Metal-Free, Site-Selective Peptide Modification by Conversion of “Customizable” Units into β-Substituted Dehydroamino Acids

Article abstract of DOI:10.1002/chem.201703758

Our site-selective modification of serine or threonine units in peptides allows the generation of β-substituted dehydroamino acids, which increase peptide resistance to hydrolysis and may improve their biological properties. Both the terminal and internal positions can be modified, and different customizable units can be activated separately. Remarkably, high Z selectivity is achieved, even at internal positions. The conversion involves a one-pot oxidative radical scission/phosphorylation process by using the low-toxicity (diacetoxyiodo)benzene/iodine system as the scission reagent. The resulting α-amino phosphonates undergo a Horner-Wadsworth-Emmons reaction to produce the dehydroamino acid derivatives (in a Z/E ratio of usually >98:2) under mild and metal-free conditions.

Full text of DOI:10.1002/chem.201703758

A Journal of
Accepted Article
Title: Metal-free, Site-Selective Peptide Modification by Conversion of
"Customizable" Units into beta-Substituted Dehydroamino Acids
Authors: Alicia Boto, Carlos Javier Saavedra, and Dacil Hernández
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.201703758
Link to VoR: http://dx.doi.org/10.1002/chem.201703758
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Metal-free, Site-Selective Peptide Modification by Conversion of
“Customizable” Units into -Substituted Dehydroamino Acids
Carlos J. Saavedra,a Dácil Hernándeza and Alicia Botoa,*[a]
Abstract: This site-selective modification of serine or threonine units
in peptides allows the generation of -substituted dehydro amino
acids, which increase peptide resistance to hydrolysis and may
improve their biological properties. Both terminal and internal
positions can be modified, and different customizable units can be
activated separately. Remarkably, high Z selectivity is achieved,
even with internal positions. The conversion involves a one-pot
oxidative radical scissionphosphorylation process, using the low-
toxicity (diacetoxyiodo)benzene-iodine system as scission reagent.
The resulting -amino phosphonates undergo a Horner-Wadsworth-
Emmons reaction to produce the dehydroamino acid derivatives
(usually Z:E>98:2) under mild and metal-free conditions.
differentiation of several similar “tagged” or “customizable”
units in the peptides. The introduction of serine (or
threonine) residues as customizable units allowed this
differentiation, since their lateral chains can be protected
with orthogonal groups. The free serine units can be
selectively transformed, while protected residues are not
affected.
In a preliminary communication, we reported the selective
conversion of serine or threonine units of di- and tripeptides
into substituted dehydroamino acids in two steps (Scheme
1).12a Thus, substrate
1 underwent the oxidative radical
scission of serine lateral chain. The resulting glycyl cation12b
was trapped by acetate ions from the reagent to give the
N,O-acetal
2. Then, a phosphorylation process converted
Introduction
acetal
2
into 2-(diethoxyphosphoryl)glycine derivative 3 12a,c
.
The introduction of dehydroamino acids into peptides can
improve their resistance to degradation and their biological
or chemical properties.1 Thus, dehydroamino acids can be
found in a variety of drugs1,2 and bioactive natural
peptides,1,3 and materials with new folded conformations.1a,4
The most usual dehydroamino acid is dehydroalanine
(Dha), which is formed by dehydration of serine or
elimination of cysteine.5 Dha is used in the selective
modification of peptides to introduce functionalized lateral
chains, for instance, by addition of alkylthiols to the
unsaturated
esters.6
The
related
compound
dehydrophenylalanine is found in peptide foldamers where
rigidity is necessary for activity.7 However, in spite of their
applications, other commercial -substituted dehydroamino
acids are scarce.8 Thus, the direct production of these
residues in peptides by site-selective modification of
‘customizable units’ should be extremely interesting.
In principle, the site-selective modification of peptides
represents an important challenge, due to the similar
reactivity of the amino acid units.9 However, in recent years
considerable progress has been achieved through the
introduction of “tagged units” (aminoacids with a convertible
group)10 or “customizable units” (amino acids whose side-
chains can be converted into very different ones, mainly by
scission-addition processes).11 An additional problem is the
Scheme 1. Conversion of customizable Ser units into
dehydroamino acids
-substituted
.
[a]
Dr. C.J Saavedra, Dr. D. Hernández, Dr. A. Boto
Instituto de Productos Naturales y Agrobiología
CSIC (Spanish Research Council)
In the following step,
reaction13
with aldehydes
dehydroamino acids with excellent Z-stereoselectivity
(conversion , Z:E > 98:2). In this way, a single peptide
a
Horner-Wadsworth-Emmons
Avda. Astrofísico Fco. Sánchez, 3; 38206-La Laguna
Tenerife, SPAIN.
Phone: +34 922 260112; Fax: +34 922 260135
alicia@ipna.csic.es
yielded -substituted

3→4
precursor was selectively converted into a variety of
derivatives.
Supporting information for this article is given via a link at the end of
the document.
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In spite of this success, the methodology needed Results and Discussion
optimization, since the scission step used toxic lead
tetraacetate. Besides, an aqueous work-up was required
between the scission and addition steps. This Article
introduces new scission conditions which avoid metal
reagents, by using the low-toxicity (diacetoxyiodo)benzene-
iodine system. In addition, they allow direct addition of the
phosphorylation reagents; no aqueous workup is needed
(simplified, one-pot procedure). Then new lateral chains
with carboxyl and cationic groups are introduced.
Our initial attempts to develop
a one-pot scission-
phosphorylation process posed considerable problems.
When the scission reaction was carried out with
(diacetoxyiodo)benzene and iodine under usual conditions
(visible light irradiation, 26 oC in DCM), followed by the
addition of a phosphorus nucleophile and a Lewis acid, a
complex mixture was formed. This result suggested a
sluggish scission step, where side-reactions took place.
To
our
satisfaction,
when
the
usual
solvent
Moreover, in our preliminary work, the process was only
performed in C-terminal positions, and it was unclear
whether good yields and stereoselectivities could be
obtained in N-terminal or internal positions, which are
usually more hindered and prone to side-reactions. In this
article, this important issue is successfully addressed.
Finally, it describes the selective activation of similar
customizable units, allowing different functionalizations at
different times, and therefore, an important diversity of the
peptide libraries.
dichloromethane was replaced by dichloroethane, which
allowed heating the reaction mixture to 80 C, the scission
o
was completed in 15
1) as substrates, the scission was performed in C-terminal
(entries 1 3), N-terminal (entry 4), and interior positions
(entry 5), followed by phosphorylation, affording satisfactory
yields of products 11 15
20 min. Using peptides 510 (Table


