Convergent synthesis of a helical, prehairpin HR1 trimer from HIV gp41

Article abstract of DOI:10.1002/psc.1262

A helical, prehairpin trimer covering the majority of the HR1 region of human immunodeficiency virus gp41 was achieved by chemically coupling three identical 51 amino acid peptides. A 1,3,5-tris(aminomethyl)-2,4,6- triethylbenzene pinwheel 'cap' was used

Full text of DOI:10.1002/psc.1262

Research Article
Received: 3 May 2010
Revised: 16 May 2010
Accepted: 17 May 2010
Published online in Wiley Interscience: 13 July 2010
(www.interscience.com) DOI 10.1002/psc.1262
Convergent synthesis of a helical,
prehairpin HR1 trimer from HIV gp41
Rong Jian Lu,∗ Catherine J. Mader, Stephen E. Schneider, Nicolai Tvermoes,
Myung-Choi Kang, John J. Dwyer,∗ Karen L. Wilson, Thomas J. Matthews,
Mary K. Delmedico and Brian Bray
A helical, prehairpin trimer covering the majority of the HR1 region of human immunodeficiency virus gp41 was achieved by
chemically coupling three identical 51 amino acid peptides. A 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene pinwheel ‘cap’
was used to trimerize the peptides by taking advantage of the unique property of triacyl fluoride and orthogonal protection
c
and deprotection. The resulting protein is fully helical, highly thermostable and soluble. Copyright ꢀ 2010 European Peptide
Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: convergent synthesis; protein synthesis; peptides; prehairpin gp41 HR1 trimer; oligomer
Introduction
introduces extraneous sequence that may preclude some of the
applications where native sequence is required. Recently, Taylor
et al., Bianchi et al. and Nakahara et al. have reported effective
solutions to the synthesis of covalent HR1 trimers (albeit only up
to 36-aa peptides) respectively [10].
We sought to stabilize a long segment of the wild type, helical
trimer using a small chemical cap. Templates have been shown
previously to reinforce intramolecular folding in designed proteins
[11–14] and the cooperative conformational network [15] in
1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene(hereaftercalledthe
pinwheel) has attracted much attention to its application for the
designofreceptorsforassessingglycoside[16], nitrate, citrate[17],
and self-assembling sieves [18] and controlling the oxygenation
level of hemoglobin [19].
In our effort to design a stable HR1 trimer, we incorporated a
1,3,5-tris (aminomethyl)-2,4,6-triethylbenzene pinwheel template
to the less structured, N-terminal end of the HR1. A Gly-Gly
spacer was used between the pinwheel and the peptide to help
maintain the integrity of the trimeric coiled coil. A glutarate spacer
was used on the pinwheel to avoid steric congestion and allow
more conformational mobility. For the first time, we were able to
engineer a 53 amino acid trimer, based on the peptide T-865 [20],
covering the majority of the HR1 region of gp41. This pinwheel
trimer not only includes the deep pocket [4c,8a,21] but the entire
HR1 groove including regions known to be important for HR2
binding and areas that mutate in response to ENF resistance [20].
The trimer is shown to be fully helical, soluble and exceptionally
stable.
Infection by human immunodeficiency virus type 1 (HIV-1)
requires fusion of the viral and cellular membranes [1]. Binding
of viral glycoproteins gp120 and gp41 to the CD4 receptor
and coreceptors (such as CCR5 and CXCR4) triggers a series of
conformational changes in gp120 and gp41 and ultimately leads
to formation of the trimer-of-hairpins structure in gp41 [2].
gp41 contains two heptad repeat regions: a trimeric coiled-coil
HR1 region which is located near the fusion peptide and the
initially unstructured HR2 region which is proximal to the viral
membrane [1]. After binding to CD4 and coreceptors, the fusion
peptide inserts into the target cell to form the fusion intermediate
state. The HR2 region then binds to hydrophobic grooves present
on the HR1 to form the six-helix bundle or trimer-of-hairpins. The
transition to the bundle state is thought to provide the energy
required to facilitate membrane fusion [1,3,5]. Both heptad repeat
regions have been proven to be attractive targets for blocking
the fusion process [4–6]. Enfuvirtide (ENF), a peptide derived from
the HR2 region and targeting the HR1, is the only fusion inhibitor
approved for clinical use [4a,4b,7].
