Novel synthesis of various orthogonally protected Cα- methyllysine analogues and biological evaluation of a Vapreotide analogue containing (S)-α-methyllysine

Article abstract of DOI:10.1039/c3ob41282b

Prochiral malonic diesters containing a quaternary carbon center have been successfully transformed into a diverse set of ^tBoc-Fmoc- α^2,2-methyllysine-OH analogues through chiral malonic half-ester intermediates obtained via enzymatic (Pig Liver Esterase, PLE) hydrolysis. The variety of chiral half-ester intermediates, which vary from 1 to 6 methylene units in the side chain, are achieved in moderate to high optical purity and in good yields. The PLE hydrolysis of malonic diesters with various side chain lengths appears to obey the Jones's PLE model according to the stereochemical configurations of the resulting chiral half-esters. The established synthetic strategy allows the construction of both enantiomers of α^2,2-methyllysine analogues, and a (S)-β^2,2- methyllysine analogue from a common synthon by straightforward manipulation of protecting groups. Two different straightforward and cost effective synthetic strategies are described for the synthesis of α^2,2-methyllysine analogues. The described strategies should find significant usefulness in preparing novel peptide libraries with unnatural lysine analogues. A Vapreotide analogue incorporating (S)-α^2,2-methyllysine was prepared. However, the Vapreotide analogue with (S)-α-methyl-α-lysine is found to lose its specific binding to somatostatin receptor subtype 2 (SSTR2).

Full text of DOI:10.1039/c3ob41282b

Organic &
Biomolecular Chemistry
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Novel synthesis of various orthogonally protected
Cα-methyllysine analogues and biological evaluation of
Cite this: Org. Biomol. Chem., 2013, 11,
6307
a Vapreotide analogue containing (S)-α-methyllysine†
Souvik Banerjee,a Walker J. Wiggins,a Jessie L. Geoghegan,a Catherine T. Anthony,b
Eugene A. Wolteringb and Douglas S. Masterson*a
Prochiral malonic diesters containing a quaternary carbon center have been successfully transformed
t
into a diverse set of Boc-Fmoc-α2,2-methyllysine-OH analogues through chiral malonic half-ester inter-
mediates obtained via enzymatic (Pig Liver Esterase, PLE) hydrolysis. The variety of chiral half-ester inter-
mediates, which vary from 1 to 6 methylene units in the side chain, are achieved in moderate to high
optical purity and in good yields. The PLE hydrolysis of malonic diesters with various side chain lengths
appears to obey the Jones’s PLE model according to the stereochemical configurations of the resulting
chiral half-esters. The established synthetic strategy allows the construction of both enantiomers of
α
2,2-methyllysine analogues, and a (S)-β2,2-methyllysine analogue from a common synthon by straight-
forward manipulation of protecting groups. Two different straightforward and cost effective synthetic strat-
egies are described for the synthesis of α2,2-methyllysine analogues. The described strategies should find
significant usefulness in preparing novel peptide libraries with unnatural lysine analogues. A Vapreotide
analogue incorporating (S)-α2,2-methyllysine was prepared. However, the Vapreotide analogue with
(S)-α-methyl-α-lysine is found to lose its specific binding to somatostatin receptor subtype 2 (SSTR2).
Received 20th June 2013,
Accepted 5th August 2013
DOI: 10.1039/c3ob41282b
www.rsc.org/obc
naturally occurring amino acids have frequently been replaced
by α,α-disubstituted non-proteinogenic amino acids in various
Introduction
In recent years there has been considerable interest in the medicinally important peptides in order to confer more
preparation and use of optically pure α,α-disubstituted-α and β metabolic stability against enzymatic and chemical degra-
nonproteinogenic amino acids in a variety of fields.1–3 These dations.4,6,7,10,13 In addition, the α,α-disubstituted amino acid
nonproteinogenic amino acids have been used successfully in residues do not undergo in vivo racemization, due to the
the construction of peptides exhibiting unique properties.4–7 absence of the alpha hydrogen.14,15 The above examples illus-
The α,α-disubstituted class of nonproteinogenic amino acids trate the reasons for the growing interest in the use of α,α-
have been used as essential building blocks in the preparation disubstituted amino acid analogues.
of complex enzyme inhibitors.3,8,9 The α,α-disubstituted
However, the construction of chiral quaternary carbon
amino acids have demonstrated improved chemical stability, centers remains a challenge to the synthetic chemist. Recently
improved hydrophobicity, controlled conformational flexibility Vogt et al.3 reviewed the most widely envisaged synthetic strat-
of the amino acid side chain, and, hence, constrained confor- egies to prepare α,α-disubstituted-α-amino acid analogues that
mational freedom of the peptides containing them.10 The include (i) the asymmetric Strecker reaction starting from aldi-
α,α-disubstituted amino acids have been observed to stabilize mines and ketimines or other Strecker related reactions, (ii)
the secondary structures of peptides by constricting the confor- electrophilic alkylation of enolates derived from oxazinones,
mational freedom of the peptide backbone.11–13 Hence, oxazolines, oxazolidines, α-acidamido-β-keto, amino acid
derived imines as chiral auxiliaries, (iii) electrophilic-α-ami-
nation of α-substituted carbonyl compounds, and (iv) stereo-
aUniversity of Southern Mississippi, 118 College Drive # 5043, Hattiesburg,
MS 39406, USA. E-mail: douglas.masterson@usm.edu; Fax: +1 (601)266-6075;
Tel: +1 (601)266-4714
bLSU Health Science Center, 1542 Tulane Ave. 734, New Orleans, LA 70112, USA.
E-mail: ewolte@lsuhsc.edu; Fax: +1 (504)-568-4633; Tel: +1 (504)-568-4750
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob41282b
specific ring opening of aziridines, epoxides and rearrangement
reactions. Green et al.16 reported the synthesis of α,α-disubsti-
tuted-α-amino acids by a Mitsunobu approach, beginning with
a chiral α,α-disubstituted-α-hydroxy ester with known stereo-
chemistry. Hartmann et al.17 reported the synthesis of optically
enriched α-methyl phenylglycine through L-proline catalyzed
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amination of racemic 2-arylpropionaldehydes, using DEAD
and DBAD. Cabrera et al.14 established the synthesis of opti-
cally pure α,α-disubstituted-α-amino acids employing organo-
catalyzed Michael addition of racemic oxazolones to
α,β-unsaturated aldehydes. Smith et al.18 presented the syn-
thesis of several α,α-disubstituted-α-amino acids from
a
common intermediate employing nucleophilic “O-alkyl
fission” ring opening of the NBn2-α-methylserine lactone,
using various organocuprates.
Although there has been extensive research on α,α-disubsti-
tuted amino acids, there are very few reports on the prepa-
ration of α,α-disubstituted lysine analogues.15,19–21 In 1988,
Seebach et al.20 reported the synthesis of α-methyl-α-lysine in
the free, unprotected, form employing the self-regeneration of
stereocenters (SRS) principle. However, Seebach’s strategy
allows synthesizing only α-methyl-α-lysine in moderate yield.
Recently, Cativiela et al. reported the synthesis of (S)-α-methyl-
α-lysine via chiral cyanopropanoate using a chiral auxiliary in
ten steps, but this strategy also allows synthesizing only
(S)-α-methyl-α-lysine in the free form.19 To the best of our knowl-
edge Chauhan is the first to report tBoc-Fmoc protected
(S)-α-methyl-α-lysine using William’s Oxazinone as a chiral auxili-
ary in eight steps.15 However, this approach utilizes an expens-
ive chiral auxiliary resulting in the ability to synthesize a single
enantiomer of the α-methyl-α-lysine derivative with 90% final
purity.
Scheme 1 General synthetic strategy.
incorporate α,α-disubstituted amino acids to impart improved
metabolic stability of SST.7 We report herein substitution of
lysine in Vapreotide with α-methyl-α-lysine, since Trp8–Lys9
bond is one of the degradation sites in native SST.36 Fig. 2
exhibits the Vapreotide analogue which was prepared and
studied for specific binding against the IMR32 cell line.
Results and discussion
Synthesis of optically enriched half-ester intermediates
Herein we report a Pig Liver Esterase (PLE) catalyzed desym-
metrization approach toward making both orthogonally pro-
tected (R)- and (S)-α-methyl-α-lysine, orthogonally protected
(S)-α-methyl-β-lysine, and orthogonally protected (S)-α-methyl-
2,3-diaminopropanoic acid from optically enriched α,α-dialkyl-
malonic half-esters. Crude PLE is inexpensive and has a
proven track record in hydrolyzing a wide variety of prochiral
quaternary malonic diesters to the corresponding optically
enriched α,α-disubstituted malonic half-esters.22–26 Our syn-
thetic strategy is convenient and flexible, allowing for the vari-
ation of the side chain of lysine analogues from 1 to
The prochiral malonic diesters (2a–f) were synthesized by
alkylation of diethyl-2-methylmalonate with the appropriate
N-(bromoalkyl)-phthalimide as shown in Scheme 2. The resulting
diesters, with the exception of 2b, were purified and isolated
in good yield. The poor yield of 2b was attributed to the dehy-
drohalogenation of 1b as evidenced by isolation of significant
quantities of alkene. Compounds 2a–2f were subjected to enzy-
matic hydrolysis using crude PLE at pH 7.4. The hydrolysis
provided enantiomerically enriched half-esters 3a–3f in good
isolated yields as shown in Scheme 2. Surprisingly, PLE was
found to provide 3a–f predominantly of the (R)-enantiomer
with substantial optical activity in all cases.
6 methylene units, synthesize both enantiomers of an α2,2
-
methyllysine analogue from the same common synthon, and
at the same time homologate the (S)-α2,2-methyllysine to the
corresponding (S)-β2,2-methyllysine. We altered the side chain
length of lysine in order to probe the effect of chain length in
PLE hydrolysis.27,28 Scheme 1 illustrates our convergent strat-
egy to synthesize various Cα-methyllysine analogues from a
common synthon type.
