Domino Process Achieves Site-Selective Peptide Modification with High Optical Purity. Applications to Chain Diversification and Peptide Ligation

Article abstract of DOI:10.1021/acs.joc.5b00932

The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes but still is a difficult synthetic challenge. Herein, we introduce natural hydroxyproline as a convertible unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, homoserine lactones, and Agl lactams, and to achieve the mild, efficient, and site-selective modification of peptides. A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both terminal and internal positions can be modified, and similar customizable units can be differentiated. The resulting products possess two reactive chains which can be manipulated independently. The versatility and scope of this process is highlighted by its application to the ligation of two peptide chains, and the generation of peptides with several chains and peptides with conformational restrictions.

Full text of DOI:10.1021/acs.joc.5b00932

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Article
Domino Process Achieves Site-Selective Peptide Modification with High
Optical Purity. Applications to Chain Diversification and Peptide Ligation
Ivan Romero-Estudillo, and Alicia Boto
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00932 • Publication Date (Web): 07 Jul 2015
Downloaded from http://pubs.acs.org on July 8, 2015
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Domino Process Achieves Site-Selective Peptide
Modification with High Optical Purity. Applications to
Chain Diversification and Peptide Ligation.
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Ivan Romero-Estudillo, and Alicia Boto*
Instituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico Francisco Sánchez 3,
38206-La Laguna, Tenerife, Spain
alicia@ipna.csic.es
RECEIVED DATE
Corresponding Author Footnote. Telephone: +34-922-260112 (Ext 267); Fax: +34 922260135.
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Abstract. The development of peptide libraries by site-selective modification of a few parent peptides
would save valuable time and materials in discovery processes, but still is a difficult synthetic
challenge. Herein we introduce natural hydroxyproline as convertible unit for the production of a
variety of optically pure aminoacids, including expensive N-alkyl aminoacids, homoserine lactones and
Agl lactams, and to achieve the mild, efficient and site-selective modification of peptides.
A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both
terminal and internal positions can be modified, and similar customizable units can be differentiated.
The resulting products possess two reactive chains which can be manipulated independently. The
versatility and scope of this process is highlighted by its application to the ligation of two peptide
chains, and the generation of peptides with several chains and peptides with conformational restrictions.
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Introduction
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The modification of peptides is a hot research topic,1 in order to provide new peptide drugs,1,2 probes
for molecular imaging1,3 and peptide catalysts for green chemistry.4 The usual procedure to create
libraries of potential peptide drugs or catalysts involves the synthesis de novo of each library member.1-4
To introduce each new amino acid, two steps are needed (deprotection of the terminal residue and a
coupling step), and therefore, this serial process is costly in time and materials, particularly with large
peptides. In addition, some peptides are quite difficult to obtain (eg. difficult sequences, macrocycles,
etc), and producing each library member will require an extra effort. Finally, since for many activity
studies only a small part of the sequence (active center, binding site) needs modification, a different
approach to create peptide libraries would be desirable.
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In this alternative strategy, a certain position/s in a parent peptide is transformed, while the others
remain unchanged (site-selective modification).5-7 In the tag-and-modify approach, a functional group
in a residue (tag) is replaced or transformed into other ones. This strategy has allowed the modification
of both peptides and proteins.8 A variation involves the use of customizable units, where the core of the
starting aminoacid is transformed into a quite different one (eg. by cleavage of the lateral chain and
replacement by new ones). 9-15
The simplest customizable unit is glycine, and thus, Kazmaier9 and Skrydstrup10 have reported their
selective enolization and reaction with electrophiles. Other customizable units are aziridine carboxylic
acids, and Van der Donk and Garner have described their opening by different nucleophiles.11
A problem in the site-selective modification of peptides is differentiating similar tags or customizable
units. Thus, when a peptide has several glycine units, the selective enolization of just one residue is a
real challenge. Important progress was achieved with the introduction of customizable units whose
reactive groups could be protected orthogonally. For instance, Steglich and Skrydstrup reported the
selective chain scission of serine or threonine residues in peptides.12 The residues with unprotected
hydroxyl groups underwent lateral chain scission, while protected residues were not affected. Since
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many orthogonal protecting groups are available, different functionalizations at different times can be
achieved.
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Recently, our group developed a sequential method for the oxidative radical scission of serine
residues in peptides, followed by the addition of C- or P-nucleophiles (conversion 1→3, Scheme 1).13,14
Although good product yields were obtained, the stereoselectivity of the reaction ranged from excellent
to low.13 In order to prepare peptides with consistently high optical purity, in a preliminary
communication15 we introduced another customizable unit, natural and low-cost 4R-hydroxy-L-proline
(Hyp).
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SCHEME 1. Site-selective conversion of customizable units Ser (previous work, left) and Hyp (this
work, right).
HO
4
3
OH
O
5
X
Z-HN
N
N
COX
H
O
R
Z-(aa)n
1
4
Z = carbamoyl, acyl, amino acyl, peptidyl
X = O-alkyl, amino acyl, peptidyl
R = H, alkyl, aryl
PhI(OAc)2
I2, hv,
Scission-oxidation process
Pb(OAc)4
O
O
+
N
X
Z-HN
R
COX
N
+
H
O
Z-(aa)n
2
5
Nu
Addition of nucleophiles
AcO
Orthogonal
"handles"
Nu
CHO
O
X
AcO
N
Z-HN
N
COX
O
H
Z-(aa)n
R
6
3
P- and C-chains
Unnatural residues
Unnatural units
Low or moderate d.e.
High optical purity
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When introduced in peptides 4 (Scheme 1), the Hyp unit could be selectively transformed using a
mild, metal-free domino process. The scission reaction proceeded regioselectively through the C4-C5
bond,16 to yield the acyliminium intermediate 5,17 which retained the stereogenic -center. The addition
of acetate ions from the reaction medium followed, yielding scission products 6 with two reactive
handles, which could be manipulated independently.
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In this Paper, a significant extension of these studies will be reported, where different scission
products 6 will be generated and transformed into a variety of optically pure compounds, from N-alkyl
amino acids to unusual -alkyl and alkenyl glycines, and peptides with rigid Agl lactams that allow
conformational constraints in the backbone. In addition, the versatility of this strategy will be
highlighted by its application to the ligation of peptides, generating larger linear or branched
derivatives.
Results and Discussion
Studies on the optical purity of the products during the scission and subsequent reductive
amination steps. As commented before, the fragmentation of Hyp units through the C4-C5 bond should
not affect the -stereogenic center, and the process would afford L-amino acids unless epimerization of
the -center took place under the reaction conditions. In order to check this point, substrates where the
Hyp unit was attached to a chiral moiety were prepared. When hydroxyproline (-)-menthyl carbamate
815 (Scheme 2) was treated with (diacetoxy)iodobenzene (DIB, PhI(OAc)2) and iodine, under irradiation
o
with visible light at 26 C, the 4-oxo-
L-homoalanine derivative 10 was formed in 80% yield. To our
satisfaction, a single diastereomer was generated, supporting the hypothesis that under the mild
reaction conditions no epimerization occurred. Interestingly, no C3-C4 scission products were isolated,
and also an N-acetoxymethyl group was formed, by reaction of the acyliminium intermediate with
acetate ions from DIB.
The scission reaction was repeated with the dipeptide Cbz-Phe-Hyp-OMe (9). In this case, the
temperature was increased to favour substrate solubility, and to reduce reaction times, despite the
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heating could promote the epimerization of both residues. However, after 1 h, the reaction afforded the
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dipeptide 11 as a single diastereomer in 82% yield.
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SCHEME 2. Studies on the stereochemical integrity of the scission and reductive amination products.
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H-Hyp-OMe (7)
ClC(O)O-menthyl
ClCOOMe,
NaHCO3 (aq)
THF, 94%
or Cbz-Phe-OH,
DIPEA, HBTU
87-91%
HO
MeO(O)C-Hyp-OMe (12)
O
OMe
DIB, I2, h
DCE, 80 oC, 1 h
N
Z
O
8 Z = COO-(-)menthyl
9 Z = (N-Cbz)-Phe
AcO
O
N
DIB, I2, h ,
CH2Cl2, 2 h
OMe
MeO
O
13 (81%)
O
NH2-R
O
OMe
NaBH4, MeOH
AcO
N
Z
N R
HN
MeO
10 Z =
(80%)
O
O
O
O
(77%)
14 R =
15 R = CH2-Ph
16 R =
(77%)
(66%)
11 Z =
(82%)
O
O
HN
O
17 R =
(68%)
Negligible epimerization; high optical purity
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The N-acetoxymethyl 4-oxo-L-homoalanine derivatives resulting from the scission could be valuable
precursors of other compounds, by controlled manipulation of the reactive N- or -chains.18 A requisite
is that such transformations proceed without epimerization of the stereogenic centers.