The success of HR1- and HR2-derived peptide fusion inhibitors
has focused much attention on gp41 as a target for the
development of small molecule fusion inhibitors as well as an
antigen for vaccine development. Efforts in this area have been
inhibited due to the difficulty in presenting the HR1 prehairpin
intermediate in a stable, soluble state that accurately represents
the viral HR1 trimer [8]. Several approaches have been used to
avoid these issues. Several groups have attempted to stabilize the
HR1 trimer, either by forming a chimera between the GCN4 trimer
and a short segment of the HR1 [9] or to substantially mutate the
peptide to stabilize the trimeric form [6]. Another approach has
beentoforma‘5-helix’bundle,whereonegrooveoftheHR1trimer
remains exposed [1]. Unfortunately, each of these approaches
∗
Correspondence to: Rong Jian Lu and John J. Dwyer, Trimeris, Inc., 3500
Paramount Parkway, Morrisville, NC 27560, USA.
E-mail: rjlu@frontierbiotech.com;proteinengineering@gmail.com
Trimeris, Inc., 3500 Paramount Parkway, Morrisville, NC 27560, USA
c
J. Pept. Sci. 2010; 16: 465–472
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

LU ET AL.
F
O
O
O
HN
O
NH2
1) Ethyl hydrogen
glutarate
F
N
H
TFFH
H2N
Pinwheel
HATU, HOAt,
DIEA, DMF
triacid
Pyridine,
DCM/DMF
NH
2) NaOH, THF,
MeOH, H2O
NH2
O
Pinwheel
triacyl fluoride
2
3
1
O
F
gp41
O
O
O
HN
O
N
H
gp
41
H-GlyGly gp41 HR1 Monomer*
De-IVDDE
4
NH
O
Pin(N-gp41)3
5
O
gp41
HR1:QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ
Scheme 1. Synthesis of Pin(N-gp41)3.
Experimental Procedures
0.03
0
Section 1: Biophysical Analysis
Analytical ultracentrifugation
The sedimentation equilibrium experiments were performed
on a Beckman Optima XL-A analytical ultracentrifuge at 4 ◦C
as described [20]. Peptide samples were diluted using 50 mM
KH2PO4, 100 mM KCl pH 7.0 and data were collected at 18 000
and 22 000 rpm at wavelengths of 235 240 and 280 nm. Weight-
averaged molecular weights were obtained by fitting each data
file individually using a single ideal species model in the Beckman-
Origin software (version 3.78 for Windows) (Figure 1). The solvent
density of the phosphate buffer (equal to 1.00894) and partial
specific volume (0.7424) were calculated using the program
SEDNTERP [20].
-0.03
1
0.8
0.6
0.4
0.2
Circular dichroism
Themidpointofthethermalunfolding transition(Tm)ofthePin(N-
gp41)3 and the HR1/HR2 complex was determined at 222 nm with
2 ◦C increments from 25 to 97 ◦C with 16 s averaging time on an
AVIV 202-01 circular dichroism spectrophotometer. The Tm was
determined to be the value that corresponded to the maximum
value of the first derivative of the thermal transition. Wavelength
scans were also performed (Figure 2) and ranged from 200 to
260 nm, with measurements at every 0.5 nm, a 4 s averaging time
and a 1.5 nm bandwidth.
5.9
5.95
Radius (cm)
6
6.05
Figure 1. Sedimentation equilibrium results for a 10 µM solution of Pin(N-
gp41)3 at 4 ◦C in 50 mM KH2PO4, 100 mM KCl pH 7.0. The line represents
the fit to the single ideal species model. For clarity, not all data points are
shown.
