A biomimetic approach to the synthesis of terpene-amino acid conjugates. The Ugi reaction in the hypothetical biosynthesis of marine natural products

Article abstract of DOI:10.3987/COM-16-13442

A unique pathway for the biosynthesis of the marine sponge terpenes, exigurin and boneratamides A-C, is proposed. Based on this proposal, a biomimetic strategy on key Ugi coupling reactions between terpene isocyanides and amino acids was developed for construction of the core structures of exigurin and boneratamides A-C.

Full text of DOI:10.3987/COM-16-13442

HETEROCYCLES, Vol. 92, No. 6, 2016, pp. -. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 22nd February, 2016, Accepted, 24th March, 2016, Published online,
DOI: 10.3987/COM-16-13442
.
A BIOMIMETIC APPROACH TO THE SYNTHESIS OF
TERPENE-AMINO ACID CONJUGATES. THE UGI REACTION IN
THE HYPOTHETICAL BIOSYNTHESIS OF MARINE NATURAL
PRODUCTS
Yoshiyasu Ichikawa,*a Kenta Saito,a Rika Mimura,a Ayumi Kitamori,a
Akihito Matsukawa,a Akifumi Ikeda,a Toshiya Masuda,b Hiyoshizo Kotsuki,a
and Keiji Nakanoa
aFaculty of Science, Kochi University, Akebono-cho Kochi 780-8520, Japan;
E-mail: ichikawa@kochi-u.ac.jp. bGraduate School of Human Life Science,
Osaka City University
Abstract – A unique pathway for the biosynthesis of the marine sponge terpenes,
exigurin and boneratamides A–C, is proposed. Based on this proposal, a
biomimetic strategy on key Ugi coupling reactions between terpene isocyanides
and amino acids was developed for construction of the core structures of exigurin
and boneratamides A–C.
INTRODUCTION
In 2003, Ohta and Ikegami at Hiroshima University reported the isolation and structural elucidation of
exigurin (1) along with (–)-10-epi-axisonitrile-3 (2) from the marine sponge Geodia exigua collected in
the Oshima Island in the Kagoshima Prefecture of Japan (Figure 1).1 While (–)-10-epi-axisonitrile-3 (2)
is a typical marine terpene isocyanide, previously isolated from the nudibranch Phylliella pustulosa,2
exigurin (1) has an unprecedented structure in which a terpene unit and sarcosine (N-methylglycine) are
joined through an amide linkage. Although Garson and Simpson suggested that (–)-10-epi-axisonitrile-3
(2) plays a possible precursor role in the biosynthesis of exigurin, questions have arisen about the
metabolic origin of this marine sponge terpene-amino acid conjugate.3
O
Me
N
CO2Me
N
N
C
H
exigurin (1)
(–)-10-epi-axisonitrile-3 (2)

Figure 1. Structures of exigurin (1) and (–)-10-epi-axisonitrile-3 (2)
In 2004, Andersen and co-workers at the University of British Columbia reported the isolation of
boneratamides A–C from the marine sponge Axinyssa aplysinoides collected in Indonesia.4 X-Ray
crystallographic analysis of the methyl ester of boneratamide A 4 established the relative stereochemistry
of boneratamide A (3) as represented in Figure 2 (only one enantiomer with C-23S absolute configuration
is shown). Boneratamide A is a terpene-amino acid conjugate in which the terpene unit and
pyroglutamic acid are connected through a 2-aminoisobutyramide moiety.
The structures of
boneratamides B and C (5) were elucidated by extensive NMR spectroscopic analysis on the
corresponding methyl esters 6, which revealed that boneratamides B and C are stereoisomers differing in
the relative configuration at either one or both of the C18 and C23 stereogenic centers. Like
boneratamide A (3), boneratamides B and C (5) are conjugates of a terpene unit and pyroglutamic acid.
O
O
O
O
18
N
N
23
23
N
N
H
H
CO2R
H
CO2R
boneratamides B and C (5): R = H
metyl esters (6): R = Me
boneratamide A (3): R = H
metyl ester (4): R = Me
Figure 2. Structures of boneratamides A–C and their methyl esters
We previously put forth the hypothesis that exigurin and boneratamides A–C might be generated in
marine organisms through a hitherto unrecognized biosynthetic pathway that involves key Ugi reactions.5
The Ugi reaction is a multicomponent reaction which combines four components including an amine,
aldehyde or ketone, carboxylic acid and isocyanide, in a one-pot manner to produce an α-acylaminoamide
II (Scheme 1). This process, known as the Ugi four-component reaction (U-4CR), has found
widespread use in both diversity and target oriented organic syntheses.6 The currently accepted
mechanism of the U-4CR starts with condensation of the amine with carbonyl compound to form a
Schiff’s base, which then reacts with the isocyanide to generate the transient O-acyl imido intermediate I.
Spontaneous O-to-N acyl migration of I results in the formation of the stable α-acylaminoamide product
II.
R3
R4
R4
R3
R4
R3
H
R2
R2
N
R2
N
N
H
R1
R5
NH2
R5
O
N
R1
OH
O
O
R1
O
C N R5
O
O
II
I

