Application of ring-closing metathesis macrocyclization to the development of Tsg101-binding antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.10.105

HIV-1 viral budding involves binding of the viral Gag^p6 protein to the ubiquitin E2 variant domain of the human tumor susceptibility gene 101 protein (Tsg101). Recognition of p6 by Tsg101 is mediated in part by a proline-rich motif that contains the sequence 'Pro-Thr-Ala-Pro' ('PTAP'). Using the p6-derived 9-mer sequence 'PEPTAPPEE', we had previously improved peptide binding affinity by employing N-alkylglycine ('peptoid') residues. The current study applies ring-closing metathesis macrocyclization strategies to Tsg101-binding peptide-peptoid hybrids as an approach to stabilize binding conformations and to observe the effects of such macrocyclization on Tsg101-binding affinity and bioavailability.

Full text of DOI:10.1016/j.bmcl.2009.10.105

Bioorganic & Medicinal Chemistry Letters 20 (2010) 318–321
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Application of ring-closing metathesis macrocyclization to the development
of Tsg101-binding antagonists
b
c
c
b
a,
*
Fa Liu a, , Andrew G. Stephen , Abdul A. Waheed , Eric O. Freed , Robert J. Fisher , Terrence R. Burke Jr.
*
a Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute-Frederick, National Institutes of Health Frederick, MD 21702, United States
b Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc. NCI-Frederick, Frederick, MD 21702, United States
c HIV Drug Resistance Program, National Cancer Institute-Frederick, National Institutes of Health Frederick, MD 21702, United States
a r t i c l e i n f o
a b s t r a c t
HIV-1 viral budding involves binding of the viral Gagp6 protein to the ubiquitin E2 variant domain of the
human tumor susceptibility gene 101 protein (Tsg101). Recognition of p6 by Tsg101 is mediated in part
by a proline-rich motif that contains the sequence ‘Pro-Thr-Ala-Pro’ (‘PTAP’). Using the p6-derived 9-mer
sequence ‘PEPTAPPEE’, we had previously improved peptide binding affinity by employing N-alkylglycine
(‘peptoid’) residues. The current study applies ring-closing metathesis macrocyclization strategies to
Tsg101-binding peptide–peptoid hybrids as an approach to stabilize binding conformations and to
observe the effects of such macrocyclization on Tsg101-binding affinity and bioavailability.
Published by Elsevier Ltd.
Article history:
Received 4 September 2009
Revised 23 October 2009
Accepted 26 October 2009
Available online 29 October 2009
Keywords:
Macrocycle
Ring-closing metathesis
Peptide mimic
Peptide
Solid-phase synthesis
Viral release is a necessary component of HIV-1 replication. To
be an efficient process, viral budding relies on recruitment of the
ubiquitin E2 variant (UEV) domain of the human tumor suscepti-
bility gene 101 protein (Tsg101) by major structural proteins of
HIV-1.1,2 This entails the direct interaction of the Tsg101 UEV do-
main with a proline-rich motif (PRM) within the viral Gagp6 pro-
tein that contains a conserved sequence, Pro-Thr/Ser-Ala-Pro
[‘P(T/S)AP’].3,4 Over-expression of the Tsg101 UEV inhibits virus
release by interfering with processing of the p6 domains and this
effect is abrogated by mutation within the P(T/S)AP binding site.
In theory, blocking this critical Tsg101-p6 interaction could pre-
vent viral budding and provide a basis for new targeted antiretro-
viral therapies.5,6 This is supported by the recent finding that
inhibition of HIV budding can be achieved by cyclic peptides that
interfere with Tsg101-Gag interactions.7
In an effort to develop Tsg101-binding inhibitors using the re-
ported p6-derived 9-mer wild-type (WT) sequence ‘P1E2P3T4A5P6P7
E8E9’, we had previously improved Tsg101-binding affinity by
applying hydrazone- and hydrazide-library techniques.8 We also
reported an oxime-based post solid-phase diversification approach
to peptide library construction.9–11 However, the peptide mimetics
resulting from this earlier work showed poor bioavailability in cell-
based experiments. Because poor cellular uptake was thought to
contribute to the low bioavailability, peptides were conjugated
with known membrane carrier antennapedia and HIV-Tat
(48–60) peptides.12 Although these constructs did exhibit apparent
enhanced cellular uptake, the Tsg101-binding affinity of the conju-
gates was seriously disrupted. Therefore, we sought an alternate
approach that could improve cell bioavailability while maintaining
Tsg101-binding affinity. We took note of the fact that cyclic ver-
sions of linear peptides can result in better cell-permeability. Addi-
tionally, appropriately restricting solution conformations through
cyclization can also afford higher affinity.13–17 The objective of
the current study was to apply RCM strategies to the WT 9-mer
peptide and to observe the effects that such macrocyclization
would have on Tsg101-binding affinity and cellular bioavailability.
