Novel branched isocyanides as useful building blocks in the Passerini-amine deprotection-acyl migration (PADAM) synthesis of potential HIV-1 protease inhibitors

Article abstract of DOI:10.1016/j.tetlet.2012.04.018

Novel branched isocyanides have been prepared from l-serine and used as building blocks in the Passerini-amine deprotection-acyl migration (PADAM) sequence for the preparation of compounds with activity against HIV-1 protease.

Full text of DOI:10.1016/j.tetlet.2012.04.018

Tetrahedron Letters 53 (2012) 3225–3229
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Novel branched isocyanides as useful building blocks in the Passerini-amine
deprotection-acyl migration (PADAM) synthesis of potential HIV-1
protease inhibitors
David Gravestock a, , Amanda L. Rousseau a,b, Anna C.U. Lourens a,à, Heinrich C. Hoppe a,§
,
Lindiwe A. Nkabinde a, Moira L. Bode a,b,
⇑
a CSIR Biosciences, Private Bag X2, Modderfontein, Johannesburg 1645, South Africa
b Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Private Bag 3, PO WITS 2050, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel branched isocyanides have been prepared from
L-serine and used as building blocks in the Passe-
Received 2 February 2012
Revised 27 March 2012
Accepted 4 April 2012
Available online 20 April 2012
rini-amine deprotection-acyl migration (PADAM) sequence for the preparation of compounds with activ-
ity against HIV-1 protease.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Multi-component coupling reaction
Passerini
Acyl migration
PADAM
HIV-1 protease
Multi-component coupling reactions (MCRs) are a versatile
means of obtaining complex molecules in a single step.1 These
reactions are extremely atom-efficient and often high-yielding.
The Passerini MCR couples an aldehyde, a carboxylic acid and an
isocyanide to yield a product containing a new stereogenic centre
and both ester and amide functionalities (step a, Scheme 1).2 An
interesting variation of the Passerini three-component reaction
has been reported, where the initial product rearranges via O,N-
migration after N-deprotection to give an N-acyl compound with
a free hydroxy group (steps b and c, Scheme 1). These sequential
reactions have been termed the Passerini-amine deprotection-acyl
migration (PADAM) approach.3 This sequence is of particular inter-
est in the synthesis of peptidomimetic compounds.4 Our interest in
this reaction arose as a means of gaining rapid entry into a library
Ph
BocHN
CHO
Ph
Ph
1
O
O
O
O
+
a
c
NHR2
RHN
NHR2
R1
R1COOH
R1
N
H
O
2
+
N
OH
6
R2
C
4
5
R=Boc
R=H
b
3
Scheme 1. PADAM approach to possible HIV-1 protease inhibitors. Reagents: (a) CH2Cl2; (b) 10 equiv TFA, CH2Cl2; (c) Et3N, CH2Cl2.
⇑
Corresponding author. Tel.: +27 11 717 6744; fax: +27 86 765 4871.
E-mail address: Moira.Bode@wits.ac.za (M.L. Bode).
Present address: Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom.
Present address: Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa.
Present address: Department of Biochemistry, Microbiology & Biotechnology, Rhodes University, PO Box 94, Grahamstown 6140, South Africa.
à
§
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.04.018

3226
D. Gravestock et al. / Tetrahedron Letters 53 (2012) 3225–3229
S2'
S1
a.
b.
Scissile bond
S1
NH2
S3
P3
S2'
P2'
O
O
P1
O
O
H
N
H
N
H
N
S
O
HN
N
O
H2N
N
H
N
H
OH
O
OH
O
P2
S2
O
P1'
S1'
O
P3'
S3'
H
O
H
O
S1'
S2
7
Figure 1. (a) Standard nomenclature for the aspartyl protease peptide substrates; and (b) the HIV-1 protease inhibitor darunavir (7) showing how the compound was
designed to fill the enzyme subsites.
of compounds with possible activity against the aspartyl protease
HIV-1 protease, particularly as the final product contains a free sec-
ondary hydroxy group, a feature common to all the FDA-approved
HIV protease inhibitors.5 The PADAM approach we envisaged for
synthesising our desired compounds 6 is shown in Scheme 1.
