Design, synthesis, and cyclization of 4-aminobutyric acid derivatives: Potential candidates as self-immolative spacers

Article abstract of DOI:10.1039/c0ob00890g

Self-immolative spacers have gained significant interest in recent years due to their utility in numerous prodrug, sensor and drug delivery systems. However, there are a very limited number of spacers that are capable of undergoing spontaneous and rapid reactions under mild conditions. To address this need, 4-aminobutyric acid derivatives were explored as a potential class of self-immolative spacers. Using a modular approach, eleven N- and α-substituted derivatives of 4-aminobutyric acid were synthesized, and their intramolecular cyclizations to γ-lactams were studied. Kinetics experiments were carried out at physiological pH and temperature, and the observed half-lives for the spacers ranged from 2 to 39 s, depending on the molecular structure. In addition, the pH dependence of the cyclization rate was also explored and it was found that cyclization still occurred rapidly at mildly acidic pH. Therefore, this class of compounds exhibits promise for incorporation into a variety of self-immolative systems where rapid cyclization reactions are desired.

Full text of DOI:10.1039/c0ob00890g

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PAPER
Design, synthesis, and cyclization of 4-aminobutyric acid derivatives: potential
candidates as self-immolative spacers†
Matthew A. DeWita and Elizabeth R. Gillies*a,b
Received 17th October 2010, Accepted 13th December 2010
DOI: 10.1039/c0ob00890g
Self-immolative spacers have gained significant interest in recent years due to their utility in numerous
prodrug, sensor and drug delivery systems. However, there are a very limited number of spacers that are
capable of undergoing spontaneous and rapid reactions under mild conditions. To address this need,
4-aminobutyric acid derivatives were explored as a potential class of self-immolative spacers. Using a
modular approach, eleven N- and a-substituted derivatives of 4-aminobutyric acid were synthesized,
and their intramolecular cyclizations to g-lactams were studied. Kinetics experiments were carried out
at physiological pH and temperature, and the observed half-lives for the spacers ranged from 2 to 39 s,
depending on the molecular structure. In addition, the pH dependence of the cyclization rate was also
explored and it was found that cyclization still occurred rapidly at mildly acidic pH. Therefore, this
class of compounds exhibits promise for incorporation into a variety of self-immolative systems where
rapid cyclization reactions are desired.
liberation of the substrate. As shown in Fig. 1, these intramolecular
Introduction
reactions generally involve electronic rearrangements such as 1,4,
1,6, or 1,8 elimination reactions2,3 or cyclizations to form highly
favored five- or six-membered rings.4,5
Chemical moieties capable of undergoing rapid and spontaneous
intramolecular reactions in response to the cleavage of a capping
or triggering unit are commonly referred to as self-immolative
spacers.1 In their typical form, these moieties comprise two reactive
termini with a capping group or trigger as one terminus and the
substrate of interest, such as a drug, fluorophore, or an additional
spacer on the other terminus. Removal of the capping group
results in an intramolecular reaction that ultimately results in the
In recent years, the interest in self-immolative spacers has
grown significantly as their application in various prodrug, sensor,
and drug delivery systems has been explored. For example, the
conjugation of self-immolative spacers to drug molecules has
created inactive prodrugs that are converted to the free and active
drugs by cleavage of the trigger upon exposure to an external
stimulus.6–15 They have also been used in the linkage of drug
molecules to small molecule or antibody targeting moieties.16–23
Sensors have been developed by using self-immolative spacers to
conjugate reporter molecules such as fluorophores or imaging
agents to peptide or enzyme sensitive triggers.24–32 The use of
these linkers in dendrimeric33–37 and oligomeric38,39 systems has
also been explored, leading to an amplified release of drugs or
reporter molecules.
aDepartment of Chemistry, The University of Western Ontario, London,
Canada. E-mail: egillie@uwo.ca; Fax: 519-661-3022; Tel: 519-661-2111
bDepartment of Chemical and Biochemical Engineering, The University of
Western Ontario,
† Electronic supplementary information (ESI) available: General proce-
dures and materials section, syntheses of compounds 6b, 8c, 9b, 9c, 10b,
10c, 11c, 12a, 12b, 12c, 12d, 13b, 13c, 13d, 17b. 1 H and 13C NMR spectra
for all synthesis products, H NMR spectra of compounds prior to and
after cyclization, kinetics data. See DOI: 10.1039/c0ob00890g
1
Fig. 1 a) Schematic of a self-immolative spacer; b) example of a 1,6 elimination reaction; c) example of a cyclization reaction.
1846 | Org. Biomol. Chem., 2011, 9, 1846–1854 This journal is The Royal Society of Chemistry 2011
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Recently, our group and others have explored the development
of linear polymers comprising self-immolative spacers. Shabat
and coworkers have explored polymers based entirely on 1,6-
elimination reactions as amplified reporters,40 enzyme sensors,41
and enzyme labels.42 Moore and coworkers have prepared mi-
crocapsules based on cross-linked versions of similar polymers.43
Our group has introduced cyclization spacers in alternation
with elimination spacers as a means of controlling the polymer
degradation rate, and have demonstrated that amphiphilic block
copolymers such as 1 (Fig. 2) comprising one self-immolative
block are capable of assembling into nanoparticles that degrade in
a controlled manner to release their cargo.44 Furthermore, we have
also developed linear polymers such as 2,45 capable of degrading
entirely by cyclization reactions in order to address the potential
toxicity of the quinone methide intermediates that are produced
during the 1,6-elimination reaction.46
Fig. 3 Target 4-aminobutyric acid derivatives for kinetic studies.
such as fluorescein, Hoechst stain and umbelliferone, contain
phenolic groups, along with the chemotherapy drug Topotecan. In
addition, many other self-immolative spacers involve phenols,1,3,5
thus allowing the new spacer to be readily alternated with other
spacers, and incorporated into polymers analogous to 1. Secondly,
some N-methylated derivatives were targeted as this has previously
been reported to significantly enhance the cyclization rate in the
case of ethylenediamine derivatives.49 Finally, substitution at the
a position allowed us to examine the Thorpe-Ingold effect50,51
and/or the reactive rotamer effect52 on these compounds. It was
also expected to slow any competing ester hydrolysis. To test the
scope of these effects, the substitution pattern and the size of the
substituents were varied from unsubstituted to an a,a-dibenzyl
derivative. A single cyclopentyl ring was also incorporated to test
the effect of a conformationally locked system. The 2-hydroxy
derivatives were designed to provide insight into inductive effects.
