A selective HIV-protease assay based on a chromogenic amino acid

Article abstract of DOI:10.1002/1522-2675(200210)85:10<3090::AID-HLCA3090>3.0.CO;2-8

(2S,3S)-2-Amino-3-hydroxy-5-(4-nitrophenoxy)pentanoic acid (5) was prepared stereoselectively as the N-Fmoc, O-(tert-butyl)-protected derivative 5a in eleven steps from ethyl (E)-4-benzyloxypent-2-enoate (6). This protected amino acid was used for the sol

Full text of DOI:10.1002/1522-2675(200210)85:10<3090::AID-HLCA3090>3.0.CO;2-8

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Helvetica Chimica Acta Vol. 85 (2002)
A Selective HIV-Protease Assay Based on a Chromogenic Amino Acid
by Fabrizio Badalassia)b), Hong Khan Nguyenc), Paolo Crottib), and Jean-Louis Reymond*a)
a) Departement f¸r Chemie und Biochemie, Universit‰t Bern, Freiestrasse 3, CH-3012 Bern (fax:
‡41316318057; e-mail: jean-louis.reymond@ioc.unibe.ch)
b
¡
) Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, I-56126 Pisa
c
¬
) Proteus SA, Parc Georges Besses, F-20000 NÓmes
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
(2S,3S)-2-Amino-3-hydroxy-5-(4-nitrophenoxy)pentanoic acid (5) was prepared stereoselectively as the N-
Fmoc, O-(tert-butyl)-protected derivative 5a in eleven steps from ethyl (E)-4-benzyloxypent-2-enoate (6). This
protected amino acid was used for the solid-phase peptide synthesis of oligopeptides, which serve as sequence-
specific chromogenic protease substrates when used in the presence of NaIO4 and bovine serum albumin. The
peptide 1 (KRAVNleÀ5ÀEANleNH2 (Nle ˆ norleucine)) allows detection of HIV-protease activity spectro-
photometrically at 405 nm.
Introduction. Enzyme assays play a crucial role in a variety of applications.
Particularly, one important assay concerns the measurement of the activity of HIV-
protease and other retroviral proteases, which provide a tool to discover and monitor
their inhibitors as anti-retroviral drugs [1]. Protease inhibitors have proven superior to
reverse-transcriptase inhibitors at reducing viral loads in the case of HIV-infections [2].
A series of cumulative mutations can render HIV-protease resistant to its inhibitors
such as indinavir. Monitoring the onset of resistance in patients enables better
adjustment of the drug cocktail by removing inhibitors to which the protease has
become resistant, thereby promoting reappearance of the wild-type-sensitive strain [3].
HIV-protease, whose function is to process the Gag-polyprotein (source of
structural viral proteins) and the Gag/Pol-polyprotein (source of enzymes for
replication, including HIV-protease itself), cleaves peptide bonds preferentially
between aromatic amino acids and proline and between pairs of hydrophobic and/or
aromatic amino acids [4]. Its activity can be measured by monitoring the decrease of
fluorescence-resonance-energy transfer (FRET) between 4-[4-(dimethylamino)phen-
yldiazo]benzoic acid (DABCYL) and 5-[(2-aminoethyl)amino]naphthalene-1-sul-
fonic acid (EDANS) attached at the N- and C-termini of the octapeptide
SQNYPIVQ, upon cleavage of the TyrÀprolyl peptide bond [5]. This assay is quite
selective but would deliver a positive signal for cleavage of any of the nine different
amide bonds in the substrate. Alternatively, the peptide KARVNleF'EANle-NH2 (F' ˆ
4-nitrophenylalanine) [6] and related sequences with 4-nitrophenylalanine either at the
P1[7] or the P1 ' [8] position are reported as chromogenic HIV-protease substrates.
Peptide cleavage is supposed to induce a detectable modulation of UV-absorbance at
peptide bonds next to 4-nitrophenylalanine, which are specifically attacked by HIV-
protease in these peptides. This method has the advantage of absolute chemoselectivity,

Helvetica Chimica Acta Vol. 85 (2002)
3091
but the signal modulation produced is only marginal, and, therefore, quite difficult to
observe. Herein, we report a related assay for HIV-protease based on the use of peptide
1 incorporating a non-natural allo-threonine analog at the amino-side of the scissile
peptide bond (Scheme 1). This amino acid undergoes a chromogenic reaction when its
amino-group is free, following an oxidation/b-elimination sequence.
Scheme 1. Principle of HIV-Protease Assay with Peptide 1
K ˆ lysine, A ˆ alanine, R ˆ arginine, Vˆ valine, Nle ˆ norleucine, E ˆ glutamate.
