Protected aminooxyprolines for expedited library synthesis: Application to Tsg101-directed proline-oxime containing peptides

Article abstract of DOI:10.1016/j.bmcl.2007.12.003

The stereoselective synthesis of aminooxy-containing proline analogues bearing Fmoc/Boc or Fmoc/Mtt protection that renders them suitable for incorporation into peptides using Fmoc protocols is reported. Acid-catalyzed unmasking at the completion of peptide synthesis yields free aminooxy-functionalities for oxime formation through reaction with libraries of aldehydes. This allows post solid-phase diversification strategies that may facilitate structure-activity relationship studies.

Full text of DOI:10.1016/j.bmcl.2007.12.003

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1096–1101
Protected aminooxyprolines for expedited
library synthesis: Application to Tsg101-directed
proline–oxime containing peptides
Fa Liu,a Andrew G. Stephen,b Robert J. Fisherb and Terrence R. Burke, Jr.a,*
aLaboratory of Medicinal Chemistry, CCR, NCI-Frederick, NIH, Building 376 Boyles Street, Frederick, MD 21702, USA
bProtein Chemistry Laboratory, SAIC-Frederick, Inc., Frederick, MD 21702, USA
Received 15 November 2007; accepted 3 December 2007
Abstract—The stereoselective synthesis of aminooxy-containing proline analogues bearing Fmoc/Boc or Fmoc/Mtt protection that
renders them suitable for incorporation into peptides using Fmoc protocols is reported. Acid-catalyzed unmasking at the completion
of peptide synthesis yields free aminooxy-functionalities for oxime formation through reaction with libraries of aldehydes. This
allows post solid-phase diversification strategies that may facilitate structure–activity relationship studies.
Published by Elsevier Ltd.
The presence of proline residues in peptides and proteins
can induce b-turn secondary structures that provide spe-
cific bioactivities.1 This is exemplified by ‘proline rich
motifs’ (PRMs) that are involved with the formation
of protein–protein complexes.2 Libraries of proline ana-
logues could serve as valuable tools for studying these
interactions. However, the preparation of peptides bear-
ing 3- and 4-substituted proline congeners has been lim-
ited by the need to synthesize each individual proline
derivative in protected form prior to peptide synthesis.3
NHR
O
NHR
O
BocHN
O
CO2H
CO2H
CO2H
N
N
N
Fmoc
Fmoc
Fmoc
(4R)-1 R = Boc
(4R)-2 R = Mtt
(4S)-1 R = Boc
(4S)-2 R = Mtt
(3S)-3
Figure 1. Structures of orthogonally protected proline analogues.
Synthesis. Commercially available (4R)-4-hydroxy-L-
proline (4) was protected as its N-Cbz, O-Bn derivative
(4R)-5 (Scheme 1).8,9 Inversion of the (4R)-stereocenter
was achieved in two steps involving the 4-nitrobenzoate
ester (6)10 under Mitsunobu conditions.11 The high cost
of (4S)-4 makes its preparation from the relatively inex-
pensive (4R)-4 attractive.12
The incorporation of suitably protected aminooxy
groups into orthogonally protected proline residues
could result in highly useful biochemical tools amenable
to diversification through oxime formation by conjuga-
tion with libraries of aldehydes following the completion
of peptide synthesis.4–6 The aminooxy-containing pro-
line analogues7 1 and 3, bearing Fmoc and Boc or 2,
having Fmoc and Mtt protection, represent examples
of proline derivatives that may be suitable for post so-
lid-phase diversification through oxime formation
(Fig. 1). Reported herein are the synthesis of these
new proline analogues and their application in a physi-
ologically relevant context where proline residues serve
as key recognition elements.
HO
HO
1. CbzCl, NaHCO3
2. BnBr, Et3N, 96%
p-Nitrobenzoic acid,
DEAD, PPh3, 100%
CO2H
CO2Bn
N
H
N
4
Cbz
(4R)-5
O
HO
NO2
NaN3,15-Crown-5, 83%
O
CO2Bn
N
CO2Bn
Cbz
N
Keywords: Proline analog; Aminooxy; Oxime; Peptide library; Tsg101.
*
Cbz 6
(4S)-5
Corresponding author. Tel.: +1 301 846 5906; fax: +1 301 846
6033; e-mail: tburke@helix.nih.gov
Scheme 1.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.12.003

F. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1096–1101
1097
fashion starting from the commercially available free
amino acid 4, (4S)-1 was obtained in 82% overall yield
(7 steps) and (4R)-1 was obtained in 53% overall yield
(9 steps).
