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Chemotactic Peptide Analogues The Role of Z-23-Didehydrophenylalanine and Phenylalanine C-Terminal Residues in Determining the Chemotactic Activity of Formylpeptides.

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143
Chemotactic Activity of Formylpeptides
Chemotactic Peptide Analogues
The Role of (2)-2,3-Didehydrophenylalanineand Phenylalanine
C-Terminal Residues in Determining the Chemotactic Activity of
Formylpeptides
Ines Torrini 'I, Giampiero Pagani Zecchini 'I, Mario Paglialunga Paradisi a)*, and Susanna Spisani b,
a'
Dipartimento di Studi Farmaceutici and Centro di Studio per la Chimica del Farmaco del CNR, Universith '' La Sapienza", 00185 Roma, Italy
b,
Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, 441 00 Ferrara, Italy
Key Words: chemotaxis; 2,3-didehydrophenylulanine;
formylpeptides
Summary
Several forrnylpeptides, analogs of the chemotactic agent HCOMet-Leu-Phe-OMe, having the HCO-Xaa-Leu-A'Phe-OMe and
HCO-Xaa-Leu-A'Phe-Phe-OMe structures (A'Phe = (Z)-2,3-didehydrophenylalanine),have been synthesized. The biological activity of these ligands has been determined on human neutrophils and
compared to that of the corresponding HCO-Xaa-Leu-Phe-OMe
derivatives not containing the unsaturated residue. The replacement of the C-terminalPhe with A'Phe causes, in all the examined
tripeptides, the loss of any biological activity. On the other hand,
the introduction into the A'Phe containing models of an additional
C-terminal Phe residue leads to the formyltetrapeptidesHCO-XaaLeu-A'Phe-Phe-OMe which show a biological activity very similar
to that exhibited by the corresponding HCO-Xaa-Leu-Phe-OMe
analogues.
HCO-Met-Leu-Phe-OMe (la)
HCO-Met-Leu-A'Phe-OMe (2a)
HCO-Met-Leu-A'Phe-Phe-OMe (3a)
HCO-Hse(Me)-Leu-Phe-OMe (lb)
HCO-Hse(Me)-Leu-A'Phe-OMe (2b)
HCO-Hse(Me)-Leu-A'Phe-Phe-OMe (3b)
HCO-Nle-Leu-Phe-OMe (lc)
HCO-Nle-Leu-A"Phe-OMe (2c)
HCO-Nle-Leu-A'Phe-Phe-OMe (3c)
HCO-Thp-Leu-Phe-OMe (Id)
HCO-Thp-Leu-A'Phe-OMe (2d)
HCO-Thp-Leu-A'Phe-Phe-OMe (3d)
Introduction
Chemotactic N-formylpeptides are involved in defense
mechanisms against bacterial infections through bindin with
specific receptors located on the neutrophil membrane$']. In
addition to the cell directed migration (chemotaxis), this
peptide-receptor interaction also elicits different biochemical
responses, including activation of radical oxygen production
and release of lysosomal enzymes. In the course of continuous studies on the structure-activity relationships in the field
of the analogs of the prototypical formyltripeptide HCO-MetLeu-Phe-OMe (fMLP-OMe), we have recently found that the
replacement of the Phe residue, at the C-terminal position,
with (Z)-2,3-didehydrophenylalanine(A'Phe) causes the loss
of biological activity toward human neutrophils[21.
On the other hand, we have also observed that the presence
of an additional C-terminal Phe residue induces a beneficial
effect on the activity of A'Phe-containing peptides"]. Thus,
the tetrapeptide HCO-Met-Leu-A'Phe-Phe-OMe (3a)L31is
able to stimulate, in contrast with the inactive tripeptides
HCO-Met-Leu-A'Phe-OMe (2a)[21 and HCO-Thp-LeuA'Phe-OMe (2d)L2] (Thp = 4-aminotetrahydrothiopyran-4carboxylic acid) , chemotaxis, superoxide anion generation,
and lysozyme release.