Results and Discussion.
Recently, we reported that a variety of hydrolytic
enzymes can be assayed spectrophotometrically with substrates that produce period-
ate-sensitive vicinal diols or amino alcohols related to 2 as hydrolysis products from
unoxidizable precursors [9]. The chemoselective oxidation by NaIO4 is followed by a
rapid b-elimination in the presence of bovine serum albumin (BSA) [10], thus yielding
either umbelliferone (ˆ 7-hydroxycoumarine) as a highly-fluorescent product or 4-
nitrophenol as a colored product (Scheme 1). Compared to assays based on direct
esters of these phenols, the key advantage of this assay is its resilience to nonspecific
processes, which allows us to address enzymatic activity with high specificity, even in
relatively crude extracts [11]. Given the general importance of proteases, we decided to
synthesize the protected form 5a of b-hydroxy-a-amino acid 5, which would be suitable
for Fmoc-based solid-phase peptide synthesis. Its incorporation into peptides would
provide a variety of sequence-specific chromogenic probes for proteases.
Since proteases are generally highly stereoselective, an enantioselective synthesis of
the l-form was required in any of the two possible diastereoisomers. We envisioned
that the (2S,3S)-diastereoisomer may be accessible from ethyl (E)-4-benzyloxypent-2-
enoate (6) via Sharpless×s asymmetric dihydroxylation with AD-mix-a as the chirality
inducing step followed by inversion at C(2) with a N-nucleophile (Scheme 2). The
functionalization with the 4-nitrophenyl group at C(5) and the introduction of a t-Bu

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Helvetica Chimica Acta Vol. 85 (2002)
protecting group on the b-OH group would be achieved along the synthesis by standard
methods.
Scheme 2. Retrosynthetic Analysis for the Target Amino Acid 5
The synthesis was realized as follows (Scheme 3). Asymmetric dihydroxylation of 6
with AD-mix-a gave diol 7 in 73% and good optical purity (95% ee) [12]. The diol
function was then protected as an acetonide to provide ester 8 (98%). The cyclic acetal
locked the conformation of the molecule and prevented any interaction between the
ester function and the OÀC(5) group, which could then be deprotected by hydro-
genation to give 9 quantitatively without any lactonization. Mitsunobu reaction to the
corresponding iodide 10 (90%) [13] and reaction with sodium 4-nitrophenolate in DMF
gave ether 11 (89%). The acetonide was then removed by acidic treatment in absolute
EtOH to give diol 12 with a disappointing yield of 70% despite many attempts to
optimize. According to a known sequence [14], this diol reacted with 2-nitro-
phenylsulfonyl chloride to give 13 (75%), which was treated with NaN3 to give azido
alcohol 14 (97%). At that stage, the OH group was protected as tert-butyl ether to give
15 via reaction with 2-methylpropene in the presence of H2SO4 (77%). The azido group
was then reduced with PPh3 to give 16. Finally, the ester function was saponified by
treatement with LiOH, and the crude amino acid was converted to the Fmoc-derivative
5a, which was isolated as pure product in 71% yield over the last three steps. The
desired chromogenic amino acid was thus obtained in a total of eleven steps and in 16%
overall yield.
Two chromogenic peptide substrates were prepared by Fmoc-solid-phase peptide
synthesis on Rink-amide polystyrene resin incorporating the protected amino acid 5a.
An HIV-protease-specific sequence 1 was obtained by simply substituting amino acid 5
for 4-nitrophenylalanine in a known HIV-protease substrate [6] to give KRAVNle-5-
1
EANleNH2 ). As a positive control, the chromogenic tetrapeptide H2N-Ala-Arg-5-
Ala-NH2 (17) was also prepared as a trypsin substrate. Both peptides were obtained
pure after reverse-phase-HPLC purification in 9 and 24% yield, respectively.
The reactivity of proteases with peptides 1 and 17 was investigated next. The
peptides were used as 1 5 m m stock solution in H2O. We examined the reactivity of the
peptides with the proteases trypsin, chymotrypsin, papain, and HIV-protease at their
respective pH-optima. The assays were conducted as endpoint measurements. Thus, the
peptides were first incubated with the proteases. Peptide cleavage was then assessed by
adjusting the pH of the samples to ca. pH 9.0, and treating with NaIO4 and BSA. The
1
)
For abbreviations of amino acids, see Scheme 1.

Helvetica Chimica Acta Vol. 85 (2002)
3093
Scheme 3. Stereoselective Synthesis of Chromogenic Amino Acid 5a
amount of 4-nitrophenol produced was then determined spectrophotometrically at
405 nm. The entire assay sequence was conveniently carried out in a total volume of
0.15 ml in individual wells of 96-well microtiter plates.
