Synthesis and bioorthogonal coupling chemistry of a novel cyclopentenone-containing unnatural tyrosine analogue

Article abstract of DOI:10.1016/j.bioorg.2007.12.006

Herein we report the synthesis of a novel amino acid with orthogonal functionality to the natural amino acid side chains. Tyrosine was O-alkylated with a cyclic 5-membered α,β-unsaturated ketone ring (5). We have established that this amino acid analogue can undergo cycloaddition reactions in aqueous media with in situ generated nitrones. Nitrone formation occurred by micellar catalysis can undergo aqueous 1,3-dipolar cycloaddition reactions with the unnatural Tyr. We also performed a linear free energy analysis of the one pot bioconjugation reaction in water using cyclopentenone as a model for the Tyr analogue and seven different aryl nitrones. We found that the Hammett ρ value was -0.94, suggesting that the reaction occurs in a concerted fashion with a slight positive charge buildup in the transition state. The Hammett ρ value also suggests that the bioconjugation reaction is tolerant of different substituents and thus may be useful for introducing novel functionality into peptides and proteins containing the Tyr analogue 5. The aqueous 1,3-dipolar cycloaddition reactions, that use nitrones to trap the O-alkylated Tyr 5, establish a novel strategy for rapid, water compatible bioconjugation reactions.

Full text of DOI:10.1016/j.bioorg.2007.12.006

Available online at www.sciencedirect.com
Bioorganic Chemistry 36 (2008) 91–97
www.elsevier.com/locate/bioorg
Synthesis and bioorthogonal coupling chemistry of a novel
cyclopentenone-containing unnatural tyrosine analogue
a,b
Gianni R. Lorello a,b,1, Marc C.B. Legault a,b,1, Bojana Rakic
,
´
Kathrine Bisgaard a, John Paul Pezacki a,b,
*
a Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ont., Canada K1A 0R6
b Department of Chemistry, University of Ottawa, Ottawa, Canada
Received 28 September 2007
Available online 12 February 2008
Abstract
Herein we report the synthesis of a novel amino acid with orthogonal functionality to the natural amino acid side chains. Tyrosine was
O-alkylated with a cyclic 5-membered a,b-unsaturated ketone ring (5). We have established that this amino acid analogue can undergo
cycloaddition reactions in aqueous media with in situ generated nitrones. Nitrone formation occurred by micellar catalysis can undergo
aqueous 1,3-dipolar cycloaddition reactions with the unnatural Tyr. We also performed a linear free energy analysis of the one pot bio-
conjugation reaction in water using cyclopentenone as a model for the Tyr analogue and seven different aryl nitrones. We found that the
Hammett q value was À0.94, suggesting that the reaction occurs in a concerted fashion with a slight positive charge buildup in the tran-
sition state. The Hammett q value also suggests that the bioconjugation reaction is tolerant of different substituents and thus may be
useful for introducing novel functionality into peptides and proteins containing the Tyr analogue 5. The aqueous 1,3-dipolar cycload-
dition reactions, that use nitrones to trap the O-alkylated Tyr 5, establish a novel strategy for rapid, water compatible bioconjugation
reactions.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Tyrosine analogue; Nitrone; Micellar catalysis; Bioconjugation reaction; [3+2] cycloadditon reaction
1. Introduction
functional properties of different molecular scaffolds [17–
20]. Ideally, these reactions are bioorthogonal in that no
side reactions occur with the endogenous functional groups
in the biological system. To date, there are several existing
bioconjugation reactions such as Huisgen cycloaddition
and Staudinger ligation, each suited to specific substrates
and applications [2]. Nucleophilic catalysis of an oxime
ligation of peptides has also been demonstrated recently
[3]. The reactive groups involved in bioorthogonal chemical
reactions of this type can be incorporated into a system of
interest either by in vitro methods or by metabolic labeling
strategies [4]. One common method for the latter is the use
of unnatural amino acids [19,20]. The addition of unnatu-
ral amino acids (UAAs) into peptides and proteins has
enabled the generation of biomolecules with novel proper-
ties and additional chemical functionality. Site specific
incorporation of UAAs has been successfully achieved
Highly selective chemical reactions that occur rapidly
and with high yield and do so in complex biological media
are important for a number of applications including the
chemical synthesis of natural and unnatural peptides and
proteins [1–5]. Some examples include the bioconjugation
of proteomic tags to target proteins within extracted prote-
omes as well as in living systems [6,7], the addition of
unnatural functionality to biomolecules such as proteins
[8–16], and for the systematic alteration of structural and
*
Corresponding author. Address: Steacie Institute for Molecular
Sciences, National Research Council of Canada, 100 Sussex Drive,
Ottawa, Ont., Canada K1A 0R6. Fax: +1 613 952 0068.
