Total Solid-Phase Synthesis of Dehydroxy Fengycin Derivatives

DOI: 10.1021/acs.joc.8b02553

Source and publish data:

Journal of Organic Chemistry p. 15297 - 15311 (2018)

Update date:2022-08-11

Topics:

Authors:

Rosés, Cristina

Camó, Cristina

Oliveras, àngel

Moll, Lluis

López, Nerea

Feliu, Lidia

Planas, Marta

Read Full Text PDF DownLoad Join now for total 90,000,000 free articles

Article abstract of DOI:10.1021/acs.joc.8b02553

A rapid and efficient solid-phase strategy for the synthesis of dehydroxy fengycins derivatives is described. This synthetic approach involved the linkage of a Tyr to a Wang resin via a Mitsunobu reaction and the elongation of the peptide sequence followed by subsequent acylation of the N-terminus of the resulting linear peptidyl resin, esterification of the phenol group of a Tyr with an Ile, and final macrolactamization. The amino acid composition as well as the presence of the N-terminal acyl group significantly influenced the stability of the macrolactone. Cyclic lipodepsipeptides with a l-Tyr³/d-Tyr⁹ configuration were more stable than those containing the Tyr residues with an opposite configuration. This work constitutes the first approach on the total solid-phase synthesis of dehydroxy fengycin derivatives.

Full text of DOI:10.1021/acs.joc.8b02553

Article

pubs.acs.org/joc

Cite This: J. Org. Chem. XXXX, XXX, XXX −XXX

Total Solid-Phase Synthesis of Dehydroxy Fengycin Derivatives

Cristina Roses, Cristina Camo, Angel Oliveras, Lluis Moll, Nerea Lopez, Lidia Feliu,*

and Marta Planas*

LIPPSO, Department of Chemistry, University of Girona, Maria Aurelia Capmany 69, 17003 Girona, Spain

* Supporting Information

ABSTRACT: A rapid and eﬃcient solid-phase strategy for the synthesis of

dehydroxy fengycins derivatives is described. This synthetic approach involved

the linkage of a Tyr to a Wang resin via a Mitsunobu reaction and the elongation

of the peptide sequence followed by subsequent acylation of the N-terminus of

the resulting linear peptidyl resin, esteriﬁcation of the phenol group of a Tyr with

an Ile, and ﬁnal macrolactamization. The amino acid composition as well as the

presence of the N-terminal acyl group signiﬁcantly inﬂuenced the stability of the

macrolactone. Cyclic lipodepsipeptides with a ^L-Tyr³/D-Tyr⁹conﬁguration were

more stable than those containing the Tyr residues with an opposite

conﬁguration. This work constitutes the ﬁrst approach on the total solid-phase

synthesis of dehydroxy fengycin derivatives.

INTRODUCTION

■

Cyclic lipodepsipeptides are a structurally diverse group of

nonribosomal metabolites typically produced by bacterial and

fungal species, such as Pseudomonas or Bacillus strains.¹⁻⁴This

class of compounds has raised interest due to their wide range

of biological activities and their structural complexity. The

main structural feature of these cyclic peptides is the

occurrence of an ester bond between the side chain of an

amino acid and the C-terminal end of the sequence. The fatty

acid moiety is commonly attached to the N-terminus of this

macrolactone. Moreover, the complexity of the structure of

these lipodepsipeptides is increased by the combination of ^L-

and ^D-amino acids as well as of other unnatural residues

present in their sequence.^5,6

Fengycins are a family of natural cyclic lipodepsipeptides

isolated from Bacillus strains.^5,6They consist of an eight-

Figure 1. Structure of fengycins A, B, and S.

residue macrocycle with a phenyl ester linkage between the

phenol group of a tyrosine (Tyr) and the α-carboxylic group of

the conﬁguration of Tyr³and Tyr⁹in fengycins and plipastatins

an isoleucine (Ile) (Figure 1). This cyclic depsipeptide bears at

its N-terminus a dipeptidyl tail acylated with a β-hydroxy fatty

acid. Diﬀerent fengycin isoforms have been described,

fengycins A and B being the most common.⁷⁻¹⁴These two

isoforms diﬀer on the amino acid at position 6, which is a ^D-

alanine (^D-Ala) in fengycin A and a D-valine (D-Val) in the B

isoform (Figure 1). Sang-Cheol et al. isolated from Bacillus

amyloliquefaciens fengycin S.¹⁵Compared to fengycin B, this

isoform bears a ^D-serine (D-Ser) at position 4 instead of a D-

allo-threonine (^D-allo-Thr). The conﬁguration of Tyr residues

at positions 3 and 9 of fengycins has been a controversial issue.

Fengycins were ﬁrst reported to incorporate a ^D-Tyr³and an L-

Tyr⁹, whereas the opposite conﬁguration (L-Tyr³/D-Tyr⁹) was

assigned to another family of related peptides named as

plipastatins.^7,16However, it has been recently conﬁrmed that

is identical, being L-Tyr³/D-Tyr⁹.¹⁷⁻²⁰Since the term

fengycins is more widely used than plipastatins, herein we

employed the former term for the peptides of this study but

using the correct conﬁguration.

Fengycin lipopeptides have attracted considerable interest

due to their strong antifungal activity and their low hemolysis

compared to other lipopeptides isolated from Bacillus

spp.^7,21,22Their signiﬁcant antifungal activity has been ascribed

to their propensity to form stable membrane-bound

aggregates.²³Moreover, they have been described as elicitors

of plant defense responses.^24,25The use of compounds with

Received: October 3, 2018

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

such activity is considered as a promising alternative approach

for the protection of crops against phytopathogens.^2,26,27

Despite their interest, to date and to the best of our

knowledge, the synthesis of fengycins has only been

accomplished by the group of Marahiel and Essen. Their

strategy comprised the solid-phase preparation of the linear

precursor and its enzymatic cyclization in solution.^5,28,29The

lack of suitable synthetic protocols for fengycins could be

attributed to the presence of a highly labile phenyl ester

function in their structure. In fact, the synthesis of cyclic

depsipeptides, either in solution or on solid phase, has only

been reported for those peptides bearing an ester bond

involving the ß-hydroxyl group of a Ser or a Thr residue.³⁰⁻³⁴

Recently, we established a convenient solid-phase strategy for

the preparation of the macrolactone of eight amino acids

present in fengycins.³⁵The key steps of the synthesis were the

formation of the phenyl ester bond and the on-resin head-to-

side-chain cyclization.

Scheme 1. Retrosynthetic Analysis of BPC838

In view of these previous results, we decided to study the

application of this methodology to the total solid-phase

synthesis of dehydroxy fengycin derivatives with the general

structure I (Figure 2). These derivatives are fengycin A, B, and

Figure 2. General structure of dehydroxy fengycin derivatives I.

S analogues that contain a ^D-Thr⁴instead of a D-allo-Thr⁴and a

dehydroxy fatty acid chain. Synthesis of analogues containing

both an ^L-Tyr³/D-Tyr⁹or a D-Tyr³/L-Tyr⁹conﬁguration was

studied. This approach would beneﬁt from the advantages

inherent to the solid-phase methodology and would allow

rapid access to a variety of fengycin analogues.

sequence and its ﬁnal acylation. As protecting groups, allyl

(All) and allyloxycarbonyl (Alloc) were selected for Tyr⁹and

Ile¹⁰, respectively, because they can be simultaneously removed

in the presence of palladium and are orthogonal with the 9-

ﬂuorenylmethoxycarbonyl (Fmoc) and tert-butyl (^tBu) groups.

The pNZ group was chosen to protect Tyr³because it has been

RESULTS AND DISCUSSION

■

Synthesis of Cyclic Lipodepsipeptides Bearing a L-

Tyr³/D-Tyr⁹. Our investigations started with the solid-phase

synthesis of the cyclic lipodepsipeptide BPC838 containing ^D-

Thr⁴, D-Val⁶, D-Tyr⁹, and an octanoyl group (Scheme 1). This

synthesis was chosen as a model to check the feasibility of our

approach. On the basis of our previous experience on the

preparation of cyclic depsipeptides, the retrosynthetic route

that we envisaged involved the following as the main steps: (i)

preparation of the heptapeptidyl resin 1 with the N-terminal ^L-

Tyr³protected with a p-nitrobenzyloxycarbonyl (pNZ) group,

(ii) ester bond formation between ^L-Tyr³and Ile¹⁰, (iv)

simultaneous deprotection of Ile¹⁰and D-Tyr⁹followed by the

on-resin cyclization, and (vi) elongation of the peptide

reported to be orthogonal to Fmoc as well as to Bu and allyl-

based protecting groups, and its removal can be achieved

under neutral conditions using SnCl₂in the presence of

catalytic amounts of acid.³⁶

According to this route, Fmoc-D-Tyr-OAll was anchored to a

Wang resin by treatment with PPh₃and diisopropylazodicar-

boxylate (DIAD) in anhydrous tetrahydrofuran (THF) under

microwave irradiation for 30 min at 60 °C (Scheme 2).³⁵The

loading of the resulting resin Fmoc-^D-Tyr(Wang)-OAll was

0.33 mmol/g as determined by the Fmoc method.³⁷In

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

Scheme 2. Synthesis of the Cyclic Peptidyl Resin 3 and Attempts of pNZ Removal

addition, a Marfey’s test conﬁrmed that the D-Tyr residue did

not racemize under these conditions.³⁸Then the peptidyl resin

pNZ-Tyr³-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-

Tyr⁹(Wang)-OAll (1) was prepared following a Fmoc/^tBu/

allyl (All) strategy. Fmoc group removal was performed using

piperidine/DMF (3:7), and the coupling of the corresponding

amino acids was mediated by N,N′-diisopropylcarbodiimide

(DIPCDI) and 2-cyano-2-(hydroxyimino)acetate (Oxyma) in

DMF. All amino acids, except for Tyr³, were incorporated as

Fmoc derivatives with the side-chain group protected with

Next, the Alloc and allyl protecting groups of resin 2 were

simultaneously removed using a catalytic amount of Pd(PPh₃)₄

in the presence of PhSiH₃in CH₂Cl₂for 4 h (Scheme 2). After

acidolytic cleavage of an aliquot of the resulting resin pNZ-

Tyr³(O-Ile¹⁰-H)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-

Tyr⁹(Wang)-OH, the corresponding peptide was obtained in

72% HPLC purity. Subsequent on-resin cyclization was

assayed using [ethyl cyano(hydroxyimino)acetato-O²]tri-(1-

pyrrolidinyl)-phosphonium hexaﬂuorophosphate (PyOxim),

Oxyma, and DIEA, yielding the cyclic peptidyl resin 3.

Treatment of an aliquot of this resin with TFA/H₂O/TIS

aﬀorded the cyclic depsipeptide pNZ-Tyr³(&)-D-Thr-Glu-D-

Val-Pro-Gln-^D-Tyr⁹-Ile¹⁰-& (4) in 31% HPLC purity, as shown

by ESI-MS where a peak at m/z 1195.5 corresponding to [M +

Na]⁺was observed (& symbol indicates the location of the

ester linkage³⁹). The linear peptide precursor was not detected

either by HPLC or by mass spectrometry. To conﬁrm the

structure of 4, the crude reaction mixture resulting from

acidolytic cleavage was treated with CH₃OH/H₂O/NH₃

(4:1:1), conditions that are known to hydrolyze ester

bonds.⁴⁰⁻⁴²HPLC and mass spectrometry analysis of the

resulting crude revealed only the presence of the linear peptide

resulting from the hydrolysis of the ester bond in 4, pNZ-Tyr³-

D-Thr-Glu-D-Val-Pro-Gln-D-Tyr⁹-Ile¹⁰-OH, and of the corre-

sponding methyl ester pNZ-Tyr³-D-Thr-Glu-D-Val-Pro-Gln-D-

Tyr⁹-Ile¹⁰-OMe. Therefore, this result demonstrated the

formation of the cyclic depsipeptide 4.

trityl (Tr) for Gln and Bu for Glu and Thr. Tyr³was

introduced as pNZ-Tyr-OH to allow the required esteriﬁcation

of the phenol group. This tyrosine derivative was prepared

from commercially available H-Tyr(O^tBu)-OH by treatment

with pNZ-N₃,³⁶followed by side-chain deprotection under

acidic conditions (68% overall yield). After completion of the

peptide sequence, acidolytic cleavage with triﬂuoroacetic acid

(TFA)/H₂O/triisopropylsilane (TIS) (95:2.5:2.5) of an

aliquot of resin 1 aﬀorded the expected peptide pNZ-Tyr-^D-

Thr-Glu-^D-Val-Pro-Gln-D-Tyr-OAll in 65% HPLC purity.

Then Alloc-Ile-OH was incorporated to 1 using the

conditions previously described for the preparation of cyclic

depsipeptides analogous to the cyclic moiety of fengycins

(Scheme 2).³⁵Therefore, esteriﬁcation of 1 with Alloc-Ile-OH

was achieved employing DIPCDI, 4-dimethylaminopiridine

(DMAP), and N,N-diisopropylethylamine (DIEA) in DMF.

Two treatments of 24 h were necessary to obtain a complete

conversion. An aliquot of the resulting resin pNZ-Tyr³(O-Ile¹⁰-

Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-

Tyr⁹(Wang)-OAll (2) was treated under acidolytic conditions

yielding the expected linear depsipeptide pNZ-Tyr(O-Ile-

Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 87% HPLC

purity.

We then attempted the pNZ group removal of resin 3 using

SnCl₂(6 M) in HCl/dioxane (1.6 mM) and DMF for 1 h at

room temperature. Unexpectedly, this deprotection was

troublesome, and even after repeating this treatment 5 more

times, the deprotected cyclic peptide BPC822 was obtained

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

Scheme 3. Alternative Retrosynthetic Analysis of BPC838

together with an important amount of the pNZ-protected

cyclic depsipeptide 4 as shown by mass spectrometry.

aﬀorded the linear lipopeptide C₇H₁₅CO-Glu-D-Orn-Tyr-D-

Thr-Glu-^D-Val-Pro-Gln-D-Tyr-OAll in 79% HPLC purity.

Next, the esteriﬁcation of 5a with Alloc-Ile-OH was performed

employing DIEA, DIPCDI, and DMAP (2 × 24 h). An aliquot

of the resulting resin C₇H₁₅CO-Glu¹(O^tBu)-D-Orn(Boc)-

Tyr³(O-Ile¹⁰-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-D-Tyr⁹(Wang)-OAll (7a) was acidolytically cleaved,

yielding the branched lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-

Tyr(O-Ile-Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 81%

HPLC purity.