Half-esters 3a–3f have been successfully resolved using
chiral HPLC techniques and the enantioselectivity was esta-
blished by integration of the appropriate chromatographic
peaks. The chiral HPLC chromatograms of the half-esters were
compared to those of racemic standards of 3a–3f prepared by
standard non-enzymatic methods.
In this article we also report a specific binding study of a
Vapreotide analogue incorporating α-methyl-α-lysine against
the IMR-32 cell line that is known to over express SSTR2 recep-
tors.29,30 Somatostatin receptors are well known to be over
expressed in a broad range of tumour cells.31 However, native
somatostatin (SST) has a short half life in vivo due to its rapid
degradation by various peptidases.7 In recent years a variety of
SST analogues have been prepared and studied for their
potency.7,30,32–35 Synthetic SST derivatives, such as Vapreotide,
have shorter peptide chains compared to native SST. To
the best of our knowledge, Prasad et al. were the first to Scheme 2 Preparation of chiral half-esters.
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The stereochemical configuration of the major enantiomer
of 3a was determined to have the (R) absolute
stereochemistry.37
The stereochemical configuration of the major enantiomer
of 3b was determined by synthesis38 as shown in Scheme 8
(ESI†). The optical activity of compound 30 was compared to
literature values in order to establish the absolute
configuration.
The configurations of 3c and 3d were determined by conver-
sion into 31a and 31b as shown in Scheme 9 (ESI†). The
optical rotations of 31a and 31b were compared with the litera-
ture values in order to determine the stereochemical configur-
ations of 3c and 3d.20
Fig. 1 Jones Active Site Model for Pig Liver Esterase.
The stereochemical configurations of 3e and 3f were also
determined by synthetic means as shown in Scheme 10
(ESI†).39–41
It is evident from Table 1 and Chart 1 that the PLE hydro-
lysis of diesters 2a–2f obey the Jones Active Site Model (JASM)
(Fig. 1).28 Diester 2c provides the highest level of optical purity
in the PLE catalyzed hydrolysis reaction. We hypothesize that
the size of the side chain is matched with the size of the large
hydrophobic pocket in the JASM. The other prochiral diesters
having a size mismatch with the large hydrophobic pocket of
the JASM results in diminished enantioselectivity with respect
to 2c.
The acid-esters (3a–f) were subjected to the Curtius
rearrangement resulting in Moz-protected (S)-α2,2-carbamates
(4a–4f) in good isolated yields as shown in Scheme 3.
Compounds 4a–4f can be considered as fully protected non-
proteinogenic amino acids.
Scheme 3 Conversion of chiral half-esters into protected amino acids.
Synthesis of an orthogonally protected (S)-α2,2-methyllysine
analogue
Scheme 4 illustrates the synthesis of tBoc-Fmoc-(S)-α2,2-methyl-
lysine-OH (7) in three steps starting with the optically enriched
synthon 3d. The bulky quaternary chiral half-ester (3d) was
directly converted into the Fmoc protected carbamate (5) by Ti(IV)
isopropoxide promoted Curtius rearrangement with good
isolated yield.42 Acid hydrolysis of the resulting carbamate (5)
hydrolyzed both the phthalimide and ethyl ester groups
leading to the free amino acid (6).43 The obtained 6 was then
converted to 7 by reaction with (Boc)2O. Hence, the optimized
Table 1 % ee of PLE hydrolyzed acid-esters
Diester
(CH2)n in the side chain
Half-ester – % ee
2a
2b
2c
2d
2e
2f
1
2
3
4
5
6
3a – 52%
3b – 92%
3c – 97%
3d – 95%
3e – 81%
3f – 64%
t
synthetic strategy is the shortest way to synthesize Boc-Fmoc-
(S)-α2,2-methyllysine-OH in overall five steps with good overall
yield (42%).
Chart 1 PLE hydrolysis assay of 3a–3f.
Scheme 4 Synthesis of tBoc-Fmoc-(S)-α2,2-methyllysine-OH.
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Scheme 5 Synthesis of tBoc-Fmoc-(R)-α2,2-methyllysine-OH.
Preparation of orthogonally protected (R)-α2,2-methyllysine
analogue
Scheme 5 illustrates the synthesis of (R)-α2,2-methyllysine ana-
logue 13 in six steps starting from 3d allowing for an enantio-
divergent synthesis of lysine analogues. In order to accomplish
the synthesis of 13, the chiral half-ester 3d was converted to
the mixed diester 8.22,24 Compound 8 was subjected to selec-
tive deprotection of the phthalimide group resulting in 9.
Compound 9 was saponified producing 10 in excellent yield.
Amino acid 10 was converted into 11 using standard reaction
conditions. The sterically hindered carboxylic acid 11 was then
converted into Fmoc protected α-amino ester 12 in good yield
with the implication of Curtius rearrangement using diphenyl-
phosphoryl azide (DPPA) and 9-fluorenylmethanol in presence
of catalytic Ti(IV) isopropoxide.42 However, the chemoselective
deprotection of the tert-butyl ester in the presence of Boc did
not proceed as desired in our hands using known literature
procedures.44–46 We believe this failure is due to the inaccessi-
bility of the sterically hindered ester by those reagents. Hence,
we had to treat the amino ester 12 with TFA to deprotect both
the tert-butyl and Boc groups followed by treatment with
(Boc)2O. However this deprotection and reprotection was a one
pot strategy that led to the (R)-α2,2-methyllysine analogue 13 in
8 steps in reasonable overall yield (30%).
Scheme 6 Synthesis of tBoc-Fmoc-(S)-α2,2-2,3-diamino propanoic acid.
α-dibenzylated aminoester (15). This failure is believed to be
due to the close proximity of phthalimido group to the bulky
quaternary center. However, the chemoselective deprotection
of the phthalimide of the dibenzylated amino ester (15) using
hydrazine resolved the problem providing 16. Saponification of
16 provides the desired 17. The free amino acid (17) was then
selectively protected with the BOC group using Boc anhydride
and NaHCO3 in H2O–dioxane system producing 18 in good iso-
lated yield. The chemoselective hydrogenolysis of 18 led to the
α-free amino acid 19 in nearly quantitative yield. The free
α-amino acid (19) was reprotected with the Fmoc group
t
leading to Boc, Fmoc protected amino acid analogue (20) in
total 10 steps in 14% overall yield.
To the best of our knowledge, Nadir et al.21 is the first
group to report the 2,3-diaminopropanoic acid in the free form
(unprotected form of 20), which is inconvenient in terms of
solid phase peptide synthesis (SPPS). This success let us syn-
thesize (S)-2,3-diaminopropanoic acid suitable for SPPS in
eight steps starting with optically enriched chiral half ester 3a.
Additionally, we have recently reported that the optical purity
of the acid-ester (3a) could be further improved to 95% ee by
substituting the crude PLE with PLE Isoenzyme 1, and 2%
EtOH as a co-solvent in the biocatalytic hydrolysis of 2a.37
Hence, this optimized synthetic strategy is able to provide
access to (S)-2,3-diaminopropanoic acid in high optical purity,
and in properly protected form for SPPS.
Synthesis of orthogonally protected (S)-α2,2
-
2,3-diaminopropanoic acid
t
Scheme 6 illustrates the synthesis of Boc-Fmoc protected (S)-
α
2,2-2,3-diaminopropanoic acid (20) in eight steps starting with
optically enriched half-ester 3a in good isolated yield. In the
first step the chiral half-ester (3a) was subjected to a Curtius
rearrangement producing the Moz protected α-amino ester
(4a). The carbamate (4a) was then treated with TFA to chemo-
Synthesis of (S)-Fmoc-β2,2-methyllysine-Boc-OH
selectively deprotect the Moz group leading to 14.23 Compound Scheme 7 illustrates the synthesis of orthogonally protected
14 was then subject to dibenzylation using excess benzyl (S)-β2,2-methyllysine (24) in eight steps. The chiral half-ester
bromide (BnBr) and K2CO3 at solvent reflux for 48 hours pro- (11), that was obtained from 3d following Scheme 5, was con-
viding 15.18 Simple base hydrolysis could not successfully verted into diazoketone (21) using standard procedures. The
drive the deprotection of the phthalimide group along diazoketone (21) was subject to photolysis resulting in the
with ester saponification in a single step starting with the γ-keto acid (22). The γ-keto acid (22) was converted into the
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analogue (25) to SSTR2. We suspect that the loss of specific
binding for SSTR2 is attributed to conformational changes of
the 25 ring resulting from the introduction of conformation-
ally constrained Cα,α-disubstituted lysine.50,51
Conclusions
We have established two convenient straightforward synthetic
strategies to prepare a variety of orthogonally protected α2,2-,
and β2,2-methyllysine analogues mediated through inexpensive
PLE hydrolysis derived acid-ester intermediates. This opti-
mized technique does not require expensive chiral auxiliaries
and reagents to generate the needed chiral quaternary carbon
center. In addition, this enantiodivergent methodology allows
to construct both D and L-isomers of the orthogonally pro-
Scheme 7 Synthesis of tBoc-Fmoc-(S)-β2,2-methyllysine-OH.
tected
α
2,2-lysine-OH starting with the enantiomerically
Fmoc protected β-amino ester (23). β-Amino ester 23 was ulti-
enriched common synthon by simple manipulation of the pro-
tecting groups. To the best of our knowledge, this is the first
time such diverse lysine analogues were synthesized in prop-
erly protected form through a common and simple synthetic
strategy. Additionally, Scheme 10 (ESI†) provides access to the
previously difficult to synthesize α,α-disubstituted amino acids
containing hydrophobic side chain in moderate to high % ee
t
mately converted into the Boc-Fmoc-(S)-β2,2-methyllysine (24)
using well established procedures.
Specific binding study of Vapreotide analogue (25)
Vapreotide® is a widely studied somatostatin analogue with
anti-neoplastic properties. Vapreotide has a higher binding
affinity to somatostatin receptor subtype 2 (SSTR2) than native
somatostatin.47,48 However, Vapreotide is prone to degradation
at the Lys–Val bond by serine proteases (Trypsin, Plasmin,
Plasma Kallikrein, etc.).36,49 To the best of our knowledge,
Rajeswaran et al. is the first to report that the introduction of
N-methyl-lysine in somatostatin analogues is tolerated and
retains the binding affinity to SSTR2.50 Hence, We have made
an effort to prepare a Vapreotide analogue 25 (Fig. 2) replacing
naturally occurring lysine with our (S)-α2,2-methyllysine (7) ana-
logue in order to study the specific binding of 25 to SSTR2.