c
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HELICAL, PREHAIRPIN HR1 TRIMER FROM HIV GP41
2-chlorotrityl chloride resin
2-chlorotrityl chloride resin
2-chlorotrityl chloride resin
Solid phase
peptide synthesis
Solid phase
peptide synthesis
Solid phase
peptide synthesis
Fmoc-NH2-GlyGly-Gln(Trt)Ala
Arg(Pbf)Gln(Trt)LeuLeuSer(tBu)
GlyIleValGln(Trt)Gln(Trt)Gln
Asn(Trt)Asn(Trt)Leu-OH
FmocLeuArg(Pbf)AlaIleGlu(tBu)
AlaGln(Trt)Gln(Trt)His(Trt)Leu
LeuGln(Trt)LeuThr(tBu)ValTrp
(Boc)Gly-OH
FmocIleLys(IvDde)Gln(Trt)LeuGln
(Trt)AlaArg(Pbf)IleLeuAlaValGlu
(tBu)Arg(Pbf)Tyr((tBu)LeuLys
(IvDde)Asp(tBu)-OH
Fmoc-AA17-33-OH
Fmoc-AA34-50-OH
Fmoc-GlyGlyAA1-16-OH
8
6
7
H-GlnNH2
H-IleLys(IvDde)Gln(Trt)LeuGln
(Trt)AlaArg(Pbf)IleLeuAlaValGlu
(tBu)Arg(Pbf)Tyr((tBu)LeuLys
(IvDde)Asp(tBu)GlnNH2
H-AA34-51NH2
9
H-LeuArg(Pbf)AlaIleGlu(tBu)AlaGln(Trt)Gln(Trt)His(Trt)LeuLeuGln(Trt)
LeuThr(tBu)ValTrp(Boc)GlyIleLys(IvDde)Gln(Trt)LeuGln(Trt)AlaArg(Pbf)IleLeu
AlaValGlu(tBu)Arg(Pbf)Tyr(tBu)LeuLys(IvDde)Asp(tBu)Gln-NH2
H-AA17-51-NH2
10
H-GlyGlyGln(Trt)AlaArg(Pbf)Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)Gln(Trt)
GlnAsn(Trt)Asn(Trt)LeuLeuArg(Pbf)AlaIleGlu(tBu)AlaGln(Trt)Gln(Trt)His(Trt)
LeuLeuGln(Trt)LeuThr(tBu)ValTrp(Boc)GlyIleLys(IvDde)Gln(Trt)LeuGln(Trt)
AlaArg(Pbf)IleLeuAlaValGlu(tBu)Arg(Pbf)Tyr(tBu)LeuLys(IvDde)Asp(tBu)Gln-NH2
H-GlyGlyAA1-51-NH2
11
global side chain deprotection
H-GlyGlyGlnAlaArgGlnLeuLeuSerGlyIleValGlnGlnGlnAsnAsnLeuLeuArgAlaIleGluAlaGlnGln
HisLeuLeuGlnLeuThrValTrpGlyIleLys(IvDde)GlnLeuGlnAlaArgIleLeuAlaValGluArgTyrLeuLys
(IvDde)AspGln-NH2
4
H-GlyGly HR1 monomer
Piewheel tracyl trifloride/purification
Pin(GlyGlyGlnAlaArgGlnLeuLeuSerGlyIleValGlnGlnGlnAsnAsnLeuLeuArgAlaIleGluAlaGlnGln
HisLeuLeuGlnLeuThrValTrpGlyIleLys(IvDde)GlnLeuGlnAlaArgIleLeuAlaValGluArgTyrLeuLys
(IvDde)AspGln-NH2)3
12
-ivDde
Pin(GlyGlyGlnAlaArgGlnLeuLeuSerGlyIleValGlnGlnGlnAsnAsnLeuLeuArgAlaIleGlu
AlaGlnGlnHisLeuLeuGlnLeuThrValTrpGlyIleLysGlnLeuGlnAlaArgIleLeuAlaValGluA
rgTyrLeuLysAspGln-NH2)3
Pin(N-gp41)3
5
Scheme 2. Synthesis of H GlyGly HR monomer (11).
Section 2: Synthesis
to ca 6.5 with HOAc. The product was precipitated with cold
methyl t-butyl ether (MTBE) (100 ml), filtered off, washed with
MTBE (3 × 25 ml) and dried on the funnel for 1 h. The crude
Synthesis of Pin(N-gp41)3 (5)
product was purified using reverse phase high-performance liquid
chromatography (RP-HPLC) (Column: Phenomenex Jupitor C4,
300 Å, 10 µm, 250×21.4 mm;mobilephase:A:0.1%trifluoroacetic
acid (TFA) in H2O; B: 0.1% TFA in acetonitrile; gradient: 55–72%
B in 63 min; flow rate: 20.0 ml min−1; detection: UV at 220 nm) to
afford pure ivDde protected product Pin(GlyGly HR1-ivdde)3 (4)
The Pin-F3 (3, 5.9 mg, 0.009 mmol) and H-GlyGly-HR1 monomer
(ivdde) (12, 532 mg, 0.082 mmol) were dissolved in dimethyl
formamide (DMF) (2 ml) at 25 ◦C (Scheme 1). Then diisopropyl
ethyl amine (DIEA) (120 µl, 0.689 mmol) was added and the
solution was stirred at 25 ◦C for 21 h. The reaction mixture
was cooled in an ice bath and then the pH was adjusted
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J. Pept. Sci. 2010; 16: 465–472 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

LU ET AL.
