Peptide bond mimicry by (E)-alkene and (Z)-fluoroalkene peptide isosteres: Synthesis and bioevaluation of α-helical anti-HIV peptide analogues

Article abstract of DOI:10.1039/b907983a

The α-helix structures of the anti-HIV fusion inhibitory peptides are stabilized by the amino acid sequence and by intrachain hydrogen bonds. The study of peptide analogues using (E)-alkene and (Z)-fluoroalkene dipeptide isosteres demonstrated the substan

Full text of DOI:10.1039/b907983a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Peptide bond mimicry by (E)-alkene and (Z)-fluoroalkene peptide isosteres:
synthesis and bioevaluation of a-helical anti-HIV peptide analogues†
Shinya Oishi,*a Hirotaka Kamitani,a Yasuyo Kodera,a Kentaro Watanabe,a Kazuya Kobayashi,a Tetsuo Narumi,a
Kenji Tomita,a Hiroaki Ohno,a Takeshi Naito,b Eiichi Kodama,b Masao Matsuokab and Nobutaka Fujii*a
Received 22nd April 2009, Accepted 30th April 2009
First published as an Advance Article on the web 4th June 2009
DOI: 10.1039/b907983a
The a-helix structures of the anti-HIV fusion inhibitory peptides are stabilized by the amino acid
sequence and by intrachain hydrogen bonds. The study of peptide analogues using (E)-alkene and
(Z)-fluoroalkene dipeptide isosteres demonstrated the substantial, yet position-dependent, contribution
of hydrogen bonds to the a-helix stability and anti-HIV bioactivity.
dipeptide in four repeat motifs (Fig. 1).6 Two potential H-bonds
may be missing when replacing the peptide bond in Lys-Lys with
Introduction
The a-helix represents one of the largest classes of secondary
=
the olefin congeners: (1) between the C O of the first Lys (i) and
structure elements found in protein and peptide structures.1 The
the N–H of the downward Glu (i+4), (2) between the N–H of the
cylindrical structures are stabilized by intrachain hydrogen bond
(H-bond) networks which are formed between the C O of residue
i and the amide N–H of the i+4 residue to generate 13-membered
pseudocyclic structures. The functional and/or interactive sur-
face(s) of the a-helix are revealed by the arrangement of the
distribution residues in the linear sequence upon folding.
=
second Lys (i+1) and the C O of the upward Glu (i-3) (Fig. 1).
=
In the case of FADI substitution, the presence of the first H-bond
was expected, because of the potential ability of a fluorine atom
to act as a H-bond acceptor.7
In order to stabilize the a-helix structure of bioactive peptides,
there are two possible approaches: (1) bridging side-chains by
covalent or non-covalent bond(s) or (2) mimicking intrachain
H-bond(s).2 Recently, we reported a novel design concept of fusion
inhibitory peptides active against HIV-1 by utilizing an X-EE-
XX-KK motif (X: original residue; E: glutamic acid; K: lysine).3,4
This motif contributes to the stabilization of the bioactive a-helix
conformation by forming two potential salt bridges between Glu
and Lys side-chains without altering the location of residues that
form the interactive surface with the viral protein gp41.4c The
peptides, named SC35EK and T-20EK, exhibit highly potent anti-
HIV activity by inhibition of the rearrangement of HIV-1 gp41 that
facilitates fusion between the host cellular and viral membranes.
In addition, a structure–activity relationship study identified a
novel amphiphilic peptide, SC29EK, with a minimal sequence for
anti-HIV activity.5 In light of its high potency of SC29EK, it
was of interest to estimate the effect of intrachain H-bond(s) on
a-helix stabilization in the presence of the X-EE-XX-KK motifs.
Accordingly, efforts herein have been undertaken to comparatively
evaluate the anti-HIV activity and biophysical properties of
SC29EK analogues containing peptide bond mimetics.
Fig. 1 Structures of (E)-alkene and (Z)-fluoroalkene dipeptide isosteres
and the potential mimicry of H-bonds stabilizing the a-helix structure.
