Toward stereocontrolled, chemoenzymatic synthesis of unnatural peptides

Article abstract of DOI:10.1016/j.tet.2008.01.103

An efficient, chemoenzymatic method for the multicomponent synthesis of unnatural tripeptides is presented. Development of a previously described procedure combines the diversity offered by multicomponent reactions with the selectivity of biocatalysts and allows the convenient introduction of varied amino acid moieties into the tripeptide scaffold, with control of the stereochemistry. Additionally, it allows the introduction of a methyl group to the amide nitrogen, leading to derivatives of N-methylated amino acids.

Full text of DOI:10.1016/j.tet.2008.01.103

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3197e3203
www.elsevier.com/locate/tet
Toward stereocontrolled, chemoenzymatic synthesis of unnatural peptides
Wiktor Szymanski a, Ryszard Ostaszewski b,
*
a Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
b Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
Received 5 October 2007; received in revised form 4 January 2008; accepted 24 January 2008
Available online 31 January 2008
Abstract
An efficient, chemoenzymatic method for the multicomponent synthesis of unnatural tripeptides is presented. Development of a previously
described procedure combines the diversity offered by multicomponent reactions with the selectivity of biocatalysts and allows the convenient
introduction of varied amino acid moieties into the tripeptide scaffold, with control of the stereochemistry. Additionally, it allows the introduc-
tion of a methyl group to the amide nitrogen, leading to derivatives of N-methylated amino acids.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Tripeptides; Passerini reaction; Enzymatic hydrolysis; Non-coded aminoacids; N-Methylated peptides
1. Introduction
were obtained in small yields and with unsatisfactory enantio-
meric excesses. The main reasons were that both the enzymatic
step and the introduction of azide moiety(precursor of the amino
group) proceeded with poor chemoselectivity.
Small peptides and their simple analogues begin to form
a group of ubiquitous compounds in medicinal chemistry.
Among them, compounds with various activities can be found,
such as anti-parasitic,1 anti-tumor2 and anti-viral3 agents, HIV-
protease inhibitors,4 calpain,5 thrombin6 and proteasome7 in-
hibitors. Modifications of N- and C-terminae were shown to
significantly increase the activity,8 while the introduction of
non-coded amino acids9 and N-alkylation of amide groups10
have been shown to improve the pharmacokinetic factors of
these compounds.
In our previous paper11 we have presented a method for the
synthesis of small peptides, in which hydrolytic enzymes were
employed for the enantioselective hydrolysis of compounds ob-
tained in the Passerini multicomponent reaction. Further trans-
formations allowed the synthesis of title compounds (Scheme
1). This method is advantageous as it combines the diversity of-
fered by multicomponent reactions with the selectivity of bioca-
talysts. The synthesis of tripeptides (R1¼ROOCCH(R)) was,
however, not fully accomplished in the cited paper, as the title
compounds, bearing an ethyl ester moiety (R1¼EtOOCCH2),
O
R3 R4
N
R1 OH
HN
R1
R2
N
H
NHPG
NHPG
1 R1
R2
O
R1 OAc
HN
O
+
6
5
NC
O
ref. 12
R1
R2
O
R3 R4
N
R2
2
R1
HN
OAc
O
rac-4
3
N
H
AcOH
R2
R2
ent-6
O
O
4
Scheme 1. Basic synthetic concept.
Here we would like to describe our efforts to overcome
these drawbacks, by introducing different protection for the
C-terminal carboxylic group.
2. Results and discussion
2.1. Synthesis of non-racemic a-hydroxyamides 5
In the first step of our studies, racemic esters of acetic acid
were prepared (Scheme 2, 4a and 4c), using the Passerini
* Corresponding author. Tel.: þ48 22 343 2120.
E-mail address: rysza@icho.edu.pl (R. Ostaszewski).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.103

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W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
multicomponent reaction, in good yields from tert-butyl iso-
cyanoacetate, acetic acid, and respective aldehyde. A group
of lipases was tested as catalysts for the stereoselective hydro-
lysis of these compounds. The group consisted of: Wheat
Germ Lipase (WGL), Novozym 435, Porcine Pancrease
Lipase (PPL), Candida rugosa Lipase (CRL), Amano AK
Lipase, Rhizopus niveus Lipase, Candida lipolytica Lipase,
Pseudomonas cepacia Lipase, and Pig Liver esterase.
(Table 1, entries 7e12), the change of the substrate proved
to be successful, as the best result was obtained in the hydro-
lysis of chloroacetic ester 4d, catalyzed by PPL. An interesting
result was also obtained in terms of the enzyme’s enantiomeric
preference. In the reaction catalyzed by WGL, different enan-
tiomers of 5b were obtained when the chloroacetic acid ester
was used instead of the acetic one (Table 1, entries 7 and 10,
respectively).
In order to assign the configuration of the obtained prod-
ucts, compounds (S)-4 and (S)-5 were synthesized from com-
mercially available, optically pure a-amino acids, by
conversion into a-hydroxy acids (NaNO2/H2SO4)13 and EDC
mediated coupling14 of thus obtained compounds with glycine
tert-butyl ester.
O
O
O
1
2
3
NC
tBuO
tBuO
O
+
+
HN
R
R2
R2
O
O
4a, R = H, R2 = CH2Ph:
4b, R = Cl, R2 = CH2Ph:
89%
92%
O
4c, R = H, R2 = CH2CH(CH3)2: 90%
4d, R = Cl, R2 = CH2CH(CH3)2: 81 %
2.2. Synthesis of primary and secondary amines 9
R
OH
The next step was to functionalize the hydroxyl group to-
ward amine derivatives. The transformation into NH2 group
was by SN2 type substitution of methanesulfonic acid esters
derived from alcohols 5 (Scheme 3). Enantiopure compounds
obtained from the correlation study were used in these synthe-
ses. Compounds (R)-9 were obtained from alcohols (S)-5 in
good overall yields (80% both for (R)-9a and b).