Scheme 1. The Ugi four component reaction (U-4CR)
In an extension of the U-4CR, bifunctional reagents in which the participating functional groups are
present in a single component, have been used to construct unique structures such as β-lactams and
iminodicarboxylic acids. In the route for β-lactam synthesis, β-amino acids serve as bifunctional
starting materials instead of the amine and carboxylic acid components (Scheme 2).7
A plausible
reaction mechanism involves initial condensation of the β-amino acid with the aldehyde to form a
protonated Schiff’s base, which then reacts with the isocyanide to form a seven-membered ring
intermediate I. Intramolecular O-to-N-acyl migration of I results in the formation of the β-lactam II.
This type of the process for β-lactam synthesis is referred to as the Ugi-four-center three-component
reaction (U-4C-3CR).8
R4
R3
R3
R4
R3 R4
C N R5
H
N
R5
O
N
HN
O
N
R5
NH2
O
O
COOH
I
O
II
Scheme 2. The Ugi-four-center three-component reaction (U-4C-3CR) for the synthesis of β-lactams
A modification of the U-4CR using amino acids as bifunctional starting materials is classified as the Ugi
five-center four-component reaction (U-5C-4CR) (Scheme 3).9
The proposed reaction mechanism of
the U-5C-4CR begins with condensation of the amino acid with the carbonyl component to form the
corresponding protonated Schiff’s base I. Addition of the isocyanide then produces the cyclic
O-acyl-imide II. High strain energy associated with the formation of α-lactam ring prevents II from
undergoing intramolecular O-to-N-acyl migration. As a result, nucleophilic attack of the fourth
component, such as methanol used as the solvent, occurs at the carboxylic carbon (the fifth reacting
center). Subsequent six-membered ring opening leads to the formation of the iminodicarboxylic acid
derivative III. It should be noted that this process creates a stereogenic center in the product III.
O
R1
H
N
R2 R1
H
R1
H
N
+
R1
O
R2
N
R2
H
R3 N C
OH
OR4
O
H2N
R3
R2
N
H
*
N
*
HOR4
R3
O
O
H
O
O
O
I
III
II
Scheme 3. The Ugi five-center four-component reaction (U-5C-4CR) forming iminodicarboxylic acid
derivatives

Taking U-4CR, U-4C-3CR and U-5C-4CR processes into consideration, analysis of the structures of
exigurin (1), boneratamide A (3), boneratamides B and C (5) led us to recognize the structural
resemblance among these terpene-amino acid conjugates. Specifically, these linked terpene-amino acid
conjugates possess a common structural motif of iminodicarboxylic acid moeity, which connects the
terpene and amino acid residues. This type of structural motif has been known as a product of the Ugi
reaction in the field of organic syntheses. Although the Ugi reaction has been used extensively in
combinatorial, diverse oriented synthesis,10 it is quite surprising for us to imagine that Nature employs a
similar approach to build up a diversity of secondary metabolites in biosynthesis. We now report a
further development and observations of our studies aiming at a biomimetic approach to the synthesis of
terpene-amino acid conjugates exigurin and boneratamides A–C.
RESULTS AND DISCUSSION
Two hypothetical biosynthetic pathways for construction of the right hand, amide portion of exigurin are
presented in Scheme 4. The key step in each route, involving a ugiase catalyzed U-5C-4CR, suggests
that exigurin may be traced back to three building blocks consisting of (–)-10-epi-axisonitrile-3 (2) and
two other simple precursors such as formaldehyde and either sarcosine or glycine. The Ugi reaction is
known to be promoted by an enzyme,11 and this precedent led us to propose a putative ugiase enzyme that
catalyzes the Ugi process in a manner reminiscent of the Ugi reaction in organic synthesis. In the
clockwise hypothetical pathway, the U-5C-4CR of (–)-10-epi-axisonitrile-3 (2) with the iminium ion
formed from formaldehyde and sarcosine generates the cyclic O-acyl imidate 7, which reacts with
methanol to furnish exigurin (1). In the other route, the U-5C-4CR of formaldehyde and glycine with
the isocyanide 2 produces 8, which is transformed to exigurin (1) by the action of methyltransferase.
Me
Me
CO2H
N
N
H
HCHO
O
Me
N
ugiase
N
O
N
O
N
C
H
CO2Me
7
MeOH
(–)-10-epi-axisonitrile-3 (2)
exigurin (1)
H N
2
CO2H
HCHO,
MeOH
O
H
methyltransferase
N
N
ugiase
H
CO2Me
8
Scheme 4. Two plausible biosynthetic pathways of exigurin (1) through the U-5C-4CR