The importance of the hydroxy group lies in the fact that HIV-1
protease inhibitors have largely been designed as transition state
mimics, where the inhibitors show structural similarity to the tet-
rahedral intermediate formed during peptide hydrolysis.6 The
aspartyl protease inhibitors have generally been designed to fill
the enzyme subsites, designated Sn or S0n, which correspond to
the hydrophobic pockets filled by amino acid side chain residues
of the natural peptidic substrate (Fig. 1a). These side chain residues
are designated as P1, P2,.....Pn moving away from the cleavage point
in one direction; and P01, P20 ,.....P0n moving in the opposite direction.
This concept is illustrated for the FDA-approved HIV-1 protease
inhibitor darunavir (7) in Figure 1b.
A library of 48 compounds was initially prepared by reacting N-
Boc-phenylalaninal 17 (Scheme 1) with commercially available
carboxylic acids 2 and isocyanides 3 in the Passerini reaction,
followed by deprotection of the Passerini product 4 and finally
O,N-acyl migration of
5 to give the desired compounds 6
(Scheme 1). Screening of these compounds for activity against
HIV-1 protease gave generally disappointing results (results not
shown). On closer examination of the structures of our first collec-
tion of compounds and comparison with the protease inhibitor in
Figure 1b, it was immediately obvious that our compounds only
possessed three groups capable of filling the enzyme subsites, in-
stead of the four (and sometimes more) groups present in the other
peptidomimetic protease inhibitors. The solution we arrived at in
order to solve this problem was to use branched isocyanides in
the PADAM sequence. This would give rise to compounds such as
8 (Fig. 2) with the requisite number of groups to occupy the
enzyme subsites, shown for comparison alongside another
NH2
Ph
Ph
O
O
O
O
R2
O
S
R1
N
H
N
H
R3
O
N
H
N
O
OH
OH
8
9
Figure 2. PADAM product 8 prepared from a branched isocyanide, and amprenavir
(9).
H
O
MeO2C
13
NHBoc
OBn
NHBoc
OBn
NHBoc
OBn
N
N
HO
R1O
R1O
g
R1O
d
e
f
OBn
OBn
17
14
15
16
R1= (a) Bn
(b)
(c)
O
O
c
O
O
O
O
H
O
HO2C
NHR
OH
HO2C
NHBoc
OBn
MeO
NHBoc
OBn
MeO
N
N
MeO
N
b
h
N
i
j
N
Me
Me
Me
OBn
OBn
10 R=H
11
12
18
19
20
a
R=Boc
k
O
O
OH
H
N
O
Ph
NHBoc
OBn
Ph
Ph
Ph
N
l
m
OBn
OBn
21
22
23
X
O
N
OBn
24
Scheme 2. Preparation of novel branched isocyanides for use in the PADAM approach. Reagents: (a) (Boc)2O, 1,4-dioxane, 1 M aq KOH, 55%; (b) (i) NaH, DMF, (ii) BnBr, 99%;
(c) K2CO3, MeI, acetone, 100%; (d) LiAlH4, THF, 84%; (e) (i) NaH, DMF, (ii) R1Br, 15a 66%, 15b 43%, 15c 49% (in THF); (f) HCOOH, Ac2O, TFA; (g) Ph3P, CCl4, EtN(iPr)2, CH2Cl2, 17a
79% from 15a, 17b 79% from 15b, 17c 51% from 15c; (h) (i) CDI, CH2Cl2, (ii) NH(OMe)MeꢀHCl, 46%; (i) HCOOH, Ac2O, TFA; (j) Ph3P, CCl4, EtN(iPr)2, CH2Cl2, 87% from 18; (k)
BnMgBr, THF, 63%; (l) HCOOH, Ac2O, TFA, 87%; (m) Ph3P, CCl4, Et3N, CH2Cl2, 42%.

D. Gravestock et al. / Tetrahedron Letters 53 (2012) 3225–3229
3227
FDA-approved protease inhibitor, amprenavir (9). Groups R2 and
R3, derived from the branched isocyanide, should in theory be able
to fill the subsites S01 and S20 (Fig. 1), and lead to better inhibition.