Synthesis
Fig. 2 Chemical structures of previously reported self-immolative poly-
The synthesis of targets 3a–j began with the previously reported
Boc-protected 4-aminobutyric acid (4).53 As shown in Scheme 1,
the phenyl ester 6a was obtained by coupling the acid 4 to phenol
using DCC in the presence of DMAP. The N-methyl derivative was
prepared by treating 4 with MeI and NaH, immediately followed
by hydrolysis of the resulting methyl ester with LiOH. The resulting
free acid 5 was then coupled to phenol using DCC and DMAP to
obtain the desired Boc-protected phenyl ester 6b.
mers incorporating cyclization spacers.44,45
In order to fully exploit self-immolative spacers in these
materials and other new applications, it is essential to have access
to spacers that react at different rates. For example, the N,N¢-
dimethylethylenediamine spacer used in both polymers 1 and 2,
cyclized slowly at pH 7.4, which resulted in polymer degradation
over a period of days to weeks. While several self-immolative
spacers based on cyclization reactions have been reported, there
are very few that cyclize rapidly under mild conditions.1,4,47,48 To
address this need and to develop a spacer that could potentially
replace the N,N¢-dimethylethylenediamine spacer in polymers
such as 1 or 2, we have investigated 4-aminobutyric acid derivatives
as a potential new class of rapidly cyclizing self-immolative
spacers. The recent incorporation of a 4-aminobutyric acid unit
into an enzymatic detection probe suggested that this class of
molecules may serve as rapidly cyclizing spacers.24 However, there
has not been a versatile synthetic strategy developed for the
preparation of various analogues, nor a comprehensive study of
their cyclization rates. Thus, described here is the design and
synthesis of eleven different derivatives of 4-aminobutyric acid,
and studies of their cyclizations.
Scheme 1 Synthesis of a-unsubstituted derivatives.
To prepare the a-monosubstituted compounds, the acid
was first converted to a t-butyl ester 7a using t-butyl-2,2,2-
trichloroacetimidate in the presence of BF3·Et2O, and then N-
methylation was performed as described above using MeI and
NaH to provide 7b (Scheme 2). Monoalkylated t-butyl esters 8a–c
were obtained by treatment of 7b with LHMDS in the presence
of LiCl at -78 ◦C, followed by the addition of the alkyl halide. In
marked contrast to the formation of the benzyl and allyl derivatives
Results and discussion
Design
The targets shown in Fig. 3 were designed with several aspects
in mind. First, a phenyl ester was selected, as it would serve
as a good model system for future applications. Many dyes,
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Scheme 2 Synthesis of a-monosubstituted derivatives.
which suffered from partial over-alkylation, monomethylation was
cleanly achieved even when a large excess of both LHMDS and
MeI were used, simplifying purification and increasing its yield
relative to the other compounds. The substituted t-butyl esters
were then converted to the free acids by first removing both the
Boc group and the ester using TFA in CH2Cl2, and then reinstalling
the Boc group on the amine. This process worked very efficiently
for all substrates, with all products being obtained with yields in
excess of 90%. The final step was formation of the phenyl ester.
When DCC was used as described above for the preparation of 6a
and b, unsatisfactory yields of the desired products were obtained,
likely due to steric hindrance at the a carbon. To circumvent
this problem, the acid was converted to a mixed anhydride using
pivaloyl chloride, and this anhydride was then reacted with phenol.
This afforded phenyl esters 10a–c in good yields, ranging from
78–86%.
The a,a-dialkylated compounds were similarly derived from the
intermediate 7b (Scheme 3). Diallyl and dibenzyl t-butyl esters 11b
& 11c were synthesized directly from 7b by treatment first with 1
eq of LHMDS and then the alkyl halide followed an hour later
by 2 eq of each, which gave clean conversion to the disubstituted
products. Synthesis of the cyclopentyl ring was done similarly
except with a single addition of the alkyl dihalide. Following the
same protocol as allylation and benzylation to generate 11a did
not prove successful, and the products obtained were a mixture
of the mono and disubstituted compounds. Resubjection of this
material also did not prove successful, even after removal of
byproducts. Similarly, 8a could not be further methylated under
these conditions. However, when the base was switched to LDA,
the second methylation occurred cleanly, affording 11a in very
good yield. Curiously, when LDA was used on 7b, a mixture
of mono and dialkylated products was once again obtained, so
it appears for this particular substrate the dimethyl t-butyl ester
could only be obtained by doing successive methylations of 7b and
then 8a, using LDA for the second methylation.
At this point a global deprotection and reinstallation of the
Boc group was carried out, again producing the N-Boc acids in
very good yields. To install the phenyl ester, it was evident that
a mixed pivaloyl anhydride would be ineffective as there would
be little steric differentiation between the two carbonyls of the
anhydride. Therefore, the best option appeared to be conversion
of the acid to an acid chloride. As the acid sensitivity of the
Boc groups was incompatible with conventional methods for
generating acid chlorides, the Ghosez reagent, 1-chloro-N,N,2-
trimethylpropenylamine, which generates the acid chloride with no
acidic byproducts, was used. This method proved quite effective,
providing the desired phenyl esters in yields ranging from 60–90%
after isolation.