The Vapreotide analogue was synthesized using properly pro-
tected (S)-α2,2-methyllysine analogue (7). The Vapreotide ana-
logue (25) was 99% pure as determined by HPLC.
via
a straightforward deamination procedure. The novel
α-methyl-α-lysine was incorporated into Vapreotide® to test the
effect of the non-proteinogenic lysine on the specific binding
of the analogue toward SSTR2 receptors. However, this simple
switch from lysine to α-methyllysine results in the loss of
specific binding to the SSTR2. Nevertheless, this synthetic
strategy should find its usefulness in constructing peptide
libraries containing these novel lysine analogues.
Experimental
General methods
THF, CH2Cl2, and DMF were dried by passage through a
column of activated alumina. All reagents were used as
received from commercial sources unless otherwise stated.
Melting points were determined in open capillary tubes and
are uncorrected. P-60 silica gel was used to conduct flash
chromatography. Silica pre-coated TLC plates were used to
perform TLC analysis. Normal phase pre-coated silica rotors
were chosen to perform radial chromatography. HRMS was
obtained using ESI/FTICR-MS and low resolution MS were
obtained by ESI/ion trap. Pig Liver Esterase (PLE) is the com-
mercially available crude preparation.
Specific binding studies of the Vapreotide analogue were
conducted against the IMR 32 human neuroblastoma cells.
However, it was observed that the Vapreotide analogue showed
no specific binding (Table 2 in ESI†) to SSTR2. Hence, a
simple switch from naturally occurring lysine to Cα,α-disubsti-
tuted lysine diminishes the specific binding of the Vapreotide
General experimental procedure for the synthesis of malonate
esters (2a–2f)
A 250 mL round bottom 3-neck flask fitted with a nitrogen
inlet, an addition funnel, and a reflux condenser was charged
with 1.2 eq. of NaH (60% dispersion in mineral oil), a stirbar,
and 100 mL of dry THF. The resulting suspension was cooled
to 0 °C in an icebath. A 50 mL solution of diethyl-2-
Fig. 2 Vapreotide analogue.
®
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methylmalonate (1 eq.) in THF was added over 30 min with Diethyl 2-(N-pentylphthalimido)-2-methyl malonate (2e)
stirring. The reaction mixture was then allowed to stir for
60 min at room temperature. A 100 mL solution of N-(bromo-
alkyl)-phthalimide (1 eq.) in THF was added over 30 min
with stirring. The reaction mixture was then heated to reflux
2e was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate, 17 g (57.4 mmol) N-(bromopentyl)-phthali-
mide and 2.74 g (68.9 mmol) NaH. The resulting yellowish
solvent for 12 h. The solution was cooled to RT, diluted with
liquid was purified by flash chromatography (30 : 70 EtOAc–
ether (300 mL), washed twice with 1 N HCl, washed with brine
hexanes), giving the pure product as a colorless liquid (14.8 g,
and dried over MgSO4. The resulting suspension was then fil-
66%). Rf = 0.42 (30% EtOAc–hexanes). IR (cm−1) = 2938, 1772,
tered and the solvent was evaporated in vacuo. The resulting
1706. 1H-NMR (CDCl3, 300 MHz): δ 7.83 (m, 2H), 7.71 (m, 2H),
yellowish liquid was purified by flash chromatography.
4.17 (q, 4H, J = 7 Hz), 3.68 (t, 2H, J = 7 Hz), 1.82 (m, 2H), 1.67
(m, 2H), 1.30 (m, 13H). 13C-NMR (CDCl3, 75 MHz): δ 172.0,
Diethyl 2-(N-methylphthalimido)-2-methyl malonate (2a)
168.0, 134.0, 132.0, 123.0, 61.0, 54.0, 38.2, 35.2, 28.5, 27.0,
24.0, 20.0, 14.0. HRMS (C21H27NO6Na+) calculated = 412.1730,
2a was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate. The resulting yellowish liquid was purified
by flash chromatography (30 : 70 EtOAc–hexanes), giving the
pure product as a white solid (12.5 g, 65%).37
found = 412.1728.
Diethyl 2-(N-hexylphthalimido)-2-methyl malonate (2f)
2f was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate, 17.8 g (57.4 mmol) N-(bromopentyl)-phthali-
mide and 2.74 g (68.9 mmol) NaH. The resulting yellowish
liquid was purified by flash chromatography (40 : 60 Et2O–
hexanes), giving the pure product as a colorless liquid (16.5 g,
71%). Rf = 0.44 (30% EtOAc–hexanes). IR (cm−1) = 2940, 1702.
1H-NMR (CDCl3, 400 MHz): δ 7.83 (m, 2H), 7.71 (m, 2H), 4.16
(m, 4H), 3.67 (m, 2H), 1.84 (m, 2H), 1.66 (m, 2H), 1.36 (m, 7H),
1.24 (m, 8H). 13C-NMR (CDCl3, 100 MHz): δ 172.0, 168.0,
134.0, 132.0, 123.0, 61.0, 54.0, 38.0, 35.0, 29.0, 28.0, 27.0, 24.0,
20.0, 14.0. HRMS (C21H27NO6Na+) calculated = 426.1893,
found = 426.1894.
Diethyl 2-(N-ethylphthalimido)-2-methyl malonate (2b)
2b was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate, 14.6 g (57.4 mmol) N-(bromoethyl)-phthali-
mide and 2.74 g (68.9 mmol) NaH. The resulting yellowish
liquid was purified by flash chromatography (18 : 82 EtOAc–
hexanes), giving the pure product as a white solid (9 g, 45%).26
Diethyl 2-(N-propylphthalimido)-2-methyl malonate (2c)
2c was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate, 15.4 g (57.4 mmol) N-(bromopropyl)-phthali-
mide and 2.74 g (68.9 mmol) NaH. The resulting yellowish
liquid was purified by flash chromatography (30 : 70 EtOAc–
hexanes), giving the pure product as a colorless liquid (12.8 g,
62%). Rf = 0.36 (30% EtOAc–hexanes). IR (cm−1) = 2981, 1772,
1705, 1614. 1H-NMR (CDCl3, 400 MHz): δ 7.84 (m, 2H), 7.72
(m, 2H), 4.16 (q, 4H, J = 7 Hz), 3.69 (t, 2H, J = 7 Hz), 1.91 (m,
2H), 1.67 (m, 2H), 1.39 (s, 3H), 1.23 (t, 6H, J = 7 Hz). 13C-NMR
(CDCl3, 100 MHz): δ 172.0, 168.0, 134.0, 132.0, 123.0, 61.0,
53.0, 38.0, 32.5, 23.3, 20.0, 14.0. HRMS (C19H23NO6Na+) calcu-
lated = 384.1423, found = 384.1406.
General experimental procedure for the formation of chiral
half-esters (3a–3f)
10 g (1 eq.) of the appropriate malonate (2a–2f) was dispersed
in 1000 mL of rapidly stirring phosphate buffer (0.1 N, pH 7.4)
containing 2% (vol/vol) EtOH as a cosolvent. The pH was
maintained using an autotitrator set to maintain a pH of 7.4
and titrate to a volume of 1 eq. NaOH (1.06 M). PLE (27 units
per mg, 90 units per mmol of the substrate) was added and
the titration was started. The hydrolysis proceeded for 1–6 days
depending on substrate. The reaction was stopped when 1 eq.
of NaOH was added. The reaction mixture was extracted 3
times with 500 mL of Et2O. The aqueous layer was then acidi-
fied to pH = 1 using 12 M HCl, extracted 8 times with Et2O.
The organic layer was dried over MgSO4, filtered, and concen-
trated in vacuo.
Diethyl 2-(N-butylphthalimido)-2-methyl malonate (2d)
2d was prepared following the general procedure for the for-
mation of diester (2a–2f) with 10 g (57.4 mmol) diethyl-2-
methylmalonate, 16.2 g (57.4 mmol) N-(bromobutyl)-phthali-
mide and 2.74 g (68.9 mmol) NaH. The resulting yellowish
liquid was purified by flash chromatography (30 : 70 EtOAc–
hexanes), giving the pure product as a white solid (15.2 g,
70%). Rf = 0.40 (30% EtOAc–hexanes). IR (cm−1) = 2950, 1702.
(R)-2-(N-Methylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
acid (3a)
3a was prepared following the general procedure for the for-
mation of half-esters (3a–3f) with 10 g (30 mmol) of 2a. An
amount of 6 g (65%) of 3a was obtained as a white solid in
52% ee.‡37
1
MP = 48 °C. H-NMR (CDCl3, 300 MHz): δ 7.85 (m, 2H), 7.71
(m, 2H), 4.17 (q, 4H, J = 7 Hz), 3.67 (t, 2H, J = 7 Hz), 1.90 (m,
2H), 1.69 (m, 2H), 1.39 (s, 3H), 1.27 (m, 8H). 13C-NMR (CDCl3,
75 MHz): δ 172.0, 168.0, 134.0, 132.0, 123.0, 61.0, 53.0, 38.0,
35.0, 29.0, 22.0, 20.0, 14.0. HRMS (C20H25NO6Na+) calculated =
398.1574, found = 398.1573.
‡The optical purity of 3a could be greatly improved to 95% using PLE isoenzyme
1, available from Enzymicals, in the biocatalytic asymmetric hydrolysis of 2a.37
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(R)-2-(N-Ethylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
acid (3b)
+3.2 (c = 2, CHCl3), 1H-NMR (CDCl3, 300 MHz): δ 7.83 (m, 2H),
7.72 (m, 2H), 4.21 (q, 2H, J = 7.19), 3.68 (t, 2H, J = 7.117), 1.86
(m, 2H), 1.69 (m, 2H), 1.44 (s, 3H), 1.34 (m, 4H), 1.27 (t, 3H, J =
7.15), 13C-NMR (CDCl3, 75 MHz): δ 178.0, 172.0, 168.4, 134.0,
132.0, 123.0, 61.4, 54.0, 38.0, 35.5, 28.3, 27.2, 24.0, 20.0, 14.0.