Results and discussion
Lys-Lys EADI8 and FADI9 were prepared by the established
procedures shown in Schemes 1 and 2, respectively. Briefly, allyl
alcohol 310 derived from a protected amino acid was converted into
Ns-amide 4. Aziridination of 4 by the Mitsunobu reaction followed
by C-1 elongation afforded the b-aziridinyl-a,b-unsaturated ester
6. Organocopper-mediated alkylation of 6 provided an a-alkyl
adduct 7 regio- and stereoselectively. Subsequent functional group
manipulations generated the expected Fmoc-protected EADI 10.
FADI synthesis began with mono-TBS-protected 1,5-
pentanediol 11. Rh-catalyzed Reformatsky–Honda reaction11
of the corresponding aldehyde gave a,a-difluoro-b-amino es-
ter 12. The simultaneous hydrogenolysis and Boc protection
followed by C-2 elongation using the Horner–Wadsworth–
Emmons reaction produced a key g,g-difluoro-a,b-enoyl sultam
14. One-pot reduction/asymmetric alkylation via transmetalation
with allyl bromide formed the FADI scaffold 15. Selective
(E)-Alkene dipeptide isostere (EADI) 1 and (Z)-fluoroalkene
dipeptide isostere (FADI) 2 of Lys-Lys were chosen as planar
peptide bond surrogates for positional scanning of each Lys-Lys
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-
ku, Kyoto, 606-8501, Japan. E-mail: soishi@pharm.kyoto-u.ac.jp, nfujii@
pharm.kyoto-u.ac.jp; Fax: +81-75-753-4570; Tel: +81-75-753-4551
bLaboratory of Virus Control, Institute for Virus Research, Kyoto University,
Sakyo-ku, Kyoto, 606-8507, Japan
† Electronic supplementary information (ESI) available: Additional ex-
perimental procedures, NMR spectra and HPLC charts. See DOI:
10.1039/b907983a
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Scheme 1 Synthesis of the Lys-Lys-type alkene dipeptide isostere. Reagents and conditions: (a) 4 N HCl/dioxane; (b) NsCl, 2,4,6-collidine, CHCl3, 65%
(2 steps); (c) DIAD, PPh3, THF/toluene, 0 ◦C, 84%; (d) O3, AcOEt, -78 ◦C, then Me2S; (e) (EtO)2P(O)CH2CO2t-Bu, LiCl, DIEA, CH3CN, 0 ◦C, 46%
(2 steps); (f) TBSO(CH2)4I, t-BuLi, CuCN, LiCl, n-pentane/Et2O/THF, -78 ◦C, 60%; (g) H2SiF6 aq., CH3CN/CH3OH, 0 ◦C; (h) CbzNHNs, PPh3,
DIAD, THF/toluene, 81% (2 steps); (i) PhSH, K2CO3, DMF; (j) Fmoc-OSu, Et3N, DMF, 84% (2 steps); (k) 4 N HCl/dioxane, 96%.
Scheme 2 Synthesis of the Lys-Lys-type fluoroalkene dipeptide isostere. Reagents and conditions: (a) DMSO, (COCl)2, Et3N, CH2Cl2, -78 ◦C;
(b) (R)-2-methoxy-1-phenylethylamine, 3 A MS, THF, 0 ◦C; (c) BrCF2CO2Et, RhCl(PPh3)3, Et2Zn, 0 ◦C, 43% (3 steps); (d) Pd(OH)2, H2, Boc2O,
˚
EtOH, 87%; (e) DIBAL-H, CH2Cl2/toluene -78 ◦C; (f) (EtO)2P(O)CH2COXs, LiCl, DIEA, CH3CN, 0 ◦C, 87% (2 steps); (g) Me2CuLi·LiI, THF/Et2O,
◦
◦
◦
◦
=
-78 C, then HMPA, then Ph3SnCl, -78 C to -40 C, then BrCH2–(E)-CH CH–CH2OTBS, -40 C, 78%; (h) 4.5% Pd/C(en), EtOH, H2; (i) aq. H2SiF6,
CH3CN/CH3OH, 78% (2 steps); (j) TsCl, Et3N, CH2Cl2; (k) NaN3, DMF; (l) PPh3, THF/H2O; (m) Cbz-OSu, Et3N, DMF, 65% (4 steps); (n) 1 N LiOH,
50% H2O2, THF/H2O; (o) TFA, CH2Cl2; (p) Fmoc-OSu, Et3N, MeCN/DMF/H2O, 64% (3 steps).
hydrogenation in the presence of Pd/C(en)12 and step-wise mod-
ifications afforded the Fmoc-protected FADI 19. The resulting
isosteres 10 and 19 were incorporated into the KK dipeptide of
SC29EK sequence by standard Fmoc-based solid-phase peptide
synthesis.