In order to evaluate the significance of the above reported
biochemical behaviour and to gain further information on the
receptor fitting requirements in correspondence of the hydro-
Arch. Pharm. Phurm. Med. Chem.
phobic pocket accommodating the Phe aromatic ring, we
report here the results obtained by comparing the biological
activity of the following four groups of N-formylpeptides:
Each group is constituted by three models possessing the
general formula HCO-Xaa-Leu-Phe-OMe (la-d), HCOXaa-Leu-A'Phe-OMe (2a-d), and HCO-Xaa-Leu-A'PhePhe-OMe (3a-dj. The synthesis of the all-saturated
tripe tides ( l a d [4-71 and of the A'Phe-containing peptides
2a,di381 and 3a7 3 1has been already reported. The new ligands
2b,c and 3b-d have been synthesized during the course of the
present work.
Results and Discussion
Chemistry
The synthesis of the N-formyltripeptides 2b,c was performed according to Scheme 1.
The DCC/HOBt peptide coupling method was used to
obtain Trt-Hse(Me)-Leu-A'Phe-OMe starting from TrtHse(Me)-OH['] and H-Leu-A';Phe-OMe[']. Mixed anhydride
method with isobutyl chloroformate was employed for the
synthesis of Boc-Nle-Leu-A'Phe-OMe starting from BocNle-OH["] and the above dipeptide. The analogs 2b,c were
obtained by treatment of the corresponding Trt- or Boc-peptides with formic acid, followed by ethyl 2-ethoxy- 1,2-dihydro- 1-quinolinecarboxylate (EEDQ).
0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1996
0365-6233/96/0303-0143$5.00 + .25/0
144
Torrini, Zecchini. Paradisi. and Spisani
Trt-Hse(Me)-OH
H-Leu-A'Phe-0Me.TFA
ii
Boc-Leu-A'Phe-OH
J.u
Tn-Hse(Me)-OH
Boc-Nle-Leu-A'Phe-OMe
Jli
A Boc-Leu-A'Phe-Phe-OMc
H-LeuA'Phe-Phe-OMe .TFA
HCO-Nle-Leu-A'Phe-OMe
2b
Tn-Hse(Me)-Leud'Phe-Phe-OMe
2c
DCC, HOBt, NMM,
II
iBuOCOCl NMM,
111
Boc-Xaa-Leu-A'Phe-Phe-OMe
HCOOH
HCO-Hse(Me)-Leu-A'Phe-Phe-OMe HCO-Xaa-Leu-A'Phe-Phe-OMe
3c: Xaa = Nle
3d: Xaa = Thp
3b
The formyltetrapeptides 3b-d were prepared according to
Scheme 2.
Scheme 2. i: OH-, H': ii: iBuOCOCI, H-Phe-OMe.HC1, NMM; iii: TE'A,
CHCI?: iv: DCC, HORt, NMM: v: iBuOCOC1. NMM; vi: HCOOH, EEDQ.
Table 1. Phy\ical and analytical data 01' N-formylpeptides 2b,c and 3h-d.
Formula
lalo
Mp, "C
Crystallization
WIvent"'
Yicldcri
Conipouiid
Solvent"i
-
2h
35 '1
2c
3b
43 hr
3C
82
+91"
188-188 5
M-EA
DMF
M
+128'
DMF
I 59- 159.5
EA
-28"
DMF
159.5-1 60
-50C
213
90
EA-I,P
xs
3d
119-121
4 3 O
M
EA
Solvents: M = methanol; EA = ethyl acetate; DMF = N,N'-dimcthylformaniidc: LP = light
petroleum; C = chloroform. Overall yield from Trt-Hse(Me)-OH. ') C,H, and N analyses were
within ? 0.4%of the theoretical values.
Table 2. Selected spectral data for peptides HCO-Xaa-Leu-A'Phe-OMe (2b,c) and HCO-Xaa-Leu-A'Phe-Phe-OMe (3b-d).
'HNMR (6)'"
Compd
IR (cm-I)
~
H-CO
Xd'l
(YCH
NH
Leu
a-CH
NH
A'Phe
NH
Phe
a-CH
YH
445
8 20 h,
972
3269,3232. 1735.
1672. 1641
9.75
3303,3257, 1713.