Scheme 2. Synthesis of racemic a-acetoxyamides 4.
Out of the examined enzymes, only WGL catalyzed the hy-
drolysis, and the stereochemical results were moderate in the
case of 4a and unsatisfactory in the case of 4c (Table 1, entries
1 and 7, respectively). Therefore a new set of racemic esters
(Scheme 2, 4b and 4d) was synthesized, using chloroacetic
acid in the Passerini reaction, to introduce a better leaving
group to the ester. The same group of enzymes as above was
tested and this time two additional enzymes PPL and CRL
were found to catalyze the hydrolysis (Table 1, entries 4e6
and 10e12).
O
O
a
tBuO
OMs
R2
b
tBuO
N3
(S)-5a,b
HN
HN
R2
(S)-7a: 98%
(R)-8a: 82%
O
O
b: 94%
b: 88%
a: R = CH2Ph
In the synthesis of enantiomerically enriched alcohol 5a
(Table 1, entries 1e6), the broadening of the enzyme spectrum
did not result in the improvement of the enantioselectivity. On
the contrary, in the stereocontrolled synthesis of alcohol 5b
b: R = CH2CH(CH3)2
O
O
O
R
R4
NHPG
d
c
tBuO
NH2
HN
tBuO
HN
HN
R2
(R)-9a: 99%
b: 99%
O
6a-f
O
Table 1
Chemoenzymatic synthesis of non-racemic compounds 5
Scheme 3. Synthesis of tripeptides 6. (a) MsCl, Et3N, DMAP, CH2Cl2, rt,
30 min; (b) NaN3, DABCO, DMAP, benzo-15-crown-5, CH2Cl2, 40 ꢀC,
24 h; (c) H2, Pd/C, methanol, 4 h; (d) Protected amino acid, EDC, HOBt,
CH2Cl2, rt, 4 h (see Table 3).
No Substrate Enzyme Time (h) Product Yield (%) eea (%) Eb
1
4a
WGL
30
(S)-5a
(R)-4a
45
54
87
64
28
2
3
4
4a
4a
4b
PPL
CRL
WGL
48
48
26
Conversion<5%
Conversion<5%
d
d
1
The synthesis (Scheme 4) of methylamine derivatives 10a
and b, precursors of N-methylated peptides, proved to be
more problematic. The method used in our previous study,
which consisted in the heating of the mesylate in the aqueous
methylamine/DMF proved unsuccessful (Table 2, entry 1),
probably due to the possible hydrolysis or aminolysis of ester
moiety.
(S)-5a
(R)-4b
(S)-5a
(R)-4b
(S)-5a
(R)-4b
(S)-5b
(R)-4c
60
16
44
50
36
50
58
38
<5
40
10
16
47
35
37
64
5
6
7
4b
4b
4c
PPL
120
26
1
4
7
CRL
WGL
41
8
9
4c
4c
PPL
CRL
WGL
48
48
20
Conversion<5%
Conversion<5%
d
d
2
O
O
O
O
R4
10 4d
11 4d
12 4d
a
(R)-5b
(S)-4d
(S)-5b
(R)-4d
(S)-5b
(R)-4d
81
16
64
32
40
32
<5
tBuO
OMs
R2
b
a
tBuO
HN
tBuO
N
63
40
85
50
69
HN
HN
HN
NHPG
R2
R2
PPL
119
45
18
9
O
O
O
(S)-7a: R = CH2Ph
(S)-7b: R = CH2CH(CH3)2
(R)-10a: 41%
b: 36%
6g-h
CRL
Determined by chiral HPLC.
Scheme 4. Synthesis of N-methylated tripeptides 6. (a) 5% MeNH2 in metha-
nol, 50 ꢀC, 24 h; (b) EDC, HOBt, CH2Cl2, rt, 4 h.
b
Calculated from the conversion-independent equation.

W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
3199
Table 2
Synthesis of 10adan optimization study
peptides. Previously reported difficulties were solved by the use
of tert-butyl ester as the protection of the C-terminus. The enzy-
matic, enantioselective hydrolysis of racemic intermediate was
optimized, where needed, by the introduction of more easily hy-
drolyzed chloroacetoxy group. Best results were obtained with
Wheat Germ Lipase and Porcine Pancrease Lipase.
For the ease of configuration determination, a set of model
peptides composed of natural amino acids was prepared to
demonstrate the synthetic potential of the method. Considering
the enormous number of commercially available aldehydes
(precursors of amino acid side chain in this method) and the
very extensive set of synthetic methods for their preparation,
we find our method to be widely applicable.
Small peptides with orthogonal protections on the ends, ob-
tained in this method, are ready for the incorporation into
larger biopolymers, using conventional methods. Compounds
with compatible protections can be easily deprotected in one
step, to yield non-natural tripeptides, compounds of great
biological relevance.
No
Reaction conditions
Yield (%)
1
2
3
4
5
6
40% aq MeNH2, DMF, 50 ꢀC, 24 h
22
7
40% aq MeNH2, DMF, cat. DMAP, 50 ꢀC, 44 h
MeNH2 hydrochloride, 2 equiv DMAP, 50 ꢀC, 96 h
33% MeNH2 in ethanol, 50 ꢀC, 24 h
29
20
41
16
5% MeNH2 in methanol, 50 ꢀC, 24 h
5% MeNH2 in methanol, 50 ꢀC, 96 h
The addition of catalytic amount of DMAP and the elonga-
tion of reaction time (Table 2, entry 2) did not result in the im-
provement of reaction outcome. To eliminate the water from
the reaction media, methylamine hydrochloride was used
with DMAP as base and DMF as solvent (Table 2, entry 3).