Anti-HIV activities of the isostere-containing peptides
20E–23E and 20F–23F were examined using the MAGI assay
(Table 1). Substitutions of the first and second N-terminal
Lys-Lys dipeptides with EADI (20E and 21E) resulted in the
loss of the anti-HIV activity (EC50 >10 mM). In contrast, the
FADI congeners exhibited weak or moderate anti-HIV activities
(20F: EC50 = 5.2 mM; 21F: EC50 = 599 nM). Both EADI and
FADI analogues with substitution at the third Lys-Lys showed
slightly lower anti-HIV potency than wild-type C295 without the
a-helix inducible XEEXXKK motifs (22E: EC50 = 865 nM; 22F:
EC50 = 663 nM). The best peptide analogues were obtained by
replacement of the C-terminal Lys-Lys with the isosteres (23E:
EC50 = 43 nM; 23F: EC50 = 37 nM); however, the potency was
lower than the original SC29EK peptide (EC50 = 2.2 nM). Similar
bioactivities of peptide 20E–23E and 20F–23F were also observed
against the other HIV-1 strains (Table 2). These observations
suggest that all the peptide bonds within the Lys-Lys and the
related H-bonding are essential for the potent anti-HIV activity of
SC29EK.
The a-helix properties of these peptides were determined by
circular dichroism (CD) analysis (Fig. 2a,b). The stable a-helix
Table 1 Sequences and anti-HIV activities of C29 and its derivatives and Tm values of the mixture with N36
EADI analogues E
FADI analogues F
Sequencea
EC50 (nM)b
Tm (◦C)c
EC50 (nM)b
Tm (◦C)c
WMEWDREINNYTSLIHSLIEESQNQQEKN C29
WEEWDKKIEEYTKKIEELIKKSEEQQKKN SC29EK
WEEWDKKIEEYTKKIEELIKKSEEQQKKN 20E/20F
308 144
2.2 0.2
>10000
>10000
865 317
51.7
67.4
43.9
40.1
62.2
64.1
—
—
—
—
44.1
49.5
60.9
64.8
5220 202
599 96
663 242
¯ ¯
WEEWDKKIEEYTKKIEELIKKSEEQQKKN 21E/21F
¯ ¯
WEEWDKKIEEYTKKIEELIKKSEEQQKKN 22E/22F
¯ ¯
WEEWDKKIEEYTKKIEELIKKSEEQQKKN 23E/23F
43
7
37
6
¯ ¯
a The underlined KK dipeptide indicates the position of the dipeptide isostere. b EC50 was determined as the concentration that blocked HIV-1 (NL4–3
strain) replication by 50%. c Tm values were defined by the midpoint of the thermal unfolding transition state as determined from [q]222 readings.
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Table 2 Anti-HIV activities of C29 and its derivatives against three HIV-1
strains
exert their anti-HIV activity by inhibiting the folding process of
the HIV-1 envelop protein gp41.
Binding affinity of SC29EK analogues to a viral protein was
determined by the thermal stability of the six-helix complexes
formed between SC29EK and N36 peptides. The melting temper-
ature (Tm), representing 50% disruption of the six-helix bundle,
was comparatively evaluated by monitoring the change in the
circular dichroism signal at 222 nm as a function of increasing
temperature (Table 1). The complexes involving peptides 20E/20F
and 21E/21F showed significantly lower thermal stability, which
correlates with the observed absent or low anti-HIV activities
of these peptides. In contrast, potent analogues 23E/23F form
stable complexes with N36 with Tm values comparable to the value
measured for SC29EK (23E: Tm = 64.1 ◦C; 23F: Tm = 64.8 ◦C).