.
Table 1. Conversion of customizable Ser and Thr units into 2-(diethoxyphosphoryl)glycine residues
Entry
Substrate
Methoda
Aminophosphonate
Yield (%)
A
B
65
72
1
A
B
48
61
2
R = H
R = H
41
A
B
61
3
R = Me
R = Me
49
A
B
68
A
B
55
70
4
5
A
B
53
75
Method A: DIB (2 equiv.), I2 (1 equiv.), dichloroethane, reflux, 20 min; then 0 oC, P(OEt)3, TMSOTf, 3 h. Method B: LTA (2 equiv.), I2 (1 equiv.), DCM,
25 °C, 1h; then aqueous work-up and solvent removal; then DCM, 0 oC, P(OEt)3, TMSOTf, 3 h.
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Interestingly, different phosphorylation conditions were
tried, and the best results were obtained with TMSOTf as
unchanged. In a later step, these ‘latent’ units could be
deprotected by conventional techniques5f and thus become
a convertible residue. In this way, a variety of side chains
can be introduced into the peptide.
the Lewis acid (which transforms in situ the N,O-acetal
2
into reactive iminium intermediates) and P(OEt)3 as the P-
nucleophile.
Once the one-pot scission-phosphorylation process was
optimized, using low-toxicity reagent (diacetoxyiodo)
benzene in the scission step, the conversion of
The success of the process in N-terminal and internal
positions is noteworthy. In internal positions, the scission
could compete with side-reactions, such as hydrogen
abstraction from nearby positions.14 Moreover, since the
addition of P-nucleophiles would be hampered by steric
hindrance, other side reactions resulting in cleavage of the
peptide chain could take place; for instance, hydrolysis of
phosphonates 1115 into dehydroamino acid derivatives
was studied. In our preliminary communication, a Horner-
Wadsworth-Emmons reaction was used for the
transformation of the C-terminal position of substrate 11
into dehydroalanines with hydrophobic
-substituents (iPr,
the N,O-acetal
addition, the iminium intermediates would be less reactive
in internal and N-terminal positions than in C-terminal
positions, because the -amide substituent is less electron-
withdrawing than a C-terminal ester group. In spite of that,
the efficient scission-addition process took place in similar
yields both in terminal (entries 1
positions.
2
or its iminium/imine derivatives. In
Ph, or (CH2)2Ph).12 However, we were also interested in the
generation of dehydroamino acids with cationic or acid
groups, such as dehydrolysine or dehydroaspartic acid,
since these residues are key for antibiotic properties (eg.
antimicrobial peptides with cationic groups), cytotoxic and
other interesting bioactivities.1,2,15 In addition, these
residues can be used to introduce fluorescent probes and
other labels in the peptide,16 and for the attachment of new
peptide chains or bioconjugation with lipids, steroids, and
many other biomolecules.17 In this Article, the introduction
of neutral, cationic or acid side-chains using substrates
4) and internal (entry 5)
On the other hand, the scission of threonine units took
place with slighty higher yields than the scission of serine
units (entry 3). Remarkably, different customizable units
can be activated independently, as shown in entries 3 and
5. Thus, while the free threonine or serine units underwent
scission, the protected Ser or Thr residues remained
12
14 and aldehydes 16a-h is addressed (Scheme 2 and
Table 2).
Scheme 2. Horner-Wadsworth-Emmons reaction in C-terminal and N-terminal positions
.
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Table 2. Conversion of customizable Ser and Thr units into 2-(diethoxyphosphoryl)glycine residues
Yield (%)a
Z:E
Entry
Aldehyde
Substrate
Product
1
16a
12
76
>98:2
R =
17a
18a
2
3
16a
16a
13
14
79
87
>98:2
93:7
19a
4
16b
12
67
>98:2
R =
17b
18b
5
6
16b
16b
13
14
83
78
>98:2
>98:2
19b
7
16c
12
62
>98:2
R =
17c
18c
8
9
16c
16c
13
14
63
57
>98:2
>98:2
19c
10
16d
12
76
>98:2
R =
17d
18d
11
12
16d
16d
13
14
62
72
92:8
19d
90:10
13
16e
12
79
>98:2
R =
17e
18e
14
15
16e
16e
13
14
84
92
>98:2
>98:2
19e
16
16f
12
82
95:5
R =
17f
18f
17
18
16f
16f
13
14
76
85
>98:2
>98:2
19f
19
16g
12
85
92:8
R =
17g
18g
20
21
16g
16g
13
14
83
93
93:7
19g
90:10
(Z = Boc) 41
(Z = H) 32
22
16h
14
>98:2
R =
19h (Z = Boc)
19i (Z = H)
aYields after purification by chromatography on silica gel
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Another important challenge was the modification of N-
terminal and internal positions, which are more hindered
and less reactive than the C-terminal ones. Indeed, the
supported that the Z-olefin was the major or exclusive
isomer.
An important concern was that under the basic conditions of
the HWE reaction, epimerization of other amino acids could
take place. To address this point, a substituent with two
HWE
conditions
developed
in
our
preliminary
communication did not work for the conversion of N-
terminal or internal residues, and a modified procedure was
developed to overcome this problem.
stereogenic centers was introduced in compounds 17
no epimers were detected by NMR or chromatography. On
the other hand, when chiral -disubstituted aldehydes were
used as reagents, their -stereogenic center could also
undergo epimerization. In order to study this possibility, the
L-proline-derived aldehyde 16g was prepared and reacted,
19f;
The reported HWE conditions12 (DBU in CH2Cl2) only gave
good results with C-terminal aminophosphonates 12 and