3b was prepared following the general procedure for the for-
mation of half-esters (3a–3f) with 10 g (29 mmol) of 2b. An
amount of 7.2 g (71%) of 3b was obtained as a white solid in
92% ee.26
HRMS (C19H23NO6Na+) calculated
384.1413.
= 384.1417, found =
(R)-2-(N-Propylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
acid (3c)
(R)-2-(N-Hexylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
acid (3f)
3c was prepared following the general procedure for the for-
mation of half-esters (3a–3f) with 10 g (28 mmol) of 2c. The
resulting half-ester was purified by flash chromatography
(40 : 60 EtOAc–hexanes) giving the product as a colorless liquid
(6.4 g, 68%). The % ee was determined to be 97% by chiral
HPLC (Diacel Chiralpak OJ-H, 4% iPrOH–hexanes, flow rate =
3f was prepared following the general procedure for the for-
mation of half-esters (3a–3f) with 10 g (25 mmol) of 2f. The
resulting half-ester was purified by flash chromatography
(40 : 60 EtOAc–hexanes) giving the pure product as a colorless
liquid (6.12 g, 61%). The % ee was determined to be 64% by
chiral HPLC (Diacel Chiralpak OJ-H, 3% iPrOH–hexanes, flow
rate = 1 mL min−1, λ = 305 nm) Rt(R) = 57.7 min (area =
13 605 958), Rt(S) = 71.7 min (area = 2 954 776). [α]2D4 = +2.05 (c =
1 mL min−1, λ = 305 nm) Rt(S) = 54.9 min (area = 130.13), Rt(R)
=
58.8 min (area = 7770.41). Rf = 0.22 (40% EtOAc–hexanes). IR
(cm−1) = 2983, 2937, 1773, 1747, 1697. [α]2D4 = +5.8 (c = 2,
MeOH). 1H-NMR (CDCl3, 400 MHz): δ 7.85 (m, 2H), 7.73 (m,
2H), 4.21 (q, 2H, J = 7 Hz), 3.71 (t, 2H, J = 7 Hz), 1.93 (m, 2H),
1.71 (m, 2H), 1.45 (s, 3H), 1.26 (t, 3H, J = 7 Hz). 13C-NMR
(CDCl3, 100 MHz): δ 176.0, 172.0, 168.0, 134.0, 132.0, 123.0,
62.0, 53.0, 38.0, 33.0, 24.0, 20.0, 14.0. HRMS (C16H17NO6Na+)
calculated = 356.3256, found = 356.3253.
1
2, CHCl3), H-NMR (CDCl3, 400 MHz): δ 7.83 (m, 2H), 7.72 (m,
2H), 4.21 (q, 2H, J = 7.19), 3.68 (t, 2H, J = 7.12), 1.86 (m, 2H),
1.69 (m, 2H), 1.44 (s, 3H), 1.34 (m, 4H), 1.27 (t, 3H, J = 7.15),
13C-NMR (CDCl3, 100 MHz): δ 177.0, 173.0, 168.4, 134.0, 132.0,
123.0, 61.3, 53.0, 38.0, 35.5, 29.2, 28.2, 26.3, 24.0, 20.0, 14.0.
HRMS (C20H25NO6Na+) calculated = 398.1574, found = 398.1572.
General experimental procedure for the formation of
(R)-2-(N-Butylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
carbamates (4a–4f)
acid (3d)
An amount of 10 g (1 eq.) of the appropriate chiral half-ester
(3a–3f) was dissolved in 50 mL dichloroethane in a 500 mL
round bottom flask with a stirbar under a N2 atmosphere. A
measured volume of Et3N (2.1 eq.) and diphenylphosphoryl-
azide (DPPA) (1.1 eq.) was added to the solution and the solu-
tion was allowed to stir at RT for 90 min. At this point the
reaction was heated to reflux solvent for 2 h. A measured
volume of para-methoxybenzyl alcohol (PMB-OH) (1.4 eq.) was
added to the reaction mixture and the reaction was continued
to reflux solvent for 12 h. The reaction was cooled and diluted
with CH2Cl2, filtered through a silica bed (1″ bed in a Buchner
funnel) and evaporated. The resulting residue was purified by
flash chromatography (40% EtOAC–hexanes) giving the pure
product as a white wax or colorless viscous oil.
3d was prepared following the general procedure for the for-
mation of half-esters (3a–3f) with 10 g (27 mmol) of 2d, The
resulting half-ester was purified by flash chromatography
(40 : 60 EtOAc–hexanes) giving the pure product as a white
solid (6.6 g, 70%). The % ee was determined to be 95% by
chiral HPLC (Diacel Chiralpak OJ-H, 4% iPrOH–hexanes, flow
rate = 1 mL min−1, λ = 305 nm) Rt(S) = 63.0 min (area = 325.24),
Rt(R) = 49.6 min (area = 11 659.65). Rf = 0.24 (40% EtOAc–
hexane). IR (cm−1) = 3250, 2943, 1718, 1696. MP = 63 °C. [α]2D3
1
= +3.3 (c = 1, CH2Cl2), H-NMR (CDCl3, 300 MHz): δ 9.82 (bs,
1H), 7.82 (m, 2H), 7.73 (m, 2H), 4.21 (q, 2H, J = 7 Hz), 3.71 (t,
2H, J = 7 Hz), 1.93 (m, 2H), 1.69 (m, 2H), 1.45 (s, 3H), 1.35 (m,
2H), 1.26 (t, 3H, J = 7 Hz), 13C-NMR (CD3Cl3, 100 MHz): δ
176.0, 174.0, 169.2, 135.3, 133.2, 124.0, 62.0, 54.3, 39.0, 36.0,
30.0, 23.0, 20.0, 14.0. HRMS (C16H17NO6Na+) calculated =
370.1261, found = 370.1256.
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-1-(1,3-dioxoisoindolin-
2-yl)propan-2-ylcarbamate (4a)
(R)-2-(N-Pentylphthalimido)-3-ethoxy-2-methyl-3-oxopropanoic
acid (3e)
4a was prepared following the general synthetic procedure for
the formation of carbamates (4a–4f). An amount of 11.7 g
3e was prepared following the general procedure for the for- (26.5 mmol, 79%) of product was obtained as a white wax. Rf =
mation of half-esters (3a–3f) with 10 g (26 mmol) of 2e. The 0.25 (40% EtOAc–hexanes). IR (cm−1) = 3368, 2958, 1774, 1708,
1
resulting half-ester was purified by flash chromatography 1612. [α]2D3 = −1.2 (c = 1.2, CHCl3). H-NMR (CDCl3, 400 MHz):
(40 : 60 EtOAc–hexanes) giving the pure product as a colorless δ 7.84 (m, 2H), 7.73 (bs, 2H), 7.31 (d, 2H, J = 9 Hz), 6.87 (d, 2H,
liquid (5.8 g, 61%). The % ee was determined to be 81% by 9 Hz), 6 (bs, 1H), 5 (q, 2H, J = 12 Hz), 4.18 (m, 2H), 4.12 (s,
chiral HPLC (Diacel Chiralpak AD-H, 3% iPrOH–hexanes, flow 2H), 3.8 (s, 3H), 1.65 (S, 3H), 1.25 (m, 3H). 13C-NMR (CDCl3,
rate = 1 mL min−1, λ = 305 nm) Rt(S) = 114.60 min (area = 100 MHz): δ 171.0, 168.5, 159.0, 155.0, 134.0, 132.0, 130.0,
1591.84), Rt(R) = 78.75 min (area = 15 497.54). Rf = 0.28 (40% 128.4, 123.0, 113.3, 66.0, 62.0, 60.2, 55.0, 43.4, 20.0, 14.0.
EtOAc–hexanes). IR (cm−1) = 3250, 2938, 1770, 1700. [α]2D4
=
HRMS (C23H24N2O7) calculated = 463.1476, found = 463.1469.
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Organic & Biomolecular Chemistry
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-4-(1,3-dioxoisoindolin-
2-yl)butan-2-ylcarbamate (4b)
3.79 (s, 3H), 3.64 (t, 2H, J = 7 Hz), 2.12 (bm, 1H), 1.76 (m, 1H),
1.64 (m, 2H), 1.55 (s, 3H), 1.27 (m, 7H). 13C-NMR (CDCl3,
100 MHz): δ 174.0, 168.0, 159.0, 155.0, 134.0, 132.0, 130.0,
129.0, 123.0, 114.0, 66.0, 61.0, 60.0, 55.0, 38.0, 36.0, 28.0, 27.0,
24.0, 23.5, 14.0. HRMS (C27H32N2O7Na+) calculated = 519.2102,
found = 519.2095.
4b was prepared following the general synthetic procedure for
the formation of carbamates (4a–4f). An amount of 12 g
(26.4 mmol, 84%) of product was obtained as a colorless
viscous oil. Rf = 0.29 (35% EtOAc–hexanes). IR (cm−1) = 3353,
1
2952, 1771, 1704, 1612. [α]2D3 = +11.3 (c = 1, CH2Cl2). H-NMR
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-8-(1,3-dioxoisoindolin-
(CDCl3, 400 MHz): δ 7.81 (m, 2H), 7.69 (m, 2H), 7.28 (m, 2H),
6.87 (m, 2H), 5.78 (bs, 1H), 4.94 (m, 2H), 4.07 (m, 2H), 3.80 (s,
3H), 3.67 (m, 2H), 2.57 (bm, 1H), 2.36 (m, 1H), 1.60 (s, 3H),
1.12 (t, 3H, J = 7 Hz), 13C-NMR (CDCl3, 100 MHz): δ 173.0,
168.0, 159.0, 154.0, 134.0, 132.0, 130.0, 128.6, 123.0, 114.0,
66.0, 62.0, 58.0, 55.0, 34.0, 33.6, 24.0, 14.0. HRMS
(C24H26N2O7Na+) calculated = 477.1638, found = 477.1635.
