L -amino acid amides via dynamic kinetic resolution

Article abstract of DOI:10.1002/adsc.201100389

Racemic natural and non-natural N-Boc-amino acid thioesters undergo enzyme-catalyzed ammoniolysis and aminolysis with concomitant base-catalyzed racemization of the non-transformed D-enantiomer with the formation of the corresponding L-amides in good yields and excellent enantiomeric purity all above 98% ee. This is the first example of a dynamic kinetic resolution of amino acid derivatives with amides formation. Copyright

Full text of DOI:10.1002/adsc.201100389

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DOI: 10.1002/adsc.201100389
l-Amino Acid Amides via Dynamic Kinetic Resolution
Davide Tessaro,a,b Lorenzo Cerioli,a Stefano Servi,a,b,* Fiorenza Viani,c
and Paola D’Arrigoa,b
a
Department of Chemistry, Materials, and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Plaza L. da Vinci
32, 20131 Milano, Italy
Fax : (+39)-02-2399-3089; e-mail: stefano.servi@polimi.it
Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano and
b
Universitꢀ degli Studi dell’Insubria, Via Mancinelli 7, 20131 Milano, Italy
ICRM, CNR, Via Mancinelli 7, 20131 Milano, Italy
c
Received: May 16, 2011; Revised: August 9, 2011; Published online: August 29, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100389.
Abstract: Racemic natural and non-natural N-Boc-
amino acid thioesters undergo enzyme-catalyzed
ammoniolysis and aminolysis with concomitant
base-catalyzed racemization of the non-transformed
d-enantiomer with the formation of the correspond-
ing l-amides in good yields and excellent enantio-
meric purity all above 98% ee. This is the first ex-
ample of a dynamic kinetic resolution of amino acid
derivatives with amides formation.
enzyme mixed catalysis leading to a dynamic kinetic
resolution (DKR) of a series of pharmaceutical inter-
mediates, primarily a-methyl propionates of the
profen family. In these studies the requirements for a
possible and efficient base-catalyzed racemization/
enzyme-catalyzed kinetic resolution have been ex-
plored and identified giving access to products by a
DKR process with high yields and ees.[5,6]
Although the nature of the thioester strongly influ-
ences the carbon acidity which allows racemization to
occur at a useful rate, a larger effect is associated to
the presence of functional groups stabilizing the inter-
mediate anion and present in position a- to the ste-
reogenic C-atom. Thus, the presence of an aromatic
ring is necessary for racemization using a trialkyl-
Keywords: amino acid amides; aminolysis; amoniol-
ysis; base-catalyzed racemization; dynamic kinetic
resolution; subtilisin; thioesters
AHCTUNGTRENNUNG
Thioesters of carboxylic acids are reactive species esters of natural and non-natural N-Boc-amino acids[7]
mimicking the activation occurring in Nature in the and found that modulation of the base can be decisive
transfer of acyl moieties. They have been used as mild in racemizing compounds with higher pKa. While tri-
activated intermediates in protein synthesis, coupling octylamine (TOA) is usually employed as a base in a
two parts through a chemical ligation[1] in the forma- biphasic water/organic solvent system, we found that
tion of cyclic peptides[2] or macrolides.[3]
compounds which were not racemizable in such a
However, in some cases they are considered to be system could be readily racemized in organic solvent
too reactive being prone to combine directly with with controlled water activity when using 1,8-diazabi-
water in an undesired hydrolytic reaction competing cycloundec-7-ene (DBU) as a base.
with the transfer of acyl group to N- or O- atoms.