The production of 4-nitrophenol could clearly be detected in the presence of
several proteases, while there was no reaction at all in the absence of enzyme, as
expected from the stability of the native peptide bond in the substrates (Table). A
reaction was clearly detected in the presence of HIV-protease with its corresponding
sequence 1. This sequence also showed a positive signal upon incubation with papain at

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Helvetica Chimica Acta Vol. 85 (2002)
its optimal pH. Although we used relatively high quantities of this enzyme in
comparison with HIV-protease, which was expressed from a plasmid at an estimated
level of 1 10 mg/ml only, it was evident that the selectivity was not achieved with this
sequence. It should be mentioned that papain activity with 1 was suppressed selectively
by addition of the oxidant NaIO4 at the start of the reaction, this probably by oxidation
of the catalytic thiol. By contrast, both HIV-protease and trypsin retained their activity
when incubated in presence of NaIO4. The reference trypsin-like sequence 17 bearing
an arginine at the P1-site reacted with trypsin only, while there was no reaction
observed with any of the other three proteases.
Table. Chromogenic Reactions of Peptides 1 and 17 with Different Proteasesa)
Protease
Conditions
Cleavage [%]
1
17
HIV-Protease
Trypsin
Chymotrypsin
Papain
100 mm AcOH (pH 4.5), 1m m EDTA, 1m m DTT
20 mm borate (pH 8.8), 2 h, 378
10 mm phosphate (pH 7.4), 160 mm NaCl, 2 h, 378
20 mm phosphate (pH 6.5), 2 h, 378
26
5
< 175
< 1
0
0
40b)
a) Conditions: 100 mm substrate 1 or 17, HIV-protease: 10 mg mlÀ1, other proteases: 0.1mg ml À1, incubation for
2 h at 378, then addition of aq. 0.2m Na2CO3 to pH 9, BSA (2 mg mlÀ1 final conc.) and NaIO4 (final conc. 1m m,
20 mm with HIV protease) and incubation for 60 min. The OD at 405 nm (0.05 0.60) was used to calculate the
4-nitrophenol conc. according to a calibration curve. b) In contrast to trypsin, papain showed no activity when
BSA and NaIO4 were added from the start, most likely due to oxidation of the catalytic thiol in this enzyme.
Conclusions. The chromogenic amino acid building block 5 has been prepared
stereoselectively in eleven steps in protected form suitable for Fmoc-based peptide
synthesis. Its incorporation into peptide sequences provides specific chromogenic
probes for proteases. The amino acid sequence may be selected at all positions relative
to the cleavage site, with the exception of the P'1-site, which is occupied by the
chromogenic amino acid. Compared with FRET-type substrates the key advantage of
this approach is that only cleavage at the desired position delivers a signal, which makes
a much higher selectivity possible. Furthermore, the 4-nitrophenol signal is very strong
and readily detected even at low conversion rates.
Whether the cross-reactivity observed between our HIV-protease sequence and
other proteases would be significant in clinical samples remains to be tested, and a
similar cross-reactivity probably occurs with other HIV-protease probes. The problem
of selectivity can be fine-tuned by adjusting the experimental conditions. Thus, the
reactivity of papain with peptide 1 is suppressed by adding IOÀ4 at the beginning of the
reaction, which oxidizes the reactive cysteine in this thiol protease. Furthemore,
working at the acidic pH of the HIV-protease should also diminish the activity of
trypsin-like proteases relative to that of HIV-protease. The main drawback of the assay
is the low reactivity of the peptide towards cleavage, which is probably due to the
presence of the unnatural and sterically demanding chromogenic amino acid 5 at the
P'1-site.

Helvetica Chimica Acta Vol. 85 (2002)
3095
This work was financially supported by the University of Bern, the Swiss National Science Foundation, The
¬
¬
Office Federal suisse de l×Education et de la Science, the european COST program (action D12), the University
¡
of Pisa and the Ministero della Universita e della Ricerca ScientifÌca e Tecnologica (MURST), Roma. P. C.
gratefully acknowledges Merck Research Laboratories for the generous financial support from 2002 ADP
Chemistry Award.