Synthetic design: RCM macrocyclization requires the insertion
into the parent peptide of two terminal alkene units that serve as
ring-closing segments. Many examples of RCM macrocyclizations
of peptides have been reported that rely on placement of alkenyl
side chains onto the
a-carbons of ring junction glycine residues.
However, our recent examination of N-substituted glycines (NSGs)
within the parent Tsg101 9-mer peptide8 suggested the potential
utility N-alkenylglycine residues as ring-forming units. The use of
NSGs is of note, because such residues constitute an important
class of peptide mimetics termed ‘peptoids’.18 Since the overall
macrocycle ring size and the insertion locations of the alkenyl
chains that would most effectively provide the best combination
of affinity, stability, and bioavailability were not known, we envi-
sioned placing the needed N-alkenylglycines within the N-terminal
and C-terminal ‘P1E2’ and ‘E8E9’ sequences, respectively. This would
* Corresponding authors.
E-mail addresses: liuf@ncifcrf.gov (F. Liu), tburke@helix.nih.gov (T.R. Burke).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.10.105

F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 318–321
319
chloride (6) with appropriate alkenyl bromides H2C@CH[CH2]nBr
(5a–5g, where n = 1–6 and 9), in the presence of triethylamine in
acetonitrile gave the corresponding N-alkenylglycine methyl esters
(7a–7g). This reaction has previously been reported using alkali or
alkali earth metal bases.22 Hydrolysis of the methyl ester (aqueous
sodium hydroxide) followed by reaction with 9-fluorenylmethoxy-
carbonyl-N-hydroxysuccinimide (Fmoc-OSu) in the presence of
NaHCO3 in aqueous dioxane gave desired reagents 8a–8g.
HCl•H2N
CO2CH3
6
Br
N
H
CO2CH3
n
n
n = 1 – 6, 9 Et3N, CH3CN, r.t.
5a – 5g
7a – 7g
i) NaOH, H2O, r.t.
N
n
Fmoc
CO2H
ii) Fmoc-OSu, NaHCO3,
dioxane, H2O, r.t.
Synthesis of macrocycles 1–4 was by solid-phase methods using
NovaSynÒ TGR resin and standard Fmoc protocols. Side chain pro-
tection of glutamic acid residues was as their tert-butyl esters. The
precursor open-chain peptides [9-(m,n)] (the use of ‘m’ and ‘n’ in
compound numbering is described in Fig. 1) were built up on the
resin and amino-terminally acylated with an N-Fmoc 5-aminova-
leric acid (N-Fmoc Ava) group. Various combinations of N-alkenyl-
glycines were substituted for residues P1 and E9 (Scheme 2). The
N-Fmoc Ava groups were then deprotected and the peptides were
acylated using fluorescein isothiocyanate (FITC). Cleavage of the
peptides from the resin (TFA) was accompanied by simultaneous
side chain deprotection. HPLC purification provided the linear
open-chain products 10-(m,n). Similar protocols were followed in
which N-alkenylglycines were used to replace residues P1 and E8;
E2 and E9; E2 and E8 to yield linear open-chain analogues 20-
(m,n), 30-(m,n) and 40-(m,n), respectively.
8a – 8g
Scheme 1. Synthesis of Fmoc-protected N-alkenylglycine residues, where a, n = 1
(over all yield 18%, based on the alkenyl bromide); b, n = 2 (13%); c, n = 3 (17%); d,
n = 4 (23%); e, n = 5 (19%); f, n = 6 (21%); g, n = 9 (22%).