13
.
On the contrary, the reaction with N-terminal
aminophosphonate 14 did not proceed to completion at
room temperature, and when heating was applied, a
complex product mixture was formed. As commented later,
the reaction at internal positions also gave poor results. A
possible explanation is that N-terminal and internal
positions are usually more hindered than C-terminal ones,
and the base could not approach well to the
aminophosphonate. In addition, the generation of the
anionic intermediate would be favored in substrates 12 and
13 compared to substrate 14, since the ester group is a
more electron-withdrawing, stabilizing substituent than the
carboxamide group.
and again, no epimers could be isolated.
Finally, the functionalization of internal positions was
studied with
aldehydes, to give products 20a

-aminophosphonate 15 and a variety of
and 20d (Scheme 3).

b
f
As happened with N-terminal positions, the conditions
reported in the literature for the HWE reaction gave
complex product mixtures. In contrast, the new procedure
with TMG gave good yields of the desired dehydroamino
acids.
Different conditions for the HWE reaction were needed, and
after some experimentation, it was found that
tetramethylguanidine in DCM gave optimal results. Since
the pKa of TMG and DBU is similar,18 the improvement
could be due to coordination of polar TMG with the peptide
substrate in the apolar solvent. As additional advantages,
TMG is less expensive and can be readily removed during
the work-up due to its water-solubility.
Once the base was optimized, the HWE reaction proceeded
well. The introduction of hydrophobic side-chains (products
1719a and 1719b) took place in most cases in good
yields and excellent Z:E selectivity (>98:2). The formation of
the valuable dehydroaspartic acid residue (products
1719c) was carried out in satisfactory yields and excellent
stereoselectivity (Z:E, >98:2).
The generation of a variety of cationic residues with cyclic,
acyclic and branched amines and heteroaromatic rings
(products 17dg, 18dg, and 19di) also took place in
good to excellent yields. The stereoselectivity depended on
the bulkiness of the new side-chain. Thus, products 18d
and 19d (R = CH2-NH-Boc) with a bulky R substituent
presented a 90:10 Z:E ratio, but in the less hindered
products 1719e (R = CH2-CH2-NH-Cbz) and 1719f (R =
CH2-CH(NHBoc)-sBu) the stereoselectivity improved (Z:E
ratio from 95:5 to >98:2). A similar result was obtained with
pyrrolidine derivatives 1719g and with the pyrrol
derivatives 19h ; thus, the bulky pyrrolidine-Boc group was

i
introduced in 19g with 90:10 Z:E ratio, while the flat pyrrole
derivative 19h presented excellent Z:E selectivity (>98:2).
Scheme 3. Horner-Wadsworth-Emmons reaction in internal positions.
The stereoselectivity of the products was studied with NOE
or NOESY experiments. In most cases, spatial correlations
Having observed the importance of steric hindrance for the
between the proton NH
(amido group of the
Z:E selectivity, a decrease in the latter was expected.
aa
dehydroamino acid) and the protons of the lateral chain of
the same residue were observed, as well as correlations
between the olefinic proton and those belonging to the
methoxy group on the C-terminal residue. These data
However, products 20b
excellent stereoselectivity (Z:E, >98:2), while the bulky
substituent iPr (in product 20a and the group
CH2CH2NHBoc (in product 20d) gave a 90:10 and 93:7 Z:E
ratio, respectively (separable mixtures).19 The chiral product
, 20d and 20f were obtained with
)
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20f was prepared to check whether epimerization of other
positions had taken place, but only one stereoisomer was
observed.
drying and evaporation, the residue was purified by
chromatography on silica gel (hexanes/EtOAc), to afford the
aminophosphonate derivatives.
-
N-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)-2-(diethoxy
The generation of Z products with remarkable purity, even
in internal positions, highlights the scope of this site-
selective modification process for the direct introduction of
valuable dehydroamino acids in peptides.
phosphoryl)glycine Methyl Ester (12). Obtained from compound
(80 mg, 0.2 mmol) according to the Scission-Oxidation-
6
Phosphorylation Procedure, Methods A or B. After purification by
rotatory chromatography (hexanes/EtOAc 40:60), compound 12
was isolated as a 1:1 diastereomer mixture (56 mg, 48% with
Method A and 71 mg, 61% with Method B): Syrup; 1H NMR (500
MHz, CDCl3, 26 °C): H 0.85-0.92 (m, 12H), 1.24-1.35 (m, 6H),
1.50-1.70 (m, 6H), 3.75/3.77 (s/s, 3H), 4.11-4.25 (m, 5H), 4.61/4.69
(m/m, 1H), 5.09 (s, 2H), 5.13/5.16 (m/m, 1H), 5.49/5.68 ([d, J = 7.9
Hz/d, J = 7.9 Hz], 1H), 6.66/6.76 ([d, J = 8.2 Hz/d, J = 7.6 Hz], 1H),
Conclusions
An interesting application of the site-selective modification
of peptides is described herein, which allows the conversion
of “customizable” serine and threonine units, which are
7.26

7.35 (m, 5H), 7.34/7.56 ([m/d, J = 8.5 Hz], 1H). 13C NMR
C 16.2 (2 CH3, d, JC,P = 5.8 Hz), 21.8
(125.7 MHz, CDCl3, 26 °C):