Thus, preparation of a second collection of compounds was
undertaken, this time using branched isocyanides in the initial
Passerini reaction. A number of possible synthetic options were
available for the preparation of branched isocyanides, but our
approach was to use an amino acid as the starting point. Amino
acids have previously been used for isocyanide preparation,8 but
these methods have generally only been applied to amino acids
with non-functionalised side chains. In our case, modification of
the starting amino acid allowed for the preparation of isocyanides
with two positions, in addition to the isocyanide, amenable to
functionalisation. Our preparation of branched isocyanides starting
from
L
-serine (10) is shown in Scheme 2. Initial N-Boc protection,
-serine (10?13) were fol-
O-benzylation and esterification of
L
lowed by LiAlH4 reduction of the methyl ester to furnish alcohol
14. This compound was reacted with a number of different alkyl
bromides to give 15a–c. Subsequent N-Boc deprotection and
formylation, combined into one step, was followed by dehydration
to give isocyanides 17a–c. Compound 20 was prepared following a
similar sequence9 after assembly of the Weinreb amide 18 using
a
b
H2N
N
H
O
N
25
26
27
Scheme 3. Preparation of adamantyl isocyanide. Reagents: (a) HCOOH, Ac2O, 75%;
(b) POCl3, Et3N, CH2Cl2, 89%.
Table 1
HIV-1 protease screening results
Ph
O
Ph
O
R1HN
NHR2
BocHN
NHR2
OR1
A
OH
B
Entry
R1
R2
Percentage residual HIV-1 protease activity at 50
lM inhibitor concn (%)
Reaction yield (%)
A
B
A
Bb
O
O
OMe
N
1
103.8 ( 2)
71.8 ( 7)
100
70
BnO
O
O
2
3
101.1 ( 11)
100.6 ( 25)
25.0 ( 0)
78.6 ( 1)
42
74
64
83
OBn
BnO
O
O
O
O
O
4
5
99.4 ( 5)
68.3a
68
86
68
82
BnO
BnO
BnO
OBn
OBn
95.4 ( 16)
72.6 ( 2)
66.1 ( 2)
6
7
8
9
99.8 ( 3)
94.7 ( 14)
49.9 ( 3)
92.4 ( 2)
99
98
68
66
66
87
48
78
O
O
O
OBn
OBn
79.1 ( 11)
85.6 ( 9)
78.5 ( 5)
BnO
BnO
O
O
O
O
O
O
O
O
BnO
BnO
BnO
BnO
O
O
O
10
11
12
94.7 ( 0)
86.4 ( 9)
96.9 ( 1)
76.7 ( 3)
92.6 ( 2)
86.9 ( 7)
99
48
45
40
100
82
a
Single data point.
Yield over two steps: deprotection and rearrangement.
b

3228
D. Gravestock et al. / Tetrahedron Letters 53 (2012) 3225–3229
1,10-carbonyldiimidazole for coupling of the carboxylic acid 12 and
the Weinreb amine.
(c) Owens, T. D.; Semple, J. E. Org. Lett. 2001, 3, 3301–3304; (d) Faure, S.;
Hjelmgaard, T.; Roche, S. P.; Aitken, D. J. Org. Lett. 2009, 11, 1167–1170.
4. (a) Banfi, L.; Guanti, G.; Riva, R.; Basso, A.; Calcagno, E. Tetrahedron Lett. 2002,
43, 4067–4069; (b) Banfi, L.; Basso, A.; Guanti, G.; Riva, R. Mol. Divers. 2003, 6,
227–235; (c) Basso, A.; Banfi, L.; Guanti, G.; Riva, R.; Tosatti, P. Synlett 2011,
2009–2012.
5. (a) De Clercq, E. Int. J. Antimicrob. Agents 2009, 33, 307–320; (b) Wensing, A. M.
J.; van Maarseveen, N. M.; Nijhuis, M. Antiviral Res. 2010, 85, 59–74.
6. Abdel-Rahman, H. M.; Al-Karamany, G. S.; El-Koussi, N. A.; Youssef, A. F.; Kiso,
Y. Curr. Med. Chem. 2002, 9, 1905–1922.
The rationale behind the preparation of isocyanide 20 was that
it presents a unique opportunity for post-MCR modification, allow-
ing for divergence at a very late stage in the synthetic sequence.
The utility of Weinreb amides in the preparation of varied ketones
and aldehydes is well known,10 and at the end of the PADAM se-
quence, the Weinreb amide functionality could conveniently be
converted to give a diverse range of compounds. A pre-MCR mod-
ification of Weinreb amide 18 was also explored in an attempt to
prepare isocyanide 24. Weinreb amide 18 was initially reacted
with benzylmagnesium bromide to give the benzyl ketone 21, fol-
lowed by deprotection and formylation to give formamide 22.
Interestingly, under typical dehydration conditions, the formamide
22 gave exclusively compound 23, the enol form of the expected
product 24, as evidenced by spectroscopic data.11 This is presum-
ably due to conjugation of the enol double bond with the isocya-
nide. In addition to the isocyanides shown in Scheme 2,
adamantyl isocyanide 27, also a branched isocyanide and not com-
mercially available at the time, was prepared from adamantyl-
amine 25 (Scheme 3).