1660, 1641
211
802
445
8 29
2c
8.01
4.27 I"
8.18 "
4.46")
8.24
3b
8.07
4.51
7.06
4.38
7.1-7.5
'' 8.19
4.92
7.1-7.5dJ
3422.3247, 1745,
1642
3c
7.93
4.3f1~'
7.54"
4.4jh'
7.0-7.5"d'8.4h
4.84
7.0-7Sd'
3431,3264, 1748.
16.59, 1631
3d
8.09
8.31 ")
4.2')
752b)
4.72
8.31
3276, 1746. 1658
'I)
dl
Boc-Xaa-OH
iv
Jiv
HCO-Hse(Me)-Leu-A'Phe-OMe
I
+
Boc-Leu-A'Phe-OMe
I
Tn-Hse(Me)-Leu-A'Phe-OMe
Scheme 1.
EEDQ
Boc-Me-OH
h'
9.10
[DblDMSO solutions for 2b and 2c: CDCli solutions for 3b-d. h'c' These assignments may be inverted
Superimpo\ecl 011 aromatic signals.
145
Chemotactic Activity of Formylpeptides
Boc-Leu-A'Phe-OMe[*I has been hydrolyzed and coupled
with H-Phe-OMe following the mixed anhydride method to
give Boc-Leu-AZPhe-Phe-OMe. Deprotection of the Boctripeptide, performed with trifluoroacetic acid (TFA)CHC13[71afforded H-Leu-AZPhe-Phe-OMe. The DCC/HOBt
peptide coupling of this key intermediate with Trt-Hse(Me)OH afforded Trt-Hse(Me)-Leu-A'Phe-Phe-OMe, while the
mixed anhydride method was used to obtain Boc-Nle-LeuAZPhe-Phe-OMeand Boc-Thp-Leu-AZPhe-Phe-OMestarting
from Boc-Nle-OH and B0c-Thp-0H[~],respectively. Finally,
usual treatment of Trt- or Boc-tetrapeptides with formic acid
and EEDQ afforded the formyl derivatives 3b-d.
Physical, analytical, and spectral data of N-formylpeptides
2b,c and 3b-d are reported in Tables 1 and 2 .
room to accommodate an additional residue[141.In the present
case the activity of the analogs HCO-Xaa-Leu-AZPhe-PheOMe is never significantly higher than that of the corresponding tripeptides HCO-Xaa-Leu-Phe-OMe. This finding,
together with the observed inactivity of the tripeptides HCOXaa-Leu-AZPhe-OMe, indicates that the presence of the
fourth residue overcomes, at least in part, the unfavourable
effect connected with the orientation of the aromatic ring
bonded at the double bond, which is observed in the unsaturated tripeptide models. This should be the consequence of a
different local conformation adopted by the AZPheresidue in
the tetrapeptide models as well as of an additional binding
interaction established by the C-terminal Phe side-chain with
the hydrophobic area of the receptor.
Biological Activity
Acknowledgement
The biological activity of compounds la-d, 2a-d, and 3a-d
has been determined on human neutrophils. The directed
migration (chemotaxis), superoxide anion production, and
lysozyme release have been measured and the results are
reported in Tables 3-5. A comparison of the biological activity of the formyltripeptides la-d with that of the analogs
2a-d, containing A'Phe in place of Phe as C-terminal residue,
clearly indicates that the introduction of the unsaturated residue causes the suppression of all the responses elicited by the
ligands la-d; the activity of the peptide l c is low but statistically significant (p < 0.05). On the other hand, the tetrapeptides 3a-d containing AZPheand Phe as third and C-terminal
residue, respectively, show a biological activity similar to that
of the tripeptides la-d containing all saturated residues (p =
0.05).It is noteworthy in this context that the formyltetrapeptide 3d maintains the selectivity exhibited by the analogs
containing the Thp residue, namely Id a n d [Thp',
Ain3]fMLP-OMe (Ain = 2-aminoindane-2-carboxylic acid),
previously evidenced by usL7].In fact, the tetrapeptide 3d is
active as chemoattractant and inactive in superoxide anion
production and lysozyme release. As to the inability of the
AZPhe containing tripeptides 2a-d to elicit biochemical responses, we have already discussed the role of the orientation
of the aromatic ring in determining the inactivity of 2dL2].In
this latter analog the aromatic ring is nearly on the same plane
of the backbone N3, C3a, C3' atoms and its orientation differs
significantly from that adopted in the crystal of the tetrapeptide 3a, where a hi h deviation from the plane of the double
bond is observed[']. The biological results reported here
support the hypothesis that the spatial orientation of the
benzylic side-chain at position 3 of the formylpeptides affects
the biolo ical activity of the analogs under study. Recent
findings['-' '1, obtained by studying fMLP-OMe analogs
containing conformationally restricted mimics of phenylalanine, confirm the crucial role of the conformation of the
side-chain phenyl ring of residue 3 in determining the biological activity.