E-mail address: John.Pezacki@nrc.ca (J.P. Pezacki).
1
These authors contributed equally to this work.
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.12.006

92
G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
using in vitro translation systems as well as using orthogo-
nal aminoacyl tRNA synthetases/tRNA pairs expressed in
bacteria [16–19] and eukaryotes [20].
the synthesis of 5 is depicted in Fig. 1. We chose to demon-
strate micellar catalyzed reactions with the unnatural tyro-
sine analog 5 because of its increased solubility in micelles,
rather than work with the free amino acid. The O-activated
N-tBoc-protected Tyr was reacted with butyl amine to pro-
duce amide 2 in 99% yield. The Mitsunobu coupling reac-
tion between 2 and (1R,4S)-cis-4-acetoxy-2-cyclopenten-1-
ol afforded olefin 3 in 43% yield. Ethanolic basic hydrolysis
of the acetal was executed to form the secondary alcohol 4
in 70% yield. Finally, PDC oxidation of 4 afforded 5 in 87%
yield (Fig. 1).
The [3+2] cycloadditions between the a,b-unsaturated
ketone functionality in 5 and nitrones were then performed
in aqueous media in the presence of SDS micelles [24]. Aryl
nitrones were efficiently generated in situ from phenyl
hydroxylamine and substituted benzaldehydes, inside
SDS micelles, as previously reported [24]. Phenyl hydroxyl
amine was prepared by reducing nitrobenzene with Zn fol-
lowing the previously reported procedure [25]. Freshly
obtained phenyl hydroxylamine was then reacted with
benzaldehyde in the presence of 0.1 M SDS with 5 min son-
ication and 2 h of stirring at room temperature. Sonication
allowed faster formation of the nitrone which was moni-
tored by TLC. We determined that a concentration of
0.1 M of SDS was sufficient to form micelles that concen-
trate the organic reagents preferentially in the hydrophobic
core of the micelle. Water molecules formed during the
reaction are expelled from micellar hydrophobic interior
leaving the nitrone product inside the micelle and promot-
ing further reaction. The [3+2] cycloaddition reaction with
Recently it has been shown that the unnatural amino
acids such as O-methyl-L-tyrosine [21,22], and O-allyl-L-
tyrosine [15], as well as a host of other UAAs can be site-
specifically incorporated into proteins with high efficiency
and fidelity [19]. Here we report the synthesis of a novel
R-(4-oxocyclopent-2-enyloxy)-L-tyrosine derivative con-
taining an a,b-unsaturated ketone functionality. We show
that this UAA can be trapped under physiological condi-
tions with aryl nitrones generated in situ utilizing micellar
catalysis [24]. This establishes a novel method for site spe-
cific functionalization of peptides and proteins in vitro that
is complementary to existing methods used for this
purpose.
