Synthesis of 1,4-Oxazepane-2,5-diones via Cyclization of Rotationally Restricted Amino Acid Precursors and Structural Reassignment of Serratin

Article abstract of DOI:10.1021/acs.joc.7b00790

Several natural products containing a 1,4-oxazepane-2,5-dione-core are known. One example is serratin, isolated from Serratia marcescens. Because of the presence of a carboxylic amide, which has a preference for a trans-conformation, and the presence of a labile lactone in this core, many synthetic methodologies commonly used for the cyclization toward medium-sized heterocycles cannot be applied. As N-acyl amino acids lacking a third substituent at nitrogen failed to undergo ring-closure, several N-protecting groups were evaluated. With the use of the removable PMB-group, an N-unsubstituted 1,4-oxazepane-2,5-dione was synthesized. Via the application of pseudoprolines (i.e. serine-derived oxazolidines as another type of protecting group), a compound with the presumed structure of the natural product serratin was obtained. As a result of the differences in spectral data, the incorrect structural assignment of the natural product serratin was identified. Instead of the predicted seven-membered heterocycle, a symmetrical serratamolide analogue is proposed to be the correct structure of serratin.

Full text of DOI:10.1021/acs.joc.7b00790

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Article
Synthesis of 1,4-oxazepane-2,5-diones via cyclization of rotationally
restricted amino acid precursors and structural reassignment of serratin
Ewout Ruysbergh, Kristof Van Hecke, Christian Victor Stevens, Norbert De Kimpe, and Sven Mangelinckx
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017
Downloaded from http://pubs.acs.org on May 23, 2017
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Synthesis of 1,4-oxazepane-2,5-diones via cyclization of rotationally
restricted amino acid precursors and structural reassignment of serratin
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Ewout Ruysbergh,§ Kristof Van Hecke,‡ Christian V. Stevens,§ Norbert De Kimpe§ and Sven
Mangelinckx§,*
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§ Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering,
Ghent University, Coupure links 653, Bꢀ9000 Ghent, Belgium
‡ XStruct, Department of Inorganic and Physical Chemistry, Faculty of Sciences, Ghent University,
Krijgslaan 281, S3, Bꢀ9000 Ghent, Belgium
* Corresponding Author: eꢀmail: Sven.Mangelinckx@UGent.be; Tel.: +32ꢀ(0)9ꢀ264.59.51
Dedicated to Prof. Al Padwa on the occasion of his 80th birthday.
Received: / Accepted: / Published:
Keywords: natural product, cyclization, heterocycle, pseudoꢀproline, 1,4ꢀoxazepane
Graphical abstract
Abstract
Several natural products containing a 1,4ꢀoxazepaneꢀ2,5ꢀdioneꢀcore are known. One example is
serratin, isolated from Serratia marcescens. Due to the presence of a carboxylic amide, which has a
preference for a trans-conformation, and the presence of a labile lactone in this core, many synthetic
methodologies commonly used for the cyclization towards mediumꢀsized heterocycles cannot be
applied. As Nꢀacyl amino acids without a third substituent at nitrogen failed to undergo ringꢀclosure,
several N-protecting groups were evaluated. With the use of the removable PMBꢀgroup, a N-
unsubstituted 1,4ꢀoxazepaneꢀ2,5ꢀdione was synthesized. Via the application of pseudoꢀprolines, i.e.
serineꢀderived oxazolidines as another type of protecting group, a compound with the presumed
structure of the natural product serratin was obtained. Due to the differences in spectral data, the
incorrect structural assignment of the natural product serratin was identified. Instead of the predicted
sevenꢀmembered heterocycle, a symmetrical serratamolide analogue is proposed to be the correct
structure of serratin.
Introduction
The synthesis of mediumꢀsized heterocycles still forms a synthetic challenge. A headꢀtoꢀtail
cyclization often fails to yield the desired sevenꢀ or eightꢀmembered heterocycles due to a combination
of energy and entropy terms. The energy term represents the increase in ring strain and unfavorable
interactions needed to overcome when the open chain form approaches the ringꢀshaped transition state.