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Therefore, the reductive amination of the scission products with primary amines was studied. This
reaction can provide high-profit -amino--lactams (Agl derivatives), as shown by the conversion of
aldehyde 13 (Scheme 2) into lactams 1417. Two enantiomeric 1-(naphthyl)ethyl amines were used to
check that no epimerization took place during the reductive amination-lactamization process, which
afforded the diastereomeric compounds 16 (2S,1’R) and 17 (2S,1’S).15 After purification of both
lactams, different diastereomer mixtures were prepared [(1’S):(1’R) 85:15, 90:10, 95:5, 98:2] and their
NMR spectra was recorded. The detection limit was about dr 98:2. With these spectra, it was checked
that the crude reductive amination mixture corresponded to (1’S):(1’R) ≥ 96:4 (see NMR series,
Supporting Information)
Creation of Agl units by reductive amination of the scission products, and application to peptide
ligation. The reductive amination-lactamization process was also studied with aminoacids and peptides
(Scheme 3). Thus, the aldehyde 13 reacted with H-Thr(tBu)-OMe to give lactam 18 in 59% yield, in
spite of the steric hindrance of the amine. When peptide 19 was used, the tripeptide 20 was formed in
82% yield as a single diastereomer.
The formation of -amino--lactams (Agl, Freidinger-Veber lactam) has proven very useful to
rigidify peptide backbones, inducing conformations that mimic -turns.1a,19 These turns are often found
in bioactive peptides which interact with their biological targets. Moreover, Agl-containing peptide
drugs often display increased stability and bioavailability, and less undesired interactions. Therefore,
the scission-reductive amination process was adapted to achieve a double goal: the ligation of small
peptides and the concomitant formation of an Agl unit to introduce conformational constraints.
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SCHEME 3. Preparation of peptides with Agl lactams.
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6
7
8
9
CO2Me
OtBu
CHO
H-Thr(tBu)-OMe,
Et3N, MeOH
AcO
MeO
N
O
O
N
26 oC, 1 h; then
O
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11
12
13
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17
18
19
20
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OMe
HN
NaBH4, 45 oC, 20 h
O
MeO
13
18 (59%)
19
, MeOH
Et3N, 26 oC, 1 h;
then NaBH4,
O
H2N
o
45 C, 20 h
( )3
Ile-OMe
NH-Boc
CO2Me
NH
O
19
1. H-Ile-OMe, DCM
NH
Boc
DIPEA, HBTU
0 to 26oC, 87%
2. H2, Pd/C, 93%
( )2
N
O
O
O
Cbz-HN
HN
( )3
OH
OMe
20
NH-Boc
21
(82%)
The process was studied with the aldehyde 11 (Scheme 4) which reacted with glycine methyl ester
under reductive conditions, affording the Agl-containing tripeptide 22 with high optical purity. When
the aldehyde 13 was treated with peptide 19 under similar conditions, the ligation of the two peptides
proceeded with lactamization, affording the desired tetrapeptide 23 in 78% yield; no epimerization was
detected.
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SCHEME 4. Ligation of peptides, forming a rigid Agl unit.
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5
6
7
8
9
O
H
N
N
OMe
HN
O
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11
12
13
14
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20
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O
O
O
22
H-Gly-OMe, MeOH, Et3N, 26°C, 1h;
then NaBH4, 45°C, 20 h, 64%
AcO
O
N
HN
O
CO2Me
O
O
11
Boc-Lys-Ile-OMe (19)
MeOH, Et3N, 26°C, 1h;
then NaBH4, 45°C, 20 h, 78%
OMe
O
O
NH
NH
Boc
O
NH
HN
N
O
O
O
23
Increasing diversity by manipulation of the N- and -chain reactive handles. Another valuable
unit in peptide chemistry is L-homoserine lactone, which reaches high market value as component of
compounds affecting quorum sensing in bacteria, and thus, microbial virulence.20 This lactone could be
readily obtained by reduction of 4-oxo-
(Scheme 5).
L
-homoalanine derivatives and intramolecular cyclization
Depending on the reaction conditions, different products can be obtained. For instance, the reduction
of aldehyde 10 with triethylsilane under Lewis acid catalysis proceeded to give the desired lactone, but
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in addition, the N-acetoxymethyl moiety was reduced to a N-methyl group, affording compound 24 as a
single diastereomer. The N-alkylated amino acids are valuable, since their introduction into peptides
alters the hydrogen bonding pattern, favouring extended conformations. This allows modulation of the
peptide activity and potency. The modified peptides also present increased bioavailability and stability
to proteolysis.1a In spite of their usefulness, there are relatively few commercially available N-methyl
aminoacids, and are quite expensive. This method allows the conversion of low-cost substrates into
valuable N-substituted amino acids.
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SCHEME 5. Formation of valuable L-homoserine lactones, including N-methyl derivatives.
CHO
AcO
O
O
Et3SiH
N
N
OMe
O
BF3OEt2
70%
O
O
O
24
O
10
O
O
O
NaBH4
AcO
MeO
N
HN
MeO
OMe
MeOH
77%
O
O
O
25
13
OH
1. DIB, I2, h
CH2Cl2, 26 oC
H
N
N
N
O
2. NaBH4, MeOH
51% (two steps)
N
CO2Me
O
Cbz
O
O
Cbz
27
26
O
AcO
N
H
N
CO2Me
O
Cbz
28
By varying the reduction conditions, the N-acetoxymethyl group can be removed. Thus, when
substrate 13 was treated with sodium borohydride in methanol, the homoserine lactone 25 was isolated
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in 77% yield. Similar reductive conditions were applied for the transformation of dipeptide 26 into the
homoserine lactone derivative 27.21 Interestingly, the intermediate aldehyde 28 was not purified, but
treated after aqueous work-up with sodium borohydride to give the final product.
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The selective manipulation of the -chain, without affecting the N-substituent, is also possible. In the
example shown in Scheme 6 (transformation 29→31),15 the homoalanine side chain was extended using
a Horner-Wadsworth-Emmons (HWE) reaction, which generated an expensive dehydrohomoglutamic
acid derivative. To our satisfaction, both the fragmentation and the HWE reaction proceeded without
epimerization to give compound 31 as a single diastereomer. In general, the orthogonal manipulation of
the - and N-chains could remarkably increase product diversity, as will be shown in the next examples.
SCHEME 6. Selective manipulation of the - chain by HWE reaction.
OH
Fmoc
Fmoc
HN
OAc
NH
N
N
CO2Me
OMe
O
O
O
OMe
O
29
31
NaH, THF
DIB, I2,
h, CH2Cl2
63%
82%
(MeO)2(O)P
CO2Me
O
OAc
O
NH
N
O
CHO
OMe
O
30
Scission of Hyp units in internal positions, and ligation to amino acids or peptides. The scission
of hydroxyproline units in internal positions represented an additional challenge, since peptide folding
could increase the probability that the reactive O-radical derived from Hyp was drawn close to aromatic
rings, carbonyl groups, or hydrogens on benzylic, tertiary or -carbons. In that case, side-reactions such
as addition of the O-radical to multiple bonds or hydrogen abstraction from nearby positions could take
place. However, if the scission was favored over side-reactions, a diversity of peptide libraries could be
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obtained. For instance, the conversion of customizable Hyp units into 4-amino homoalanines could be
applied to the generation of antibiotic peptides,1,2 where a balance of non polar and cationic residues is
crucial for antimicrobial activity while avoiding hemolysis.22-24
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The cleavage of Hyp in internal positions and its conversion to 4-amino homoalanines was studied
with a model tetrapeptide, which was prepared from compounds 7 and 3225 as shown in Scheme 7.
SCHEME 7. Scission of Hyp units in internal positions for the preparation of antimicrobials.
H-Hyp-OCH3 (7)
1) Cbz-Phe-OH, DCM
Boc-Pro-Ile-OMe (32)
TFA, DCM
HBTU, DIPEA
2) NaOH, MeOH
OH
N
H
N
Cbz
NH
HN
O
OCH3
O
OH
O
O
33 (86%)
34 (99%)
DIPEA, HBTU, DCM
HO
N
N
O
NH
O
O
OCH3
HN
O
O
O
35 (89%)
DIB, I2, h DCE, 80 oC
45 min, 64%
X
AcO
N
N
O
NH
O
O
OCH3
O
HN
O
O
36 X = CHO
37 X =
morpholine, Et3N,
DCE; NaBH(OAc)3
86%
O
N
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The coupling of dipeptides 33 and 3426 afforded substrate 3515 in very good yield. The scission of the
tetrapeptide 35 under treatment with DIB and iodine was promoted by heating, in part for solubility
reasons. The reaction afforded the aldehyde 36 (64%), which was readily purified and converted into a
4-morpholino homoalanine derivative 37 using a reductive amination reaction (86%). The morpholino-
containing residue is used in commercial antimicrobials such as cobicistat.23
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In order to study the selectivity of the scission when two or more Hyp units were present, a model
peptide was prepared as shown in Scheme 8 (conversions 7→3927 and 39→40).