Despite the longer reaction time, the yield improved only
slightly. We turned therefore to alcoholic solutions of methyl-
amine. Ethanolic solution with high concentration was used
and the yield was only 20% (Table 2, entry 4). The 5% meth-
anolic solution on the other hand gave the best result (Table 2,
entry 5), the yield was 41% and the formation of side products
was minimized, which simplified the purification. The elonga-
tion of reaction time (Table 2, entry 6) did not result in the im-
provement of yield, probably causing a severe decomposition
of the ester moiety. Using the optimized reaction conditions,
the methylamine derivative (R)-10b was synthesized with
36% yield (Scheme 4).
4. Experimental
4.1. General
Optical rotations were measured with a JASCO DIP-360
polarimeter. NMR spectra were measured with a Varian 200
GEMINI and Varian 400 GEMINI spectrometer, with TMS
as internal standard. TLCs were performed with silica gel 60
(230e400 mesh, Merck) and silica gel 60 PF254 (Merck).
HPLC experiments were carried in three variants: A: DAICEL
CHIRACEL OD-H column with a pre-column, eluent: hexane/
i-PrOH 95:5 (v/v), flow: 1 mL/min; B: DAICEL CHIRACEL
OD-H column with a pre-column, eluent: hexane/i-PrOH 9:1
(v/v), flow: 1 mL/min; C; CHIRACEL OJ-H column with a
pre-column, eluent: hexane/i-PrOH 9:1 (v/v), flow: 1 mL/
min; CHN analyses were performed on PerkineElmer 240
Elemental Analyzer. MS spectra were recorded on an API 365
(SCIEX) apparatus.
2.3. Synthesis of title peptides 6
Target tripeptide analogues were prepared by EDC medi-
ated coupling of amines 9 with model amino acids (glycine
for 6a, b, d, e, g, h, and (S)-alanine for 6c and f), bearing a benz-
yloxycarbonyl (CBz) or tert-butoxycarbonyl (Boc) protecting
groups on the nitrogen atom (Schemes 3 and 4). Yields of
compounds 6 are shown in Table 3.
All of the target compounds were obtained in analytically
pure form with very high yields (>95%) in case of the coup-
ling of primary amines (6aef). The N-methylated peptides
(6g and h) were obtained in lower yields, probably due to
steric hindrance.
4.2. General procedure for the synthesis of Passerini reaction
products rac-4
Table 3
Synthesis of compounds 6
To a 1 M solution of acetic acid (1 equiv) in CH2Cl2 was
added aldehyde (1.1 equiv) and then isocyanide (1.1 equiv)
at room temperature. After 24 h, the solvent was evaporated
and the product was purified by flash chromatography (silica
gel, hexane/ethyl acetate, 4:1 v/v).
6
R2
R3
R4
PG
Yield (%)
a
b
c
d
e
f
CH2Ph
CH2Ph
CH2Ph
H
H
Boc
CBz
Boc
Boc
CBz
Boc
CBz
CBz
90
97
99
95
99
99
58
47
H
H
H
CH3
H
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2Ph
H
4.2.1. tert-Butyl rac-(2-acetoxy-3-phenyl-propionylamino)-
acetate (rac-4a)
H
H
H
CH3
H
g
h
CH3
CH3
Yield: 89% of white crystals; Rf¼0.40 (ethyl acetate/hexane
7:3, v/v); mp 99e101 ꢀC (ethyl acetate/hexane). 1H NMR
(400 MHz, CDCl3): d 1.45 (s, 9H, (CH3)3), 2.06 (s, 3H,
CH3CO), 3.09 (dd, 1H, J¼14.4, 7.6 Hz, PhCH2), 3.23 (dd,
1H, J¼14.4, 4.4 Hz, PhCH2), 3.81e3.95 (m, 2H, CH2NH),
5.40 (dd, 1H, J¼7.6, 4.4 Hz, CHOAc), 6.47 (br s, 1H, NH),
7.15e7.28 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3):
CH2CH(CH3)2
H
3. Summary
In this paper we present the development of our chemoenzy-
matic, multicomponent methodology for the synthesis of small

3200
W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
d 20.8, 28.0, 37.7, 41.6, 74.1, 82.5, 126.9, 128.3, 129.4, 135.9,
168.4, 169.1, 169.3. HRMS (ESI, [MþNa]þ) 344.1453
(C17H23NO5Na: 344.1468). Retention time of enantiomers:
tS¼7.93, tR¼9.93 (variant B).
4.3.1. tert-Butyl (R)-(2-acetoxy-3-phenyl-propionylamino)-
acetate ((R)-4a)
[a]2D0 þ32.0 (c 0.76, CHCl3, for the enantiopure compound
obtained in the correlation study). Other analyses consistent
with rac-4a.
4.2.2. tert-Butyl rac-(2-(chloroacetoxy)-3-phenyl-
propionylamino)acetate (rac-4b)
4.3.2. tert-Butyl (R)-(2-(chloroacetoxy)-3-phenyl-
propionylamino)acetate ((R)-4b)
Yield: 92% of yellow oil; Rf¼0.20 (ethyl acetate/hexane
8:2, v/v). 1H NMR (200 MHz, CDCl3): d 1.46 (s, 9H,
(CH3)3), 3.14 (dd, 1H, J¼14.4, 7.8 Hz, PhCH2), 3.30 (dd,
1H, J¼14.4, 4.6 Hz, PhCH2), 3.81e3.95 (m, 2H, CH2NH),
4.01e4.07 (m, 2H, CH2Cl), 5.48 (dd, 1H, J¼7.8, 4.6 Hz,
CHOC(O)), 6.51 (br s, 1H, NH), 7.16e7.30 (m, 5H, ArH).