A positive effect on the bioactivity of the incorporation of
a fourth amino acid residue at the C-terminal position of the
chemotactic native ligand fMLP-OMe has been previously
de~cribed['~-'~];
the significant increase of the biological
activity exhibited by the resulting formyltetrapeptides containing the Phe residue at the third position has suggested that
the corresponding hydrophobic receptor area has sufficient
Arch. Phurm. Pharm. Med. Chem. 329,143-148 (1996)
We thank Prof. Gino Lucente for helpful discussion.
Experimental Part
Peptide Synthesis
Melting points: Buchi oil bath apparatus, uncorrected.- Optical rotations:
Schmidt-Haensch Polartronic D polarimeter, 1 dm cell, 20 "C.- IR spectra
(KBr disks): Perkin-Elmer 983 spectrophotometer.- 'H NMR (in CDCI3,
unless otherwise noted): Varian XL-300 spectrometer, TMS int. stand.Column chromdtographies: Merck silica gel 60 (230-400 mesh) (1 :40).PLC: silica gel Merck 60 F254 plates.- Drying agent: Na2S04.- Light
petroleum: 40-60 "C bp fraction.- Elemental analyses: Servizio Microanalisi
del CNR, Area della Ricerca di Roma, Montelibretti, Italy.
The abbreviations used are as follows: Boc, terr-butoxycarbonyl; DCC,
dicyclohexylcarbodiimide; HOBt, 1 -hydroxybenzotriazole; NMM, Nmethylmorpholine; Trt, triphenylmethyl (trityl).
Trt-Hse(Me)-Leu-A'Phe-OMe
To a chilled solution of N-Trt-0-methyl-L-homo~erine'~'
(0.233 g, 0.62
mmol) and HOBt (0.192 g, 1.24 mmol) in dry DMF (1.3 ml) was added DCC
(0.128 g, 0.62 mmol). The mixture was kept at -5 "C for 15 min, at room
temp. for 20 min, and then mixed with a solution of H,Leu-A'PheOMe.TFA'*' (0.25 g, 0.62 mmol) in dry DMF (1.3 ml), neutralized with
NMM, and cooled at -5 "C. The mixture was stirred at room temp. for 6.5 h,
filtered from the precipitate N,h'-dicyclohexylurea, and the solvent evaporated in vucuo. The remaining residue was taken up with ethyl acetate,
washed with 2% aqueous citric acid, water, 5% aqueous NaHC03, water and
dried. The organic layers were evaporated to give the crude title compound
(0.224 g), which was used without further purification for subsequent formylation.