Experimental Part
General. All reagents were purchased from Aldrich or Fluka and used without further purification. All
reactions were controlled by TLC on Alugram SIL G/UV254 silica-gel sheets (Macherey-Nagel) with detection by
UVor with 0.5% phosphomolybdic acid soln. in 95% EtOH. Silica gel 60 (Macherey-Nagel, 230 400 mesh) was
used for flash chromatography (FC). Anal. RP-HPLC was done on a Waters 600 Controller with a Waters 996
photodiode-array detector with a Vydac 218TP-54 (C18, pore size 300 ä, 0.45 Â 22 cm) column, and four
different eluents: A (0.1% TFA in H2O), B (H2O/MeCN 50 :50), C (H2O), and D (MeCN/H2O/TFA
60 :40 :0.1). Prep. HPLC was performed on a Waters Prep-LC and Delta Prep-4000 with a Waters 486 tunable
absorbance detector. The ee of diol 7 was determined by analysis on a chiral HPLC column OD-H (Daicel:
25 cm  0.46 cm i.d.; hexane/i-PrOH 85 :15). M.ps. were determined on a Kofler apparatus and are uncorrected.
OR were measured with a Perkin-Elmer 241 digital polarimeter with a 1-dm cell. IR Spectra: Mattson 3000 FT-
IR spectrophotometer; n in cmÀ1
.
1H- and 13C-NMR Spectra: Bruker AC-200 or -300; d ˆ in ppm, J in Hz.
Ethyl (2R,3S)-5-Benzyloxy-2,3-dihydroxypentanoate (7). A soln. of AD-mix-a [12] (34.51 g), NaHCO3
(6.21g, 73.95 mmol), and MeSO 2NH2 (2.34 g, 24.65 mmol) in t-BuOH/H2O 1:1(250 ml) was stirred at r.t. until
both phases were clear and then cooled to 08. Ethyl (E)-5-benzyloxypent-2-enoate (6) [15] (5.77 g, 24.65 mmol)
was added and the slurry was stirred at 08 for 24 h (TLC). Solid Na2S2O5 (37.0 g) was added at 08, and the
resulting mixture was warmed to r.t. and stirred for 1h. Extraction with AcOEt and evaporation of the washed
(2n NaOH) org. soln. gave a solid crude residue, which was subjected to FC (hexane/AcOEt 7:3; Rf 0.2) to give
20
D
pure 7 (4.79 g, 73%, 95% ee). White waxy solid. ‰aŠ ˆ À4.5 (c ˆ 0.42, CHCl3). IR (neat): 3446s (OH), 3387s
(OH), 1726s (CˆO). 1H-NMR (200 MHz, CDCl3): 7.43 7.17 (m, 5 H); 4.53 (s, 2 H); 4.28 (q, J ˆ 7.2, 2 H);
4.14 4.07 (m, 1H); 4.07 3.97 (br. s, 1H); 3.81 3.59 ( m, 2 H); 2.15 1.93 (m, 1H); 1.93 1.72 ( m, 1H); 1.31(t,
J ˆ 7.1, 3 H ) . 13C-NMR (50 MHz, CDCl3): 173.16; 137.85; 128.43; 127.72; 127.68; 73.56; 73.25; 71.37; 67.96; 61.87,
33.25; 14.13. Anal. calc. for C14H20O5: C 62.67, H 7.51; found: C 62.51, H 7.33.
Ethyl (4R,5S)-5-(2-Benzyloxyethyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (8). A soln. of 7 (2.40 g,
8.95 mmol) in anh. acetone (32 ml) was treated with 2,2-dimethoxypropane (9.33 g, 89.55 mmol, 10.98 ml) and
TsOH (12 mg), and the resulting mixture was stirred at r.t. for 1.5 h. Na2CO3 (0.57 g) was added, and stirring was
prolonged for 10 min. Evaporation of the filtered (Celite) org. soln. afforded a crude product consisting of
practically pure 8 (2.71g, 98%), which was used in the next step without further purification. An anal. sample
20
D
was purified by FC (hexane/AcOEt 8 :2; Rf 0.3) to give pure 8. Colorless oil. ‰aŠ ˆ À16.0 (c ˆ 0.70, CHCl3). IR
(neat): 1757 s (CˆO), 1734 s (CˆO). 1H-NMR (300 MHz, CDCl3): 7.40 7.23 (m, 5 H); 4.51( s, 2 H); 4.41 4.25
(m, 1H); 4.25 4.09 ( m, 1H); 4.21( q, J ˆ 7.2, 2 H); 3.72 3.54 (m, 2 H); 2.15 2.03 (m, 1H); 2.03 1.89 ( m,
1H); 1.45 ( s, 3 H); 1.43 (s, 3 H); 1.30 (t, J ˆ 7.1, 3 H ) . 13C-NMR (50 MHz, CDCl3): 170.62; 138.28; 128.35; 127.51;
110.70; 79.14; 76.41; 72.96; 66.61; 61.32; 33.60; 27.10; 25.69; 14.11. Anal. calc. for C17H24O5: C 66.21, H 7.84;
found: C 66.10, H 7.79.