SCHEME 8. Site-selective modification of one Hyp unit in an internal position.
In substrate 40 the internal Hyp residue had a free hydroxyl group, while the C-terminal Hyp unit was
protected. When the scission reaction was carried out, only the unprotected Hyp residue was cleaved,
affording the aldehyde 41 in high optical purity. In this case, no heating was necessary to promote the
scission. The cleavage of the internal Hyp unit is interesting from a conformational standpoint, since it
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decreases the system rigidity, can alter elements of the secondary structure (such as turns) and finally,
the presence or removal of the N-acetoxymethyl groups can influence the number of hydrogen bonds.
On the other side, the attachment of the scission products to amino acids or peptides would allow the
creation of new interactions or folding patterns.28 The ligation studies were performed with aldehyde 41
(Scheme 9), which underwent a reductive amination with proline benzyl ester, yielding the tetrapeptide
42 in 83% yield.
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SCHEME 9. Peptide ligation after selective scission of one Hyp unit in an internal position.
H-Pro-OBn
DCE, Et3N, 26°C
10 min; then
CO2Bn
N
OTBS
NaBH(OAc)3, 6 h
AcO
83%
N
N
H
N
O
CO2Me
Cbz
O
O
OTBS
42
AcO
N
N
H
N
O
CO2Me
Cbz
O
O
Boc-HN
41
CO2Me
N
H
( )2
Boc-Lys-Ile-OMe
MeOH, Et3N
26°C, 1h,
Z
N
OTBS
then NaBH4
45°C, 18 h
75%
N
H
N
H
N
O
CO2Me
Cbz
O
43
Z = H
ClCO2Me, THF
NaHCO3 (ac), 97%
44 Z = CO2Me
The reductive amination also allows ligation of two peptide chains. Thus, when the aldehyde 41 was
treated with Boc-Lys-Ile-OMe under reductive conditions, the pentapeptide 43 was obtained in 76%
yield. In order to reduce its polarity and facilitate its manipulation, the peptide was carbamoylated,
affording compound 44 in 97% yield. The creation of libraries of peptides with several chains would
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facilitate the identification of drugs with multiple interactions with the biological target and/or
decreased proteolysis in vivo, as well as the development of peptide catalysts with improved substrate
recognition
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The applications of this methodology to create libraries of antimicrobial and catalytic peptides will be
reported in due course.
Conclusions. Natural and inexpensive hydroxyproline has been introduced as customizable unit for
the generation of a diversity of amino acids with high optical purity, including valuable N-alkyl amino
acids, homoserine lactones, Freidinger-Veber lactams, 4-aminohomoalanines, and -alkylglycines.
Hydroxyproline underwent a mild, metal-free domino scission-oxidation-addition of acetate ions
process, generating a residue with two reactive chains (the -lateral chain and the N-acetoxymethyl
group) which can be transformed independently.
The customizable unit was also used for the mild, site-selective modification of peptides, an strategy
that allowed the creation of peptide libraries from a single (or a few) parent peptides, saving valuable
time and materials in discovery processes. The reactive group in the Hyp unit can be protected with
orthogonal groups, allowing differenciation between several customizable units in the same peptide.
The free residues would be transformed, while protected ones would remain unchanged.
The process is versatile, and thus, when the scission of C-terminal positions was followed by a
reductive amination, a double goal was achieved: the ligation of small peptides and the concomitant
formation of an Agl unit (Freidinger-Veber lactam) to introduce conformational constraints. When the
scission was performed on internal positions of the peptide, the reductive amination provided potential
antimicrobial compounds; moreover, the ligation of two peptide chains gave compounds capable of
multiple interactions with biological or catalysis targets.
On the other side, the manipulation of the N-acetoxymethyl moiety allowed the release of the free
amino group group or the formation of N-methylated units, which in turn allows modulation of the
peptide conformation and activity.
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Experimental Section.
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General Remarks: General Methods. Commercially available reagents and solvents were analytical
grade or were purified by standard procedures prior to use. All reactions involving air- or moisture-
sensitive materials were carried out under nitrogen atmosphere. Three alternative spray reagents for
TLC analysis were used: (a) 0.5% vanillin in H2SO4-EtOH (4:1); (b) 0.25% ninhydrin in ethanol and (c)
Fleet’s reagent [Ce(SO4)2 (0.5 g) and ammonium phosphomolybdate hydrate (2.5 g) in H2SO4 (5 mL)
and water (65 mL)]. Once sprayed, the TLC was heated until development of color.
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Merck silica gel 60 PF254 and 60 (0.063-0.2 mm) were used for rotatory chromatography and column
chromatography, respectively. Melting points were determined with a hot-stage apparatus and are
o
uncorrected. Optical rotations were measured at the sodium line at ambient temperature (26 C). Mass
spectra were carried out using electrospray ionization techniques (ESI) or electronic impact (EI), the
latter was determined at 70 eV using an ion trap mass analyzer. Nuclear Magnetic Resonance spectra
1
were determined at 500 or 400 MHz for H NMR and 125.7 or 100 MHz for 13C NMR in the presence
o
o
of tetramethylsilane (TMS) as internal standard, at 25 C or 70 C, as stated for each case. Due to
rotamer equilibrium, the resolution of some NMR spectra at 26 oC was low (formation of broad bands);
o
1
for those cases, the spectra at 70 C are provided. H NMR references: CDCl3 (H 7.26), CD3OD (H
13
3.31); C NMR references: CDCl3 (C 77.0), CD3OD (C 49.0). Abbreviations (in the NMR spectra):
br b, broad band; br d, broad doublet, etc
The aminoacid and peptide derivatives H-Pro-OBn∙HCl, H-Trp-OEt∙HCl, H-Thr(tBu)-OMe∙HCl, H-
Hyp-OMe∙HCl, H-Pro-OMe∙HCl, Boc-Val-OH, Cbz-Leu-OH, Cbz-Phe-OH and Boc-Thr(Bn)-Ala-Phe-
OH are commercial reagents.
N-[(1R,2S,5R)-Menthyloxycarbonyl]-4R-hydroxy-L-proline methyl ester (8):15 4R-hydroxy-L-
proline methyl ester hydrochloride (0.600 g, 3.3 mmol) was added to a saturated aqueous NaHCO3
o
solution (10.0 mL). After cooling at 0 C, a solution of ()-menthyl chloroformate (0.85 mL, 3.96
mmol) in THF (10.0 mL) was added. The mixture was allowed to reach 26 oC and stirred for 3 h. Then
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it was poured into water and extracted with EtOAc, and the organic layer was dried over sodium sulfate
and filtered. The solvent was removed under vacuum and the residue was purified by column
chromatography (hexane-EtOAc 80:20) to give menthyl carbamate (8) (1.014 g, 94%) as an oil: []D =
99.27 (c 0.37, CHCl3); 1H NMR (500 MHz, CDCl3, 70 oC): H 0.81 (d, J = 7.0 Hz, 3H), 0.800.91 (m,
2H), 0.89 (d, J = 5.9 Hz, 3H), 0.90 (3H, d, J = 6.9 Hz), 1.07 (m, 1H), 1.33 (m, 1H), 1.46 (m, 1H),
1.641.73 (m, 2H), 1.85 (br b, 1H, OH), 1.93 (m, 1H), 2.04 (m, 1H), 2.11 (ddd, J = 5.1, 7.0, 13.3 Hz,
1H), 2.27 (1H, m), 3.52 (m, 1H), 3.67 (dd, J = 4.7, 11.7 Hz, 1H), 3.71 (s, 3H), 4.44 (m, 1H), 4.48 (m,
1H), 4.55 (ddd, J = 4.4, 10.7, 11.0 Hz, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 16.9, 20.7, 21.9,
24.2, 26.8, 31.5, 34.5, 38.8, 41.7, 47.7, 52.0, 54.8, 57.9, 69.4, 75.9, 154.2, 173.1. IR (CHCl3): 3417,
1746, 1694, 1420 cm–1. HRMS (EI) calcd for C17H29NO5 (M+) 327.2046, found 327.2053. Anal. Calcd
for C17H29NO5: C, 62.36; H, 8.93; N, 4.28; found: C, 62.30; H, 8.80; N, 4.35.