As shown in Scheme 4, the synthesis of the a-hydroxy targets be-
gan with Boc-protected S-4-amino-2-hydroxybutyric acid54 (14).
This acid was treated with MeI in the presence of Cs2CO3 to afford
the desired methyl ester 15 in very good yield. The next step was
protection of the 2-hydroxy group, for which we selected a second
Boc group. This group was chosen because it could be attached
easily in high yields, and cleavage could occur simultaneously
with the N-Boc group, thus removing an additional deprotection
step from the reaction sequence. This Boc group was installed
Scheme 3 Synthesis of a,a-disubstituted derivatives.
1848 | Org. Biomol. Chem., 2011, 9, 1846–1854
Scheme 4 Synthesis of a-hydroxy substituted derivatives.
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Table 1 Rate constants and corresponding half lives for the intramolec-
ular cyclizations of 3a–k
using Boc2O in the presence of catalytic DMAP, affording di-
Boc protected compound 16a in 90% yield. To generate the target
lacking an N-methyl group, 16a was treated with LiOH to cleave
the methyl ester, and the corresponding acid was converted to
phenyl ester 17a in 70% yield using the mixed anhydride method
described above. The N-methyl derivative was synthesized by first
methylating 16a using MeI and NaH, and then following the same
procedure as above to obtain the phenyl ester 17b.
Substrate
Rate constant (s-1)
Half-life (s)
3a
3b
3c
3d
3e
3f
3g
3h
3i
0.0179 0.0035
0.0708 0.0090
0.116 0.014
0.127 0.017
0.104 0.024
0.1334 0.0031
0.1338 0.0059
0.0276 0.0024
0.3516 0.0026
0.172 0.022
0.233 0.013
39
9.8
6.0
5.5
6.7
5.2
5.2
25
2.0
4.0
3.0
Kinetics
UV-visible spectroscopy was evaluated as a possible analytical tool
for measuring the cyclization kinetics. The UV-visible spectra of
phenol and a representative phenyl ester 6a are shown in Fig. 4.
While there is some overlap between the two spectra, at 276 nm the
phenol is strongly absorbing while the phenyl ester is only weakly
absorbing. Therefore, it was possible to perform kinetic studies
measuring the phenol released upon cyclization by monitoring
the absorbance at 276 nm.
3j
3k
out at the physiological temperature of 37 ◦C. The pH was verified
after cyclization, and no change was observed. The linearity of
ln[A]0/[A] versus time graphs where [A] is the concentration of
the starting material suggested that the cyclizations followed first
order or pseudo first order kinetics (see ESI†). Rate constants
were calculated as the slopes of the these graphs. The reported
rate constants are the average of those obtained over a minimum
of 3 experimental runs (Table 1). Reported errors correspond to
the calculated standard deviations of these runs. From the average
rate constant, the half-life for each spacer was also calculated.
In each case, the structure of the cyclized product was verified
by NMR spectroscopy and mass spectrometry (see ESI†). There
was no evidence of background ester hydrolysis for any of the
substrates.
All of the spacers cyclized quite rapidly with the half lives
ranging from 2 to 39 s. When comparing 3a and 3b, it is clear
that methylating the amine had a dramatic effect on the rate,
with the half-life being reduced by a factor of approximately
four. Consistent with the Thorpe-Ingold50,51 and reactive rotamer
effects,52 a substitution further increased the rate of cyclization,
as all of the monosubstituted spacers 3c, 3d, and 3e reacted
faster than the unsubstituted spacer 3b and cyclized at similar
rates. The a,a-disubstituted spacers 3f and 3g cyclized at similar
rates to their monosubstituted analogues. However, the a,a-
dibenzyl substituted spacer 3h exhibited a nearly 4-fold decrease
in rate, suggesting that steric crowding proximal to the ester
impedes cyclization. In contrast, the a-cyclopentyl substituted
compound 3i exhibited the fastest rate of cyclization, indicating
that conformational rigidity can play a role. Finally, both of
the a-hydroxy substituted spacers 3j and 3k cyclized faster than
the a-aliphatic substituted derivatives. Interestingly, the dramatic
increase in rate caused by N-methylation that was observed for
3a versus 3b was not noted for 3j versus 3k. The rate did increase,
indicating that there was still an effect, but it appears that the most
significant contribution was from the a-hydroxy substituent.
Fig. 4 UV-visible absorption spectra of phenol and a representative
phenyl ester 6a.
All of the target molecules were stored in their Boc-protected
forms and were deprotected immediately prior to kinetic studies
by treatment with TFA as shown in Scheme 5. Removal of the
Boc group was verified by 1H NMR spectroscopy. In most cases,
protonation of the amine in the form of the TFA salt was sufficient
to inhibit cyclization prior to and during NMR spectroscopy,
but occasionally some cyclization was observed (see ESI†). The
kinetic studies were performed by dissolving the deprotected
substrates in i-PrOH, then diluting this solution ten-fold with
0.1 M pH 7.4 phosphate buffer. The measurements were carried
Scheme 5 Deprotection and subsequent cyclization of 4-aminobutyric acid derivatives.
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Table 2 Rate constants and corresponding half lives for the intramolec-
ular cyclizations of 3i at different pHs
under a N2 atmosphere using flame or vacuum-dried glassware.
Column chromatography was performed using silica gel (0.063–
0.200 mm size, 70–230 mesh). H NMR spectra were obtained
1
pH
Rate constant (s-1)
Half-life (s)
at 400 MHz and 13C NMR spectra were obtained at 100 MHz.