Reaction of t-butyl isocyanide (9) with sarcosine (10) and aqueous formaldehyde was selected to evaluate
the feasibility of the Ugi processes that take place in the hypothetical biosynthetic pathway (Scheme 5).
The U-5C-4CR of these substances in methanol took place smoothly at room temperature to form 11
albeit in low yield (24%). After unsuccessful attempts to improve the yields of this process, the
alternative hypothetical biosynthesis scenario shown in Scheme 4 was explored. Since the use of
glycine as a substrate of the Ugi reaction leads to over-Ugi products,12 N-protected glycine was utilized as
the amino acid substrate. To our delight, the U-5C-4CR using t-butyl isocyanide (9), N-benzylglycine
(12) and paraformaldehyde generated amide 13 in 73% yield. Removal of the benzyl group in 13
followed by the reductive amination of 14 under the Eschweiler-Clarke reaction conditions furnished 11
in 60% yield in two steps.
Me
O
Me
N
HO2C
NH
10
C
N
N
aq. HCHO, MeOH
(24%)
H
CO2Me
9
11
CH2Ph
12
HO2C
NH·HCl
(HCHO)n, Et3N
MeOH
aq. HCHO
H2, Pd(OH)2
EtOH
O
CH2Ph
N
O
H
HCO2H
N
N
N
(60%, 2 steps)
(73%)
H
H
CO2Me
CO2Me
13
14
Scheme 5. Model synthesis of the right part of exigurin
We next turned our attention to synthesize a terpene-amino acid conjugate, which possesses the structural
motif corresponding to the right part of exigurin (Scheme 6). The terpene isocyanide 15, prepared
from (–)-menthol by a method we previously developed,12a was treated with N-benzyl glycine 12 and
paraformaldehyde in methanol under similar conditions to those depicted in Scheme 5. This U-5C-4CR
proceeded uneventfully to afford 16 in 87% yield. Hydrogenolysis of the benzylamine in 16 produced
17, which was subjected to the Eschweiler-Clarke reaction conditions. To our surprise, we observed a
considerable amount of a byproduct, the structure of which was tentatively assigned as cyclic amide 19.
Separation of 19 was difficult due to its similar TLC behavior with 18. In contrast to the low-yield
observed for reaction of t-butyl isocyanide (9), the U-5C-4CR of the terpene isocyanide 15 with sarcosine
13
(10) and aqueous formaldehyde produced 18 in 40% yield. 1H and C NMR data for exigurin (1),
model compound 11 and despiro-exigurin 18 are summarized in Table 1, which shows that the NMR data
of both 11 and 18 are in good accord with those reported for exigurin (1). Thus, although the yield is
modest, synthesis of despiro-exigurin 18 having the right structure found in exigurin was achieved
through operationally simple one-pot U-5C-4CR process.