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N-[N-(Benzyloxycarbonyl)phenylalanyl]-4R-hydroxy-L-proline methyl ester (9): A solution of
Cbz-Phe-OH (0.700 g, 2.34 mmol) and H-Hyp-OMe•HCl (0.552 g, 3.04 mmol) in dichloromethane (30
mL) at 0°C, was treated with DIPEA (0.8 mL, 4.6 mmol) and HBTU (0.978 g, 2.57 mmol). After 1 h,
the reaction mixture was allowed to reach room temperature and stirred for 1 h. Then it was poured into
saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with 5% aqueous
HCl and with water, dried over anhydrous sodium sulfate, filtered and evaporated under vacuum. The
residue was purified by column chromatography on silica gel (hexane/EtOAc, 40:60), yielding the
1
dipeptide (9) (0.868 g, 87%), as a white foam: []D = 38.0 (c 0.43, CHCl3). H NMR (500 MHz,
CDCl3, 70 oC): H 1.98 (ddd, J = 4.7, 7.9, 12.9 Hz, 1H), 2.08 (br b, 1H), 2.23 (br dd., J = 9.5, 10.7 Hz,
1H), 2.95 (dd, J = 6.3, 13.6 Hz, 1H), 3.10 (dd, J = 7.6, 13.9 Hz, 1H), 3.22 (dd, J = 4.1, 11.0 Hz, 1H),
3.69 (br b, 1H), 3.73 (s, 3H), 4.40 (m, 1H), 4.63 (dd, J = 7.9, 8.2 Hz, 1H), 4.67 (m, 1H), 5.01 (d, J =
13
12.3 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 5.53 (br b, 1H), 7.207.35 (m, 10H). C NMR (125.7 MHz,
CDCl3, 70 oC): C 37.7, 39.1, 52.1, 54.0, 55.2, 57.9, 67.1, 70.3, 127.0, 128.0, 128.5, 129.7, 136.2, 136.5,
170.8, 172.1; one (C) signal corresponding to the carbamate group was not clearly observed. IR
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(CHCl3) 3405, 1742, 1722, 1710, 1641, 1631 cm–1. HRMS (EI) calcd for C23H26N2O6 (M+) 426.1791,
found 426.1800.
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9
General procedure for the scission of 4-hydroxyproline derivatives: A solution of the substrate (1
mmol) in dichloromethane (10 mL) was treated with (diacetoxyiodo)benzene (DIB, 0.644 g, 2.0 mmol)
and iodine (0.127 g, 0.5 mmol). The reaction mixture was irradiated with visible light (80 W tungsten-
filament lamp) at 26 oC until disappearance of the starting material (2.5-4 h); then was poured into 10%
aqueous Na2S2O3, and extracted with CH2Cl2. The organic layer was dried over sodium sulfate and
filtered, then the solvent was removed under vacuum and the residue was purified by column
chromatography (hexane/EtOAc), yielding N-acetoxymethyl-4-oxohomoalanine derivatives.
(2S) N-(Acetoxymethyl)-N-[(1R,2S,5R)-menthyloxycarbonyl]-4-oxo-L-homoalanine methyl ester
(10): Obtained from hydroxyproline derivative (8) (0.327 g, 1.0 mmol) according to the General
Scission Procedure. After 2.5 h, the reaction mixture was extracted and purified by column
chromatography (hexane/EtOAc 85:15), yielding compound (10) (0.308 g, 80%) as a yellowish oil: []D
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= 95.7 (c 0.87, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.78 (d, J = 6.9 Hz, 3H), 0.86 (m, 1H),
0.89 (d, J = 7.3 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.981.10 (m, 2H), 1.37 (m, 1H), 1.47 (m, 1H),
1.641.72 (m, 2H), 1.83 (m, 1H), 2.02 (s, 3H), 2.03 (m, 1H), 2.95 (m, 1H), 3.31 (dd, J = 6.0, 17.9 Hz,
1H), 3.70 (s, 3H), 4.63 (br ddd, J = 4.1, 10.7, 10.8 Hz, 1H), 4.86 (dd, J = 6.6, 6.7 Hz, 1H), 5.40 (d, J =
11.1 Hz, 1H), 5.43 (brd, J = 11.4 Hz, 1H), 9.75 (s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 16.3,
20.5, 20.6, 21.8, 23.7, 26.4, 31.4, 34.2, 41.1, 44.8, 47.4, 52.4, 55.5, 72.4, 77.2, 154.8, 170.2, 170.4,
197.9. IR (CHCl3): 1741, 1707, 1458, 1420 cm–1. HRMS (EI) calcd for C18H28NO7 [M+  Me]
370.1866, found 370.1861. Anal. Calcd for C19H31NO7: C, 59.20; H, 8.11; N, 3.63; found: C, 59.21; H,
8.16; N, 3.70.
N-Acetoxymethyl-N-(N-benzyloxycarbonyl-L-phenylalanyl)-4-oxo-L-homoalanine methyl ester
(11): A solution of the dipeptide Cbz-Phe-Hyp-OMe (9) (86.0 mg, 0.2 mmol) in dry dichloroethane (4
mL) was treated with DIB (130.0 mg, 0.4 mmol) and iodine (51.0 mg, 0.2 mmol). The mixture was
o
stirred at 80 C for 1 h, under irradiation with visible light. Then it was poured into 10% aqueous
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Na2S2O3 and extracted with CH2Cl2. After drying and filtering the organic layer in the usual way, the
solvent was evaporated under vacuum, and the residue was purified by rotatory chromatography
(hexane/EtOAc 70:30), affording the aldehyde 11 (80.0 mg, 82%) as a white foam: []D = 28.6 (c
0.42, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 1.97 (s, 3H), 2.78 (dd, J = 7.2, 18.5 Hz, 1H), 2.95
(dd, J = 6.3, 13.5 Hz, 1H), 3.03 (dd, J = 7.7, 13.6 Hz, 1H), 3.27 (dd, J = 5.2, 18.4 Hz, 1H), 3.62 (s, 3H),
4.76 (dd, J = 6.0, 6.2 Hz, 1H), 5.02 (dd, J = 7.9, 7.8 Hz, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.09 (d, J = 12.4
Hz, 1H), 5.20 (br d, J = 12.1 Hz, 1H), 5.305.45 (m, 2H), 7.107.40 (m, 10H), 9.62 (s, 1H). 13C NMR
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(125.7 MHz, CDCl3, 70 oC): C 20.5, 39.9, 44.0, 52.5, 52.6, 55.7, 67.0, 72.0, 127.2, 128.0, 128.1, 128.5,
128.6, 129.5, 135.9, 136.6, 155.5, 169.6, 170.2, 173.3, 197.9. IR (CHCl3) 3327, 1744, 1721, 1670, 1522
cm–1. HRMS (EI) calcd for C23H25N2O6 (M+  CO2Me) 425.1713, found 425.1698; calcd for C7H7
([PhCH2]+) 91.0548, found 91.0550. Anal. Calcd for C25H28N2O8: C, 61.98; H, 5.83; N, 5.78; found: C,
61.63; H, 5.99; N, 5.94.
N-[(Methyloxycarbonyl]-4R-hydroxy-L-proline methyl ester (12) has been previously reported.29
(2S)-N-Methoxycarbonyl-N-(acetoxymethyl)-4-oxo-L-homoalanine methyl ester (13): Obtained
from (4R) N-methoxycarbonyl-4-hydroxy-L-proline methyl ester (12) (0.203 g, 1.0 mmol) according to
the General Scission Procedure. After 2.5 h, the reaction mixture was extracted and purified by column
chromatography (hexane/EtOAc 80:20), yielding compound (13) (0.212 g, 81%), as a yellowish oil:
1
o
[]D = 84.5 (c 0.35, CHCl3). H NMR (500 MHz, CDCl3, 70 C): H 2.04 (s, 3H), 3.02 (m, 1H), 3.30
(dd, J = 5.7, 18.3 Hz, 1H), 3.72 (s, 3H), 3.75 (s, 3H), 4.90 (dd, J = 5.7, 5.7 Hz, 1H), 5.43 (s, 2H), 9.75
(s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 20.7, 44.7, 52.7, 53.4, 55.6, 72.5, 155.6, 170.2, 170.6,
198.0. IR (CHCl3): 1728, 1480, 1441 cm–1. HRMS (EI) calcd for C8H12NO6 [M+  CH2CHO] 218.0665,
found 218.0657. Anal. Calcd for C10H15NO7: C, 45.98; H, 5.79; N, 5.36; found: C, 45.67; H, 5.91; N,
5.46.
General procedure for the reductive amination-lactamization: A solution of aldehyde (0.20
mmol) in dry methanol (3.0 mL) was treated with the amine (0.28 mmol) and triethylamine (38.0 µL,
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0.27 mmol). After 1 h at 26 ºC, sodium borohydride (10.0 mg, 0.26 mmol) was added and the reaction
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mixture was warmed to 45 C for 20 h. Then the mixture was allowed to reach room temperature and
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poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and filtered. The solvent was evaporated under vacuum and the residue was
purified by rotatory chromatography (hexane/EtOAc), yielding the corresponding -amino lactam.
(S) 1-Allyl-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (14): A solution of aldehyde 13 (53.0
mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with allylamine (20.0 µL, 0.27 mmol) following
the General procedure of reductive amination-lactamization. The residue was purified by rotatory
chromatography (hexane/EtOAc 40:60), yielding lactam 14 (31.0 mg, 77%) as a white solid: m.p.