The entropy term is linked with the probability of the two chain terminals coming close enough to
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interact.1 The resistance towards cyclization is even more profound when a carboxylic amide bond is
present. The preference of an amide for a trans-conformation removes both termini of the linear
precursor from each other’s proximity, impeding cyclization.2 As mediumꢀsized lactams constitute a
class with a high potential for drug applications, many synthetic efforts have been devoted to this type
of mediumꢀsized heterocycles. NꢀSubstitution of the amide and dilute reaction conditions are often
applied,3 but methods using solid support,4 or that rely on a Staudinger ligation for ring closure,5 are
described as well. Another possibility is the use of pincer auxiliaries, fulfilling both a tethering and
templating role.6
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Although several methods are available for the synthesis of 1,4ꢀdiazepaneꢀ2,5ꢀdiones,3, 5ꢀ7 to the best
of our knowledge, no general method is known for the synthesis of 1,4ꢀoxazepaneꢀ2,5ꢀdiones.
However, several natural products have been isolated containing this sevenꢀmembered core. One
example is callipeltin L (1) (Figure 1), belonging to a group of antifungal peptides, produced by the
marine sponge Latrunculia sp.8 Compound 2 with a similar lactone core was isolated from the
methanolysis mixture of the marine immunosuppressant lipopeptide microcolin A and shows
relatively potent immunosuppressive activity as well.9 Also inducamide C (3), isolated from a
chemically induced mutant strain of Streptomyces sp. and exhibiting modest cytotoxicity, contains the
same 1,4ꢀoxazepaneꢀ2,5ꢀdione core.10 Finally, a bacterial metabolite was isolated from the gramꢀ
negative bacterium Serratia marcescens and identified as serratin (4a), but its biological activities
have not been evaluated yet.11 Bacteria belonging to the Serratia genus, are known to produce the
cyclodepsipeptides serratamolide AꢀF composed out of similar building blocks as serratin.12 These
macrocyclic compounds do not only possess antimycobacterial activity, but can induce cell cycle
arrest and proapoptotic effects in breast cancer cells.13
Figure 1: 1,4ꢀOxazepaneꢀ2,5ꢀdione core-containing natural products 1,8 2,9 3,10 and 4a.11
Due to the instability of the lactone bond, it is often difficult to isolate sufficient amounts of these
natural products to fully assess their biological activities. However, the same lactone bond offers the
distinct advantage of rendering the molecule neutral by masking the carboxylic acid unit, allowing
better membrane permeability whereafter degradation or nucleophilic attack within the cell releases
the active molecule.14
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The goal of this study was to develop a method for the synthesis of 1,4ꢀoxazepaneꢀ2,5ꢀdiones and to
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apply this methodology on the synthesis of the natural product serratin (4a).
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Results and discussion
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Our synthetic efforts were focused in the first instance on the synthesis of the natural product serratin
(4a) as it contains the targeted sevenꢀmembered core. The exact biosynthetic origin of serratin (4a) is
unknown, so the possibility was evaluated that the sevenꢀmembered ring arises from a spontaneous
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rearrangement of a secondary bacterial metabolite. For Nꢀ3ꢀoxoacylꢀLꢀhomoserine lactones 5, a type
of Nꢀacylated homoserine lactones (AHLs) that gramꢀnegative bacteria use as signal molecules to
regulate different phenotypes in a cellꢀdensity controlled manner in a phenomenon called quorum
sensing (QS), it is known that these molecules can rearrange via a Claisenꢀlike condensation to
tetramic acids 6 (Scheme 1).15 These compouds 6 possess interesting biological properties such as iron
chelation and antimicrobial effects.15 Another type of QS signal molecules, Nꢀ(3ꢀhydroxyacyl)ꢀ
Lꢀ
homoserine lactones 7, could participate in a similar rearrangement to 1,4ꢀoxazepaneꢀ2,5ꢀdiones 8.16
This type of reactivity has been suggested by the difference in heat stability of the autoinducers of
Aliivibrio fischeri (previously designated as Vibrio fischeri), Nꢀ(3ꢀoxohexanoyl)ꢀ
Lꢀhomoserine lactone
5a (R = Pr), and the autoinducer of Vibrio harveyi, Nꢀ(3ꢀhydroxybutanoyl)ꢀ ꢀhomoserine lactone 7a
L
(R = Me). While heating a medium containing the autoinducer 5a of A. fischeri at 100 °C for 5
minutes did not have an effect on the bioluminescence inducing activity of this quorum sensing signal
molecule, applying the same treatment on a medium containing the autoinducer 7a of V. harveyi,
caused a complete deactivation of the bioluminescence inducing activity.17 Not surprisingly, both
autoinducers lose their QS stimulating properties at high pH due to ringꢀopening.