The N-terminal tryptophan-rich domain (WRD) of inhibitory
peptides such as C34 is essential for binding to the cavity formed by
the N36 coiled-coil.11 H-Bonds linked by the first and second Lys-
Lys peptide bonds in SC29EK would reinforce the arrangement of
these tryptophans. Interestingly, less potent anti-HIV activity of
peptide 22E/22F was observed compared with C29, whereas the
complexes with N36 showed higher thermal stability. This result
suggests that the loss of crucial H-bonds could reduce the anti-
HIV activity, even though the X-EE-XX-KK motifs apparently
aid the conformational stability of the six-helix bundle.
EC50 (nM)a
Peptides
NL4–3
IIIB
396 83
6.5 0.9
>10000
>10000
>10000
3010 554
5110 2,750
2200 712
153 27
Ba-L
42
1.9 0.2
>10000
5580 1920
>10000
600 302
2630 386
527 95
C29
SC29EK
20E
308 144
2.2 0.2
>10000
5220 202
>10000
599 96
865 317
663 242
8
20F
21E
21F
22E
22F
23E
37
43
6
7
33
51
2
7
23F
237 16
a EC50 is the concentration that blocks HIV-1 replication by 50%.
structure of SC29EK was disrupted by a single substitution of
the second or third Lys-Lys peptide bond with the isosteres in
21E/21F and 22E/22F. This suggests that the contribution of the
H-bonds to the stability of the a-helix is likely to be superior
to the multiple introductions of the X-EE-XX-KK motifs at
these positions. Conversely, the effects of N- and C-terminal
substitution were less significant as observed in 20E/20F and
23E/23F. This may be rationalized by the fact that these peptide
bonds of SC29EK are positioned at the edge of the helix and
that C-terminal Lys-Lys is involved in only upward H-bonding
through the donor N–H moiety. CD spectra of SC29EK analogues
in the presence of an interactive counterpart N36 indicated the
formation of stable six-helix coiled-coil structures (Fig. 2c,d).13
This observation supports the concept that SC29EK analogues
In terms of the mimicking ability of the two-peptide-bond
isosteres, FADI peptides 20F–23F exhibited slightly more potent
anti-HIV activity and formed more stable complexes with N36
(except for 22F). Although a fluoroalkene with a large dipole
moment imperfectly reproduces the H-bonds needed for a-helix
stabilization, FADI is an appropriate peptide bond surrogate to
investigate structural requirements in bioactive peptides.
Fig. 2 CD spectra of EADI- and FADI-containing SC29EK analogues in the absence (a,b) and presence (c,d) of N36.
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3.21 (4H, m), 4.01–4.21 (2H, m), 4.30–4.54 (2H, m), 4.81–5.26
(6H, m), 5.30–5.81 (3H, m), 7.19–7.41 (13H, m), 7.52–7.59 (2H,
m), 7.74 (2H, d, J 7.5); dC (125 MHz; CDCl3) 22.5, 24.0, 29.2, 29.3,
31.5, 34.3, 40.6 (2C), 47.1, 48.4, 52.3, 63.7, 66.4 (2C), 119.8 (2C),
124.9 (2C), 126.7 (2C), 126.9 (2C), 127.5 (2C), 127.7, 127.9, 128.3,
129.2 (2C), 129.3 (2C), 129.5, 133.3, 134.2, 136.5, 141.1, 143.7
(2C), 143.8 (2C), 155.8, 156.3, 156.4, 178.0; m/z (FAB) 782.3201
([M + H]+, C44H49ClN3O8 requires 782.3208).
Conclusions
The effects of H-bonds on the stability of the a-helix of an HIV-1
fusion inhibitor were investigated by positional-scanning of the
Lys-Lys dipeptides using EADI and FADI. As demonstrated by
CD analysis of the SC29EK analogues, H-bonds in the middle
of the sequence contribute significantly to the stabilization of the
a-helix. In contrast, the effect of H-bonds on the anti-HIV activity
of the peptides depends on the distance from the crucial interactive
domain. As such, we have shown that EADI and FADI can be
used for conformational evaluation of bioactive and/or functional
a-helical peptides.