2. Results and discussion
Previous studies have established that [3+2] cycloaddi-
tions involving organic nitrones and electron deficient ole-
fins occur efficiently in aqueous solutions with the aid of
micellar catalysis [23]. In order to adapt this reaction to
bioconjugate chemistry, we first sought to develop a suit-
able UAA containing an electron deficient olefin that
would be reactive enough to efficiently undergo cycloaddi-
tion reactions with nitrones but not so reactive that it
would undergo addition reactions with the side chains of
cystiene residues. We chose to make R-(4-oxocyclopent-2-
enyloxy)-L-tyrosine 5 and the approach that we took for
OAc
OH
OH
O
H
N
c
a
b
H
N
O
O
H
N
O
O
O
O
N
O
O
O
N
H
O
O
O
N
H
3
1
2
OH
O
O
O
d
H
N
H
N
O
O
O
O
O
N
H
O
N
H
4
5
Fig. 1. Synthesis of unnatural O-alkylated Tyr derivative 5. (a) Butyl amine, DCM, reflux, 1 h; (b) (1R,4S)-cis-4-acetoxy-2-cylcopenten-1-ol, DIAD, PPh3,
THF, rt, 24 h; (c) 1 M LiOH, THF, rt, 24 h; (d) PDC, DCM, rt, 7 h.

G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
93
the modified Tyr alkene 5 was performed in situ. The
desired reaction product 6 was observed, however only
13% of the product could be successfully isolated after 2
days of stirring at 37 °C (Fig. 2). We determined that 5
was completely recoverable from the latter reaction and
thus was stable to the reaction conditions.
5 can be performed for longer reaction times and at higher
temperatures, however these conditions would be impracti-
cal for in vivo applications.
Although the L-tyrosine derivative 5 was observed to
undergo cycloaddition reactions with nitrones slowly under
physiological conditions, 5 is also a Michael acceptor that
can potentially react with endogenous biological nucleo-
philes such as thiol groups via 1,4-nucleophilic addition.
To determine if such addition reactions would occur com-
petitively with nitrone cycloaddition, we performed compe-
tition experiments between modified Tyr substrate 5, the
diphenyl nitrone, and glutathione (up to 2 lM). We moni-
tored the product formation by LC/MS. After 48 h stirring
at 37 °C, this competitive study showed exclusively the
[3+2] nitrone cycloaddition product with no thiol 1,4-addi-
tion product observed. This observation indicates that the
unnatural tyrosine analogue 5 is suitable for bioconjugate
chemistry because it displays reactivity that is orthogonal
to the natural functionality of proteins and other cellular
materials [6].
Secondly, we investigated substituent effects of aryl nit-
rones on the relative rates of 1,3-dipolar cycloaddition
reaction with cyclopentenone, a model for 5. We chose to
perform this study with a model system rather than 5
because of the slower reaction times for 5 relative to those
of cyclopentenone, probably because of differences in solu-
bility in the micelles used for catalysis. Specifically, we per-
formed competition reactions involving the cycloaddition
of diphenyl nitrone and a series of nitrones substituted with
substituent X at the para position on the phenyl ring with a
limiting amount of cyclopentenone (Fig. 3), at 37 °C. In
one pot, an excess amount of phenyl hydroxylamine was
reacted with equimolar concentrations of benzaldehyde
and the para-substituted benzaldehyde for 90 min in
0.1 M SDS. The excess phenyl hydroxylamine was used
to ensure complete formation of both nitrones. For the
[3+2] cycloaddition reaction, less than 1 equivalent of
cyclopentenone was used. This reaction was performed
for 48 h at 37 °C at which time complete conversion to
products was observed. The reaction products were then
analyzed by LC/MS. The relative rate constants for the
competing cycloaddition reactions were determined from
the relative peak heights or ion yields for the products in
the LC/MS (see experimental section for further details).
The seven substituted benzaldehydes (R = –OH;
–N(CH3)2; –NO2; –CH3; –Cl; –CN; –OCH3) were tested
To optimize the yield of 6 we systematically varied the
reaction conditions including reaction time, temperature,
and ratios of reactants and catalyst. While optimizing the
yield of this reaction we discovered that, even after pro-
longed reaction times, only a maximum of ꢀ38% yield
was achievable even after 12–14 days with no decomposi-
tion of unreacted 5. We hypothesized that product inhibi-
tion of the micelle catalyzed cycloaddition step was
occurring and thus limiting the yield. Product inhibition
did not occur for the micelle catalyzed nitrone formation
step since we could observe full formation of the nitrone
by LC–MS. To test this hypothesis we conducted reactions
at 37 °C with a molar excess (0.025 M SDS and a ꢀ100-
fold reduction in the concentration of the other starting
materials) of SDS and observed yields of ꢀ84% of 6.
