Synthesis and NMR elucidation of pentacycloundecane-derived hydroxy acid peptides as potential anti-HIV-1 agents

Article abstract of DOI:10.1007/s11224-012-0164-2

The synthesis and NMR elucidation of eight novel peptides incorporating the pentacycloundecane (PCU)-derived hydroxy acid are reported. The PCU cage amino acids were synthesized as racemates and the incorporation of the PCU-derived hydroxy acid with natural (S)-amino acids produced inseparable diastereomeric peptides. A series of overlapping signals from the cage and that of the peptide side chain was observed in the ¹H- and ¹³C-NMR spectra, complicating the elucidation thereof. Two-dimensional NMR techniques proved to be a very useful tool in overcoming these difficulties. These compounds are potential HIV protease inhibitors.

Full text of DOI:10.1007/s11224-012-0164-2

Struct Chem
DOI 10.1007/s11224-012-0164-2
ORIGINAL RESEARCH
Synthesis and NMR elucidation of pentacycloundecane-derived
hydroxy acid peptides as potential anti-HIV-1 agents
•
Rajshekhar Karpoormath Fernando Albericio
•
•
Thavendran Govender Glenn E. M. Maguire
•
Hendrik G. Kruger
Received: 19 July 2012 / Accepted: 8 November 2012
Ó Springer Science+Business Media New York 2012
Abstract The synthesis and NMR elucidation of eight
novel peptides incorporating the pentacycloundecane
(PCU)-derived hydroxy acid are reported. The PCU cage
amino acids were synthesized as racemates and the incor-
poration of the PCU-derived hydroxy acid with natural (S)-
amino acids produced inseparable diastereomeric peptides.
A series of overlapping signals from the cage and that of
Keywords 2D NMR ꢀ Pentacycloundecane ꢀ PCU ꢀ
Cage peptides ꢀ HIV inhibitors
Introduction
Polycyclic cage compounds have been studied extensively
over the past few decades [1–24]. South African authors
have contributed considerably toward utilizing NMR
spectroscopy as a tool to determine the structures of these
cage compounds, several of which have found application
in medicinal chemistry [10, 15, 19, 24–45]. The assign-
ments of 1H spectra of the cage compounds becomes more
challenging due to broad unresolved overlapping reso-
nances as a result of through-space effects, geminal and
vicinal proton–proton coupling, and long-range proton–
proton interactions. Our research group has extensively
used two-dimensional (2D) NMR spectroscopic techniques
to study the structure of the cage skeleton in relation to its
side ‘‘arms’’ [19, 24, 37–45]. As a part of our ongoing
research program, the synthesis of eight pentacyclounde-
cane (PCU)-derived hydroxy acid peptides (1–8) was
attempted (Fig. 1).
1
the peptide side chain was observed in the H- and 13C-
NMR spectra, complicating the elucidation thereof. Two-
dimensional NMR techniques proved to be a very useful
tool in overcoming these difficulties. These compounds are
potential HIV protease inhibitors.
Electronic supplementary material The online version of this
article (doi:10.1007/s11224-012-0164-2) contains supplementary
material, which is available to authorized users.
R. Karpoormath
School of Pharmaceutical Sciences, University
of KwaZulu-Natal, Durban 4001, South Africa
F. Albericio ꢀ T. Govender ꢀ G. E. M. Maguire ꢀ
H. G. Kruger (&)
Catalysis and Peptide Research Unit, School of Health Sciences,
University of KwaZulu-Natal, Durban 4001, South Africa
e-mail: kruger@ukzn.ac.za
The use of cage compounds in drug design has found
several applications in the pharmaceutical drug discovery
research [11, 15, 16, 19–24, 44–48]. Cage-based com-
pounds have been reported to improve drug lipophilicity; it
also serves as a transport medium in facilitating drug
delivery through cell membranes such as the blood–brain
barrier (BBB) and the central nervous system (CNS) [2, 4,
23, 49]. The cage compounds increase the duration of drug
action by retarding the action of drug metabolism [13, 50].
In this study, the PCU hydroxy acid [33] incorporated
into short peptides was used to mimic an irreversible non-
scissile ether-bridged type isostere as potential HIV-1
F. Albericio
Institute for Research in Biomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028 Barcelona, Spain
F. Albericio
CIBER-BBN, Networking Centre on Bioengineering,
Biomaterials and Nanomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028 Barcelona, Spain
F. Albericio
Department of Organic Chemistry, University of Barcelona,
´ ´
Martı i Franques 1-11, 08028 Barcelona, Spain
123

Struct Chem
Ha Hs
Ha Hs
4
4
Ha Hs
4
5
3
5
3
5
3
6 2
6 2
6 2
10
9
25d
9
10
9
10
O
O
7 1
O
25c
25b
25c
25b
O
7 1
O
8
11
12
7 1
O
8
11
12
8
11
12
HO
HO
HO
13
NH
14 (S)
13
NH
13
NH
O
O
25a
24
O
H
N
H
20
(S)
20
H
20
14
15
17
(S1) 8 N
19
16
16
14
15
17
23
O
16
(S)
(S1) 8 N
19
18
(S)
22
23
19
OH
OH
22b
22
15
17
O
O
22c
22d
O
21
21
21
22a
22b
22c
1
2
3
Ha Hs
Ha Hs
Ha Hs
4
4
4
5
3
5
3
5
3
6 2
6 2
6 2
9
10
9
9
10
10
O
O
O
7 1
O
7 1
O
7 1
O
8
11
12
8
11
12
8
11
12
HO
HO
HO
13
13
13
25
26
NH
O
NH
O
NH
O
H
(S)
H
H
19
19
(S)
14
(S)
(S)
14
14
23
24
24
26
16 N 18
OH
26
O
(S)
17 N (S)
OH
17 N
18
27 OH
(S)
24
(S)
25
22
19
23
20
23
20
15
17
N
N 25
N
18
15
15
16a
16a
H
H
H
O
O
O
O
O
16b
16b
16b
16b
20
21
21
22
21
22
16c
16c
16c
16c
16d
16d
4
5
6
Ha Hs
Ha Hs
4
4
5
3
5
3
6 2
6 2
30c
30b
27c
9
9
10
10
30b
30a
29
30d
30c
O
27b
27a
26
27d
27c
O
7 1
O
7 1
O
8
11
12
8
11
12
HO
HO
13
NH
(S)
13
26
O
NH
O
27b
OH
H
H
19
19
(S)
14
15
14
24
N
24
N
27
O
(S) 23
20
28
17 N
18
O
17 N (S)
(S)
25
(S)
23
20
28
18
15
25
16a
H
16a
H
O
O
O
16b
16c
16b
16c
16b
16b
21
22
21
22
16c
16c
16d
16d
8
7
Fig. 1 Structures of compounds 1-8
protease inhibitors analogs to a non-cage ether reported by
Ghosh et al. [51]. It was shown that the cage ether bridge
(C9–C8–O–C11–C10) is virtually non-hydrolyzable under
conditions that were harsher than physiological conditions
[33]. Compounds 1, 5, and 6 (Fig. 1) gave promising
in vitro activity against HIV-1 wild-type C-SA protease
enzyme with IC50 values ranging from 0.6 to 2 lm [22].