Boc-Nle-Leu-ALPhe-OMe
Isobutyl chloroformate (0.11 ml, 0.78 mmol) was added at -1.5 "C to a
stirred solution of Boc-Nle-OH"ol (0.182 g, 0.78 mmol) and NMM (0.10 ml,
0.93 mmol) in dry CH2C12 (6.4 ml). The temp. was kept at -1 5 "C for 15 min,
then a cold solution of H.Leu-A'Phc-OMe.TFA (0.31.5 g, 0.78 mmol) and
NMM (0.09 ml, 0.78 mmol) in dry DMF (0.8 ml) was added. The mixture
was stirred at room temp. for 24 h, and then evaporated under vacuum. The
residue was dissolved in ethyl acetatc and washed with 5% aqueous KHS04,
water, satd. aqueous NaHC03, and brine. The organic phase was dried and
evaporated to give a residue (0.393g) which was chromatographed on a silica
column. Elution with CHzC12-ether (955) gave 0.318 g (81%) of the title
compound, mp 130-133 "C (EtOAc-light petroleum).- [ a ] D = -63" (c 1.0,
CHCI?).-IR: 3283; 1727; 1688; 1643; 1514cm-'.- 'HNMR: 6=0.80-1.00
[m, YH, (CH3)zCH and Nle E-CH~],1.21-1.87 [m, 18H, (CH?)z-CH-W2,
Nle p-, y-, 6-CHz, and C(CH3)3 (s at 1.40ppm)], 3.80 (s, 3H, COOCH3), 4.05
(m, lH, Nle a-CH), 4.63 (m, IH, Leu a-CH), 5.04 (d, J = 7.5 Hz, lH, Nle
NH), 6.74 (d, J = 7.5 Hz, lH, Leu NH), 7.24-7.50 (m, 6H, aromatic and
146
Torrini. Zecchini, Paradisi. and Spisani
ALPheP-CH), 8 I 1 (\. 1H. A'Phc NH) Anal (CnHiiNi06) (503 6) calcd
C 6 4 4 H 8 21 N 8 3, found C 6 4 2 H 8 32 N 8 2
-
12H, aromatic, Phe NH, and ALPhe P-CH), 8.36 (a, IH. A'Phe NH).-Anal.
( C ~ ~ H ~ S N (680.8)
~ O ~ Scalcd
)
C 63.5 H 7.1 1 N 8.2: found C 63.3 H 7.46 N
8.0.
Hoe-Lei~~A'Phc.-Phr-OMe
Isobutyl chloroformate (0.74 inl. 5.4 mmol) war added at -15 "C to a stirred
solutioii of the dipeptide Boc-Leu-A'Phe-OH (2.304 g, 5.4 mmol), obtained
by alkaline hydrolysisi7' for one day of the corresponding methyl ester'", in
dry DMF (10.8 ml) containing NMM (0.713 ml. 6.48 mmol). The tcmp. was
kept at -15 "C for 15 min , thcn a chillcd solution of H-Phe-OMe.HCI
( I . 164 g. 5.4 mmol) and NMM (0.59 inl, 5.4 minol) in dry DMF (10.8 ml)
was added. The mixture was stirred at room temp. for 24 h, and then
evaporated under vacuum. The residue was dissolved in ethyl acetate and
washed with 5% aqueous KHSOI. water, satd. aqueous NaHCO3, and brine.
The organic phase was dried and evaporated to givc the title compound in
quantitative yield, nip 149.5-150 "C (EtOAc-n-hexme).- Lalo=+lo" (c 1 .O.
CHCI?).- IR : 3357; 3231; 1747: 1721: 1658 cm-'.- 'H NMR: 6 = 0.94
[apparent t. hH. (CH>)KHI, 1.43 1s. 9H, C(CH?)?I, 1.45-1.77 [m. 3H,
(CH3)2-CN-CH2], 3.20 (m, 2H, Phc P-CHz), 3.70 (s. 3H, COOCH?). 4.16
(in, I H, LCUa-CH), 4.92 (m, 2H, Phe a-CH and Leu NH), 6.90 (unresolved
d, I H, Phe NH). 7.13-7.46 (m, 6H. aromatic and A'Phe P-CH), 7.70 (s, 1 H.
A'Phe NH).-Anal. (C~OH~CIN~O(>)
(573.6) calcd C 67.0 H 7.31 N 7.8: found
C 67.3 H 7.24 N 7.8.
H-Lrl,-A'Phr-Phr.-OMe. TFA
Boc-Leu-A'Phe-Phe-OMe (0.887 g, 1.65 mmol) wah dissolved in a mixture
of TFA and dry CHCli (I: I , 2 ml) and stirred at room temp. for 4 h. The
organic solvent was removed under reduced pressure and the foamy residuc
(0.91 g), obtained by treatment with dry ether, was used without further
puri lication.