Ethyl (4R,5S)-5-(2-Hydroxyethyl)-2,2-dimethyl-1,3-dioxolidine-4-carboxylate (9). A soln. of 8 (2.69 g,
8.75 mmol) in abs. EtOH (60 ml) and in the presence of 10% Pd/C (0.54 g) was vigorously stirred under H2 at r.t.
for 18 h. Evaporation of the filtered (Celite) org. soln. afforded a crude liquid product consisting of practically
pure 9 (1.91 g, 99%), which was used in the next step without further purification. An anal. sample of 9 was
20
D
purified by FC (hexane/AcOEt 1:1; Rf 0.3) to give pure 9. Colorless liquid. ‰aŠ ˆ À10.0 (c ˆ 0.48, CHCl3). IR
(neat): 3446s (OH), 1753s (CˆO), 1734s (CˆO). 1H-NMR (300 MHz, CDCl3): 4.32 4.19 (m, 4 H); 3.83 (t, J ˆ
5.7, 2 H); 2.12 2.00 (m, 1 H); 1.98 1.89 (m, 1H); 1.47 ( s, 3 H); 1 .43 (s, 3 H); 1 .30 (t, J ˆ 7.2, 3 H). 13C-NMR
(75 MHz, CDCl3): 170.75; 111.01; 79.00; 77.85; 61.52; 60.01; 35.82; 27.05; 25.55; 14.13. Anal. calc. for C10H18O5: C
55.03, H 8.31; found: C 55.29, H 8.56.
Ethyl (4R,5S)-5-(2-Iodoethyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (10). A soln. of PPh3 (3.127 g,
11.92 mmol) in anh. CH2Cl2 (37 ml) under N2, was treated with 1H-imidazole (0.81g, 11.92 mmol) and then with
I2 (3.03 g, 11.92 mmol) [13]. A soln. of 9 (2.00 g, 9.17 mmol) in anh. CH2Cl2 (9 ml) was added, and the mixture
was stirred at r.t. for 2 h. Evaporation of the solvent gave a crude product, which was filtered through a short
20
D
silica-gel column (hexane/AcOEt 9 :1; Rf 0.5) to give pure 10 (2.70 g, 90%). Yellow oil. ‰aŠ ˆ À30.0 (c ˆ 0.39,

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Helvetica Chimica Acta Vol. 85 (2002)
CHCl3). IR (neat): 1759s (CˆO), 1730s (CˆO). 1H-NMR (300 MHz, CDCl3): 4.25 (q, J ˆ 7.1, 2 H); 4.21 4.16
(m, 1H); 4.13 ( d, J ˆ 7.3, 1H); 3.36 3.20 ( m, 2 H); 2.37 2.26 (m, 1H); 2.24 2.11( m, 1 H); 1.45 (s, 3 H); 1.44
(s, 3 H); 1.31 (t, J ˆ 7.2, 3 H). 13C-NMR (75 MHz, CDCl3): 170.72; 126.89; 111.64; 79.07; 78.77; 61.92; 38.13;
27.45; 25.99; 14.58; 0.89. Anal. calc. for C10H17IO4: C 36.30, H 5.22; found: C 36.17, H 5.01.
Ethyl (4R,5S)-2,2-Dimethyl-5-[2-(4-nitrophenoxy)ethyl]-1,3-dioxolane-4-carboxylate (11). A soln. of 10
(2.680 g, 8.17 mmol) in anh. DMF (32 ml) was treated with sodium 4-nitrophenolate (1.710 g, 10.62 mmol), and
the mixture was stirred at 508 for 16 h. Aq. workup (Et2O/H2O) and evaporation of the org. phase afforded a
crude product consisting of practically pure 11 (2.46 g, 89%), which was used in the next step without further
purification. An anal. sample of 11 was purified by FC (hexane/AcOEt 8 :2; Rf 0.3) to give pure 11. Pale yellow
20
oil. ‰aŠ ˆ À19.4 (c ˆ 0.52, CHCl3). IR (neat): 1755s (CˆO), 1732 s (CˆO). 1H-NMR (300 MHz, CDCl3): 8.20
D
(d, J ˆ 9.2, 2 H); 6.96 (m, J ˆ 9.2, 2 H); 4.38 4.32 (m, 1H); 4.30 4.16 ( m, 5 H); 2.40 2.29 (m, 1H); 2.23 2.12
(m, 1H); 1.47 ( s, 3 H); 1.44 (s, 3 H); 1 .27 (t, J ˆ 7.0, 3 H). 13C-NMR (75 MHz, CDCl3): 170.32; 163.66; 141.64;
125.87; 114.43; 111.15; 79.04; 75.71; 65.15; 61.48; 33.10; 27.09; 25.68; 14.12. Anal. calc. for C16H21NO7: C 56.63, H
6.24, N 4.13; found: C 56.32, H 6.08, N 3.92.