O
1. NH2NH2-H2O
2. Boc2O, Et3N
(4R): 81%; (4S): 96%
N-Hydroxyphthalimide,
DEAD,PPh3
(4R): 95%; (4S): 97%
N
O
O
5
CO2Bn
N
N-Boc-protection of aminooxy functionality is suitable
for Fmoc-based solid-phase protocols used in combina-
tion with moderately labile acid-sensitive resins, where
removal of the Boc groups is concomitant with cleav-
age of the peptide from the resin. However, when
deprotection of the aminooxy group is desired without
cleavage of the peptide from the solid-phase resin,
more highly acid-labile methyltrityl (Mtt) protection
(2) is appropriate. Toward this objective, the isomeric
phthalimido-protected aminooxy intermediates (4R)-7
and (4S)-7 were treated with methylhydrazine, then di-
rectly reacted with 4-methyltrityl chloride to give the
Mtt protected compounds (4R)-9 and (4S)-9 (Scheme
3).20,21 Simultaneous hydrogenolytic cleavage of both
the amino and carboxylic protecting groups (PdC/H2
in EtOAc:MeOH) and subsequent treatment with
Fmoc-OSu provided the final products (4S)-222 and
(4R)-223 in 77% overall yield (7 steps) and 63% overall
yield (9 steps), respectively, starting from commercially
available 4.
Cbz
7
NHBoc
O
1. H2, Pd•C;
2. Fmoc-Osu, NaHCO3
(4R): 86%; (4S): 92%
1
CO2Bn
N
Cbz
8
Scheme 2.
Mitsunobu transformation of the protected isomeric
(4R)- and (4S)-4-hydroxyprolines (5)13 was achieved
smoothly with inversion of C4 stereochemistry to give
the corresponding (4S)- and (4R)-N-phthalimido-
aminooxy compounds (7) in nearly quantitative yield
(Scheme 2).14,15
The phthalimido groups of 7 were cleaved at room
temperature using hydrazine hydrate and the free
aminooxy primary amines were directly protected by
treatment with Boc anhydride and triethylamine.
Hydrogenation of the resulting derivatives (8)16,17 over
PdC removed both the O-Bn and N-Cbz protecting
groups to yield the free amino acids, which were con-
verted in situ to their N-Fmoc forms (1).18,19 In this
Preparation of the N-Boc derivatized isomeric 3-
substituted products (3) began by protection of com-
mercially available (3S)-3-hydroxy-L-proline (10) using
the conditions described above for the conversion of 4
to (4R)-5. The attempted Mitsunobu transformation
of the resulting (3S)-1124 using N-hydroxyphthalimide
under a variety of reaction conditions predominately
gave the corresponding a,b-elimination material with
no desired product. With the epimeric (3R)-11, pre-
pared in near quantitative yield from (3S)-11 by a
two-step p-nitrobenzoyl ester stereo-inversion se-
quence,25,26 a low yield (10%) of the desired 3-phtha-
limidooxy analogue (3S)-13 could be obtained.27,28
This allowed subsequent conversion to the final prod-
uct (3S)-3 (Scheme 4).29
1.MeNH2NH2;
1. H2, Pd/C;
2. FmocOsu, NaHCO3
(4R): 91%; (4S): 91%
2. 4-methyltrityl Chloride,
DIPEA;
NHMtt
O
(4R): 92%; (4S): 91%
7
2
CO2Bn
N
Cbz
9
Scheme 3.
OH
CO2H
OH
p-Nitrobenzoic acid,
DEAD, PPh3, 100%
1. CbzCl, NaHCO3
2. BnBr, Et3N, 90%
CO2Bn
N
H
N
Cbz
(3S)-11
NO2
10
OH
N-Hydroxyphthalimide,
DEAD,PPh3, 10%
NaN3,15-Crown-5,
83%
O
O
CO2Bn
N
CO2Bn
N
Cbz
Cbz
(3R)-11
12
O
BocHN
O
1. H2, Pd•C;
2. Fmoc-Osu, NaHCO3
93%
1. NH2NH2-H2O
2. Boc2O, Et3N, 83%
N
O
O
CO2Bn
(3S)-3
N
CO2Bn
Cbz
N
13
Cbz
14
Scheme 4.