The stereochemistry of 6 was elucidated using a combi-
nation of NOE experiments of resonances associated with
the hydrogens of the 5-membered ring. High conversions
could be achieved, however, the solubility of 5 within the
SDS micelle is likely a limiting factor for the reaction times
which typically required 48–72 h. The low reactivity of 5
towards diphenyl nitrone indicates that the functionaliza-
tion of peptides and proteins containing 5 via [3+2] cyclo-
addition reactions with nitrones will likely be limited to
in vitro synthesis. Peptide conjugates prepared in vitro using
O
H
Ph(R)
N
O
Ph
O
O
H
a, b
H
H
N
O
O
O
N
H
6
Fig. 2. Synthesis of 1,3-dipolar cycloaddition adduct 6. (a) PhCHO,
PhNHOH, 0.1 M SDS, sonication, rt, 2 h; (b) 5, 37 °C, 2 days.
CHO
O
O
NHOH
O
*
N
N
R(H)
*
*
37°C, 48 h
R(H)
0.1 M SDS
O
(H)R
Fig. 3. General reaction scheme depicting nitrone synthesis and 1,3-dipolar cycloaddition reaction. R = –OH, –Me, –OMe, –NO2, –Cl, –CN, –N(CH3)2.

94
G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
in duplicates. The relation between the substituent on the
phenyl ring and the rate of reaction with the [3+2] cycload-
dition reaction is presented in the Hammet plot and plot
fits the data best using rp parameters (see Fig. 4). The plot
gives a q of À0.94. This value of q is consistent with a tran-
sition state that has modestly more localized positive
charge than the reactant and is consistent with a concerted
rather than a stepwise mechanism for the micelle catalyzed
cycloaddition.
For the plot in Fig. 4, the cycloaddition products of two
analogs did not agree well with the line of best fit. These
were the products formed from reaction with 4-hydroxy-
benzaldehyde and 4-chlorobenzaldehyde. For the former,
the pKa of the hydroxyl group is ꢀ7.6 which in aqueous
solution is largely deprotonated. We interpret the lower
than expected reactivity of 4-hydroxybenzaldehyde in the
one pot reaction to be due to an unfavorable solubility of
the charged species inside the non-polar interior of the
micelle. This lowers the rate constant for the reaction with
the olefin as indicated in Fig. 5. For the case of 4-chloro-
benzaldehyde, significant dimerization was observed dur-
ing the nitrone formation reaction [26–30] that led to a
lower ratio of rate constants. However, when the olefin
was added to the nitrone-forming reaction after 10 min,
negligible dimer was observed and the ratio of rate con-
stants for kX/kH fit the line in the plot in Fig. 4. The q value
representing the slope of the line of best fit is small indicat-
ing that the micelle catalyzed [3+2] cycloaddition in water
displays little sensitivity towards substituent effects. This
indicates that the reaction can be used with a variety of
substrates and that these reactions should proceed with
similar efficiency.
2
1
-N(Me)2
-Me
0
-Cl
-OMe
-CN
-1
-OH
-NO2
-2
-1.5
-1
-0.5
0
σ
0.5
1
1.5
Fig. 4. Hammett plot depicting the substituent effect on the one pot 1,3-
dipolar cycloaddition reaction. The open circles represent the data points
obtained after a 2-h pre-incubation of the aldehydes with an excess of
hydroxylamine followed by the addition of olefin. The solid triangle
represents the data point for 4-chlorobenzaldehyde following only a
10 min pre-incubation of aldehydes with hydroxylamine followed by the
addition of olefin. The solid circle represents the data point for
4-hydroxybenzaldehyde that deviates significantly from the linear free
energy relationship. Extrapolation of points shows a q value of À0.94
(r = 0.98).