(CH3), 22.0/22.1 (CH3), 22.7/22.8 (CH3), 22.9 (CH3), 24.5 (CH),
24.6 (CH), 41.1 (CH2), 41.2 (CH2), 50.5/50.6 (CH, [d, JC,P = 146 Hz/
d, JC,P = 147.2 Hz), 51.4/51.5 (CH), 53.0/53.1 (CH3), 53.4/53.5 (CH),
63.6 (CH2, d, JC,P = 7.4 Hz), 63.9 (CH2, d, JC,P = 6.4 Hz), 67.0 (CH2),
128.0 (2 CH), 128.1 (CH), 128.4 (2 CH), 136.2 (C), 156.3 (C),
commercial and inexpensive amino acids, into valuable -
substituted dehydroamino acids. Since the serine unit can
be protected with orthogonal groups, similar units can be
activated separately; only the free residues are
transformed.
166.8/166.9 (C), 171.8 (C), 172.3/172.5 (C). IR (CHCl3): max
=
3426, 3318, 1747, 1712, 1678, 1506 cm–1. MS (EI): m/z= 585 (M+,
<1), 91 ([CH2Ph]+, 100); HRMS (EI) calcd for C27H44N3O9P
585.2815, found 585.2804; calcd for C7H7 91.0548, found 91.0549;
Anal. Calcd for C27H44N3O9P: C, 55.37; H, 7.57; N, 7.18. Found: C,
55.14; H, 7.48; N, 7.00.
For the first time,
a
sequential serine scission-
phosphorylation process is achieved, and the process is
carried out with low-toxicity hypervalent iodine reagents.
The resulting
-amino phosphonates are transformed into
the -substituted dehydroamino acids by using a Horner-

General Procedures for the Horner-Wadsworth-Emmons
Wadsworth-Emmons reaction. Since the usual HWE
conditions did not work for N-terminal and internal positions,
a modified procedure with TMG was developed, which
afforded good to excellent yields and Z:E selectivities. In
addition to hydrophobic chains, cationic or acid residues
(dehydrolysine, dehydroaspartic, and other units) have
been introduced without epimerization of adjacent amino
acids. Therefore, this methodology could be very valuable
for the transformation of a few starting peptides into
libraries of rigid analogues of bioactive or catalytic peptides,
saving time and materials in discovery processes.
Reaction. To a solution of the
CH2Cl2 (2 mL) was
diazabicyclo[5.4.0]undec-7-ene (DBU) or 1,1,3,3-tetramethyl-
guanidine (TMG) (2.5 equiv) in dry CH2Cl2 (1 mL). The reaction
mixture was stirred for 10 min, and then was added the aldehyde (2

-phosphonate (0.1

0.2 mmol) in dry
of 1,8-
added solution
a
equiv) in dry CH2Cl2 (1 mL). After stirring for 15 min72 h, the
solution was poured into saturated aqueous NaHCO3 and extracted
with CH2Cl2. The organic layer was dried over sodium sulfate,
filtered and evaporated under vacuum. The residue was purified by
chromatography on silica gel (hexanes/EtOAc) affording the
dehydroamino acid derivatives.
(Z)-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)--dehydro-
leucine Methyl Ester (17a). Obtained from the amino phosphonate
(
12) (117 mg, 0.2 mmol), isobutyraldehyde (37 μL, 29 mg, 0.4
Experimental Section
mmol) and DBU (75 L, 76 mg, 0.5 mmol), according to the General

HWE Procedure (16 h of reaction). After purification by rotatory
General
Procedure
for
the
ScissionOxidation
chromatography (hexanes/EtOAc 70:30), compound (17a) (76 mg,

Phosphorylation Process. Method A. To a solution of the
76%, Z:E >98:2) was isolated as a syrup; [ D = 55 (c 0.76,
CHCl3); H NMR (500 MHz, CDCl3, 26 °C): H 0.91 (d, J = 6.3 Hz,
3H), 0.92 (d, J = 6.3 Hz, 6H), 0.94 (d, J = 6.4 Hz, 3H), 1.02 (d, J =
]
1
starting material (0.2 mmol) in dry dichloroethane (8 mL) was added
iodine (51 mg, 0.2 mmol) and diacetoxyiodobenzene (DIB) (129 mg,
0.4 mmol). The reaction mixture was refluxed for 20 min. Then the
6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H), 1.471.60 (m, 2H), 1.621.70
(m, 4H), 1.77 (m, 1H), 2.56 (m, 1H), 3.73 (s, 3H), 4.20 (m, 1H), 4.55
o
reaction mixture was cooled to 0 C and triethylphosphite (174

L,
166 mg, 1 mmol) and TMSOTf (109
L, 133 mg, 0.6 mmol) were
(m, 1H), 5.09 (s, 2H), 5.25 (d, J = 7.9 Hz, 1H), 6.51 (d, J = 10.4 Hz,
added. The reaction mixture was stirred for 3 h, then it was poured
into saturated aqueous NaHCO3 and extracted with CH2Cl2. After
usual solvent drying and evaporation, the residue was purified by
1H), 6.55 (d, J = 7.6 Hz, 1H), 7.30
13C NMR (125.7 MHz, CDCl3, 26 °C):
22.9 (2 CH3), 24.7 (CH3), 24.8 (2 × CH), 27.9 (CH), 40.3 (CH2),
41.2 (CH2), 51.8 (CH), 52.3 (CH3), 53.8 (CH), 67.3 (CH2), 123.1 (C),
128.2 (CH), 128.3 (2 CH), 128.6 (2 CH), 136.0 (C), 146.1 (CH),
156.4 (C), 165.0 (C), 170.7 (C), 172.5 (C). IR (CHCl3): max= 3425,
3337, 1717, 1678, 1503 cm–1. MS (EI): m/z= 503 (M+, 1), 460 (M+ 
CHMe2, 3), 91 ([CH2Ph]+, 100). HRMS (EI) calcd for C27H41N3O6
503.2995, found 503.2997; calcd for C7H7 910548, found 91.0547;
Anal. Calcd for C27H41N3O6: C, 64.39; H, 8.21; N, 8.34. Found: C,
64.38; H, 8.52; N, 8.14.