aqueous sodium cyanide stirred in a mixture of water and
acetic acid resulted in a mixture of cyanohydrins (10 and
11) [33]. This cyanohydrin mixture, when treated with
concentrated hydrochloric acid, gave the tri-hydroxy car-
boxylic acid 12 [33]. The resulting tri-hydroxy carboxylic
acid 12 was refluxed in 1,4-dioxane to give the desired
hydroxy acid 13 as a racemate. These reactions are outlined
in Scheme 1.
Compounds 1–8 were obtained by coupling the corre-
sponding short peptides to 13 in the presence of coupling
reagents HATU, DIPEA in DMF at ambient temperatures
as shown in Scheme 2. It should be noted that the resulting
cage peptides are mixtures of two diastereomers as the
PCU hydroxy cage acid was synthesized as a racemate.
The products 1–8 (Fig. 1) were isolated and purified as
described in the ‘‘Experimental’’ section. It was not
Synthesis
The PCU peptide derivatives 1–8 were synthesized from
the cage ether 13, with Cookson’s dione 9 [3, 7, 52] as the
starting material. The latter can be easily obtained from the
photocyclization of the Diels–Alder adduct of cyclopenta-
diene and p-benzoquinone. Treatment of the dione 9 with
123

Struct Chem
i
ii
+
HO
CN
HO
CN
COOH
O
O
O
O
HO
OH HO
9
10
11
12
iii
O
HO
O
OH
13
Scheme 1 Synthesis of 5-hydroxy-4- oxahexacyclo[5.4.1.02,6.03,10.05,9.08,11]dodecane -3-carboxylic acid 13; i NaCN, CH3COOH, H2O, 0 °C;
ii conc. HCl, reflux; iii 1,4-dioxane, reflux
the case of H-4s a pair of doublets is observed resonating at
i
1.82 and 1.84 ppm with a J value of 6.0 Hz. Similar obser-
vations are recorded for compounds 5 and 6, which can
perhaps be attributed to a side-chain conformational effect.
For compound 3, the H-4s signal at 1.83 ppm (J = 9.4 Hz)
appears as a triplet due to coalescence of the pair of doublets.
In PCU cage compounds, proton H-4a (H-4s) normally
exhibits COSY and NOESY/ROESY interactions with H-3/5
and H-2/6 (H-3/5 and H-9/10), respectively, which has proven
to be a convenient handle in solving the NMR spectra of these
compounds. A similar interaction is also observed between
H-2/6 and H-1/7. HMBC, NOESY, and ROESY techniques
are useful methods to determine the position of the ‘‘arms’’/
substituents in relation to the cage moiety especially when the
cage is asymmetric.
O
O
R
HO
HO
O
O
OH
1-8 R=Peptide
13
Scheme 2 Synthesis of 5-hydroxy-4-oxahexacyclo[5.4.1.02,6.03,10
5,9.08,11]dodecane peptide analogs. i HATU, DIPEA, DMF, rt
.
0
possible to separate the diastereomeric mixtures by pre-
parative HPLC. The correctness of the cage peptides were
confirmed with standard NMR and MS techniques. The
synthesized cage peptides were obtained in high purity
([98 %) which was determined with HPLC. These syn-
thesized compounds 1–8 are polar and lipophilic in nature
with CLogP values -0.76, 1.08, 1.17, -1.03, 0.46, 0.81,
2.65, and 2.74, respectively.
In particular, both H-4a (1.47 ppm) and H-4s
(1.83 ppm) of 1 display COSY correlations to the reso-
nances at 2.52 and 2.46 ppm. These resonances were
assigned to H-3/H-5. As the molecule is asymmetric, the
specific assignment is still pending. In addition, the H-4a
resonance shows a NOESY correlation to 2.66 and
2.68 ppm (H-2/H-6) and the H-4s resonance to 2.52 and
2.71 ppm (H-9/H-10). In order to distinguish between the
proton resonances of H-9 and H-10, a HMBC correlation
between carbonyl resonance C-12 (170.5 ppm) and the
protons resonance at 2.71 ppm indicated that this reso-
nance is associated to H-10. The position of H-10 was
further confirmed as it displayed COSY correlation to a
proton signal at 2.52 ppm, which was assigned to H-9. The
carbonyl resonance of C-12 also displays a HMBC corre-
lation to H-10 and H-1 (2.75 ppm). The latter in turn
showed a COSY correlation to H-2 (2.66 ppm). A COSY
correlation between the resonance at 2.52 ppm (also H-9)
and the signal of H-10 as well as H-2 was observed,
indicating that the proton signal of H-3 and H-9 are over-
lapping. This was further confirmed by the HMBC spec-
trum, wherein the carbon peaks of C-3 (46.1 ppm) and C-9
Results and discussion
The PCU cage structures show complex overlapping of the
protons as the PCU cage derivatives (1–8) are diastereo-
meric, asymmetric, and non-meso compounds which further
complicates the structural elucidation of these compounds.
Due to the diastereomeric nature of these compounds, all the
13C NMR signals of these compounds (1–8) are split. It is
known from the literature that the proton NMR spectrum of
PCU cage compounds usually show a pair of doublets
(geminal protons) with an AB spin resonance at approxi-
mately 1.5 and 1.8 ppm, having a coupling constant of about
&10 Hz. The assignments for all PCU cage compounds 1–8
are presented in Tables 1 and 2.