10000
0
frozen and lyophilized to give Pin(N-gp41)3 (5, 37 mg) in 88%
yield and as a single peak in the HPLC chromatogram purity 100%
by LC-MS. MS(Da): calculated monoisotope for C834H1389N255O231
18673.48, found 18674.84 (Figures 3 and 4).
-10000
-20000
-30000
-40000
Synthesis of pinwheel triacid (2): 1,2,3-tri(N-methylpentanoic
acidamide)-2,4,6-triethylbenzene (pinwheel triacid)
A 100 ml round bottom flask was charged with 20 ml of anhydrous
DMF under an inert atmosphere, and ethyl hydrogen glutarate
(761 mg, 4.752 mmol) was added. The resulting solution was
cooled to 0 ◦C in an ice-water bath, and N-[(dimethylamino)-
1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmeth-
anaminium hexafluorophosphate N-oxide (HATU) (1.807 g,
4.752 mmol), 1-hydroxy-7-azabenzotriazole (HOAt) (646 mg,
4.751 mmol) and DIEA (2.55 ml, 17.28 mmol) were added and
stirred at 0 ◦C for 10 min. Then 1,3,5-tris(aminomethyl)-2,4,6-
triethylbenzene trihydrochloride (517 mg, 1.44 mmol) [22] was
added. The reaction was stirred at 0 ◦C for 1 h and then at 25 ◦C
for 24 h. Half of the solvent was removed under reduced pressure
and the remaining solids were dissolved in 70 ml of CH2Cl2 and
washed with cold 0.5 N HCl (2 × 20 ml), H2O (2 × 20 ml), cold
0.5 N NaOH (2 × 20 ml) and brine (2 × 20 ml). The organic layer
was collected and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure and the resulting crude product
was dried under high vacuum overnight. The crude product was
dissolved in 9 ml of tetrahydrofuran (THF) and 3 ml of MeOH
200
210
220
230
240
250
260
wavelength (nm)
Figure 2. Wavelength scan of 6.6 µM Pin(N-gp41)3
( ) and 20 µM T865
•
(
) in phosphate buffered saline (PBS) pH 6.0 at 25◦C as determined by
◦
circular dichroism. The helicity of Pin(N-gp41)3 and T865 was found to be
90% and 97%, respectively.
as a fluffy white solid in 25% yield (45 mg). MS(Da) calculated for
C912H1497N255O243 19910.24, found 19909.65. To a 100 ml round
bottom flask were charged Pin(GlyGly HR1-ivdde)3 (4, 45 mg,
0.00226 mmol) and 5% hydrazine in DMF (4 ml). The solution was
stirred for 2 h, then cooled to 0–5 ◦C and HOAc (5–6 drops) was
added bringing the pH to 6.5. To the cooled solution was added
MTBE (50 ml) resulting in precipitation of the trimer. The solid was
collected by filtration, washed with MTBE (3×15 ml) and dissolved
in 20% acetonitrile in water (10 ml, 0.1% TFA). The solution was
TIC
T15.8
1.0E+4
100
90
80
70
60
50
40
30
20
10
0
T17.8
T18.9
T2.4
T2.5
T29.2
T21.0
21
T24.4
0
35
0
7
14
28
UV
1.0
100
90
80
70
60
50
40
30
20
10
0
T15.6
T32.5
T30.5
T26.7
T21.0
T12.2
T11.0
T0.5
0
35
0
7
14
21
28
Retention Time (Min)
´
Figure 3. HPLC-MS spectrum of Pin(N-gp41)3. LC column: keystone BetBasic C18, 2.0 × 150 mm (3 µm/150 Å). Mobile phase: (A) 0.1% formic acid in
water; (B) 0.1% formic acid in acetonitrile. MS: positive ion mode (+); scanning 450–2500 m/z every 2 s; Sciex heater at 400 ◦C. LC: UV detector at 220 nM
with flow 0.20 ml min−1. This figure is available in colour online at www.interscience.wiley.com/journal/jpepsci.