Selective removal of both Alloc and allyl protecting groups

in 7a with Pd(PPh₃)₃and PhSiH₃in CH₂Cl₂for 4 h followed

by exposure of an aliquot of the resulting resin 8a to TFA/

H₂O/TIS aﬀorded the expected linear peptide in 76% HPLC

purity (Scheme 4). Resin 8a was then treated with Oxyma,

PyOxim, and DIEA in DMF for 24 h to prompt its cyclization.

Acidolytic cleavage of the resulting resin aﬀorded the desired

cyclic lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-Tyr(&)-D-Thr-

Glu-^D-Val-Pro-Gln-D-Tyr-Ile-& (BPC838) in 33% HPLC

purity. Analysis by mass spectrometry of the crude reaction

mixture showed the formation of BPC838 as a major product.

Interestingly, only traces of the linear peptide resulting from

hydrolysis of the macrolactone were observed. Puriﬁcation by

reverse-phase column chromatography (RP-HPLC) yielded

BPC838 in >99% HPLC purity. The structure of this peptide

was veriﬁed by 1D and 2D NMR experiments. This result

On the basis of these results, we envisaged an alternative

route to obtain the cyclic lipodepsipeptide BPC838 (Scheme

3). In this approach, instead of building the macrolactone and

then incorporating the tail, we planned the synthesis of the

linear peptidyl resin 5a already bearing the acylated dipeptidyl

tail. Then this resin would be esteriﬁed at Tyr³with Ile¹⁰and

ﬁnally cyclized to render the desired cyclic lipodepsipeptide.

For this purpose, as depicted in Scheme 4, we ﬁrst

synthesized the linear peptidyl resin Fmoc-Glu¹(O^tBu)-D-

Orn(Boc)-Tyr³-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-

Tyr⁹(Wang)-OAll (6) through side-chain anchoring of Fmoc-

D-Tyr-OAll to a Wang resin followed by sequential Fmoc

removal and coupling steps using the conditions mentioned

above for resin 1. Orn and Tyr³were incorporated as Fmoc-

Orn(Boc)-OH and Fmoc-Tyr-OH, respectively. An aliquot of

the resulting linear peptidyl resin 6 was treated with TFA/

H₂O/TIS (95:2.5:2.5) for 2 h, aﬀording the linear peptide

Fmoc-Glu-^D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in

74% HPLC purity.

With peptidyl resin 6 in hand, we proceeded to its acylation

with octanoic acid (Scheme 4). Thus, after Fmoc group

removal octanoic acid was incorporated using Oxyma/DIPCDI

in DMF under stirring at room temperature overnight.

Acidolytic treatment of an aliquot of the resulting resin 5a

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

Scheme 4. Structure and General Synthetic Approach of Cyclic Lipodepsipeptides BPC838, BPC854, and BPC856

demonstrated the suitability of this strategy for the total solid-

phase synthesis of dehydroxy fengycin derivatives.

tide. These peptides were fully characterized by NMR

spectroscopy.

This methodology was then extended to the preparation of

cyclic lipodepsipeptides BPC854 and BPC856 bearing a lauric

and palmitic acid, respectively (Scheme 4). For this purpose,

after Fmoc removal of resin 6 acylation of the resulting free

amino group was performed using lauric and palmitic acid in

the presence of DIPCDI and Oxyma. The corresponding

lipopeptidyl resins 5b and 5c were acydolitically cleaved,

yielding the expected lipopeptides in 90% and 93% HPLC

purity, respectively. Subsequent esteriﬁcation of 5b and 5c with

Alloc-Ile-OH followed by allyl and Alloc group removal and

on-resin cyclization provided, after cleavage with TFA/H₂O/

TIS (95:2.5:2.5), the cyclic lipodepsipeptides C₁₁H₂₃CO-Glu-

D-Orn-Tyr(&)-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-&

(BPC854) and C₁₅H₃₁CO-Glu-D-Orn-Tyr(&)-D-Thr-Glu-D-

Val-Pro-Gln-^D-Tyr-Ile-& (BPC856) in 49% and 50% HPLC

purity, respectively. Both crude peptides were puriﬁed by RP-

HPLC and characterized by mass spectrometry, yielding

BPC854 and BPC856 in good purities (92% HPLC purity).

To conﬁrm the presence of the ester bond, these cyclic

lipodepsipeptides were subjected to basic hydrolysis using

CH₃OH/H₂O/NH₃(4:1:1). HPLC and mass spectrometry

analysis of the resulting crudes revealed only the presence of

the linear peptide resulting from the hydrolysis of the ester

bond, demonstrating the formation of the cyclic lipodepsipep-

Similarly, we prepared analogues of BPC838 diﬀering on the

amino acids at positions 4 and 6 (Figure 3). In particular,

BPC840 (^D-Ser⁴, D-Val⁶, D-Tyr⁹), BPC842 (D-Thr⁴, D-Ala⁶, D-

Tyr⁹), and BPC844 (D-Ser⁴, D-Ala⁶, D-Tyr⁹) were synthesized

following the above methodology and obtained in 35%, 26%,

and 21% HPLC purity, respectively. ESI-MS analysis showed

Figure 3. Structure of cyclic lipodepsipeptides BPC840, BPC842,

and BPC844.

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

the presence of the corresponding cyclic lipodepsipeptide as

the major product. RP-HPLC puriﬁcation aﬀorded these

peptides in purities > 94%. Their structure was characterized

by NMR.

BPC858 (^D-Tyr³, D-Thr⁴, D-Val⁶, lauroyl), and BPC860 (D-

Tyr³, D-Thr⁴, D-Val⁶, palmitoyl).

These cyclic lipodepsipeptides were synthesized according

to the protocol described in Scheme 4. After TFA cleavage,

mass spectrometry analysis of the crude reaction mixtures

showed the presence of the expected peptide along with a

substantial amount of a byproduct with a m/z corresponding

to [M + H₂O]⁺. This byproduct could correspond to the linear

precursor or to the linear compound resulting from the

hydrolysis of the ester bond of the ﬁnal cyclic depsipeptide. To

verify the identity of this byproduct, the crude reaction

mixtures were subjected to CH₃OH/H₂O/NH₃(4:1:1).⁴⁰⁻⁴²

As depicted in Scheme 5, if the linear precursor is present in

the crudes, hydrolysis of the ester bond would lead to the

release of the Ile residue (Scheme 5A). However, when the

crudes were subjected to hydrolysis only peaks at [M + H₂O]⁺

and at [M + CH₃OH]⁺were observed (Scheme 5B). This

result demonstrated that the cyclizations were complete and

that the cyclic lipodepsipeptides hydrolyzed during the

cleavage or the HPLC analysis. The crude reaction mixtures

from the synthesis of BPC858 and BPC860 were puriﬁed by

reverse-phase column chromatography and were obtained in

78% and 70% HPLC purity, respectively.

Synthesis of Cyclic Lipodepsipeptides Bearing ^D-Tyr³/

L-Tyr⁹. Taking into account the diﬀerent conﬁguration

reported for Tyr³and Tyr⁹for natural fengycins, we decided

to prepare cyclic lipodepsipeptides incorporating a ^D-Tyr³and

an ^L-Tyr⁹(Figure 4). In particular, we attempted the synthesis

In our previous studies on the synthesis of the macrolactone

of eight amino acids present in fengycins, compounds bearing

the same conﬁguration at the Tyr residues as the cyclic

lipodepsipeptides depicted in Figure 4 were obtained together

with a dimeric product and the linear precursor prior to

cyclization.³⁵The results obtained herein point out, on one

hand, that the presence of the fatty acid tail in the linear

precursor may favor its cyclization and, on the other hand, that

the resulting macrolactone is less stable than the one

containing the Tyr residues with an opposite conﬁguration

(i.e., BPC846 vs BPC838). Moreover, these ﬁndings further

Figure 4. Structure of cyclic lipodepsipeptides BPC846, BPC848,

BPC850, BPC852, BPC858, and BPC860.

of BPC846 (^D-Tyr³, D-Thr⁴, D-Val⁶, octanoyl), BPC848 (D-

Tyr³, D-Ser⁴, D-Val⁶, octanoyl), BPC850 (D-Tyr³, D-Thr⁴, D-

Ala⁶, octanoyl), BPC852 (D-Tyr³, D-Ser⁴, D-Ala⁶, octanoyl),

Scheme 5. Hydrolysis Reaction of the Crude Reaction Mixtures from the Synthesis of the Cyclic Lipodepsipeptides: (A)

Hydrolysis of the Linear Precursors; (B) Hydrolysis of the Cyclic Lipodepsipeptides

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

calibrated using sodium formate. The instrument was operated in

positive ESI(+) ion mode.

support the recent hypothesis of Honma and co-workers that

postulated ^L-Tyr³/D-Tyr⁹as the correct fengycin conﬁgu-

ration.¹⁹

NMR spectra (¹H NMR, ¹³C NMR) were recorded with a 400 or

300 MHz Bruker Ultrashield Avance spectrometer. Chemical shifts

were reported as δ values (ppm) directly referenced to undeuterated

residual solvent signal (i.e., DMSO-d₆δ = 2.50 ppm). The following

multiplicity abbreviations are used: (d) doublet, (dd) double doublet,

(td) triple doublet, (m) multiplet, and (br) broad peak. 2D-COSY

and 2D-TOCSY experiments were carried out to assign the ¹H NMR

peaks of the cyclic lipodepsipeptides.

CONCLUSION

■

In summary, here we describe an eﬃcient solid-phase approach

for the total synthesis of dehydroxy derivatives of fengycins A,

B, and S. The synthesis was accomplished using a Fmoc/^tBu/

allyl strategy, the key steps being the formation of a phenyl

ester and the ﬁnal macrolactamization. This study revealed the

signiﬁcance of the conﬁguration of the Tyr residues on the

stability of the macrolactone and showed that the most stable

compounds were those containing an ^L-Tyr³and a D-Tyr⁹. This

study represents the ﬁrst approach on the total solid-phase

synthesis of fengycin derivatives and would allow rapid access

to a large variety of analogues.⁴³

IR spectra were recorded with a Bruker Alpha FT-IR spectrometer

equipped with a Bruker platinum ATR adaptador, and wavenumbers

(ν) are expressed in cm⁻¹.

The “&” symbol was used as an indicator of the ester chemical

bond to facilitate the view of cyclic depsipeptide formulas. On the

one-line formula, the “&” symbol indicates both the location of one

end of a chemical bond and the point to which this bond is attached.

This symbol has already been used in the nomenclature of other cyclic

depsipeptides.³⁹

Allyl N^α-(9-ﬂuorenylmethyloxycarbonyl)-D-tyrosinate, allyl N^α-(9-

ﬂuorenylmethyloxycarbonyl)-^L-tyrosinate, and N^α-allyloxycarbonyl-L-

isoleucine were prepared according to previously reported proce-

dures.³⁵

EXPERIMENTAL SECTION

■

General Methods. Peptide synthesis was performed manually in a

polypropylene syringe ﬁtted with a polyethylene porous disc. Solvents

and soluble reagents were removed in vacuo. Wang resin was

purchased from Fluka. Amino acid derivatives and other chemical

reagents were purchased from IrisBiotech, Sigma-Aldrich, or Panreac

and used without further puriﬁcation, unless otherwise noted.

Solvents were purchased from Sigma-Aldrich, Sharlau, VidraFoc,

SDS, or VWR International. All organic solvents were synthesis grade

except for CH₃CN, which was multisolvent HPLC grade. Solvents

were puriﬁed and dried by an activated alumina puriﬁcation system

(mBraun SPS-800) or by conventional distillation techniques. H₂O

was deionized and ﬁltered by a COT Millipore Q-gradient system

(COT < 3 ppb) with a resistivity of 18 MΩ·cm⁻¹.

Synthesis of Amino Acids. N^α-(p-Nitrobenzyloxycarbonyl)-O-

tert-butyl-^L-tyrosine.³⁶NaN₃(164.37 mg, 2.53 mmol) was dissolved

in H₂O (0.66 mL). and the mixture was added to a solution of pNz-Cl

(468.3 mL, 2.11 mmol) in 1,4-dioxane (0.92 mL). The mixture was

stirred at room temperature for 2 h until the formation of p-

nitrobenzylazidoformate (pNz-N₃). Next, a solution of H-L-Tyr(^tBu)-

OH (500 mg, 2.11 mmol) in 1% Na₂CO₃/1,4-dioxane (1:1, 2.63 mL)

was added dropwise. The resulting white suspension was stirred for 48

h at room temperature, keeping the pH between 8 and 10 by addition

of 10% Na₂CO_3.The progress of the reaction was monitored by TLC.

Once the reaction was completed, H₂O (30 mL) was added and the

resulting suspension was washed with tert-butyl methyl ether (3 × 15

mL). The aqueous portion was acidiﬁed to pH 2 with 3 N HCl, and a

precipitate appeared, which was ﬁltered oﬀ and dried to yield pNz-^L-

Microwave-assisted reactions were carried out on a Discover S-

Class CEM Corp. Microwave equipped with an Explorer-Autosampler

and controlled with the Synergy software. The equipment was

provided with a multispeed magnetic stirring system with adjustable

speed and an automated power control system based on temperature

feedback through a volume-independent noninvasive infrared sensor

control which ranges from 0 to 300 W. The work temperature range is

from 40 to 300 °C with a heating rate of 2−6 °C/s. The reactions

were performed in 35 mL reaction vessels whose volume can range

from 2 to 25 mL.

Compounds were analyzed under standard analytical high-

performance liquid chromatography (HPLC) conditions using a

Dionex liquid chromatography instrument composed of an UV/vis

Dionex UVD170U detector, a P680 Dionex bomb, and an ASI-100

Dionex automatic injector and using the CHROMELEON 6.60

software. The analysis was carried out with a Kromasil 100 C₁₈(4.6

mm × 40 mm, 3.5 μm) column with a 2−100% B linear gradient over

7 min at a ﬂow rate of 1 mL/min. Solvent A was 0.1% aq TFA, and

solvent B was 0.1% TFA in CH₃CN. Detection was performed at 220

nm. Purity was estimated with the integrated area under peaks.

Peptide puriﬁcations were carried on a reverse-phase column

chromatography CombiFlash Rf200.