2-yl)octan-2-ylcarbamate (4f)
4f was prepared following the general synthetic procedure for
the formation of carbamates (4a–4f). An amount of 11.3 g
(22 mmol, 83%) of product was obtained as colorless viscous
oil. Rf = 0.34 (35% EtOAc–hexanes). IR (cm−1) = 3366, 2936,
2859, 1770, 1703, 1612. [α]2D3 = −1.7 (c = 1.4, CH2Cl2). H-NMR
1
(CDCl3, 400 MHz): δ 7.74 (m, 2H), 7.62 (m, 2H), 7.22 (d, 2H, J =
7 Hz), 6.80 (d, 2H, J = 8 Hz), 5.51 (bs, 1H), 4.91 (s, 2H), 4.11
(m, 2H), 3.73 (s, 3H), 3.58 (t, 2H, J = 7 Hz), 2.04 (m, 1H), 1.67
(m, 1H), 1.56 (m, 2H), 1.47 (s, 3H), 1.20 (m, 8H), 0.98 (bm,
1H). 13C-NMR (CDCl3, 100 MHz): δ 173.0, 167.0, 157.0, 154.0,
133.0, 131.0, 129.0, 128.0, 122.0, 113.0, 65.0, 60.0, 59.0, 54.0,
37.0, 36.0, 28.0, 27.0, 26.0, 23.0, 22.0, 13.0. HRMS
(C28H34N2O7Na+) calculated = 533.2258, found = 533.2251.
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-5-(1,3-dioxoisoindolin-
2-yl)pentan-2-ylcarbamate (4c)
4c was prepared following the general synthetic procedure for
the formation of carbamates (4a–4f). An amount of 11.8 g
(25.2 mmol, 84%) of product was obtained as a colorless
viscous oil. Rf = 0.27 (35% EtOAc–hexane). IR (cm−1) = 3359,
1
2939, 1770, 1702, 1612. [α]2D3 = −6.0 (c = 0.8, CH2Cl2). H-NMR
(CDCl3, 400 MHz): δ 7.82 (m, 2H), 7.70 (m, 2H), 7.28 (m, 2H),
6.88 (m, 2H), 5.63 (bs, 1H), 4.95 (s, 2H), 4.16 (m, 2H), 3.79 (s,
3H), 3.65 (m, 2H), 2.26 (m, 1H), 1.84 (m, 1H), 1.69 (m, 1H),
1.54 (s, 3H), 1.48 (m, 1H), 1.21 (t, 3H, J = 7 Hz). 13C-NMR
(CDCl3, 100 MHz): δ 174, 168, 160, 154, 134, 132, 130, 128,
123, 114, 66, 62, 60, 55, 38, 34, 23.5, 23.4, 14. HRMS
(C25H28N2O7Na+) calculated = 491.1789, found = 491.1782.
Synthesis of (9H-fluoren-9-yl) methyl (S)-2-(ethoxycarbonyl)-
6-(1,3-dioxoisoindolin-2-yl)hexan-2-ylcarbamate (5)
A volume of 320 µL Et3N (2.3 mmol) was added to a solution of
0.7 g (1.9 mmol) 3d in 25 mL dichloroethane under a N2 atmos-
phere. A volume of 460 µL DPPA (2 mmol) was added to the
reaction mixture. The mixture was allowed to stir at RT for 2 h.
The mixture was then heated to reflux solvent for 3 h. The reac-
tion was cooled and washed with saturated NH4Cl solution.
The organic layer was dried over MgSO4, filtered, and evapor-
ated under reduced pressure giving the crude isocyanate. The
isocyanate was dissolved in dry toluene under a N2 atmosphere.
An amount of 0.75 g (3.8 mmol) 9-fluorenylmethanol and a
volume of 66 µL Ti(IV) isopropoxide was added to the solution.
The mixture was heated to 80 °C for 12 h. The mixture was
cooled and the toluene was evaporated under reduced pressure
giving the crude product. The residue was purified by chrom-
atography (10% hexanes–CH2Cl2) giving 0.95 g 5 (1.75 mmol,
92%) as a white solid. Rf = 0.29 (10% hexanes–CH2Cl2). IR
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-6-(1,3-dioxoisoindolin-
2-yl)hexan-2-ylcarbamate (4d)
4d was prepared following the general synthetic procedure for
the formation of carbamates (4a–4f). An amount of 11.5 g
(24 mmol, 83%) of product was obtained as a colorless viscous
oil. Rf = 0.31 (35% EtOAc–hexanes). IR (cm−1) = 3360, 2958,
1768, 1701, 1612. [α]D23 = −1.5 (c = 1.5, CHCl3). 1H-NMR (CDCl3,
400 MHz): δ 7.82 (m, 2H), 7.69 (m, 2H), 7.30 (m, 2H), 6.88 (m,
2H), 5.62 (bs, 1H), 4.99 (s, 2H), 4.17 (m, 2H), 3.8 (s, 3H), 3.63
(t, 2H, J = 7 Hz), 2.17 (m, 1H), 1.83 (m, 1H), 1.65 (m, 2H), 1.56
(s, 3H), 1.33 (m, 1H), 1.23 (t, 3H, J = 7 Hz), 1.12 (m, 1H).
13C-NMR (CDCl3, 100 MHz): δ 174.0, 168.0, 159.5, 154.6, 134.0,
132.1, 130.0, 128.7, 123.2, 114.0, 66.2, 62.0, 60.0, 55.3, 38.0,
36.0, 28.4, 23.4, 21.4, 14.1. HRMS (C26H30N2O7Na+) calculated
= 505.1945, found = 505.1930.
(cm−1) = 3365, 2940, 1769, 1704, 1613, 1504. MP = 81 °C, [α]2D2
=
−14.3 (c = 1, CHCl3). 1H-NMR (CDCl3, 400 MHz): δ 7.78 (m,
4H), 7.64 (m, 4H), 7.40 (t, 2H, J = 7 Hz), 7.32 (t, 2H, J = 7 Hz),
5.72 (bs, 1H), 4.35 (bm, 2H), 4.20 (bm, 3H), 3.65 (bm, 2H), 2.21
(bm, 1H), 1.85 (bm, 1H), 1.60 (m, 5H), 1.35 (bm, 1H), 1.24 (bm,
3H), 1.13 (bm, 1H). 13C-NMR (CDCl3, 100 MHz): δ 174.0, 168.0,
144.0, 141.0, 134.0, 132.0, 128.0, 127.0, 125.0, 123.0, 120.0,
66.0, 62.0, 60.0, 47.0, 37.5, 36.0, 28.0, 23.4, 21.0, 14.0. HRMS
4-Methoxybenzyl-(S)-2-(ethoxycarbonyl)-7-(1,3-dioxoisoindolin-
2-yl)heptan-2-ylcarbamate (4e)
4e was prepared following the general synthetic procedure for (C32H32N2O6Na+) calculated = 563.2152, observed = 563.2144.
the formation of carbamates (4a–4f). An amount of 11.5 g
Fluorenylmethyloxycarbonylamino-2-methylhexanoic acid·HCl (6)
(23 mmol, 82%) of product was obtained as a colorless viscous
oil. Rf = 0.33 (35% EtOAc–hexanes), IR (cm−1) = 3367, 2938, 6 was synthesized from 5 following a literature published pro-
1
1770, 1703, 1612. [α]2D3 = +1.4 (c = 1, CH2Cl2). H-NMR (CDCl3, cedure.52 An amount of 0.9 g (1.7 mmol) 5 was dissolved in
400 MHz): δ 7.83 (m, 2H), 7.70 (m, 2H), 7.28 (d, 2H, J = 9 Hz), 12 mL 1,4-dioxane. A volume of 12 mL 5 N HCl was added to
6.87 (m, 2H, J = 9 Hz), 5.63 (bs, 1H), 4.99 (s, 2H), 4.18 (m, 2H), the solution. The solution was heated to reflux solvent for
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24 h. At which time the reaction was found to be completed by The solution was heated to reflux solvent for 6 h. The reaction
ESI-MS (ESI†). The solution was concentrated under reduced mixture was found to turn turbid and a white precipitate
pressure and the residue was taken for the next step without formed within 2 h of reflux. The reaction was monitored by
further purification.
TLC. The reaction was cooled to RT and the MeOH was
removed in vacuo. The residue was taken up in CH2Cl2 and the
white precipitate was filtered off. The CH2Cl2 was evaporated
Synthesis of (S)-tBoc-Fmoc-α-methyl-α-lysine-OH (7)
An amount of 0.25 g NaHCO3 (3 mmol) was added to a solu- under reduced pressure giving 6.75 g (24.7 mmol, 95%) of 9 as
tion of 0.6 g of 6 (∼1.5 mmol) in 15 mL of water with stirring. a colorless oil. Rf = 0.16 (5% MeOH–CH2Cl2), IR (cm−1) = 3395,
The solution was cooled to 0 °C. A solution of 0.65 g (Boc)2O 2977, 2934, 2867, 1723, 1654. 1H-NMR (CDCl3, 400 MHz): δ
(3 mmol) in 15 mL 1,4-dioxane was added to the reaction 4.17 (q, 2H, J = 7 Hz), 2.70 (t, 2H, J = 7 Hz), 1.82 (m, 2H), 1.53
mixture over 20 min. The reaction was allowed to stir at 0 °C (bs, 2H), 1.45 (m, 11H), 1.35 (s, 3H), 1.27 (m, 5H). 13C-NMR
for an hour and then at ambient temperature for 12 h. The (CDCl3, 100 MHz): δ 172.0, 171.0, 81.0, 61.0, 54.0, 42.0, 35.0,
mixture was given pentane wash to remove excess (Boc)2O. The 34.0, 28.0, 21.0, 19.0, 14.0. HRMS (C14H27NO4Na+) calculated =
reaction mixture was diluted with 30 mL of water, acidified to 296.1832, observed = 296.1828.
pH 4 with 2 M HCl, and extracted (3 × 50 mL) with Et2O. The
combined ether layer was washed with brine, dried over
MgSO4, evaporated under reduced pressure giving the crude
Synthesis of (S)-2-(tert-butoxycarbonyl)-6-amino-2-
methylhexanoic acid (10)
product as light yellow oil. The residue was purified by radial An amount of 1.76 g LiOH (73.5 mmol) was added in a solu-
chromatography using 5% MeOH–CH2Cl2 giving 0.78
g
tion of 6.7 g 9 (24.5 mmol) in 30 mL of 3 : 7 EtOH–H2O
(1.6 mmol, 94% over two steps) of product as a white solid. Rf mixture. The solution was allowed to stir for 48 h at RT. The
= 0.33 (5% MeOH–CH2Cl2). IR (cm−1) = 3350, 2941, 1681, 1504. solvents were evaporated under reduced pressure upon com-
MP = 95 °C. [α]2D2 = +14.4 (c = 1, CHCl3), 1H-NMR (CD3OD, pletion as determined by TLC (5% MeOH–CH2Cl2). The
400 MHz): δ 7.69 (d, 2H, J = 8 Hz), 7.55 (d, 2H, J = 8 Hz), 7.28 residue was triturated with MeOH to precipitate excess LiOH.