9294 ºC (EtOAc/hexane); []D = 9.2 (c 0.3, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.89 (m,
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1H), 2.61 (m, 1H), 3.253.35 (m, 2H), 3.67 (s, 3H), 3.86 (dd, J = 6.2, 15.2 Hz, 1H), 3.93 (dd, J = 6.0,
15.2 Hz, 1H), 4.22 (dd, J = 9.2, 9.3 Hz, 1H), 5.18 (dddd, J = 1.3, 1.3, 1.4, 8.8 Hz, 1H), 5.205.22 (m,
o
1H), 5.52 (br b, 1H), 5.70 (m, 1H). 13C NMR (125.7 MHz, CDCl3, 26 C): C 27.7, 43.6, 45.8, 52.2,
52.8, 118.4, 131.6, 157.0, 171.8. IR (CHCl3) 3426, 1722, 1695, 1513 cm–1. HRMS (EI) calcd for
C9H14N2O3 [M+] 198.1004, found 198.1011; calcd for C7H9NO (M+ NH2CO2Me) 123.0684, found
123.0682. Anal. Calcd for C9H14N2O3: C, 54.53; H, 7.12; N, 14.13; found: C, 54.35; H, 7.20; N, 14.36.
(S) 1-Benzyl-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (15): A solution of aldehyde 13 (53.0
mg, 0.20 mmol) in dry methanol (3 mL) was treated with benzylamine (30.0 µL, 0.28 mmol) following
the General procedure of reductive amination-lactamization. The residue was purified by rotatory
chromatography (hexane/EtOAc 50:50), yielding lactam 15 (39.0 mg, 77%) as a white solid: m.p. 121-
123 ºC (EtOAc/hexane); []D = 7.96 (c 0.33, CHCl3). 1H NMR (500 MHz, CDCl3 with TMS, 26 oC):
H 1.86 (m, 1H), 2.61 (m, 1H), 3.183.25 (m, 2H), 3.70 (s, 3H), 4.26 (m, 1H), 4.46 (d, J = 15 Hz, 1H),
13
4.51 (d, J = 14.7 Hz, 1H), 5.45 (br b, 1H, NH), 7.23 (br.d., J = 7.8 Hz, 2H), 7.277.36 (m, 3H). C
o
NMR (125.7 MHz, CDCl3, 26 C): C 27.7, 43.3, 47.1, 52.2, 52.8, 127.7, 128.0, 128.7, 135.7, 157.0,
171.9. IR (CHCl3) 1722, 1697, 1510 cm–1. HRMS (EI) calcd for C13H16N2O3 [M+] 248.1161, found
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248.1162; calcd for C11H11NO (M+  NH2CO2Me) 173.0841, found 173.0846. Anal. Calcd for
C13H16N2O3: C, 62.89; H, 6.50; N, 11.28; found: C, 63.11; H, 6.75; N, 10.95.
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(1'R,3S) 1-[1'-(2-Naphthalenyl)ethyl)-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (16): A
solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with (R)-(+)-1-(2-
naphthyl)ethylamine (48.0 mg, 0.28 mmol) and triethylamine (38.0 µL, 0.27 mmol). After stirring for 1
h at 26 ºC, sodium borohydride (10.0 mg, 0.26 mmol) was added, and then the mixture was heated to 45
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ºC for 20 h. After cooling to 26 C, it was poured into water and extracted with EtOAc. The organic
layer was dried and filtered as usual, the solvent was evaporated under vacuum and the residue was
purified by rotatory chromatography (hexane/EtOAc 50:50), yielding lactam 16 (42.0 mg, 66%) as a
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white solid: m.p. 148150 ºC (EtOAc/hexane); []D = +217.9 (c 0.36, CHCl3). H NMR (500 MHz,
CDCl3, 26 oC): H 1.66 (d, J = 7.1 Hz, 3H), 1.85 (m, 1H), 2.52 (m, 1H), 2.85 (ddd, J = 6.8, 6.8, 9.8 Hz,
1H), 3.32 (dd, J = 9.3, 9.7 Hz, 1H), 3.69 (s, 3H), 4.22 (m, 1H), 5.56 (br.b., 1H), 5.64 (ddd, J = 7.0, 7.0,
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7.1 Hz, 1H), 7.35 (dd, J = 1.5, 8.6 Hz, 1H), 7.457.52 (m, 2H), 7.72 (s, 1H), 7.787.88 (m, 3H). C
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NMR (125.7 MHz, CDCl3, 26 C): C 15.9, 27.7, 39.4, 50.0, 52.2, 53.2, 125.5, 126.1, 126.3, 127.6,
127.9, 128.5, 132.8, 133.1, 136.9, 157.0, 171.6. IR (CHCl3): 3426, 3018, 1721, 1688, 1511 cm–1. HRMS
(EI) calcd for C18H20N2O3 [M+] 312.1474, found 312.1472. Anal. Calcd for C18H20N2O3: C, 69.21; H,
6.45; N, 8.97; found: C, 68.96; H, 6.42; N, 8.79.
(1'S,3S)
1-[1'-(2-Naphthalenyl)ethyl)-3-(N-methoxycarbonyl)amino-2-pyrrolidinone
(17):
Obtained from aldehyde 13 (53.0 mg, 0.20 mmol) and (S)-()-1-(2-naphthyl)ethylamine (48.0 mg, 0.28
mmol) according to the previous procedure. After work-up and rotatory chromatography purification
(hexane/EtOAc 50:50), lactam 17 was obtained (45.0 mg, 68%) as a slighty yellowish solid: m.p. 194-
197 ºC (EtOAc/hexane); []D = 207.8 (c 0.34, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.63 (d,
J = 7.1 Hz, 3H), 1.70 (m, 1H), 2.58 (m, 1H), 2.99 (dd, J = 9.3, 9.4 Hz, 1H), 3.28 (ddd, J = 6.4, 10.0,
10.0 Hz, 1H), 3.70 (s, 3H), 4.30 (m, 1H), 5.50 (br. b., 1H), 5.62 (ddd, J = 7.0, 7.1, 7.1 Hz, 1H), 7.39 (dd,
J = 1.7, 8.6 Hz, 1H), 7.457.51 (m, 2H), 7.73 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.797.84 (m, 2H). 13C
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NMR (125.7 MHz, CDCl3, 26 C): C 16.2, 28.2, 39.3, 49.8, 52.3, 53.3, 125.4, 125.5, 126.2, 126.4,
127.6, 128.0, 128.6, 132.8, 133.2, 137.1, 157.1, 171.6. IR (CHCl3): 3424, 1724, 1692 cm–1. HRMS (EI)
calcd for C18H20N2O3 [M+] 312.1474, found 312.1474.
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Experiments to determine the stereoselectivity of the process. The pure isomers 16 and 17 do not
only have different spectroscopic and physical data, but also present different Rf (0.45 for 16 and 0.39
for 17. Therefore, in case that both isomers were formed in significant amounts, they could be separated
by chromatography. Careful purification of the mixture yielded only one diastereomer in each case.
However, in the crude product mixture, traces of a minor diastereomer could be detected (dr ≥ 96:4).
In order to check the accuracy of the 1H NMR to determine the diastereomer ratio by integration of the
signals, mixtures of the purified isomers 16 and 17 were prepared, by weighting different amounts of
1
the pure isomers, and mixing them in CDCl3. H NMR spectra of the 85:15, 90:10, 95:5 and 98:2
(16):(17)-mixtures were recorded, and we checked that the integral ratio corresponded to the real isomer
1
ratio. In the 98:2 mixture the minor isomer could hardly be detected by H NMR, due to NMR
sensitivity limit (although the sensitivity is often superior to other detection techniques).30 In the next
section (NMR spectra) records of these experiments are displayed.