The geminal bridge methylene proton (H-4a) for the PCU
hydroxy peptide 1 register as a doublet (AB system) at
1.47 ppm (H-4a) with a coupling constant of 10.4 Hz, but in
123

Struct Chem
Table 1 1H NMR data for PCU hydroxy peptides 1–8
Atom
1 d1HB,C
2 d1HA,C
3 d1HB,C
4 d1HA,C
5 d1HB,C
6 d1HB,C
7 d1HA,C
8 d1HA,C
1
2.75
2.66
2.52
2.77
2.65
2.51
2.72
2.58
2.45
2.76
2.63
2.47
2.73
2.59
2.44
2.73
2.59
2.46
2.71
2.57
2.43
2.72
2
2.58
3
2.45
4a
1.47 d,
1.47 d,
1.47 d,
1.48 d,
1.44 d,
1.45 d,
1.44 d,
1.44 d,
J = 10.4 Hz
J = 10.1 Hz
J = 9.3 Hz
J = 10.2 Hz
J = 10.2 Hz
J = 10.1 Hz
J = 10.2 Hz
J = 10.1 Hz
1.79 d,
J = 10.1 Hz
4s
1.82/1.84 d,
J = 6.0 Hz,
H-4 s/4sbD
1.83 d,
1.82 t,
1.83 d,
1.79/1.80 d,
J = 6.0 Hz,
H-4 s/4sbD
1.79/1.80 d,
J = 6.1 Hz,
H-4 s/4sbD
1.78
J = 10.1 Hz
J = 9.3 Hz,
H-4 sE
J = 10.2 Hz
d,J = 10.2 Hz
5
2.46
2.68
2.48
–
2.49
2.66
2.51
–
2.44
2.64
2.47
–
2.47
2.67
2.50
–
2.45
2.65
2.44
–
2.45
2.67
2.42
–
2.43
2.66
2.46
–
2.44
2.64
2.45
–
6
7
8
9
2.52
2.71
–
2.55
2.69
–
2.49
2.66
–
2.51
2.68
–
2.47
2.65
–
2.48
2.65
–
2.45
2.66
–
2.50
2.64
–
10
11
12
13
–
–
–
–
–
–
–
–
6.91/6.93 d,
J = 3.1 Hz,
NH-13a/13bD
6.95 d,
6.88/6.90 d,
J = 5.7 Hz,
NH-13a/13bD
7.48/7.50 d,
J = 3.2 Hz,
NH-13a/13bD
7.40 t,
7.44 t,
7.38/7.41 d,
J = 5.5 Hz,
NH-13a/13bD
7.44/7.46 d,
J = 6.3 Hz,
NH-13a/13bD
J = 9.3 Hz
J = 9.0 Hz,
NH-13E
J = 9.0 Hz,
NH-13E
14
15
16/
4.25
1.97
4.16
4.24
1.93
4.37
1.22
–
4.62
4.60
4.61
4.60
1.92
2.99
2.99
2.90/2.98
7.08–7.16
2.97
0.79/0.81 d,
J = 6.7 Hz,
H-16a/16bF
0.73-0.77
0.74/0.77 d,
J = 6.7 Hz,
H-16a/16bF
7.08–7.23
7.11–7.24
7.10–7.22
16b2d
17
18
19
20
21
0.86/0.88 d,
J = 6.7 Hz,
H-17a/17bF
0.73-0.77
–
0.82/0.84 d,
J = 6.7 Hz,
H-17a/17bF
7.81/7.87 d,
J = 9.0 Hz,
NH-17a/17bD
–
–
–
–
–
–
4.21
8.01/8.05 d,
J = 9.0 Hz,
NH-18a/18bD
7.97 d/8.02,
J = 9.0 Hz,
NH-18a/18bD
7.93/7.98 d,
J = 9.1 Hz,
NH-18a/18bD
8.01/8.06 d,
J = 9.0 Hz,
NH-18a/18bD
8.44 t,
8.40 t,
8.60/8.61 d,
J = 6.8 Hz,
NH-19a/19bD
1.94
4.24
1.97
0.87
4.23
4.25
1.96
0.83
4.23
1.92
0.83
J = 8.0 Hz,
NH-19E
J = 7.1 Hz,
NH-19E
4.19 q,
4.44
4.33
0.82/0.84 d,
J = 3.9 Hz,
H-20a/20bF
1.96
J = 7.4 Hz
1.26/1.27 d,
J = 5.2 Hz,
H-21a/21bF
2.85/3.07
1.30/1.31 d,
J = 5.2 Hz,
H-21a/21bF
–
0.87/0.88 d,
J = 3.4 Hz,
H-21a/21bF
0.86 t,
J = 6.0 Hz,
H-21E
22/
22b2d
–
–
–
7.17-7.27
–
0.87
–
0.89/0.90 d,
J = 4.6 Hz,
H-22a/22bF
0.83
–
0.83
–
23
24
25/
–
–
–
8.25/8.27 d,
J = 3.4 Hz,
NH-23a/23bD
–
5.10 d,
4.21
8.36
8.36 t,
8.33 d,
8.55/8.57 d,
J = 4.6 Hz,
NH-24a/24bD
J = 1.3 Hz
J = 6.3 Hz,
H-24E
J = 7.2 Hz
–
–
–
–
–
–
7.30-7.37
1.25
3.78
4.20
4.45
4.35
25b2d
26
–
–
–
–
–
–
–
–
–
1.29 d,
2.92/3.05
7.17-7.25
–
1.32 d,
J = 7.2 Hz
J = 7.2 Hz
27/
27b2d
–
–
–
–
28
123

Struct Chem
Table 1 continued
Atom
1 d1HB,C
2 d1HA,C
3 d1HB,C
4 d1HA,C
5 d1HB,C
6 d1HB,C
7 d1HA,C
8 d1HA,C
29
–
–
–
–
–
–
–
–
–
–
–
–
5.11
30/
30b2d
7.33–7.37
A
B
C
D
E
F
400 MHz
600 MHz
Solvent (CD3)2SO
Two separate resonances potentially due to a side-chain conformational effect
Due to coalescence of the pair of doublets, the signal appears as a triplet
The methyl protons appear as a pair of doublets
(56.2 ppm) displayed correlation to the same proton signal
resonating at 2.52 ppm (H-3). Since now the position of
H-3 (2.52 ppm) is confirmed, therefore, the resonance at
2.46 ppm was attributed to H-5. In addition to the COSY
correlation between H-1 and H-2, a further correlation
between H-1 and 2.48 ppm allowed this resonance to be
assigned to H-7. In the 13C NMR spectrum, the quaternary
carbon signals appearing at 118.2 and 89.75 ppm were
assigned to C-8 and C-11 based on their deshielding effect
and chemical environment. All the remaining cage carbon
signals were obtained from the HSQC spectrum.