7.37 (m, 5H), 7.57 (br s, 1H).

C 21.6 (2 CH3), 22.0 (CH3),


chromatography on silica gel (hexanes/EtOAc), to afford the
aminophosphonate derivatives.
-



Method B. To a solution of the starting material (0.2 mmol) in dry
dichloromethane (8 mL) was added iodine (51 mg, 0.2 mmol) and
lead tetraacetate (LTA, 178 mg, 0.4 mmol). The reaction mixture
was stirred for 1 h at room temperature (26 oC) under irradiation
with visible light (80-W tungsten-filament lamp). Then the reaction
mixture was poured into 10% aqueous Na2S2O3 and extracted with
CH2Cl2, and the solvent was dried and evaporated. The unpurified
residue was dissolved in dry CH2Cl2 (8 mL), the solution was cooled
(Z)-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)--dehydro-5-
(phenyl)norvaline Methyl Ester (17b). Obtained from the amino
phosphonate (12) (117 mg, 0.2 mmol), hydrocinnamaldehyde (53
to 0 oC and triethylphosphite (174
TMSOTf (109 L, 133 mg, 0.6 mmol) were added. The reaction
L, 166 mg, 1 mmol) and

μL, 54 mg, 0.4 mmol) and DBU (75
L, 76 mg, 0.5 mmol),
mixture was stirred for 3 h, then it was poured into saturated
aqueous NaHCO3 and extracted with CH2Cl2. After usual solvent
according to the General HWE Procedure (16 h of reaction). After
purification by column chromatography (hexanes/EtOAc 40:60),
This article is protected by copyright. All rights reserved.

10.1002/chem.201703758
Chemistry - A European Journal
FULL PAPER
compound (17b) (76 mg, 67%, Z:E >98:2) was isolated as a syrup;
[(benzyloxycarbonyl)amino] propionaldehyde (83 mg, 0.4 mmol)
[
 D
]
=

50 (c 0.26, CHCl3); 1H NMR (500 MHz, CDCl3, 26 oC):H
and DBU (75
Procedure (16 h

L, 76 mg, 0.5 mmol), according to the General HWE
of reaction). After purification by rotatory
0.81
2.65


0.87 (m, 12H), 1.38
2.72 (m, 2H), 3.64 (s, 3H), 4.13 (m, 1H), 4.45 (ddd, J = 6.0,

1.71 (m, 6H), 2.35

2.40 (m, 2H),
chromatography (hexanes/EtOAc 65:35), compound (17e) (101 mg,
79%, Z:E >98:2) was isolated as a crystalline solid; m.p. 158-159 oC
8.2, 8.2 Hz, 1H), 4.98 (d, J = 11 Hz, 1H), 5.03 (d, J = 12.0 Hz, 1H),
5.24 (br d, J = 7.6 Hz, 1H), 6.58 (br d, J = 8.2 Hz, 1H), 6.62 (dd, J =
(neat); [ D
oC): H 0.89
1.76 (m, 1H), 2.30
4.17 (m, 1H), 4.52 (ddd, J = 5.7, 8.2, 8.5 Hz, 1H), 5.07 (s, 2H), 5.08
(s, 2H), 5.28 (br b, 1H), 5.58 (br b, 1H), 6.62 6.67 (m, 2H),
7.26
7.38 (m, 10H), 7.87 (br b, 1H). 13C NMR (100.6 MHz, CDCl3,
26 oC):
C 21.8 (2 CH3), 22.9 (2 CH3), 24.8 (2 CH), 28.8 (CH2),
39.6 (CH2), 40.2 (CH2), 41.0 (CH2), 52.1 (CH), 52.4 (CH3), 54.0
(CH), 66.6 (CH2), 67.4 (CH2), 126.8 (C), 128.0 (4 CH), 128.1
(CH), 128.3 (CH), 128.4 (2 CH), 128.6 (2 CH), 135.4 (CH),
136.0 (C), 136.7 (C), 156.5 (C), 156.6 (C), 164.4 (C), 170.6 (C),
172.7 (C). IR (CHCl3):
max= 3426, 3346, 1715, 1505 cm–1. MS (EI):
] = 
51 (c 0.30, CHCl3); 1H NMR (500 MHz, CDCl3, 26
7.3, 7.3 Hz, 1H), 7.10

7.14 (m, 3H), 7.19

7.30 (m, 7H), 7.50 (br b,

0.94 (m, 12H), 1.48
1.59 (m, 2H), 1.61
1.66 (m, 3H),
1H). 13C NMR (125.7 MHz, CDCl3, 26 oC): C 21.8 (CH3), 22.0
(CH3), 22.8 (CH3), 22.9 (CH3), 24.7 (2 CH), 30.2 (CH2), 34.1
(CH2), 40.6 (CH2), 41.1 (CH2), 51.8 (CH), 52.3 (CH3), 53.6 (CH),

2.40 (m, 2H), 3.27
3.35 (m, 2H), 3.74 (s, 3H),

67.2 (CH2), 125.3 (C), 126.1 (CH), 128.1 (2
128.4 (2 CH), 128.5 (4 CH), 136.0 (C), 137.8 (CH), 140.9 (C),
156.4 (C), 164.6 (C), 170.4 (C), 172.5 (C). IR (CHCl3): max= 3422,
1716, 1504 cm–1. MS (EI): m/z= 565 (M+, <1), 91 ([CH2Ph]+, 100).
HRMS (EI) calcd for C32H43N3O6 [M+] 565.3152, found 565.3165;
calcd for C7H7 910548, found 91.0545. Anal. Calcd for C32H43N3O6:
C, 67.94; H, 7.66; N, 7.43. Found: C, 68.00; H, 7.51; N, 7.47.