T~t-H.sr~(Me)-Lcri-A'Plif~-P
he-OMr
The tetrapeptide Trt-Hse(Me)-Leu-A'Phe-Phe-OMe was prepared following the same procedure used to obtain Trt-Hse(Me)-Lcu-ALPhe-OMestarting
from Trt-Hse(Me)-OH (0.201 g, 0.536 mmol) and an equimolar ainount of
H.Leu-A'Phe-Phe-OMeTFA. Usual workup afforded the crude title compound which was then convertcd in the corresponding N-forinyl derivative
without further purification.
Boc.-N/e-Lc~ri-A'Phr-Phe-OMe
The title Boc-protected tetrapeptide was prepared following thc aamc
procedure adopted to obtain the tripeptide Boc-Nle-Leu-A'Phe-OMe starting
from Boc-Nle-OH (0.208 g, 0.9 mmol) and H-Leu-ALPhe-Phe-OMe.TFA
(0.496 g, 0.9 mmol). Usual workup affordcd a residue which was chromatographed on a silica column. Elution with CHKlz-ether (9: 1 ) gave 0.375
(64%) of the title compound, mp 129-132 "C (EtOAc-light petroleum).[aln=-32"(c 1,0,CHCI~).-IR:3310:3287: 1726: 1687; 1644: 1526cm-'.'H IVMR: 6= 0.80-1.00 [m, 9H, (CH1)zCH and Nle E-CH~],1.25-1.5J2 [in,
I8H. (CH3)z-CH-CHz. Nle [3-, y.6-CH2, and C(CH?)j(sat I .35 ppm)],3.20
(m, 2H, Phe P-CH?), 3.70 (s. 3H, COOCHI), 3.89 (in, IH. Nle a-CH'I, 4.37
(m, IH, Leu a-CH), 4.92 (m,2H, Phe a-CH and Nle NH), 6.62 (d. J = 5.5
Hz.lH,LeuNH),7.11 (d.J=7Hz,I€I,PheNH),7.18-7.45(m,IIH.
aromatic and A'Phe P-CH), 8.01 (s, IH, ALPheNH).- Anal. (C16HxiN407)
(650.8) calcd C 66.4 H 7.73 N 8.6; found C 66.3 H 7.82 N 8.4.
Genrrcil Proceduwfor the Syn/hesis ofFor.iii!.l/)ei?.i,litle\2b,c (ind 3h-d
The N-Trt- or N-Boc-protected peptide (1 mmol) was dissolved in 98%
formic acid (4 ml) and the mixture was stirred at room temp. for 24 h. After
, residue was dissolved in
removal of the excess of formic acid in ~ x m mthe
dry chloroform (4 ml) and EEDQ ( I .2 mmol) wa\ added. The solution was
stirred at room temp. for 5 h and the product was precipitated by n-hcxane.
The solid obtained was washed several times with ether and dried to give the
pure N-formyl derivatives 2b, 212, and 3c or the nearly homogeneous derivatives 3b and 3d. The tetrapeptides 3b and 3d were further purified by P I X
using CH2ChMeOH (955) and EtOAc-n-hexane (9: 1 ) as eluant, respectively.
Hiolojircoi Assuj
Ce1l.s
Human peripheral blood neutrophils were purified cmploying the standard
techniques of dextran (Pharmacia) sedimentation. centrifugation on FicollPaque (Pharmacia), and hypotonic lysis of red cells. The cells were washed
twice and resuspended in KRPG (Krebs-Ringer phosphate containing 0. I%,
$r/v glucose, pH 7.4) at a concentration of 50x10' cells/ml. The percentage
of neutrophils was 98-100'10 pure.
Rundom L,ocmnotion
Random locomotion was performed with 48-well microchemotaxis chamber (Bio Probe, Italy) and thc migration into the filter was evaluated by the
method of leading-front'17'.The actual control random movement is 32 p m
k 3 SE of ten separate experiments done in duplicate.
Cheiizotaxis
In order to study the potential chemotactic activity, each peptide was added
to the lower compartment of the chemotaxis chamber. Peptides were diluted
from a stock solution (lo-' M in DMSO) with KRPG containing I mg/ml of
bovine wrum albumin (Orha Behringwerke, FKG) and used at concentrations
ranging from 10-"M to 1 O-s M. Data were expressed in terms ofchemotactic
index, which is the ratio: (migration toward test attractant minus migration
toward the buffer)/migration toward the buffer; the reported values are the
mean of six separate experiments done in duplicate. Standard errors are in
the 0.02-0.09 chemotactic index range (Table 3 ) .