(t, 2H, J = 8 Hz), 7.21 (t, 2H, J = 8 Hz), 4.22 (d, 2H, J = 7 Hz), The MeOH layer was evaporated under reduced pressure giving
4.11 (t, 1H, J = 7 Hz), 2.92 (t, 2H, J = 7 Hz), 1.77 (bs, 2H), 1.33 5.76 g of 10 (96%, 23.5 mmol) as a white wax. Rf = 0.10 (5%
(m, 16H). 13C-NMR (CD3OD, 100 MHz): 176.0, 157.0, 155.0, MeOH–CH2Cl2). IR (cm−1) = 3297, 2961, 2937, 1541, 1448.
144.0, 143.9, 141.0, 127.0, 126.7.0, 125.0, 119.0, 78.0, 66.0, 1H-NMR (CD3OD, 400 MHz): δ 2.64 (t, 2H, J = 7 Hz), 1.79 (m,
59.0, 40.0, 36.0, 29.0, 27.0, 21.0, 20.0. HRMS (C27H34N2O6Na+) 2H), 1.46 (m, 11H), 1.29 (m, 5H). 13C-NMR (CD3OD, 100 MHz):
calculated = 505.2309, observed = 505.2296.
δ 178.0, 175.0, 80.0, 56.0, 41.0, 36.0, 33.0, 26.0, 21.0, 20.0.
ESI-MS (C12H23NO4Na+) calculated 268.3, observed 268.2.
Synthesis of (S)-1-tert-butyl 3-ethyl 2-[4-(1,3-dioxoisoindolin-
2-yl)butyl]-2-methylmalonate (8)
Synthesis of (S)-2-(tert-butoxycarbonyl)-6-(tert-
butyloxycarbonylamino)-2-methylhexanoic acid (11)
A volume of 3 mL conc. H2SO4 was added to a solution of 10 g
3d (29 mmol) in 100 mL CH2Cl2 in a 250 mL sealed tube. The An amount of 3.9 g NaHCO3 (46.5 mmol) was added to a solu-
solution was cooled to −7 °C in an ice salt bath. A volume of tion of 5.7 g of 10 (23.2 mmol) in 20 mL H2O. The solution
50 mL of condensed isobutylene was added to the solution. was cooled to 0 °C. A solution of 6 g of (Boc)2O (28 mmol) in
The tube was capped tightly and allowed to stir over night at 20 mL 1,4-dioxane was added drop wise to the reaction
RT. The tube was uncapped and allowed to stir for 2 h at mixture. The reaction was allowed to stir at 0 °C for an hour
ambient pressure to allow the excess isobutylene to evaporate. and then brought to RT. The reaction was allowed to stir at RT
The solution was diluted with CH2Cl2 and gently washed three for 12 h. The reaction mixture was extracted with pentane. The
times with 1 N NaOH (50 mL). The CH2Cl2 layer was dried over aqueous layer was acidified to pH 4 using 2 N HCl and
MgSO4, evaporated under reduced pressure, and purified by extracted three times with Et2O (50 mL). The combined ether
chromatography (40% EtOAC–hexanes) giving 10.8
g of layer was dried over MgSO4, evaporated under reduced
product (26.7 mmol, 92%) as a colorless liquid. Rf = 0.60 (40% pressure, and purified by chromatography (40% EtOAc–
1
EtOAc–hexanes), IR (cm−1) = 2977, 2937, 1771, 1707. H-NMR hexanes) giving 7.6 g (22 mmol, 95%) of 11 as a colorless
(CDCl3, 400 MHz): δ 7.84 (m, 2H), 7.71 (m, 2H), 4.16 (q, 2H, J = viscous oil. Rf = 0.54 (40% EtOAc–hexanes), IR (cm−1) = 3380,
1
7 Hz), 3.68 (t, 2H, J = 7 Hz), 1.85 (m, 2H), 1.69 (m, 2H), 1.42 (s, 2976, 2934, 1706, 1522. H-NMR (CDCl3, 400 MHz): δ 4.61 (bs,
9H), 1.28 (m, 8H). 13C-NMR (CDCl3, 100 MHz): δ 172.0, 171.0, 1H), 3.12 (bm, 2H), 1.84 (m, 2H), 1.46 (m, 23H), 1.29 (m, 2H).
168.0, 134.0, 132.0, 123.0, 81.0, 61.0, 54.0, 38.0, 35.0, 29.0, 13C-NMR (CDCl3, 100 MHz): δ 177.0, 172.0, 156.0, 82.0, 79.0,
28.0, 22.0, 20.0, 14.0. HRMS (C22H29NO6Na+) calculated = 54.0, 40.0, 35.0, 30.0, 28.4, 27.8, 22.0, 20.0. HRMS
426.1887, observed = 426.1873.
(C17H31NO6Na+) calculated = 368.2043, observed = 368.2035.
Synthesis of (S)-1-tert-butyl 3-ethyl 2-(4-aminobutyl)-2-
methylmalonate (9)
Synthesis of (R)-tert-butyl-2-(9-fluorenylmethylamino)-6-
(tert-butyloxycarbonylamino)-2-methylhexanoate (12)
A volume of 2.8 mL (31.4 mmol) N2H4·H2O (35% in H2O) was A volume of 2.7 mL of Et3N (19.4 mmol) was added to a solu-
added in a solution of 10.5 g 8 (26 mmol) in 60 mL MeOH. tion of 5.6 g 11 (16.2 mmol) in 60 mL dichloroethane under a
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N2 atmosphere. A volume of 3.9 mL (17.3 mmol) of DPPA was H2O, and dried over MgSO4. The residue was purified by flash
added to the reaction mixture. The mixture was allowed to stir chromatography (5% MeOH–CH2Cl2) giving 5.7 g (20.6 mmol,
at RT for 2 h. The mixture was heated to reflux solvent for 3 h. 90%) of 14 as a white wax. Rf = 0.64 (5% MeOH–CH2Cl2), IR
The reaction was cooled and the organic layer was extracted (cm−1) = 3391, 3325, 3000, 2959, 1770, 1731, 1704, 1557.
with saturated NH4Cl solution, dried over MgSO4 and evapor- 1H-NMR (CDCl3, 400 MHz): δ 7.85 (m, 2H), 7.73 (m, 2H), 4.20
ated under reduced pressure giving the isocyanate. The isocya- (m, 2H), 3.91 (m, 2H), 1.77 (bs, 2H), 1.41 (s, 3H), 1.29 (t, 3H,
nate was taken up in dry toluene under a N2 atmosphere. An J = 7 Hz), 13C-NMR (CDCl3, 100 MHz): δ 175.0, 169.0, 134.0,
amount of 6.4 g (32.4 mmol) 9-fluorenylmethanol was added 132.0, 123.0, 61.0, 58.0, 46.0, 24.0, 14.0. HRMS
to the solution along with 300 µL of Ti(IV) isopropoxide. The (C14H16N2O4Na+) calculated = 299.1002, observed = 299.1002.
solution was heated to 80 °C over night. The reaction was
Synthesis of (S)-ethyl 2-(dibenzylamino)-2-
[(1,3-dioxoisoindolin-2-yl)methyl]propanoate (15)
cooled and the organic layer was evaporated under reduced
pressure. The residue was then purified by chromatography
(CH2Cl2) giving 7.75 g (14.4 mmol, 89%) of 12 as colorless wax. An amount of 4.2 g (15.2 mmol) of 14 was dissolved in 60 mL
An amount of 50 mg of 12 was further purified by reversed of distilled acetonitrile in a 250 mL three necked flask under a
phase HPLC (40% CH3CN–H2O to 100% CH3CN in 15 min at N2 atmosphere. An amount of 12.6 g (91.2 mmol) of K2CO3
262 nm, Rt = 16.6 min) giving 35 mg of pure 12 as a colorless was added with stirring. A volume of 9 mL (76 mmol) of BnBr
oil. Rf = 0.57 (CH2Cl2), IR (cm−1) = 3359, 2975, 2931, 1707, was added drop wise. The reaction mixture was heated to
1
1516. H-NMR (CDCl3, 400 MHz): δ 7.76 (d, 2H, J = 8 Hz), 7.61 reflux solvent for 12 h. The reaction mixture was diluted with
(d, 2H, J = 8 Hz), 7.40 (t, 2H, J = 7 Hz), 7.32 (t, 2H, J = 7 Hz), 50 mL of H2O and the solution was extracted with ether three
5.82 (bs, 1H), 4.47 (m, 2H), 4.18 (t, 1H, J = 7 Hz), 4.02 (bs, 1H), times. The ether layer was washed with H2O, washed with
3.07 (bm, 2H), 2.23 (bm, 1H), 1.74 (bm, 1H), 1.45 (m, 25H). brine, dried over MgSO4 and evaporated. The residue was then
13C-NMR (CDCl3, 100 MHz): δ 173.2, 156.03, 154.3, 143.9, purified by flash chromatography (30% EtOAc–hexanes) giving
141.4, 127.6, 127.0, 125.1, 120.0, 82.2, 79.2, 66.3, 60.1, 47.2, 5.6 g (12.3 mmol, 81%) of 15 as a white solid. Rf = 0.59 (30%
40.1, 35.8, 29.7, 28.3, 27.9, 23.7, 21.2. HRMS (C31H42N2O6Na+) EtOAc–hexanes), IR (cm−1) = 2970, 1770, 1713, 1620. MP =
calculated = 561.2935, observed = 561.2924.