Ethyl (2R,3S)-2,3-Dihydroxy-5-(4-nitrophenoxy)pentanoate (12). A soln. of 11 (0.83 g, 2.439 mmol) in abs.
EtOH (40 ml) was treated with TsOH (0.23 g, 1.22 mmol), and the mixture was stirred at 508 for 20 h. After
cooling, Na2CO3 (0.232 g) was added, and the mixture was stirred at r.t. for 10 min. Evaporation of the org.
solvent afforded a crude product, which was subjected to FC (hexane/AcOEt 1:1; Rf 0.39) to give pure 12
20
D
(0.51 g, 70%). White solid. M.p. 103 1048. ‰aŠ ˆ À29.5 (c ˆ 0.22, CHCl3). IR (nujol): 3391s (OH), 3277s
(OH), 1730s (CˆO). 1H-NMR (300 MHz, CDCl3): 8.20 (d, J ˆ 9.2, 2 H); 6.97 (d, J ˆ 9.2, 2 H); 4.35 4.18 (m,
5 H); 4.14 (dd, J ˆ 4.78, 2.21, 1 H); 2.19 2.10 (m, 2 H); 1 .33 t(, J ˆ 7.2, 3 H). 13C-NMR (50 MHz, CDCl3):
172.97; 163.78; 141.62; 125.92; 114.49; 73.28; 69.29; 65.43; 62.45; 33.31; 14.17. Anal. calc. for C13H17NO7: C 52.17,
H 5.73, N 4.68; found: C 52.33, H 6.01, N 4.74.
Ethyl (2R,3S)-3-Hydroxy-5-(4-nitrophenoxy)-2-[(2-nitrophenylsulfonyl)oxy]pentanoate (13). A soln. of 12
(1.38 g, 4.63 mmol) in anh. pyridine (23 ml) was treated at 08 with 2-nitrophenylsulfonyl chloride (1.03 g,
4.63 mmol), and the mixture was left at this temp. for 24 h [14]. After dilution in Et2O, evaporation of the
washed (H2O, 1n aq. HCl, sat. aq. NaCl) org. soln. gave a crude product, which was purified by FC (hexane/
20
D
AcOEt 6 :4; Rf 0.3) to give pure 13 (1.68 g, 75%). Pale yellow solid. M.p. 111 1128. ‰aŠ ˆ À27.0 (c ˆ 0.3,
CHCl3). IR (nujol): 3493 s (OH), 1715 s (CˆO). 1H-NMR (300 MHz, CDCl3): 8.40 (d, J ˆ 8.5, 2 H); 8.18 (d,
J ˆ 9.2, 4 H); 6.96 (d, J ˆ 9.2, 2 H); 5.11 (d, J ˆ 2.94, 1H); 4.49 4.44 ( m, 1H); 4.32 4.07 ( m, 4 H); 2.16 2.09
(m, 2 H); 1.21 (t, J ˆ 7.2, 3 H). 13C-NMR (75 MHz, CDCl3): 167.04; 164.10; 151.65; 142.53; 142.51; 130.25;
126.63; 124.97; 115.15; 81.61; 69.22; 65.44; 63.31; 33.21; 14.68. Anal. calc. for C19H20N2O11S: C 47.11, H 4.16, N
5.78; found: C 47.32, H 4.29, N 5.60.
Ethyl (2S,3S)-2-Azido-3-hydroxy-5-(4-nitrophenoxy)pentanoate (14). A soln. of 13 (1.21 g, 2.51 mmol) in
anh. DMF (43 ml) was treated with NaN3 (1.01 g, 15.61 mmol), and the mixture was stirred under N at 508 for
2
24 h. After cooling, dilution with Et2O and evaporation of the washed (sat. aq. NaCl) mixture afforded a crude
product (0.810 g, 97%) consisting of practically pure 14, which was used in the next step without further
purification. An anal. sample of 14 was subjected to FC (hexane/AcOEt 6 :4; Rf 0.35) to give pure 14. Pale
20
yellow oil. ‰aŠ ˆ À47.6 (c ˆ 0.42, CHCl3). IR (neat): 3509m (OH), 2114s (N3), 1738s (CˆO). 1H-NMR
D
(300 MHz, CDCl3): 8.20 (d, J ˆ 9.2, 2 H); 6.97 (d, J ˆ 9.2, 2 H); 4.42 4.12 (m, 5 H); 4.05 (d, J ˆ 5.9, 1H); 2.18
2.06 (m, 1H); 2.10 1.97 ( m, 1H); 1.35 ( t, J ˆ 7.2, 3 H). 13C-NMR (50 MHz, CDCl3): 168.94; 163.68; 141.77;
126.03; 114.53; 68.73; 66.11; 65.12; 62.47; 32.32; 14.24. Anal. calc. for C13H16N4O6: C 48.15, H 4.97, N 17.28;
found: C 48.26, H 5.21, N 17.37.