(2R,5S,3Z)-5-[N-(tert-Butoxycarbonyl)amino]-2-[(E)-4-(tert-
butyldimethylsiloxy)but-2-enyl]-9-(tert-butyldimethylsiloxy)-4-
fluoronon-3-enoyl (S)-sultam (15). To a suspension of CuI
(180 mg, 0.94 mmol) in THF (4.8 cm3) at -78 ◦C under argon was
added dropwise a solution of MeLi·LiBr complex in Et2O (1.5 M,
1.3 cm3, 1.89 mmol), and the mixture was stirred for 10 min at
0 ◦C. To the solution of the above organocopper reagent at -78 ◦C
was added dropwise a solution of the N-enoyl sultam 14 (150 mg,
0.24 mmol) in THF (4.8 cm3). The mixture was stirred for 30 min
at -78 ◦C and HMPA (0.66 cm3, 3.78 mmol) was added dropwise
to the mixture. After stirring for 30 min at -78 ◦C, a solution
of triphenyltin chloride (182 mg, 0.47 mmol) in THF (3.0 cm3)
was added dropwise, and the mixture was then stirred for 30 min
Experimental section
Synthesis
tert-Butyl (2R,5S,3E)-2-[4-(tert-butyldimethylsiloxy)butyl]-9-
[N -(2-chlorobenzyloxycarbonyl)amino]-5-[N -(2-nitrophenylsul-
fonyl)amino]non-3-enoate (7). To a stirred solution of TBSO-
(CH2)4I (236 mg, 0.75 mmol) in dry Et2O (0.5 cm3), was added
dropwise 1.59 M t-BuLi in Et2O solution (1.0 cm3, 1.58 mmol)
under Ar at -78 ◦C. After being stirred at this temperature
for 30 min, the mixture was stirred at 0 ◦C for 30 min. To a
stirred solution of CuCN (61 mg, 0.61 mmol) and LiCl (52 mg,
1.23 mmol) in dry THF (0.8 cm3), was added dropwise the
above 0.5 ◦M TBSO(CH2)4Li in THF solution◦(1.2 cm3) under
Ar at -78 C, and the mixture was stirred at 0 C for 10 min. A
solution of aziridinyl enoate 6 (91 mg, 0.15 mmol) in dry THF
(1.0 cm3) was added dropwise to the above mixture at -78 ◦C
with stirring, and the stirring was continued for 1.5 h followed
by quenching with saturated NH4Cl/28% NH4OH solution (1/1,
2.0 cm3). The mixture was washed with H2O and brine and dried
over MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexane-EtOAc (3:1)
◦
at -40 C. (E)-(4-Bromobut-2-enyloxy)(tert-butyl)dimethylsilane
(501 mg, 1.89 mmol) in THF (3.0 cm3) was added dropwise and the
mixture was stirred for 20 h at -40 ◦C. The reaction was quenched
at -40 ◦C by addition of a saturated NH4Cl/28% NH4OH
solution (1/1, 6.0 cm3) and the mixture was stirred at room
temperature for additional 30 min. The mixture was extracted
with Et2O and the extract was washed with brine and dried over
MgSO4. Concentration under reduced pressure followed by flash
chromatography over silica gel with n-hexane-EtOAc (5:1) gave
the title compound 15 (148 mg, 78% yield) as a colorless oil; [a]25
D
-47.1 (c 1.00 in CHCl3); nmax/cm-1 3317 (NHCO), 1693 (CO); dH
(500 MHz; CDCl3) 0.03 (6H, s), 0.04 (6H, s), 0.88 (9H, s), 0.89 (9H,
s), 0.96 (3H, s), 1.15 (3H, s), 1.24–1.64 (17H, m), 1.83–1.91 (3H,
m), 2.02–2.05 (2H, m), 2.33–2.37 (1H, m), 2.51–2.55 (1H, m), 3.41
(1H, d, J 13.7), 3.49 (1H, d, J 13.7), 3.58 (2H, t, J 6.3), 3.86 (1H,
t, J 6.3), 4.06 (2H, d, J 3.4), 4.12–4.21 (2H, m), 4.60–4.72 (1H, m),
4.97 (1H, dd, J 36.7 and 8.6), 5.58 (2H, m); dC (125 MHz; CDCl3)
-5.3 (4C), 18.2, 18.3, 19.8, 20.7, 21.9, 25.9 (6C), 26.4, 28.3 (3C),
32.2, 32.3, 32.8, 36.9, 38.3, 41.0, 44.6, 47.6, 48.2, 51.6 (d, J 27.6),
53.0, 62.8, 63.5, 65.1, 79.4, 103.3 (d, J 12.0), 125.9, 132.7, 154.8,
158.6 (d, J 261.5) 172.2; dF (470 MHz; CDCl3) -119.1–-119.8
(m); m/z (FAB) 801.4732 ([M + H]+, C40H74FN2O7SSi2 requires
801.4739).