13C NMR (50 MHz, CDCl3): d 28.4, 38.1, 40.8, 42.2, 76.0,
83.1, 127.5, 128.9, 129.8, 135.6, 166.1, 168.6. HRMS (ESI,
[MþNa]þ) 378.1088 (C17H22NO5NaCl: 378.1079). Retention
time of enantiomers: tS¼8.18, tR¼10.90 (variant B).
[a]2D0 þ7.4 (c 1.0, CHCl3, for the compound obtained in
the enzymatic step, 35% ee). Other analyses consistent with
rac-4b.
4.3.3. tert-Butyl (R)-{[2-(acetoxy)-4-methylopentanoyl]-
amino}acetate ((R)-4c)
[a]2D0 þ22.2 (c 1.0, CHCl3, for the enantiopure compound
obtained in the correlation study). Other analyses consistent
with rac-4c.
4.2.3. tert-Butyl rac-{[2-(acetoxy)-4-methylopentanoyl]-
amino}acetate (rac-4c)
4.3.4. tert-Butyl rac-{[2-(chloroacetoxy)-4-methylo-
pentanoyl]amino}acetate ((R)-4d)
Yield: 90% of white crystals; Rf¼0.44 (ethyl acetate/hexane
7:3, v/v); mp 78e80 ꢀC (ethyl acetate/hexane). Anal.
C14H25NO5 requires: C, 58.52%; H, 8.77%; N, 4.87%. Found:
C, 58.57%; H, 8.79%; N, 4.76%. 1H NMR (400 MHz, CDCl3):
d 0.92 (d, J¼6.0 Hz, 3H, CH3CH), 0.93 (d, J¼6.0 Hz, 3H,
CH3CH), 1.47 (s, 9H, (CH3)3C), 1.67e1.76 (m, 3H, CHCH2),
2.16 (s, 3H, CH3CO), 3.91e3.93 (m, 2H, CH2NH), 5.21e
5.25 (m, 1H, CHOAc), 6.50 (br s, 1H, NH). 13C NMR
(100 MHz, CDCl3): d 20.9, 21.7, 23.0, 24.5, 28.0, 40.8, 41.7,
72.6, 82.5, 168.7, 169.8, 170.3. Retention time of enantiomers:
tR¼6.91, tS¼7.45 (variant A).
[a]2D0 þ8.6 (c 0.43, CHCl3, for the compound obtained in
the enzymatic step, 85% ee). Other analyses consistent with
rac-4d.
4.3.5. tert-Butyl (S)-(2-(hydroxy)-3-phenyl-propionylamino)-
acetate ((S)-5a)
White crystals; Rf¼0.61 (ethyl acetate/hexane 4:6, v/v); mp
95e96 ꢀC (ethyl acetate/hexane); [a]D20 ꢁ69.4 (c 1.0, CHCl3,
for the enantiopure compound obtained in the correlation
study). Anal. C15H21NO4 requires: C, 64.50%; H, 7.58%; N,
5.01%. Found: C, 64.44%; H, 7.59%; N, 4.95. 1H NMR
(400 MHz, CDCl3): d 1.47 (s, 9H, (CH3)3), 2.62 (br s, 1H,
OH), 2.85 (dd, 1H, J¼14.1, 9.2 Hz, PhCH2), 3.25 (dd, 1H,
J¼14.1, 3.6 Hz, PhCH2), 3.81e4.01 (m, 2H, CH2NH), 4.32
(dd, 1H, J¼9.2, 3.6 Hz, CHOH), 7.03 (br s, 1H, NH), 7.24e
7.34 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3): d 11.4,
28.0, 40.8, 41.5, 72.9, 82.4, 126.9, 128.7, 129.5, 136.9,
168.8, 172.8. Retention time of enantiomers: tR¼14.05, tS¼
15.25 (variant A).
4.2.4. tert-Butyl rac-{[2-(chloroacetoxy)-4-methylo-
pentanoyl]-amino}acetate (rac-4d)
Yield: 81% of white crystals; Rf¼0.50 (ethyl acetate/hexane
7:3, v/v); mp 79e80 ꢀC (ethyl acetate/hexane). Anal.
C14H24ClNO5 requires: C, 52.25%; H, 7.52%; N, 4.35%. Found:
C, 52.09%; H, 7.66%; N, 4.26%. 1H NMR (200 MHz, CDCl3):
d 0.92 (d, J¼6.0 Hz, 6H, (CH3)2CH), 1.47 (s, 9H, (CH3)3C),
1.60e1.85 (m, 3H, CHCH2), 3.92e3.94 (m, 2H, CH2NH),
4.15e4.16 (m, 2H, ClCH2), 5.28e5.34 (m, 1H, CHOAc), 6.52
(br s, 1H, NH). 13C NMR (50 MHz, CDCl3): d 22.1, 23.4,
24.9, 28.4, 41.0, 41.1, 42.2, 74.6, 83.0, 169.7. Retention time
of enantiomers: tR¼8.16, tS¼9.14 (variant B).
4.3.6. tert-Butyl (S)-{[2-(hydroxy)-4-methylopentanoyl]-
amino}acetate ((S)-5b)
Yellow oil; Rf¼0.45 (ethyl acetate/hexane 5:5, v/v); [a]D20
ꢁ37.5 (c 1.0, CHCl3, for the enantiopure compound obtained
1
in the correlation study). H NMR (200 MHz, CDCl3): d 0.94
4.3. General procedure for the enzymatic hydrolysis of
Passerini reaction products
(d, J¼6.6 Hz, 6H, CH3CH), 1.47 (s, 9H, (CH3)3C), 1.52e1.86
(m, 3H, CHCH2), 3.12 (br s, 1H, OH), 3.80e4.06 (m, 2H,
CH2NH), 4.17 (dd, 1H, J¼9.2, 4.0 Hz, CHOH), 7.06 (br s,
1H, NH). 13C NMR (50 MHz, CDCl3): d 21.7, 23.8, 24.8,
28.4, 41.9, 44.0, 71.0, 82.8, 169.5, 175.2. HRMS (ESI,
[MþNa]þ) 268.1508 (C12H23NO4Na: 258.1519). Retention
time of enantiomers: tS¼5.22, tR¼6.19 (variant C).