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HELICAL, PREHAIRPIN HR1 TRIMER FROM HIV GP41
Mariner Spec +458:481 (T +15.61:16.40) ASC=>NR(4.00)[BP = 1246.2, 158]
1246.13
157.8
100
90
80
70
60
50
40
30
20
10
0
1336.16
1168.30
1438.58
1099.60
1699.90
1558.43
1341.11
1446.90
1038.57
1869.77
1565.21
1708.96
935.69
1213.81
1269.4
0
2500.0
449.0
859.2
1679.6
2089.8
Mass (m/z)
Figure 4. MS spectrum of Pin(GlyGlyHR1)3 from 15.61 to 16.40 min for the peak 2 RT15.8 min in the total ion current (TIC). This figure is available in colour
online at www.interscience.wiley.com/journal/jpepsci.
and cooled in an ice-water bath. Sodium hydroxide (218 mg,
5.46 mmol) in 3 ml of water was added dropwise and the solution
was stirred at 25 ◦C for 5 h. The THF and methanol were removed
under reduced pressure and the aqueous layer was cooled in an
ice bath and the pH adjusted to 1–2 by slow addition of 1 N
HCl. The crude product was purified by RP-HPLC (Phenomenex
Jupiter C18, 300 Å, 10 µ, 250 × 21.4 mm, A: 0.1% TFA in H2O; B:
0.1% TFA in acetonitrile; gradient 20–50% B in 40 min) to afford
pure product as a white solid in 49% yield (414 mg). 1H NMR
(400 MHz, DMSO-d6) δ: 12.01 (br s, 3H), 7.78 (t, 3H, J = 4.5 Hz),
4.26 (d, 6H, J = 4.0 Hz), 2.64–2.62 (q, 6H, J = 7.5 Hz), 2.20–2.16
(t, 6H, J = 7.3 Hz), 2.12–2.08 (t, 6H, J = 7.4 Hz), 1.71–1.67 (p,
6H, J = 7.4 Hz), 1.05 (t, 9H, J = 7.4 Hz). [MH]+ calculated for
C30H45N3O9 592.31, found 592.13.
in 59.1% yield (253 mg). 1H NMR (400 MHz, DMSO-d6) δ: 7.85 (br,
3H), 4.26 (b, 6H), 2.68–2.61 (m, 12H), 2.30–2.27 (m, 6H), 1.77 (t, 6H,
J = 7.2 Hz), 1.06 (t, 9H, J = 7.3 Hz). IR: 1850 cm−1
.
Synthesis of H-GlyGly HR1 (ivdde) monomer (12)
A 500 ml round bottom flask containing trifluoroacetic acid
(85.5 ml), dithiothreitol (8.55 g), 8.55g phenol and water
(4.28 ml) was purged with nitrogen for 5 min. The solution was
cooled to 0–5 ◦C for 10 min and H-GlyGlyGln(Trt)AlaArg(Pbf)
Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)Gln(Trt)GlnAsn(Trt)Asn
(Trt)LeuLeuArg(Pbf)AlaIleGlu(tBu)AlaGln(Trt)Gln(Trt)His(Trt)Leul
LeuGln(Trt)LeuThr(tBu)ValTrp(Boc)GlyIleLys(ivDde)Gln(Trt)LeuGln
(Trt)AlaArg(Pbf)IleLeuAlaValGlu(tBu)Arg(Pbf)Tyr(tBu)LeuLys(iv
Dde)Asp(tBu)GlnNH2 (2.1 g, 0.19 mmol) was added. The reaction
was removed from the ice bath and stirred at 25 ◦C for 4 h. The
reaction mixture was then cooled to 0–5 ◦C and cold MTBE
(400 ml) was added over 20 min with rapid stirring. The resulting
slurry was stirred for an additional 30 min, the solids filtered off,
washed with MTBE (3×50 ml), and dried on the funnel for 1 h. The
solids were suspended in 60 ml of 50 : 50 H2O : acetonitrile and the
pH adjusted to ca 7.5 with 0.35 N NaHCO3. When all the solids had
dissolved, the pH was lowered back to ca 4 with acetic acid (HOAc),
and the resulting cloudy suspension was stirred at 25 ◦C overnight
to effect decarboxylation of the tryptophans. The solvent was
lyophilized off to afford a fluffy ivory colored crude product that
was then purified by RP-HPLC (Column: Phenomenex Jupiter C18,
Synthesis of pinwheel triacyl fluoride (3): 1,2,3-tri(N-methyl(pentanyl
fluoride)amide)-2,4,6-triethylbenzene(Pin-F3)
To a solution of pinwheel triacid (0.426 g, 0.72 mmol), pyridine
(0.175 ml, 2.16 mmol), anhydrous CH2Cl2 (20 ml), and DMF (4 ml)
the fluoro-N,N,Nꢁ,Nꢁ-tetramethylformamidinium hexafluorophos-
phate (TFFH) (0.856 g, 3.24 mmol) was added under an inert
atmosphere. The reaction was stirred at 25 ◦C for 20 h and then di-
luted with 250 ml of CH2Cl2 and washed with ice water (3×50 ml).