MeO2C
MeO2C
N
O
N
O
HO2C
12
NH·HCl
H2
Pd(OH)2
H
CH2Ph
CH2Ph
N
H
N
H
N
(HCHO)n, Et3N, MeOH
(87%)
C
17
15
16
MeHN
CO2H
10
aq. HCHO
HCO2H
O
O
Me
N
aq. HCHO, MeOH
(40%)
N
H
N
N
CO2Me
CO2Me
19
18
Scheme 6. Synthesis of despiro-exigurin 18.
Table 1.
1H and 13C NMR data for exigurin (1), model compounds 11 and 18
O
Me
N
O
Me
N
O
Me
N
N
17
18
19
OMe
N
H
N
H
16
H
O
OMe
O
OMe
O
18
11
exigurin (1)
13C NMR
(C6D6, 125 MHz)
1H NMR (C6D6, 500 MHz)
1*
11
18
1*
11
18
16
17
168.98
61.55
43.19
58.41
169.1
61.9
42.7
58.2
171.0
51.1
168.5
61.5
43.0
58.3
170.9
51.2
17
2.92 (2H, s) 2.98 (2H, s)
2.02 (3H, s) 2.03 (3H, s)
3.05 (2H, s)
17-NMe
2.09 (3H, s)
2.85
17-NMe
18
2.84
(2H, s)
2.75–2.86
(2H, s)
18
(2H, s)
19
170.64
51.03
19-OMe
3.26 (3H, s) 3.23 (3H, s)
3.28 (3H, s)
19-OMe
* NMR data of 1 are reproduced from the ref. 1a.
Two retrosynthetic scenarios, based on the hypothetical biosynthesis pathway, can be envisaged for the
synthesis of boneratamides A–C (Scheme 7). A plausible intermediate in the biosynthetic route to
boneratamides A–C is axisonitrile-3 (20), a terpene isocyanide previously isolated from the marine
sponge Axinella cannabina.13 Based on this proposal, boneratamides A–C can be envisioned to arise from
three building blocks including axisonitrile-3 (20), either acetone or acetaldehyde, and glutamic acid (21).
Moreover, because U-4C-3CR of β-amino acids leads to β-lactams (Scheme 2), we surmised that

corresponding reactions of γ-amino acids such as glutamic acid would produce γ-lactam ring systems
present in boneratamides A–C. Another possible retrosynthetic scenario for the preparation of
boneratamides A–C follows a stepwise sequence starting with U-5C-4CR of glutamic acid (21) as a
bifunctional reagent and water as the fourth component to form 22, which subsequently cyclizes to form
γ-lactam core structure in boneratamides A–C.
O
O
O
U-4C-3CR
ugiase
O
or
N
H
N
N
H
CO2H
C
CO2H
R
NH2
HO2C
boneratamides A–C: R = Me or H
axisonitrile-3 (20)
21
CO2H
R
C
c
y
c
4
-
C
5
-
U
l
a
O
H
s
e
e
s
a
i
g
N
u
N
H
CO2H
R
22: R = Me or H
Scheme 7. Two possible retro-synthetic scenarios for biosynthesis of boneratamides A–C
Initial studies probing these scenarios focused on the synthesis of the right part of boneratamide A
employing t-butyl isocyanide (9) as a model substrate (Scheme 8). We observed that U-4C-3CR of 9,
acetone (10 equiv) and α-methyl L-glutamate (23) (1.0 equiv) in methanol proceeds smoothly at 50 °C to
produce the γ-lactam 25 (69%) that possesses the structural motif found in boneratamide A methyl ester
(4) (Scheme 8, entry A). A plausible mechanism for formation of 25 involves generation of the oxazepinone
O
O
O
C
N
O
O
MeOH
9
N
CO2R
24
N
N
N
H
H
HO2C
CO2R
CO2R
NH2
25: R = Me
26: R = H
23: R = Me
21: R = H
entry
yield (%)
product
69
A
B
25
19 and 10
25 and 26
Scheme 8. One-pot synthesis of the right-hand amide portion of boneratamide A