Ester rac-4 (55 mg) was dissolved in 10 mL of solvent (wa-
ter/Et2O 8:2, v/v). Enzyme (9 mg) was added in one portion to
the suspension and stirred on a shaker at 300 rpm at room tem-
perature for the amount of time given in Table 1. Extraction
with ethyl acetate, concentration in vacuo, and purification
of the resulting residue by flash chromatography (silica gel,
hexane/ethyl acetate) afforded enaniomerically enriched ester
4 and alcohol 5. The yields and enantiomeric excesses are
given in Table 1.
4.4. Synthesis of compounds 6aef
The transformation of alcohols (S)-5a and (S)-5b into
amides 6aef is further exemplified by the synthesis of 6a.

W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
3201
4.4.1. Methanesulfonic acid (S)-1-benzyl-2-[(4-tert-
223.1080 (C11H15N2O3Na: 223.1077). IR (CHCl3) n: 3363,
3316, 2979, 2933, 1741, 1665, 1526, 1368, 1227, 1158,
butoxycarbonyl)methylamino]-2-oxoethyl ester ((S)-7a)
Solution of alcohol (S)-5a (186 mg, 0.67 mmol, from corre-
lation study12) in CH2Cl2 (6 mL) was cooled to ꢁ50 ꢀC. Tri-
ethylamine (280 mL, 2.00 mmol) was added in one portion and
then methanesulfonyl chloride (103 mL, 1.33 mmol, solution
in 1.0 mL of CH2Cl2) was added dropwise. The mixture was
stirred for 60 min at room temperature, the solvent was evap-
orated, and the product was purified by flash chromatography
(silica gel, hexane/ethyl acetate 85:15, v/v). Yield: 99%,
233 mg of white crystals; Rf¼0.81 (hexane/ethyl acetate 4:6,
v/v); mp 94e96 ꢀC (ethyl acetate/hexane); [a]2D0 ꢁ72.6 (c
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 1.47 (s, 9H,
(CH3)3), 2.47 (s, CH3SO2), 3.04 (dd, 1H, J¼14.4, 9.6 Hz,
PhCH2), 3.45 (dd, 1H, J¼14.4, 3.6 Hz, PhCH2), 3.88e4.02
(m, 2H, CH2NH), 5.14 (dd, 1H, J¼10.0, 3.6 Hz, CHOMs),
6.82 (br s, 1H, NH), 7.27e7.33 (m, 5H, ArH). 13C NMR
(100 MHz, CDCl3): d 28.0, 37.8, 38.6, 41.9, 81.4, 82.6,
127.5, 128.8, 129.7, 135.6, 167.8, 168.0. HRMS (ESI,
[MþNa]þ) 380.1156 (C16H23NO6NaS: 380.1138).
702 cmꢁ1
.
4.4.4. BocGly-(R)-Phe-GlyOtBu (6a)
To a solution of amine (R)-9a (50 mg, 0.18 mmol) in CH2Cl2
(8.0 mL) were added Boc-protected glycine (32 mg,
0.18 mmol) and 1-hydroxybenzotriazole (37 mg, 0.27 mmol).
The solution was cooled to 0 ꢀC and EDC (39 mg, 0.20 mmol)
was added in one portion. After 4 h the solvent was evaporated
and the residue was taken up in ethyl acetate (15 mL). This so-
lution was washed with 5% aqueous citric acid solution
(2ꢂ5 mL), saturated NaHCO3 solution (2ꢂ5 mL), and brine
(5 mL). The organic phase was dried (MgSO4) and evaporated
to yield the analytically pure compound. Yield: 90% of colorless
oil; Rf¼0.35 (hexane/ethyl acetate 4:6, v/v); [a]2D0 þ0.9 (c 1.0,
1
CHCl3). H NMR (400 MHz, CDCl3): d 1.43 (s, 9H, (CH3)3),
1.44 (s, 9H, (CH3)3), 3.05e3.15 (m, 2H, PhCH2), 3.72e3.80
(m, 2H, Gly-CH2), 3.78e3.96 (m, 2H, Gly-CH2), 4.72e4.78
(m, 1H, Phe-CH), 5.24 (br s, 1H, NHBoc), 6.67 (br s, 1H,
NH), 6.82 (d, 1H, J¼8.1 Hz, NH), 7.16e7.30 (m, 5H, ArH).
13C NMR (100 MHz, CDCl3): d 28.0, 28.2, 38.0, 41.9, 54.1,
82.2, 127.0, 128.6, 129.2, 129.7, 136.6, 168.4, 169.6, 170.7.
HRMS (ESI, [MþNa]þ) 458.2262 (C22H33N3O6Na:
458.2262). IR (CHCl3) n: 3312, 2979, 2934, 1741, 1715,
4.4.2. tert-Butyl (R)-(2-azido-3-phenyl-propionylamino)-
acetate ((R)-8a)
To a solution of methanesulfonic acid ester (S)-7a (207 mg,
0.58 mmol) in CH2Cl2 (10.0 mL) were added 1,4-diaza-bicy-
clo[2.2.2]octane (104 mg, 0.93 mmol), 4-dimethylaminopyri-
dine (10 mg, 0.08 mmol), sodium azide (76 mg, 1.16 mmol),
and benzo-15-crown-5 (10 mg, 0.04 mmol). The mixture was
stirred and refluxed. After 24 h the product was purified by flash
chromatography (silica gel, hexane/ethyl acetate 3:1, v/v).