Scheme 1: Described rearrangement of Nꢀ(3ꢀoxoacyl)ꢀL
ꢀhomoserine lactones 5 to tetramic acids 6,15
and the attempted rearrangement of another type of QS signal molecules, Nꢀ(3ꢀhydroxyacyl)ꢀ
Lꢀ
homoserine lactones 7, to 1,4ꢀoxazepaneꢀ2,5ꢀdiones 8.
Although rearrangement product 8 possesses a 2ꢀhydroxyethyl group instead of the hydroxymethyl
substituent in serratin (4a), it was decided to evaluate this route with Nꢀ(3ꢀhydroxyhexanoyl)ꢀ
L
ꢀ
homoserine lactone 7b (R = Pr)18 by mimicking the reaction conditions (heating at 100 °C for 5 min)
described by Eberhard.17 However, the formation of the desired sevenꢀmembered ring 8b (R = Pr) was
never observed (Scheme 1) and the starting material 7b was fully recovered. When the reaction time
was prolonged, elimination and hydrolysis products were observed as well. Reaction conditions suited
for the nucleophilic attack of the βꢀhydroxy group, might give rise to compound 8b, but then the
resulting primary hydroxy group could reattack the sevenꢀmembered lactone, and form the more stable
fiveꢀmembered lactone ring, yielding once again starting material 7b.
As the lipoamino acid Nꢀ(3ꢀhydroxydecanoyl)ꢀLꢀserine, or serratamic acid, has been isolated from
alkaline extracts of S. marcescens cultures,19 presumably formed by the hydrolysis of serratamolides,
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serratamic acidꢀanalogue 12a was evaluated as the possible origin of serratin (4a). This compound 12a
was synthesized via a 1ꢀethylꢀ3ꢀ[3ꢀ(dimethylamino)propyl]carbodiimide (EDC)ꢀmediated coupling
reaction of βꢀhydroxynonanoic acid 9a with serine ester 10a, followed by alkaline hydrolysis (Scheme
2). For the ringꢀclosure, reaction with 1 equiv of EDC and 1 equiv of Et3N in water was tested but no
reaction was observed. Repeating this reaction with EDC with a catalytic amount of DMAP in
dichloromethane yielded a complex reaction mixture.20 Stirring overnight in acetic anhydride also
failed to yield any of the desired ringꢀclosed product 4a.20
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Scheme 2: Synthesis of Nꢀ(3ꢀhydroxyacyl) amino acids 12a-d and ring closure towards 1,4ꢀ
oxazepaneꢀ2,5ꢀdiones 4a-d.
The apparent lack of cyclization of the serine derivative 12a (Scheme 2), can be explained by the fact
that the amide bond strongly prefers a transꢀconformation, while for the desired cyclization to occur, a
cisꢀamide bond is needed. 2D NOESY analysis of methyl ester 11a revealed that indeed only the
transꢀconformer was present (SI, Figure S1).21 In the case of larger heterocycles, such as fourteenꢀ
membered rings, trans- amides can be included and this was used for the synthesis of a serratamolide
analogue, but during this synthesis sevenꢀmembered rings were never observed.22 Prolineꢀcontaining
peptides typically contain an elevated amount of cisꢀamide bonds.23 Therefore, the prolineꢀcontaining
analogue 12b was synthesized and the cyclization was reevaluated (Scheme 2).20 To our delight,
bicyclic structure 4b was formed in all the cyclization conditions tested.