CH), 128.2 (CH),










m/z= 638 (M+, <1), 91 ([CH2Ph]+, 100). HRMS (EI) calcd for
C34H46N4O8 638.3316, found 638.3318; calcd for C7H7 91.0548,
found 91.0551. Anal. Calcd for C34H46N4O8: C, 63.93; H, 7.26; N,
8.77. Found: C, 63.65; H, 7.06; N, 8.55.
(Z)-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)--dehydro-4-
ethyl-1-methyl aspartate (17c). Obtained from the amino
phosphonate (12) (117 mg, 0.2 mmol), ethyl glioxalate solution 50%
in toluene (53 μL, 54 mg, 0.4 mmol) and DBU (75
L, 76 mg, 0.5
mmol), according to the General HWE Procedure (15 min of
reaction). After purification by rotatory chromatography
Methyl
(Z)-2-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)-5-((tert-
butoxycarbonyl)amino)-6-methyl-2-octenoate (17f). Obtained
from the amino phosphonate (12) (117 mg, 0.2 mmol), (3S)-tert-
butoxycarbonyl-amino-(4S)-methyl-hexanal (91.6 mg, 0.4 mmol)
and DBU (75
Procedure (16
(hexanes/EtOAc 85:15), compound (17c) (66 mg, 62%, Z:E >98:2)
o
was isolated as a solid; m.p. 158-159 C (neat); [ D
]
=

45 (c 0.22,
0.96 (m, 12H),
1.80 (m, 6H), 3.83 (s, 3H), 4.20
1
CHCl3); H NMR (500 MHz, CDCl3, 26 °C): H 0.90


L, 76 mg, 0.5 mmol), according to the General HWE
of reaction). After purification by rotatory
1.28 (dd, J = 6.9, 7.3 Hz, 3H), 1.53

h
(ddd, J = 6.9, 6.9, 7.3 Hz, 2H), 4.25 (m, 1H), 4.58 (m, 1H), 5.09 (d, J
= 12.3 Hz, 1H), 5.12 (d, J = 13 Hz, 1H), 5.15 (br b, 1H), 5.54 (s,
chromatography (hexanes/EtOAc, 70:30), compound (17f) (54.3
mg, 82%, Z:E 95:5) was isolated as a crystalline solid; m.p.
1H), 6.53 (br s, 1H), 7.30

7.38 (m, 5H), 10.59 (br b, 1H); 13C NMR
oC (EtOAc/n-hexane); [

]D =
(500 MHz, MeOD, 26 oC): H 0.82
0.94 (m, 12H), 1.07 (m, 1H),
1.41 (s, 9H), 1.38 1.48 (m, 3H), 1.52 (dd, J = 7.4, 7.5 Hz, 1H),
1.61 1.71 (m, 4H), 2.21 2.30 (m, 2H), 3.52 (m, 1H), 3.68 (s, 3H),

54 (c 0.51, CHCl3); 1H NMR
(100.6 MHz, CDCl3, 26 °C): C 14.1 (CH3), 21.8 (CH3), 21.9 (CH3),
22.9 (CH3), 23.0 (CH3), 24.6 (CH), 24.8 (CH), 40.6 (CH2), 40.7
(CH2), 52.1 (CH), 52.9 (CH3), 53.4 (CH), 61.0 (CH2), 67.2 (CH2),




103.3 (CH), 128.0 (2
142.8 (C), 156.4 (C), 164.0 (C), 167.5 (C), 170.3 (C), 172.4 (C). IR
(CHCl3):
max= 3430, 3297, 1741, 1715, 1702, 1685, 1505 cm–1. MS
(EI): m/z= 533 (M+, <1), 91 ([CH2Ph]+, 100). HRMS (EI) calcd for
C27H39N3O8 533.2737, found 533.2742, calcd for C7H7 91.0548,
found 91.0545. Anal. Calcd for C27H39N3O8: C, 60.77; H, 7.37; N,
7.87. Found: C, 60.55; H, 7.34; N, 7.82.

CH), 128.3 (CH), 128.6 (2

CH), 136.1 (C),
4.11 (dd, J = 7.3, 7.6 Hz, 1H), 4.42 (dd, J = 7.5, 7.6 Hz, 1H), 4.50
(br s, 1H), 5.05 (d, J = 12.9 Hz, 1H), 5.07 (d, J = 12.7 Hz, 1H), 6.66

(dd, J = 6.6, 7.9 Hz, 1H), 7.25
7.32 (m, 5H). 13C NMR (125.7 MHz,
o
MeOD, 26 C): C 11.9 (CH3), 15.6 (CH3), 21.9 (CH3), 22.1 (CH3),
23.36 (CH3), 23.43 (CH3), 25.9 (CH2), 26.5 (2 CH), 28.8 (3 CH3),


31.2 (CH2), 40.4 (CH), 41.6 (CH2), 41.9 (CH2), 52.6 (CH3), 53.3
(CH), 55.2 (CH), 55.3 (CH), 67.9 (CH2), 80.0 (C), 128.4 (C), 129.0
(2
 CH), 129.1 (CH), 129.5 (2  CH), 138.0 (CH), 138.7 (C), 158.2
Methyl
butoxycarbonyl)amino-2-butenoate (17d). Obtained from the
amino phosphonate 12 (117 mg, 0.1 mmol), N-tert-
butoxycarbonyl-2-amino ethanal (31.9 mg, 0.2 mmol) and DBU (75
according to the General HWE Procedure
(Z)-2-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)-4-(tert-
(C), 158.8 (C), 166.0 (C), 173.7 (C), 175.4 (C). IR (CHCl3): max
=
3428, 2964, 1695, 1500, 1415 cm–1. HRMS (ESI) calcd for
C35H55DN4O8Na [M(D)+ + Na] 684.4059, found 684.4044; calcd for
C35H56N4O8Na [M+ + Na] 683.3996, found 683.3995. Anal. Calcd for
C35H55DN4O8: C, 63.52; H, 8.68; N, 8.47. Found: C, 63.44; H, 9.04;
N, 8.55.
(
)
(16 h of reaction). After purification by column chromatography
(hexanes/EtOAc 50:50), compound (17d) (44.6 mg, 76%, Z:E >
98:2) was isolated as
(EtOAc/hexane); [ D
CDCl3, 26 °C)H 0.92 (d, J = 6.0 Hz, 6H), 0.95 (d, J = 6.0 Hz, 6H),
1.43 (s, 9H), 1.52 1.80 (m, 6H), 3.65 3.82 (m, 2H), 3.75 (s, 3H),
4.19 (m, 1H), 4.56 (m, 1H), 5.09 (s, 2H), 5.13 (br b, 1H), 5.26 (d, J =
a
crystalline solid; m.p. 153