Supetno-yidcAnion
(02-J
Production
0 2 - release was monitored continuously in a therinostattcd spectrophotometer as superoxidc dismutase-inhibitable reduction of ferricytochrome c (Sigma, USA), as described elsewhere"X'. At zero time, different
amounts (lo-' - 2 x lo-' M) of each peptide wcre added and ahsorhance
change accompanying cytochrome c reduction was monitored at 550 nm.
Neutrophils were incubated with 5 @in1 cytochalasin B (Sigma) for 5 min
prior to activation by peptides. Rcsults were expressed as net nmoles of
O2-D x 10' cells/5 min and are the mean of six separate experiments donc
in duplicate. Standard errors are in 0 . 1 4 nmoles 0 2 - rangc (Table 4).
B~~~-T~II)-LEM-A'P~~,-I'IIF-OMP
Eniyine ASSUJ
The title compound was synthesized in 55% yield following the same
procedure and chromatographic purification described for Boc-Nle-Leurriiig from B0c-Thp-0H'~'(0.78 mmol) and an equimolar ainount of H-Leu-ALPhe-Phe-OMe.TFA, mp 170-1 74 " C (dcc.)
(EtOAc-light petrolcum).- [ a111
= +6" ( c 1 .O, CHCh).- IR: 3262; 1746: 1658:
1625: 1536 ciii-'.- 'H NMR: 6 = 0.92 and 0.96 [two d, J = 6.5 Hz. 6H,
(CH3)2CH], I .24 [ s,9H, C(CH3)?], 1.50-2.80 Lm. I 1 H, ( C H 8 ) K H - C H z and
two CHZ-CHz-S], 3.19 and 3.24 ( A and B of ail ABX, J = 6. 6.5, and 14 Hz,
2H. Phe P-CH2). 3.70 (s, 3H. COOCH1). 4.38 (in,IH, Leu a-CH), 4.94 (m.
2H. Phe a-CH and Thp NH), 6.65 (d. J = 6 Hz. 1 H, Leu NH). 7.16-7.50 (m.
Release of neutro hi1 granule enzymes was evaluated by determining
lysoryme activit$Ix[ this was quantified ncphelometrically by the rate of
lysis of cell wall suspension of Micrc~coccirs!,.vocfeikticus (Sigma). Enzyme
release was expressed as a net percentage of total enzyme content released
by0,l"rTritonX-100.Totalenzyineactivitywas
85+1 pg/l x lO'cells/min.
To study the degranulation-inducin& activity of each peptide, neutrophils
were first incubatcd with cytochalasin B for 15 min at 37 "C and then in the
2 x 10--' M for a
prescncc of each peptide in a final concentration of
further 15 min. The value\ are the mean of five scparate experiments done
in duplicate. Standard errors are in 1-68 range (Table 5 ) .
-
147
Chemotactic Activity of Formylpeptides
Table 3. Chemotactic activity of formylpeptides la-d, 2 a 4 , and 3a-d.
Formylpeptide
Chemotactic index
-12
-1 1
-10
-9
-8
-7
-6
-5
la
2a
3a
0.50
0.01
0.30
0.90
0.03
0.58
1.05
0.10
0.58
1.15
0.12
0.70
1.os
0.34
0.4.5
0.85
0.38
0.44
0.62
0.29
0.13
0.36
0.08
0.05
lb
2b
3b
1.oo
0.25
0.84
1.20
0.26
0.86
1.15
0.28
0.96
1.10
0.28
1.02
0.91
0.25
0.95
0.65
0.23
0.57
0.20
0.20
0.43
0.10
0.20
0.29
lc
2c
3c
0.17
0
0.35
0.36
0
0.40
0.43
0
0.47
0.55
0
0.50
0.58
0
0.60
0.40
0
0.62
0.20
0
0.30
0
0
0.30
Id
2d
3d
0.50
0.80
0.30
0.81
1.02
0.34
0.83
1.10
0.17
0.86
1.10
0.13
0.41
0.82
0.13
0.41
0.31
0.13
0.21
0.27
0.19
0.26
bdM1
0.20
0.56
Table 4. Superoxide anion production activated by formylpeptides la-d, 2 a 4 , and 3a-d.