NMR chemical shifts are reported in ppm and are calibrated
against residual solvent signals of CDCl3 (d 7.26, 77.36). Coupling
constants are expressed in Hertz (Hz). Infrared spectra were
obtained as films from CH2Cl2 on NaCl plates. High-resolution
mass spectrometry (HRMS) was performed using a Finnigan
MAT 8400 electron impact (EI) or a Micromass LCT electrospray
ionization time-of-flight (ESI) mass spectrometer. UV-visible
spectrscopy experiments were carried out using a Varian Cary
300 Bio UV-visible spectrophotometer.
7.4
7.0
6.0
5.0
4.0
0.3516 0.0026
0.1822 0.0188
0.0616 0.0085
0.0199 0.0015
0.0091 0.0012
2.0
3.8
11
35
76
It was also of interest to investigate the effect of pH on the
cyclization rate. This was of interest in considering the potential
application of these new spacers in areas such as drug delivery. For
example, certain drug delivery targets such as tumors,55,56 inflamed
tissues,57 and intracellular compartments such as endosomes and
lysosomes58 are known to exhibit mildly acidic pHs and it would
be desirable that the cyclization rate not be dramatically slowed in
these environments. To explore this, the cyclization of the spacer 3i
was investigated at pHs 4.0, 5.0, 6.0 and 7.0 following the method
previously described. As shown in Table 2, there was indeed
a pronounced decrease in the cyclization rate with decreasing
pH. Nevertheless, the cyclization was still rapid even at pH 4 with a
half life of 76 s. In comparison, the N, N-dimethylethylenediamine
spacer previously employed44,45 has a reported half life of greater
than 15 days at pH 4.2 and 37 ◦C. Therefore, these 4-aminobutyric
acid spacers appear to be more promising for a wider range of
physiological environments.
Synthesis of acid 5. To a flask containing 453 (0.250 g,
1.23 mmol), DMF (12 mL) and MeI (0.23◦mL, 3.69 mmol)
were added, and the solution was cooled to 0 C. NaH (0.128 g,
3.21 mmol) was suspended in DMF (1 mL) and the suspension
was added dropwise to the reaction mixture. The resulting mixture
was stirred for 4 h. A second portion of MeI (0.10 mL, 1.61 mmol)
was then added followed by additional NaH (0.070 g, 2.92 mmol)
suspended in DMF (0.5 mL) and the reaction mixture was stirred
2 h. The solvent was removed in vacuo, and the crude material
was taken up in CH2Cl2 and poured onto H2O. The product was
extracted with CH2Cl2. The combined organic layers were dried
over MgSO4, filtered, and the solvent was removed in vacuo. The
resulting oil was taken up in 1 : 1 THF : H2O (12 mL), LiOH·H2O
(0.104 g, 2.48 mmol) was added and then the solution was stirred
overnight. The solution was poured into 1 M HCl, and the
product was extracted with EtOAc. The combined organic layers
were dried over MgSO4, filtered, and the solvent was removed.
The crude product was purified by column chromatography (3 : 2
cyclohexane : EtOAc), yielding 5 (0.195 g, 73%) as a clear, colorless
oil. nmax/cm-1 3205, 2980, 2937, 1738, 1698, 1490, 1457, 1401,
1367.1H NMR (CDCl3): d 11.08 (br s, 1H), 3.35–3.22 (m, 2H),
2.85 (s, 3H), 2.36 (t, J = 7.2, 2H), 1.85 (quint, J = 6.8, 2H), 1.45
(s, 9H). 13C NMR (CDCl3): d 178.1, 155.9, 79.6, 47.9 & 47.5
(rotamers), 34.0, 31.0 & 30.8 (rotamers), 28.2, 22.7. HRMS: calc’d
[M]+ (C10H19NO4): 217.1314. Found: (EI) 217.1318.
Conclusion
A
new series of self-immolative spacers derived from 4-
aminobutyric acid was developed. A modular synthetic approach
was used for the preparation of eleven different derivatives.
These derivatives allowed the effects of N-methylation and a-
substitution to be explored. As expected, N-methylation led
to enhanced cyclization rates. a-Substitution led to enhanced
cyclization rates when the substituents were not too bulky but
large groups such as benzyl slowed the cyclization. Electron
withdrawing groups or conformationally restricted groups at the a
position accelerated the rate. Overall, all of the target compounds
exhibited rapid cyclization kinetics with half lives of less than
one minute at pH 7.4 and 37 ◦C. In addition, cyclization still
occurred rapidly at mildly acid pH. This suggests that these spacers
should be of great utility in systems where a rapid release of
the substrate even at acidic pHs is required. Furthermore, the
versatile synthetic approach should allow the introduction of
additional functionalities and also for their incorporation into
a range of chemical systems, allowing for many new applications.
Progress towards such applications is currently underway and will
be reported in due course.
Synthesis of phenyl ester 6a and general DCC mediated ester-
ification procedure. Compound 453 (0.409 g, 2.01 mmol) was
dissolved in CH2Cl2 (20 mL). Phenol (0.228 g, 2.45 mmol, 1.22
eq), DCC (0.623 g, 3.02 mmol, 1.5 eq) and DMAP (0.0236 g,
0.193 mmol, 0.09 eq) were added, and the solution was stirred
for 1 h. The precipitate was filtered off and rinsed with CH2Cl2.
The filtrate was poured into 1 M NaOH and the product was
extracted with CH2Cl2. The combined organic layers were dried
over MgSO4, filtered, and the solvent was removed in vacuo. The
crude material was purified by column chromatography (85 : 15
cyclohexane : EtOAc), affording 6a (0.536 g, 95%) as a white solid.