Tyr(^tBu)-OH as a white solid (653.3 mg, 75% yield). H NMR (400

MHz, CD₃OD) δ (ppm): 1.31 (s, 9H, (CH₃)₃), 2.87 (dd, J = 9.6, 14.0

Hz, 1H, CH₂-β), 3.20 (dd, J = 4.6, 14.0 Hz, 1H, CH₂-β), 4.39 (dd, J =

4.6, 9.6 Hz, 1H, CH-α), 5.09 (d, J = 13.8 Hz, 1H, OCH₂), 5.19 (d, J =

13.8 Hz, 1H, OCH₂), 6.88 (d, J = 8.6 Hz, 2H, CH_Arom3-Tyr), 7.14 (d,

J = 8.6 Hz, 2H, CH_Arom2-Tyr), 7.45 (d, J = 8.8 Hz, 2H, CH_Arom2-pNz),

8.20 (d, J = 8.8 Hz, 2H, CH_Arom3-pNz). ¹³C{¹H} NMR (400 MHz,

CD₃OD) δ (ppm): 29.2 ((CH₃)₃), 38.3 (CH₂-β), 57.3 (CH-α), 65.9

(OCH₂), 79.4 (C(CH₃)₃), 124.5, 125.1 (CH_Arom3-Tyr, CH_Arom3

pNz), 128.9, 130.9 (CH_Arom2-Tyr, CH_Arom2-pNz), 133.9 (C_Arom1-Tyr),

146.1, 148.8 (C_Arom1-pNz, C_Arom4-pNz), 155.3 (OC_Arom4-Tyr), 157.8

(HN−CO), 175.7 (COOH).

N^α-(p-Nitrobenzyloxycarbonyl)-L-tyrosine. This compound was

prepared following the procedure described for Fmoc-^L-Tyr-OAll

starting from pNz-^L-Tyr(^tBu)-OH (613.3 mg, 1.47 mmol). pNz-L-

Tyr-OH was obtained quantitatively as a white solid (478.1 mg, 90%

yield). IR (neat): 3325 (COO-H_st), 1697 (CO_st), 1608, 1512

(NO_{2 st as}), 1441, 1343 (NO_{2 st sim}), 1201, 1105, 1059 cm⁻¹; ¹H NMR

(400 MHz, CD₃OD) δ (ppm): 2.82 (dd, J = 9.6, 14.0 Hz, 1H, CH₂-

β), 3.13 (dd, J = 4.8, 14.0 Hz, 1H, CH₂-β), 4.38 (dd, J = 4.8, 9.6 Hz,

1H, CH-α), 5.11 (d, J = 14.0 Hz, 1H, OCH₂), 5.19 (d, J = 14.0 Hz,

1H, OCH₂), 6.70 (d, J = 8.4 Hz, 2H, CH_Arom3-Tyr), 7.05 (d, J = 8.4

Hz, 2H, CH_Arom2-Tyr), 7.45 (d, J = 8.8 Hz, 2H, CH_Arom2-pNz), 8.19

(d, J = 8.8 Hz, 2H, CH_Arom3-pNz). ¹³C{¹H} NMR (400 MHz,

CD₃OD) δ (ppm): 37.9 (CH₂-β), 57.0 (CH-α), 66.0 (OCH₂), 116.2

(CH_Arom3-Tyr), 124.5 (CH_Arom3-pNz), 128.8, 129.2 (CH_Arom2-Tyr,

MS (ESI) were recorded with an Esquire 6000 ESI Bruker ion Trap

LC/MS instrument equipped with an electrospray ion source (Serveis

Tecnics de recerca, University of Girona). The instrument was

operated in both positive ESI(+) and negative ESI(−) ion modes.

Samples (5 μL) were introduced into the mass spectrometer ion

source directly through a 1200 Series Agilent HPLC autosampler. The

mobile phase (80:20 CH₃CN/H₂O at a ﬂow rate of 100 μL/min) was

delivered by an Agilent HPLC pump. Nitrogen was employed as both

drying and nebulizing gas.

CH_Arom2-pNz), 131.3 (C_Arom1-Tyr), 146.1, 148.8 (C_Arom1-pNz, C_Arom4

pNz), 157.3, 157.9 (OC_Arom4-Tyr, HN−CO), 175.2 (COOH). MS

(ESI) m/z: 361.0 [M + H]⁺, 383.0 [M + Na]⁺.

HRMS analyses were performed under conditions of ESI with a

Bruker MicroTOF-QII instrument using a hybrid quadrupole time-of-

N^α-(9-Fluorenylmethyloxycarbonyl)-D-tyrosine. The commercial

amino acid Fmoc-^D-Tyr(O^tBu)-OH (0.6 g, 1.31 mmol) was dissolved

in TFA/CH₂Cl₂(1:1) and stirred at room temperature for 3 h. The

solution was concentrated to dryness followed by repeated washings

ﬂight mass spectrometer (Serveis Tecnics de Recerca, University of

Girona). Samples were introduced into the mass spectrometer ion

source by direct infusion through a syringe pump and were externally

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

and evaporations with diethyl ether. The resulting residue was

digested in pentane to yield Fmoc-^D-Tyr-OH as a white powder (0.58

g, 97% yield). IR (neat): 3308 (OH_st), 1685 (CO_st), 1541, 1515

(arC-C_st, NH_δ), 1450 (CH_2δ), 1230 (arC-O_st), 735 (CH_2γ) cm⁻¹. ¹H

NMR (400 MHz, DMSO-d₆) δ (ppm): 2.74 (dd, J = 10.8, 13.6 Hz,

1H, CH₂-β), 2.95 (dd, J = 4.0, 13.6 Hz, 1H, CH₂-β), 4.05−4.11 (m,

1H, CH-Fmoc), 4.15−4.21 (m, 3H, OCH₂-Fmoc, CH-α), 6.66 (d, J =

8.4 Hz, 2H, CH_Arom6-Tyr), 7.06 (d, J = 8.4 Hz, 2H, CH_Arom5-Tyr),

7.27−7.34 (m, 2H, CH_Arom13-Fmoc), 7.41 (td, J = 3.2, 7.6 Hz, 2H,

CH_Arom14-Fmoc), 7.63−7.71 (m, 3H, CH_Arom12-Fmoc, OH), 7.88 (d, J

= 7.6 Hz, 2H, CH_Arom15-Fmoc) 9.22 (br, 1H, NH), 12.71 (br, 1H,

COOH). ¹³C{¹H} NMR (300 MHz, DMSO-d₆) δ (ppm): 35.2

(CH₂-β), 46.1 (CH-Fmoc), 55.4 (CH-α), 65.1 (OCH₂-Fmoc), 114.5

(CH_Arom6-Tyr), 119.6, 124.8, 126.6, 127.1, 127.5 (CH_Arom12-Fmoc,

CH_Arom13-Fmoc, CH_Arom14-Fmoc, CH_Arom15-Fmoc, C_q‑Arom4-Tyr),

(hydroxyimino) acetate (Oxyma) (4 equiv) and N,N′-diisopropylcar-

bodiimide (DIPCDI) (4 equiv) in DMF for 3 h under stirring at room

temperature. The completion of each coupling was monitored by a

Kaiser test⁴⁵or a chloranil test.⁴⁶The Fmoc protecting group was

removed by treating the resin with piperidine/DMF (3:7, 1 × 2 + 3 ×

10 min). After each coupling and deprotection step, the resin was

washed with DMF (6 × 1 min) and CH₂Cl₂(3 × 1 min) and air

dried.

To obtain the lipopeptidyl resins, each peptidyl resin was subjected

to N-terminal Fmoc removal as described above. After washings, the

resin was treated with the corresponding fatty acid (3 equiv), Oxyma

(3 equiv), and DIPCDI (3 equiv) in DMF under overnight stirring at

room temperature. The resin was then washed with DMF (6 × 1 min)

and CH₂Cl₂(3 × 1 min) and air dried. Completion of the reaction

was checked with the Kaiser test.⁴⁵An aliquot of each resulting

lipopeptidyl resin was treated with triﬂuoroacetic acid (TFA)/H₂O/

triisopropylsilane (TIS) (95:2.5:2.5) for 2 h at room temperature.

Following TFA evaporation, the resulting crude lipopeptide was

precipitated with diethyl ether and then decanted to give a white solid

that was taken up in H₂O/CH₃CN (1:1), lyophilized, and analyzed by

HPLC and mass spectrometry.

129.5 (CH_Arom5-Tyr), 140.2, 143.2 (C_q‑Arom11-Fmoc_,C_q‑Arom16

Fmoc), 155.4, 155.4 (OC_q‑Arom7-Tyr, NH−CO), 173.0 (O−C

O). MS (ESI) m/z: 404.0 [M + H]⁺, 426.1 [M + Na]⁺.

N^α-(9-Fluorenylmethyloxycarbonyl)-L-tyrosine. Starting from the

commercially available Fmoc-^L-Tyr(O^tBu)-OH (0.5 g, 1.09 mmol),

this compound was prepared following the procedure described above

for Fmoc-^D-Tyr-OH. Fmoc-L-Tyr-OH was obtained as a white

powder (0.43 g, 96% yield). IR (neat): 3354 (OH_st), 1655 (C

O_st), 1538, 1514 (arC-C_st, NH_δ), 1449 (CH_2δ), 1256, 1220 (arC-O_st),

Linear Peptidyl Resin pNZ-Tyr-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-

Gln(Tr)-^D-Tyr(Wang)-OAll (1). Starting from Fmoc-D-Tyr(Wang)-

OAll, this peptidyl resin was prepared following the above general

procedure. Acidolytic cleavage of an aliquot of 1 aﬀorded pNZ-Tyr-^D-

Thr-Glu-^D-Val-Pro-Gln-D-Tyr-OAll in 65% purity. HPLC (λ = 220

nm) t_R= 7.28 min. MS (ESI) m/z(+): 1118.5 [M + H]⁺, 1140.4 [M +

Na]⁺, 1156.4 [M + K]⁺.

735 (CH_2γ) cm⁻¹. H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.75

(dd, J = 10.4, 13.8 Hz, 1H, CH₂-β), 2.95 (dd, J = 4.4, 13.8 Hz, 1H,

CH₂-β), 4.05−4.11 (m, 1H, CH-Fmoc), 4.15−4.22 (m, 3H, OCH₂-

Fmoc, CH-α), 6.66 (d, J = 8.4 Hz, 2H, CH_Arom6-Tyr), 7.06 (d, J = 8.4

Hz, 2H, CH_Arom5-Tyr), 7.27−7.34 (m, 2H, CH_Arom13-Fmoc), 7.39−

7.43 (m, 2H, CH_Arom14-Fmoc), 7.63−7.68 (m, 3H, CH_Arom12-Fmoc,

OH), 7.88 (d, J = 7.2 Hz, 2H, CH_Arom15-Fmoc), 9.20 (br, 1H, NH),

12.68 (br, 1H, COOH). ¹³C{¹H} NMR (400 MHz, DMSO-d₆) δ

(ppm): 36.2 (CH₂-β), 47.0 (CH-Fmoc), 56.3 (CH-α), 66.1 (OCH₂-

Fmoc), 115.4 (CH_Arom6-Tyr), 120.5, 125.7, 127.5, 128.1, 128.4

(CH_Arom12-Fmoc, CH_Arom13-Fmoc, CH_Arom14-Fmoc, CH_Arom15-Fmoc,

C_q‑Arom4-Tyr), 130.48 (CH_Arom5-Tyr), 141.1, 144.2 (C_q‑Arom11-Fmoc_,

C_q‑Arom16-Fmoc), 156.3, 156.4 (OC_q‑Arom7‑Tyr, NH−CO), 173.9

(O−CO). MS (ESI) m/z: 404.0 [M + H]⁺, 426.1 [M + Na]⁺.

Solid-Phase Synthesis of Cyclic Lipodepsipeptides. Syn-

thesis of Peptidyl Resins 1 and 6 and of the Linear Lipopeptidyl

Resins. Wang resin (450 mg, loading 1.1 mmol/g, 100−200 mesh,

cross-linked with 1% DVB) was placed in a microwave tube and

swollen in dry tetrahydrofuran (THF) for 30 min. A solution of

Fmoc-^L-Tyr-OAll or Fmoc-D-Tyr-OAll (878.11 mg, 1.98 mmol) and

triphenylphosphine (PPh₃) (519.35 mg, 1.98 mmol) in dry THF (3

mL) was added to the resin, and the resulting mixture was cooled at 0

°C. A solution of diisopropylazodicarboxylate (DIAD, 390 μL, 1.98

mmol) in dry THF (0.5 mL) was added dropwise, and the mixture

was stirred 1 h at 0 °C. The resulting mixture was subjected to

microwave irradiaton at 60 °C for 30 min. Then the resin was

transferred to a polypropylene syringe and washed sequentially with

THF (3 × 2 min), CH₂Cl₂(3 × 2 min), DMF (3 × 2 min), DMF/

H₂O (1:1, 3 × 2 min), DMF (3 × 2 min), CH₃OH (3 × 2 min),

CH₂Cl₂(3 × 2 min), and diethyl ether (3 × 2 min) and dried in

vacuo. The loading of the resulting resins Fmoc-^L-Tyr(Wang)-OAll

and Fmoc-^D-Tyr(Wang)-OAll was determined using the Fmoc test,

being 0.20 and 0.29 mmol/g, respectively.³⁷The racemization of the

anchored amino acid was evaluated using Marfey’s reagent.³⁸Finally,

the resin was acetylated with acetic anhydride/pyridine/CH₂Cl₂

(86:7:7, 2 × 30 min). The resin was washed with CH₂Cl₂(3 × 2

min), DMF (3 × 2 min), CH₃OH (3 × 1 min), CH₂Cl₂(3 × 2 min),

and diethyl ether (3 × 2 min) and dried in vacuo.⁴⁴

Linear Peptidyl Resin Fmoc-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-Thr(^tBu)-

Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (6). Starting from

Fmoc-^D-Tyr(Wang)-OAll, this peptidyl resin was prepared following

the above general procedure. Acidolytic cleavage of an aliquot of 1

aﬀorded Fmoc-Glu-^D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll

in 74% purity. HPLC (λ = 220 nm) t_R= 7.23 min.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5a). This

peptidyl resin was prepared from 6 following the above procedure

using octanoic acid as fatty acid. Acidolytic cleavage of an aliquot of

5a aﬀorded C₇H₁₅CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-

Tyr-OAll in 79% purity. HPLC (λ = 220 nm) t_R= 7.09 min. MS

(ESI) m/z(+): 654.8 [M + 2H]²⁺, 1308.7 [M + H]⁺, 1330.6 [M +

Na]⁺. MS (ESI) m/z(−): 1306.7 [M − H]⁻, 1328.6 [M + Na −

2H]⁻.