In this article we describe the combination of base
They behave as excellent acyl donors in transesteri- catalysis and enzyme catalysis for transformation of
fication, transamidation and hydrolysis reactions, cat- N-Boc-amino acid thioesters to obtain the corre-
alyzed by hydrolytic enzymes, lipases and proteases. sponding primary or secondary amides with complete
Many examples of lipase-catalyzed kinetic resolution deracemization of the racemic starting substrates. Ac-
employing thioesters as acyl donors both in hydrolysis cording to our best knowledge, this is the first DKR
and transesterification have appeared.[4]
leading to the direct formation of primary and secon-
Thioesters have another peculiar chemical reactivi- dary l-amino acid amides.
ty: they are considerably more C-acidic than the cor-
Enzymatic Amide Formation: Amide bond forma-
responding carboxylates, oxo esters or amides. This tion is a labor intensive process often involved in the
reactivity towards bases has been exploited in a base- synthesis of fine chemicals. Aminolysis of esters and
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Davide Tessaro et al.
COMMUNICATIONS
thioesters is a practical method of amide synthesis
avoiding more intensive carboxylate activation.[8]
In recent evaluations of chemical processes trying
to comply to the rule of GMF and green chemistry,
amide bond formation was identified as one of the
most utilized and problematic synthetic steps in the
pharmaceutical industry. From a detailed study it was
found that N-acylation reactions for amide formation
were used in more than 50% of current syntheses of
drug candidates.[9]
Clearly, the enzymatic procedure even when requir-
ing carboxylate activation (esters as intermediates)
avoids the need of condensing agents necessary for
chemical amide formation. Lipases are often consid-
ered for amoniolysis/aminolysis with the argument
that the reverse amide hydrolysis is not efficiently cat-
alyzed by these enzymes. Moreover, the enantioselec-
tivity of lipases can be modulated through substrate
engineering and reaction engineering or by selecting
the enzyme among the great diversity of these cata-
lysts allowing one to have access to compounds of
both stereochemical preference.[10,11]
Figure 1. DKR of N-Boc-amino acid thioesters.
Proteases, in contrast, have well defined stereo-
chemical preferences, particularly towards amino acid
derivatives and the problem of shifting reactions to- TOA was not effective in promoting the racemization
wards reverse-hydrolysis requires a specific solu- of these compounds nor were the bases that we plan-
tion.[12,13]
ned to employ as nucleophiles in the transamidation
In particular, subtilisin, almost a shelf reagent procedure, namely ammonia, propylamine and ben-
under the name of Alcalaseꢂ, (alcalase is an endopro- zylamine. Rates of racemization in t-BuOH were de-
tease from Bacillus licheniformis. Its main component pendent on the base concentration: when at least
is subtilisin A) has been applied in ammoniolysis re- 1.2 equiv. of base were used, the rate of racemization
actions in water,[14] in genetically modified form[15] was not the rate-determining step of the DKR transa-
and in organic solvents[16] for the enzyme-catalyzed midation.
formation of amides and peptide bonds.[17] The stabili-
Competing Enzymatic Hydrolysis: Due to the rela-
ty of this protease in organic solvents and in the pres- tively high reactivity of thioesters, the competing hy-
ence of bases makes it particularly suitable for the drolysis reaction must be suppressed or minimized.
purpose of catalyzing amminolysis reactions with con- While the enzyme-catalyzed hydrolytic step is much
comitant base-catalyzed racemization of the substrate. slower than the corresponding amoniolysis/aminolysis
Since we have previously shown that in the presence reaction when water is competing with a stronger nu-
of water and strong bases like DBU, non-enzyme cat- cleophile, the spontaneous chemical hydrolysis is also
alyzed hydrolysis takes place, non-aqueous conditions dependent on the water concentration and can signifi-
have to be employed. Therefore, t-BuOH proved to cantly compete with the enzyme-catalyzed reaction.
have the required solvent properties together with Therefore tBuOH containing 0.08% water was used.
non-nucleophilic behaviour. The strong specificity of However, after the addition of the enzymatic prepa-
subtilisin towards amino acids of the l-configuration rate the water content raised to 1.5%, not always op-
is beneficial but at the same time, limiting, since timal for selectivity. In a previous work, enzymatic hy-
access to compounds of the d-series in a DKR process drolysis of N-Boc-phenylglycine thioesters[18] in DKR
is not possible.