The organic layer was collected and dried over MgSO4. The sol-
vent was removed under reduced pressure and the residue was
re-crystallized with CH2Cl2 affording pure product as a white solid
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J. Pept. Sci. 2010; 16: 465–472 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

LU ET AL.
300 Å, 10 µ, 250 × 21.4 mm; mobile phase: A: 0.1% TFA in H2O;
B: 0.1% TFA in acetonitrile; gradient: 40–60% B in 45 min; flow
rate: 20.0 ml min−1; detection: UV at 220 nm) to afford 0.62 g of H-
GlyGlyGlnAlaArgGlnLeuLeuSerGlyIleValGlnGlnGlnAsnAsnLeuLeu
ArgAlaIleGluAlaGlnGlnHisLeulLeuGlnLeuThrValTrpGlyIleLys(iv
Dde)GlnLeuGlnAlaArgIleLeuAlaValGluArgTyrLeuLys(ivDde)Asp
GlnNH2 (12, H-GlyGly HR1 monomer) as a fluffy white solid in
49% yield and a single peak in the HPLC (Method D). MS(Da):
calculated monoisotope for C294H486N84O79 6457.65, found
6457.79 (Scheme 2).
procedure for the synthesis of intermediates 6–10 is available in
supporting information.
Results and Discussion
We envisioned synthesis of the Pin(N-gp41)3 (5) from the pinwheel
triacid (2) and the H-GlyGly HR1 monomer, where the internal
lysines at positions 37 and 51 were protected. The triaminomethyl
pinwheel (1) was coupled to the activated ester of ethyl hydrogen
glutarate through standard peptide coupling conditions (HATU,
HOAt,DIEA,DMF)[23].Followingsaponificationinmethanol/water
and purification by RP-HPLC, the pinwheel triacid (2) was isolated
as a white solid in 49% yield.
Synthesis of H-GlyGlyAA1-51 NH2 (11, fully protected)
A
500 ml round bottom flask was charged with Fmoc
Attempts to synthesize the H-GlyGly HR1 peptide by linear
solid phase peptide synthesis (SPPS) using Fmoc protected amino
acids proved to be difficult and failed to produce the desired
quantities of material. A three fragment approach to building the
H-GlyGly-HR1 peptide, similar to that used for the synthesis of ENF,
was developed [24]. The three side chain protected fragments,
FmocGlyGly-AA1-16OH, FmocAA17-33OH and FmocAA34-50OH,
were built by SPPS on the super-acid labile 2-chlorotrityl chloride
resin. The ε-amines of the two lysines in FmocAA34-50OH
were protected with (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-
3-methylbutyl (ivDde), a functionality that is stable toward the
basic conditions used to remove Fmoc in peptide synthesis as well
as the acidic conditions used to remove the side chain protecting
groups on the other amino acids [25]. The protected fragments
were assembled in solution phase condensations to provide
side chain protected H-GlyGly-AA1-51 NH2. Global side chain
deprotection of H-GlyGly-AA1-51 NH2 with a cocktail composed
oftrifluoroaceticacid:water:dithiothreitol:phenol(80 : 4 : 8 : 8vol,
vol, wt, wt) followed by chromatographic purification produced
the desired H-GlyGly HR1 monomer with the internal lysines
protected. The inclusion of phenol in the cocktail was essential
to avoid extensive trifluoroacetylation of the peptide during side
chain deprotection.