1098
F. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1096–1101
Table 1. Tsg101-binding affinities of proline–oxime containing peptides determined as in Ref. 32
NX
COOH
HO
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
N
N
NH2
N
N
H
N
N
H
S
O
O
O
O
O
O
O
O
COOH
Y
COOH
Y =
X = H2
HO
O
OH
X = R
(19)
(4S)-aminooxy-Pro (15)
(4R)-aminooxy-Pro (16)
(3S)-aminooxy-Pro (17)
R CHO (18)
(20)
AcOH
(21)
X
IC50 (lM)
20
Peptide: 19
55 [15]
21
H2
43 [16]
21 [17]
nd
42
54
41
41
23
a
b
c
HOOC
75
83
27
26
16
20
d
HO
nd
48
64
nd
26
30
26
29
25
20
15
22
O
e
O
f
g
N
h
NC
nd
nd
20
12
22
18
i
O2N
O
j
58
42
13
17
12
12
k
l
N

F. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1096–1101
1099
Table 1 (continued)
X
IC50 (lM)
Peptide: 19
20
21
O2N
91
21
16
m
N
nd
93
25
16
16
19
11
22
n
HO
HOOC
o
H3CO
114
H3CO
p
F
F
F
F
139
32
19
F
q
BnO
54
30
27
18
17
r
nd
s
H3CO
HO
nd
23
13
t
nd, not determined.
Application of aminooxy-proline analogues to peptide li-
brary synthesis. In order to demonstrate the utility of
post solid-phase diversification using aminooxy-proline
analogues, we employed a Tsg101-binding peptide sys-
tem. The UEV (ubiquitin E2 variant) domain of the hu-
man protein Tsg101 (tumor susceptibility gene 101) is
recruited by major structural proteins of HIV-1 to facil-
itate viral budding. This involves the direct interaction
of the Tsg101 UEV domain with a Pro-Thr-Ala-Pro
(‘PTAP’) sequence in the viral Gagp6 protein.30,31 Using
a p6–derived sequence, we had previously replaced crit-
ical Pro residues with hydrazone- and hydrazide-con-
taining N-substituted glycines as peptoid surrogates
that allowed the expedited synthesis of libraries of
Tsg101-directed binding antagonists.32 In our current
study, we utilized the wild-type p6-derived ‘PEP-
TAPPEE’ sequence to examine libraries of peptides hav-
ing proline–oximes situated in place of the Pro7 residue
(underlined).33,34
nally protected residues described above. TFA-
mediated cleavage of peptides from the resin gave
the globally deprotected aminooxy-proline containing
peptides 15–17 (Table 1) which were purified by
HPLC. Condensation of 15–17 with a series of alde-
hydes (18a–t) was conducted overnight in DMSO with
AcOH and a molar ratio of peptide to aldehyde of
1:1. As indicated by HPLC, the reactions went to
completion, yielding proline–oxime containing peptides
19–21 in sufficient purity (>90%) for direct biological
evaluation (Table 1).35 Certain of these peptides (for
example, 21o; IC50 = 11 lM) exhibited up to fivefold
higher Tsg101-binding affinity than wild-type peptide
(Kd = 54 lM),36 indicating the potential utility of the
approach.
Conclusions. Reported herein are the design and synthe-
sis of 3- and 4-aminooxy-substituted proline analogues
suitably protected for Fmoc-based peptide synthesis.
As demonstrated by application in a Tsg101-binding
system, these non-natural amino acid analogues may
be highly useful for post solid-phase diversification
Libraries were generated from parent aminooxy-pro-
line containing peptides prepared using the orthogo-

1100
F. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1096–1101
2H), 7.38–7.28 (m, 5H), 7.22–7.15 (m, 5H), 5.58 (m, 1H),
5.25–5.02 (m, 4H), 4.73 (dd, J = 9.2, 1.6 Hz, 0.5H), 4.63
(dd, J = 9.2, 1.6 Hz, 0.5H), 3.92–3.85 (m, 2H), 2.66–2.46
(m, 2 H). ESI-MS (+ve) m/z: 527.1 (M+Na)+. HR-ESI/
APCI MS cacld for C27H25N2O8 (M+H)+: 505.1611,
Found: 505.1606.
strategies that could facilitate rapid structure-activity
relationship studies, potentially leading to new biologi-
cally active motifs.