83 °C. 1H-NMR (CDCl3, 400 MHz): δ 7.83 (m, 2H), 7.71 (m,
2H), 7.34 (d, 4H, J = 8 Hz), 7.12 (m, 6H), 4.23 (q, 2H, J = 7 Hz),
3.95 (m, 6H), 1.35 (m, 6H). 13C-NMR (CDCl3, 100 MHz): δ
Synthesis of (R)-tBoc-Fmoc-α-methyl-α-lysine-OH (13)
An amount of 2 g (3.7 mmol) of 12 was dissolved in 20 mL of 174.0, 168.0, 141.0, 134.0, 132.0, 128.3, 128.0, 126.0, 123.0,
1 : 1 TFA–CH2Cl2. The solution was allowed to stir for 12 h at 68.0, 61.0, 55.0, 44.0, 19.0, 14.0. HRMS (C28H28N2O4Na+) =
RT under a N2 atmosphere. The reaction was monitored by 479.1941, observed = 479.1938.
TLC (5% MeOH–CH2Cl2) and ESI-mass spectrometry for com-
(S)-Ethyl-3-amino-2-(dibenzylamino)-2-methylpropanoate (16)
pletion. At which point the TFA–CH2Cl2 layer was evaporated
under reduced pressure giving free amino acid. The residue An amount of 3.2 g (7 mmol) of 15 was dissolved in 20 mL of
was taken up in 15 mL of H2O and 0.76 g (9 mmol) of NaHCO3 (8 : 2) MeOH and CH2Cl2. A volume of 1.7 mL (21 mmol) of
was added to the solution slowly to control the effervescence. N2H4 (35% in H2O) was added. The solution was heated to
The mixture was cooled to 0 °C. A solution of 0.96 g reflux solvent for 3 h. The formation of a white precipitate
(4.4 mmol) (Boc)2O in 15 mL 1,4-dioxane was added to the indicated the completion of the reaction. The reaction mixture
mixture slowly at 0 °C. The reaction was allowed to stir at 0 °C was filtered and the filtrate was evaporated giving 2 g
for an hour. The reaction was then allowed to warm to RT and (6.5 mmol, 92%) of 16 as a yellowish oil. Rf = 0.65 (5% MeOH–
stir for 12 h. The reaction mixture was extracted with pentane CH2Cl2). IR (cm−1) = 2979, 1717, 1601. 1H-NMR (CDCl3,
to remove excess (Boc)2O. The aqueous layer was then acidified 400 MHz): δ 7.21 (m, 10H), 4.16 (m, 2H), 3.84 (m, 4H), 2.95 (s,
to pH 4 with 2 N HCl, extracted three times with Et2O (50 mL). 2H), 1.35 (s, 3H), 1.31 (t, 3H, J = 7 Hz), 1.20 (bs, 2H), 13C-NMR
The combined ether layer was dried over MgSO4, evaporated (CDCl3, 100 MHz): δ 174.0, 141.0, 128.4, 128.0, 126.0, 69.0,
under reduced pressure and purified by chromatography (5% 60.0, 55.0, 48.0, 20.0, 14.0. HRMS (C20H26N2O2Na+) calculated
MeOH–CH2Cl2) giving 1.52 g (3.15 mmol, 85% over two steps) = 349.1886, observed = 349.1873.
of 13 as a white solid similar to 7. All characterization data of
(S)-Ethyl-3-amino-2-(dibenzylamino)-2-methylpropanoic acid
13 complied with the data for 7. The polarimetry reading con-
(17)
firmed 13 as the enantiomer to 7. [α]2D2 = −11.5 (c = 1, CHCl3).
An amount of 1.41 g (4.3 mmol) of 16 was dissolved in 25 mL
of EtOH. An amount of 0.52 g (12.9 mmol) of well crushed
NaOH pellets were added. The solution was heated to reflux
Synthesis of (S)-ethyl 2-amino-2-((1,3-dioxoisoindolin-2-yl)-
methyl) propanoate (14)
An amount of 10 g (23 mmol) 4a was dissolved in 60 mL of solvent for 4 h. The EtOH layer was acidified to pH 2, evapor-
methylene chloride and 10 mL TFA was added. The solution ated to dryness under high vacuum, and triturated with
was stirred for 1 h. The solution became dark purple in color. MeOH. The MeOH layer was neutralized with solid NaHCO3,
A volume of 100 mL H2O was added to the solution and the filtered, and evaporated under reduced pressure giving 1.16 g
organic layer was washed with NaHCO3 solution, washed with (3.9 mmol, 90%) of 17 as yellowish wax. 1H-NMR (CD3OD,
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400 MHz): δ 7.18 (m, 10H), 3.91 (m, 4H), 2.83 (m, 2H), 1.87 (s, MeOH–CH2Cl2), IR (cm−1) = 3317, 2974, 1694, 1513. MP =
3H). 13C-NMR (CD3OD, 100 MHz): δ 182.0, 144.0, 129.6, 129.0, 82 °C. [α]2D2 = −10.5 (c = 1, CHCl3), 1H-NMR (CD3OD,
127.0, 70.0, 56.0, 22.0. HRMS (C18H22N2O2Na+) calculated = 400 MHz): δ 7.80 (d, 2H, J = 7 Hz), 7.68 (d, 2H, J = 7 Hz), 7.39
321.1573, observed = 321.1573.
(t, 2H, J = 7 Hz), 7.31 (t, 2H, J = 7 Hz), 4.31 (bs, 2H), 4.22 (t, 1H,
J = 7 Hz), 3.55 (m, 2H), 1.44 (s, 12H). 13C-NMR (CD3OD,
100 MHz): δ 159.0, 157.0, 145.4, 145.3, 143.0, 129.0, 128.0,
126.4, 126.3, 121.0, 80.0, 68.0, 55.0, 46.0, 28.0, 21.0. HRMS
Synthesis of (S)-2-(dibenzylamino)-3-(tert-
butyloxycarbonylamino)-2-methylpropanoic acid (18)
A solution of 1 g of 17 (3.4 mmol) in 10 mL water was placed (C24H28N2O6Na+) calculated = 463.1839, observed = 463.1835.
in a 50 mL round bottom flask. An amount of 0.56 g
(6.7 mmol) of NaHCO3 (2 eq.) was added with stirring. The
solution was cooled to 0 °C. A solution of 0.98 g (4.7 mmol)
Synthesis of (S)-tert-butyl 2-tert-butyloxyaminobutyl-4-diazo-2-
methyl-3-oxobutanoate (21)
(Boc)2O (1.4 eq.) in 10 mL 1,4 dioxane was added drop wise. Acid 11 (3 g, 8.7 mmol) was dissolved in 10 mL THF and
The reaction was allowed to stir at 0 °C for an hour. The reac- cooled to −25 °C. A measured 1 equivalent of Et3N (1.2 mL,
tion was then allowed to warm to RT overnight. The reaction 8.7 mmol) and 1.05 equivalents of ClCO2Me (710 µL,
mixture was diluted with 15 mL H2O, acidified to pH 4 with 9.1 mmol) was added drop wise to the THF solution. The
NaHSO4, and extracted twice with Et2O. The combined ether mixture was stirred for 2 h giving rise to the mixed anhydride,
layer was washed with water (5 × 30 mL), washed with brine, which was taken immediately for the next step. The resulting
dried over MgSO4, and evaporated. The residue was purified by white suspension of the mixed anhydride was allowed to warm
chromatography (40% EtOAc–hexanes) giving 1.2 g (3 mmol, to 0 °C and a solution of dry diazomethane (2 equivalent,
91%) of 18 as a white solid. Rf = 0.28 (40% EtOAc–hexane). IR 17.4 mmol) in Et2O was carefully added. The reaction mixture
(cm−1) = 2977, 1698, 1494. MP = 58 °C. 1H-NMR (CDCl3, was allowed to stir for 12 h in the dark at 0 °C. Excess diazo-
400 MHz): δ 7.21 (m, 10H), 5.44 (bs, 1H), 4.09 (m, 4H), 3.64 methane was removed by passing N2 through the solution for
(m, 2H), 1.42 (m, 12H). 13C-NMR (CDCl3, 100 MHz): δ 175.0, 30 min. The reaction mixture was then diluted with Et2O,
156.0, 137.0, 128.8, 128.5, 127.7, 79.0, 71.0, 55.0, 44.0, 28.0, washed with saturated NaHCO3, saturated NH4Cl, and brine.
20.0. HRMS [C23H30N2O4Na+] calculated = 421.2097, observed The organic layer was dried over MgSO4, and concentrated
= 421.2094.
under reduced pressure. The resulting residue was purified by
chromatography (1 : 1 Et2O–hexanes) giving 2.62 g (7.1 mmol,
82%) of 21 as a clear yellowish oil. Rf = 0.42 (1 : 1 Et2O–
hexanes). IR (cm−1) = 3381, 2976, 2934, 2110, 1704, 1517.
(S)-2-Amino-3-(tert-butyloxycarbonylamino)-2-methylpropanoic
acid (19)
A solution of 1 g (2.5 mmol) of 18 in 25 mL MeOH was placed 1H-NMR (CDCl3, 400 MHz): δ 5.41 (s, 1H), 4.58 (bs, 1H), 3.09
in a pressure bottle. An amount of 0.2 g (20% by weight) of Pd-C (bm, 2H), 1.85 (m, 1H), 1.71 (m, 1H), 1.45 (m, 21H), 1.28 (m,
was added to the bottle. The solution was placed on a Parr 4H). HRMS (C18H31N3O5Na+) 392.2156, observed = 392.2153.
shaker hydrogenation apparatus and allowed to shake with
(S)-3-tert-Butyloxyaminobutyl-4-tert-butyloxy-3-methyl-4-
oxobutanoic acid (22)
30 psi hydrogen gas for 12 h. The reaction mixture was filtered
through a Celite bed to remove the catalyst. The filtrate was
evaporated giving 19. An amount of 0.5 g (2.3 mmol, 92%) of An amount of 2.5 g 21 (6.8 mmol) was dissolved in 15 mL 3 : 7
19 was obtained as a white wax. Rf = 0.3 (5% MeOH–CH2Cl2). H2O–THF in a 50 mL round bottom flask. The flask was
IR (cm−1) = 2977, 1701, 1602, 1508. MP = 204 °C. 1H-NMR purged with N2 and the resulting solution was photolyzed with
(CD3OD, 400 MHz): δ 3.44 (s, 2H), 1.47 (m, 12H). 13C-NMR a Hanovia lamp (500 W) at a distance of approximately 10 cm.