Experimental
n
max/cm-1 3080, 3068, 3010, 2981, 2936, 2866, 1762, 1697, 1596,
General procedures and materials. All reagents were purchased
from commercial sources and used without further purification
unless otherwise noted. Anhydrous DMF and THF were obtained
from a solvent purification system. Anhydrous CH2Cl2 and NEt3
were distilled over CaH2. Diisopropylamine was distilled over
MgSO4. Unless otherwise stated, all reactions were performed
1523, 1494, 1483, 1367. 1H NMR (CDCl3): d 7.42–7.35 (m, 2H),
7.27–7.22 (m, 2H), 7.12–7.07 (m, 1H), 4.67 (br s, 1H), 3.27 (quartet,
J = 6.6 Hz, 2H), 2.62 (t, J = 7.4, 2H), 1.95 (quint, J = 7.0 Hz,
2H), 1.46 (s, 9H).13C NMR (CDCl3): d 171.7, 155.9, 150.5, 129.2,
125.7, 121.4, 79.1, 39.7, 31.5, 28.3, 25.2. HRMS: calc’d [M+H]+
(C15H22NO4): 280.1543. Found: (EI) 280.1549.
1850 | Org. Biomol. Chem., 2011, 9, 1846–1854
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Synthesis of tert-butyl ester 7a. Compound 453 (1.00 g,
4.92 mmol) was dissolved in CH2Cl2 (15 mL). t-Butyl-2,2,2-
trichloroacetimidate (1.76 mL, 9.83 mmol, 2.0 eq) and BF3·Et2O
(0.100 mL, 0.707 mmol, 0.14 eq) were added and the reaction
mixture was stirred for 45 min. The solution was then filtered to
remove the precipitate and the precipitate was rinsed with CH2Cl2.
The filtrate was poured into a saturated solution of Na2CO3, and
the product was extracted into CH2Cl2. The combined organic
layers were dried over MgSO4 and filtered, and the solvent was
removed in vacuo. The material was taken up in cyclohexane and
filtered a second time. The solvent was removed in vacuo, yielding
7a (1.22 g, 96%) as a clear, colourless oil. nmax/cm-1 2980, 2937,
1718, 1716, 1525, 1456, 1393, 1368. 1H NMR (CDCl3): d 4.73 (br s,
1H), 3.10 (quart, J-6.3 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.73 (quint,
J = 7.0 Hz, 2H), 1.40 (s, 9H), 1.39 (s, 9H). 13C NMR (CDCl3): d
172.6, 155.7, 80.3, 79.0, 39.9, 32.8, 28.3, 28.0, 25.2. HRMS: calc’d
[M]+ (C13H25NO4): 259.1784. Found: (EI) 259.1779.
dissolved in THF (3 mL), and then LiCl (0.048 g, 1.128 mmol)
was added and the solution was cooled to -78 ◦C. LHMDS
(1.0 M solution in THF, 0.93 mL, 0.93 mmol) was added slowly,
and the solution was stirred 30 min. Allyl bromide (0.068 mL,
0.786 mmol) was then added dropwise, and the solution was
stirred for 30 min at -78 ◦C, then warmed to RT and stirred for an
additional 90 min. The reaction mixture was then poured into 1 M
HCl, and the product was extracted into EtOAc. The combined
organic layers were dried over MgSO4, filtered, and the solvent was
removed in vacuo. Purification by column chromatography (99 : 1
cyclohexane : EtOAc → 19 : 1 cyclohexane : EtOAc) to provide 8b
(0.162 g, 69%) as a thick, colorless oil. nmax/cm-1 2978, 2935, 1729,
1700, 1482, 1458, 1394, 1367. 1H NMR (CDCl3): d 5.81–5.68 (m,
1H), 5.15–4.98 (m, 2H), 3.39–3.06 (m, 2H), 2.84 (s, 3H), 2.40–2.17
(m, 3H), 1.88–1.76 (m, 1H), 1.69–1.57 (m, 1H), 1.46 (s, 9H), 1.45
(s, 9H). 13C NMR (CDCl3): d 174.1, 155.5, 135.2, 116.7, 80.3, 79.2,
47.0 & 46.4 (rotamers), 43.3, 36.4, 34.1, 29.6 & 29.3 (rotamers),
28.3, 28.0. HRMS: calc’d [M]+ (C17H31NO4): 313.2253. Found:
(EI) 313.2259.
Synthesis of N-methyl tert-butyl ester 7b. To a flask containing
7a (1.11 g, 4.28 mmol) were added DMF (20 mL) and MeI
(0.29 mL, 4.66 mmol), and the solution was cooled to 0 ◦C. NaH
(0.103 g, 4.31 mmol) was suspended in DMF (2 mL) and added
dropwise to the reaction mixture. The solution was stirred for
1 h, then a second equivalent each of MeI and NaH were
added as above, and the solution was stirred overnight. The
solution was poured into a 1 : 1 mixture of H2O : saturated brine,
and the product was extracted into CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and the solvent
was removed in vacuo. Purification by column chromatography
(19 : 1 cyclohexane : EtOAc → 9 : 1 cyclohexane : EtOAc) yielded
7b (0.990 g, 84%) as a thin, colourless oil. nmax/cm-1 2979, 2937,
1731, 1700, 1482, 1458, 1395, 1367.1H NMR (CDCl3): d 3.23 (t,
J = 7.4 Hz, 2H), 2.85 (s, 3H), 2.22 (t, J = 7.6 Hz, 2H), 1.79 (quint,
J = 7.6 Hz, 2H), 1.46 (s, 9H), 1.45 (s, 9H). 13C NMR (CDCl3): d
172.4, 155.6, 80.1, 79.1, 48.1 & 47.6 (rotamers), 34.0, 32.5, 28.3,
28.0, 23.3 & 22.9 (rotamers). HRMS: calc’d [M]+ (C14H27NO4):