The first system for the DKR transamidation proce- MTBE solvent system in the presence of trioctyl-
dure was then a subtilisin-catalyzed kinetic resolution amine as the racemizing agent. It is therefore expect-
of the thioesters using ammonia or amines as nucleo- ed that in the present conditions (100 mg substrate,
philes (Figure 1). 15 mL t-BuOH, 22 mM) with a stronger base (DBU)
conditions was performed using a biphasic water/
AHCTUNGTRENNUNG
Base-Catalyzed Racemization: Base-catalyzed rac- and a substantial amount of water (80 mM), concur-
emization is the second necessary key step for the rent hydrolysis could take place.
completion of the cycle. On the basis of previous cal-
However, it has been observed that the hydrolysis
culations[7] the racemization of the selected substrates rates of thioester and oxoester in basic solution are
1a–3a was effected using DBU as a base. In fact, very similar while the aminolysis of a thioester with a
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l-Amino Acid Amides via Dynamic Kinetic Resolution
Table 1. Non-enzymatic depletion of substrate 1a (40 mM in
t-BuOH).
Table 2. Depletion of substrate 1a (40 mM in t-BuOH,
1.2 equiv. DBU, 100 mg Alcalase® CLEA).
Nucleophile
NH3
Equiv.
Unreacted 1a [%]
Nucleophile Equiv.
Unreacted 1a [%]
1 h 2 h 5 h 25 h
Amides [ee %]
1 h
2 h
5 h
25 h
1.5
3
6
1.2
2.4
4.8
1.2
2.4
4.8
100
100
100
96
89
74
83
59
64
100
100
100
91
86
55
74
46
77
100
99
99
85
68
28
54
26
7
96
95
90
55
3
0
14
1
NH3
2
41 36 20 20
1b (98)
1c (99)
1d (98)
n-PrNH2
BnNH2
1.2
1.2
21 11
17
0
0
0
0
4
n-PrNH2
proving that in these conditions, base-catalyzed race-
mization was occurring at the required rate, the chem-
ical amidation was largely suppressed because of the
effective catalytic activity of the protease which
works with a practically complete enantioselectivity.
The reaction was repeated with all the three nucleo-
BnNH2
0
primary amine to form an amide is more than 100- philes and the data collected (Table 2) compared with
fold faster than the reaction of the corresponding those from non-enzymatic reactions (Table 1).
oxo
G
The results observed both in terms of isolated
tease-catalyzed aminolysis reaction should be much yields (ca. 80%) and ee (>98%) proved that in all the
faster than the corresponding hydrolysis and that this three reactions the base-catalyzed racemization is
reaction will not compete with the enzyme-catalyzed active, with no or very limited competition from
ammoniolysis. From preliminary results on the same chemical aminolysis which would otherwise affect the
substrate, N-Boc-Phe ethyl thioester, complete hy- ee, and with no or very limited competition from
drolysis in t-BuOH with subtilisin CLEA occurred in chemical hydrolysis which would otherwise affect the
about 24 h, while under comparable conditions the chemical yields. The phenylalanine derivative 1a was
aminolysis went to completion in 4–5 h (Table 1). No efficiently deracemized to give the corresponding
hydrolysis product was observed in the reaction mix- amides in high yields and almost absolute stereoselec-
tures after complete aminolysis. Therefore, no at- tivity. Substrates 2a and 3a, which were transformed
temps to use a solvent with a lower water content at a much reduced rate, required adjustment of the
were made.