(2S,3S)-3-(terc-Butoxy)-2-((S)-3-((methoxycarbonyl)amino)-2-oxo-1-pyrrolidinyl)butanoate
(18): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with H-
Thr(OtBu)-OMe•HCl (64.0 mg, 0.28 mmol), following the General procedure of reductive amination-
lactamization. The residue was purified by rotatory chromatography (hexane/EtOAc 50:50), yielding
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lactam 18 (39.0 mg, 59%) as a yellowish oil. []D = +34.0 (c 0.32, CHCl3). H NMR (500 MHz,
CDCl3, 26 oC): H 1.12 (d, J = 6.3 Hz, 3H), 1.15 (s, 9H), 1.88 (m, 1H), 2.64 (ddd, J = 6.0, 6.7, 12.3 Hz,
1H), 3.52 (ddd, J = 6.1, 10.6, 10.7 Hz, 1H), 3.69 (s, 3H), 3.70 (s, 3H), 3.86 (br dd., J = 8.8, 10.7 Hz,
1H), 4.31 (m, 1H), 4.42 (dddd, J = 2.9, 6.3, 6.5, 6.6 Hz, 1H), 4.77 (d, J = 2.9 Hz, 1H), 5.16 (br b, 1H).
o
13C NMR (125.7 MHz, CDCl3, 26 C): C 20.7, 28.7, 29.8, 44.4, 51.8, 52.2, 52.6, 60.4, 68.3, 74.2,
157.1, 169.7, 173.7. IR (CHCl3) 1729, 1703, 1698 cm–1. HRMS (EI) calcd for C14H23N2O6 [M+  Me]
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315.1556, found 315.1551; calcd for C8H10N2O4 (M+  OMe  CH(Me)OtBu) 198.0641, found
198.0640. Anal. Calcd for C15H26N2O6: C, 54.53; H, 7.93; N, 8.48; found: C, 54.60; H, 8.08; N, 8.42.
Synthesis of compound (19) and its precursor (45). N2-(tert-Butoxycarbonyl)-N6-
(benzyloxycarbonyl)-L-lysyl-L-isoleucine methyl ester (45): A solution of Boc-Lys(Cbz)-OH (21)
(1.00 g, 2.6 mmol) and H-Ile-OMe·HCl (0.614 g, 3.38 mmol) in dichloromethane (30 mL) at 0 °C was
treated with DIPEA (0.89 mL, 5.1 mmol), and HBTU (1.096 g, 2.89 mmol). The reaction mixture was
stirred at 0 °C for 1 h and then was allowed to reach room temperature and stirred for 1 h more. Then it
was poured over saturated aqueous NaHCO3, and the organic layer was extracted with EtOAc and
washed with 10% aqueous HCl. The organic layer was dried and filtered as usual, and the solvent was
removed under vacuum. The residue was purified by column chromatography on silica gel
(hexane/EtOAc, 60:40), yielding dipeptide 45 (1.145 g, 87%), as a white foam: []D = 8.0 (c 0.36,
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CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.89 (dd, J = 6.5, 6.7 Hz, 3H), 0.91 (d, J = 6.4 Hz, 3H),
1.18 (m, 1H), 1.341.47 (m, 3H), 1.42 (s, 9H), 1.471.57 (m, 2H), 1.62 (m, 1H), 1.82 (m, 1H), 1.89 (m,
1H), 3.123.22 (m, 2H), 3.68 (s, 3H), 4.10 (m, 1H), 4.55 (d, J = 5.0, 8.6 Hz, 1H), 5.08 (br b, 1H, NH),
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5.09 (br s, 2H), 5.23 (br b, 1H, NH), 6.69 (br b, 1H, NH), 7.257.35 (m, 5H). C NMR (125.7 MHz,
CDCl3, 26 oC): C 11.5, 15.5, 22.5, 25.0, 28.3, 29.4, 31.6, 37.7, 40.5, 52.1, 54.3, 56.5, 66.6, 80.0, 128.1,
128.5, 136.7, 155.8, 156.6, 172.0, 172.3. IR (CHCl3) 3324, 1722, 1703, 1694, 1666, 1530 cm–1. HRMS
(EI) calcd for C26H41N3O7 [M+] 507.2945, found 507.2939; calcd for C7H7 ([PhCH2]+) 91.0548, found
91.0545. Anal. Calcd for C26H41N3O7: C, 61.52; H, 8.14; N, 8.28; found: C, 61.15; H, 8.42; N, 8.14.
N2-(tert-Butoxycarbonyl)-L-lysyl-L-isoleucine methyl ester (19): A solution of the dipeptide 45
(1.00 g, 1.97 mmol) in methanol (30 mL) was treated with 10% Pd/C (200 mg), and the reaction
mixture was stirred under hydrogen for 15 h. Then it was filtered through celite, and the solvent was
evaporated under vacuum, affording the dipeptide 19 (0.685 g, 93%), as a yellowish oil: []D = 15.0 (c
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0.35, CHCl3). H NMR (500 MHz, CD3OD, 26 C): H 0.93 (d, J = 7.2 Hz, 3H), 0.95 (dd, J = 6.4, 6.8
Hz, 3H), 1.27 (m, 1H), 1.401.55 (m, 5H), 1.46 (s, 9H), 1.61 (m, 1H), 1.75 (m, 1H), 1.91 (m, 1H), 2.68
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(dd, J = 7.1, 7.1 Hz, 2H), 3.73 (s, 3H), 4.09 (br dd, J = 6.5, 7.1 Hz, 1H), 4.41 (d, J = 5.9 Hz, 1H). C
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C18H35N3O5Na ([M  Na]+) 396.2474, found 396.2463.
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N2-(Methoxycarbonyl)-2-(4-(2-oxo-1-pyrrolidinyl)butyl)-L-glycyl-L-isoleucine methyl ester (20):
A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with Boc-Lys-
Ile-OMe (19) (105.0 mg, 0.28 mmol), following the General procedure of reductive amination-
lactamization. The residue was purified by rotatory chromatography (hexane/EtOAc, 10:90), affording
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lactam 20 (84.0 mg, 82 %) as a yellowish oil: []D = 12.5 (c 0.38, CHCl3). H NMR (500 MHz,
CDCl3, 70 oC): H 0.86 (dd, J = 7.4, 7.5 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H), 1.16 (m, 1H), 1.32 (m, 2H),
1.39 (m, 9H), 1.40 (m, 1H), 1.54 (m, 2H), 1.58 (m, 1H), 1.751.87 (m, 3H), 2.54 (m, 1H), 3.223.33
(m, 4H), 3.62 (s, 3H), 3.67 (s, 3H), 4.00 (m, 1H), 4.16 (br dd, J = 8.2, 8.3 Hz, 1H), 4.47 (dd, J = 5.1, 8.5
Hz, 1H), 5.13 (br d, J = 7.6 Hz, 1H), 5.52 (br b, 1H), 6.68 (br d, J = 8.2 Hz, 1H). 13C NMR (125.7 MHz,
CDCl3, 70 oC): C 11.3, 15.4, 22.4, 25.1, 26.4, 27.8, 28.2, 31.4, 37.7, 42.4, 43.8, 51.7, 52.0, 52.9, 54.6,
56.6, 79.9, 155.7, 157.0, 171.8, 172.0. IR (CHCl3) 3306, 2965, 1721, 1708, 1690, 1681, 1668, 1530 cm–
1. HRMS (EI) calcd for C24H42N4O8 (M+) 514.3003, found 514.3013.
Methyl 2-(3S-[N-(benzyloxycarbonyl)phenylalanyl]amino-2-oxo-1-pyrrolidinyl)acetate (22): A
solution of aldehyde 11 (50.0 mg, 0.10 mmol) in dry methanol (2.0 mL) was treated with H-Gly-
OMe•HCl (18.0 mg, 0.14 mmol) and Et3N (20.0 L, 14 mmol), following the General Procedure for
reductive amination-lactamization. After the usual work-up, the residue was purified by rotatory
chromatography (hexane/EtOAc, 30:70), affording product 22 (30.0 mg, 64%), as a yellowish foam:
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6.4, 12.5 Hz, 1H), 3.06 (dd, J = 7.0, 14.1 Hz, 1H), 3.11 (dd, J = 6.4, 14.0 Hz, 1H), 3.34 (dd, J = 8.4, 9.2
Hz, 1H), 3.46 (ddd, J = 6.8, 9.3, 9.4 Hz, 1H), 3.72 (s, 3H), 3.95 (d, J = 17.5 Hz, 1H), 4.12 (d, J = 17.5
Hz, 1H), 4.31 (m, 1H), 4.44 (ddd, J = 6.9, 6.9, 7.5 Hz, 1H), 5.05 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4
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Hz, 1H), 5.30 (d, J = 7.1 Hz, 1H), 6.41 (br b, 1H), 7.177.32 (m, 10H). 13C NMR (125.7 MHz, CDCl3,
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70 oC): C 27.6, 38.7, 44.5, 44.9, 51.4, 52.2, 56.3, 67.1, 127.1, 128.0, 128.1, 128.5, 128.7, 129.5, 136.4,
136.5, 155.9, 168.6, 171.6, 172.3. IR (CHCl3) 3296, 1744, 1703, 1698, 1680, 1537 cm–1. HRMS (EI)
calcd for C24H27N3O6 (M+) 453.1900, found 453.1885; calcd for C7H7 ([PhCH2]+) 91.0548, found
91.0545. Anal. Calcd for C24H27N3O6: C, 63.56; H, 6.00; N, 9.27; found: C, 63.47; H, 6.28; N, 9.56.
2-(4-(3R-(N-Benzyloxycarbonyl-L-phenylalanyl)amino-2-oxo-1-pyrrolidinyl)butyl)-2-N-(tert-
butoxycarbonyl)-L-glycyl-L-isoleucine methyl ester (23): A solution of the aldehyde 11 (50.0 mg,
0.10 mmol) in dry methanol (2.0 mL), was treated with Boc-Lys-IIe-OMe (19) (53.0 mg, 0.14 mmol)
and Et3N (20.0 L, 0.14 mmol), following the General Procedure for reductive amination-lactamization.