allow RCM macrocyclization with maintenance of the critical
‘P3T4A5P6’ recognition region. By employing a range of chain
lengths in the N-alkenyl side chains, a series of macrocycles (1–
4) could be constructed that would vary in ring size and location
of ring-closure attachments (Fig. 1). In this fashion, we could ex-
plore a variety of macrocycle structures in an attempt to arrive
at optimum configurations. A similar generation of libraries of
macrocyclic peptides has previously been described as an approach
at interrogating bioactive conformations.19–21
Synthesis: The requisite ring junction-forming N-alkenylglycine
residues were prepared as their N-Fmoc-protected derivatives
(8a–8g, shown in Scheme 1). Treatment of methyl glycinate hydro-
Macrocyclizations of intermediates 9-(m,n) were conducted on
the resin at room temperature in CH2Cl2 using Hoveyda-Grubbs 2nd
FITC–Ava
CO2H
m
O
n
O
S
O
O
O
H
N
O
N
HO
NH2
N
H
N
H
N
Pro-Thr-Ala-Pro-Pro
N
H
O
O
O
CO2H
1-(m,n)
OH
m = 1, 6, 9; n = 1-6
m
n
O
O
O
O
H
H
N
N
NH2
FITC-Ava
N
Pro-Thr-Ala-Pro-Pro
N
O
O
CO2H
CO2H
2-(m,n)
m = 1, 6; n = 1-6
CO2H
m
n
O
O
O
N
N
N
NH2
Pro-Thr-Ala-Pro-Pro
N
H
FITC–Ava
O
O
O
3-(m,n)
m = 1, 6; n = 1-6
m
n
O
O
O
H
N
N
N
NH2
Pro-Thr-Ala-Pro-Pro
N
FITC–Ava
O
O
O
4-(m,n)
CO2H
m = 1, 6; n = 1-6
Figure 1. Structures of macrocycles prepared in the current study. Amino-terminal tagging with fluorescein isothiocyanate (FITC) permitted the performance of fluorescence
anisotropy-based Tsg101-binding assays.

320
F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 318–321
t
CO2 Bu
m
n
O
O
O
O
H
N
N
N
FmocHN
SPPS
N
Pro-Thr-Ala-Pro-Pro
N
H
H
O
O
H2N
t
CO2 Bu
NovaSyn® TGR
Amide Resin
9-(m,n)
CO2H
m
n
O
O
O
O
H
i) piperidine, DMF
N
N
NH2
FITC-Ava
N
Pro-Thr-Ala-Pro-Pro
N
H
O
O
ii) FITC
iii) TFA,iPr3SiH, H2O
CO2H
1'-(m,n)
Scheme 2. Solid-phase synthesis of open-chain macrocycles 10-(m,n).
Table 1
generation RCM catalyst [(1,3-bis-(2,4,6-trimethylphenyl)-2-
imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthe-
nium]23 (Scheme 3). Except for substrates containing short alkenyl
side chains (the peptide–peptoid hybrids containing either two resi-
dues of 8a or one residue each of 8a and 8b), the open-chain precur-
sors were completely consumed in the RCM reaction. The efficiency
of ring-closure was sequence-dependent, with members from the
2-series giving the highest percentage of expected ring-closed prod-
uct. Finally, amino terminal acylation with FITC as described above
and cleavage of peptides from the resin provided the target macrocy-
clic peptides 1-(m,n), 2-(m,n), 3-(m,n), and 4-(m,n) (Fig. 1). RCM is
known to provide mixtures of alkenyl E/Z geometric isomers during
themetathesisreaction. PreviouslyreportedexamplesofRCMmacro-
cyclizations performed on large peptide substrates have highlighted
the difficulties of obtaining defined E/Z geometries at the ring-form-
ing alkenyl junction.19,24–27 Consistent with these prior studies, our
current work does not explicitly define the geometries of RCM ring-
closure, nor does it extrapolate conformations of the resulting
macrocycles.
Tsg101-binding affinities of selected compoundsa
No.
Kd
(lM)
1-(6,4)
1-(6,5)
1-(6,6)
1-(9,2)
1-(9,3)
1-(9,4)
1-(9,5)
1-(9,6)
3-(6,1)
3-(6,2)
3-(6,5)
3-(6,6)
38
24
19
17
11
22
32
23
47
44
35
31
a
Binding affinities were determined using a fluorescence anisotropy assay as
described in Ref. 7.
than the WT peptide and 10-fold higher affinity than the open-
chain analogue 10-(1,1) (Kd = 120
l
M).
Cellular uptake: An objective of the current study was to exam-
ine the effects of macrocyclization on cellular uptake. For -helices,
Tsg101-binding affinities: Determination of Tsg101-binding affin-
ities was conducted using a fluorescence anisotropy assay.8 Open-
chain peptide–peptoid hybrids 10, 20, and 30 exhibited affinities
a
RCM macrocyclization can lead to more cell-permeable ligands.28–33
This effect has been attributed to several factors that include in-
creased lipophilicity imparted by the ring-closing alkenyl hydrocar-
bon chain. It has also been shown that peptoids can serve as
platforms for enhanced cellular uptake.34,35 This is due in part to
the replacement of peptide backbone amide hydrogens by N-alkyl
groups. Because macrocycles prepared within the current series con-
tain both peptoid bonds and ring-closing alkenyl hydrocarbon
chains, we anticipated an increase in cellular uptake. The inclusion
similar to the WT parent 9-mer peptide (Kd = 50–60 l
M).8 Macro-
cyclic peptide–peptoid hybrids 2 and 4 showed significantly re-
duced binding affinities, potentially indicating induced
conformations that were unfavorable for binding. Certain members
of the 1 and 3 series macrocycles showed binding affinities greater
than those of the WT peptide (Table 1). The highest affinities were
provided by 1-(6,6) (Kd = 19
lM), 1-(9,2) (Kd = 17 lM), and 1-(9,3)
(Kd = 11 M). The latter analogue showed fivefold higher affinity
l
t
CO2 Bu
m
Hoveyda – Grubbs 2nd
generation catalyst
n
O
O
O
O
H
N
N
N
FITC-Ava
N
Pro-Thr-Ala-Pro-Pro
N
H
9-(m,n)
H
O
CH2Cl2, r.t.