conditions in order to obtain high ee, at the expense
Competing Chemical Amoniolysis/Aminolysis: of isolated yields. In particular, a higher amount of
Table 1 summarizes the data concerning the transami- enzyme increased the enzymatic reaction rates so de-
dation of substrate 1a in the presence of different creasing the chemical amidation but accelerating also
concentrations of the three nucleophiles that are the the concurrent hydrolysis reaction. Table 3 summariz-
object of this study: ammonia, propylamine, benzyl- es the experimental results. Surprisingly, no amide
A
was obtained with compound 3a and ammonia. Ex-
The spontaneous chemical reaction of the reactive periments with different enzyme preparations (subtili-
thioesters with these nitrogen nucleophiles is not neg- sin immobilized on different supports) proved to be
ligible, and is dependent on their concentration and much less efficient. Moreover, the cross-linked
on the nature of the nucleophile. Thus, while the enzyme can be recovered and reused with complete
effect of ammonia is very limited, with the two pri- recovery of activity employing a simple protocol.
mary amines the amide formation can go to comple-
In conclusion, this method appears to be of general
tion when 5 equiv. of amine are used in 25 h. Needless applicability[20] for the preparation of l-amides start-
to say, in these conditions no enantiomer discrimina- ing from a racemic N-Boc-amino acid thioester with
tion can occur. Therefore, the catalytic efficiency of good yields and excellent optical purity.
the biocatalyst is crucial. The effect of the competing
non-enzyme-catalyzed reaction could be controlled if
the lower concentrations of amines were applied
(Table 1).
Experimental Section
DKR of N-Boc-AA Thioesters in Ammoniolysis–
Aminolysis: In a first experiment, a 40 mM solution
of substrate 1a (8 mL) in t-BuOH with 1.2 equiv.
DBU as the racemization catalyst and 100 mg subtili-
sin CLEA were treated with 1.5 equiv. of propylamine
and the reaction mixture was stirred at 308C. After
5 h the reaction was complete and the corresponding
General Remarks
All chemicals were purchased from Sigma–Aldrich. All sol-
vents were of analytical grade. Silica gel 60 F254 plates
(Merck) were used for analytical TLC. Detection was ach-
ieved with UV light followed by I2, ninhydrin or potassium
permanganate staining. HPLC analysis were performed on a
Merck apparatus equipped with an UV detector, fitted with
l-propylamide isolated in 85% yield and 98% ee, a Chiralcel OD (length 250 mm; diameter 4.6 mm; pore size
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COMMUNICATIONS
Table 3. Experimental results.
Substrate Product (isolated
yields)
Product
[ee%]
DBU
[equiv.]
Enzyme
[mg]
Amine
[equiv.]
Reaction time t-BuOH vol.
[h]
[mL]
1a
1a
1a
2a
2a
2a
3a
3a
3a
1b (82)
1c (76)
1d (89)
2b (60)
2c (63)
2d (87)
–
>98
99
>98
99
99
99
–
98
96
2
4
3
2
4
2
–
2
2
100
100
200
100
400
400
–
2
7
7
5
30
24
24
–
30
30
3.5
7
1.2
1.2
3
1.2
1.2
–
3.5
3.5
14
14
–
3c (65)
3d (69)
200
100
1.2
1.2
7
7
1
5 mm; eluent: hexane/2-propanol), H NMR spectra were re-
corded on a Bruker ARX 400 instrument operating at the
1H resonance frequency of 400 MHz. Chemical shifts (d,
ppm) are reported relative to tetramethylsilane (TMS) as in-
ternal standard. All spectra were recorded in DMSO-d6, or
CDCl3 at 305 K. Optical rotations were determined with a
Propol Digital Polarimeter Dr Kenchen, and [a]D values are
given in units of degcm2 gÀ1 at 258C.