In conclusion, we have prepared a novel O-alkylated
tyrosine analogue with a cyclic 5-membered a,b-unsatu-
O
NHOH
+
CHO
OH
O
O
CHO
OH
NHOH
*
N
0.1 M SDS
N
+
*
*
O
OH
HO
+
H2O
-H+
+H+
-H+
-H+
+H+
+H+
O
O
NHOH
*
N
*
*
X
X
O
O
CHO
O
O
N
O
Fig. 5. General reaction scheme depicting the proposed mechanism by which nitrone synthesis and 1,3-dipolar cycloaddition reaction occur for the
reaction involving 4-hydroxybenzaldehyde (R = ÀOH) that results in its lower reactivity.

G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
95
rated ketone ring amino acid with orthogonal function-
3.1. Synthesis
ality to the natural amino acid side chains. We have
shown this amino acid analogue can undergo cycloaddi-
tion reactions in aqueous media with in situ generated
nitrones in one pot reactions and that these reactions
may be useful in the chemical synthesis of unnatural
proteins. We evaluated the linear free energy relation-
ships of the one pot bioconjugation reaction and deter-
mined that the reaction is relatively insensitive to
substituent indicating that a variety of substrates should
be compatible with the reaction conditions described.
We are currently exploring utility of this chemistry
towards the systematic modifications of peptides and
proteins.
3.1.1. N-(Boc-L-tyrosyloxy)butylamine (2)
Butylamine (1.35 ml, 13.65 mmol) was added to a solu-
tion of N-(Boc-L-tyrosyloxy)succinimide (2.58 g, 6.82 mmol)
in dry DCM (20 ml). The reaction mixture was refluxed for
1 h. Ten microliters of water was added and the resulting
mixture was extracted with DCM, dried over anhydrous
MgSO4, filtered and concentrated in vacuo. Purification by
flash chromatography (heptane/EtOAc 1:1) afforded the
desired product (2.30 g, 99.9%). TLC (heptane/EtOAc 1:1)
1
Rf 0.18. H NMR (400 MHz CDCl3) 7.06 (d, J = 8.4 Hz,
2H), 6.77 (d, J = 8.4 Hz, 2H), 6.07 (br s, 1H), 5.22 (br s,
1H), 4.28 (s, 1H), 3.25–3.13 (m, 2H), 3.07–2.92 (m, 2H),
1.44 (s, 9H), 1.41–1.33 (m, 2H), 1.29–1.19 (m, 2H), 0.89 (t,
J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 172.0,
156.1, 155.7, 130.7, 128.3, 80.8, 60.9, 56.7, 39.7, 38.2, 31.7,
28.7, 21.5, 21.0, 20.3, 14.6, 14.0. MS (ESI+) m/z = 337.3
(MH)+.
3. Materials and methods
All chemicals were obtained from commercial sources
and used without further purification unless indicated.