Ethyl (2S,3S)-2-Azido-3-(tert-butyloxy)-5-(4-nitrophenoxy)pentanoate (15).
A soln. of 14 (1.075 g,
3.23 mmol) in anh. CH2Cl2 (9 ml), placed in a 25-ml pressure tube cooled at À 508, was treated with liquid 2-
methylpropene (8 ml) and 98% H2SO4 (34 ml). After the tube was plugged, the temp. was raised to 08, and the
mixture was then stirred at r.t. for 72 h. The mixture was cooled again to 08 and, after neutralization (solid
NaHCO3), was stirred at r.t. while N2 was blown through the soln. to remove the unreacted 2-methylpropene.
After taking up the residue with CH2Cl2, evaporation of the washed (H2O) org. phase gave a crude product
(1.33 g), which was subjected to FC (hexane/AcOEt 9 :1; Rf 0.25) to give pure 15 (0.972 g, 77%). Pale yellow
20
solid. M.p. 45 498. ‰aŠ ˆ À9.92 (c ˆ 1.23, CHCl3). IR (nujol): 2110s (N3), 1751s (CˆO). 1H-NMR (300 MHz,
D
CDCl3): 8.20 (d, J ˆ 9.2, 2 H); 6.94 (d, J ˆ 9.2, 2 H); 4.39 4.10 (m, 6 H); 2.15 2.04 (m, 1H); 1.90 1.79 ( m,
1H); 1.31( t, J ˆ 7.2, 3 H); 1.22 (s, 9 H). 13C-NMR (75 MHz, CDCl3): 168.63; 164.32; 142.28; 126.62; 114.99;
76.13; 69.62; 67.84; 65.16; 62.60; 31.40; 29.11; 14.84. Anal. calc. for C17H24N4O6: C 53.68, H 6.36, N 14.73; found:
C 53.93, H 6.48, N 14.96.

Helvetica Chimica Acta Vol. 85 (2002)
3097
Ethyl (2S,3S)-Amino-3-(tert-butyloxy)-5-(4-nitrophenoxy)pentanoate (16).
A
soln. of 15 (0.57 g,
1.50 mmol) in THF (20 ml) was treated with PPh3 (0.39 g, 1.50 mmol) and H2O (2.5 ml), and the mixture was
stirred at r.t. for 2 h, until N2 evolution stopped, and was then stirred at 608 for 19 h. After cooling, dilution in
Et2O and evaporation of the washed (sat. aq. NaCl) org. soln. gave a crude mixture (0.95 g) consisting of Ph3PO
and 16 (1H-NMR) as the only reaction product, which was used in the next step without further purification.
1H-NMR (300 MHz, CDCl3): 8.20 (d, J ˆ 9.2, 2 H); 6.95 (d, J ˆ 9.2, 2 H); 4.30 4.08 (m, 5 H); 3.87 (d, J ˆ 4.6,
1H); 2.10 1.92 ( m, 1H); 1.88 1.70 ( m, 1H); 1.38 ( t, J ˆ 7.2, 3 H); 1.2 (s, 9 H).
(2S,3S)-3-(tert-Butyloxy)-2-{N-[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-5-(4-nitrophenoxy)pentanoic
Acid (5a). A soln. of the above described crude mixture (0.474 g) consisting of 16 (0.75 mmol) and Ph3PO in 1,4-
dioxane (2 ml) was treated with a soln. of LiOH (0.054 g, 2.25 mmol) in H2O (2 ml), and the mixture was stirred
at r.t. for 2 h. After dilution with H2O (3 ml) and neutralization (5n HCl), solid Na2CO3 (0.525 g) was added to
achieve pH ˆ 8 9. The mixture was cooled to 08, treated with a soln. of FmocONSu (ˆ N-[(9H-fluoren-9-
ylmethoxy)carbonyloxy]succinimide; 0.28 g, 0.83 mmol) in 1,4-dioxane (1.5 ml) and then stirred at r.t. for 18 h.