O
t
CO2 Bu
10-(m,n)
i) piperidine, DMF
1-(m,n)
ii) FITC
iii) TFA,iPr3SiH, H2O
Scheme 3. Solid-phase synthesis of target macrocycles 1-(m,n).

F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 318–321
321
Table 2
Supplementary data
Uptake of selected peptides into HeLa cellsa
No.
Relative uptakeb
Reaction yields and analytical data for products 8a–8g, mass
spectral data for peptides and peptide–peptoid hybrids and
Tsg101-binding affinities. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.10.105.
WT 9-mer
10-(1,1)
20-(1,1)
30-(1,1)
40-(1,1)
1-(1,3)
1-(6,2)
1-(6,6)
2-(1,3)
2-(6,2)
2-(6,6)
10-(6,6)
20-(6,6)
30-(6,6)
40-(6,6)
3-(1,3)
3-(6,2)
3-(6,6)
4-(1,3)
4-(6,2)
4-(6,6)
74 34
74 14
79 30
98 32
74 22
175 60
100 44
328 75
146 44
100 42
194 31
35 17
40 12
42 12
45 14
229 54
77 23
199 57
272 72
76 34
200 65
References and notes
1. von Schwedler, U. K.; Stuchell, M.; Mueller, B.; Ward, D. M.; Chung, H.-Y.;
Morita, E.; Wang, H. E.; Davis, T.; He, G.-P.; Cimbora, D. M.; Scott, A.;
Kraeusslich, H.-G.; Kaplan, J.; Morham, S. G.; Sundquist, W. I. Cell 2003, 114,
701.
2. Mazze, F. M.; Degreve, L. Acta Virologica 2006, 50, 75.
3. Garrus, J. E.; von Schwedler, U. K.; Pornillos, O. W.; Morham, S. G.; Zavitz, K. H.;
Wang, H. E.; Wettstein, D. A.; Stray, K. M.; Cote, M.; Rich, R. L.; Myszka, D. G.;
Sundquist, W. I. Cell 2001, 107, 55.
4. Freed, E. O. Trends Microbiol. 2003, 11, 56.
5. Turpin, J. A. Exp. Rev. Anti-Infect. Ther. 2003, 1, 97.
6. Reeves, J. D.; Piefer, A. J. Drugs 2005, 65, 1747.
7. Tavassoli, A.; Lu, Q.; Gam, J.; Pan, H.; Benkovic, S. J.; Cohen, S. ACS Chem. Biol.
2008, 3, 757.
8. Liu, F.; Stephen, A. G.; Adamson, C. S.; Gousset, K.; Aman, M. J.; Freed, E. O.;
Fisher, R. J.; Burke, T. R., Jr. Org. Lett. 2006, 8, 5165.
9. Liu, F.; Stephen, A. G.; Waheed, A. A.; Aman, M. J.; Freed, E. O.; Fisher, R. J.;
Burke, T. R., Jr. ChemBioChem 2008, 9, 2000.
a
Data generated as described in the experimental procedures.
Values calculated as the average fluorescence intensities quantified using
b
softWoRx 3.7.0 software from cells treated with the indicated peptides. At least 20
cells from 4 to 6 different fields each containing approximately 5 cells were
quantified for fluorescent intensities. Data are shown as means average deviation.
10. Liu, F.; Thomas, J.; Burke, T. R., Jr. Synthesis 2008, 2432.
11. Liu, F.; Stephen, A. G.; Fisher, R. J.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2008,
18, 1096.
12. von Heijne, G. J. Membrane Biol. 1990, 115, 195.
13. Piserchio, A.; Salinas, G. D.; Li, T.; Marshall, J.; Spaller, M. R.; Mierke, D. F. Chem.
Biol. 2004, 11, 469.
of FITC tags on the synthetic peptide–peptoid hybrids permitted
measurement of cellular uptake by fluorescence microscopy.