Acknowledgments
11. Gomez-Vidal, J. A.; Silverman, R. B. Org. Lett. 2001, 3,
2481.
This research was supported in part by the Intramural
Research Program of the NIH, Center for Cancer Re-
search, NCI-Frederick and the National Cancer Insti-
tute, National Institutes of Health, under contract
N01-CO-12400. The content of this publication does
not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does
mention of trade names, commercial products, or orga-
nizations imply endorsement by the U.S. Government.
12. Gomez-Vidal, J.; Forrester, M. T.; Silverman, R. B. Org.
Lett. 2001, 3, 2477.
20
1
13. (4S)-5: ½aꢀ ꢁ 17:1 (c 2.56, CHCl3). H NMR (400 MHz,
CDCl3) d D7.35–7.24 (m, 10H), 5.30–5.00 (m, 4H), 4.49 (dd,
J = 9.6, 1.6 Hz, 0.4H), 4.42 (dd, J = 9.6, 1.6 Hz, 0.6H),
4.34 (m, 1H), 3.72–3.57 (m, 2H), 3.27 (br s, 1H), 2.29 (m,
1H), 2.12 (m, 1H). 13C NMR (100 MHz, CDCl3) d 174.12,
174.04, 154.98, 154.31, 136.36, 136.25, 135.23, 135.04,
128.58, 128.48, 128.44, 128.37, 128.27, 128.15, 128.08,
128.03, 127.90, 127.84, 70.97, 70.00, 67.46, 67.42, 67.38,
67.34, 67.25, 58.25, 57.88, 55.91, 55.57, 38.68, 37.80.
20
1
14. (4S)-7: Mp 130–132 °C. ½aꢀD ꢁ 4:20 (c 0.88, CHCl3). H
NMR (400 MHz, CDCl3) d 7.84–7.81 (m, 2H), 7.77–7.74
(m, 2H), 7.45–7.23 (m, 10H), 5.33–4.99 (m, 4H), 4.94 (m,
1H), 4.65 (dd, J = 10.0, 2.0 Hz, 0.5H), 4.58 (dd, J = 9.6,
2.0 Hz, 0.5H), 4.05 (m, 1H), 3.92 (m, 1H), 2.70 (m, 0.5H),
2.66 (m, 0.5H), 2.45 (m, 1H). ESI-MS (+ve) m/z: 523.1
(M+Na)+. HR-ESI/APCI MS cacld for C28H24N2NaO7
(M+Na)+: 523.1481, Found: 523.1475.
References and notes
1. (a) Mauger, A. B. J. Natural Prod. 1996, 59, 1205; (b)
Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.;
Scharpe, S. FASEB J. 1995, 9, 736; (c) Ruzza, P.; Siligardi,
G.; Donella-Deana, A.; Calderan, A.; Hussain, R.;
Rubini, C.; Cesaro, L.; Osler, A.; Guiotto, A.; Pinna, L.
A.; Borin, G. J. Peptide Sci. 2006, 12, 462; (d) Reimer, U.;
Scherer, G.; Drewello, M.; Kruber, S.; Schutkowski, M.;
Fischer, G. J. Mol. Biol. 1998, 279, 449.
20
1
15. (4R)-7: Mp. 124–126°C. ½aꢀD ꢁ 44:1 (c 1.09, CHCl3). H
NMR (400 MHz, CDCl3) d 7.85–7.82 (m, 2H), 7.77–7.75
(m, 2H), 7.40–7.18 (m, 10H), 5.26–5.14 (m, 3 H), 4.99–4.96
(m, 2H), 4.78 (m, 1H), 4.08 (m, 1H), 3.75 (dd, J = 12.8,
4.0 Hz, 1H), 2.71 (m, 1H), 2.15 (m, 1H). ESI-MS (+ve)
m/z: 523.1 (M+Na)+. HR-ESI/APCI MS cacld for
C28H24N2NaO7 (M+Na)+: 523.1481, Found: 523.1473.
2. Ball, L. J.; Kuhne, R.; Schneider-Mergener, J.; Oschkinat,
H. Angew. Chem., Int. Ed. 2005, 44, 2852.
3. (a) Jacquot, Y.; Broutin, I.; Miclet, E.; Nicaise, M.;
Lequin, O.; Goasdoue, N.; Joss, C.; Karoyan, P.; Desma-
dril, M.; Ducruix, A.; Lavielle, S. Bioorg. Med. Chem.