155 °C
17f-Z:17f-E Mixture: (Z:E 4:1, fraction enriched in the E-isomer).
1
]
=

19 (c 0.40, CHCl3); 1H NMR (500 MHz,
Only relevant signals of the E-isomer are described: H NMR (500
o
MHz, MeOD, 26 C): H 2.51 (m, 1H), 2.63 (m, 1H), 3.48 (m, 1H),


6.16 (dd, J = 7.4, 7.7 Hz, 1H).
(Z)-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)--dehydro-[3-(2-
N-tert-butoxycarbonyl-pyrrolidinyl)]alanine Methyl Ester (17g).
Obtained from the amino phosphonate (12) (117 mg, 0.2 mmol), N-
tert-butoxycarbonyl-pyrrolidine-2-carbaldehyde (79.6 mg, 0.4 mmol)
7.0 Hz, 1H), 6.51 (m, 1H), 6.56 (d, J = 6.0 Hz, 1H), 7.287.38 (m,
5H), 8.40 (br s, 1H). 13C NMR (125.7 MHz, CDCl3, 26 °C) C 21.87
(CH3), 21.91 (CH3), 22.87 (CH3), 22.91 (CH3), 24.7 (CH), 24.8 (CH),
28.4 (3
 CH3), 37.9 (CH2), 41.0 (CH2 × 2), 52.1 (CH), 52.5 (CH3),
and DBU (75
Procedure (16

L, 76 mg, 0.5 mmol), according to the General HWE
of reaction). After purification by column
53.7 (CH), 67.3 (CH2), 79.9 (C), 127.1 (C), 128.1 (2 CH), 128.3
(CH), 128.6 (2 CH), 131.3 (CH), 136.0 (C), 156.3 (C), 156.4 (C),
h
chromatography (hexanes/EtOAc 70:30), compounds 17g-Z (44.6
mg, 78%) and 17g-E (3.9 mg, 7%) were isolated (Z:E 90:10).
164.5 (C), 170.9 (C), 172.4 (C). IR (CHCl3):

max= 3429, 3324,
CH2NHBoc, 2),
1708, 1504, 1213 cm–1. MS (EI): m/z= 460 (M+

Compound 17g-Z was isolated as a crystalline solid; m.p. 148
°C (EtOAc/hexane); []D = 96

150
(c 1.0, CHCl3); H NMR (500 MHz,
CDCl3, 26 °C):H 0.90 (d, J = 6.0 Hz, 6H), 0.98 (d, J = 6.0 Hz, 6H),
1.43 (s, 9H), 1.46 1.96 (m, 9H), 2.05 (m, 1H), 3.23 3.36 (m, 2H),
248 ([BnOCONHCH(CH2CHMe2)-CO]+, 3), 91 ([CH2Ph]+, 100).
HRMS (EI) calcd for C24H34N3O6 460.2448, found 460.2449; calcd
for C14H18O3N 248.1287, found 248.1283; calcd for C7H7 910548,
found 91.0548. Anal. Calcd for C30H46N4O8: C, 61.00; H, 7.85; N,
9.48. Found: C, 60.98; H, 7.70; N, 9.64.
1


3.75 (s, 3H), 4.22 (m, 1H), 4.49 (m, 1H), 4.60 (m, 1H), 5.09 (s, 2H),
5.26 (d, J = 8.3 Hz, 1H), 5.94 (d, J = 9.5 Hz, 1H), 6.58 (d, J = 8.0
Hz, 1H), 7.27
7.35 (m, 5H), 10.2 (s, 1H). 1H NMR (400 MHz,
CD3OD, 26 °C). Rotamer mixture, some signals are splittedH 0.92
(d, J = 6.5 Hz, 3H), 0.94 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.7 Hz,
(Z)-(N-Benzyloxycarbonyl-L-leucyl-L-leucyl)--dehydro-5-(N-
benzyloxycarbonyl)ornithine methyl ester (17e). Obtained from
the amino phosphonate
(
12)
(117 mg, 0.2 mmol),
This article is protected by copyright. All rights reserved.

10.1002/chem.201703758
Chemistry - A European Journal
FULL PAPER
3H), 0.98 (d, J = 6.8 Hz, 3H), 1.41/1.46 (s/s, 9H), 1.50

2.30 (m,
Waite, Biochemistry 2016, 55, 743−750; e) T. Kuranaga, H. Mutoh, Y.
Sesoko, T. Goto, S. Matsunaga, M. Inoue, J. Am. Chem. Soc. 2015,
137, 9443−9451; f) C. Wang, J. Sperry, Tetrahedron, 2014, 70, 3430-
3439; g) A. Schmitz, S. Kehraus, T. F. Schꢀberle, E. Neu, C. Almeida,
M. Roth, G. M. Konig, J. Nat. Prod. 2014, 77, 159−163.
10H), 3.38 (dd, J = 6.4, 6.5 Hz, 2H), 3.73 (s, 3H), 4.19 (m, 1H),
4.35 4.58 (m, 2H), 5.09 (s, 2H), 6.25/6.51 ([d, J = 8.8 Hz/ d, J = 7.0