The H-21 methyl signal shows a correlation with the
chiral methine proton H-20 appearing as a quartet signal at
4.19 ppm (J = 7.40 Hz). H-20 further shows a COSY cor-
relation to a triplet proton signal at 8.44 ppm (H-19,
J = 8.04 Hz). This pair of doublets coalesces into a triplet
signal at 8.44 ppm. All the compounds 1–8 give perfect pairs
of doublets for the corresponding NH proton signals. From
literature, it is known that NH proton signals in linear pep-
tides usually appear as doublets [53]. As these PCU cage
peptides are a mixture of diastereomers, the amide protons
appear as a pair of doublets. The remaining carbonyl peak
registers at 173.8 ppm in the 13C NMR spectrum and was
assigned to C-22. This was further confirmed by HMBC
spectra wherein H-21 displays correlation to the terminal
carbonyl carbon C-22. All other assignments were further
verified utilizing HSQC and HMBC spectra. ROESY cor-
relations were observed between H-13 of the peptide chain
and the PCU cage protons (H-1, H-2 and H-10). Further to
this, the isopropyl methyl protons H-16/H-17 displayed
ROESY correlations to H-1, H-4a, and H-4s.
The next section describes the NMR correlations for the
rest of the peptide sequence attached to the PCU cage of
compound 1. The methyl (alanine) and isopropyl methyl
(valine) carbons register at 17.1, 17.8, and 19.1 ppm and
were identified from the APT 13C NMR spectrum. The car-
bon signal at 17.1 ppm exhibits HSQC correlations to the 1H
NMR signals appearing as a pair doublets at 1.26 and
1.27 ppm (J = 5.20 Hz), similarly the isopropyl methyl
carbon signals 17.8 and 19.1 ppm display correlations to two
pairs of doublets at 0.79/0.81 and 0.86/0.88 ppm with a
coupling constant approximately &6.7 Hz. The isopropyl
methyl groups are also split due to the asymmetric nature of
the environment close by. Hence, these side-chain methyl
protons were assigned to H-21 (1.26/1.27 ppm), H-16 (0.79/
0.81 ppm), and H-17 (0.86/0.88 ppm), respectively. The
H-15 methine signal at 1.97 ppm was identified by a COSY
correlation to the isopropyl methyl protons H-16 and H-17.
This H-15 resonance in turn shows a COSY interaction with
the chiral H-14 methine proton signal appearing as a multi-
plet at 4.25 ppm. The chiral H-14 proton further displays
COSY correlation to H-13 (NH) a doublet signal resonating
at 6.91 ppm (J = 4.1 Hz). For this resonance, a pair of
doublets is seen because of the diastereomeric nature of the
compound, which is further validated by the APT 13C NMR
spectrum where the carbonyl carbon (C-12) is registered as a
split signal. The H-15 resonance shows a HMBC correlation
to a carbonyl carbon registered at 170.2 ppm. This was then
assigned to C-18.
In NOESY experiments, the nuclear Overhauser effect
(NOE) depends on the correlation time sc of the compound
and the observation frequency x. As the NOE changes sign
at xsc & 1.12, leading to little or no NOE observations for
medium-sized compounds (MW &1,000 g mol-1) [54].
This results in poor correlation signals and also prevents
the extraction of distance information from the NOESY
spectra. In order to overcome this problem, ROESY
experiments were carried out for compounds 1, 4, 5, 6, and
7 to look for interactions between closely related protons.
As ROESY spectrum yields negative cross-peaks (corre-
sponding to positive Overhauser enhancements) for all
values of xsc [55–57]. For the remainder of the compounds
2–8, the same procedure was used for the elucidation of the
cage signals. This discussion for the remaining compounds
will be omitted for the sake of brevity. For the peptide side
chains, only differences to previous compounds will be
highlighted. The assignments for compound 1 are sum-
marized in Tables 1 and 2.