gave the title compound 7 (72 mg, 60%) as a colorless oil; [a]23
D
-73.2 (c 0.87 in CHCl3); nmax/cm-1 3349 (NHCO), 1725 (CO); dH
(500 MHz; CDCl3) 0.04 (6H, s), 0.89 (9H, s), 1.08–1.64 (21H,
m), 2.64 (1H, dt, J 8.0 and 6.3), 3.07–3.21 (2H, m), 3.55 (2H, t,
J 6.3), 3.86–3.96 (1H, m), 4.92 (1H, br s), 5.21 (2H, s), 5.26 (1H,
dd, J 15.5 and 7.5), 5.39 (1H, d, J 8.0), 5.40 (1H, dd, J 15.5 and
8.0), 7.22–7.30 (2H, m), 7.37 (1H, dd, J 5.7 and 2.3), 7.42 (1H,
dd, J 5.7 and 2.3), 7.65–7.74 (2H, m), 7.83 (1H, dd, J 6.9 and
2.3), 8.09 (1H, dd, J 6.9 and 2.3); dC (100 MHz; CDCl3) -5.3
(2C), 18.3, 22.5, 23.3, 25.9, 28.0 (3C), 29.3 (3C), 32.3, 32.6, 35.4,
40.6, 49.3, 56.7, 62.8, 63.9, 80.6, 125.3, 126.9, 129.3, 129.5, 129.8,
130.9 (2C), 131.2, 132.8, 133.3, 133.5, 134.3, 135.1, 147.8, 156.2,
172.8; m/z (FAB) 782.3246 ([M + H]+, C37H57ClN3O9SSi requires
782.3273).
(2R,5S,3Z)-2-{4-[N -(Benzyloxycarbonyl)amino]butyl}-9-[N -
(benzyloxycarbonyl)amino]-5-[N -(9-fluorenylmethoxycarbonyl)-
amino]-4-fluoronon-3-enoic acid (19). To a solution of the sultam
18 (376 mg, 0.34 mmol) and aqueous 50% H2O2 (0.12 cm3,
1.75 mmol) in THF/H2O (5/1, 6.0 cm3) at 0 ◦C was added
aqueous 1 N LiOH (0.67 cm3, 0.67 mmol), and the mixture was
stirred at room temperature for 2 h. After being diluted with
EtOAc (20 cm3), the mixture was washed with 0.1 N HCl and
dried over MgSO4. Concentration under reduced pressure gave the
corresponding acid, which was used in the next reaction without
purification. To a solution of the above acid in CH2Cl2 (15 cm3)
at 0 ◦C was added TFA (4.0 cm3), and the mixture was stirred at
room temperature for 0.5 h. Concentration under reduced pressure
gave an oily residue, which was dissolved in MeCN/DMF/H2O
(2R,5S,3E)-2-{4-[N-(tert-Butoxycarbonyl)amino]butyl}-9-[N-
(2-chlorobenzyloxycarbonyl)amino]-5-[N -(9-fluorenylmethoxy-
carbonyl)amino]non-3-enoic acid (10). To the Fmoc-protected
amine 9 (435 mg, 0.52 mmol) was added 4 N HCl/dioxane
(5.0 cm3) at 0 ◦C, and the mixture was stirred for 20 h at room
temperature. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexane-EtOAc (1:1)
gave the title compound 10 (391 mg, 96%) as a semisolid; [a]22
D
-18.0 (c 0.87 in CHCl3); nmax/cm-1 3324 (NHCO), 1703 (CO); dH
(500 MHz; CDCl3) 1.09–1.81 (12H, m), 2.85–3.01 (1H, m), 3.03–
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(10/9/1, 20 cm3). Fmoc-OSu (159 mg, 0.472 mmol) and Et3N
(0.094 cm3, 0.675 mmol) were added to the mixture at 0 ◦C,
and the mixture was stirred at room temperature for 12 h. After
being diluted with EtOAc (70 cm3), the reaction mixture was
washed with 1 N HCl and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography over
silica gel with n-hexane-EtOAc (1:1) gave the title compound
Measurement of CD spectra
Peptides were incubated at 37 ◦C for 30 min (the final con-
centrations of peptides were 10 mM in 5 mM HEPES buffer,
pH 7.2). CD spectra were acquired on a Jasco spectropolarime-
ter (Model J-710, Jasco Inc., Tokyo, Japan) at 25 ◦C as the
average of 8 scans. Thermal unfolding at intervals of 0.5 ◦C
was performed after a 0.25-min equilibration at the desired
temperature and an integration time of 1.0 s. The mid point of
the thermal unfolding transition (melting temperature, Tm) of
each complex was determined from the maximum of the first
derivative, with respect to the reciprocal of the temperature, of the
[q]222 values.