To evaluate if the imposed rigidity, caused by the cyclic structure of proline, was really necessary for
the cyclization, the highest yielding cyclization conditions were applied to sarcosine derivative 12c
(Scheme 2). The existence of both cisꢀ and transꢀisomers of compound 12c in solution was apparent
by the presence of both conformers observed in 1H and 13C NMR (CDCl3, SI Figure S2ꢀA and S3ꢀA).24
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No such conformers were observed in the H and 13C NMR spectra of derivative 12a. The cyclic
product 4c, with a similar sevenꢀmembered core as callipeltin L (1), was obtained in 62% yield. In the
1H and 13C NMR spectrum, no different isomers were observed (CDCl3, SI Figure S2ꢀB and S3ꢀB),
consistent with the formation of a more rigid, cyclic structure without the possibility of cis/transꢀ
isomerization. As a proof of concept, the reaction was repeated with the glycine derivative 12d under
identical reaction conditions, but no cyclization was observed (Scheme 2). As the ringꢀclosing reaction
only proceeds with high difficulty, a spontaneous, nonꢀenzymatical rearrangement can be excluded for
the origin of serratin (4a).
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From the abovementioned results, it is obvious that the Nꢀ(3ꢀhydroxyacyl) amino acid derivative needs
to be forced in the correct conformation for cyclization to occur. This necessity was also observed
during the synthesis of 1,4ꢀoxazepanꢀ5ꢀones.21 The introduction of a third substituent at nitrogen, as is
the case for the proline and sarcosine derivatives 12b and 12c, altered the cis/transꢀratio, and resulted
in cyclization. To obtain the natural product serratin (4a), this additional group at nitrogen should be a
removable one. Due to the lability of the desired lactone, many protecting groups, which can be
applied for the synthesis of sevenꢀmembered lactams, cannot be used for this cyclization as the
reaction conditions needed for their removal after the cyclization step, will destroy the lactone bond.
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As a benzyl group can be removed via hydrogenolysis, this Nꢀprotecting group was evaluated as a
possible solution. Reductive amination of benzaldehyde 13a with methyl glycinate 10d yielded the Nꢀ
benzylꢀprotected methyl ester of glycine 14a, which was coupled to βꢀhydroxynonanoic acid 9a.
Alkaline hydrolysis, followed by cyclization in dilute reaction conditions gave the desired Nꢀbenzylꢀ
protected sevenꢀmembered ringꢀcontaining compound 17a (Scheme 3).
To remove the Nꢀprotecting group, several reaction conditions were tested but none was able to deliver
the desired compound 4e (SI, Table S1).25 Similar difficulties to remove a benzyl group from a
carboxylic amide were also encountered by Williams et al. while debenzylating a diketopiperazine.26
Scheme 3: Synthesis of Nꢀbenzyl and NꢀPMBꢀprotected sevenꢀmembered lactones 17a and 17b and
CANꢀmediated deprotection of 17b to 4e.
As the synthesis of the Nꢀbenzylꢀprotected sevenꢀmembered coreꢀcontaining compound 17a was
successful, while the Nꢀdeprotection proved to be problematic, the pꢀmethoxybenzyl (PMB) protecting
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group was evaluated as a more labile protecting group. Reductive amination of glycine methyl ester
hydrochloride 10d with anisaldehyde 13b gave NꢀPMBꢀprotected methyl glycinate 14b in a good
yield (Scheme 3).27 The same Nꢀacylation and cyclization conditions used for the synthesis of the Nꢀ
benzyl derivative 17a were applied, to yield the NꢀPMBꢀprotected sevenꢀmembered ringꢀcontaining
compound 17b (Scheme 3).