123

Struct Chem
Table 2 13C NMR chemical shifts of compounds 1–8
Atom 1 d13CB,C
2 d13CA,C
3 d13CB,C
4 d13CA,C
5 d13CB,C
6 d13CB,C
7 d13CA,C
8 d13CA,C
1
48.9 (CH)
42.2 (CH)
46.1 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
41.1 (CH)
46.3 (CH)
118.2 (C)
56.2 (CH)
58.6 (CH)
89.8 (C)
48.7 (CH)
42.1 (CH)
46.0 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
40.9 (CH)
46.2 (CH)
118.1 (C)
56.4 (CH)
58.5 (CH)
89.6 (C)
48.2 (CH)
42.2 (CH)
46.2 (CH)
42.8 (CH2)
42.8 (CH2)
42.8 (CH)
41.0 (CH)
46.4 (CH)
118.2 (C)
56.2 (CH)
57.9 (CH)
89.7 (C)
48.4 (CH)
42.2 (CH)
46.1 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
41.0 (CH)
46.3 (CH)
118.2 (C)
56.2 (CH)
58.2 (CH)
89.5 (C)
48.1 (CH)
42.1 (CH)
46.2 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
40.9 (CH)
46.2 (CH)
118.1 (C)
56.1 (CH)
57.9 (CH)
89.6 (C)
48.2 (CH)
42.1 (CH)
46.1 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
40.9 (CH)
46.3 (CH)
118.2 (C)
56.1 (CH)
57.9 (CH)
89.5 (C)
48.1 (CH)
42.0 (CH)
46.0 (CH)
42.8 (CH2)
42.8 (CH2)
42.7 (CH)
41.0 (CH)
46.3 (CH)
118.1 (C)
56.1 (CH)
58.2 (CH)
89.5 (C)
48.2 (CH)
42.1 (CH)
46.1 (CH)
42.8 (CH2)
42.8 (CH2)
42.8 (CH)
41.0 (CH)
46.4 (CH)
118.2 (C)
56.2 (CH)
57.9 (CH)
89.6 (C)
2
3
4a
4s
5
6
7
8
9
10
11
12
13
14
15
16/
170.5 (C)
–
170.4 (C)
–
170.5 (C)
–
170.3 (C)
–
170.5 (C)
–
170.4 (C)
–
170.7 (C)
–
170.5 (C)
–
56.4 (CH)
31.5 (CH)
56.5 (CH)
31.4 (CH)
55.9 (CH)
31.5 (CH)
47.8 (CH)
18.6 (CH3)
171.8 (C)
52.9 (CH)
37.3 (CH2)
137.3 (C)
53.1 (CH)
37.4 (CH2)
137.3 (C)
52.9 (CH)
37.4 (CH2)
137.4 (C)
53.1 (CH)
37.3 (CH2)
137.4 (C)
17.8/19.1
(CH3)
17.8/19.0
(CH3)
17.7/19.0
(CH3)
16a
16b
16c
16d
17
–
–
–
–
–
–
–
–
–
–
–
–
–
129.3 (CH)
127.9 (CH)
126.2 (CH)
170.3 (C)
129.2 (CH)
127.9 (CH)
126.2 (CH)
170.2 (C)
129.2 (CH)
128.1 (CH)
126.3 (CH)
170.3 (C)
129.2 (CH)
128.0 (CH)
126.2 (CH)
170.3 (C)
17.8/19.1
(CH3)
17.8/19.0
(CH3)
17.7/19.0
(CH3)
18
19
20
170.1 (C)
–
170.4 (C)
–
170.4
–
56.8 (CH)
31.1 (CH)
–
–
–
–
57.2 (CH)
30.9 (CH)
57.0 (CH)
31.1 (CH)
57.0 (CH)
31.1 (CH)
56.9 (CH)
31.1 (CH)
47.2 (CH)
53.3 (CH)
47.6 (CH)
17.9/19.1
(CH3)
21
17.1 (CH3)
173.9 (C)
36.6 (CH2)
137.4 (C)
16.7 (CH3)
172.1 (C)
17.9/19.1
(CH3)
18.0/19.0
(CH3)
18.1/19.0
(CH3)
17.9/19.1
(CH3)
18.1/19.0
(CH3)
22/
170.3 (C)
18.0/19.0
(CH3)
18.1/19.0
(CH3)
17.9/19.1
(CH3)
18.1/19.0
(CH3)
22a
22b
22c
22d
23
–
–
–
–
–
–
128.9 (CH)
128.1 (CH)
126.3 (CH)
172.7 (C)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
171.0 (C)
–
170.5 (C)
–
170.5 (C)
–
170.6 (C)
–
24
66.0 (CH2)
138.8 (C)
47.4 (CH)
17.0 (CH3)
25/
–
40.5 (CH2)
47.4 (CH)
53.4 (CH)
47.6 (CH)
25a
25b
25c
25d
26
–
–
–
–
–
–
–
–
–
–
127.8 (CH)
–
–
–
–
–
128.0 (CH)
–
–
–
–
–
128.4 (CH)
–
–
–
–
–
–
–
173.9 (C)
–
171.1 (C)
–
16.9 (CH3)
173.9 (C)
36.5 (CH2)
137.4 (C)
16.7 (CH3)
172.2 (C)
27/
27a
27b
27c
27d
28
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
128.9 (CH)
127.8 (CH)
126.2 (CH)
172.7 (C)
–
–
–
–
–
29
30a
65.9 (CH2)
135.9 (C)
–
123

Struct Chem
Table 2 continued
Atom 1 d13CB,C
2 d13CA,C
3 d13CB,C
4 d13CA,C
5 d13CB,C
6 d13CB,C
7 d13CA,C
8 d13CA,C
30b
30c
30d
A
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
127.8 (CH)
128.4 (CH)
127.9 (CH)
100 MHz for 13C
150 MHz for 13C
Solvent (CD3)2SO
B
C
1
For compound 2, the aromatic protons in the H NMR
cage protons H-5, H-7, and H-9, whereas in compound 6
H-13 displays ROESY correlations to the proton signals
H-3, H-10, H-21/22, and H-26. For compound 7, the aro-
matic protons H-16 and H-27 display ROESY correlations
to the isopropyl methyl protons H-21/22. Similarly, in
compound 8, the isopropyl protons H-21/22 display NO-
ESY correlations to the aromatic protons H-30 and the cage
spectra are not well resolved, but the carbons for the aro-
matic region in the 13C NMR spectra are very well sepa-
rated. In the HMBC spectrum, the chiral methine proton
H-20 (4.44 ppm) displays a correlation to the quaternary
carbon C-22a (137.4 ppm) of the phenyl ring and the dia-
stereomeric methylene protons H-21a/H-21b (2.85/
3.07 ppm) show correlations to the aromatic carbon C-22b
(128.9 ppm). Carbon signals for C-22c (128.1 ppm) and
C-22d (126.3 ppm) were assigned [58, 59] based on the
intensity of the peaks.
1
proton H-1. The H- and 13C-NMR peak assignments of
compounds 4–8 are given in Tables 1 and 2.
The carbonyl signal at 170.4 ppm for compound 2
shows HMBC correlations to various proton signals (H-20,
H-19, H-15, H-14, H-13, H-10, and H-1), which confirms
the overlapping of the carbonyl carbon signals of C-18 and
C-12. The methodology to elucidate the remaining signals
of the peptide side chain was similar to that described for
compound 1. NOESY correlations were observed between
H-13 and H-1. The aromatic protons (H-22) displayed
through space correlations to H-9 and H-6 of the cage
protons. The assignments of the peaks were further con-
firmed by HMBC and HSQC spectra. These assignments
are presented in Tables 1 and 2.
In the 1H NMR spectra of compound 3, a doublet
(diastereomeric) methylene proton signal (H-24) resonating
at 5.10 ppm (J = 1.38 Hz) and integrating to two protons
shows HMBC correlation to C-22 (172.1 ppm). H-24 fur-
ther shows HMBC correlations to a quaternary and aro-
matic carbon signals registered at 138.8 and 127.8 ppm,
which were assigned to C-25a and C-25b, respectively.