2007, 15, 1439; (b) Kim, W.; Hardcastle, K. I.; Conticello,
V. P. Angew. Chem., Int. Ed. 2006, 45, 8141; (c) Taylor, C.
M.; Hardre, R.; Edwards, P. J. B. J. Org. Chem. 2005, 70,
1306.
4. (a) Johnson, S. M.; Petrassi, H. M.; Palaninathan, S. K.;
Mohamedmohaideen, N. N.; Purkey, H. E.; Nichols, C.;
Chiang, K. P.; Walkup, T.; Sacchettini, J. C.; Sharpless,
K. B.; Kelly, J. W. J. Med. Chem. 2005, 48, 1576; (b) Lees,
A.; Sen, G.; LopezAcosta, A. Vaccine 2006, 24, 716; (c)
Nazarpack-Kandlousy, N.; Chernushevich, I. V.; Meng,
L.; Yang, Y.; Eliseev, A. V. J. Am. Chem. Soc. 2000, 122,
3358; (d) Su, S.; Acquilano, D. E.; Arumugasamy, J.;
Beeler, A. B.; Eastwood, E. L.; Giguere, J. R.; Lan, P.;
Lei, X.; Min, G. K.; Yeager, A. R.; Zhou, Y.; Panek, J. S.;
Snyder, J. K.; Schaus, S. E.; Porco, J. A., Jr. Org. Lett.
2005, 7, 2751.
5. Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475.
6. Baindur, N.; Harris, S. M.; Labroo, V. M.; Combinatorial
non-peptide libraries, U.S. Patent No. 5,646,285, July 8,
1997 (Zymogenetics, Inc., USA).
7. The synthesis of unprotected (2S,4S)-4-aminooxyproline
hydrochloride has been reported: Phuket, S. R. N.;
Trifonov, L. S.; Yu, C. X.; Worthen, D. R.; Crooks, P.
A.; Rosenthal, G. A.; Freeman, J. W. Drug Dev. Res.
1999, 47, 170.
20
1
16. (4S)-8: ½aꢀ ꢁ 37:6 (c 1.27, CHCl3). H NMR (400 MHz,
CDCl3) dD7.35–7.23 (m, 10H), 6.82 (s, 0.50H), 6.74 (s,
0.50H), 5.23–4.99 (m, 4H), 4.56–4.45 (m, 2H), 3.78 (d,
J = 12.8 Hz, 0.5H), 3.73 (d, J = 12.8 Hz, 0.5H), 3.59 (dd,
J = 12.8, 4.8 Hz, 0.5H), 3.56 (dd, J = 12.8, 4.8 Hz, 0.5H),
2.51 (d, J = 4.8 Hz, 0.5H), 2.48 (d, J = 4.8 Hz, 0.5H), 2.21
(ddd, J = 18.8, 9.2, 4.8 Hz, 0.5H), 2.16 (ddd, J = 18.8, 9.2,
4.8 Hz, 0.5H), 1.41 (s, 4.5H), 1.40 (s, 4.5H). ESI-MS (+ve)
m/z: 493.1 (M+Na)+. HR-ESI/APCI MS cacld for
C25H30N2NaO7 (M+Na)+: 493.1951, Found: 493.1955.
20
17. (4R)-8: ½aꢀ ꢁ 30:4 (c 1.40, CHCl3). 1H NMR (400 MHz,
CDCl3) d D7.85 (s, 0.56H), 7.67 (s, 0.44H), 7.30–7.10 (m,
10H), 5.19–5.09 (m, 2H), 5.02–4.94 (m, 2H), 4.55–4.45 (m,
2H), 3.93 (d, J = 12.4 Hz, 0.56H), 3.82 (d, J = 12.4 Hz,
0.44H), 3.56 (m, 1H), 2.55 (m, 1H), 1.99 (m, 1H), 1.41 (s,
9H). ESI-MS (+ve) m/z: 493.1 (M+Na)+. HR-ESI/APCI
MS cacld for C25H30N2NaO7 (M+Na)+: 493.1951, Found:
493.1968.