273.1940. Found: (EI) 273.1947.
Synthesis of acid 9a and general deprotection-N-Boc reprotection
procedure. Under an air atmosphere, a flask was charged with 8a
(0.108 g, 0.376 mmol), and then 1 : 1 TFA:CH2Cl2 (2 mL) was
added and the solution was stirred for 2 h. The solvent was then
removed in vacuo, and CH2Cl2 was added and removed 3 times
to help further remove residual TFA. After thorough drying, the
material was taken up in 1 : 1 dioxane : 0.5 M NaOH solution
(2 mL), and the pH was adjusted to approximately 12 using
1 M NaOH. Di-t-butyldicarbonate (0.470 mmol, 1.25 eq) was
added, and the solution was stirred overnight. The material was
poured onto 1 : 1 1 M HCl : saturated brine, and the product was
extracted into EtOAc. The combined organic layers were dried
over MgSO4, filtered, and the solvent was removed in vacuo. The
crude material was purified by column chromatography (85 : 15
cyclohexane : EtOAc → 70 : 30 cyclohexane : EtOAc) to provide
9a (79.5 mg, 91%) as a clear, colorless oil. nmax/cm-1 3472, 3237,
2979, 2941, 1701, 1687, 1488, 1467, 1403, 1368. 1H NMR (CDCl3):
d 10.24 (br s, 1H), 3.52–3.11 (m, 2H), 2.85 (s, 3H), 2.46 (sextet, J =
7.0 Hz, 1H), 2.02–1.82 (m, 1H), 1.71–1.55 (m, 1H), 1.46 (s, 9H),
1.23 (d, J = 7.4 Hz, 3H). 13C NMR (CDCl3): d 181.3, 156.0, 79.9,
46.7, 36.7, 34.0, 31.2, 28.3, 17.0. 3472, 3237, 2979, 2941, 1701,
1687, 1488, 1467, 1403, 1368. HRMS: calc’d [M]+ (C11H21NO4):
231.1471. Found: (EI) 231.1464.
Synthesis of a-methyl tert-butyl ester 8a. Compound 7b
(0.188 g, 0.687 mmol) was dissolved in THF (2 mL), and then
LiCl (0.044 g, 0.104 mmol) was added and the solution was cooled
to -78 ◦C. MeI (0.43 mL, 6.91 mmol) was added, followed by
dropwise addition of LHMDS (1.0 M solution in THF, 2.00 mL,
2.00 mmol). The resulting solution was stirred for 30 min at -78 ◦C,
then warmed to RT and stirred an additional 90 min. The reaction
mixture was then poured into 1 M HCl and the product was
extracted into EtOAc. The combined organic layers were dried
over MgSO4, filtered, and the solvent was removed in vacuo. The
crude material was purified by column chromatography (19 : 1
cyclohexane : EtOAc) to afford 8a (0.174 g, 88%) as a pale yellow
oil. nmax/cm-1 2979, 2937, 1729, 1700, 1482, 1460, 1395, 1367.1H
NMR (CDCl3): d 3.37–3.11 (m, 2H), 2.84 (s, 3H), 2.31 (sextet, J =
7.0 Hz, 1H), 1.88 (sextet, J = 7.0 Hz, 1H), 1.63–1.51 (m, 1H), 1.46
(s, 9H), 1.45 (s, 9H), 1.15 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3): d
175.5, 155.6, 80.0, 79.2, 46.9 & 46.5 (rotamers), 37.9, 34.1, 31.7 &
31.2 (rotamers), 28.4, 28.0, 17.1. HRMS: calc’d [M]+ (C15H29NO4):
287.2097. Found: (EI) 287.2085.
Synthesis of phenyl ester 10a and general pivaloyl chloride medi-
ated esterification procedure. The acid 9a (0.181 g, 0.783 mmol)
was dissolved in CH2Cl2 (8 mL). Freshly distilled NEt3 (0.27 mL,
1.94 mmol) and pivaloyl chloride (0.12 mL, 0.975 mmol) were
added and the solution was stirred for 30 min. Phenol (0.115 g,
1.22 mmol) and DMAP (0.012 g, 98.2 mmol) were added and
the solution was stirred overnight. The solution was poured
into 1 M HCl and the product was extracted into CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and
the solvent was removed. The crude material was purified by
column chromatography (9 : 1 cyclohexane : EtOAc) to provide
10a (0.187 g, 78%) as a colorless oil. nmax/cm-1 3040, 2979, 2940,
1
1758, 1695, 1495, 1481, 1398, 1366. H NMR (CDCl3): d 7.39
(dd, J = 7.8 & 8.2 Hz, 2H), 7.23 (dd, J = 7.0 & 7.4 Hz, 1H), 7.09 (d,
J = 7.8 Hz, 2H), 3.37–3.23 (m, 2H), 2.88 (s, 3H), 2.70 (sextet, J =
7.0 Hz, 1H), 2.16–2.03 (m, 1H), 1.80–1.67 (m, 1H), 1.47 (s, 9H),
Synthesis of a-allyl tert-butyl ester 8b and general monoalky-
lation procedure. Compound 7b (0.204 g, 0.747 mmol) was
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1.36 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3): d 174.9, 155.6, 150.7,
129.3, 125.6, 121.4, 79.3, 46.7 & 46.3 (rotamers), 37.0, 31.6 &
31.0 (rotamers), 28.3, 17.1. HRMS: calc’d [M+H]+ (C17H26NO4):
308.1856. Found: (EI) 308.1860.
3.22–3.08 (m, 2H), 2.83 (s, 3H), 2.14–2.02 (m, 2H), 1.86–1.74 (m,
2H), 1.69–1.58 (m, 4H), 1.53–1.39 (m, 2H), 1.46 (s, 9H), 1.45 (s,
9H). 13C NMR (CDCl3): d 176.3, 155.4, 79.8, 79.0, 52.7, 46.2 &
45.9 (rotamers), 35.6, 36.1, 34.0, 28.4, 27.9, 24.8. HRMS: calc’d
[M+H]+ (C18H34NO4): 328.2482. Found: (EI) 328.2482.