7. N-Boc-phenylalaninal was prepared from N-Boc-phenylalanine by LiAlH4
reduction of the Weinreb amide of N-Boc-phenylalanine.
_
8. Berłozecki, S.; Szyman´ ski, W.; Ostaszewski, R. Synth. Commun. 2008, 38, 2714–
2721.
9. (S)-3-(Benzyloxy)-2-formamido-N-methoxy-N-methylpropanamide (19). HCO2H
(18.8 ml) and Ac2O (4.63 ml, 49.0 mmol) were stirred together for 5 min
at room temperature and then added under N2 to (S)-tert-butyl (3-(benzyloxy)-
1-(methoxy(methyl)amino)-1-oxopropan-2-yl)carbamate 18 (5.53 g, 16.34
mmol). To this was added dropwise trifluoroacetic acid (3.77 ml, 49.0 mmol)
and the mixture was stirred for 1.5 h at room temperature. Brine (200 ml),
EtOAc (200 ml) and solid NaHCO3 (50 g) were added to the reaction mixture.
After effervescence had ceased, H2O (100 ml) was added and the organic layer
separated. The aqueous layer was extracted again with EtOAc (2 ꢁ 100 ml) and
combined organic layers dried over MgSO4. The solvent was removed in vacuo
to give (S)-3-(benzyloxy)-2-formamido-N-methoxy-N-methylpropanamide
(19) as a colourless gum (4.95 g) that was used in the next step without
further purification.
(S)-3-(Benzyloxy)-2-isocyano-N-methoxy-N-methylpropanamide (20). (S)-3-
These branched isocyanides (17a–c, 20, 23 and 27) were reacted
with N-Boc-phenylalaninal 1 and commercially available carbox-
ylic acids 2 according to Scheme 1. Disappointingly, isocyanide
17c appeared to decompose during the Passerini reaction and no
desired product was isolated. Reactions using compound 23 re-
turned only starting material. The other isocyanides were success-
fully reacted and the N-Boc-O-acyl compounds 4 were deprotected
using TFA and subsequent rearrangement of 5 to the desired prod-
ucts 6 was carried out in the presence of Et3N.12 Both the Passerini
4 and PADAM 6 products were screened in a fluorescence-based13
HIV-1 protease inhibition assay.14
Results from the screening show clearly that the products from
the Passerini reaction (column A, Table 1) were inactive against
HIV-1 protease, while the rearranged products (column B, Table 1)
were generally more active. This result is expected when consider-
ing that the rearranged products have the secondary hydroxy
groups considered essential for activity against HIV-1 protease.
The only exception to this is the Passerini product of entry 8, where
significant inhibitory activity was observed. The rearranged prod-
uct prepared from N-Boc-phenylalaninal, tert-butyl acetic acid
and adamantyl isocyanide showed the best inhibitory activity of
all the compounds tested (Table 1, entry 2). Interestingly, adaman-
tyl-based compounds are showing increasing utility in the treat-
ment of many different conditions, including as antiviral agents.15
In conclusion, a method has been demonstrated for the prepara-
tion of highly functionalised isocyanides that can be used in multi-
component reactions to gain rapid entry to complex molecules,
which may then be further functionalised. A number of different
MCRs utilise isocyanide building blocks and thus the type of
branched isocyanides reported here should find use in a range of
applications. Utilisation of these branched isocyanides in other
MCR applications is currently under investigation.
(Benzyloxy)-2-formamido-N-methoxy-N-methylpropanamide
19
(4.35 g,
16.34 mmol), PPh3 (6.43 g, 24.5 mmol), DIPEA (4.27 ml, 24.5 mmol) and CCl4
(2.37 ml, 24.5 mmol) were dissolved in CH2Cl2 (70 ml) and stirred under N2
overnight at room temperature. The reaction mixture was concentrated by
removal of two-thirds of the solvent in vacuo and the remaining mixture
purified by column chromatography (elution with EtOAc:hexane) to yield (S)-
3-(benzyloxy)-2-isocyano-N-methoxy-N-methylpropanamide (20) as a clear
viscous oil (3.53 g, 87% from 18). 1H NMR (400 MHz, CDCl3) d 7.40–7.24 (m,
5H), 4.85 (t, J = 6.3 Hz, 1H), 4.59 (s, 2H), 3.88–3.77 (m, 2H), 3.71 (s, 3H), 3.21 (s,
3H); 13C NMR (100 MHz, CDCl3) d 164.24, 159.76, 136.98, 128.36, 127.88,
127.67, 73.52, 69.14, 61.60, 53.38, 32.36.