20
1
18. (4S)-1: ½aꢀ ꢁ 26:4 (c 0.65, CHCl3). H NMR (400 MHz,
CDCl3) d D7.86–7.55 (m, 6H), 7.42–7.35 (m, 2H), 7.33–7.29
(m, 2H), 4.61–4.33 (m, 4H), 4.22 (m, 1H), 3.92 (d,
J = 12.8 Hz, 0.4H), 3.78 (d, J = 12.4 Hz, 0.6H), 3.50 (m,
1H), 2.79 (d, J = 14.8 Hz, 0.6H), 2.50 (d, J = 14.4 Hz,
0.4H), 2.36 (m, 0.4H), 2.16 (m, 0.6H), 1.46 (s, 9H). ESI-
MS (+ve) m/z: 491.1 (M+Na)+. HR-ESI/APCI MS cacld
for C25H28N2NaO7 (M+Na)+: 491.1794, Found: 491.1788.
20
19. (4R)-1: ½aꢀ ꢁ 41:2 (c 0.97, CHCl3). 1H NMR (400 MHz,
8. (4R)-4-hydroxy-L-proline is available from Sigma-Aldrich;
(3S)-3-hydroxy-L-proline is available from Acros
Organics.
CDCl3) dD7.76 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 7.6 Hz,
1H), 7.59–7.51 (m, 2H), 7.41–7.25 (m, 4.35H), 7.18
( 0.65H), 4.53–4.33 (m, 4H), 4.24 (t, J = 6.8 Hz, 0.65H),
4.13 (t, J = 6.8 Hz, 0.35H), 3.96 (d, J = 12.6 Hz, 0.35H),
3.85 (d, J = 12.6 Hz, 0.65H), 3.53 (m, 1H), 2.23 (m,
0.65H), 2.10 (m, 0.35H), 1.49 (s, 5.5H), 1.46 (s, 3.5H). ESI-
MS (+ve) m/z: 491.1 (M+Na)+. HR-ESI/APCI MS cacld
for C25H28N2NaO7 (M+Na)+: 491.1794, Found: 491.1800.
20
9. (4R)-5: ½aꢀ ꢁ 53:2 (c 2.02, CHCl3). 1H NMR (400 MHz,
CDCl3) dD7.32–7.18 (m, 10H), 5.23–5.11 (m, 2H), 5.01–
4.95 (m, 2H), 4.53 (m, 1H), 4.40 (m, 1H), 3.65–3.51 (m,
2H), 2.91 (br s, 1H), 2.26 (m, 1H), 2.02 (m, 1H).
20
10. 6: ½aꢀD ꢁ 63:8 (c 1.36, CHCl3). 1H NMR (400 MHz,
CDCl3) d 8.16 (AB, JAB = 8.8, 2.0 Hz, 2H), 8.03–7.97 (m,

F. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1096–1101
1101
20
20. (4R)-9: ½aꢀ ꢁ 30:5 (c 0.94, CHCl3). 1H NMR (400 MHz,
HR-ESI/APCI MS cacld for C27H24N2NaO8 (M+Na)+:
527.1430, Found: 527.1428.
CDCl3) dD7.31–7.19 (m, 19H), 7.16 (m, 1H), 7.10 (d,
J = 9.0 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.44 (s, 0.5H),
6.41 (s, 0.5H), 5.19–4.93 (m, 4H), 4.35 (t, J = 8.0 Hz,
0.5H), 4.24 (t, J = 8.0 Hz, 0.5H), 4.10 (m, 1H), 3.85 (d,
J = 12.0 Hz, 0.5H), 3.67 (d, J = 12.0 Hz, 0.5H), 3.47 (dd,
J = 8.0, 4.4 Hz, 0.5H), 3.44 (dd, J = 8.0, 4.4 Hz, 0.5H),
2.90 (s, 3 H), 2.25 (m, 1H), 1.87 (dd, J = 8.2, 5.0 Hz, 0.5H),
1.83 (dd, J = 8.2, 5.0 Hz, 0.5H). ESI-MS (+ve) m/z: 649.3
(M+Na)+. HR-ESI/APCI MS cacld for C40H38N2NaO5
(M+Na)+: 649.2678, Found: 649.2674.
20
26. (3R)-11: ½aꢀD ꢁ 33:0 (c 1.10, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.34–7.22 (m, 10H), 5.25–5.00 (m,
4H), 4.56 (m, 1H), 4.48 (d, J = 6.8 Hz, 0.4H), 4.44 (d,
J = 6.8 Hz, 0.6H), 3.68 (m, 1H), 3.51 (m, 1H), 2.85 (d,
J = 5.0 Hz, 0.6H), 2.79 (d, J = 5.0 Hz, 0.4H), 2.04 (m, 2H).