Synthesis of a,a-dimethyl tert-butyl ester 11a. A flask was
charged with freshly distilled NH(iPr)2 (0.25 mL, 1.77 mmol)
Synthesis of phenyl ester 13a and general Ghosez reagent
mediated esterification procedure. Compound 12a (0.153 g,
0.624 mmol) was dissolved in CH2Cl2 (6 mL). 1-Chloro-N,N,2-
trimethylpropenylamine (0.12 mL, 0.907 mmol) was added and
the solution was stirred for 1 h. Phenol (0.118 g, 1.25 mmol) and
distilled NEt3 (0.18 mL, 1.29 mmol) were added and the solution
was stirred overnight. The reaction mixture was poured into 1 M
HCl and the product was extracted into CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and the solvent
was removed. Purification by column chromatography (97 : 3
cyclohexane : EtOAc) yielded 13a (0.181 g, 90%) as a colorless oil.
◦
and THF (1 mL), and the solution was cooled to -78 C. BuLi
(2.5 M solution in hexane, 0.69 mL, 1.73 mmol) was added and the
solution was stirred for 15 min. This solution was then transferred
via canula to a flask containing 8a (0.165 g, 0.574 mmol) and LiCl
(0.041 g, 0.967 mmol) in THF (2 mL) and the solution was stirred
for 15 min. MeI (0.22 mL, 3.53 mmol) was added dropwise and
the solution was stirred overnight. The reaction mixture was then
poured onto 1 M HCl, and the product was extracted into EtOAc.
The combined organic layers were dried over MgSO4, filtered, and
the solvent was removed in vacuo. The product was further purified
by column chromatography (19 : 1 cyclohexane : EtOAc) to afford
11a (0.165 g, 95%) as a pale yellow oil. nmax/cm-1 2979, 2953, 1700,
1654, 1480, 1458, 1394, 1367. 1H NMR (CDCl3): d 3.25–3.12 (m,
2H), 2.84 (s, 3H), 1.75–1.66 (m, 2H), 1.46 (s, 9H), 1.45 (s, 9H),
1.60 (s, 6H). 13C NMR (CDCl3): d 176.6, 155.5, 79.9, 79.1, 45.4,
41.2, 37.8, 33.9, 28.4, 27.9, 25.1. HRMS: calc’d [M]+ (C16H31NO4):
301.2253. Found: (EI) 301.2246.
n
max/cm-1 3107, 3078, 3073, 2978, 2934, 2898, 1752, 1699, 1594,
1
1494, 1485, 1471, 1428, 1394, 1368. H NMR (CDCl3): d 7.39
(dd, J = 7.8 & 8.2 Hz, 2H), 7.25 (dd, J = 8.2 & 7.4 Hz, 1H), 7.07 (d,
J = 7.8 Hz, 2H), 3.37–3.29 (m, 2H), 2.88 (s, 3H), 1.97–1.87 (m, 2H),
1.47 (s, 9H), 1.38 (s, 6H). 13C NMR (CDCl3): d 175.7, 155.4, 150.8,
129.3, 125.6, 121.4, 79.3, 45.3 & 45.0 (rotamers), 41.1, 37.8 & 37.0
(rotamers), 34.1, 28.4, 25.1. HRMS: calc’d [M+H]+ (C18H28NO4):
322.2013. Found: (EI) 322.2013.
Synthesis of a,a-diallyl tert-butyl ester 11b and general di-
alkylation procedure. Compound 7b (0.232 g, 0.849 mmol)
was ◦dissolved in THF (4 mL) and the solution was cooled to
-78 C. LHMDS (1.0 M solution in THF, 0.85 mL, 0.85 mmol)
was added slowly and the solution was stirred for 15 min. Allyl
bromide (0.074 mL, 0.855 mmol) was added dropwise, and the
solution was stirred for 1 h. A second addition of LHMDS
(1.70 mL, 1.70 mmol) and allyl bromide (0.13 mL, 1.73 mmol) was
performed and the solution was stirred overnight. The solution
was poured into 1 M HCl and the product was extracted into
EtOAc. The combined organic layers were dried over MgSO4,
filtered, and the solvent was removed in vacuo. Further purification
by column chromatography (97 : 3 cyclohexane : EtOAc) afforded
11b (0.280 g, 93%) as a clear, colorless oil. nmax/cm-1 3081, 2979,
Synthesis of methyl ester 15. Compound 14 (3.31 g, 15.1 mmol)
was dissolved in DMF (150 mL). Cs2CO3 (6.17 g, 18.9 mmol)
was added, and then MeI (0.99 mL, 15.9 mmol) was added
slowly and the solution was stirred for 90 min. The reaction
mixture was poured into 1 : 1 H2O : saturated brine, and the
product was extracted into CH2Cl2. The combined organic layers
were dried over MgSO4, filtered, and the solvent was removed
in vacuo. Column chromatography (7 : 3 cyclohexane : EtOAc →
1 : 1 cyclohexane : EtOAc) yielded 15 (3.05 g, 87%) as a yellow oil.
n
max/cm-1 3390, 2980, 2952, 1754, 1697, 1527, 1455, 1394, 1368.