Yield: 82%, 158 mg of colorless oil; Rf¼0.56 (hexane/ethyl ac-
etate 6:4, v/v); [a]2D0 ꢁ34.1 (c 1.0, CHCl3). 1H NMR (400 MHz,
CDCl3): d 1.47 (s, 9H, (CH3)3), 3.00 (dd, 1H, J¼14.0, 9.0 Hz,
PhCH2), 3.39 (dd, 1H, J¼14.0, 4.0 Hz, PhCH2), 3.84e4.01
(m, 2H, CH2NH), 4.22 (dd, 1H, J¼9.0, 4.0 Hz, NHCH2), 6.77
(br s, 1H, NH), 7.24e7.37 (m, 5H, ArH). 13C NMR
(100 MHz, CDCl3): d 28.0, 38.8, 41.9, 65.6, 82.6, 127.2,
128.7, 128.8, 129.4, 129.7, 136.2, 168.4, 168.7. HRMS (ESI,
[MþNa]þ) 327.1435 (C15H20N4O3Na: 327.1428). IR (CHCl3)
n: 3317, 2980, 2115, 1742, 1667, 1532, 1369, 1250, 1226,
1660, 1527, 1499, 1368, 1249, 1228, 1161, 753, 701 cmꢁ1
.
In an analogous manner, compounds 6beh were
synthesized.
4.4.5. CBzGly-(R)-Phe-GlyOtBu (6b)
Yield: 97% of colorless oil; Rf¼0.36 (hexane/ethyl acetate
1
4:6, v/v); [a]2D0 þ0.6 (c 1.0, CHCl3). H NMR (200 MHz,
CDCl3): d 1.43 (s, 9H, (CH3)3), 3.05e3.15 (m, 2H, PhCH2),
3.72e3.80 (m, 2H, Gly-CH2), 3.78e3.96 (m, 2H, Gly-CH2),
4.72e4.84 (m, 1H, BnCH), 5.09 (s, 2H, PhCH2O), 5.70 (br s,
1H, NHCBz), 6.78 (br s, 1H, NH), 7.04 (d, 1H, J¼8.2 Hz,
NH), 7.14e7.43 (m, 5H, ArH). 13C NMR (50 MHz, CDCl3):
d 28.3, 38.5, 42.3, 44.8, 54.6, 67.5, 82.7, 127.3, 128.4,
128.5, 128.8, 128.9, 129.6, 130.0, 136.6, 167.8, 168.8, 169.7,
171.3. HRMS (ESI, [MþNa]þ) 492.2084 (C25H31N3O6Na:
492.2105). IR (CHCl3) n: 3302, 1729, 1656, 1531, 1368,
1157, 701 cmꢁ1
.
1229, 1157, 753, 699 cmꢁ1
.
4.4.3. tert-Butyl (R)-(2-amino-3-phenyl-propionylamino)-
acetate ((R)-9a)
Azide (R)-8a (92 mg, 0.30 mmol) was dissolved in methanol
(8 mL). To this mixture 10% Pd/C (10 mg) was added and hy-
drogen was fluxed through the solution (from a rubber balloon
through a needle) for the period of 4 h. Then the reaction mixture
was filtered through a bed of Celite and the solvent was evapo-
rated. Yield 99%, 83 mg of colorless oil. [a]2D0 þ28.5 (c 1.0,
4.4.6. Boc-(S)-Ala-(R)-Phe-GlyOtBu (6c)
Yield: 99% of colorless oil; Rf¼0.44 (hexane/ethyl acetate
1
4:6, v/v); [a]2D0 ꢁ2.2 (c 1.0, CHCl3). H NMR (200 MHz,
CDCl3): d 1.27 (d, 3H, J¼7.2 Hz, CH3CH), 1.43 (s, 9H,
(CH3)3), 1.44 (s, 9H, (CH3)3), 3.05e3.15 (m, 2H, PhCH2),
3.76e4.20 (m, 4H, 2ꢂGly-CH2), 4.76e4.86 (m, 1H, Phe-
CH), 5.24 (br s, 1H, NHBoc), 6.90 (br s, 1H, NH), 6.95 (s,
1H, NH), 7.27e7.37 (m, 5H, ArH). 13C NMR (50 MHz,
CDCl3): d 28.4, 29.6, 31.3, 38.0, 42.3, 54.4, 82.5, 127.3,
128.9, 129.6, 136.8, 168.8, 171.3, 173.4. HRMS (ESI,
[MþNa]þ) 472.2426 (C23H35N3O6Na: 472.2418). IR (Nujol)
n: 3299, 1748, 1676, 1657, 1651, 1531, 1454, 1367, 1225,
1
CHCl3). H NMR (400 MHz, CDCl3): d 1.48 (s, 9H, (CH3)3),
1.82 (br s, 2H, NH2), 2.69 (dd, 1H, J¼13.7, 9.9 Hz, PhCH2),
3.32 (dd, 1H, J¼13.7, 3.8 Hz, PhCH2), 3.66 (dd, 1H, J¼9.8,
3.8 Hz, NHCH), 3.92e4.01 (m, 2H, CH2NH), 7.22e7.33 (m,
5H, ArH), 7.78 (br s, 1H, NH). 13C NMR (100 MHz, CDCl3):
d 28.0, 40.8, 41.7, 56.4, 82.2, 126.8, 128.7, 129.3, 129.7,
137.9, 169.1, 174.4. HRMS (positive ESI, [MꢁC(CH3)3þH]þ)
1162 cmꢁ1
.

3202
W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
4.4.7. BocGly-(R)-Leu-GlyOtBu (6d)
1-hydroxybenzotriazole (22 mg, 0.15 mmol). The solution
was cooled to 0 ꢀC and EDC (21 mg, 0.11 mmol) was added
in one portion. After 4 h the solvent was evaporated and the
residue was taken up in ethyl acetate (15 mL). This solution
was washed with 5% aqueous citric acid solution (2ꢂ5 mL),
saturated NaHCO3 solution (2ꢂ5 mL), and brine (5 mL).