These assignments were further confirmed from the 13C-
APT spectra. Based on the intensity of the peaks [58, 59],
the carbon signals registered at 128.0 and 128.4 ppm were
assigned to C-25c and C-25d. Through-space interactions
were observed in the NOESY spectrum between the PCU
cage protons (H-4a, H-1, H-2, and H-10) and the methyl
protons of the peptide chain (H-16 and H-17). The 1H- and
13C-NMR assignments are shown in Tables 1 and 2.
For compound 4, no ROESY correlations were observed
between the PCU cage protons and the peptide chain. In
ROESY spectrum of compound 5, the aromatic protons
H-16 displayed through space correlation with the PCU
Conclusion
The synthesis and complete NMR elucidation of eight
novel PCU cage peptides were successfully carried out.
The synthesis was achieved utilizing known protocols from
the literature. The NOESY and ROESY experiments
showed interesting through space correlations between the
cage side chain and the PCU cage protons. Compounds 1–8
are all non-separable diastereomers and are unsymmetrical
in nature, which complicated the assignments of PCU cage
protons. However, 2D NMR spectroscopy proved to be a
crucial tool for effective structural elucidation of these
compounds in spite of major overlapping of proton signals.
All the 13C carbon signals for these compounds are split.
Incorporation of the PCU hydroxy cage acid 13 into the
peptides as potential HIV-1 protease inhibitors was
successful.
Experimental
Analysis was performed on an Agilent 1100 HPLC (Waters
Xbridge C18 150 mm 9 4.6 mm 9 5 ls) coupled to a UV
detector (215 nm) and an Agilent VL ion trap mass spec-
trophotometer in the positive mode. Semi-preparative
HPLC was carried out on a Shimadzu 8A instrument (Ace
C18 150 mm 9 21.2 mm 9 5 ls) with a UV/Vis detector
(215 nm) and automated fraction collector. A two-buffer
system was employed, utilizing formic acid as the ion-
pairing agent. Buffer A consisted of 0.1 % formic acid/
H2O (v/v) and buffer B consisted of 0.1 % formic acid/
123

Struct Chem
acetonitrile (v/v). High-resolution electron spray ionization
mass spectroscopic (HR-ESI-MS) analysis was performed
on a Bruker MicroTOF QII mass spectrometer in positive
mode with an internal calibration. Microwave couplings
were conducted on a Discovery CEM Liberty microwave
peptide synthesizer. The coupling conditions of the peptide
synthesizer were adapted from literature [19, 20]. Optical
rotations were measured at room temperature in dry
methanol using a Perkin Elmer Polarimeter-Model 341. All
IR spectra were recorded on a Perkin Elmer Spectrum 100
instrument with a universal ATR attachment.
spectral widths for the 13C spectra are 12335.526,
33333.332 Hz (HSQC and HMBC) for 1, 3, 5, and 6;
5813.954, 16666.666 (HSQC), 5813.954, 27932.961 Hz
(HMBC) for 2; 5952.381, 16666.666 (HSQC); 5952.381,
27932.961 Hz (HMBC) for 4; 5747.126; 16666.666 Hz
(HSQC), 5747.126, 27932.961 Hz (HMBC) for 7; and
3703.704 Hz, 16666.666 Hz (HSQC), 3703.704 Hz,
27932.961 Hz (HMBC) for 8. The number of data points per
spectrum in F2 are 2048 (COSY, NOESY/ROESY for 1–8),
2048 (HSQC for 1, 3, 5, and 6), 1024 (HSQC for 2, 4, 7, and
8), and 4096 (HMBC for 1–8), whereas the number of time-
incremented spectra in F1 were 128 (COSY), 256 (NOESY
for 2–4 and 8), 512 (ROESY for 5–7 and 1), 128 (HMBC),
and 256 (HSQC) for compounds 1–8; relaxation delay was
3.0 s (COSY and HMBC for 1, 3, 5, and 6), 1.5 s (COSY for
2, 4, 7, and 8), 1.5 s (ROESY for 1 and 5–7), 1.5 s (HSQC
and HMBC for 2, 4, 7, and 8), 2.0 s (NOESY for 2–4 and 8)
and 1.0 s (HSQC for 1, 3, 5, and 6). The spectra acquired in
phase-sensitive mode for all compounds are NOESY/
ROESY and HSQC; the spectra for all compounds were
acquired in absolute value mode. Gradients were used
for COSY, HSQC, and HMBC spectra of all compounds.
All NMR spectra are available as Supplementary Material.
All the amino acids, resins, and coupling reagents are
commercially available and were purchased from GL
Biochem (Shanghai) Ltd. Analytical grade solvents for
synthesis were procured from Sigma-Aldrich (South
Africa).
The 1H- and 13C-NMR data for compounds 2, 4, 7, and 8
were recorded on a Bruker AVANCE III 400 MHz spec-
trometer and compounds 1, 3, 5, and 6 were analyzed using
a Bruker AVANCE III 600 MHz when higher sensitivity
was required. The chemical shifts were referenced to the
solvent peak d = 2.50 ppm (1H) and 39.9 ppm (13C) for
1
(CD3)2SO at room temperature. The H-NMR spectra for
compounds 1, 3, 5, and 6 were recorded at a transmitter
frequency of 600.100 MHz, with a spectral width of
12,335.526 Hz; acquisition time of 1.328 s, pulse width of
13.57 ls (1 and 6) and 13.75 ls (3 and 5); relaxation delay
of 1.0 s and scans of 8 and 16 for compounds 1 and 3, 5, 6,
1
respectively. For compounds 2, 4, 7, and 8, the H-NMR
spectra were recorded at a transmitter frequency of
400.220 MHz, with a spectral width of 8223.685 Hz;
acquisition time of 1.992 s (2, 7, and 8) and 3.98 s (4);
pulse width of 10.00 ls; relaxation delay of 1.0 s and scans
of 16.