Linear Lipopeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5b). This

peptidyl resin was prepared from 6 following the above procedure

using lauric acid as fatty acid. Acidolytic cleavage of an aliquot of 5b

aﬀorded C₁₁H₂₃CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-

OAll in 90% purity. HPLC (λ = 220 nm) t_R= 7.91 min. MS (ESI)

m/z(+): 683.3 [M + 2H]²⁺, 1364.8 [M + H]⁺. MS (ESI) m/z(−):

1362.7 [M − H]⁻, 1384.6 [M + Na − 2H]⁻.

Linear Lipopeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5c). This

peptidyl resin was prepared from 6 following the above procedure

using palmitic acid as fatty acid. Acidolytic cleavage of an aliquot of 5c

aﬀorded C₁₅H₃₁CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-

OAll in 93% purity. HPLC (λ = 220 nm) t_R= 8.84 min. MS (ESI)

m/z(+): 1420.8 [M + H]⁺. MS (ESI) m/z(−): 1418.7 [M − H]⁻,

1440.7 [M + Na − 2H]⁻.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll. Starting

from Fmoc-^D-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr-D-Ser-Glu-D-Val-Pro-Gln-D-Tyr-OAll in

67% purity. HPLC (λ = 220 nm) t_R= 6.97 min. MS (ESI) m/

z(+): 647.8 [M + 2H]²⁺, 1294.6 [M + H]⁺. MS (ESI) m/z(−):

1292.6 [M − H]⁻, 1314.5 [M + Na − 2H]⁻.

Fmoc-L-Tyr(Wang)-OAll and Fmoc-D-Tyr(Wang)-OAll resins

were elongated by the solid-phase method following a standard

Fmoc chemistry. Fmoc-Gln(Tr)-OH, Fmoc-Pro-OH, Fmoc-^D-Val-

OH, Fmoc-^D-Ala-OH, Fmoc-Glu(O^tBu)-OH, Fmoc-D-Thr(^tBu)-OH,

Fmoc-^D-Ser(^tBu)-OH, Fmoc-Tyr-OH, Fmoc-D-Tyr-OH, Fmoc-D-

Orn(Boc)-OH, and pNZ-Tyr-OH were used as amino acid

derivatives. The coupling of the corresponding protected amino

acid (4 equiv) was carried out in the presence of ethyl 2-cyano-2-

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-OAll. Starting

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

from Fmoc-D-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Ala-Pro-Gln-D-Tyr-OAll in

64% purity. HPLC (λ = 220 nm) t_R= 6.89 min. MS (ESI) m/

z(+): 641.3 [M + 2H]²⁺, 1280.6 [M + H]⁺. MS (ESI) m/z(−):

1278.6 [M − H]⁻, 1300.5 [M + Na − 2H]⁻.

Synthesis of Linear Depsipeptidyl Resins Containing a

Phenyl Ester. Each peptidyl resin was treated with Alloc-^L-Ile-OH (7

equiv), DIEA (1.4 equiv), DIPCDI (7 equiv), and DMAP (0.7 equiv)

in DMF for 24 h at room temperature. This treatment was repeated

twice. After each treatment, the resin was washed with DMF (6 × 1

min) and CH₂Cl₂(3 × 1 min) and air dried. An aliquot of the each

resulting resin was treated with TFA/H₂O/TIS (95:2.5:2.5) for 2 h at

room temperature. Following TFA evaporation and diethyl ether

extraction, the crude peptide was dissolved in H₂O/CH₃CN,

lyophilized, analyzed by HPLC, and characterized by mass

spectrometry.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-D-

Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-OAll. Starting

from Fmoc-^D-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr-D-Ser-Glu-D-Ala-Pro-Gln-D-Tyr-OAll in

71% purity. HPLC (λ = 220 nm) t_R= 6.84 min. MS (ESI) m/

z(+): 633.7 [M + 2H]²⁺, 1266.6 [M + H]⁺. MS (ESI) m/z(−):

1264.6 [M − H]⁻, 1286.5 [M + Na − 2H]⁻.

Linear Depsipeptidyl Resin pNZ-Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (2). Starting from 1, this

depsipeptidyl resin was prepared according to the general procedure

described above. Acidolytic cleavage of an aliquot of 2 aﬀorded pNZ-

Tyr(O-Ile-Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 87% purity.

HPLC (λ = 220 nm) t_R= 8.21 min. MS (ESI) m/z(+): 1315.6 [M +

H]⁺, 1337.6 [M + Na]⁺, 1353.5 [M + K]⁺.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-OAll in

69% purity. HPLC (λ = 220 nm) t_R= 7.00 min. MS (ESI) m/

z(+): 1308.6 [M + H]⁺. MS (ESI) m/z(−): 1306.6 [M − H]⁻.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-Glu-D-Val-Pro-Gln-Tyr-OAll in

67% purity. HPLC (λ = 220 nm) t_R= 6.97 min. MS (ESI) m/

z(+): 647.7 [M + 2H]²⁺, 1294.6 [M + H]⁺, 1316.6 [M + Na]⁺. MS

(ESI) m/z(−): 1292.6 [M − H]⁻, 1314.5 [M + Na − 2H]⁻.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Ala-Pro-Gln-Tyr-OAll in

68% purity. HPLC (λ = 220 nm) t_R= 6.89 min. MS (ESI) m/

z(+): 1280.6 [M + H]⁺, 1302.6 [M + Na]⁺. MS (ESI) m/z(−):

1278.6 [M − H]⁻, 1300.5 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll (7a). Starting from 5a, this depsipeptidyl resin was

prepared according to the general procedure described above.

Acidolytic cleavage of an aliquot of 7a aﬀorded C₇H₁₅CO-Glu-D-

Orn-Tyr(O-Ile-Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 81%

purity. HPLC (λ = 220 nm) t_R= 7.82 min. MS (ESI) m/z(+):

753.3 [M + 2H]²⁺, 1505.7 [M + H]⁺. MS (ESI) m/z(−): 1503.7 [M

− H]⁻, 1525.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll (7b). Starting from 5b, this depsipeptidyl resin was

prepared according to the general procedure described above.

Acidolytic cleavage of an aliquot of 7b aﬀorded C₁₁H₂₃CO-Glu-D-

Orn-Tyr(O-Ile-Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 88%

purity. HPLC (λ = 220 nm) t_R= 8.72 min. MS (ESI) m/z(+):

781.4 [M + 2H]²⁺, 1561.9 [M + H]⁺. MS (ESI) m/z(−): 1559.8 [M

− H]⁻, 1581.7 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll (7c). Starting from 5c, this depsipeptidyl resin was

prepared according to the general procedure described above.

Acidolytic cleavage of an aliquot of 7c aﬀorded C₁₅H₃₁CO-Glu-D-

Orn-Tyr(O-Ile-Alloc)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-OAll in 88%

purity. HPLC (λ = 220 nm) t_R= 9.74 min. MS (ESI) m/z(+):

809.5 [M + 2H]²⁺, 1617.9 [M + H]⁺. MS (ESI) m/z(−): 1615.8 [M

− H]⁻, 1637.8 [M + Na − 2H]⁻.

Linear Lipopeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using octanoic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-Glu-D-Ala-Pro-Gln-Tyr-OAll in

67% purity. HPLC (λ = 220 nm) t_R= 6.85 min. MS (ESI) m/

z(+): 1266.6 [M + H]⁺, 1288.6 [M + Na]⁺. MS (ESI) m/z(−):

1264.6 [M − H]⁻, 1286.5 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-

D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll, this

depsipeptidyl resin was prepared according to the general procedure

described above. Acidolytic cleavage of an aliquot of the resulting

resin aﬀorded C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-Alloc)-D-Ser-Glu-D-

Linear Lipopeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using lauric acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₁₁H₂₃CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-OAll in

66% purity. HPLC (λ = 220 nm) t_R= 8.14 min. MS (ESI) m/

z(+): 1364.7 [M + H]⁺, 1386.7 [M + Na]⁺. MS (ESI) m/z(−):

1362.7 [M − H]⁻, 1384.6 [M + Na − 2H]⁻.

Val-Pro-Gln-^D-Tyr-OAll in 81% purity. HPLC (λ = 220 nm) t_R

7.72 min. MS (ESI) m/z(+): 746.3 [M + 2H]²⁺, 1491.7 [M + H]⁺.

MS (ESI) m/z(−): 1489.7 [M − H]⁻, 1512.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-

D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-OAll, this

depsipeptidyl resin was prepared according to the general procedure

described above. Acidolytic cleavage of an aliquot of the resulting

resin aﬀorded C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-Alloc)-D-Thr-Glu-D-

Linear Lipopeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-

D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll. Starting

from Fmoc-Tyr(Wang)-OAll, this peptidyl resin was prepared

following the above general procedure using palmitic acid as fatty

acid. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₁₅H₃₁CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-OAll in

79% purity. HPLC (λ = 220 nm) t_R= 9.11 min. MS (ESI) m/

z(+): 710.8 [M + 2H]²⁺, 1420.8 [M + H]⁺, 1442.8 [M + Na]⁺. MS

(ESI) m/z(−): 708.8 [M − 2H]²⁻, 1418.7 [M − H]⁻, 1440.7 [M +

Na − 2H]⁻.

Ala-Pro-Gln-^D-Tyr-OAll in 77% purity. HPLC (λ = 220 nm) t_R

7.68 min. MS (ESI) m/z(+): 739.8 [M + 2H]²⁺, 1477.7 [M + H]⁺.

MS (ESI) m/z(−): 1475.6 [M − H]⁻, 1497.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr-

(Wang)-OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-Tyr-

D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-OAll, this

depsipeptidyl resin was prepared according to the general procedure

described above. Acidolytic cleavage of an aliquot of the resulting

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

resin aﬀorded C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-Alloc)-D-Ser-Glu-D-

resulting peptidyl resin was exposed to acidolytic conditions of TFA/

H₂O/TIS (95:2.5:2.5) for 2 h at room temperature. Following TFA

evaporation and diethyl ether extraction, the crude peptide was

dissolved in H₂O/CH₃CN, lyophilized, analyzed by HPLC, and

characterized by mass spectrometry.

Ala-Pro-Gln-^D-Tyr-OAll in 77% purity. HPLC (λ = 220 nm) t_R

7.60 min. MS (ESI) m/z(+): 732.3 [M + 2H]²⁺, 1463.7 [M + H]⁺.

MS (ESI) m/z(−): 1461.6 [M − H]⁻, 1483.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-

OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-D-Thr-

(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll, this depsipep-

tidyl resin was prepared according to the general procedure described

above. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-Thr-Glu-D-Val-Pro-Gln-

Tyr-OAll in 79% purity. HPLC (λ = 220 nm) t_R= 7.94 min. MS

(ESI) m/z(+): 753.8 [M + 2H]²⁺, 1505.8 [M + H]⁺. MS (ESI) m/

z(−): 1503.7 [M − H]⁻, 1526.7 [M + Na − 2H]⁻.

Linear Depsipeptidyl Resin pNZ-Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OH. This peptidyl resin was

obtained from 2 following the above general procedure. Acidolytic

cleavage of an aliquot of this resin aﬀorded pNZ-Tyr(O-Ile-H)-^D-Thr-

Glu-^D-Val-Pro-Gln-D-Tyr-OH in 72% purity. HPLC (λ = 220 nm) t_R

= 6.77 min. MS (ESI) m/z(+): 615.2 [M + H + K]²⁺, 1191.5 [M +

H]⁺, 1213.5 [M + Na]⁺, 1229.5 [M + K]⁺.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-

OH (8a). This peptidyl resin was obtained from 7a following the

above general procedure. Acidolytic cleavage of an aliquot of 8a

aﬀorded C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-

Gln-^D-Tyr-OH (BPC837) in 76% purity. HPLC (λ = 220 nm) t_R=

6.68 min. MS (ESI) m/z(+): 691.3 [M + 2H]²⁺, 1381.6 [M + H]⁺.

MS (ESI) m/z(−): 1379.6 [M − H]⁻, 1401.6 [M + Na − 2H]⁻

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-

OH. This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-^D-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Ser-Glu-D-Val-Pro-Gln-D-Tyr-

OH (BPC839) in 86% purity. HPLC (λ = 220 nm) t_R= 6.65 min.

MS (ESI) m/z(+): 684.3 [M + 2H]²⁺, 1367.6 [M + H]⁺. MS (ESI)

m/z(−): 1365.6 [M − H]⁻, 1387.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-

OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-D-Ser-

(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll, this depsipep-

tidyl resin was prepared according to the general procedure described

above. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-Ser-Glu-D-Val-Pro-Gln-

Tyr-OAll in 77% purity. HPLC (λ = 220 nm) t_R= 7.89 min. MS

(ESI) m/z(+): 746.3 [M + 2H]²⁺, 1491.7 [M + H]⁺. MS (ESI) m/

z(−): 1489.7 [M − H]⁻, 1511.7 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-

OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-D-Thr-

(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OAll, this depsipep-

tidyl resin was prepared according to the general procedure described

above. Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-Thr-Glu-D-Ala-Pro-Gln-

Tyr-OAll in 83% purity. HPLC (λ = 220 nm) t_R= 7.85 min. MS

(ESI) m/z(+): 739.8 [M + 2H]²⁺, 1477.7 [M + H]⁺. MS (ESI) m/

z(−): 1475.7 [M − H]⁻, 1498.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-

OH. This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-Tyr(O-Ile-Alloc)-^D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln-

(Tr)-^D-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Thr-Glu-D-Ala-Pro-Gln-D-Tyr-

OH (BPC841) in 82% purity. HPLC (λ = 220 nm) t_R= 6.59 min.

MS (ESI) m/z(+): 677.3 [M + 2H]²⁺, 1353.6 [M + H]⁺. MS (ESI)

m/z(−): 1351.6 [M − H]⁻, 1373.5 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-

OAll. Starting from C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr-D-Ser-

(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OAll, this depsipep-

tidyl resin was prepared according to the general procedure described

above. Acidolytic cleavage of an aliquot of the resulting peptidyl resin

aﬀorded C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-Ser-Glu-D-Ala-

Pro-Gln-Tyr-OAll in 83% purity. HPLC (λ = 220 nm) t_R= 7.79

min. MS (ESI) m/z(+): 732.3 [M + 2H]²⁺, 1463.7 [M + H]⁺. MS

(ESI) m/z(−): 1461.7 [M − H]⁻, 1483.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-

D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr-

(Wang)-OAll. Starting from C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll,

this depsipeptidyl resin was prepared according to the general

procedure described above. Acidolytic cleavage of an aliquot of the

resulting resin aﬀorded C₁₁H₂₃CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-

Thr-Glu-^D-Val-Pro-Gln-Tyr-OAll in 82% purity. HPLC (λ = 220 nm)

t_R= 9.07 min. MS (ESI) m/z(+): 1562.0 [M + H]⁺, 1583.9 [M +

Na]⁺. MS (ESI) m/z(−): 1559.9 [M − H]⁻, 1581.8 [M + Na −

2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-D-Tyr(Wang)-

OH. This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-Tyr(O-Ile-Alloc)-^D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln-

(Tr)-^D-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Ser-Glu-D-Ala-Pro-Gln-D-Tyr-

OH (BPC843) in 69% purity. HPLC (λ = 220 nm) t_R= 6.55 min.