intermediate 24, which then undergoes intramolecular O-to-N-acyl migration. In the case of the
U-4C-3CR using glutamic acid 21 as the substrate, a mixture of methyl ester 25 and carboxylic acid 26
was isolated in 19% and 10% yield, respectively (entry B). Note that the structure of free carboxylic
acid in 26 corresponds to the right hand structure of boneratamide A.
We next explored the synthesis of the despiro analogue of boneratamide A employing a terpene
isocyanide 27,14 which was prepared from (+)-menthol (Scheme 9). The U-4C-3CR of terpene
isocyanide 27 using reaction conditions similar to those described in Scheme 8 furnished the desired
despiro-boneratamide A 28 in 67% yield. It should be noted that in this highly efficient one-pot process,
conjugation of terpene with amino acid took place along with γ-lactam ring formation. 1H and 13C NMR
analysis of 28 shows an approximately 1:1 mixture of amide rotamers. Fortunately, we obtained a nice
crystalline 28, and X-ray crystallographic analysis of 28 leads to an unambiguous assignment of its
structure.
CO2Me
O
O
23
NH2
HO2C
O
N
N
N
C
H
CO2Me
(67%)
MeOH
28
27
28 [X-ray structure]
Scheme 9. Synthesis of despiro-boneratamide A 28
Synthesis of the right-hand portion of boneratamides B and C was explored using the reaction conditions
established during the model synthesis of boneratamide A (Scheme 10). Disappointingly, the
U-4C-3CR employing t-butyl isocyanide (9), acetaldehyde and α-methyl L-glutamate 23 produced a 1:1
mixture of products 29 in low yields. Moreover, the diastereomers display similar TLC behavior, which
hampered separation and identification.
O
O
O
MeOH
(22%)
CO2Me
NH2
HO2C
N C
N
N
H
H
H
CO2Me
9
23
29
Scheme 10. Attempted synthesis of the right-hand portion of boneratamides B and C
In order to improve yields and to obtain each stereoisomer 29 in pure form, we turned our attention to an
alternative stepwise approach, which corresponds to the second hypothetical biosynthetic route depicted

in Scheme 7. In this way, the U-5C-4CR of t-butyl isocyanide (9), acetaldehyde and γ-methyl
L-glutamate (30) provided a 3:1 mixture of products 31a and 31b in 37% yield (Scheme 11). To our
delight, this mixture could be separated and each respective isomer was cyclized by heating in toluene to
furnish 29a and 29b in 77% and 71% respective yields. Fortunately, the major stereoisomer 29a is
crystalline and the newly formed stereogenic center in 29a was firmly secured by X-ray crystallographic
analysis to assign the R absolute configuration.
Scheme 11. Model synthesis of the right portions of boneratimides B and C via a stepwise approach
We next investigated to synthesize despiro analogues of boneratamides B and C using U-4C-3CR
(Scheme 12). Unfortunately, the U-4C-3CR of terpene isocyanide 27, acetaldehyde and α-methyl L
-glutamate 23 produced an intractable tarry reaction products and none of the desired 33 was detected by
1H NMR and TLC analysis of the crude products. One of the isolable and identified by-products was 32,
which presumably arose via the Passerini reaction of isocyanide 27 with acetaldehyde followed by the
Cannizzaro reaction.
O
O
MeOH
O
O
H
N
N
CO2Me
NH2
H2OC
N
N
C
H
H
CO2Me
H
OAc
23
32
27
33
Scheme 12. Unsuccessful U-4C-3CR for the synthesis of despiro analogues of boneratamides B and C
The stepwise U-5C-4CR approach was then employed to synthesize despiro analogues of boneratamides
B and C 33 (Scheme 13). In this route, reaction of the terpene isocyanide 27 using conditions similar to

those described in Scheme 11 afforded an inseparable mixture of the products 34 in 28% yield. Heating
a solution of this mixture 34 in refluxing toluene facilitated cyclization to form a 3:2 mixture of δ-lactams
33a and 33b in 80% yield. Repeated chromatographic separations gave isomer 33a as a crystalline
substance. X-Ray crystallographic analysis confirmed the structure of 33a and the newly formed
stereocenter corresponding to the C18 positions of boneratamides B and C was assigned to be R
configuration.
Scheme 13. Synthesis of despiro analogues of boneratamides B and C
The comparison of the NMR data of 33a and 33b with those of natural boneratamide B and C methyl
1
13
esters is summarized in Table 2. The H and C NMR data for the methyl esters of boneratamides B
and C were found to compare favourably with those of the despiro analogues 33a and 33b. However,
characteristic resonances that would enable differentiation of boneratamides B and C as well as 33a and
33b were not present in the spectra.
Although the assignment of the configurations to the C18
stereocenters in natural boneratamides B and C remains unclear, X-ray analysis of 33a coupled with
comparison of NMR data in Table 2 confirms the proposed structures of natural boneratamides B and C.
CONCLUSIONS
Motivated by the belief that ‘molecules in nature obey the rules of organic chemistry and follow
nonenzymatic mechanism’,15 the Ugi chemistry presumably carried out by marine organisms was
explored on the bench in the laboratory. Our research endeavour along this line led to the synthesis of
terpene-amino acid conjugates that have identical structural motifs found in exigurin and boneratamides
A–C in a manner pertinent to our proposed biogenetic pathway. In particular, the synthesis of despiro