After cooling to 08, 5n HCl was added to achieve pH ˆ 2, and the mixture was extracted with Et2O. Evaporation
of the combined Et2O extracts gave a crude product, which was purified by FC (AcOEt/hexane/AcOH
4 :5.9 :0.1; Rf 0.26) to give pure 5a (0.293, 71%), which, after trituration with CH2Cl2/hexane, was obtained as a
20
pale yellow solid. M.p. 107 1098. ‰aŠ ˆ ‡14.87 (c ˆ 0.39, CHCl3). IR (nujol): 1717s, 1668s. 1H-NMR
D
(300 MHz, CDCl3): 8.17 (d, J ˆ 8.8, 2 H); 7.75 (d, J ˆ 7.7, 2 H); 7.58 (d, J ˆ 7.0, 2 H); 7.39 (t, J ˆ 7.3, 2 H); 7.30 (d,
J ˆ 7.3, 2 H); 6.93 (d, J ˆ 7.7, 2 H); 4.75 4.63 (m, 1H); 4.72 ( d, J ˆ 7.0, 2 H); 4.23 (t, J ˆ 6.8, 2 H); 4.14 (br. s,
2 H); 2.12 1.88 (m, 2 H); 1 .20 s(, 9 H). 13C-NMR (75 MHz, CDCl3): 173.80; 163. 49; 156.26; 143.62; 141.67;
141.31; 127.76; 127.05; 125.91; 124.98; 120.01; 114.39; 75.38; 68.61; 67.24; 64.70; 58.18; 47.14; 31.66; 28.29. EI-MS
(pos.): m/z (relative intensity): 549 (4), 493 (15), 271 (11), 179 (100), 178 (48). Anal. calc. for C30H32N2O8: C
65.68, H 5.88, N 5.11; found: C 65.83, H 6.11, N 5.29.
1
Solid-Phase Peptide Synthesis (SPPS). General. Nonapeptide KRAVNle-5-EANleNH2 ) (1; HIV-protease
substrate) and tetrapeptide H2N-Ala-Arg-5-Ala-NH2 (17; trypsin substrate) were prepared by SPPS with Rink-
resin (Bachem) (200 mg, 0.47 mmol gÀ1, 200 400 mesh). The following protected amino acids, purchased from
Novabiochem or Bachem, were used: Fmoc-Nle, Fmoc-Ala, Fmoc-Glu(t-BuO)-OH, Fmoc-Val, Fmoc-
Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH. The peptides were prepared according to a standard protocol for Fmoc-
SPPS and purified by prep. reverse-phase HPLC.
Nonapeptide 1. Nonapeptide 1 was obtained as a white solid (9%) (tR ˆ 27.7 min, A/D 80 :20 20 :80 with
1% minÀ1 gradient). The structure of the peptide was confirmed by 1H-NMR (300 MHz, CD3OD) and ES-MS
‡
(pos.; 1150 ([M À H] )).
Tetrapeptide 17. Tetrapeptide 17 was obtained as a white solid (24%) (tR 18.6 min, A/D 95 :5 35 :65 with
1% minÀ1 gradient). The structure of the peptide was confirmed by 1H-NMR (300 MHz, CD3OD) and ES-MS
‡
(pos.; 567 ([M À H] )).
Enzyme Measurements. The peptides 1 and 17 were diluted from 1m m stock solns. in deionized milliQ-
filtered H2O. The proteases trypsin (from hog pancreas, 1650 U/mg, Fluka 93615), a-chymotrypsin (from bovine
pancreas, 37 U/mg, Sigma SC-4129), and papain (from Papaya latex, 30 U/mg, Sigma SR-4762) were diluted
from 1mg ml À1 stock solns. in phosphate buffer saline (PBS, 10 mm phosphate pH 7.2, 160 mm NaCl). HIV-
¾
Protease was obtained by in vitro expression from a plasmid by means of the Phenomics expression system
(Proteus SA, NÓmes, France), and the soln. also contained glycerol, which required additional IOÀ4 for detection
¬
(see below). The reactions were run in polystyrene 96-well half-area cell-culture plates and recorded on a
Spectramax 250 microplate spectrophotometer (Molecular Devices). Reactions were initiated by addition of the
substrate stock soln. (10 ml) to the prediluted enzyme soln. (90 ml) in their respective buffer (see the Table), and
incubated at 378 for 2 h. The pH was then adjusted to pH 9 by addition of 0.2m Na2CO3 (20 ml), BSA (final conc.
2 mg mlÀ1) and NaIO4 (final conc. 1m m, 20 mm with HIV protease). The assay was then incubated at 258 for
60 min, and the OD was recorded at 405 nm as endpoint. The OD at 405 nm (0.05 0.60) was used to calculate
the 4-nitrophenol conc. according to a calibration curve under the same conditions.
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ReceivedJune 3, 2002