GlyGlyGln(Trt)AlaArg(Pbf)Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)
Gln(Trt)GlnAsn(Trt)Asn(Trt)LeuLeuArg(Pbf)AlaIleGlu(tBu)AlaGln
(Trt)Gln(Trt)His(Trt)LeulLeuGln(Trt)LeuThr(tBu)ValTrp(Boc)GlyIle
Lys(ivDde)Gln(Trt)LeuGln(Trt)AlaArg(Pbf)IleLeuAlaValGlu(tBu)Arg
(Pbf)Tyr(tBu)LeuLys(ivDde)Asp(tBu)GlnNH2 (10.5g, 0.95 mmol),
DMF (60 ml) and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU)
(0.10 ml, 0.67 mmol). The solution was stirred at ambient temper-
ature for 90 min and DCM was added (100 ml). The solution was
transferred to a separator funnel and 0.1 N HCl (100 ml) was added.
The aqueous layer was discarded and the process repeated. The
process was repeated two more times with water (2 × 100 ml).
The organic solution was dried over MgSO4, then filter and
concentrated using a rotary evaporator. The resulting residue was
cooled to 0–5 ◦C and MTBE (300 ml) was added to precipitate the
protected peptide. The suspension was stirred for 2 h, collected by
filtration, washed with MTBE (2 × 100 ml) and dried affording H-
GlyGlyGln(Trt)AlaArg(Pbf)Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)
Gln(Trt)GlnAsn(Trt)Asn(Trt)LeuLeuArg(Pbf)AlaIleGlu(tBu)AlaGln
(Trt)Gln(Trt)His(Trt)LeulLeuGln(Trt)LeuThr(tBu)ValTrp(Boc)GlyIle
Lys(ivDde)Gln(Trt)LeuGln(Trt)AlaArg(Pbf)IleLeuAlaValGlu(tBu)Arg
(Pbf)Tyr(tBu)LeuLys(ivDde)Asp(tBu)GlnNH2 (9.75 g) in 94.8% yield.
Method C HPLC purity, 69.8A%. MS(Da): calculated monoisotope
for C603H774N84O93S4, 10807.73, found 10807.88.
Initial attempts at formation of the pinwheel HR1 trimer
from H-GlyGly HR1 and pinwheel triacid activated as the
N-hydroxysuccinimide, paranitrophenyl, pentafluorophenyl, N-
hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole esters in
DMF produced pin(GlyGly HR1)1 as the main product and <1%
of the desired trimer. DMF competes with the peptide in reacting
with the activated esters, presumably forming a Villsmeyer type
reagent, which in turn formylates (after exposure to water) the
N-terminus of the peptide.
Treatment of the pinwheel triacid (2) with Carpino’s reagent
[26], TFFH yielded the pinwheel triacyl fluoride (3). Condensation
of the more stable pinwheel triacyl fluoride with an excess of
H-GlyGly HR1 in DMF afforded a mixture of the desired pinwheel
trimer as well as dimers and monomers. The trimer was isolated by
preparative HPLC and treated with hydrazine to remove the ivDde
protecting groups on the internal lysines to give Pin(N-gp41)3 in
22% yield.
Circular dichroism was used to characterize the trimeric
pinwheelcompound. AsshowninFigure 2, thewavelengthscanof
Pin(N-gp41)3 at6.6 µM inPBSpH6.0isindicativeofahelicalspecies
and we determined that the construct is approximately 90%
helical. Figure 5 shows that the thermal stability of Pin(N-gp41)3
is substantially higher than the peptide T865. At 6.6 and 0.5 µM,
helical structure is maintained to 95 ◦C, as compared to T865
which cooperatively unfolds at temperatures well below 95 ◦C.
Synthesis of Fmoc-GlyGlyAA1-51 NH2 (fully protected)
A 500 ml round bottom flask was charged with FmocGlyG-
lyGln(Trt)AlaArg(Pbf)Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)Gln
(Trt)GlnAsn(Trt)Asn(Trt)LeuOH (6, 3.79 g, 0.97 mmol), H-LeuArg
(Pbf)AlaIleGlu(tBu)AlaGln(Trt)Gln(Trt)His(Trt)LeulLeuGln(Trt)Leu
Thr(tBu)ValTrp(Boc)GlyIleLys(ivDde)Gln(Trt)LeuGln(Trt)AlaArg
(Pbf)IleLeuAlaValGlu(tBu)Arg(Pbf)Tyr(tBu)LeuLys(ivDde)Asp(tBu)
GlnNH2(10, 7.14 g, 1.00 mmol), HOAt (0.359 g, 2.91 mmol), DMF
(130 ml) and DIEA (0.675 ml, 3.88 mmol). The solution was
cooled to 0–5 ◦C and HATU (0.405 g, 1.07 mmol) was added.