analogue of boneratamide A 28 showed remarkable efficiency to assemble multicomponents in a one-pot
process. The results of the current study shed light on a long-standing mystery relating to the
biosynthetic origin of exigurin and boneratamides. The Ugi type reaction is not confined to the realm of
organic synthesis, and it is now apparent that conjugation of terpenes with amino acids by the Ugi
reaction is one of the Nature’s strategies to expand the diversity of natural products.
Table 2. Selected 1H and 13C NMR data for boneratamide B and C methyl esters (6a and 6b) and
despiro analogues 33a and 33b
O
O
18
N
13C NMR (C6D6,125 MHz)
23
N
6a*
6b*
33a
H
33b
CO2Me
H
17
18
170.2
170.0
53.5
170.5
52.2
169.6
metyl esters of boneratamides
53.3
175.6
58.8
52.6
176.0
58.0
B and C (6a and 6b)
20
175.9
58.1
175.7
59.2
23
O
O
24
173.4
14.1
172.4
14.4
174.0
15.3
172.4
14.2
N
N
H
25
R2
R1
CO2Me
OMe
51.9
52.0
52.2
52.1
33a: R1 = Me, R2 = H
33b: R1 = H, R2 = Me
1H NMR (C6D6, 500 MHz)
6a*
6.91
6b*
7.20
33a
7.16
33b
7.29
d, J = 10.9 Hz
16
18
d, J = 10.8 Hz
d, J = 10.9 Hz
overlapped with C6H6
4.46
4.52
4.61
4.60
q, J = 7.2 Hz
q, J = 7.3 Hz
q, J = 7.0 Hz
q, J =7.0 Hz
21
21
1.87
1.84
1.85
1.86
ddd, J = 16.6, 9.6, 2.4 Hz ddd, J = 16.8, 9.6, 3.4 Hz brdd, J = 16.5, 10.0 Hz ddd, J = 16.5, 9.5, 3.5 Hz
2.26
m
2.27
m
2.24
2.27
dt, J = 16.5, 10.0 Hz
dq, J = 16.5, 9.5 Hz
23
25
4.11
3.86
4.37
brd, J = 10.0 Hz
4.49
dd, J = 9.5, 1.8 Hz
dd, J = 8.8, 2.8 Hz
dd, J = 8.0, 3.5 Hz
1.37
1.34
1.42
1.26
d, J = 7.2 Hz
d, J = 7.2 Hz
d, J = 7.0 Hz
d, J = 7.0 Hz
OMe
3.22
s
3.34
s
3.25
s
3.38
s
* NMR data of methyl esters of bonerataimde B and C (6a and 6b) are reproduced from the ref. 4.