14.326; HPLC analysis (hexane/2-propanol=400/1.5, flow=
1.2 mLminÀ1, T=308 K): peaks d: 26.4 min; l: 19.5 min.
l-N-Boc-Phe-SEt (l-1a): White solid; mp 79–818C; [a]D:
À24.4 (c 1.0, 2-propanol, 208C).
d,l-N-Boc-homoPhe-SEt (d,l-2a): White solid; mp 85–
868C; chemical formula: C17H25NO3S; exact mass: 323.16;
molecular eeight: 323.45; MS: m/z=323.16 (100.0%), 324.16
(18.8%), 325.15 (4.5%), 325.16 (2.4%), 324.15 (1.2%); ele-
mental analysis: C 63.13, H 7.79, N 4.33, O 14.84, S 9.91;
1H NMR: d=1.248 (t, J=7.415 Hz, 3H), 1.463 (s, 9H), 1.8–
1.959 (m, 1H), 2.1–2.26 (m, 1H), 2.6–2.8 (m, 2H), 2.876 (q,
J=7.415 Hz, 2H), 4.382 (br s,1H), 4.960 (br s,1H), 7.179 (m,
2.5H), 7.276 (m, 2.5H); 13C NMR: d=201.08, 155.03,
140.57, 128.44, 128.25, 126.02, 79.95, 60.22, 34.36, 31.57,
28.20, 23.03, 14.37; HPLC analysis (hexane/2-propanol=99/
1, flow=1.2 mLminÀ1, T=308 K): peaks d: 13.5 min; l:
32.3 min.
Enzymes
Immobilized subtilisin from Bacillus subtilis Fluka (2.4 U/g)
and Alcalaseꢂ 3.0 T from Novozymes (3.0 U/g) were found
to be equivalent in terms of activity and selectivity. Cross-
linked subtilisin from B. subtilis CLEAꢂ was purchased
from CLEA technologies (450 U/g) and was preferentially
used in all experiments described.
l-N-Boc-homoPhe-SEt (l-2a); [a]D: À16.5 (c 1.0, 2-propa-
nol, 208C); white solid; mp 78–798C.
General Procedure for d,l-N-Boc-amino Acid
Thioethyl Esters Synthesis
d,l-N-Boc-norLeu-SEt (d,l-3a): White solid; mp 37–
388C; chemical formula: C13H25NO3S; exact mass: 275.16;
molecular weight: 275.41; MS: m/z=275.16 (100.0%),
276.16 (14.5%), 277.15 (4.5%), 277.16 (1.7%), 276.15
(1.2%); elemental analysis: C 56.69, H 9.15, N 5.09, O 17.43,
To a stirred solution of d,l-N-Boc-amino acid (12 mmol) in
dichloromethane (90 mL), in an ice bath, DMAP
(0.1 equiv.) and ethanethiol were added. Then, a solution of
DCC (1.1 equiv.) in dichloromethane (10 mL) was added
dropwise at room temperature. After a short incubation
period, a crystalline white precipitate began to form. After
30 min, the ice bath was removed and the reaction was left
to go to completion after a further overnight stirring period
at room temperature. The reaction mixture was filtered in
order to eliminate the white solid (N,N’-dicyclohexylurea)
and the filtrate was evaporated under reduced pressure. The
residue was then purified by column chromatography on
silica gel using hexane/ethyl acetate (95:5) as eluent to give
the desired amino acid thioester in high yields (from 80 to
90%). Starting from the N-Boc-amino acid of l-configura-
tion, enantiomerically pure thiol esters were obtained.
1
S, 11.64; H NMR: d=0.901 (t, J=7.3 Hz, 3H), 1.250 (t, J=
7.312 Hz, 3H), 1.289–1.416 (br s, 4H), 1.459 (s, 9H),
1.599(br s, 1.5H), 2.2 (br s, 0.5H), 2.88 (q, J=7.63 Hz, 2H),
4.316 (br s, 1H), 4.902 (br s,1H); 13C NMR: d=201.34,
155.10, 79.70, 60.40, 32.32, 28.14, 27.27, 22.89, ?22.10, 14.30,
13.56; HPLC analysis (hexane/2-propanol=99/1, flow=
1.2 mLminÀ1, T=308 K): peaks d: 6 min; l: 7.4 min.
l-N-Boc-norLeu-SEt (l-3a): White solid; mp 49–508C;
[a]D: À20.3 (c 1.0, 2-propanol, 208C).