MS (ESI) m/z(+): 670.3 [M + 2H]²⁺, 1339.6 [M + H]⁺. MS (ESI)

m/z(−): 1337.6 [M − H]⁻, 1359.6 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH.

This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-Gln-Tyr-

OH (BPC845) in 87% purity. HPLC (λ = 220 nm) t_R= 6.82 min.

MS (ESI) m/z(+): 1268.6 [M − Ile + H]⁺, 1381.7 [M + H]⁺. MS

(ESI) m/z(−): 1266.6 [M − Ile − H]⁻, 1379.6 [M − H]⁻, 1401.6 [M

+ Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-

D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr-

(Wang)-OAll. Starting from C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OAll,

this depsipeptidyl resin was prepared according to the general

procedure described above. Acidolytic cleavage of an aliquot of the

resulting resin aﬀorded C₁₅H₃₁CO-Glu-D-Orn-D-Tyr(O-Ile-Alloc)-D-

Thr-Glu-^D-Val-Pro-Gln-Tyr-OAll in 69% purity. HPLC (λ = 220 nm)

t_R= 10.13 min. MS (ESI) m/z(+): 1618.0 [M + H]⁺, 1639.9 [M +

Na]⁺. MS (ESI) m/z(−): 1616.9 [M − H]⁻, 1638.8 [M + Na −

2H]⁻.

General Method for Allyl/Alloc Removal. Each peptidyl resin

was treated with Pd(PPh₃)₃(0.1 equiv) and PhSiH₃(10 equiv) in

CH₂Cl₂under nitrogen for 4 h. After this time, the resulting resin was

washed with THF (3 × 15 s), CH₂Cl₂(3 × 2 min), DMF (10 × 1

min), and CH₂Cl₂(3 × 2 min) and air dried. An aliquot of each

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-H)-^D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH.

This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Ser-Glu-D-Val-Pro-Gln-Tyr-

OH (BPC847) in 93% purity. HPLC (λ = 220 nm) t_R= 6.78 min.

MS (ESI) m/z(+): 1254.6 [M − Ile + H]⁺, 1367.7 [M + H]⁺. MS

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

(ESI) m/z(−): 1252.5 [M − Ile − H]⁻, 1365.6 [M − H]⁻, 1387.6 [M

+ Na − 2H]⁻.

resulting peptide was cleaved from the resin with TFA/H₂O/TIS

(95:2.5:2.5) for 2 h at room temperature. Following TFA evaporation

and diethyl ether extraction, the crude peptide was dissolved in H₂O/

CH₃CN, lyophilized, and analyzed by HPLC and mass spectrometry.

Cyclic Depsipeptide pNZ-Tyr(&)-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-

Ile-& (4). Starting from pNZ-Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-

D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OH, on-resin cyclization followed

by acidolytic cleavage aﬀorded the cyclic depsipeptide pNZ-Tyr(&)-

D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-& (4) in 31% purity. HPLC (λ =

220 nm) t_R= 7.55 min. MS (ESI) m/z(+): 1195.5 [M + Na]⁺, 1211.4

[M + K]⁺. MS (ESI) m/z(−): 1171.5 [M − H]⁻.

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OH.

This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Thr-Glu-D-Ala-Pro-Gln-Tyr-

OH (BPC849) in 79% purity. HPLC (λ = 220 nm) t_R= 6.73 min.

MS (ESI) m/z(+): 677.4 [M + 2H]²⁺, 1240.6 [M − Ile + H]⁺, 1353.7

[M + H]⁺. MS (ESI) m/z(−): 1238.5 [M − Ile − H]⁻, 1351.6 [M −

H]⁻, 1373.6 [M + Na − 2H]⁻.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-Tyr(&)-D-Thr-Glu-D-

Val-Pro-Gln-^D-Tyr-Ile-& (BPC838). Starting from 8a, on-resin

cyclization followed by acidolytic cleavage aﬀorded the cyclic

lipodepsipeptide BPC838 in 33% purity. HPLC (λ = 220 nm) t_R=

7.26 min. MS (ESI) m/z(+): 1363.6 [M + H]⁺. MS (ESI) m/z(−):

1361.6 [M − H]⁻. The crude peptide was puriﬁed by RP-HPLC

obtaining BPC838 in >99% purity. HPLC (λ = 220 nm) t_R= 7.45

Linear Lipodepsipeptidyl Resin C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-

Tyr(O-Ile-H)-^D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OH.

This peptidyl resin was obtained from C₇H₁₅CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₇H₁₅CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Ser-Glu-D-Ala-Pro-Gln-Tyr-

OH (BPC851) in 78% purity. HPLC (λ = 220 nm) t_R= 6.67 min.

MS (ESI) m/z(+): 1226.6 [M − Ile + H]⁺, 1339.7 [M + H]⁺, 1361.6

[M + Na]⁺. MS (ESI) m/z(−): 1224.5 [M − Ile − H]⁻, 1337.6 [M −

H]⁻, 1359.6 [M + Na − 2H]⁻.

min. H NMR (400 MHz, DMSO-d₆+ D₂O) δ (ppm): 0.72−0.87

(m, 18 H), 1.15−1.20 (m, 14 H), 1.41−1.45 (m, 4 H), 1.55−1.60 (m,

3 H), 1.69−1.76 (m, 4 H), 1.82−1.98 (m, 4 H), 2.06−2.26 (m, 10

H), 2.67−2.77 (m, 3 H), 2.86−2.88 (m, 2 H), 3.16−3.27 (m, 2 H),

3.50−3.53 (m, 1 H), 3.97−4.00 (m, 1 H), 4.17−4.26 (m, 3 H), 4.31−

4.39 (m, 1 H), 4.44−4.47 (m, 1 H), 4.55−4.63 (m, 2 H), 6.63 (d, 2

H, J = 8.0 Hz), 6.84 (d, 2 H, J = 8.0 Hz), 6.99−7.04 (m, 4 H). MS

(ESI) m/z(+): 682.3 [M + 2H]²⁺, 693.3 [M + Na + H]²⁺, 1363.8 [M

+ H]⁺, 1385.8 [M + Na]⁺. MS (ESI) m/z(−): 1361.8 [M − H]⁻,

1383.6 [M + Na − 2H]⁻. HRMS (ESI) m/z(+): calcd for

C₆₆H₉₉N₁₂O₁₉1363.7144, found 1363.7130; calcd for

C₆₆H₉₈N₁₂NaO₁₉1385.6963, found 1385.6934; calcd for

C₆₆H₉₉N₁₂NaO₁₉693.3518, found 693.3505; calcd for

C₆₆H₉₈N₁₂Na₂O₁₉704.3428, found 704.3408.

Linear Lipodepsipeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-

OH (8b). This peptidyl resin was obtained from 7b following the

above general procedure. Acidolytic cleavage of an aliquot of 8b

aﬀorded C₁₁H₂₃CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-

Gln-^D-Tyr-OH (BPC853) in 86% purity. HPLC (λ = 220 nm) t_R=

7.45 min. MS (ESI) m/z(+): 719.4 [M + 2H]²⁺, 1437.7 [M + H]⁺.

MS (ESI) m/z(−): 1436.6 [M − H]⁻, 1457.6 [M + Na − 2H]⁻

Linear Lipodepsipeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-

Tyr(O-Ile-H)-^D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-

OH (8c). This peptidyl resin was obtained from 7c following the above

general procedure. Acidolytic cleavage of an aliquot of 8c aﬀorded

C₁₅H₃₁CO-Glu-D-Orn-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-Gln-D-

Tyr-OH (BPC855) in 89% purity. HPLC (λ = 220 nm) t_R= 8.23

min. MS (ESI) m/z(+): 747.4 [M + 2H]²⁺, 1493.8 [M + H]⁺. MS

(ESI) m/z(−): 1491.7 [M − H]⁻, 1513.7 [M + Na − 2H]⁻

Linear Lipodepsipeptidyl Resin C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-

D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-

OH. This peptidyl resin was obtained from C₁₁H₂₃CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₁₁H₂₃CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-Gln-

Tyr-OH (BPC857) in 62% purity. HPLC (λ = 220 nm) t_R= 7.84

min. MS (ESI) m/z(+): 719.4 [M + 2H]²⁺, 1325.8 [M − Ile + H]⁺,

1437.9 [M + H]⁺, 1459.9 [M + Na]⁺. MS (ESI) m/z(−): 1322.7 [M

− Ile − H]⁻, 1435.8 [M − H]⁻, 1457.8 [M + Na − 2H]⁻.

Linear Lipodepsipeptidyl Resin C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-

D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-

OH. This peptidyl resin was obtained from C₁₅H₃₁CO-Glu(O^tBu)-D-

Orn(Boc)-^D-Tyr(O-Ile-Alloc)-D-Thr(^tBu)-Glu(O^tBu)-D-Val-Pro-Gln-

(Tr)-Tyr(Wang)-OAll following the above general procedure.

Acidolytic cleavage of an aliquot of the resulting resin aﬀorded

C₁₅H₃₁CO-Glu-D-Orn-D-Tyr(O-Ile-H)-D-Thr-Glu-D-Val-Pro-Gln-

Tyr-OH (BPC859) in 64% purity. HPLC (λ = 220 nm) t_R= 8.69

min. MS (ESI) m/z(+): 747.5 [M + 2H]²⁺, 758.4 [M + Na + H]²⁺,

1380.9 [M − Ile + H]⁺, 1494.0 [M + H]⁺, 1516.9 [M + Na]⁺. MS

(ESI) m/z(−): 1378.8 [M − Ile + H]⁻, 1491.9 [M − H]⁻, 1513.9 [M

+ Na − 2H]⁻.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-Tyr(&)-D-Ser-Glu-D-

Val-Pro-Gln-^D-Tyr-Ile-& (BPC840). Starting from C₇H₁₅CO-Glu-

(O^tBu)-D-Orn(Boc)-Tyr(O-Ile-H)-D-Ser(^tBu)-Glu(O^tBu)-D-Val-Pro-

Gln(Tr)-^D-Tyr(Wang)-OH, on-resin cyclization followed by acid-

olytic cleavage aﬀorded the cyclic lipodepsipeptide BPC840 in 35%

purity. HPLC (λ = 220 nm) t_R= 7.25 min. MS (ESI) m/z(+): 1349.6

[M + H]⁺. MS (ESI) m/z(−): 1347.6 [M − H]⁻. The crude peptide

was puriﬁed by RP-HPLC obtaining BPC840 in >99% purity. HPLC

(λ = 220 nm) t_R= 7.22 min. ¹H NMR (400 MHz, DMSO-d₆+ D₂O)

δ (ppm): 0.73−0.86 (m, 15 H), 1.14−1.21 (m, 12 H), 1.41−1.48 (m,

4 H), 1.55−1.61 (m, 3 H), 1.68−1.90 (m, 10 H), 2.00−2.33 (m, 8

H), 2.67−2.77 (m, 2 H), 2.85−2.87 (m, 2 H), 3.19−3.33 (m, 4 H),

3.97−3.99 (m, 1 H), 4.12−4.26 (m, 6 H), 4.35−4.46 (m, 2 H), 4.56−

4.60 (m, 1 H), 4.64−4.75 (m, 1 H), 6.63 (d, 2 H, J = 8.0 Hz), 6.86 (d,

2 H, J = 8.0 Hz), 6.95−7.04 (m, 4 H). MS (ESI) m/z(+): 675.3 [M +

2H]²⁺, 1349.7 [M + H]⁺. MS (ESI) m/z(−): 1347.6 [M − H]⁻.

HRMS (ESI) m/z(+): calcd for C₆₅H₉₇N₁₂O₁₉1349.6987, found

1349.6962; calcd for C₆₅H₉₆N₁₂NaO₁₉1371.6807, found 1371.6804;

calcd for C₆₅H₉₇N₁₂NaO₁₉686.3440, found 686.3431; calcd for

C₆₅H₉₆N₁₂Na₂O₁₉697.3350, found 697.3342.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-Tyr(&)-D-Thr-Glu-D-

Ala-Pro-Gln-^D-Tyr-Ile-& (BPC842). Starting from C₇H₁₅CO-Glu-

(O^tBu)-D-Orn(Boc)-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu(O^tBu)-D-Ala-Pro-

Gln(Tr)-^D-Tyr(Wang)-OH, on-resin cyclization followed by acid-

olytic cleavage aﬀorded the cyclic lipodepsipeptide BPC842 in 26%

purity. HPLC (λ = 220 nm) t_R= 7.13 min. MS (ESI) m/z(+): 1335.6

[M + H]⁺. MS (ESI) m/z(−): 1333.6 [M − H]⁻. The crude peptide

was puriﬁed by RP-HPLC obtaining BPC842 in >99% purity. HPLC

(λ = 220 nm) t_R= 7.26 min. ¹H NMR (400 MHz, DMSO-d₆+ D₂O)