EXPERIMENTAL
U-5C-4-CR for the synthesis of despiro-exigurin 18. To a solution of sarcosine (10) (135 mg, 1.51
mmol) in MeOH (20.0 mL) at room temperature was added aqueous formaldehyde (37%, 0.10 mL, 1.33
mmol). After stirring at 50 °C for 20 min, terpene isocyanide 15 (100 mg, 0.61 mmol) was added. The
solution was stirred at 50 °C for 20 min, and concentrated under reduced pressure. The resulting residue
was treated with water, and the aqueous layers were extracted with AcOEt (× 3). The combined organic
layers were dried (Na2SO4) and concentrated under reduced pressure. The crude residue (131 mg) was
subjected to silica gel chromatography (AcOEt/hexane 1:1, containing 1% isopropylamine) to afford
methyl ester 18 (72 mg, 40%) as a colorless oil: [α]D26 +22.3 (c 1.00, CHCl3); IR (NaCl) νmax 3473, 3352,
2950, 2922, 1747, 1679, 1517, 1457, 1199 cm－1; 1H NMR (CDCl3, 500 MHz) δ 0.86 (d, J = 7.0 Hz, 3H),
0.88 (d, J = 7.0 Hz, 3H), 0.89 (d, J = 7.0 Hz, 3H), 0.91–1.16 (m, 6H), 1.22–1.35 (m, 1H), 1.38–1.52 (m,
1H), 1.75 (dt, J = 13.0, 3.0 Hz, 1H), 1.82–1.88 (m, J = 13.0 Hz, 2H), 2.42 (s, 3H), 3.16 (s, 2H), 3.32 (s,
2H), 3.71 (s, 3H), 4.32 (dd, J = 9.0, 3.0 Hz, 1H), 7.56 (brd, J = 9.0 Hz, 1H); 1H NMR (C6D6, 500 MHz) δ
0.92–0.72 (m, 8H), 0.98 (dt, J = 13.0, 3.0 Hz, 1H), 1.06 (d, J = 7.0 Hz, 3H), 1.18 (ddt, J = 17.0, 8.0, 3.0
Hz, 1H), 1.40–1.52 (m, 1H), 1.53–1.68 (m, 2H), 1.72 (brd, J = 13.0 Hz, 1H), 1.98–2.09 (m, 4H), 2.80 (d,
J = 17.0 Hz, 1H), 2.82 (d, J = 17.0 Hz, 1H), 2.98 (d, J = 17.0 Hz, 1H), 3.01 (d, J = 14.0 Hz, 1H), 3.23 (s,
3H), 4.65 (dq, J = 13.0, 7.0 Hz, 1H), 7.59 (brd, J = 7.0 Hz, 1H);13C NMR (CDCl3, 125 MHz) δ 20.6, 20.9,
13
22.2, 25.2, 26.7, 29.5, 34.7, 40.1, 43.1, 45.4, 46.2, 51.5, 58.4, 60.9, 169.1, 170.8; C NMR (C6D6, 125
MHz) δ 21.0, 21.3, 22.6, 25.7, 27.1, 30.0, 35.3, 40.9, 43.0, 45.6, 46.8, 51.2, 58.3, 61.5, 168.5, 170.9.
HRMS(ESI): m/z calcd for C16H30N2O3Na[M+Na]+ 321.2154, found 321.2155.
U-4C-3CR for the synthesis of despiro-boneratamide A 28. To a solution of L-glutamic acid
1-methyl ester (23) (219 mg, 1.36 mmol) and acetone (0.70 mL, 9.1 mmol) in MeOH (15.0 mL) was
added isocyanide 27 (150 mg, 0.91 mmol). After stirring at room temperature for 1.5 h, the solution was
heated at 50 °C for 3 days, followed by refluxing for 2 days. The reaction mixture was concentrated
under reduced pressure, and the residue was dissolved in AcOEt and water. The organic layer was
separated, and the aqueous layer was extracted with AcOEt. The combined organic extracts were dried
(Na2SO4) and concentrated to afford a residue, which was subjected to silica gel chromatography
(AcOEt/hexane 1:1 to 4:1) to furnish γ-lactam 28 (222 mg, 67%): mp 139–140 °C (recrystallized from
hexane); [α]D25 –24.9 (c 1.00, CHCl3); IR (KBr) νmax 3370, 2950, 1731, 1664, 1528, 1221 cm－1;1H NMR
(CDCl3, 500 MHz) δ 0.82–1.90 (m, 26H), 2.09 (m, 1H), 2.20 (dq, J = 13.2, 2.9 Hz, 1H ), {2.37 (m)}, 3.15
(d, J = 13.2 Hz, 3H), 3.74 (ddd, J = 20.0, 9.7, 1.7 Hz, 1H), 4.71 (m, 1H), 7.50 (d, J = 9.2 Hz, 1H), {7.60
(d, J = 8.0 Hz)}; 13C NMR (C6D6, 125 MHz) δ 21.1, (21.4), 21.6, (21.7), 22.9, (22.9), 23.4, (23.6), (24.4),
24.6, 25.3, (25.5), (25.8), 25.9, (27.1), 27.3, 29.8, (29.8), (30.0), 30.2, 35.6, (35.6), 40.5, (40.6), 46.4,

(46.6), 46.8, (47.0), (52.36), 52.45, 57.4, (57.8), 60.3, (60.5), (172.2), 172.4, 173.7, (174.2), (175.6),
176.1. HRMS(ESI): m/z calcd for C20H34N2O4Na [M+Na]+ 389.2416, found 389.2401; HRMS(ESI): m/z
calcd for C20H34N2O4K [M+K]+ 405.2156, found 405.2155.
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