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For the removal of the PMBꢀprotecting group, several reaction conditions were tested (Table 1). Both
2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (DDQ) and ceric ammonium nitrate (CAN), the reagents
commonly used for PMBꢀdeprotection, were evaluated under different reaction conditions, alongside
other reagents.28
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Table 1: Reaction conditions evaluated to remove the PMBꢀprotecting group of 17b.28
Entry Reaction conditions
Result
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2 equiv DDQ, CH2Cl2:H2O 9:1, r.t., 8 h
ꢀ[a]
5 equiv CAN, MeCN:H2O 4:1, 0 °C to r.t., 2 h
5 equiv CAN, MeCN:H2O (NaOAc/HOAc buffer, pH 5.2), 0 °C,
2 h
4e (15%)[b]
4e (18%)[b] and 17b (41%)[b]
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10 equiv CAN, MeCN:H2O 4:1, 0 °C, 1 h
5 equiv CAN, EtOAc:H2O 4:1, 0 °C, 2 h
10 equiv CAN, EtOAc:H2O 4:1, 0 °C, 2 h
5 equiv CAN, tBuOH:CH2Cl2 4:1, r.t., 2 h
5 equiv CAN, MeCN:H2O 99:1, r.t., o.n.
4 equiv PhI(OAc)2, MeOH, r.t., o.n.
BF3.OEt2, r.t., o.n.
4e (30%)[b] and 17b (6%)[b]
4e (36%)[b] and 17b (28%)[b]
4e (32%)[b] and 17b (33%)[b]
ꢀ[c]
4e (10%)[b] and 17b (70%)[b]
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ꢀ[a]
ꢀ[d]
ꢀ[e]
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BF3.OEt2, 128 °C, 6 h
[a] Solvolysis of 17b. [b] Quantitative data were obtained after extraction of the reaction mixture with ethyl acetate, followed
by a washing step with an aqueous saturated sodium bicarbonate solution to remove ringꢀopened degradation products. The
crude yield of the different products was determined via integration of the 1H NMR spectrum. [c] Deprotection followed by
solvolysis or solvolysis followed by deprotection. [d] No reaction. [e] Complex reaction mixture.
As the deprotection with 5 equivalents of CAN in a solvent mixture of ethyl acetate and water in a 4:1ꢀ
ratio gave the best result (Table 1, entry 5), this reaction was repeated on a larger scale to give the
pure, fully deprotected, sevenꢀmembered lactone 4e in 14% yield after purification via column
chromatography (Scheme 3). This rather low yield can be attributed to several factors. Firstly, the
reaction time is too short to allow complete conversion, which is obvious from the recovery of the Nꢀ
PMBꢀprotected lactone 17b. Secondly, when the product 4e is formed, lactonolysis can occur by the
water present as coꢀsolvent, which is needed for the deprotection. This lactonolysis can also open the
starting material 17b, but this route seems to be slower. Thirdly, reactions with CAN often give rise to
a laborious workꢀup due to the difficult separation of the turbid aquatic phase and the organic phase.
As the synthesis of the deprotected sevenꢀmembered core 4e was successfully completed, the focus
was put on the synthesis of serratin (4a), differing only from lactone 4e by the presence of a
hydroxymethyl group. However, when the Nꢀacylation reaction was repeated with methyl N-PMBꢀ
serinate, O-acylation instead of Nꢀacylation was observed. To avoid this unwanted reaction, the
reaction sequence was repeated with Oꢀbenzylserine but when the cyclization was attempted in the
final step, a complex reaction mixture was obtained instead of the desired heterocycle. This lack of
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cyclization could be caused by steric factors. A similar observation was made by Imramovský et al.
during a coupling reaction of Nꢀbenzyloxycarbonylꢀprotected amino acids with a salicylanilide.29
When NꢀCbzꢀglycine and NꢀCbzꢀalanine were used, a sevenꢀmembered ring was formed. This type of
cyclization was not observed when valine or phenylalanine were used. Another possibility was a postꢀ
cyclization modification step, but the introduction of a hydroxymethyl group via reaction of Nꢀmethyl
derivative 4c as a test substrate with formaldehyde failed in all reaction conditions tested (15 equiv
KHCO3, 11 equiv paraformaldehyde, DMF, r.t., o.n.; 1 equiv LDA, dry THF, N2, ꢀ78 °C, 1 h followed
by 6 equiv paraformaldehyde, ꢀ78°C, 3 h to r.t., o.n.; 1 equiv LDA, dry THF, N2, ꢀ78 °C, 1 h followed
by formaldehyde (g) (formed by dry heating of paraformaldehyde at 170 °C), ꢀ78°C, 3 h to r.t., o.n.).30
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Another option for the synthesis of serratin (4a) was via the use of pseudoꢀprolines (ΨPro). These
oxazolidines are formed via reaction of serine (and threonine) with aldehydes or ketones. These cyclic
structures are unstable under acidic conditions, but can be acylated in alkaline environment and
isolated as such. Pseudoꢀprolines are commonly used to alter the solubility of peptides by disrupting
secondary structure formation or to facilitate the cyclization of small peptides.31 Oxazolidine 18b was
synthesized by heating the hydrochloride of the methyl ester of Lꢀserine 10a with pivaldehyde 13c and
triethylamine with continuous removal of water (Scheme 4). This oxazolidine 18b was obtained as a
3:2ꢀmixture of diastereomers in 71% yield.32
Scheme 4: Synthesis of oxazolidineꢀcontaining bicyclic structures 21a-c.