Analytical thin-layer chromatography was performed on
commercial glass plates pre-coated (250 lm layer thick-
ness) with silica gel 60 F254 (E. Merck). The TLC spots
were viewed under ultraviolet light (254 nm) and by heat-
ing the TLC plate after treatment with a solution of
ammonium molybdate in 10% aqueous H2SO4. Conven-
tional flash column chromatography, using Silicycle Ultra
Pure Silica Gel (230–400 mesh), was performed to purify
all compounds. Removal of organic solvents was per-
3.1.2. (1S,4S)-4-Acetoxy-(4-(2-tert-butoxycarbonylamino-
2-butylcarbamoyl-ethyl)-phenoxy)-2-cyclopentene (3)
N-(Boc-L-tyrosyloxy)butylamine 2 (0.79 g, 2.36 mmol)
was dissolved in dry THF (12 ml) and (1R,4S)-cis-4-acet-
oxy-2-cyclopenten-1-ol (0.40 g, 2.84 mmol), DIAD (0.93
ml, 4.71 mmol) and PPh3 (1.24, 4.71 mmol) were added suc-
cessively at room temperature. The reaction mixture was
stirred at room temperature under the argon atmosphere
for 24 h. Water was added and the resulting mixture was
extracted with EtOAc, dried over anhydrous MgSO4, filtered
and concentrated in vacuo. Purification by flash chromatog-
raphy (heptane/EtOAc 1:1) afforded the desired product
(0.47 g, 43%). TLC (heptane/EtOAc 1:1) Rf 0.31. 1H NMR
(400 MHz, CDCl3), d 7.11 (d, J = 8.4 Hz, 2H), 6.81 (d,
J = 8.4 Hz, 2H), 6.26–6.22 (m, 1H), 6.18–6.14 (m, 1H),
5.88–5.80 (m, 1H), 5.70 (br s, 1H), 5.47–5.42 (m, 1H), 5.07
(br s, 1H), 4.27–4.16 (m, 1H), 3.22–3.09 (m, 2H), 3.09–2.88
(m, 2H), 2.39–2.24 (m, 2H), 2.05 (s, 3H), 1.41 (s, 9H), 1.38–
1.30 (m, 2H), 1.28–1.17 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, CDCl3) d 171.6, 157.3, 136.5, 135.3,
130.9, 130.8, 81.4, 79.0, 60.8, 39.6, 38.3, 38.2, 31.8, 31.0,
28.7, 21.5, 21.4, 20.3, 19.5, 14.6, 14.0. MS (ESI+) m/
z = 461.3 (MH)+.
formed by roto-evaporation on a Buchi R-114 Rotovapor
¨
using a Buchi B-178 vacuum system. Trace solvents were
removed on a high vacuum pump. The 1H and 13C
nuclear magnetic resonance (1H NMR) spectra were
recorded at 400 MHz and at 100 MHz, respectively, on
a Bruker DRX-400 spectrometer. The nuclear magnetic
resonance spectra (NMR) of all compounds were mea-
sured in a solution of deuterated chloroform (CDCl3).
The chemical shifts were reported in parts per million
(ppm) downfield from tetramethylsilane (dscale). The mul-
tiplicity, coupling constants (J in Hz), and number of pro-
tons were indicated in parentheses after each chemical
shift. HPLC/MS analysis was performed with Waters
Alliance 2795 liquid chromatograph equipped with
Waters 996 PDA diode array detector and connected to
Micromass ZQ2000 mass spectrometer equipped with
pneumatically assisted electrospray ionization source,
operating in positive mode. The Waters SunFire C18
(2.1 Â 100 mm, 3.5 lm) column was used. Samples were
run on a Waters Alliance 2795 LC Column, with the col-
umn temperature at 20 °C. HPLC was performed with
gradient elution of acetonitrile/water both with 0.1% for-
mic acid. Flow rate was 0.2 ml/min and all the eluent was
directed first to diode array detector and then to mass
spectrometer. The source temperature was set at 80 °C,
desolvation gas temperature was set at 200 °C an electro-
spray capillary was set at 3.5 kV with a cone voltage set at
10 V. Data were collected in single ion recording (SIR)
mode.
3.1.3. (1R,4R)-4-Hydroxy-(4-(2-tert-butoxycarbonylamino-
2-butylcarbamoyl-ethyl)-phenoxy)-2-cyclopentene (4)
(1R,4R)-4-Acetoxy-(4-(2-tert-butoxycarbonylamino-2-
butylcarbamoyl-ethyl)-phenoxy)-2-cyclopentene 3 (0.47 g,
1.02 mmol) was dissolved in THF (10 ml), followed by
addition of 1 M LiOH (10 ml). The reaction mixture was
stirred at room temperature for 24 h. The solvent was then
evaporated under reduced pressure and the residue was dis-
solved in ethyl acetate, washed with water, dried over anhy-
drous MgSO4, filtered and concentrated in vacuo.
