boc-L-methionyl-tauryl-L-alanine methyl ester (4b).
Cbz-tauryl-L-alanine methyl ester 3b (0.413 g, 1.2 mmol)
was N-deprotected according to the above general pro-
cedure (method b). The dipeptide, as trifluoroacetate salt
(white solid), was acylated by Boc-L-methionine
(0.300 g, 1.2 mmol) according to the carbodiimide cou-
pling general procedure. Silica gel flash chromatography
(CH2Cl2/MeOH 99:1) gave the pure product as a color-
less oil (0.397 g, 75%). [a]D ꢀ11ꢁ. IR m: 3428, 1732,
ꢀ30ꢁ. IR m: 3430, 3034, 1733, 1707, 1645 cmꢀ1
.
1H
NMR d: 1.42 [9H, s, C(CH3)3], 1.76 and 1.93 (2H, two
m, Met b-CH2), 2.11 (3H, s, S-CH3), 2.54 (2H, m, Met
c-CH2), 2.94–3.13 [7H, m, Phe b-CH2, Tau a-CH2
and N-CH3 (s at 3.11)], 3.35 (1H, m, 1H of Tau
b-CH2), 3.75 (3H, s, COOCH3), 4.13 (1H, m, 1H of
Tau b-CH2), 4.44 (1H, m, Met a-CH), 4.71 (1H, m,
Phe a-CH), 5.33 (1H, d, J = 6.7 Hz, Met NH), 5.66
(1H, d, J = 8.3 Hz, Phe NH), 7.23–7.46 (5H, m, aromat-
ics). Anal. Calcd for C23H37N3O7S2: C, 51.96; H, 7.01;
N, 7.90. Found: C, 52.04; H, 7.15; N, 7.82.
1690 cmꢀ1 1H NMR d: 1.40 [9H, s, C(CH3)3], 1.55
.
(3H, d, J = 8.2 Hz, Ala CH3), 1.80 and 1.93 (2H, two
m, Met b-CH2), 2.13 (3H, s, S-CH3), 2.59 (2H, m, Met
c-CH2), 3.28 (2H, m, Tau a-CH2), 3.62 and 3.76 (2H,
m, Tau b-CH2), 3.82 (3H, s, COOCH3), 4.19 (1H, m,
Met a-CH), 4.32 (1H, m, Ala a-CH), 5.28 (1H, d,
J = 8.0 Hz, Met NH), 6.19 (1H, d, J = 7.4 Hz, Ala
NH), 7.56 (1H, poorly resolved t, Tau NH). Anal. Calcd
for C16H31N3O7S2: C, 43.52; H, 7.08; N, 9.52. Found: C,
43.22; H, 7.15; N, 9.41.
4.5.6.
Boc-L-methionyl-homotauryl-L-phenylalanine
4.5.3. Boc-L-phenylalanyl-tauryl-L-methionine methyl
ester (4c). Cbz-tauryl-L-methionine methyl ester 3c
(0.578 g, 1.43 mmol) was N-deprotected according to
the above general procedure (method a). The dipeptide,
as hydrobromide (white solid), was acylated by Boc-L-
phenylalanine (0.380 g, 1.43 mmol) according to the car-
bodiimide coupling general procedure. Crystallization
from diethyl ether/hexane gave the pure product as a
white solid (0.547 g, 74%). [a]D +21ꢁ (2% CHCl3). Mp
methyl ester (4f). Cbz-homotauryl-L-phenylalanine
methyl ester 3f (0.148 g, 0.34 mmol) was N-deprotected
according to the above general procedure (method b).
The dipeptide, as trifluoroacetate salt (white solid),
was acylated by Boc-L-methionine (0.086 g, 0.34 mmol)
according to the carbodiimide coupling general proce-
dure. Silica gel flash chromatography (CHCl3/MeOH
98:2) gave the pure product as a pale yellow oil.
(0.154 g, 85%). [a]D ꢀ26ꢁ. IR m: 3343, 3039, 1676 cmꢀ1
.