After the usual work-up, the residue was purified by rotatory chromatography (hexane/EtOAc, 80:20),
affording product 23 (59.0 mg, 78%), as a yellowish foam: []D = 18.0 (c 0.36, CHCl3). 1H NMR (500
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MHz, CDCl3, 70 C): H 0.88 (dd, J = 7.4, 7.6 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 1.20 (m, 1H), 1.30-
1.50 (m, 3H), 1.42 (s, 9H), 1.51-1.69 (m, 3H), 1.78-1.90 (m, 3H), 2.54 (m, 1H), 3.03 (dd, J = 7.2, 14
Hz, 1H), 3.14 (dd, J = 6.0, 14 Hz, 1H), 3.18 (m, 1H), 3.21-3.31 (m, 2H), 3.42 (m, 1H), 3.70 (s, 3H),
4.04 (m, 1H), 4.30 (m, 1H), 4.49 (m, 1H), 4.52 (dd, J = 5.1, 8.5 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 5.08
(d, J = 12.4 Hz, 1H), 5.18 (br b, 1H, NH), 5.52 (br b, 1H, NH), 6.73 (br d, J = 7.7 Hz, 1H), 6.91 (br b,
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1H, NH), 7.16-7.33 (m, 10H); 13C NMR (125.7 MHz, CDCl3, 70 C): C 11.4, 15.5, 22.3, 25.2, 26.3,
27.1, 28.3, 31.4, 37.8, 38.8, 42.2, 44.0, 51.7, 51.9, 54.8, 56.7, 66.9, 80.1, 126.8, 127.8, 127.9, 128.4,
128.5, 129.4, 136.5, 136.6, 155.8, 171.6, 171.9, 172.0, 172.0. IR (CHCl3) 3306, 1712, 1694, 1682, 1668,
1652, 1660, 1538 cm–1. HRMS (EI) calcd for C39H55N5O9Na (M+  Na) 760.3879, found 760.3885.
Anal. Calcd for C39H55N5O9: C, 63.48; H, 7.51; N, 9.49; found: C, 63.54; H, 7.71; N, 9.80.
N-[(1R,2S,5R)-menthyloxycarbonyl]-N-methyl-L-homoserine lactone (24): To a solution of
aldehyde 10 (39.0 mg, 0.10 mmol) in dry dichloromethane (2.0 mL) were added BF3·OEt2 (25.0 µL, 0.2
mmol) and Et3SiH (40.0 µL, 0.25 mmol). The mixture was stirred at 26 ºC for 16 h, and then was
poured into saturated aqueous NaHCO3 and extracted with dichloromethane. After drying and
evaporating, the residue was purified by rotatory chromatography (hexane/EtOAc, 80:20), yielding the
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NMR (500 MHz, CDCl3, 70 C): H 0.81 (d, J = 7.0 Hz, 3H), 0.87 (m, 1H), 0.91 (d, J = 6.7 Hz, 3H),
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0.92 (d, J = 7.0 Hz, 3H), 0.98 (ddd, J = 11.4, 11.7, 12 Hz, 1H), 1.08 (dddd, J = 3.2, 12.6, 12.9, 13.0 Hz,
1H), 1.39 (br dd, J = 11.0, 12.0 Hz, 1H), 1.50 (m, 1H), 1.661.73 (m, 2H), 1.92 (m, 1H), 2.09 (m, 1H),
2.38 (m, 1H), 2.45 (m, 1H), 2.92 (s, 3H), 4.23 (ddd, J = 7.0, 9.5, 9.8 Hz, 1H), 4.43 (ddd, J = 2.6, 9.2, 9.2
Hz, 1H), 4.63 (ddd, J = 4.4, 10.7, 11.1 Hz, 1H), 4.71 (br dd, J = 9.2, 9.8 Hz, 1H). 13C NMR (125.7
MHz, CDCl3, 70 oC): C 16.6, 20.8, 21.9, 24.0, 26.0, 26.6, 31.5, 32.4, 34.5, 41.6, 47.7, 56.4, 65.1, 76.6,
156.1, 173.4. HRMS (EI) calcd for C16H27NO4 [M+] 297.1940, found 297.1946. Anal. Calcd for
C16H27NO4: C, 64.62; H, 9.15; N, 4.71; found: C, 64.39; H, 9.14; N, 4.67.
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N-(Methoxycarbonyl)-L-homoserine lactone (25): A solution of aldehyde 13 (53 mg, 0.20 mmol)
in dry methanol (3.0 mL) was treated with sodium borohydride (10.0 mg, 0.26 mmol) and stirred at 26
oC for 4 h. Then the reaction mixture was poured into water and extracted with EtOAc. The organic
layer was dried, filtered and concentrated under vacuum. The residue was purified by rotatory
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NMR (500 MHz, CDCl3, 26 C): H 2.22 (m, 1H), 2.74 (m, 1H), 3.70 (s, 3H), 4.25 (m, 1H), 4.20-4.30
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(m, 2H), 5.44 (br b, 1H, NH). C NMR (125.7 MHz, CDCl3, 26 C): C 30.3, 50.5, 52.6, 65.7, 156.7,
175.0. HRMS (EI) calcd for C6H9NO4 (M+) 159.0532, found 159.0531; calcd for C5H9NO2 (M+  CO2)
115.0633, found 115.0634. Anal. Calcd for C6H9NO4: C, 45.28; H, 5.70; N, 8.80; found: C, 45.13; H,
5.80; N, 8.56.
N-(N-Benzyloxycarbonyl-L-prolyl)-4R-hydroxy-L-proline methyl ester (26): A solution of Cbz-
Pro-OH (0.600 g, 2.41 mmol) and H-Hyp-OMe·HCl (0.479 g, 2.65 mmol) in dichloromethane (30.0
mL) at 0 °C, was treated with DIPEA (0.82 mL, 4.71 mmol), HOBt·H2O (0.404 g, 2.64 mmol) and
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EDAC (0.506 g, 2.64 mmol). After 1 h at 0 C, the reaction mixture was allowed to reach room
temperature and stirred for 20 h. Then it was poured into saturated aqueous NaHCO3 and extracted with
EtOAc. The organic layer was washed with 10% aqueous HCl and with water, dried over anhydrous
sodium sulfate, filtered and evaporated under vacuum. The residue was purified by column
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chromatography on silica gel (hexane/EtOAc, 40:60), yielding the dipeptide Cbz-Pro-Hyp-OMe (26)
(0.753 g, 83%), as a white foam: []D = 98.9 (c 0.18, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC) 3:1
rotamer mixture: H 1.88 (m, 1H), 1.96 (ddd, J = 4.6, 8.5, 13.3 Hz, 1H), 1.982.20 (m, 3H), 2.34 (m,
1H), 3.50 (m, 1H), 3.543.68 (m, 2H), 3.70/3.71 (s, 3H), 4.01 (d, J = 10.9 Hz, 1H), 4.434.53 (m, 2H),
4.70 (dd, J = 8.3, 8.3 Hz, 1H), 5.03/5.04 ([d, J = 12.5 Hz/ d, J = 12.4 Hz], 1H), 5.10/5.14 ([d, J = 12.3
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Hz/ d, J = 12.5 Hz], 1H), 7.30-7.34 (m, 5H). 13C NMR (125.7 MHz, CDCl3, 26 C): C 23.5/24.3,
29.3/30.1, 37.0/37.6, 46.9/47.2, 52.3, 54.4/54.9, 57.6, 57.9, 67.0/67.2, 70.3/70.5, 127.7, 128.0, 128.5,
136.5/136.9, 154.2/155.2, 171.3/171.6, 172.6/172.9. IR (CHCl3) 3440, 1744, 1684, 1658, 1449, 1436
cm–1. HRMS (EI) calcd for C19H24N2O6 (M+) 376.1634, found 376.1628; calcd for C7H7 ([OCH2Ph]+)
91.0548, found 91.0552. Anal. Calcd for C19H24N2O6: C, 60.63; H, 6.43; N, 7.44; found: C, 60.31; H,
6.53; N, 7.28.