ESI-MS (+ve) m/z: 378.1 (M+Na)+. HR-ESI/APCI MS
cacld for C20H21NNaO5 (M+Na)+: 378.1317, Found:
378.1312.
20
27. 13: ½aꢀD þ 1:25 (c 1.65, CHCl3). 1H NMR (400 MHz,
20
1
21. (4S)-9: ½aꢀ ꢁ 25:5 (c 1.00, CHCl3). H NMR (400 MHz,
CDCl3) dD7.33–7.30 (m, 2H), 7.25–7.18 (m, 16H), 7.15–
7.02 (m, 6H), 6.40 (s, 0.5H), 6.38 (s, 0.5H), 5.18–5.05 (m,
2H), 4.96 (d, J = 11.6 Hz, 1H), 4.87 (d, J = 12.8 Hz, 0.5H),
4.75 (d, J = 12.4 Hz, 0.5H), 4.51 (dd, J = 8.0, 1.2 Hz,
0.5H), 4.40 (dd, J = 8.0, 1.2 Hz, 0.5H), 4.07 (m, 0.5H),
4.03 (m, 0.5H), 3.70 (d, J = 12.4 Hz, 0.5H), 3.62 (d,
J = 12.0 Hz, 0.5H), 3.45 (dd, J = 12.4, 4.8 Hz, 0.5H), 3.40
(dd, J = 12.4, 4.8 Hz, 0.5H), 2.39 (t, J = 12.0 Hz, 1H), 2.28
(s, 3H), 2.00 (m, 1H). ESI-MS (+ve) m/z: 649.2 (M+Na)+.
HR-ESI/APCI MS cacld for C40H38N2NaO5 (M+Na)+:
649.2678, Found: 649.2665.
CDCl3) d 7.84–7.81 (m, 2 H), 7.78–7.75 (m, 2 H), 7.41–
7.25 (m, 9H), 7.16 (m, 1H), 5.21–4.83 (m, 6H), 3.87 (m,
1H), 3.78 (m, 1H), 2.32 (m, 1H), 2.17 (m, 1H). ESI-MS
(+ve) m/z: 523.1 (M+Na)+. HR-ESI/APCI MS cacld for
C28H24N2NaO7 (M+Na)+: 523.1481, Found:523.1481.
20
28. 14: ½aꢀD ꢁ 17:2 (c 0.74, CHCl3). 1H NMR (400 MHz,
CDCl3) d 7.32–7.19 (m, 11H), 5.20–5.10 (m, 2H), 5.06–
4.98 (m, 2H), 4.71 (s, 0.45H), 462 (s, 0.55H), 4.48 (m, 1H),
3.70–3.56 (m, 2H), 2.13 (m, 1H), 2.02 (m, 1H), 1.41 (s, H).
ESI-MS (+ve) m/z: 493.1 (M+Na)+. HR-ESI/APCI MS
cacld for C25H30N2NaO7 (M+Na)+: 493.1951, Found:
493.1968.
20
20
1
1
22. (4S)-2: ½aꢀ ꢁ 17:9 (c 0.79, CHCl3). H NMR (400 MHz,
CDCl3) dD7.72–7.62 (m, 2 H), 7.50–7.36 (m, 2 H), 7.36–
7.28 (m, 2 H), 7.28–7.12 (m, 14H), 7.10–6.6 (m, 5H), 4.40–
4.00 (m, 4H), 3.90 (m, 1H), 3.40 (m, 1H), 3.26 (m, 1H),
2.60 (m, 1H), 2.27 (s, 3 H) 1.90 (m, 1H). ESI-MS (+ve)
m/z: 647.2 (M + Na)+. HR-ESI/APCI MS cacld for
C40H36N2NaO5 (M+Na)+: 647.2522, Found: 647.2518.