95–97 ꢁC. IR m: 3432, 3037, 1731, 1684 cmꢀ1. H NMR
1H NMR d: 1.45 [9H, s, C(CH3)3], 1.82 (2H, m, HTau
b-CH2), 1.90 (1H, m, 1H of Met b-CH2), 2.06 [4H, m,
1H of Met b-CH2 and S-CH3 (s at 2.11)], 2.53 (2H, m,
Met c-CH2), 2.77 (2H, m, HTau a-CH2), 3.00–3.39
(4H, three m, Phe b-CH2 and HTau c-CH2), 3.78 (3H,
s, COOCH3), 4.19 (1H, m, Met a-CH), 4.40 (1H, m,
Phe a-CH), 5.06 (1H, d, J = 9.0 Hz, Phe NH), 5.18
(1H, d, J = 6.6 Hz, Met NH), 6.52 (1H, poorly resolved
t, HTau NH), 7.19–7.36 (5H, m, aromatics). Anal.
Calcd for C23H37N3O7S2: C, 51.96; H, 7.01; N, 7.90.
Found: C, 51.74; H, 7.08; N, 7.59.
1
d: 1.45 [9H, s, C(CH3)3], 2.00–2.12 [4H, m, 1H of Met
b-CH2 and S-CH3 (s at 2.10)], 2.18 (1H, m, 1H of Met
b-CH2), 2.66 (2H, m, Met b-CH2), 3.07 and 3.17 (4H,
two m, Phe b-CH2 and Tau a-CH2), 3.52 and 3.89 (2H,
two m, Tau b-CH2), 3.84 (3H, s, COOCH3), 4.30 (1H,
m, Phe a-CH), 4.35 (1H, m, Met a-CH), 5.11 (1H, d,
J = 6.8 Hz, Phe NH), 6.14 (1H, d, J = 8.1 Hz, Met NH),
7.23–7.34 (5H, m, aromatics), 7.36 (1H, poorly resolved
t, Tau NH). Anal. Calcd for C23H35N3O7S2Æ0.5 H2O: C,
50.17; H, 6.89; N, 7.98. Found: C, 50.28; H, 6.66; N, 7.95.
4.5.4. Boc-L-methionyl-tauryl-L-(N-methyl)phenylalanine
methyl ester (4d). Cbz-tauryl-(N-methyl)-L-phenylala-
nine methyl ester 3d (0.260 g, 0.60 mmol) was N-depro-
tected according to the above general procedure
(method b). The dipeptide, as trifluoroacetate salt (white
solid), was acylated by Boc-L-methionine (0.150 g,
0.60 mmol), according to the carbodiimide coupling gen-
eral procedure. Colorless oil (0.280 g, 88%). [a]D ꢀ14ꢁ.
4.6. General procedure for preparation of the N-formyl
derivatives 5a–f
The N-Boc derivative (1.0 mmol) was dissolved in for-
mic acid (3 mL) and the mixture was allowed to stand
at room temperature overnight. After removal of the
excess formic acid under vacuum, the residue was dis-
solved in dry DMF (2 mL). EEDQ 97% (1.2 mmol)
was added and the solution was stirred at room temper-
ature for 24 h. Evaporation under reduced pressure
afforded the crude product.
1
IR m: 3435, 3039, 1735 cmꢀ1. H NMR d: 1.45 [9H, s,
C(CH3)3], 1.75 and 1.88 (2H, two m, Met b-CH2), 2.09
(3H, s, S-CH3), 2.48–2.63 (3H, m, Met c-CH2 and 1H
of Tau a-CH2), 2.70–2.93 [4H, m, 1H of Tau a-CH2
and N-CH3 (s at 2.91)], 2.95, and 3.36 (2H, AB of on
ABX, J = 5.5, 10.2, and 14.2 Hz, Phe b-CH2), 3.45
(2H, m, Tau b-CH2), 3.79 (3H, s, COOCH3), 4.22 (1H,
4.6.1. Formyl-L-alanyl-tauryl-L-phenylalanine methyl
ester (5a). From Boc-L-alanyl-tauryl-L-phenylalanine
methyl ester 4a (0.283 g, 0.61 mmol). Silica gel flash

2650
C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
chromatography (CH2Cl2/MeOH 98:2) gave the pure
product as a white foam. (0.188 g, 80%). [a]D ꢀ10ꢁ.