General Procedure for the Determination of
Racemization Kinetics of l-N-Boc-amino Acid
Thioesters
d,l-N-Boc-Phe-SEt (d,l-1a): White solid; mp 79–808C;
chemical formula: C16H23NO3S exact mass: 309.14; molecu-
lar weight: 309.42; MS: m/z=309.14 (100.0%), 310.14
(18.6%), 311,14 (5.3%), 311,15 (1.5%); elemental analysis:
C 62.11, H 7.49, N 4.53, O 15.51, S 10.36; 1H NMR: d=
1.236 (t, J=7.465 Hz, 3H), 1.4 (s, 9H), 2.874 (q, J=
Approximately 20 mg of substrate were dissolved in 2 mL of
t-BuOH in a polarimetric tube and the rotation value was
recorded. 0.5 equiv. of diazabicycloundecene (DBU) were
then added and the decrease in optical rotation value was
recorded during 30 min at 408C.
7.383 Hz, 2H), 3.04 (br s, 1H), 3.06–3.178ACTHNUGRTENUNG(m, 1H), 4.62 (br
s, 1H), 4.87 (br s, 1H), 7.135–7.19 (m, 2H), 7.205–7.31 (m,
3H); 13C NMR: d=200.799, 154.854, 135.825, 129.243,
128.344, 126.870, 80.010, 60.944, 38.355, 28.163, 23.156,
Control experiments proved that no change in the abso-
lute rotation values in time was observed when the substrate
was the N-Boc-amino acid or with the thioesters in the ab-
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l-Amino Acid Amides via Dynamic Kinetic Resolution
sence of a base: krac =l-1a 4.63ꢃ10À1 secÀ1; l-2a 1.15ꢃ
10À1 secÀ1; l-3a 6.7ꢃ10À2 secÀ1.
l-N-Boc-homophenylalanine Propylamide (2c): Com-
pound 2c was obtained as a solid (reaction time 24 h) in
62.5% yield (61.9 mg): HPLC analysis (Chiralcel OD,
hexane/2-propanol 98:2, flow rate=1.00 mLminÀ1, l=
210 nm): tR =15.13 min; enantiomeric excess 99.04%; mp
978C (hexane/i-Pr2O); [a]20: À7.7 (c 1,MeOH); 1H NMR
(400 MHz, CDCl3) d=0.74D(t, 3H), 1.44 (s, 9H), 1.50 (dq,
2H), 1.92–2.12 (ddt, 2H), 2.67 (t, 2H), 3.19 (m, 2H), 4.12 (s,
1H), 5.36 (s, 1H), 6.50 (s, 1H), 7.15–7.28 (m, 5H); 13C NMR
(400 MHz, CDCl3) d=11.3, 22.7, 28.3, 31.9, 34.2, 41.1, 54.2,
79.9, 126.0, 128.3, 128.4, 141.0, 155.8, 172.0; m/z=391.2,
calcd. for C22H28N2O3 [M + Na]+: 391.2.
l-N-Boc-homophenylalanine Benzylamide (2d): Com-
pound 2d was obtained as a solid (reaction time 24 h) in
87.1% yield (99.3 mg): HPLC analysis (Chiralcel OD,
hexane/2-propanol 97:3, flow rate=1.00 mLminÀ1, l=
210 nm): tR =20.93 min; enantiomeric excess 99.16%; mp
General Procedure for the Enzymatic Syntesis of N-
Boc-amino Acid Amides
N-Boc-amino acid thioester l-3a (100 mg) was added to a
mixture containing t-BuOH (3.5–14 mL), Alcalaseꢂ-CLEA
(100–400 mg), amine (1.2–3 equiv.) and DBU (2–4 equiv.).
The mixture was shaken at 378C for 1–30 h. The reaction
mixture was centrifuged for 10 min: the liquid phase was
separated from the Alcalaseꢂ-CLEA. The solid was washed
with EtOH (2ꢃ3.5 mL) and separated by centrifugation.