N-(N-Benzyloxycarbonyl-L-prolyl)-L-homoserine lactone (27): Obtained from the dipeptide (26)
(0.376 g, 1.0 mmol) using the General procedure of radical scission-oxidation. After 4 h, the reaction
mixture underwent the usual work-up, yielding a gum which contained the aldehyde intermediate [N-
(N-benzyloxycarbonyl-L-prolyl)-N-acetoxymethyl-4-oxo-L-homoalanine methyl ester (28)]. This
aldehyde was not purified, but dissolved in dry methanol (8 mL) and treated with sodium borohydride
(50.0 mg, 1.2 mmol). The reaction mixture was warmed to 45 °C for 20 h, and then was cooled to room
temperature, poured into water and extracted with EtOAc. The organic layer was dried, filtered and
concentrated as usual, and the residue was purified by rotatory chromatography (hexane/EtOAc, 50:50),
affording the lactone 27 (0.169 g, 51% for the two steps), as a yellowish foam: []D = 68.8 (c 0.2,
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CHCl3). H NMR (500 MHz, CDCl3, 70 C): H 1.89 (m, 1H), 1.942.14 (m, 3H), 2.28 (m, 1H), 2.65
(m, 1H), 3.483.58 (m, 2H), 4.20 (ddd, J = 6.3, 9.3, 10.6 Hz, 1H), 4.324.44 (m, 3H), 5.13 (d, J = 12.4
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Hz, 1H), 5.18 (d, J = 12.4 Hz, 1H), 6.95 (br b, 1H, NH), 7.277.40 (m, 5H). C NMR (125.7 MHz,
CDCl3, 70 oC): C 24.3, 29.8, 47.3, 49.2, 60.6, 65.7, 67.5, 128.0, 128.1, 128.5, 136.5, 172.5, 174.5; one
signal, corresponding to the carbamate, was not clearly observed. IR (CHCl3) 3419, 1783, 1729, 1688
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cm–1. HRMS (EI) calcd for C17H20N2O5 (M+) 332.1372, found 332.1375; calcd for C7H7 ([CH2Ph]+)
91.0548, found 91.0545.
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N-(N-Fluorenylmethyloxycarbonyl-L-leucyl)-4R-hydroxy-L-proline methyl ester (29): To a
solution of Fmoc-Leu-OH (700 mg, 1.98 mmol) and H-Hyp-OMe·HCl (400 mg, 2.20 mmol) in
dichloromethane (30 mL) at 0°C were added DIPEA (0.67 mL, 3.85 mmol), HOBt (333 mg, 2.17
mmol) and EDAC (417 mg, 2.17 mmol). The reaction mixture was stirred at 0°C for 1 h and then was
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allowed to reach 26 C and was stirred for other 20 h. Then it was poured into saturated aqueous
NaHCO3, and the organic layer was washed with 10% aqueous HCl. After usual drying and solvent
evaporation, the residue was purified by column chromatography (hexane/EtOAc, 60:40), yielding the
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dipeptide 29 (771 mg, 81%), as a white foam: []D = 58.13 (c 0.16, CHCl3). H NMR (400 MHz,
CDCl3, 70 oC): H 0.96 (d, J = 6.7 Hz, 3H, Me), 0.98 (d, J = 6.5 Hz, 3H), 1.501.65 (m, 2H), 1.75 (m,
1H), 2.01 (m, 1H), 2.31 (br dd, J = 8.2, 13.4 Hz, 1H), 2.89 (br b, 1H, OH), 3.603.75 (m, 1H), 3.72 (s,
3H), 3.91 (d, J = 11.1 Hz, 1H), 4.17 (dd, J = 7.2, 7.5 Hz, 1H), 4.254.40 (m, 2H), 4.454.55 (m, 2H),
4.66 (dd, J = 8.2, 8.4 Hz, 1H), 5.59 (d, J = 8.5 Hz, 1H), 7.29 (dd, J = 7.4, 7.5 Hz, 2H), 7.38 (dd, J = 7.5,
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7.5 Hz, 2H), 7.557.57 (m, 2H), 7.74 (d, J = 7.5 Hz, 2H). C NMR (100.6 MHz, CDCl3, 26 C): C
22.0, 23.1, 24.5, 37.5, 41.6, 47.1, 50.9, 52.2, 55.2, 57.6, 67.2, 70.3, 119.9, 125.1, 127.1, 127.7, 141.3,
143.7/143.8, 156.5, 171.8, 172.4. IR (CHCl3): 3430, 1745, 1711, 1650, 1511 cm–1. HRMS (EI) calcd for
C27H32N2O6 [M+], 480.2260, found 480.2242. Anal. Calcd for C27H32N2O6: C, 67.48; H, 6.71; N, 5.83;
found: C, 67.78; H, 6.67; N, 5.55.
N-Acetoxymethyl-N-(N-fluorenylmethyloxycarbonyl-L-leucyl)-4-oxo-L-homoalanine
methyl
ester (30): Obtained from dipeptide 29 (0.480 g, 1.0 mmol) according to the General Scission
Procedure. After 4 h, the reaction mixture was extracted and purified by column chromatography
(hexane/EtOAc 70:30), yielding compound 30 (0.339 g, 63%), as a yellowish gum: []D = 35.74 (c
0.15, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.94 (d, J = 6.7 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H),
1.51 (m, 2H), 1.68 (m, 1H), 2.09 (s, 3H), 3.03 (dd, J = 7.5, 18.8 Hz, 1H), 3.39 (dd, J = 5.3, 18.6 Hz,
1H), 3.66 (s, 3H), 4.22 (dd, J = 7.1, 7.2 Hz, 1H), 4.36 (dd, J = 7.1, 10.2 Hz, 1H), 4.41 (dd, J = 7.2, 10.5
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Hz, 1H), 4.804.92 (m, 2H), 5.34 (d, J = 8.9 Hz, 1H), 5.46 (d, J = 12.2 Hz, 1H), 5.64 (d, J = 12.3 Hz,
1H), 7.31 (dd, J = 7.5, 7.5 Hz, 2H), 7.40 (dd, J = 7.3, 7.6 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.76 (d, J =
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7.6 Hz, 2H), 9.75 (s, 1H). C NMR (125.7 MHz, CDCl3, 26 C): C 20.6, 21.7, 23.3, 24.7, 42.7, 43.9,
47.2, 49.8, 52.7, 55.5, 67.0, 72.1, 120.0, 125.1, 127.1, 127.7, 141.3, 143.8, 155.9, 169.7, 170.4, 174.7,
198.4. IR (CHCl3): 3430, 1745, 1724, 1669, 1570 cm–1. HRMS (EI) calcd for C29H34N2O8 [M+]
538.2315, found 538.2332. Anal. Calcd for C29H34N2O8: C, 64.67; H, 6.36; N, 5.20; found: C, 64.51; H,
6.19; N, 5.54.
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Dimethyl N-Acetoxymethyl-N-(N-fluorenylmethyloxycarbonyl-L-leucyl)-N-acetoxymethyl-4,5-
dehydro-L-homoglutamate (31): A solution of methyl 2-(dimethoxyphosphoryl)acetate (27.0 mg, 0.15
mmol) in dry THF (1.0 mL) was slowly added to a suspension of sodium hydride (60 % in mineral oil,
6.0 mg, 0.15 mmol) in dry THF (1.5 mL) at 10 °C. After stirring for 45 minutes, a solution of aldehyde
30 (54.0 mg, 0.10 mmol) in dry THF (1 mL) was added dropwise, and the mixture was stirred at 10 °C
for 35 min. Then it was poured into water and extracted with diethyl ether. The organic layer was dried,
filtered and evaporated as usual, and the residue was purified by rotatory chromatography
(hexane/EtOAc, 80:20), yielding product 31 (49.0 mg, 82%) as a yellowish syrup: []D = 22.30 (c
0.38, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.94 (d, J = 6.7 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H),
1.421.56 (m, 2H), 1.70 (m, 1H), 2.10 (s, 3H), 2.92 (dd, J = 7.3, 7.4 Hz, 2H), 3.67 (s, 3H), 3.68 (s, 3H),
4.21 (dd, J = 7.2, 7.2 Hz, 1H), 4.34 (dd, J = 7.0, 10.6 Hz, 1H), 4.40 (dd, J = 7.4, 10.6 Hz, 1H), 4.55 (dd,
J = 7.0, 8.3 Hz, 1H), 4.83 (ddd, J = 4.2, 9.1, 9.4 Hz, 1H), 5.34 (d, J = 9.1 Hz, 1H), 5.43 (d, J = 12.4 Hz,
1H), 5.55 (d, J = 12.4 Hz, 1H), 5.85 (d, J = 15.6 Hz, 1H), 6.80 (ddd, J = 7.6, 8.1, 15.6 Hz, 1H), 7.30 (dd,
J = 7.5, 7.5 Hz, 2H), 7.39 (dd, J = 7.4, 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.6 Hz, 2H).
13C NMR (125.7 MHz, CDCl3, 26 oC): C 20.6, 21.6, 23.2, 24.7, 31.6, 42.9, 47.2, 49.9, 51.4, 52.6, 59.6,
67.1, 71.7, 120.0, 124.0, 125.1, 127.0, 127.7, 141.3, 143.8, 143.9, 156.5, 166.3, 169.9, 170.5, 174.9. IR
(CHCl3): 3431, 1745, 1721, 1674, 1511 cm–1. HRMS (EI) calcd for C32H38N2O9 [M+] 594.2577, found
594.2573. Anal. Calcd for C32H38N2O9: C, 64.63; H, 6.44; N, 4.71; found: C, 64.75; H, 6.68; N, 4.88.
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