J = 8.3 Hz, Met NH), 6.82 (1H, t, J = 4.8 Hz, Tau
NH), 7.23–7.35 (5H, m, aromatics), 8.18 (1H, s, HCO).
Anal. Calcd for C19H29N3O6S2Æ0.5 H2O: C, 48.70; H,
6.45; N, 8.97. Found: C, 48.92; H, 6.29; N, 8.62.
Mp 126–128 ꢁC. IR m: 3412, 1742, 1673 cmꢀ1
.
1H
NMR d: 1. 35 (3H, d, J = 6.9 Hz, Ala CH3), 2.83 and
2.90 (2H, two m, Tau a-CH2), 3.00 and 3.16 (2H, A
and B of an ABX, J = 5.0, 8.9, and 13.7 Hz, Phe b-
CH2) 3.47 and 3.56 (2H, two m, Tau b-CH2), 3.76
(3H, s, COOCH3), 4.40 (1H, m, Phe a-CH), 4.47 (1H,
m, Ala a-CH), 5.86 (1H, d, J = 9.0 Hz, Phe NH), 6.51
(1 H, d, J = 8.0 Hz, Ala NH), 7.09 (1 H, poorly resolved
t, Tau NH), 7.19–7.32 (5H, m, aromatics), 8.08 (1H, s,
HCO). Anal. Calcd for C16H23N3O6S: C, 49.86; H,
6.01; N, 10.90. Found: C, 49.58; H, 5.96; N, 10.96.
4.6.5. Formyl-L-methionyl-(N-methyl)tauryl-L-phenylala-
nine methyl ester (5e). From Boc-L-methionyl-(N-meth-
yl)tauryl-L-phenylalanine methyl ester 4e (0.150 g,
0.28 mmol). Silica gel flash chromatography (CHCl3/
MeOH 98:2) gave the pure product as a colorless oil.
(0.090 g, 70%). [a]D ꢀ22ꢁ. IR m: 3411, 1744, 1684,
1
1644 cmꢀ1. H NMR d: 1.85 and 2.10 (2H, two m, Met
b-CH2), 2.14 (3H, s, S-CH3), 2.58 (2H, m, Met c-CH2),
2.99–3.20 (4H, m, Tau a-CH2 and Phe b-CH2), 3.15
(3H, s, N-CH3), 3.55 (2H, m, Tau b-CH2), 3.80 (3H, s,
COOCH3), 4.49 (1H, m, Phe a-CH), 5.17 (1H, m, Met
a-CH), 5.73 (1H, d, J = 9.0 Hz, Phe NH), 6.39 (1H, d,
J = 8.0 Hz, Met NH), 7.24–7.49 (5H, m, aromatics),
8.19 (1H, s, HCO). Anal. Calcd for C19H29N3O6S2: C,
49.66; H, 6.36; N, 9.14. Found: C, 49.38; H, 6.13; N, 9.02.
4.6.2. Formyl-L-methionyl-tauryl-L-alanine methyl ester
(5b). From Boc-L-methionyl-tauryl-L-alanine methyl es-
ter 4b (0.200 g, 0.45 mmol). Silica gel flash chromatogra-
phy (CH2Cl2/MeOH 98:2) gave the pure product, which
was triturated with hexane. White solid. (0.139 g, 84%).
[a]D ꢀ50ꢁ. Mp 98–100 ꢁC. IR m: 3415, 1739, 1674 cmꢀ1
.
1H NMR d: 1.50 (3H, d, J = 7.2 Hz, Ala CH3),
1.97–2.17 (2H, m, Met b-CH2), 2.12 (3H, s, S-CH3),
2.58 (2H, t, J = 7.1 Hz, Met c-CH2), 3.26 (2H, m, Tau
a-CH2), 3.65, and 3.87 (2H, two m, Tau b-CH2), 3.80
(3H, s, COOCH3), 4.28 (1H, m, Ala a-CH), 4.60 (1H,
m, Met a-CH), 6.11 (1H, d, J = 8.4 Hz, Ala NH), 6.77
(1H, d, J = 8.2 Hz, Met NH), 7.57 (1H, t, J = 5.2 Hz,
Tau NH), 8.18 (1H, s, HCO). Anal. Calcd for
C12H23N3O6S2: C, 39.01; H, 6.27; N, 11.37. Found: C,
38.87; H, 6.05; N, 11.12.