The organic phases were pooled and dried under vacuum.
The residue was dissolved in MTBE and washed with HCl
0.1M (3ꢃ5 mL). The organic phase was dried (Na2SO4) and
evaporated under vacuum. The product obtained was dis-
solved and crystallized in suitable solvents.
888C (hexane/AcOEt); [a]2D0: À14.1
(c 1,MeOH); 1H NMR
ACHTUNGTRENNUNG
(400 MHz, CDCl3): d=1.40 (s, 9H), 1.92–2.17 (ddt, 2H),
2.76 (t, 2H), 4.12 (s, 1H), 4.41 (ddd, 2H), 5.12 (s, 1H), 6.55
(s, 1H) , 7.12–7.34 (m, 9H); 13C NMR (400 MHz, CDCl3):
d=28.3, 31.9, 33.9, 43.4, 54.2, 80.2, 126.1, 127.5, 127.6, 128.4,
128.5, 128.7, 138.0, 140.8, 155.7, 171.8; MS: m/z=391.2,
calcd. for C22H28N2O3 [M+Na]+: 391.2.
l-N-Boc-phenylalanine Amide (1b): Compound 1b was
obtained as a solid (reaction time 7 h) in 82% yield (70 mg):
HPLC analysis (Chiralcel OD, hexane/2-propanol 95:5, flow
rate=1.00 mLminÀ1, l=210 nm): tR =13.83 min; enantio-
meric excess 98.3%; mp 1478C (AcOEt/hexane); [a]2D0: 10.74
1
(c 1, MeOH); H NMR (400 MHz, DMSO-d6): d=1.30 (s,
l-N-Boc-norleucine Propylamide (3c): Compound 3c was
obtained as a solid (reaction time 30 h) in 65% yield
(93 mg); mp 74–758C; HPLC analysis (Chiralcel OD,
hexane/2-propanol 99:1, flow rate=1.00 mLminÀ1, l=
210 nm): tR =15.71 min; enantiomeric excess 98.36%; [a]2D0:
9H), 2.74 (dd, 1H), 2.97 (dd, 1H), 3.98 (s, 1H), 4.10 (s, 1H),
6.68 (d, 1H), 6.95 (d, 1H), 7.18 (m, 1H), 7.22–7.32 (m, 5H);
13C NMR(400 MHz, DMSO-d6): d=33.8, 59.9, 60.4, 83.1,
130.4, 132.3, 133.5, 143.4, 160.3, 178.8; MS: m/z=287.0,
calcd. for C14H21N3O2 [M+Na]+: 287.1.
1
À14.5 (c 1, MeOH); H NMR (400 MHz, CDCl3): d=0.82
l-N-Boc-phenylalanine Propylamide (1c): Compound 1c
was obtained as a solid (reaction time 7 h) in 76% yield
(75.7 mg): HPLC analysis (Chiralcel OD, hexane/2-propanol
98:2, flow rate=1.00 mLminÀ1, l=254 nm): tR =15.51 min;
enantiomeric excess >99%; mp 1138C (CH2Cl2/i-Pr2O);
(m, 6H), 1.26 (m, 4H), 1.37 (m, 9H), 1.46 (m, 2H), 1.53 (m,
1H), 1.75 (m, 1H), 3.14 (m, 2H), 3.94 (m, 1H), 5.02 (s, 1H),
6.24 (s, 1H); 13C NMR (400 MHz, CDCl3): d=11.2, 13.7,
22.3, 22.6, 27.7, 28.2, 32.4, 41.0, 54.6, 79.7, 155.7, 172.3; MS:
m/z=273.0, calcd for C15H30N2O3 [M+H]+: 273.2.