δ (ppm): 0.71−0.89 (m, 12 H), 1.10−1.22 (m, 15 H), 1.43−1.45 (m,

4 H), 1.57−1.61 (m, 2 H), 1.71−1.89 (m, 8 H), 1.99−2.22 (m, 10

H), 2.69−2.76 (m, 2 H), 2.84−2.95 (m, 2 H), 3.22−3.25 (m, 1 H),

3.50−3.55 (m, 2 H), 3.98−4.01 (m, 2 H), 4.10−4.14 (m, 1 H), 4.18−

4.24 (m, 2 H), 4.38−4.42 (m, 1 H), 4.57−4.61 (m, 1 H), 4.62−4.69

(m, 1 H), 4.81−4.90 (m, 1 H), 6.63 (d, 2 H, J = 8.0 Hz), 6.84 (d, 2 H,

J = 8.0 Hz), 6.94 (d, 2 H, J = 8.0 Hz), 7.05 (d, 2 H, J = 8.0 Hz). MS

(ESI) m/z(+): 668.3 [M + 2H]²⁺, 1335.7 [M + H]⁺. MS (ESI) m/

z(−): 1333.6 [M − H]⁻. HRMS (ESI) m/z(+): calcd for

Synthesis of the Cyclic Lipodepsipeptides. On-resin cycliza-

tion of each linear peptidyl resin was mediated by Oxyma (5 equiv),

[ethyl cyano(hydroxyimino)acetato-O²-tri-(1-pyrrolidinyl)-phospho-

nium hexaﬂuorophosphate (PyOxim) (5 equiv), and DIEA (10

equiv) in DMF. The reaction mixture was stirred at room temperature

for 24 h. The resin was then washed with DMF (6 × 1 min), CH₂Cl₂

(3 × 1 min), and diethyl ether (3 × 2 min) and dried in vacuo. The

completion of the cyclization was checked with the Kaiser test.⁴⁵The

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

C₆₄H₉₅N₁₂O₁₉1335.6831, found 1335.6855; calcd for

C₆₄H₉₅N₁₂NaO₁₉679.3362, found 679.3379; calcd for

C₆₄H₉₄N₁₂Na₂O₁₉690.3271, found 690.3301.

product from the hydrolysis of the macrolactone C₇H₁₅CO-Glu-D-

Orn-^D-Tyr-D-Thr-Glu-D-Ala-Pro-Gln-Tyr-Ile-OH (16% purity, t_R

6.93 min) [3:4 ratio by MS (ESI)]. MS (ESI) m/z(+): 677.3 [M +

H₂O + 2H]²⁺, 1335.7 [M + H]⁺, 1353.7 [M + H₂O + H]⁺, 1375.7 [M

+ H₂O + Na]⁺. MS (ESI) m/z(−): 675.1 [M + H₂O-2H]²⁻, 1333.6

[M − H]⁻, 1351.6 [M + H₂O − H]⁻, 1373.6 [M + H₂O + Na −

2H]⁻. The crude mixture was puriﬁed by RP-HPLC obtaining

BPC850 (48% purity, t_R= 6.96 min) together with the linear product

from the hydrolysis of the macrolactone (52% purity, t_R= 6.84 min).

MS (ESI) m/z(+): 668.8 [M + 2H]²⁺, 1335.7 [M + H]⁺, 1353.6 [M +

H₂O + H]⁺. MS (ESI) m/z(−): 1333.6 [M − H]⁻, 1351.6 [M + H₂O

− H]⁻. HRMS (ESI) m/z(+): calcd for C₆₄H₉₅N₁₂O₁₉1335.6831,

found 1335.6812; calcd for C₆₄H₉₄N₁₂NaO₁₉1357.6650, found

1357.6648; calcd for C₆₄H₉₇N₁₂O₂₀1353.6937, found 1353.6909;

calcd for C₆₄H₉₆N₁₂NaO₂₀1375.6756, found 1375.6727.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-D-Tyr(&)-D-Ser-Glu-

D-Ala-Pro-Gln-Tyr-Ile-& (BPC852). Starting from the peptidyl resin

C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Ser(^tBu)-Glu-

(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC852 (11% purity, t_R= 7.06 min) and the linear

product from the hydrolysis of the macrolactone C₇H₁₅CO-Glu-D-

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-Tyr(&)-D-Ser-Glu-D-

Ala-Pro-Gln-^D-Tyr-Ile-& (BPC844). Starting from C₇H₁₅CO-Glu-

(O^tBu)-D-Orn(Boc)-Tyr(O-Ile-H)-D-Ser(^tBu)-Glu(O^tBu)-D-Ala-Pro-

Gln(Tr)-^D-Tyr(Wang)-OH, on-resin cyclization followed by acid-

olytic cleavage aﬀorded the cyclic lipodepsipeptide BPC844 in 21%

purity. HPLC (λ = 220 nm) t_R= 7.13 min. H NMR (400 MHz,

DMSO-d₆+ D₂O) δ (ppm): 0.73−0.76 (m, 6 H), 0.83−0.85 (m, 3

H), 1.10−1.20 (m, 15 H), 1.40−1.45 (m, 4 H), 1.56−1.62 (m, 3 H),

1.73−1.88 (m, 8 H), 2.07−2.24 (m, 10 H), 2.71−2.74 (m, 2 H),

2.87−2.91 (m, 2 H), 3.19−3.33 (m, 2 H), 3.47−3.53 (m, 2 H), 3.95−

3.97 (m, 1 H), 4.11−4.24 (m, 5 H), 4.36−4.40 (m, 1 H), 4.56−4.62

(m, 2 H), 4.86−4.87 (m, 1 H), 6.63 (d, 2 H, J = 8.0 Hz), 6.86 (d, 2 H,

J = 8.0 Hz), 6.94 (d, 2 H, J = 8.0 Hz), 7.06 (d, 2 H, J = 8.0 Hz). MS

(ESI) m/z(+): 1321.6 [M + H]⁺. MS (ESI) m/z(−): 1319.6 [M −

H]⁻. The crude peptide was puriﬁed by RP-HPLC obtaining BPC844

in 94% purity. HPLC (λ = 220 nm) t_R= 7.12 min. MS (ESI) m/z(+):

661.8 [M + 2H]²⁺, 1321.7 [M + H]⁺. HRMS (ESI) m/z(+): calcd for

C₆₃H₉₃N₁₂O₁₉1321.6674, found 1321.6701; calcd for

C₆₃H₉₂N₁₂NaO₁₉1343.6494, found 1343.6521.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-D-Tyr(&)-D-Thr-Glu-

D-Val-Pro-Gln-Tyr-Ile-& (BPC846). Starting from the peptidyl resin

C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC846 (17% purity, t_R= 7.19 min) and the linear

product from the hydrolysis of the macrolactone C₇H₁₅CO-Glu-D-

Orn-^D-Tyr-D-Ser-Glu-D-Ala-Pro-Gln-Tyr-Ile-OH (18% purity, t_R

6.88 min) [1:2 ratio by MS (ESI)]. MS (ESI) m/z(+): 1321.7 [M +

H]⁺, 1339.7 [M + H₂O + H]⁺. MS (ESI) m/z(−): 1319.6 [M − H]⁻,

1337.6 [M + H₂O − H]⁻, 1359.6 [M + H₂O + Na − 2H]⁻. The

crude mixture was puriﬁed by RP-HPLC obtaining BPC852 (81%

purity, t_R= 6.95 min) together with the linear product from the

hydrolysis of the macrolactone (19% purity, t_R= 6.78 min). MS (ESI)

m/z(+): 1321.6 [M + H]⁺, 1339.6 [M + H₂O + H]⁺. MS (ESI) m/

z(−): 1319.6 [M − H]⁻, 1337.6 [M + H₂O − H]⁻. HRMS (ESI) m/

z(+): calcd for C₆₃H₉₃N₁₂O₁₉1321.6674, found 1321.6653; calcd for

C₆₃H₉₂N₁₂NaO₁₉1343.6494, found 1343.6510; calcd for

C₆₃H₉₅N₁₂O₂₀1339.6780, found 1339.6800; calcd for

C₆₃H₉₄N₁₂NaO₂₀1361.6600, found 1361.6581.

Cyclic Lipodepsipeptide C₁₁H₂₃CO-Glu-D-Orn-Tyr(&)-D-Thr-Glu-D-

Val-Pro-Gln-^D-Tyr-Ile-& (BPC854). Starting from 8b, on-resin

cyclization followed by acidolytic cleavage aﬀorded the cyclic

lipodepsipeptide BPC854 in 49% purity. HPLC (λ = 220 nm) t_R=

8.21 min. MS (ESI) m/z(+): 1420.8 [M + H]⁺. MS (ESI) m/z(−):

1417.8 [M − H]⁻. The crude peptide was puriﬁed by RP-HPLC

obtaining BPC854 in 92% purity. HPLC (λ = 220 nm) t_R= 7.99 min.

¹H NMR (400 MHz, DMSO-d₆+ D₂O) δ (ppm): 0.71−0.85 (m, 18

Orn-^D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH (17% purity, t_R

7.04 min) [1:1 ratio by MS (ESI)]. MS (ESI) m/z(+): 1363.7 [M +

H]⁺, 1381.7 [M + H₂O + H]⁺, 1403.7 [M + H₂O + Na]⁺. MS (ESI)

m/z(−): 1361.7 [M − H]⁻, 1379.6 [M + H₂O − H]⁻, 1402.6 [M +

H₂O + Na − 2H]⁻. The crude mixture was puriﬁed by RP-HPLC

obtaining BPC846 (59% purity, t_R= 7.11 min) together with the

linear product from the hydrolysis of the macrolactone (37% purity, t_R

= 6.97 min). MS (ESI) m/z(+): 682.3 [M + 2H]²⁺, 691.3 [M + H₂O

+ 2H]²⁺, 1363.7 [M + H]⁺, 1381.7 [M + H₂O + H]⁺. MS (ESI) m/

z(−): 1361.6 [M − H]⁻, 1379.6 [M + H₂O − H]⁻. HRMS (ESI) m/

z(+): calcd for C₆₆H₉₉N₁₂O₁₉1363.7144, found 1363.7118; calcd for

C₆₆H₉₈N₁₂NaO₁₉1385.6963, found 1385.6984; calcd for

C₆₆H₁₀₁N₁₂O₂₀1381.7250, found 1381.7242; calcd for

C₆₆H₁₀₀N₁₂NaO₂₀1403.7069, found 1403.7063.

H), 1.14−1.19 (m, 18 H), 1.40−1.58 (m, 6 H), 1.61−1.74 (m, 4 H),

1.87−2.25 (m, 14 H), 2.65−2.76 (m, 3 H), 2.80−2.85 (m, 2 H),

3.02−3.21 (m, 2 H), 3.26−3.34 (m, 1 H), 3.48−3.50 (m, 1 H), 3.80−

3.87 (m, 2 H), 3.96−4.02 (m, 2 H), 4.11−4.25 (m, 4 H), 4.42−4.46

(m, 1 H), 4.56−4.60 (m, 2 H), 6.61 (d, 2 H, J = 8.0 Hz), 6.83 (d, 2 H,

J = 8.0 Hz), 6.99−7.03 (m, 4 H). MS (ESI) m/z(+): 1419.8 [M +

H]⁺. MS (ESI) m/z(−): 1417.7 [M − H]⁻. HRMS (ESI) m/z(+):

calcd for C₇₀H₁₀₇N₁₂O₁₉1419.7770, found 1419.7763; calcd for

C₇₀H₁₀₆N₁₂NaO₁₉1441.7589, found 1441.7593.

Cyclic Lipodepsipeptide C₁₅H₃₁CO-Glu-D-Orn-Tyr(&)-D-Thr-Glu-D-

Val-Pro-Gln-^D-Tyr-Ile-& (BPC856). Starting from 8c, on-resin

cyclization followed by acidolytic cleavage aﬀorded the cyclic

lipodepsipeptide BPC856 in 50% purity. HPLC (λ = 220 nm) t_R=

9.25 min. MS (ESI) m/z(+): 1475.8 [M + H]⁺, 1497.8 [M + Na]⁺.

MS (ESI) m/z(−): 1473.8 [M − H]⁻. The crude peptide was puriﬁed

by RP-HPLC yielding BPC856 in 92% purity. HPLC (λ = 220 nm) t_R

= 9.23 min. ¹H NMR (400 MHz, DMSO-d₆+ D₂O) δ (ppm): 0.79−

0.89 (m, 18 H), 1.14−1.22 (m, 28 H), 1.40−1.50 (m, 6 H), 1.56−

1.63 (m, 2 H), 1.69−1.77 (m, 2 H), 1.84−2.33 (m, 14 H), 2.68−2.73

(m, 4 H), 2.88−2.90 (m, 2 H), 3.26−3.37 (m, 2 H), 3.60−3.63 (m, 1

H), 4.00−4.04 (m, 2 H), 4.11−4.17 (m, 2 H), 4.23−4.28 (m, 2 H),

4.43−4.46 (m, 1 H), 4.48−4.51 (m, 1 H), 4.56−4.60 (m, 1 H), 4.70−

4.73 (m, 1 H), 6.63 (d, 2 H, J = 8.0 Hz), 6.83 (d, 2 H, J = 8.0 Hz),

6.93 (d, 2 H, J = 8.0 Hz), 7.04 (d, 2 H, J = 8.0 Hz). MS (ESI) m/

z(+): 738.4 [M + 2H]²⁺, 749.9 [M + Na + H]²⁺, 1475.9 [M + H]⁺,

1497.9 [M + Na]⁺. MS (ESI) m/z(−): 1473.9 [M − H]⁻, 1495.8 [M

+ Na − 2H]⁻. HRMS (ESI) m/z(+): calcd for C₇₄H₁₁₅N₁₂O₁₉

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-D-Tyr(&)-D-Ser-Glu-

D-Val-Pro-Gln-Tyr-Ile-& (BPC848). Starting from the peptidyl resin

C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Ser(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC848 (10% purity, t_R= 7.18 min) and the linear

product from the hydrolysis of the macrolactone C₇H₁₅CO-Glu-D-

Orn-^D-Tyr-D-Ser-Glu-D-Val-Pro-Gln-Tyr-Ile-OH (17% purity, t_R

7.00 min) [3:4 ratio by MS (ESI)]. MS (ESI) m/z(+): 685.4 [M +

H₂O + 2H]²⁺, 1350.7 [M + H]⁺, 1367.7 [M + H₂O + H]⁺. MS (ESI)

m/z(−): 682.2 [M + H₂O-2H]²⁻, 1348.6 [M − H]⁻, 1366.7 [M +

H₂O − H]⁻, 1387.6 [M + H₂O + Na − 2H]⁻. The crude mixture was

puriﬁed by RP-HPLC obtaining BPC848 (38% purity, t_R= 7.09 min)

together with the linear product from the hydrolysis of the

macrolactone (62% purity, t_R= 6.93 min). MS (ESI) m/z(+):

675.3 [M + 2H]²⁺, 1349.7 [M + H]⁺, 1367.7 [M + H₂O + H]⁺. MS

(ESI) m/z(−): 1347.6 [M − H]⁻, 1365.6 [M + H₂O − H]⁻. HRMS

(ESI) m/z(+): calcd for C₆₅H₉₇N₁₂O₁₉1349.6987, found 1349.6999;

calcd for C₆₅H₉₆N₁₂NaO₁₉1371.6807, found 1371.6827; calcd for

C₆₅H₉₉N₁₂O₂₀1367.7093, found 1367.7079; calcd for

C₆₅H₉₈N₁₂NaO₂₀1389.6913, found 1389.6902.