4.6.6. Formyl-L-methionyl-homotauryl-L-phenylalanine
methyl ester (5f). From Boc-L-methionyl-homotauryl-
L-phenylalanine methyl ester 4f (0.110 g, 0.21 mmol).
Silica gel flash chromatography (CHCl3/MeOH 98:2)
gave the pure product as a colorless oil. (0.051 g,
53%). [a]D = ꢀ49ꢁ. IR m: 2957, 1743, 1669 cmꢀ1
.
1H
NMR d: 1.78 (2H, m, HTau b-CH2), 1.95 and 2.08
(2H, two m, Met b-CH2), 2.10 (3H, s, S-CH3), 2.51
(2H, m, Met b-CH2), 2.73 (2H, m, HTau a-CH2),
2.90–341 (4H, three m, Phe b-CH2 and HTau c-CH2),
3.78 (3H, s, COOCH3), 4.37 (1H, m, Phe a-CH), 4.61
(1H, m, Met a-CH), 5.47 (1H, poorly resolved d, Phe
NH), 6.65 (1H, d, J = 8.9 Hz, Met NH), 6.78 (1H, t,
J = 4.3 Hz, HTau NH), 7.20–7.34 (5H, m, aromatics),
8.17 (1H, s, HCO). Anal. Calcd for C19H29N3O6S2: C,
49.66; H, 6.36; N, 9.14. Found: C, 49.77; H, 6.18; N,
8.96.
4.6.3. Formyl-L-phenylalanyl-tauryl-L-methionine methyl
ester (5c). From Boc-L-phenylalanyl-tauryl-L-methio-
nine methyl ester 4c (0.200 g, 0.38 mmol). Silica gel flash
chromatography (CHCl3/MeOH 99:1) gave the pure
product, which was crystallized from EtOAc/ hexane.
White solid. (0.166 g, 98%). [a]D + 26ꢁ. Mp 90–92 ꢁC.
IR m: 3415, 1739, 1675 cmꢀ1 1H NMR d: 1.89-2.15
.
[5H, m, Met b-CH2 and S-CH3 (s at 2.11)], 2.65 (2H,
m, Met c-CH2), 3.03–3.25 (4H, m, Tau a-CH2, and
Phe b-CH2), 3.54 and 3.75 (2H, two m, Tau b-CH2),
3.81 (3H, s, COOCH3), 4.34 (1H, m, Met a-CH), 4.65
(1H, s, Phe a-CH), 6.10 (1H, d, J = 9.4 Hz, Met NH),
6.50 (1H, d, J = 7.4 Hz, Phe NH), 7.18–7.32 (5H, m,
aromatics), 7.34 (1H, poorly resolved t, Tau NH), 8.10
(1H, s, HCO). Anal. Calcd for C18H27N3O6S2ÆH2O: C,
46.64; H, 6.311; N, 9.06. Found: C, 46.67; H, 6.28; N,
9.25.
4.7. Biological assays. Cell preparation
Cells were obtained from the blood of healthy subjects,
and human peripheral blood neutrophils were purified
by using the standard techniques of dextran (Pharmacia,
Uppsala, Sweden) sedimentation, centrifugation on
Ficoll–Paque (Pharmacia), and hypotonic lysis of
contaminating red cells. Cells were washed twice and
resuspended in Krebs–Ringer phosphate (KRPG), pH
7.4, at a final concentration of 50 · 106 cells/ml and
kept at room temperature until used. Neutrophils were
98–100% viable, as determined using the Trypan blue
exclusion test.
4.6.4. Formyl-L-methionyl-tauryl-(N-methyl)-L-phenyl-
alanine methyl ester (5d). From Boc-L-methionyl-tau-
ryl-L-(N-methyl)phenylalanine methyl ester 4d (0.160 g,
0.30 mmol). Silica gel flash chromatography (CHCl3/
MeOH 98:2) gave the pure product, which was crystal-
lized from EtOAc/hexane. White solid. (0.088 g, 55%).