29. (3S)-3: ½aꢀD ꢁ 33:0 (c 0.60, CHCl3). H NMR (400 MHz,
CDCl3) 8.85 (br s, 1H), d 7.77–7.70 (m, 2.6H), 7.56–7.50
(m, 2.4H), 7.37–7.30 (m, 2H), 7.29–7.23 (m, 2H), 4.67–4.52
(m, 2H), 4.44 (m, 1H), 4.35 (m, 1H), 4.21 (t, J = 7.2 Hz,
0.6H), 4.12 (t, J = 7.2 Hz, 0.4 Hz), 3.69–3.58 (m, 2H),
2.23–2.00 (m, 2H), 1.44 (s, 9H). ESI-MS (+ve) m/z: 491.1
(M+Na)+. HR-ESI/APCI MS cacld for C25H28N2NaO7
(M+Na)+: 491.1794, Found: 491.1800.
30. Demirov, D. G.; Freed, E. O. Virus Res. 2004, 106, 87.
31. Mazze, F. M.; Degreve, L. Acta Virol. (English Edition)
2006, 50, 75.
32. 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.
33. Substitution of the Pro7 residue by alanine results in
significant reduction of Tsg101-binding affinity, indicating
the importance of the 7-position.
34. N-Terminal labeling with fluorosceine isothiocyanate
linked via an aminovaleric acid spacer (FITC-Ava-) was
employed.
35. A mixture of HPLC-purified aminooxy-proline containing
peptide (15–17) (15 mM in DMSO, 10 lL), aldehdye (18a–
18t) (15 mM in DMSO, 10 lL) and acetic acid (70 mM in
DMSO, 10 lL) was gently agitated at room temperature
(overnight). Examination by HPLC showed the reactions
had gone to completion to produce oxime products in
P90% purity. Crude reaction mixtures were used directly
for biological evaluation.
36. Based on a fluorescence anisotropy binding assay require-
ment of 20 mL of 5 mM peptide inhibitor solution,
0.20 mmol of TGR solid-phase resin is theoretically
sufficient to prepare a 740 member oxime library.
20
23. (4R)-2: ½aꢀ ꢁ 35:6 (c 0.64, CHCl3). 1H NMR (400 MHz,
CDCl3) dD7.71 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz,
1H), 7.54 (d, J = 7.2 Hz, 0.5H), 7.48 (d, J = 7.2 Hz, 0.5H),
7.42 (dd, J = 12.6, 7.4 Hz, 1H),7.35 (t, J = 7.6 Hz, 1H),
7.30 (dd, J = 11.8, 2.2 Hz, 1H), 7.25–7.16 (m, 13 H), 7.13
(d, J = 8.4 Hz, 2 H), 7.08–7.02 (m, 3 H), 6.92 (d, J = ,
0.5H), 6.82 (d, J = , 0.5H), 4.31–3.99 (m, 5H), 3.80 (d,
J = 12.0 Hz, 0.3 H), 3.60 (d, J = 12.0 Hz, 0.7H), 3.32 (dd,
J = 11.8, 4.2 Hz, 0.7H), 3.24 (dd, J = 12.2, 4.2 Hz, 0.3 H),
2.30 (s, 3 H), 2.20 (m, 1H), 1.87 (m, 0.7H), 1.73 (m, 0.3 H).
ESI-MS (+ve) m/z: 647.2 (M+Na)+. HR-ESI/APCI MS
cacld for C40H36N2NaO5 (M+Na)+: 647.2522, Found:
647.2511.
20
24. (3S)-11: ½aꢀD ꢁ 24:7 (c 1.64, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.38–7.20 (m, 10H), 5.22–5.13 (m,
2 H), 5.09–5.00 (m, 2 H), 4.45 (m, 1H), 4.42 (m, 0.5H),
4.33 (m, 0.5H), 3.75–3.62 (m, 2 H), 2.24 (br s, 1H), 2.08
(m, 1H), 1.91 (m, 1H). ESI-MS (+ve) m/z: 356.1 (M+H)+.
HR-ESI/APCI MS cacld for C20H21NNaO5 (M+Na)+:
378.1317, Found: 378.1316.
20
25. 12: ½aꢀD ꢁ 91:4 (c 0.80, CHCl3). 1H NMR (400 MHz,
CDCl3) d 8.14 (t, J = 9.2 Hz, 2 H), 7.93 (t, J = 9.2 Hz, 2
H), 7.41–7.26 (m, 5H), 7.15–7.07 (m, 5H), 5.75 (m, 1H),
5.24–5.05 (m, 3 H), 4.98–4.78 (m, 2 H), 3.85–3.69 (m, 2 H),
2.38–2.25 (m, 2 H). ESI-MS (+ve) m/z: 527.1 (M+Na)+.