19 (267.3 mg, 65% yield) as a semisolid; [a]24 -19.6 (c 1.13 in
D
DMSO); nmax/cm-1 3333 (OH), 1693 (CO); dH (500 MHz; DMSO-
d6) 1.04–1.70 (12H, m), 2.89–3.02 (4H, m), 3.21 (1H, dt, J 9.7
and 7.5), 3.98–4.10 (1H, m), 4.22 (1H, t, J 6.9), 4.30 (2H, d, J
6.9), 4.85 (1H, dd, J 37.2 and 9.7), 4.99 (4H, s), 7.19–7.44 (16H,
m), 7.65–7.74 (3H, m), 7.89 (2H, d, J 7.5), 12.35 (1H, br s); dC
(125 MHz; DMSO-d6) 22.6, 23.7, 28.9, 29.0, 30.9, 31.9, 40.0, 40.1,
40.3, 46.6, 51.3 (d, J 31.2), 65.0, 65.1, 65.4, 104.0 (d, J 12.0),
120.0 (2C), 125.1 (2C), 127.0 (2C), 127.6 (2C), 127.6 (4C), 127.7
(2C), 128.3 (4C), 137.2 (2C), 140.7 (2C), 143.7, 143.8, 155.6, 156.0
(2C), 159.4 (d, J 257.9), 174.4; dF (470 MHz; DMSO-d6) -117.9–
-118.5 (m); m/z (FAB) 766.3512 ([M + H]+, C44H49FN3O8 requires
766.3504).
Acknowledgements
This work was supported by the Science and Technology Incu-
bation Program in Advanced Regions from Japan Science and
Technology Agency, Grant-in-Aids for Scientific Research from
MEXT, Japan, and Health and Labour Sciences Research Grants
(Research on HIV/AIDS). K.T. and T.N. are grateful for the JSPS
Research Fellowships for Young Scientists.
General procedure for preparation of peptide by Fmoc-SPPS
R
The protected peptide chains were constructed on the Novasynꢀ
References
TGR resin (0.26 mmol g-1, 96 mg, 0.025 mmol). t-Bu ester for
Asp and Glu; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Pbf) for Arg; t-Bu for Thr, Tyr and Ser; Boc for Lys; and
Trt for Gln, Asn and His were employed for side-chain protec-
tion. Fmoc-amino acids (0.075 mmol) were coupled by using
N,N¢-diisopropylcarbodiimide (DIC; 0.012 cm3, 0.075 mmol)
and N-hydroxybenzotriazole monohydrate (HOBt·H2O, 11.5 mg,
0.075 mmol) in DMF for 2 h. Coupling of dipeptide isosteres
(EADI 10: 49 mg, 0.063 mmol; FADI 19, 48 mg, 0.063 mmol)
was carried out with DIC and HOBt·H2O for 12 h. The pep-
tide resins were treated with 1 M TMSBr-thioanisole/TFA in
the presence of m-cresol and 1,2-ethanedithiol as scavengers.
The reaction mixture was precipitated with diethyl ether. The
resulting powder was collected by centrifugation and then washed
three times with diethyl ether. The crude product was purified
by preparative HPLC to afford the expected peptides as a
colorless powder. The purity of each compound was assessed
analytical RP-HPLC prior to the CD analysis and biological
testing (>98%).
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