Formylpeptide
nmoles 0 2 -9
-8
-7
-6
-5
4.7
la
2a
3a
3
2
27
7
2
38
44
3
51
43
3
45
37
3
33
20
2
25
lb
2b
3b
3
0
1
6
0
15
10
0
35
38
0
47
53
16
38
30
20
33
lc
2c
3c
0
0
1
0
0
2
10
0
12
26
0
16
25
0
18
20
0
15
Id
2d
3d
3
3
0
4
2
0
4
2
0
5
2
0
4
2
0
2
2
0
WMI
Table 5. Lysozyme release stimulated by formylpeptides la-d, 2 a 4 , and 3a-d
Formylpeptide
% Lysozyme release
-9
-8
-7
-6
-5
-4.7
la
2a
3a
35
6
20
54
10
48
59
10
57
67
20
57
62
32
54
48
23
20
lb
2b
3b
18
0
29
26
7
36
36
4
45
55
10
46
53
25
52
40
29
46
lc
2c
3c
4
5
8
14
3
15
22
4
20
30
4
25
28
5
21
27
7
15
Id
2d
3d
3
3
2
7
6
3
7
7
3
8
6
3
9
6
5
5
4
5
10g[M]
Arch. Phann. Pharm.Med. Cheni. 329,143-148 (1996)
Torrini. Zecchini, Paradiai, and Spisani
Stutis I i c . d Anal?.\ I.\
The nonp;irametric Wilcoxon test was used in the \tatistical evaluation of
differences between Zroupa.
References
P.M. Murphy, Annu. Rev. Iwnnunol. 1994, 12, 593-633.
191 K. Rarlos. D. Papaioannou, P. Cordopatis, D. Theodoropoulos, Tetruhcdmn 1983, 39, 475478.
[lo] G.M. Bonora, C. Toniolo, Mukr-omol. C‘11em 1978, 179, 1453-1463.
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Reeker, S. Polinelli. W.H.J. Boesten, H.E. Shoemaker, E.M. Meijer, J.
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Riopolwzerr 1994, 34, 1291-I 302.
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Gavuz70, F. Marza. G. Pochetti, S. Spisani, Tetruliedron 1993, 49,
489-496.
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Peptidr Prorrin Rex 1987, 20, 525-532.
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Z. 1’hy.siol. Clzenr. 1981, 362. 163-174.
I. Torrini, G. Pagani Zecchini, M. Paglialunga Paradisi, G. Lucente. E.
Gavuzro, F. M a u a , G. Pochetti. S. Spisani, A.L. Giuliani, I t i t . J.
Pc.,oritfe Prorein Rex 1991, 38, 495-504.
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[I41 R.J. Freer, A.R. Day, N. Muthukumaraswamy, D. Pinon, A. Wu, H.J.
Showell, E.L. Becker, Riochetni.srq 1982, 21, 257-263.
[ 151 P.A. Kaj, A.R. Dentino. Z. Situ, M.J. Levine, Ahsrructs of Ptrpers
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Annual Meeting of American Crystallographic Association. Univerqity
of Pittsburgh, 1992, Abstract PA19.
j l h ] A. Rot,L.E. Henderson,T.D. Cope1and.E.J. Leonard. Proc. Not/. A c d .
Sci. USA 1987,84.7967-797 I.
1171 S.H. Zigmond, J.G. Hirsch. .I. Exp. Mrd. 1973, 137. 387-410.
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R. Gavioli. S. Traniello, Inflnmniutiori 1992, 16, 147-158.
Received: October 30, 1995 [FP069]
Ardr. P/zat-nz. Phumi. Med. Cheni. 329, 143-148 (1996)
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determinism, residue, phenylalanine, terminal, activity, role, chemotactic, didehydrophenylalanine, formylpeptides, peptide, analogues
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