4.7.1. Random locomotion. Random locomotion was
performed with 48-well microchemotaxis chamber (Bio
Probe, Milan, Italy) and migration into the filter was
evaluated by the leading-front method 21. The actual
control random movement is 35 3 lm SE of ten sepa-
rate experiments performed in duplicate.
[a]D ꢀ25ꢁ. Mp 73-75 ꢁC. IR m: 3417, 1740, 1673 cmꢀ1
.
1H NMR d: 1.85-2.20 [5H, m, Met b-CH2 and S-CH3
(s at 2.10)], 2.45–2.70 (3H, m, Met c-CH2 and 1H of
Tau a-CH2), 2.78–2.85 (4H, m, 1H of Tau a-CH2 and
N-CH3), 2.93, and 3.36 (2H, A and B of an ABX,
J = 5.2, 10.3, and 14.3 Hz, Phe b-CH2), 3.48 (2H, m,
Tau b-CH2), 3.79 (3H, s, COOCH3), 4.63 (1H, m, Met
a-CH), 4.85 (1H, m, Phe a-CH), 6.47 (1H, d,
4.7.2. Chemotaxis. Each peptide was added to the lower
compartment of the chemotaxis chamber. Peptides were
diluted from a stock solution (10ꢀ2 M in DMSO) with

C. Giordano et al. / Bioorg. Med. Chem. 14 (2006) 2642–2652
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expressed in terms of chemotactic index (C.I.), which
is the ratio: (migration toward test attractant ꢀ migra-
tion toward the buffer)/migration toward the buffer;
the values are means of six separate experiments
performed in duplicate. Standard errors are in the
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4.7.3. Superoxide anion (O2ꢀ) production. This anion
was measured by the superoxide dismutase-inhibitable
reduction of ferricytochrome c (Sigma, USA) modified
for microplate-based assays. Tests were carried out in a
final volume of 200 ml containing 4 · 105 neutrophils,
100 nmol cytochrome c, and KRPG. At zero time dif-
ferent amounts (10ꢀ10–8 · 10ꢀ5 M) of each peptide
were added and the plates were incubated into a micro-
plate reader (Ceres 900, Bio-TeK Instruments, Inc.)
with the compartment temperature set at 37 ꢁC. Absor-
bance was recorded at 550 and 468 nm. The difference
in absorbance at tꢀhe two wavelengths was used to cal-
culate nmol of O2 produced using an absorptivity for
cytochrome c of 18.5 mMꢀ1 cmꢀ1. Neutrophils were
incubated with 5 lg/ml cytochalasin B (Sigma) for
5 min prior to activation bꢀy peptides. Results were ex-
pressed as net nmol of O2 per 1 · 106 cells per 5 min
and are means of six separate experiments performeꢀd
in duplicate. Standard errors are in 0.1–4 nmol O2
range.
4.7.4. Enzyme assay. The release of neutrophil granule
enzymes was evaluated by determination of lysozyme
activity, modified for microplate-based assays. Cells,
3 · 106/well, were first incubated in triplicate wells of
microplates with 5 lg/ml cytochalasin B at 37 ꢁC for
15 min and then in the presence of each peptide at a final
concentration of 10ꢀ10–8 · 10ꢀ5 M for a 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 lys-
odeikticus. The reaction rate was measured using a
microplate reader at 465 nm. Enzyme release was ex-
pressed as a net percentage of total enzyme content re-
leased by 0.1% Triton X-100. Total enzyme activity
was 85 1 mg per 1 · 107 cells/min. The values are
means of five separate experiments done in duplicate.
Standard errors are in the range 1–6%.
23. Lajoie, G.; Kraus, J.-L. Peptides 1984, 5, 653.
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Acknowledgments
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This work was supported in part by Fondazione Cassa
di Risparmio di Ferrara. We are grateful to Banca del
Sangue di Ferrara for providing fresh blood.
26. Spisani, S.; Breveglieri, A.; Fabbri, E.; Vertuani, G.;
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