(CD3OD, 100 MHz): δ 174.0, 158.0, 79.0, 61.0, 46.0, 27.0, 19.0. The photolysis was allowed to proceed for 48 h. At that point
HRMS (C9H18N2O4Na+) calculated = 241.1159, 241.1158.
the reaction was found to be completed as evident by TLC. The
clear and colorless solution was concentrated under reduced
pressure and the water layer was extracted three times with
Et2O. The combined Et2O layer was washed with brine, dried
Synthesis of (S)-2-(9-fluorenylmethyloxycarbonylamine)-3-
(tert-butyloxycarbonylamino)-2-methylpropanoic acid (20)
An amount of 0.35 g of NaHCO3 (4.1 mmol) was added to a over MgSO4, and evaporated under reduced pressure. The
solution of 0.45 g 19 (2.1 mmol) in 15 mL water with stirring. residue was purified by chromatography (30% EtOAc–hexanes)
The solution was cooled 0 °C. A solution of 1.1 g Fmoc-Osu giving 1.83 g of 22 (5.1 mmol, 75%) as a yellowish oil. The
(3.2 mmol) in 15 mL 1,4-dioxane was added to the reaction 1H-NMR and the HRMS is highly indicative of the product.
mixture over 20 min. The reaction was allowed to stir at 0 °C Hence, the product was taken for the next step without further
for an hour and then at ambient temperature for 12 h. At that purification. Rf = 0.18 (30% EtOAc–hexanes), IR (cm−1) = 3364,
1
point the reaction was diluted with 30 mL of water, acidified to 2977, 2936, 1709, 1521. H-NMR (CDCl3, 400 MHz): δ 4.54 (bs,
pH 4 with 4 M HCl, extracted (3 × 50 mL) with Et2O. The com- 1H), 3.10 (bm, 2H), 2.73 (m, 1H), 2.38 (m, 1H), 1.55 (m, 22H),
bined ether layer was washed with brine, dried over MgSO4, 1.20 (m, 5H). 13C-NMR (CDCl3, 100 MHz): δ 176.2, 175.2,
and evaporated under reduced pressure. The residue was puri- 155.9, 80.6, 72.4, 44.4, 42.4, 40.1, 38.8, 30.0, 28.4, 27.8, 21.8,
fied by radial chromatography using 5% MeOH–CH2Cl2 giving 21.4. HRMS (C18H33NO6Na+) calculated = 382.2200, observed =
0.86 g (1.96 mmol, 85%) of 20 as a white solid. Rf = 0.41 (5% 382.2196.
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(S)-tert-Butyl-2-tert-butyloxycarbonylaminobutyl-3-
(m, 2H), 7.38 (t, 2H, J = 7 Hz), 7.30 (t, 2H, J = 7 Hz), 6.37 (bm,
(9-fluorenylmethyloxycarbonylamino)-2-methylpropanoate (23) 1H), 5.42 (bm, 1H), 4.58 (m, 1H), 4.35 (m, 1H), 4.21 (m, 1H),
3.36 (m, 2H), 3.09 (bm, 2H), 1.41 (m, 18H). 13C-NMR (CDCl3,
A volume of 0.79 mL Et3N was added to a solution of 1.7 g of
100 MHz): δ 179.5, 156.9, 156.2, 143.6, 141.2, 127.7, 127.1,
22 (4.7 mmol) in 25 mL dichloroethane under N2 atmosphere.
125.1, 120.0, 79.3, 66.8, 47.3, 46.8, 40.0, 36.7, 36.0, 30.4, 28.4,
A volume of 1.2 mL (5.2 mmol) DPPA was added to the reaction
21.3, 20.4. HRMS (C28H36N2O6Na+) calculated = 519.2965,
mixture. The reaction was allowed to stir at RT for 2 h. The
mixture was heated to reflux solvent for 3 h. The mixture was
observed = 519.2459.
cooled and the organic layer was extracted with saturated
Synthesis and specific binding of Vapreotide analogue (25)
against IMR 32 cell line
NH4Cl solution, dried over MgSO4, and evaporated under
reduced pressure giving the isocyanate. The isocyanate was
taken up in dry toluene under N2 atmosphere. An amount of
1.84 g (9.4 mmol) 9-fluorenylmethanol was added to the solu-
tion along with 100 μL of Ti(IV) isopropoxide. The reaction was
heated to 80 °C over night. The reaction was cooled and the
organic layer was evaporated under reduced pressure. The
residue was then purified by chromatography (CH2Cl2 to 3%
MeOH–CH2Cl2) giving 2 g 23 (3.76 mmol, 80%) as sticky light
yellowish wax. The product was found too sticky to dry the
solvent all the way. It was characterized by 1H-NMR and
HRMS. The product was taken for the next step without
further attempt to purify it. Rf = 0.78 (3% MeOH–CH2Cl2). IR
The Vapreotide analogue (25) was synthesized in collaboration
with New England Peptide (Gardner, MA) using properly pro-
tected (S)-α2,2-methyllysine analogue (7) prepared in our labo-
ratories as described above. The Vapreotide analogue was
determined to be 99% pure by HPLC.
Binding of the Vapreotide analogue (25) was conducted
against IMR 32 human neuroblastoma cells. These cells over
express SSTR2 receptors. In order to perform the binding
assays four groups of triplicate wells were studied (n = 12
total). Each well contained 500 000 IMR 32 cells in 2 mL of
media. These wells also contained 100 000 counts of 111In-
pentetreotide. Three wells were competed with 10−6 M octreotide
and three wells were competed with 25. All 12 wells were incu-
bated at 37 °C for 20 hours. Cells were harvested, washed and
counted in a gamma counter. However, gamma counter result
revealed that the Vapreotide analogue 25 has no specific
binding for SSTR2 (Table 2 in ESI†).
1
(cm−1) = 3340, 2975, 2933, 1756, 1688, 1513. H-NMR (CDCl3,
400 MHz): δ 7.76 (d, 2H, J = 7 Hz), 7.59 (d, 2H, J = 7 Hz), 7.39
(t, 2H, J = 7 Hz), 7.30 (t, 2H, J = 7 Hz), 4.60 (bm, 2H, J = 7 Hz),
4.37 (m, 2H), 4.22 (t, 1H, J = 7 Hz), 3.38 (m, 1H), 3.24 (m, 1H),
3.09 (m, 2H), 1.46 (bm, 6H), 1.44 (bm, 21H). HRMS
(C32H44N2O6Na+) calculated = 575.3091, observed = 575.3083.
Synthesis of (S)-Fmoc-α-methyl-β2,2-lysine-Boc-OH (24)
An amount of 1.5 g of 23 (2.7 mmol) was dissolved in 20 mL of
1 : 1 TFA–CH2Cl2. The solution was allowed to stir for 12 h at
RT under N2 atmosphere. The reaction was monitored by TLC
(5% MeOH–CH2Cl2) and ESI-mass spectrometry for the com-
pletion. At which point the TFA–CH2Cl2 layer was evaporated
under reduced pressure giving free amino acid. The residue
was taken up in 15 mL of H2O and 0.54 g (6.5 mmol) NaHCO3
was added to the solution slowly to control the effervescence.
The mixture was cooled to 0 °C. A solution of 0.71 g (Boc)2O
(3.2 mmol) in 15 mL 1,4-dioxane was added to the mixture
slowly at 0 °C. The reaction was allowed to stir at 0 °C for an
hour. The reaction was then allowed to warm to RT and stir for
12 h. The reaction mixture was extracted with pentane to
remove excess (Boc)2O. The aqueous layer was then acidified to
pH 4 with 2 N HCl, extracted three times with Et2O (50 mL).
The combines ether layer was dried over MgSO4, evaporated
under reduced pressure, purified by chromatography (5%
MeOH–CH2Cl2) giving 1.21 g of 24 (2.44 mmol, 90% over two
steps) as a white solid after purification by flash chromato-
graphy (CH2Cl2 to 5% MeOH–CH2Cl2). Rf = 0.50 (5% MeOH–
CH2Cl2). IR (cm−1) = 3338, 2940, 1693, 1518. [α]2D3 = −6.0 (c =
0.7, CHCl3). MP = 73 °C. 1H-NMR (CD3OD, 400 MHz): δ 7.81
(d, 2H, J = 7 Hz), 7.66 (d, 2H, J = 7 Hz), 7.40 (t, 2H, J = 7 Hz),
7.32 (t, 2H, J = 7 Hz), 4.36 (m, 2H), 6.23 (m, 1H), 3.03 (t, 2H, J =
7 Hz), 1.61 (m, 1H), 1.44 (m, 12H), 1.30 (m, 2H), 1.13 (s, 3H).
1H-NMR (CDCl3, 400 MHz): δ 7.75 (d, 2H, J = 7 Hz), 7.58
Acknowledgements
DSM would like to thank the National Science Foundation for
a CAREER award (MCB-0844478). We are thankful to the
National Science Foundation for instrument grants providing
mass spectrometry and NMR facilities used in this research
(CHE-0639208, DBI-0619455, CHE-0840390) and the Depart-
ment of Chemistry and Biochemistry at USM for continued
support of our programs.
Notes and references
1 C. Cativiela and M. D. Díaz-de-Villegas, Tetrahedron:
Asymmetry, 2000, 11, 645–732.
2 Y. Ohfune and T. Shinada, Eur. J. Org. Chem., 2005, 5127–
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