Cyclic Lipodepsipeptide C₇H₁₅CO-Glu-D-Orn-D-Tyr(&)-D-Thr-Glu-

D-Ala-Pro-Gln-Tyr-Ile-& (BPC850). Starting from the peptidyl resin

C₇H₁₅CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu-

(O^tBu)-D-Ala-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC850 (14% purity, t_R= 7.07 min) and the linear

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

1475.8396, found 1475.8376; calcd for C₇₄H₁₁₄N₁₂NaO₁₉1497.8215,

found 1497.8227.

methodology described above. C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-

Glu-^D-Val-Pro-Gln-Tyr-Ile-OH (t_R= 7.00 min, 28% HPLC purity)

together with the methyl ester derivative C₇H₁₅CO-Glu-D-Orn-D-Tyr-

D-Ser-Glu-D-Val-Pro-Gln-Tyr-Ile-OMe (t_R= 7.20 min, 27% HPLC

purity) were obtained. MS (ESI) m/z(+): 1367.7 [M + H]⁺, 1381.7

[M(OMe) + H]⁺, 1389.7 [M + Na]⁺. MS (ESI) m/z(−): 1365.6 [M

− H]⁻, 1379.7 [M(OMe) − H]⁻, 1387.6 [M + Na − 2H]⁻.

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Ala-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC850 was performed following the

methodology described above. C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-

Glu-^D-Ala-Pro-Gln-Tyr-Ile-OH (t_R= 6.92 min, 35% HPLC purity)

together with the methyl ester derivative C₇H₁₅CO-Glu-D-Orn-D-Tyr-

D-Thr-Glu-D-Ala-Pro-Gln-Tyr-Ile-OMe (t_R= 7.12 min, 23% HPLC

purity) were obtained. MS (ESI) m/z(+): 1353.6 [M + H]⁺, 1368.6

[M(OMe) + H]⁺, 1375.7 [M + Na]⁺. MS (ESI) m/z(−): 1351.6 [M

− H]⁻, 1365.6 [M(OMe) − H]⁻, 1387.5 [M(OMe) + Na − 2H]⁻.

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-Glu-D-Ala-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC852 was performed following the

methodology described above. C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-

Glu-^D-Ala-Pro-Gln-Tyr-Ile-OH (t_R= 6.87 min, 33% HPLC purity)

together with the methyl ester derivative C₇H₁₅CO-Glu-D-Orn-D-Tyr-

D-Ser-Glu-D-Ala-Pro-Gln-Tyr-Ile-OMe (t_R= 7.07 min, 30% HPLC

purity) were obtained. MS (ESI) m/z(+): 1339.7 [M + H]⁺, 1353.6

[M(OMe) + H]⁺, 1361.7 [M + Na]⁺. MS (ESI) m/z(−): 1337.6 [M

− H]⁻, 1351.7 [M(OMe) − H]⁻, 1359.5 [M + Na − 2H]⁻, 1373.5

[M(OMe) + Na − 2H]⁻.

C₁₁H₂₃CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OH.

The hydrolysis of the peptide BPC854 was performed following the

methodology described above. C₁₁H₂₃CO-Glu-D-Orn-Tyr-D-Thr-Glu-

D-Val-Pro-Gln-D-Tyr-Ile-OH (t_R= 7.99 min, 30% HPLC purity)

together with the methyl ester derivative C₁₁H₂₃CO-Glu-D-Orn-Tyr-

D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OMe (t_R= 8.09 min, 16% HPLC

purity) were obtained. MS (ESI) m/z(+): 726.4 [M(OMe) + 2H]²⁺,

1437.8 [M + H]⁺, 1451.8 [M(OMe) + H]⁺. MS (ESI) m/z(−):

1435.7 [M − H]⁻, 1449.8 [M(OMe) − H]⁻, 1472.7 [M(OMe) + Na

− 2H]⁻.

C₁₅H₃₁CO-Glu-D-Orn-Tyr-D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OH.

The hydrolysis of the peptide BPC856 was performed following the

methodology described above. C₁₅H₃₁CO-Glu-D-Orn-Tyr-D-Thr-Glu-

D-Val-Pro-Gln-D-Tyr-Ile-OH (t_R= 8.96 min, 20% HPLC purity)

together with the methyl ester derivative C₁₅H₃₁CO-Glu-D-Orn-Tyr-

D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OMe (t_R= 9.05 min, 19% HPLC

purity) were obtained. MS (ESI) m/z(+): 754.9 [M(OMe) + 2H]²⁺,

1493.9 [M + H]⁺, 1507.9 [M(OMe) + H]⁺. MS (ESI) m/z(−):

1491.8 [M − H]⁻, 1505.8 [M(OMe) − H]⁻, 1527.7 [M(OMe) + Na

− 2H]⁻.

Cyclic Lipodepsipeptide C₁₁H₂₃CO-Glu-D-Orn-D-Tyr(&)-D-Thr-Glu-

D-Val-Pro-Gln-Tyr-Ile-& (BPC858). Starting from the peptidyl resin

C₁₁H₂₃CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC858 (16% purity, t_R= 8.39 min) and the linear

product from the hydrolysis of the macrolactone C₁₁H₂₃CO-Glu-D-

Orn-^D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH (21% purity, t_R

8.23 min) [2.5:4 ratio by MS (ESI)]. MS (ESI) m/z(+): 1419.9 [M +

H]⁺, 1437.9 [M + H₂O + H]⁺, 1459.8 [M + H₂O + Na]⁺. MS (ESI)

m/z(−): 1418.8 [M − H]⁻, 1435.8 [M + H₂O − H]⁻, 1457.8 [M +

H₂O + Na − 2H]⁻. The mixture was puriﬁed by reverse-phase

column chromatography. The cyclic lipodepsipeptide BPC858 was

obtained in 78% purity. HPLC (λ = 220 nm) t_R= 8.14 min. MS (ESI)

m/z(+): 1419.9 [M + H]⁺, 1441.9 [M + Na]⁺. MS (ESI) m/z(−):

1417.8 [M − H]⁻, 1439.7 [M + Na − 2H]⁻. C₁₁H₂₃CO-Glu-D-Orn-D-

Tyr-^D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH was obtained in 91%

purity. HPLC (λ = 220 nm) t_R= 7.99 min. MS (ESI) m/z(+):

1437.9 [M + H]⁺, 1459.8 [M + Na]⁺. MS (ESI) m/z(−): 1435.8 [M

− H]⁻, 1457.8 [M + Na − 2H]⁻.

Cyclic Lipodepsipeptide C₁₅H₃₁CO-Glu-D-Orn-D-Tyr(&)-D-Thr-Glu-

D-Val-Pro-Gln-Tyr-Ile-& (BPC860). Starting from the peptidyl resin

C₁₅H₃₁CO-Glu(O^tBu)-D-Orn(Boc)-D-Tyr(O-Ile-H)-D-Thr(^tBu)-Glu-

(O^tBu)-D-Val-Pro-Gln(Tr)-Tyr(Wang)-OH, on-resin cyclization fol-

lowed by acidolytic cleavage aﬀorded a mixture of the cyclic

lipodepsipeptide BPC860 (24% purity, t_R= 9.04 min) and the linear

product from the hydrolysis of the macrolactone C₁₅H₃₁CO-Glu-D-

Orn-^D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH (25% purity, t_R

8.88 min) [2:3 ratio by MS (ESI)]. MS (ESI) m/z(+): 1475.8 [M +

H]⁺, 1493.8 [M + H₂O + H]⁺, 1515.8 [M + H₂O + Na]⁺. MS (ESI)

m/z(−): 1473.7 [M − H]⁻, 1491.7 [M + H₂O − H]⁻, 1514.7 [M +

Na − 2H]⁻. The mixture was puriﬁed by reverse-phase column

chromatography. Cyclic lipodepsipeptide BPC860 was obtained in

70% purity. HPLC (λ = 220 nm) t_R= 9.06 min. MS (ESI) m/z(+):

1476.0 [M + H]⁺, 1497.9 [M + Na]⁺. MS (ESI) m/z(−): 1473.9 [M

− H]⁻, 1495.8 [M + Na − 2H]⁻. C₁₅H₃₁CO-Glu-D-Orn-D-Tyr-D-Thr-

Glu-^D-Val-Pro-Gln-Tyr-Ile-OH was obtained in 78% purity. HPLC (λ

= 220 nm) t_R= 8.89 min. MS (ESI) m/z(+): 1494.0 [M + H]⁺,

1516.0 [M + Na]⁺. MS (ESI) m/z(−): 1491.9 [M − H]⁻, 1513.9 [M

+ Na − 2H]⁻.

General Method for the Hydrolysis of Cyclic Depsipeptides.

Cyclic depsipeptide 4 and cyclic lipodepsipeptides BPC846, BPC848,

BPC850, BPC852, BPC854, BPC856, BPC858, and BPC860 were

treated with a solution of CH₃OH/H₂O/NH₃(4:1:1) for 24 h at

room temperature.⁴²Next, the mixture was evaporated and the crude

residue was dissolved in H₂O/CH₃CN, lyophilized, analyzed by

HPLC and characterized by mass spectrometry.

C₁₁H₂₃CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC858 was performed following the

methodology described above. C₁₁H₂₃CO-Glu-D-Orn-D-Tyr-D-Thr-

Glu-^D-Val-Pro-Gln-Tyr-Ile-OH (t_R= 7.92 min, 43% HPLC purity)

together with the methyl ester derivative C₁₁H₂₃CO-Glu-D-Orn-D-

Tyr-^D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OMe (t_R= 8.05 min, 13%

HPLC purity) were obtained. MS (ESI) m/z(+): 1437.9 [M + H]⁺,

1451.9 [M(OMe) + H]⁺, 1459.9 [M + Na]⁺. MS (ESI) m/z(−):

1435.8 [M − H]⁻, 1449.8 [M(OMe) − H]⁻, 1457.7 [M + Na −

2H]⁻, 1471.7 [M(OMe) + Na − 2H]⁻.

C₁₅H₃₁CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC860 was performed following the

methodology described above. C₁₅H₃₁CO-Glu-D-Orn-D-Tyr-D-Thr-

Glu-^D-Val-Pro-Gln-Tyr-Ile-OH (t_R= 8.87 min, 38% HPLC purity)

together with the methyl ester derivative C₁₅H₃₁CO-Glu-D-Orn-D-

Tyr-^D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OMe (t_R= 8.94 min, 19%

HPLC purity) were obtained. MS (ESI) m/z(+): 1493.9 [M + H]⁺,

1507.9 [M(OMe) + H]⁺, 1515.8 [M + Na]⁺. MS (ESI) m/z(−):

1491.9 [M − H]⁻, 1505.9 [M(OMe) − H]⁻, 1513.8 [M + Na −

2H]⁻.

pNZ-Tyr-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OH. The hydrolysis of

the peptide 4 was performed following the methodology described

above. pNZ-Tyr-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OH (t_R= 7.34

min, 26% HPLC purity) together with the methyl ester derivative

pNZ-Tyr-^D-Thr-Glu-D-Val-Pro-Gln-D-Tyr-Ile-OMe (t_R= 7.49 min,

20% HPLC purity) were obtained. MS (ESI) m/z(+): 1191.5 [M +

H]⁺, 1205.5 [M(OMe) + H]⁺, 1213.5 [M + Na]⁺, 1227.5 [M(OMe)

+ Na]⁺. MS (ESI) m/z(−): 1189.5 [M − H]⁻, 1203.5 [M(OMe) −

H]⁻.

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC846 was performed following the

methodology described above. C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Thr-

Glu-^D-Val-Pro-Gln-Tyr-Ile-OH (t_R= 7.04 min, 30% HPLC purity)

together with the methyl ester derivative C₇H₁₅CO-Glu-D-Orn-D-Tyr-

D-Thr-Glu-D-Val-Pro-Gln-Tyr-Ile-OMe (t_R= 7.24 min, 30% HPLC

purity) were obtained. MS (ESI) m/z(+): 1381.7 [M + H]⁺, 1395.7

[M(OMe) + H]⁺. MS (ESI) m/z(−): 1379.6 [M − H]⁻, 1393.7

[M(OMe) − H]⁻, 1401.6 [M + Na − 2H]⁻, 1415.6 [M(OMe) + Na

− 2H]⁻.

C₇H₁₅CO-Glu-D-Orn-D-Tyr-D-Ser-Glu-D-Val-Pro-Gln-Tyr-Ile-OH.

The hydrolysis of the peptide BPC848 was performed following the

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

(12) Pathak, K. V.; Keharia, H.; Gupta, K.; Thakur, S. S.; Balaram, P.

Lipopeptides from the Banyan Endophyte, Bacillus Subtilis K1: Mass

Spectrometric Characterization of a Library of Fengycins. J. Am. Soc.

Mass Spectrom. 2012, 23, 1716−1728.

ASSOCIATED CONTENT

■

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.joc.8b02553.

(13) Villegas-Escobar, V.; Ceballos, I.; Mira, J. J.; Argel, L. E.; Orduz

Peralta, S.; Romero-Tabarez, M. Fengycin C Produced by Bacillus

Subtilis EA-CB0015. J. Nat. Prod. 2013, 76, 503−509.

Spectral data of amino acids; HPLC and ESI-MS of

linear peptides, depsipeptides, and lipodepsipeptides;

HPLC, ESI-MS, HRMS, and 1D and 2D NMR of cyclic

lipodepsipeptides (PDF)

(14) Sa, R.-B.; An, X.; Sui, J.-K.; Wang, X.-H.; Ji, C.; Wang, C.-Q.;

Li, Q.; Hu, Y.-R.; Liu, X. Purification and Structural Characterization

of Fengycin Homologues Produced by Bacillus subtilis from Poplar

Wood Bark. Australas. Plant Pathol. 2018, 47, 259−268.

(15) Sang-Cheol, L.; Kim, S.-H.; Park, I.-H.; Chung, S.-Y.; Chandra,

M. S.; Yong-Lark, C. Isolation, Purification, and Characterization of

Novel Fengycin S from Bacillus Amyloliquefaciens LSC04 Degrading-

Crude Oil. Biotechnol. Bioprocess Eng. 2010, 15, 246−253.