1
[a]2D0: 2.89 (c 1.5, MeOH); H NMR (400 MHz, CDCl3): d=
l-N-Boc-norleucine Benzylamide (3d): Compound 3d was
obtained as a solid (reaction time 20 h) in 69% yield
(100 mg); HPLC analysis (Chiralcel OD, hexane/2-propanol
98:2, flow rate=1.00 mLminÀ1, l=210 nm): tR =14.59 min;
enantiomeric excess 96.06%; mp 708C (AcOEt/hexane);
0.80 (t, 3H), 1.40 (m, 11H), 3.03 (m, 2H), 3.12 (m, 2H),
4.28 (dd, 1H), 5.12 (s, 1H), 5.80 (s, 1H), 7.18–7.30 (m, 5H);
13C NMR(400 MHz, CDCl3): d=11.1, 22.5, 28.2, 38.7, 41.1,
56.1, 80.1, 126.8, 128.6, 129.2, 136.8, 155.4, 171.1; MS: m/z=
307.0, calcd. for C17H26N2O3 [M+H]+: 307.2.
1
[a]2D0: À19.5 (c 1, MeOH); H NMR (400 MHz, CDCl3): d=
N-Boc-l-phenylalanine Benzylamide (1d): Compound 1d
was obtained as a solid (reaction time 5 h) in 89% yield
(102 mg): HPLC analysis (Chiralcel OD, hexane/2-propanol
98:2, flow rate=1.00 mLminÀ1, l=254 nm): tR =24.00 min;
enantiomeric excess 98.3%; mp 1288C (AcOEt/i-Pr2O);
0.88 (t, 3H), 1.32 (m, 4H), 1.41 (s, 9H), 1.86 (m, 2H), 4.06
(d, 1H), 4.43 (ddd, 2H), 4.99 (s, 1H), 6.51 (s, 1H), 7.21–7.34
(m, 5H); 13C NMR (400 MHz, CDCl3): d=13.8, 22.3, 27.7,
28.2, 32.3, 43.2, 54.7, 79.8, 127.2, 127.5, 128.5, 138.2, 155.7,
172.3; MS: m/z=321.1, calcd. for C18H28N2O3 [M + H]+:
321.2.
1
[a]2D0: À2.7 (c 1, MeOH); H NMR (400 MHz, CDCl3): d=
1.40 (s, 9H), 3.08 (ddd, 2H), 4.34 (m, 3H), 5.02 (s, 1H), 6.06
(s, 1H); 7.08–7.30 (m, 10H); 13C NMR (400 MHz, CDCl3):
d=28.1, 38.7, 43.3, 50.0, 80.2, 126.7, 127.2, 127.5, 128.4,
129.2, 136.6, 137.6, 155.6, 171.6; MS: m/z=355.1, calcd. for
C12H26N2O3 [M+H]+: 355.2.
Acknowledgements
l-N-Boc-homophenylalanine Amide (2b): Compound 2b
was obtained as a solid (reaction time 30 h) in 59.9% yield
(49.1 mg): HPLC analysis (Chiralcel OD, hexane/2-propanol
93:7, flow rate=1.00 mLminÀ1, l=210 nm): tR =19.96 min;
enantiomeric excess 98.94%; mp 135–1368C, (AcOEt/i-
Pr2O); [a]2D0: +4.8 (c 0.5, CHCl3); 1H NMR (400 MHz,
Support from COST Action CM0701-CASCAT (Cascade
Chemo-Enzymatic Processes: New Synergies between
Chemistry and Biochemistry) is gratefully aknowledged.
CDCl3): d=1.44 (s, 9H), 1.94, 2.16 (ddt, 2H), 2.70 (t, 2H), References
4.12 (s, 1H), 5.16 (s, 1H), 5.72 (s, 1H), 6.22 (s, 1H), 7.15–
7.30 (m, 5H); 13C NMR(400 MHz, CDCl3): d=28.3, 31.8,
33.9, 53.8, 80.3, 126.2, 128.3, 128.8, 140.8, 155.7, 174.5; MS:
m/z=301.2, calcd. for C15H22N2O3 [M+Na]+: 301.2.
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2338
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Adv. Synth. Catal. 2011, 353, 2333 – 2338