Accordingly, a subset of peptide–peptoid hybrids was examined
for cellular uptake. All macrocyclic analogues showed increased cel-
lular uptake relative to control WT 9-mer peptide (Table 2). The best
cellular uptake was shown by macrocycles having larger ring sizes,
with 1-(6,6) showing the greatest uptake. In contrast, the corre-
sponding ring-open macrocycle precursors did not show appreciable
increased cell-permeability as compared to the WT 9-mer peptide.
This may indicate that increased hydrophobicity imparted by the
alkenyl chains is not the sole reason for increased cell-permeability
of the macrocycles.
Our current Letter details the design, synthesis and biological
evaluation of four sets of RCM-derived macrocyclic peptide–peptoid
hybrids. Several distinct families were defined by different position-
ing of the ring-closing segments within the peptide backbones. To
explore conformational effects of macrocyclization, the ring-closing
alkenyl chains were varied to provide a total of 11 gradually enlarg-
ing macrocycles for each family. This gave ring sizes from 23 to 39
members. The best cellular uptakes were shown by larger ring sizes.
Peptide–peptoid hybrid 1-(6,6) (total ring size of 39) exhibited the
14. Horswill, A. R.; Benkovic, S. J. Cell Cycle 2005, 4, 552.
15. Jiang, S.; Li, Z.; Ding, K.; Roller, P. P. Curr. Org. Chem. 2008, 12, 1502.
16. Linde, Y.; Ovadia, O.; Safrai, E.; Xiang, Z.; Portillo, F. P.; Shalev, D. E.; Haskell-
Luevano, C.; Hoffman, A.; Gilon, C. Biopolymers 2008, 90, 671.
17. Antos, J. M.; Popp, M. W. L.; Ernst, R.; Chew, G. L.; Spooner, E.; Ploegh, H. L. J.
Biol. Chem. 2009, 284, 16028.
18. Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.;
Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlow, C. K.; Spellmeyer, D. C.;
Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Proc. Nat. Acad. Sci.
U.S.A. 1992, 89, 9367.
19. Reichwein, J. F.; Wels, B.; Kruijtzer, J. A. W.; Versluis, C.; Liskamp, R. M. J. Angew.
Chem., Int. Ed. 1999, 38, 3684.
20. Reichwein, J. F.; Versluis, C.; Liskamp, R. M. J. J. Org. Chem. 2000, 65, 6187.
21. Davies, J. S. J. Pept. Sci. 2003, 9, 471.
22. Cho, J. H.; Kim, B. M. Tetrahedron Lett. 2002, 43, 1273.
23. Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H. J. Am. Chem.
Soc. 1999, 121, 791.
24. Chapman, R. N.; Dimartino, G.; Arora, P. S. J. Am. Chem. Soc. 2004, 126, 12252.
25. Bernal, F.; Tyler, A. F.; Korsmeyer, S. J.; Walensky, L. D.; Verdine, G. L. J. Am.
Chem. Soc. 2007, 129, 2456.
26. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.;
Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 1466.
27. Chapman, R.; John, L.; Kulp, I.; Patgiri, A.; Kallenbach, N. R.; Bracken, C.; Arora,
P. S. Biochemistry 2008, 47, 4189.
28. Henchey, L. K.; Jochim, A. L.; Arora, P. S. Curr. Opin. Chem. Biol. 2008, 12, 692–
697.
29. Bird, G. H.; Bernal, F.; Pitter, K.; Walensky, L. D. Methods Enzymol. 2008, 446,
369–386.
30. Kutchukian, P. S.; Yang, J. S.; Verdine, G. L.; Shakhnovich, E. I. J. Am. Chem. Soc.
2009, 131, 4622–4627.
best combination of Tsg101-binding affinity (Kd = 19 lM) and cellu-
lar uptake. Work is in progress to evaluate the antiviral efficacy of
macrocycles such as 1-(6,6) in whole cell systems.
31. Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891.
32. Wang, D.; Liao, W.; Arora, P. S. Angew. Chem. Int. Ed. 2005, 44, 6525.
33. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.;
Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 1446.
34. Unciti-Broceta, A.; Diezmann, F.; Ou-Yang, C. Y.; Fara, M. A.; Bradley, M. Bioorg.
Med. Chem. 2009, 17, 959.
Acknowledgements
This Work was supported in part by the Intramural Research
Program of the NIH, Center for Cancer Research, NCI-Frederick
and the National Cancer Institute, National Institutes of Health, un-
der contract N01-CO-12400.
35. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.;
Rothbard, J. B. Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 13003.