Purification by flash chromatography (heptane/EtOAc
2:3) afforded the desired product (0.29 g, 70%). TLC (hep-
1
tane/EtOAc 2:3) Rf 0.11. H NMR (400 MHz, CDCl3), d

96
G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
7.11 (d, J = 8.4 Hz, 2 H), 6.81 (d, J = 8.4 Hz, 2H), 6.18–
6.12 (m, 2H), 5.68 (br s, 1H), 5.48–5.43 (m, 1H), 5.17–
4.98 (m, 2H), 4.26–4.15 (m, 1H), 3.22–3.09 (m, 2H),
3.08–2.86 (m, 2H), 2.37–2.27 (m, 1H), 2.22–2.14 (m, 1H),
1.42 (s, 9H), 1.42–1.29 (m, 2H), 1.28–1.16 (m, 2H), 0.87
(t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) d
171.5, 157.4, 139.6, 133.9, 130.8, 129.4, 115.8, 81.9, 80.6,
76.4, 70.5, 60.8, 56.5, 41.5, 39.6, 38.2, 31.8, 28.6, 22.5,
22.3, 20.3, 14.6, 14.0. MS (ESI+) m/z = 419.4 (MH)+.
116.44, 115.81, 84.86, 75.14, 73.47, 60.93, 45.40, 39.58,
38.19, 31.85, 28.71, 20.32, 14.10. MS (ESI+) m/z = 614.4
(MH)+.
3.2. General procedure for [3+2] cycloaddition reactions
under micellar catalysis
5.0 equivalents (0.618 mmol) of the variable aldehyde
were added to a vial containing 5.0 equivalents of benzalde-
hyde (63 ll, 0.618 mmol) and phenylhydroxylamine
(162 mg, 1.483 mmol, 12 equiv). A 0.1 M solution of SDS
(2 mL, 0.2 mmol) was then added [22]. The mixture was
sonicated for 5 min at room temperature. A Teflon coated
magnetic stirring bar was added and the mixture was stir-
red for 1.5 h to permit nitrone formation. At this point;
1.0 equivalent of 2-cyclopentene-1-one was added and the
reaction was allowed to stir for 48 h at 37 °C. The products
were extracted with ethyl acetate, and the organic layer was
washed twice with brine. The organic phase was then dried
over magnesium sulfate and filtered, and the ratio of prod-
ucts was then analyzed by LC–MS.
3.1.4. (1R)-4-Oxo-(4-(2-tert-butoxycarbonylamino-2-butylc-
arbamoyl-ethyl)-phenoxy)-2-cyclopentene (5)
PDC (0.54 g, 1.43 mmol) was added to a room
temperature solution of (1R,4R)-4-hydroxy-(4-(2-tert-but-
oxycarbonylamino-2-butylcarbamoyl-ethyl)-phenoxy)-2-cyclo-
pentene 4 (0.29 g, 0.69 mmol) in dry DCM (25 ml). The
resulting reaction mixture was stirred for 7 h and then
diluted with Et2O (50 ml) and filtered through celite. Puri-
fication by flash chromatography afforded the desired
product (0.25 g, 87%). TLC (heptane/EtOAc 2:3) Rf 0.33.
1H NMR (400 MHz, CDCl3) 7.64 (dd, J = 5.7, 2.3 Hz,
1 H), 7.10 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H),
6.31 (dd, J = 5.7, 1.1 Hz, 1H), 6.19 (br s, 1H), 5.40–5.35
(m, 1H), 5.27 (br s, 1H), 4.25 (s, 1H), 3.25–3.03 (m, 2H),
3.00–2.88 (m, 2H), 2.82 (dd, J = 18.4, 6.0 Hz, 1H), 2.36
(dd, J = 18.4, 2.0 Hz, 1H), 1.36 (s, 9H), 1.34–1.28 (m,
2H), 1.27–1.10 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). 13C
NMR (100 MHz, CDCl3) 205.4, 171.5,159.9, 156.7, 136.9,
131.1, 115.8, 75.6, 42.2, 39.6, 38.2, 31.8, 28.7, 20.4, 14.1.