Hz], 1H), 7.25-7.40 (m, 5H). 13C NMR (125.7 MHz, CDCl3, 26 °C):
C 21.8 (CH3), 22.3 (CH3), 22.7 (CH3), 23.0 (CH3), 24.0 (CH), 24.6
(CH2), 28.0 (CH), 28.4 (3 CH3), 31.1 (CH2), 41.9 (CH2), 42.9
(CH2), 46.5 (CH2), 51.6 (CH), 52.2 (CH3), 53.1 (CH), 53.5 (CH),
66.9 (CH2), 80.5 (C), 127.97 (2 CH), 128.04 (CH), 128.4 (2 CH),
129.9 (CH), 130.1 (C), 136.3 (C), 155.6 (C), 156.1 (C), 164.5 (C),
[4]
a) H. Vilaꢁa, A. C. L. Hortelꢂo, E. M. S. Castanheira, M. J. R. P.
Queiroz, L. Hilliou, I. W. Hamley, J. A. Martins, P. M. Ferreira,
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A. Krezel, K. Haremza, M. Makowski, P. Kafarski, J. Mol. Struct. 2013,
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Freund, S. B. Tsogoeva, Peptides for Asymmetric Catalysis, in Catalytic
Methods in Asymmetric Synthesis, (Eds.: M. Gruttadauria, F.
Giacalone), John Wiley and Sons, Hoboken, NJ, USA, 2011.
171.1 (C), 171.8 (C). IR (CHCl3):
max= 3425, 3020, 1726, 1670,
1500, 1404, 1168 cm–1. MS (EI): m/z= 630 (M+, 53), 91 ([CH2Ph]+,
100). HRMS (EI) calcd for C33H50N4O8 630.3629, found 630.3654;
calcd for C7H7 91.0548, found, 91.0549. Anal. Calcd for
C33H50N4O8: C, 62.84; H, 7.99; N, 8.88. Found: C, 62.72; H, 7.80;
N, 8.74.
17g-E isomer: [ D
CD3OD, 26 °C): A rotamer mixture was observed, and some signals
are splitted, H 0.87 1.02 (m, 12H), 1.37/1.46 (s, 9H), 1.40 1.85 (m,
10H), 3.33 3.43 (m, 2H), 3.73 (s, 3H), 4.20 (m, 1H), 5.09 (br s, 2H),
6.28/6.50 (br d, J = 9.8 Hz/ brb, 1H), 7.28
7.38 (m, 5H). 13C NMR
(100.6 MHz, CD3OD, 26 °C): C 21.1 (CH3), 22.2 (CH3), 23.5 (CH3),
] = 
37.9 (c 0.14, CHCl3); 1H NMR (400 MHz,






23.6 (CH3), 24.9 (CH), 25.3 (CH), 26.0 (CH2), 29.0 (3 CH3),
32.4/33.4 (CH2), 42.0 (CH2), 42.2 (CH2), 47.7 (CH2), 52.9 (CH3),
54.1 (CH), 55.2 (CH), 55.3 (CH), 56.6 (CH), 67.9 (CH2), 81.6 (C),
129.0 (2 CH), 129.2 (CH), 129.6 (2 CH), 136.8 (C), 138.4 (C),
156.7 (C), 158.7 (C), 166.3 (C), 174.2 (C), 175.6 (C). IR (CHCl3):
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a) J. Chandrasekar, A. C. Wylder, S. K. Silverman, J. Am. Chem. Soc.
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a) For the introduction of new lateral chains, see: L. M. Repka, J. R.
Chekan, S. K. Nair, W. A. van der Donk, Chem. Rev., 2017, 117, 5457-
5520; b) A. Dumas, L. Lercher, C. D. Spicer, B. G. Davis, Chem. Sci.
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See also: Y. Zhang, R. Yang, J. Huang, Q. Liang, Y. Guo, W. Bian, L.
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Bartolucci, S. Lucarini, G. Piersanti, Eur. J. Org. Chem. 2015, 3352–
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Castanheira, C. G. Frost, Eur. J. Org. Chem. 2013, 550–556.

max= 3421, 1718, 1698, 1683, 1670 cm–1. MS (EI): m/z= 631 (M+ +
H, <1), 91 ([CH2Ph]+, 100). HRMS (EI) calcd for C33H51N4O8
631.3707, found 631.3720; calcd for C7H7 91.0548, found 91.0545.
Anal. Calcd for C33H50N4O8: C, 62.84; H, 7.99; N, 8.88. Found: C,
63.05; H, 8.29; N, 8.74.
[6]
Acknowledgements
This work was supported by the Research Program SAF-
2013-48399-R, Plan Estatal de I+D, Ministerio de Economía
y Competitividad, Spain, and European Social Funds
(FSE). DH and CJS thank CSIC (Spanish Research
Council) and Gobierno de Canarias, respectively, for their
postdoctoral research contracts. DH also thanks her current
contract financed by Cabildo de Tenerife, Program TF INNOVA
2016-21 (with MEDI & FDCAN Funds).
[7]
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Keywords: Synthesis (org.)• Domino Process • Sustainable
Chemistry • Peptides • Selectivity
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́
[19] In some cases, the high Z/E stereoselectivity could be due to
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Entry for the Table of Contents
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Carlos J. Saavedra, Dácil Hernández,
Alicia Boto*
Page No. – Page No.
Metal-free, Site-Selective Peptide
Modification by Conversion of
“Customizable” Units into -
Substituted Dehydroamino Acids
Direct route achieved! A metal-free, site-selective modification of serine or
threonine units in peptides that allows the generation of -substituted dehydro
amino acids, which increase peptide resistance to hydrolysis and may improve their
biological properties. Both terminal and internal positions can be modified, and
different customizable units can be activated separately. Remarkably, high Z
selectivity is achieved, even with internal positions.
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