The solution was stirred at 0–5 ◦C for 10 min then warmed
to ambient temperature and stirred an additional 1 h. The
solution was cooled to <10 ◦C and water (200 ml) was added
to precipitate the product. The resulting suspension was stirred
at <10 ◦C for 30 min. The solid was collected by filtration,
washed with water (3 × 75 ml) and dried to give FmocGlyG-
lyGln(Trt)AlaArg(Pbf)Gln(Trt)LeuLeuSer(tBu)GlyIleValGln(Trt)Gln
(Trt)GlnAsn(Trt)Asn(Trt)LeuLeuArg(Pbf)AlaIleGlu(tBu)AlaGln(Trt)
Gln(Trt)His(Trt)LeulLeuGln(Trt)LeuThr(tBu)ValTrp(Boc)GlyIleLys(iv
Dde)Gln(Trt)LeuGln(Trt)AlaArg(Pbf)IleLeuAlaValGlu(tBu)Arg(Pbf)
Tyr(tBu)LeuLys(ivDde)Asp(tBu)GlnNH2 (10.6 g) in 99% yield.
MS(Da): calculated monoisotope for C618H784N84O95S4, 11029.80,
found 11029.87. Method C HPLC purity, 58A%. The experimental
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 465–472

HELICAL, PREHAIRPIN HR1 TRIMER FROM HIV GP41
0
-10000
-20000
-30000
-40000
-50000
0
-10000
-20000
-30000
-40000
0
20
40
60
80
100
120
20
30
40
50
60
70
80
90
100
Temperature (C)
Temperature (C)
Figure 6. Thermal stability of Pin(N-gp41)3 trimer alone (ꢀ) and Pin(N-
gp41)3/T649 bundle (ꢂ). Data was obtained at 1 µM concentrations in PBS
pH 7.0.
Figure 5. Thermal unfolding transitions of Pin(N-gp41)3 at 6.6 µM ( ),
•
Pin(N-gp41)3 at 0.5 µM (ꢀ), T865 at 20 µM ( ) and T865 at 1 µM (ꢁ)
◦
as determined by circular dichroism. Thermal melts were done in PBS
pH 6.0 (higher concentrations) or pH 7.0 (lower concentrations) and
at 25 ◦C.
largest oligomer prepared by chemical synthesis via a convergent
synthetic approach, taking advantage of the unique property
of triacyl fluoride and orthogonal protection and deprotection.
This will enable the design and preparation of a wide range of
template-assembled oligomeric protein analogs for functional,
immunogenic and therapeutic research.
Therefore, Pin(N-gp41)3 is almost fully helical and substantially
more thermostable than the self-associated peptide.
To further investigate the enhancement in HR1 stability,
we repeated the thermal unfolding experiment in the pres-
ence of 5
M urea. The observed Tm for T-865 in 5 M
urea was found to be 55 ◦C (not shown). In contrast, Pin(N-
gp41)3 has a Tm of 86 ◦C in 5 M urea, further demonstrating
that the covalent trimer has significantly enhanced thermal
stability.
Supporting information
Supporting information may be found in the online version of this
article.
Although it is assumed from the crystallographic structures that
the HR1 is trimeric [1,6], it has been demonstrated in solution
that the peptide T-865 self-associates into a tetrameric species
[20]. We analyzed Pin(N-gp41)3 by analytical ultracentrifugation
and found that the weight-averaged molecular weight at 10 µM
to be 17 525 +/− 440 Da. This molecular weight is in good
agreement with the predicted mass of a trimer (18673.48),
demonstrating that the covalent construct is a trimeric species, as
designed.
To investigate how well HR2 peptides could bind to the
engineered trimer, we performed peptide mixing experiments
using circular dichroism. Although Pin(N-gp41)3 is highly helical,
HR2 peptides are unstructured in solution and adopt a helical
conformation upon binding. We used the HR2 peptide T-649, a
peptide closely related to C34 [20], and the observed helicity of the
mixture demonstrates a significant increase in helical structure,
whichisindicativeoftheformationofthesix-helixbundle.Thedata
for the interaction between Pin(N-gp41)3 and T649 is shown in
Figure 6. A loss of structure is observed at high Tm for T-649, which
is likely due to the loss of structure of the HR2 as it dissociates from
the HR1 trimer, consistent with observations from our previous
work [20].
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