The organic phase was dried (MgSO4) and evaporated to yield
the analytically pure compound. Yield: 58% of colorless oil;
Rf¼0.35 (hexane/ethyl acetate 4:6, v/v); [a]1D9 þ52.0 (c 1.0,
CHCl3). 1H NMR (200 MHz, CDCl3): d 1.43 (s, 9H,
(CH3)3), 2.89 (s, 3H, CH3N), 3.00 (dd, 1H, J¼14.4, 9.2 Hz,
PhCH2CH), 3.34 (dd, 1H, J¼14.6, 6.8 Hz, PhCH2CH),
3.67e3.92 (m, 2H, Gly-CH2), 3.91e4.07 (m, 2H, Gly-CH2),
5.10 (s, 2H, PhCH2O), 5.36 (dd, 1H, J¼9.2, 6.8 Hz,
PhCH2CH), 5.70 (br s, 1H, NHCBz), 6.57 (br s, 1H, NH),
7.18e7.44 (m, 5H, ArH). 13C NMR (50 MHz, CDCl3):
d 28.4, 34.0, 42.3, 58.3, 67.3, 82.6, 112.2, 116.4, 126.9,
127.1, 127.7, 128.4, 128.9, 129.0, 159.9, 168.8, 170.0.
HRMS (ESI, [MþNa]þ) 472.2437 (C23H35N3O6Na:
472.2418). IR (CHCl3) n: 3329, 2930, 1727, 1651, 1531,
Yield: 95% of colorless oil; Rf¼0.30 (hexane/ethyl acetate
5:5, v/v); [a]2D0 þ25.8 (c 1.0, CHCl3). H NMR (200 MHz,
1
CDCl3): d 0.89e0.93 (m, 6H, CH3CH), 1.42 (s, 9H, (CH3)3C),
1.44 (s, 9H, (CH3)3C), 1.52e1.78 (m, 3H, CHCH2), 3.78e
3.92 (m, 4H, 2ꢂGly-2H), 4.48e4.60 (m, 1H, Leu-H), 5.37 (br
s, 1H, NHBoc), 6.82e6.95 (m, 2H, 2NH). 13C NMR
(50 MHz, CDCl3): d 22.2, 23.3, 25.0, 28.4, 28.6, 31.3, 41.3,
42.3, 51.8, 82.5, 156.5, 169.0, 170.1, 172.4. HRMS (ESI,
[MþNa]þ) 424.2423 (C19H35N3O6Na: 424.2418). IR (Nujol)
n: 3251, 1750, 1661, 1528, 1226, 1168 cmꢁ1
4.4.8. CBzGly-(R)-Leu-GlyOtBu (6e)
.
Yield: 99% of colorless oil; Rf¼0.32 (hexane/ethyl acetate
1
5:5, v/v); [a]2D0 þ21.0 (c 1.0, CHCl3). H NMR (200 MHz,
CDCl3): d 0.89e0.93 (m, 6H, CH3CH), 1.44 (s, 9H,
(CH3)3C), 1.52e1.78 (m, 3H, CHCH2), 3.78e4.00 (m, 4H,
2ꢂGly-2H), 4.48e4.61 (m, 1H, Leu-H), 5.10 (s, 2H,
PhCH2), 5.73 (br s, 1H, NHBoc), 6.82e6.88 (m, 2H, 2NH),
7.30e7.40 (m, 5H, ArH). 13C NMR (50 MHz, CDCl3):
d 22.3, 23.2, 25.1, 28.4, 41.4, 42.3, 51.9, 67.5, 82.7, 128.4,
128.5, 128.9, 136.4, 168.0, 169.6, 172.3. HRMS (ESI,
[MþNa]þ) 458.2259 (C22H33N3O6Na: 458.2262). IR
(CHCl3) n: 3300, 2958, 1729, 1652, 1537, 1368, 1236, 1156,
1455, 1368, 1250, 1157 cmꢁ1
.
4.5.2. CBzGly-(N-Me)-(R)-Leu-GlyOtBu (6h)
Yield: 47% of yellow oil; Rf¼0.38 (hexane/ethyl acetate 5:5,
v/v); [a]1D9 þ83.9 (c 0.57, CHCl3). 1H NMR (200 MHz, CDCl3):
d 0.90 (d, 3H, J¼9.6 Hz, CH3CH), 0.93 (d, 3H, J¼9.6 Hz,
CH3CH), 1.44 (s, 9H, (CH3)3C), 1.60e1.78 (m, 3H, CHCH2),
2.90 (s, 3H, CH3N), 3.78e4.05 (m, 4H, 2ꢂGly-2H), 5.05e
5.20 (m, 1H, Leu-H), 5.10 (s, 2H, PhCH2), 5.80 (br s, 1H,
NHBoc), 6.47 (br s, 1H, NH), 7.32e7.47 (m, 5H, ArH). 13C
NMR (50 MHz, CDCl3): d 22.3, 23.4, 25.2, 28.4, 30.0, 36.4,
42.2, 43.3, 55.1, 64.1, 67.3, 113.2, 119.4, 128.4, 128.8, 160.4,
167.5, 167.7, 169.5. HRMS (ESI, [MþNa]þ) 506.2254
(C25H33N3O6Na: 506.2262). IR (CHCl3) n: 3330, 2957, 2932,
754 cmꢁ1
.