1H NMR (CDCl3): d 4.89 (br s, 1H), 4.26 (dd, J = 3.9 & 8.1 Hz,
1H), 3.79 (s, 3H), 3.43–3.20 (m, 2H), 2.07–1.96 (m, 1H), 1.89–1.77
(m, 1H), 1.44 (s, 9H). 13C NMR (CDCl3): d 174.9, 156.2, 79.1,
68.4, 52.1, 36.5, 33.7, 28.1. HRMS: calc’d [M+H]+ (C10H20O5):
234.1336. Found (EI) 234.1335.
1
2936, 1723, 1700, 1483, 1458, 1393, 1367. H NMR (CDCl3): d
5.83–5.66 (m, 2H), 5.15–5.05 (m, 4H), 3.27–3.10 (m, 2H), 2.82 (s,
3H), 2.30 (d, J = 7.4 Hz, 4H), 1.76–1.68 (m, 2H), 1.46 (s, 18H). 13
C
Synthesis of O-Boc methyl ester 16a. Compound 15 (0.491 g,
2.11 mmol) was dissolved in THF (20 mL). Di-t-butyldicarbonate
(0.577 g, 2.64 mmol) and DMAP (0.030 g, 0.25 mmol) were
added and the solution was stirred for 1 h. The solution was
then poured into 1 M HCl and the product was extracted into
CH2Cl2. The combined organic layers were dried over MgSO4,
filtered, and the solvent was removed in vacuo. Purification by
column chromatography (9 : 1 cyclohexane : EtOAc) afforded 16a
(0.635 g, 91%) as a thick, colorless oil. nmax/cm-1 3408, 2982, 2958,
NMR (CDCl3): d 174.4, 155.4, 133.4, 118.3, 80.6, 79.3, 47.7, 44.6
& 44.2 (rotamers), 39.0, 34.0, 32.2 & 31.8 (rotamers), 28.4, 28.0.
HRMS: calc’d [M]+ (C20H35NO4): 353.2566. Found: (EI) 353.3557.
Synthesis of a-cyclopentyl tert-butyl ester 11d. Compound 7b
(0.142 g, 0.519 mmol) was dissolved in THF (25 mL) and the
solution was cooled to -78 ◦C. LHMDS (1.0 M soln in THF,
0.84 mL, 0.84 mmol) was added and the solution was stirred
for 15 min. 1,4-Dibromobutane (0.070 mL, 0.586 mmol) was
added dropwise and the solution was stirred 30 min. A second
equivalent of LHMDS was added and the solution was stirred
overnight. The reaction mixture was poured into 1 M HCl and
the product was extracted 3 times with EtOAc. The combined
organic layers were dried over MgSO4, filtered, and the solvent
was removed in vacuo. Purification by column chromatography
(98 : 2 cyclohexane : EtOAc → 97 : 3 cyclohexane : EtOAc) yielded
11d (0.078 g, 46%) as a clear, colorless oil. nmax/cm-1 2976, 2953,
1
1749, 1718, 1521, 1458, 1369. H NMR (CDCl3): d 4.90 (br s,
1H), 4.80 (dd, J = 5.0 & 7.0 Hz, 1H), 3.64 (s, 3H), 3.23–3.04
(m, 2H), 2.02–1.83 (m, 2H), 1.37 (s, 9H), 1.31 (s, 9H). 13C NMR
(CDCl3): d 170.3, 155.4, 152.5, 82.7, 78.9, 72.0, 52.0, 36.2, 31.1,
28.1, 27.3. HRMS: calc’d [M+H]+ (C15H28NO7): 334.1866. Found:
(EI) 334.1874.
Synthesis of N-methyl O-Boc methyl ester 16b. Compound
16a (0.133 g, 0.399 mmol) was dissolved in DMF (2 mL), and the
solution was cooled to 0 ◦C. MeI (0.25 mL, 4.01 mmol) was added,
1
2875, 1724, 1701, 1482, 1456, 1393, 1367. H NMR (CDCl3): d
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and NaH (0.010 g, 0.429 mmol) suspended in DMF (0.5 mL) was
added dropwise to the reaction mixture. The solution was stirred
for 1 h and then a second equivalent of NaH was added as above
and the solution was stirred a second hour. The reaction mixture
was then poured onto 1 : 1 1 M HCl : saturated brine, and the
product was extracted into CH2Cl2. The combined organic layers
were dried over MgSO4, filtered, and the solvent was removed
in vacuo. Further purification by column chromatography (96 : 4
cyclohexane : EtOAc) yielded 16b (0.103 g, 74%) as a colourless
oil. nmax/cm-1 2981, 2937, 1748, 1700, 1483, 1460, 1396, 1369. 1H
NMR (CDCl3): d 4.81 (dd, J = 4.3 & 8.6 Hz, 1H), 3.71 (s, 3H),
3.56–3.31 (m, 1H), 3.26–3.15 (m, 1H), 2.80 (s, 3H), 2.14–1.89 (m,
2H), 1.44 (s, 9H), 1.40 (s, 9H). 13C NMR (CDCl3): d 170.4, 155.4,
152.7, 82.9, 79.5, 72.0, 52.2, 44.9, 34.5 & 34.2 (rotamers), 29.5 &
29.1 (rotamers), 28.2, 27.5. HRMS: calc’d [M+H]+ (C16H30NO7):
348.2017. Found: (EI) 348.2015.
was taken as 100% conversion (verified by NMR). From this,
the % conversion with respect to time was calculated. To obtain
the first order rate constants, ln[A]0/[A] versus time was plotted
where [A]0/[A] effectively corresponds to 100/(100% conversion).
Phosphate buffers (0.1 M) were used for pHs 6.0, 7.0, and 7.4.
Acetate buffers (0.1 M) were used for pHs 4.0 and 5.0.
Acknowledgements
We thank the Natural Sciences and Engineering Research Council
of Canada for funding this work.
Notes and references
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from a methyl to phenyl ester. Under an air atmosphere, a flask
was charged with 16a (0.168 g, 0.503 mmol) and the material
was dissolved in 1 : 1 THF–H2O (5 mL). LiOH·H2O (0.0264 g,
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