10. (a) Balasubramaniam, S.; Aidhen, I. S. Synthesis 2008, 3707–3738; (b) Sibi, M. P.
Org. Prep. Proced. Int. 1993, 25, 15–40.
11. Spectroscopic data for 4-(benzyloxy)-3-isocyano-1-phenylbut-2-en-2-ol (23).
1H NMR (400 MHz, CDCl3) d 7.73 (s, 1H), 7.37–7.15 (m, 10H), 4.58 (s, 2H), 4.47
(s, 2H), 4.02 (s, 2H); 13C NMR (100 MHz, CDCl3) d 149.62, 148.69, 137.93,
136.80, 132.15, 128.62, 128.51, 128.37, 127.82, 127.67, 126.81, 72.49, 63.51,
31.00; HRMS (ESI): m/z: 280.1339 [M+H]+; calcd for C18H18NO2: 280.1339.
12. The method for the Passerini reaction and subsequent deprotection and acyl
migration is exemplified by the reaction between N-Boc-phenylalaninal, tert-
butyl acetic acid and adamantyl isocyanide 27.
(3S)-1-(Adamantan-1-ylamino)-3-[(tert-butoxycarbonyl)amino]-1-oxo-4-phe-
nylbutan-2-yl 3,3-dimethylbutanoate.
isocyanide (27) (0.161 g,
Adamantyl
1.0 mmol), tBuCH2COOH (0.128 g, 1.1 mmol) and N-Boc-phenylalaninal
(0.250 g, 1.0 mmol) were stirred in dry CH2Cl2 (3 ml) at room temperature
for 3 days. The solvent was removed in vacuo and purification by column
chromatography (elution CHCl3/MeOH, 49:1 to 9:1) afforded the Passerini
product as a mixture of two diastereomers (0.223 g, 42%). 1H NMR (400 MHz,
CDCl3) d 7.48–6.93 (m, 5H), 5.86–5.52 (m, 1H), 5.33–4.60 (m, 2H), 4.24 (s, 1H),
3.05–2.52 (m, 2H), 2.24 (d, J = 19.6, 2H), 2.13–1.79 (m, 9H), 1.79–1.47 (m, 6H),
1.47–1.15 (m, 9H), 1.15–0.71 (m, 9H); 13C NMR (100 MHz, CDCl3) d 171.09,
170.09, 167.02, 166.67, 155.35, 154.81, 137.45, 129.22, 129.10, 128.41, 128.33,
126.50, 126.39, 79.20, 73.90, 73.46, 53.32, 52.95, 52.26, 52.21, 47.79, 47.49,
41.36, 37.80, 37.49, 36.18, 30.89, 29.60, 29.32, 28.20. HRMS (ESI): m/z:
527.3483 [M+H]+; calcd for C31H47N2O5: 527.3485.
(3S)-N-(Adamantan-1-yl)-3-(3,3-dimethylbutanamido)-2-hydroxy-4-phenylb-
utanamide. (3S)-1-(Adamantan-1-ylamino)-3-[(tert-butoxycarbonyl)amino]-
1-oxo-4-phenylbutan-2-yl 3,3-dimethylbutanoate (0.177 g, 0.335 mmol) was
dissolved in CH2Cl2 (0.8 ml) and TFA (0.26 ml, 3.35 mmol) was added. The
reaction mixture was stirred at room temperature for 2 h, after which the
solvent and most of the TFA were removed in vacuo. The residue was re-
dissolved in CH2Cl2 (0.8 ml) and Et3 N (0.30 ml) was added. This was stirred at
room temperature for 1 h after which H2O (5 ml) and additional CH2Cl2 (10 ml)
were added. The layers were separated and the aqueous layer was extracted
with CH2Cl2 (2 ꢁ 5 ml). The combined organic layer was dried over MgSO4 and
the solvent removed in vacuo. Column chromatography (elution CHCl3/MeOH,
49:1) afforded the PADAM product as a mixture of two diastereomers (0.091 g,
64%). Restricted rotation about an amide bond for diastereomer B gave rise to
two rotamers, apparent from the 1H and 13C NMR spectra. Only one amide
proton signal for each diastereomer was observed in the 1 H spectrum.