The 13C-NMR spectra for compounds 1, 3, 5, and 6 were
recorded at 150.910 MHz, with a spectral width of
36,057.69 Hz; acquisition time of 0.908 s; pulse width of
9.00 ls; relaxation delay of 2 s and scans of 8160, 1600, 4800
(7, and 8), respectively. All the previous data were obtained
from Bruker AVANCE III 600 MHz instrument. For com-
pounds 2, 4, 7, and 8, the 13C-NMR spectra were recorded at
100.645 MHz, with a spectral width of 24,038.461 Hz;
acquisition time of 1.363 s; pulse width of 8.40 ls; relaxation
delay of 2 s and scans of 1024 (2, 4 and 8), 512 (7), respec-
tively, on AVANCE III 400 MHz NMR instrument.
All 2D experimental data obtained on the Bruker
AVANCE III 600 MHz for compounds 1, 3, 5, and 6 were as
follows: 90° pulse width of 10.00 ls (3), 13.75 ls (3 and 5)
and 13.57 ls (6), respectively, for all spectra. Spectral
General procedure for the synthesis of 5-hydroxy-
4-oxahexacyclododecane-3-carboxylic acid, compound 13
Compound 13 was synthesized according to the previously
reported method [33].
General procedure for manual SPPS loading of first
amino acid to the 2-chlorotrityl chloride resin [19, 20]
Activated 2-chlorotrityl chloride resin (1 g, 1.33 mmol)
was swelled in dry DCM (10 mL) for 10 min in a sintered
glass reaction vessel. Fmoc-amino acid-OH (3.99 mmol) in
DCM (10 mL) and N,N-diisopropylethylamine (DIPEA)
(6.65 mmol) were added to the resin and were mixed for
2 h with a stream of nitrogen bubbles. The solvent was
removed by filtration and the resin was washed with DCM
(3 9 10 mL). A few resin beads (*5 mg) were removed
from the reaction vessel and dried under vacuum for an
hour. A solution of 20 % piperidine in DMF was added to
the dried resin beads (1 mg) and left to stand for 20 min.
General washing procedure for manual SPPS: After each
step of SPPS, the resin was washed with DCM
(3 9 10 mL), DMF (3 9 10 mL), and DCM (3 9 10 mL).
1
widths for H spectra 1, 3, 5, and 6 are 12,335.526 Hz
(COSY), 72,11.539 Hz (ROESY), 7,075.472 Hz (for 3,
NOESY), 12,335.526 Hz (HSQC), 9,615.385 Hz (HMBC).
For compounds 2, 4, 7, and 8 all 2D experimental data
obtained on Bruker AVANCE III 400 MHz were as follows:
90° pulse width of 10.00 ls for all spectra. The 1H spectral
widths for all the 2D experiments (COSY, ROESY, HSQC,
and HMBC) are 5813.954 Hz (2), 5952.381 Hz (4),
5747.126 Hz (7), and 3703.704 Hz (8), respectively. The
123

Struct Chem
General procedure for the synthesis of peptides using
microwave power
Compound 1
A colorless oil (76 %). Retention time: 14.13 min (HPLC),
[a]2D0 = -20.00 (c 0.20 in MeOD); IR (neat): 3288.6,
2970.5, 1726.1, 1638.0, 1529.2, 1454.7, 1326.9, 1292.2,
1204.7, 1137.1, 992.5, 950.2, 906.4, 868.8, 641.7,
521.5 cm-1; HR-ESI-MS (m/z): calculated for C20H26N2
NaO6 ([M?Na]23?) 413.1683 found 413.1687. The NMR
data for 1 are presented in Tables 1 and 2.
Stock solutions of amino acids (0.2 mM), DIPEA (1 mM),
and O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexa-
fluoro-phosphate (HBTU) (2 mM) were prepared and added
into their appropriate reaction vessels on the peptide syn-
thesizer. All peptides were synthesized on a 0.5 mmol scale
using microwave power (see Table 1 of the Supplementary
information). The standard coupling method for arginine
(Arg) [60] was used for attachment of the second amino acid
to the resin (1 g) preloaded with the first amino acid. The
standard coupling method was used for subsequent reactions
and for Fmoc deprotection [19, 20, 61]. The washing pro-
cedures were similar to those used for manual SPPS. Final
cleavage from the resin was performed manually. The resin-
bound peptide was washed with DCM (3 9 10 mL) in a
reaction vessel while nitrogen was bubbled through the
solution. A cleavage mixture of 0.5 % (v/v) TFA and 95 %
(v/v) DCM was added to the dried resin while nitrogen was
bubbled through the solution for 10 min. The resin was
washed three times with the cleavage mixture and the
cleaved peptide in solution was separated from the resin by
filtration and collected in a flask containing water (100 mL).
The filtrate was extracted several times with DCM to
remove the peptide from the water layer. DCM was removed
under reduced pressure using a Teflon pump at 40 °C and
affording the peptide as a white powder.
Compound 2
A white solid (62 %). Retention time: 15.23 min (HPLC),
melting point: 129–130 °C, [a]2D0 = -11.76 (ca. 0.17 in
MeOD); IR (neat): 3300.5, 2967.2, 2868.8, 1725.5, 1640.5,
1527.3, 1455.4, 1326.0, 1292.2, 1204.9, 1138.1, 1105.6,
991.9, 949.5, 905.6, 868.6, 740.6, 698.9, 521.6, 495.4 cm-1
;
HR-ESI-MS (m/z): calculated for C26H30N2O6 ([M?H]?)
467.2177 found 467.2156. The NMR data for 2 are listed in
Tables 1 and 2.
Compound 4
A pale white solid (48 %). Retention time: 14.28 min
(HPLC), melting point: 159–160 °C, [a]2D0 = -41.67
(c 0.12 in MeOD); IR (neat): 3293.0, 2969.8, 1725.6,
1639.4, 1527.3, 1454.4, 1330.9, 1292.1, 1206.3, 1138.7,
993.7, 951.8, 905.6, 868.9, 789.5, 641.5, 521.7 cm-1; HR-
ESI-MS (m/z): calculated for C23H31N3O7 ([M?H]?)
462.2235 found 462.2269. The NMR data for 4 are pre-
sented in Tables 1 and 2.