4.4.9. Boc-(S)-Ala-(R)-Leu-GlyOtBu (6f)
Yield: 99% of white crystals; Rf¼0.43 (hexane/ethyl acetate
5:5, v/v); mp 185e187 ꢀC (ethyl acetate/hexane); [a]2D0 þ18.4
(c 1.0, CHCl3). 1H NMR (200 MHz, CDCl3): d 0.89e0.93 (m,
6H, CH3CH), 1.34 (d, J¼7.0 Hz, 3H, Ala-CH3), 1.42 (s, 9H,
(CH3)3C), 1.44 (s, 9H, (CH3)3C), 1.52e1.78 (m, 3H, CHCH2),
3.78e4.00 (m, 4H, 2ꢂGly-2H), 4.05e4.22 (m, 1H, 2ꢂAla-H),
4.42e4.58 (m, 1H, Leu-H), 5.24 (d, J¼7.0 Hz, 1H, NHBoc),
6.83 (d, J¼9.2 Hz, 1H, Leu-NH), 6.97 (br s, H, Gly-NH).
13C NMR (50 MHz, CDCl3): d 18.5, 22.1, 23.4, 25.1,
28.4, 28.6, 41.0, 42.3, 51.8, 80.6, 82.4, 155.9, 169.0, 172.4,
173.4. HRMS (ESI, [MþNa]þ) 438.2562 (C20H37N3O6Na:
438.2575). IR (Nujol) n: 3290, 3240, 1749, 1677, 1652,
1727, 1651, 1530, 1368, 1251, 1158 cmꢁ1
.
Acknowledgements
This work was financially supported by Polish State Com-
mittee for Scientific Research, Grant PBZeKBN 126/T09/07.
1568, 1532, 1455, 1366, 1226, 1168 cmꢁ1
.
4.5. Synthesis of compounds 6aeh
References and notes
The optimized transformation of alcohols (S)-5a and (S)-5b
into N-methylated amides 6g and h, respectively is further
exemplified by the synthesis of 6g.
1. Valente, C.; Guedes, R. C.; Moreira, R.; Iley, J.; Gutc, I.; Rosenthal, P. J.
Bioorg. Med. Chem. Lett. 2006, 16, 4115e4119.
2. Nunes, M.; Kaplan, J.; Wooters, J.; Hari, M.; Minnick, A. A., Jr.; May,
M. K.; Shi, C.; Musto, S.; Beyer, C.; Krishnamurthy, G.; Qiu, Y.; Loganzo,
F.; Ayral-Kaloustian, S.; Zask, A.; Greenberger, L. M. Biochemistry 2005,
44.
4.5.1. CBzGly-(N-Me)-(R)-Phe-GlyOtBu (6g)
A solution of methanesulfonic acid ester (S)-7a (60 mg,
0.17 mmol) in methanol (containing 5 wt% of methylamine)
was stirred for 24 h in 50 ꢀC. The volatiles were then evapo-
rated and the crude product was purified by flash chromatog-
raphy (silica gel, ethyl acetate/methanol 9:1, v/v). HRMS
(ESI, [MþH]þ) 293.1857 (C16H25N2O3: 293.1860). Obtained
compound (R)-10a was used in further synthesis. To a solution
of amine (R)-10a (28 mg, 0.09 mmol) in CH2Cl2 (4.0 mL)
were added CBz-protected glycine (21 mg, 0.10 mmol) and
3. Perni, R. B.; Chandorkar, G.; Cottrell, K. M.; Gates, C. A.; Lin, C.; Lin,
K.; Luong, Y.-P.; Maxwell, J. P.; Murcko, M. A.; Pitlik, J.; Rao, G.;
Schairer, W. C.; Van Drie, J.; Wei, J. Bioorg. Med. Chem. Lett. 2007,
17, 3406e3411.
4. Priem, G.; Rocheblave, L.; De Michelis, C.; Courcambeck, J.; Kraus, J. L.
J. Chem. Soc., Perkin Trans. 1 2000, 819e824.
5. Shirasaki, Y.; Nakamura, M.; Yamaguchi, M.; Miyashita, H.; Sakai, O.;
Inoue, J. J. Med. Chem. 2006, 49, 3926e3932.
6. Hanessian, S.; Ersmark, K.; Wang, X.; Del Valle, J. R.; Blomberg, N.;
Xuec, Y.; Fjellstrom, O. Bioorg. Med. Chem. Lett. 2007, 17, 3480e3485.
¨

W. Szymanski, R. Ostaszewski / Tetrahedron 64 (2008) 3197e3203
3203
´
7. van Swieten, P. F.; Samuel, E.; Hernandez, R. O.; van den Nieuwendijk,
10. Aurelio, L.; Borwnlee, R. T. C.; Hughes, A. B. Chem. Rev. 2004, 104,
5823e5846.
A. M. C. H.; Leeuwenburgh, M. A.; van der Marel, G. A.; Kessler,
B. M.; Overkleefta, H. S.; Kisselev, A. F. Bioorg. Med. Chem. Lett.
2007, 17, 3402e3405.
´
11. Szymanski, W.; Zwolinska, M.; Ostaszewski, R. Tetrahedron 2007, 63,
´
7647e7653.
´
8. Shirasaki, Y.; Miyashita, H.; Yamaguchi, M. Bioorg. Med. Chem. 2006,
14, 5691e5698.
12. Szymanski, W.; Ostaszewski, R. Tetrahedron: Asymmetry 2006, 17,
2667e2671.
9. Larsen, S. D.; Barf, T.; Liljebris, C.; May, P. D.; Ogg, D.; O’Sullivan, T. J.;
Palazuk, B. J.; Schostarez, H. J.; Stevens, F. C.; Bleasdale, J. E. J. Med.
Chem. 2002, 45, 598e622.
13. Cahiez, G.; Metais, E. Tetrahedron Lett. 1995, 36, 6449e6452.
14. Akullian, L. C.; Snapper, M. L.; Hoveyda, A. H. J. Am. Chem. Soc. 2006,
128, 6532e6533.