Diastereomer A: 1H NMR (400 MHz, CDCl3) d 7.33–7.13 (m, 5H), 6.91 (d, J = 8.3
Hz, 1H), 6.76 (s, 1H), 4.28–4.16 (m, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.15 (dd,
J = 13.9, 6.4 Hz, 1H), 2.92 (dd, J = 13.8, 9.3 Hz, 1H), 2.12–2.03 (m, 3H), 2.03–1.97
(m, 6H), 1.96 (d, J = 4.4 Hz, 2H), 1.71–1.64 (m, 6H), 0.90 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 173.67, 171.86, 138.18, 129.12, 128.34, 126.37, 73.31,
Acknowledgement
The authors wish to thank the Innovation Fund (now the Tech-
nology Innovation Agency, South Africa) for financial support.
References and notes
1. (a) Dömling, A.; Ugi, I. Angew. Chem., Int. Ed. 2000, 39, 3168–3210; (b) Wang, S.-
X.; Wang, M.-X.; Wang, D.-X.; Zhu, J. Angew. Chem., Int. Ed. 2008, 47, 388–391.
2. (a) Dömling, A. Chem. Rev. 2006, 106, 17–89; (b) Maeda, S.; Komagawa, S.;
Uchiyama, M.; Morokuma, K. Angew. Chem., Int. Ed. 2011, 50, 644–649.
3. (a) Banfi, L.; Guanti, G.; Riva, R. Chem. Commun. 2000, 985–986; (b) Owens, T.
D.; Araldi, G.-L.; Nutt, R. F.; Semple, J. E. Tetrahedron Lett. 2001, 42, 6271–6274;
55.45, 51.67, 49.99, 41.41, 36.23, 35.47, 30.66, 29.66, 29.32. Diastereomer B: 1
NMR (400 MHz, CDCl3) d 7.33–7.13 (m, 5H), 6.50 (br s, 1H), 6.25 and 6.15 (br d,
J = 6.9 Hz and br s, 1H), 4.97 and 4.14 (d, J = 5.0 Hz and d, J = 1.5 Hz, 1H),
H

D. Gravestock et al. / Tetrahedron Letters 53 (2012) 3225–3229
3229
4.28–4.16 and 3.48–3.41 (2 ꢁ m, 1H), 3.13–3.07 and 3.00 (dd, J = 14.3, 10.3 Hz
compounds at a final concentration of 50
37 °C with 9.7 U/ L enzyme and 2.0
(0.1 M NaOAc, 1.0 M NaCl, 1.0 mM EDTA, 1.0 mM DTT, 0.1% BSA, 5% DMSO, pH
4.7). Incubations were carried out in a total volume of 200 L in microtitre
plates. The extent of peptide cleavage was determined by measuring EDANS
fluorescence at Ex340 nm/Em480 nm in Tecan Infinite F500 plate reader.
Percentage residual enzyme activity was calculated from the 480 nm
fluorescence of test wells relative to control wells (no inhibitor added) after
subtracting background fluorescence (wells containing no enzyme). All
experiments were performed in duplicate.
l
M were incubated for 20 min at
and dd, J = 14.0, 4.5 Hz, 1H), 2.92 and 2.56 (dd, J = 13.8, 9.3 Hz and dd, J = 13.6,
9.8 Hz, 1H), 2.32 and 1.95–1.88 (d, J = 2.7 Hz and m, 2H), 2.12–2.03 (m, 3H),
2.03–1.97 (m, 6H), 1.71–1.64 (m, 6H), 1.08 and 0.87 (2 ꢁ s, 9H); 13C NMR
(100 MHz, CDCl3) d 174.61, 171.01, 170.85, 167.01, 138.35, 137.94, 129.24,
129.04, 128.53, 128.34, 126.55, 126.48, 77.20, 75.07, 57.32, 54.07, 52.10, 51.64,
49.99, 47.83, 41.45, 39.49, 36.23, 35.60, 35.47, 30.99, 30.69, 29.57, 29.34. HRMS
(ESI): m/z: 427.2958 [M+H]+; calcd for C26H39N2O3: 427.2961.
l
lM substrate peptide in assay buffer
l
a
13. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1990, 247, 954–
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14. The protease assay is based on the cleavage of a fluorogenic peptide substrate
containing terminal DABCYL and EDANS moieties (Sigma–Aldrich, South
Africa) by recombinant HIV-1 protease (Bachem, Switzerland). Test
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