(16) Schneider, J.; Taraz, K.; Budzikiewicz, H.; Deleu, M.; Thonart,

P.; Jacques, P. The Structure of Two Fengycins from Bacillus Subtilis

S499. Z. Naturforsch., C: J. Biosci. 1999, 54, 859−866.

(17) Nishikiori, T.; Naganawa, H.; Muraoka, Y.; Aoyagi, T.;

Umezawa, H. Plipastatins: New Inhibitors of Phospholipase A2,

Produced by Bacillus Cereus BMG302-fF67. III. Structural

Elucidation of Plipastatins. J. Antibiot. 1986, 39, 755−761.

(18) Volpon, L.; Besson, F.; Lancelin, J. NMR Structure of

Antibiotics Plipastatins A and B from Bacillus Subtilis Inhibitors of

Phospholipase A2. FEBS Lett. 2000, 485, 76−80.

AUTHOR INFORMATION

■

Corresponding Authors

*E-mail: lidia.feliu@udg.edu.

*E-mail: marta.planas@udg.edu.

ORCID

Lidia Feliu: 0000-0001-9792-6106

Marta Planas: 0000-0003-4988-4970

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

C.R. is the recipient of a predoctoral fellowship from the

MINECO of Spain. This work was ﬁnanced by grants

AGL2012-39880-C02-02, AGL2015-69876-C2-2-R (MINE-

CO/FEDER, EU), and MPCUdG2016/038. The authors

(19) Honma, M.; Tanaka, K.; Konno, K.; Tsuge, K.; Okuno, T.;

Hashimoto, M. Termination of the Structural Confusion between

Plipastatin A1 and Fengycin IX. Bioorg. Med. Chem. 2012, 20, 3793−

3798.

(20) Ma, Z.; Hu, J. Plipastatin A1 Produced by a Marine Sediment-

Derived Bacillus amyloliquefaciens SH-B74 Contributes to the

Control of Gray Mold Disease in Tomato. 3 Biotech 2018, 8, 125.

(21) Deleu, M.; Paquot, M.; Nylander, T. Effect of Fengycin, a

Lipopeptide Produced by Bacillus Subtilis, on Model Biomembranes.

Biophys. J. 2008, 94, 2667−2679.

acknowledge the Serveis Tecnics de Recerca of the University

of Girona for mass spectrometry analysis and NMR experi-

ments.

REFERENCES

■

(1) Stein, T. Bacillus Subtilis Antibiotics: Structures, Syntheses and

Specific Functions. Mol. Microbiol. 2005, 56, 845−857.

(2) Ongena, M.; Jacques, P. Bacillus Lipopeptides: Versatile

Weapons for Plant Disease Biocontrol. Trends Microbiol. 2008, 16,

115−125.

(3) Raaijmakers, J. M.; De Bruijn, I.; Nybroe, O.; Ongena, M.

Natural Functions of Lipopeptides from Bacillus and Pseudomonas:

More than Surfactants and Antibiotics. FEMS Microbiol. Rev. 2010,

34, 1037−1062.

(22) Zhao, H.; Shao, D.; Jiang, C.; Shi, J.; Li, Q.; Huang, Q.; Rajoka,

M. S. R.; Yang, H.; Jin, M. Biological activity of lipopeptides from

Bacillus. Appl. Microbiol. Biotechnol. 2017, 101, 5951−5960.

(23) Sur, S.; Romo, T. D.; Grossfield, A. Selectivity and Mechanism

of Fengycin, an Antimicrobial Lipopeptide, from Molecular

Dynamics. J. Phys. Chem. B 2018, 122, 2219−2226.

(24) Ongena, M.; Jacques, P.; Toure, Y.; Destain, J.; Jabrane, A.;

Thonart, P. Involvement of Fengycin-Type Lipopeptides in the

Multifaceted Biocontrol Potential of Bacillus Subtilis. Appl. Microbiol.

Biotechnol. 2005, 69, 29−38.

(4) Bionda, N.; Cudic, P. Cyclic Lipodepsipeptides in Novel

Antimicrobial Drug Discovery. Croat. Chem. Acta 2011, 84, 315−329.

(5) Sieber, S. A.; Marahiel, M. A. Learning from Nature’s Drug

Factories : Nonribosomal Synthesis of Macrocyclic Peptides. J.

Bacteriol. 2003, 185, 7036−7043.

(25) Ongena, M.; Jourdan, E.; Adam, A.; Paquot, M.; Brans, A.;

Joris, B.; Arpigny, J.-L.; Thonart, P. Surfactin and Fengycin

Lipopeptides of Bacillus Subtilis as Elicitors of Induced Systemic

Resistance in Plants. Environ. Microbiol. 2007, 9, 1084−1090.

(26) Emmert, E. A. B.; Handelsman, J. Biocontrol of Plant Disease:

A (Gram-) Positive Perspective. FEMS Microbiol. Lett. 1999, 171, 1−

(6) Sarabia, F.; Chammaa, S.; Sanchez Ruiz, A.; Martín Ortiz, L.;

Lopez Herrera, F. J. Chemistry and Biology of Cyclic Depsipeptides

of Medicinal and Biological Interest. Curr. Med. Chem. 2004, 11,

1309−1332.

(7) Vanittanakom, N.; Loeffler, W.; Koch, U.; Jung, G. J. Fengycin-

A Novel Antifungal Lipopeptide Antibiotic Produced by Bacillus

Subtilis F-29−3. J. Antibiot. 1986, 39, 888−901.

(8) Chen, H.; Wang, L.; Su, C. X.; Gong, G. H.; Wang, P.; Yu, Z. L.

Isolation and Characterization of Lipopeptide Antibiotics Produced

by Bacillus Subtilis. Lett. Appl. Microbiol. 2008, 47, 180−186.

(9) Bie, X.; Lu, Z.; Lu, F. Identification of Fengycin Homologues

from Bacillus Subtilis with ESI-MS/CID. J. Microbiol. Methods 2009,

79, 272−278.

(10) Pueyo, M. T.; Bloch, C.; Carmona-Ribeiro, A. M.; di Mascio, P.

Lipopeptides Produced by a Soil Bacillus Megaterium Strain. Microb.

Ecol. 2009, 57, 367−378.

(27) Li, X.-Y.; Mao, Z.-C.; Wang, Y.-H.; Wu, Y.-X.; He, Y.-Q.; Long,

C.-L. Diversity and Active Mechanism of Fengycin-Type Cyclo-

peptides from Bacillus subtilis XF-1 Against Plasmodiophora

brassicae. J. Microbiol. Biotechnol. 2013, 23, 313−321.

(28) Samel, S. A.; Wagner, B.; Marahiel, M. A.; Essen, L.-O. The

Thioesterase Domain of the Fengycin Biosynthesis Cluster: A

Structural Base for the Macrocyclization of a Non-Ribosomal

Lipopeptide. J. Mol. Biol. 2006, 359, 876−889.

(29) Sieber, S. A.; Tao, J.; Walsh, C. T.; Marahiel, M. A. Peptidyl

thiophenols as substrates for nonribosomal peptide cyclases. Angew.

Chem., Int. Ed. 2004, 43, 493−498.

(30) Hur, J.; Jang, J.; Sim, J.; Son, W. S.; Ahn, H.-C.; Kim, T. S.;

Shin, Y.-H.; Lim, C.; Lee, S.; An, H.; Kim, S.-H.; Oh, D.-C.; Jo, E.-K.;

Jang, J.; Lee, J.; Suh, Y.-G. Conformation-Enabled Total Syntheses of

Ohmyungsamycins A and B and Structural Revision of Ohmyungsa-

mycin B. Angew. Chem., Int. Ed. 2018, 57, 3069−3073.

(11) Pecci, Y.; Rivardo, F.; Martinotti, M. G.; Allegrone, G. LC/ESI-

MS/MS Characterisation of Lipopeptide Biosurfactants Produced by

the Bacillus Licheniformis V9T14 Strain. J. Mass Spectrom. 2010, 45,

772−778.

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

(31) Liu, J.; Zhu, X.; Kim, S. J.; Zhang, W. Antimycin-type

depsipeptides: discovery, biosynthesis, chemical synthesis, and

bioactivities. Nat. Prod. Rep. 2016, 33, 1146−1165.

(32) Guo, C.; Mandalapu, D.; Ji, X.; Gao, J.; Zhang, Q. Chemistry

and Biology of Teixobactin. Chem. - Eur. J. 2018, 24, 5406−5422.

(33) Cruz, L. J.; Francesch, A.; Cuevas, C.; Albericio, F. Synthesis

and Structure−Activity Relationship of Cytotoxic Marine Cyclo-

depsipeptide IB-01212 Analogues. ChemMedChem 2007, 2, 1076−

1084.

(34) Hall, E. A.; Kuru, E.; VanNieuwenhze, M. S. Solid-Phase

Synthesis of Lysobactin (Katanosin B): Insights into Structure and

Function. Org. Lett. 2012, 14, 2730−2733.

(35) Roses, C.; Camo, C.; Vogels, K.; Planas, M.; Feliu, L. Solid-

Phase Synthesis of Cyclic Depsipeptides Containing a Tyrosine

Phenyl Ester Bond. Org. Lett. 2016, 18, 4140−4143.

(36) Isidro-Llobet, A.; Guasch-Camell, J.; Alvarez, M.; Albericio, F.

p-Nitrobenzyloxycarbonyl (pNZ) as a Temporary Nα-Protecting

Group in Orthogonal Solid-Phase Peptide Synthesis − Avoiding

Diketopiperazine and Aspartimide Formation. Eur. J. Org. Chem.

2005, 2005, 3031−3039.

(37) Grant, G. A. Synthetic Peptides. A User’s Guide; W. H. Freeman

and Co., 1992.

(38) Adamson, J. G.; Hoang, T.; Crivici, A.; Lajoie, G. A. Use of

Marfey’s Reagent to Quantitate Racemization upon Anchoring of

Amino Acids to Solid Supports for Peptide Synthesis. Anal. Biochem.

1992, 202, 210−214.

(39) Gracia, C.; Isidro-llobet, A.; Cruz, L. J.; Acosta, G. A.; Alvarez,

M.; Cuevas, C.; Giralt, E.; Albericio, F. Convergent Approaches for

the Synthesis of the Antitumoral Peptide, Kahalalide F. Study of

Orthogonal Protecting Groups. J. Org. Chem. 2006, 71, 7196−7204.

(40) Kuroda, J.; Fukai, T.; Nomura, T. Collision-Induced

Dissociation of Ring-Opened Cyclic Depsipeptides with a Guanidino

Group by Electrospray Ionization/ion Trap Mass Spectrometry. J.

Mass Spectrom. 2001, 36, 30−37.

(41) Stawikowski, M.; Cudic, P. A Novel Strategy for the Solid-

Phase Synthesis of Cyclic Lipodepsipeptides. Tetrahedron Lett. 2006,

47, 8587−8590.

(42) Cochrane, J. R.; Yoon, D. H.; McErlean, C. S. P.; Jolliffe, K. a. A

Macrolactonization Approach to the Total Synthesis of the

Antimicrobial Cyclic Depsipeptide LI-F04a and Diastereoisomeric

Analogues. Beilstein J. Org. Chem. 2012, 8, 1344−1351.

(43) Parts of this manuscript are adapted from the Ph.D. dissertation

of Cristina Roses: Solid-Phase Synthesis of Cell-Penetrating γ-

Peptide/Antimicrobial Peptide Conjugates and of Cyclic Lip-

odepsipeptides Derived from Fengycins. Ph.D. Dissertation; Uni-

versitat de Girona, 2015.

(44) Thutewohl, M.; Waldmann, H. Solid-Phase Synthesis of a

Pepticinnamin E Library. Bioorg. Med. Chem. 2003, 11, 2591−2615.

(45) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Color

Test for Detection of Free Terminal Amino Groups in the Solid-

Phase Synthesis of Peptides. Anal. Biochem. 1970, 34, 595−598.

(46) Vojkovsky, T. Detection of Secondary Amines on Solid Phase.

Pept. Res. 1995, 8, 236−237.

DOI: 10.1021/acs.joc.8b02553

J. Org. Chem. XXXX, XXX, XXX−XXX

Products guided by the article

Product name:4-nitrobenzyl carbonazidate

Cas No:71731-66-3

R&D Labs maybe for 71731-66-3

Suzhou ATL Chemical Co.,ltd

Contact:+86-0512-69209969

Address:Room 317,Lushan Road,Suzhou New District,Jiangsu Province,China.
Wuhan HengHeDa Pharm Co., Ltd

Contact:86-27-84888681

Address:Wuhan economic & technology development zone
Dalian Join King Biochemical Tech. Co., Ltd.

Contact:0411 39216206

Address:814 First State Blvd
Shenzhen Hongyuan Import & Export Co., Ltd.

Contact:0755-26407171

Address:Shenzhen Hongyuan Chemical New Materials Technology Co., Ltd.
LIAOYANG WANRONG CHEMICALS COMPANY LIMITED

Contact:86-419-2390789

Address:XINLI VILLAGE , DONG NINGWEI COUNTY,TAIZIHE DISTRICT, LIAOYANG , LIAONING

Relevant to this article

p-Tyrosol: a new synthetic method and new types of pharmacological activity

Doi:10.1007/s11172-015-1140-y
(2015)
Laser-power-dependent Coordination and Photo-oxidation of Zinc Tetraphenylporphin in Alkyl Chlorides probed by Resonance Raman Spectroscopy

Doi:10.1039/ft9928802853
(1992)
Erratum: Five-bond cleavage in copper-catalyzed skeletal rearrangement of O-propargyl arylaldoximes to β-lactams (Journal of the American Chemical Society (2009) 131 (2804-2805) DOI: 10.1021/ja900133m)

Doi:10.1021/ja201074e
(2011)
Rhodium(III)-catalysed decarbonylative annulation through C-H activation: Expedient access to aminoisocoumarins by weak coordination

Doi:10.1039/c8cc07167e
(2018)
Synthesis and crystal structures of N,N'-bis(5-fluoro-2-hydroxybenzylidene) ethane-1,2-diamine and its dinuclear manganese(III) complex with antibacterial activities

Doi:10.1080/00958972.2013.859253
(2013)
Method for the microbiological production of optical antipodes of 6-hydroxynicotine

Doi:10.1515/znb-1969-0133
(1969)

Article Doi

Total Solid-Phase Synthesis of Dehydroxy Fengycin Derivatives

DOI: 10.1021/acs.joc.8b02553

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.joc.8b02553

Full text of DOI:10.1021/acs.joc.8b02553

Products guided by the article

R&D Labs maybe for 71731-66-3

Relevant to this article

Hot Product