General procedure for coupling of peptides to
5-hydroxy-4-oxahexacyclododecane-3-carboxylic acid
to yield compounds 1–8
Compound 5
The cleaved peptide (1.2 eq.) was dissolved in DCM
(3 mL) followed by the addition of 5-hydroxy-4-oxahex-
acyclododecane-3-carboxylic acid [33] (1 equiv), HATU
(2.5 equiv) in DMF (7 mL), and DIPEA (3 equiv) as a
base. The mixture was left to stir at room temperature for
24 h. The product was evaporated to dryness under vacuum
using a Teflon pump attached to the Buchi rotary evapo-
rator, which was maintained at 40 °C. A cleavage mixture
(10 mL) of 95 % (v/v) TFA and 5 % (v/v) DCM was added
to the coupled peptide and stirred for 24 h at room tem-
perature to remove the N-Boc protecting group. The TFA
was removed from the mixture by bubbling air through the
peptide and the remaining DCM was removed under vac-
uum at 30 °C. The product was obtained as yellow oil
which was purified by preparative HPLC [buffer solution
A: 0.1 % formic acid/H2O (v/v) and buffer solution B:
0.1 % formic acid/acetonitrile (v/v)].
A pale yellow oil (54 %). Retention time: 15.28 min
(HPLC), [a]2D0 = -27.27 (c 0.22 in MeOD); IR (neat):
3288.5, 2965.7, 1728.2, 1638.4, 1524.0, 1327.6, 1206.3,
1138.62, 949.5, 905.3, 699.0 cm-1; HR-ESI-MS (m/z):
calculated for C28H33N3NaO7 ([M?Na]23?) 546.2110 found
546.2190. The NMR data for 5 are listed in Tables 1 and 2.
Compound 6
A pale yellow oil (42 %). Retention time: 15.35 min
(HPLC), [a]2D0 = -23.81 (c 0.21 in MeOD); IR (neat):
3277.7, 2966.7, 1721.5, 1637.6, 1528.4, 1454.6, 1350.3,
1226.6, 1141.49, 911.2, 743.8, 698.9 cm-1; HR-ESI-MS
(m/z): calculated for C29H35N3NaO7 ([M?Na]23?
)
560.2367 found 560.2351. The NMR data for 5 are given in
Tables 1 and 2.
123

Struct Chem
Compound 7
References
1. Gerzon K, Kau D (1967) J Med Chem 10:189–199
2. Nagasawa HT, Elberlin Ja, Shirota FN (1973) J Med Chem
16:823–826
3. Marchand AP, Allen RW (1974) J Org Chem 39:1596
4. Nagasawa HT, Elberling JA, Shirota FN (1975) J Med Chem
18:826–830
5. Craze GA, Watt I (1981) J Chem Soc Perkin Trans 2:175–184
6. Griffin GW, Marchand AP (1989) Chem Rev 89:997–1010
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9. Marchand AP (1989) Advances in theoretically interesting mol-
ecules. In: Thummel RP (ed) A research annual, vol 1. JAI Press,
Greenwich, p 467
A yellow oil (42 %). Retention time: 15.62 min (HPLC),
[a]2D0 = -33.33 (ca. 0.21 in MeOD); IR (neat): 3287.8,
2964.4, 1714.9, 1640.5, 1524.1, 1327.2, 1205.2, 1138.4,
737.7, 697.0 cm-1. HR-ESI-MS (m/z): calculated for
C35H39N3NaO7 ([M?Na]23?) 636.2719 found 636.2680.
The NMR data are presented in Tables 1 and 2.
General procedure [62] for the synthesis of benzyl-
protected 5-hydroxy-4-oxahexacyclododecane
peptides, 3 and 8
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(2004) Magn Reson Chem 42:617–623
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(2009) Eur J Med Chem 44:4297–4305
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21:38–41
5-Hydroxy-4-oxahexacyclododecane peptide (1 eq) and
benzylbromide (2.5 eq) were dissolved in DMF (8 mL).
Cesium carbonate (2 eq) was added to the solution at
ambient temperature. After stirring for 2 h, saturated
aqueous sodium bicarbonate (100 mL) was added to the
solution and extracted with ethyl acetate (3 9 100 mL).
The combined organic phases were washed with 5 %
aqueous citrate and saturated aqueous sodium chloride
(100 mL) and dried over sodium sulfate. The solvent was
evaporated, and the crude peptide was precipitated in cold
ether and dried under reduced pressure. This crude product
was further purified by preparative HPLC [buffer solution
A: 0.1 % formic acid/H2O (v/v) and buffer solution B:
0.1 % formic acid/acetonitrile (v/v)].
19. Makatini MM, Petzold K, Sriharsha SN, Soliman MES, Honar-
parvar B, Arvidsson PI, Sayed Y, Govender P, Maguire GEM,
Compound 3
Kruger HG, Govender
21:2274–2277
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MES, Honarparvar B, Parboosing R, Naidoo A, Arvidsson PI,
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T (2011) Bioorg Med Chem Lett
A pale yellow oil (51 %). Retention time: 15.90 min
(HPLC), [a]2D0 = -13.33 (c 0.21 in MeOD); IR (neat):
3286.7, 2966.8, 1742.8, 1643.5, 1524.4, 1455.5, 1347.2,
1203.2, 1160.4, 1138.8, 1052.4, 991.3, 949.3, 904.7, 738.0,
697.3, 640.9 cm-1; HR-ESI-MS (m/z): calculated for
C27H32N2NaO6 ([M?Na]23?) 503.2153 found 503.2150.
The NMR data for 3 are listed in Tables 1 and 2.
Compound 8
A yellow oil (46 %). Retention time: 15.68 min (HPLC),
[a]2D0 = -19.05 (c 0.21 in MeOD); IR (neat): 3289.0,
2965.3, 1720.5, 1641.6, 1523.6, 1326.3, 1206.3, 1138.5,
906.4, 698.1 cm-1; HR-ESI-MS (m/z): calculated for
C36H41N3NaO7 ([M ? Na]23?) 650.2837 found 650.2828.
The NMR data for 8 are listed in Tables 1 and 2.
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Acknowledgments This research was supported by NRF (SA) TG
(GUN: 66319), HGK (GUN: 69728), HGK and PIA (SA-Sweden
bilateral grant), Aspen Pharmacare and University of KwaZulu-Natal
for financial support. We thank Mr. Dilip Jagjivan (UKZN, South
Africa) for his assistance with the NMR experiments.
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