MS (ESI+) m/z = 417.3 (MH)+.
3.3. Inlet methods for monitoring kinetics in [3+2]
cycloaddition reactions by LC/MS
para-substituent on benzaldehyde: –N(CH3)2 and
–OCH3. Total run time: 30 min. Mobile phase consisting
of 60% water/0.1% formic acid [A] and 40% MeCN/0.1%
formic acid [B] for 10 min followed by a gradual change
in gradient over the next 10 min to 45% [A] 55% [B], fol-
lowed by a change to 5% [A] 95% [B] at 21 min and run
at this ratio for 9 min.
3.1.5. {1-Butylcarbamoyl-2-[4-(4-oxo-2,3-diphenyl-hexa-
hydro-cyclopenta[d]isoxazol-6-yloxy)-phenyl]-ethyl }-
carbamic acid tert-butyl ester (6)
para-substituent on benzaldehyde: –NO2. Total run
time: 30 min. Mobile phase consisting of 50% water/0.1%
formic acid [A] and 50% MeCN/0.1% formic acid [B]. Gra-
dient is changed over a 20 min period to 45% [A] 55% [B].
Gradient is then changed to 5% [A] 95% [B] over a 5 min
period and run for another 5 min using this gradient.
para-substituent on benzaldehyde: –CH3, –Cl, –OH,
–CN. Total run time: 35 min. Mobile phase consisting of
49% water/0.1% formic acid [A] and 51% MeCN/0.1% for-
mic acid [B] for 10 min. Gradient is then changed over
10 min to 42% [A] 58% [B]. Gradient is then changed to
5% [A] 95% [B] over 1 min and sample is run for 9 min at
this gradient. The gradient is then changed to 90% [A]
10% [B] over a 1 min period and the sample is run for
another 5 min. The plots were generated using sigma values
from the literature [29,30] using GraFit version 4.0 (Erith-
acus Software Ltd., Surrey, UK).
Benzaldehyde (0.12 ml, 1.20 mmol) and phenylhydroxy-
amine (0.13 g, 1.20 mmol) were dissolved in 0.1 M SDS
(1 ml). The reaction mixture was sonicated for 5 min and
then stirred at room temperature for 2 h before compound
5 (0.10 g, 0.24 mmol) was added. The resulting reaction
mixture was stirred for 2 days at 37 °C. Saturated aqueous
NaCl solution was added and the product was extracted
into EtOAc, dried over anhydrous MgSO4, filtered and
concentrated in vacuo. Purification by preparative HPLC
(NovaPak C18 19 Â 300 mm) gradient 10–95% acetonitrile
in water, afforded the desired product (19 mg, 13%). The
stereochemistry of 6 was determined using NOE experi-
ments involving the hydrogens of the 5-membered ring.
1H NMR (400 MHz, CDCl3) 7.45–7.38 (m, 2H), 7.39–
7.33 (m, 2H), 7.32–7.23 (m, 3H), 7.18–7.12 (m, 2H),
7.10–7.05 (m, 2H), 7.05–6.99 (m, 1H), 6.88–6.82 (m, 2H),
5.75 (br s, 1H), 5.16–5.03 (m, 3H), 5.00 (d, J = 6.7 Hz,
1H), 4.27–4.15 (m, 1H), 3.67–3.60 (m, 1H), 3.24–3.08 (m,
2H), 3.06–2.93 (m, 2H), 2.58 (dd, J = 18.2, 6.5 Hz, 1H),
2.42–2.33 (m, 1H), 1.41 (s, 9H), 1.39–1.30 (m, 2H), 1.27–
1.16 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR
(100 MHz, CDCl3) 211.34, 171.37, 155.84, 149.76, 136.62,
131.09, 129.73, 129.50, 129.04, 128.49, 127.81, 123.79,
Acknowledgments
We thank Don Leek and Malgosia Daroszewska for
their assistance with NMRs and mass spectrometry,
respectively.

G.R. Lorello et al. / Bioorganic Chemistry 36 (2008) 91–97
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