Several procedures were evaluated for the Nꢀacylation of compound 18b (SI, Table S2). Formation of
the mixed anhydride of βꢀhydroxynonanoic acid 9a via reaction with isobutyl chloroformate, followed
by reaction with the methyl ester of oxazolidine 18b proved to be the best procedure to obtain
compound 19b (SI, Table S2, entry 4).33 The Nꢀacylated (2ꢀtbutyl)oxazolidine 19b possessed a C2,C4ꢀ
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cisꢀrelation, although the starting oxazolidine 18b was obtained as a 3:2 diastereomeric mixture. Ringꢀ
tautomerism allows equilibration to the more stable product with the tBuꢀgroup in a quasiꢀaxial
position.34 Subsequent alkaline hydrolysis of the acylated 2ꢀtBuꢀoxazolidine 19b, followed by
cyclization in dilute reaction conditions yielded bicyclic structure 21b (Scheme 4).
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Bicyclic compound 21b was obtained as a 1:1 mixture of diastereomers. The diastereomers (RSS)ꢀ21b
and (RRS)ꢀ21b were separated via column chromatography followed by recrystallization. One of the
two diastereomers remained an amorphous powder, while the other one formed needleꢀlike crystals,
allowing structure confirmation and stereochemistry determination via Xꢀray diffraction analysis
(Figure 2). The crystals belong to the orthorhombic Sohnke space group P212121 and hence contain
only one enantiomer, being (RSS)ꢀ21b with a cisꢀrelationship of the substituents.
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O
A)
H
O
(S)
(S)
O
N
(R)
O
21b
(RSS)-
O
B)
HO
O
(S)
(S)
HN
O
4a
(SS)-
Figure 2: A) Molecular structure of (RSS)-21b, showing thermal displacement ellipsoid, drawn at the
30% probability level. The positional disorder of the C6H13 alkyl chain is shown in yellow. B)
Stereochemistry of serratin (SS)ꢀ4a based upon comparison with theoretical calculations of the 13C
NMR chemical shift.11
Both diastereomers showed quite different chemical shifts. The crystalline diastereomer (RSS)ꢀ21b,
with both substituents in a cisꢀrelationship and the RSSꢀstereochemistry showed a signal for CHꢀO at
75.2 ppm (CDCl3) in the 13C NMR spectrum and the attached hydrogen atom CH-O showed a
1
multiplet at 4.71ꢀ4.83 ppm (CDCl3) in the H NMR spectrum. For the other diastereomer (RRS)ꢀ21b,
with a RRSꢀstereochemistry, the corresponding signals were at 79.7 ppm (CDCl3, 13C NMR) and 4.68ꢀ
1
4.79 ppm (CDCl3, H NMR). The signal around 70 ppm in the 13C NMR spectrum is quite
characteristic and is present in all 1,4ꢀoxazepaneꢀ2,5ꢀdiones described in literature (SI, Table S3).8ꢀ11,
20, 29, 35
Another difference was the shift of the CH2 adjacent to the CHꢀO moiety. For diastereomer
(RSS)ꢀ21b the corresponding signals were a dd at 2.85 ppm (CDCl3) and a dd at 2.97 ppm (CDCl3),
while for the other diastereomer (RRS)ꢀ21b a d at 2.72 ppm (CDCl3) and a dd at 3.14 ppm (CDCl3)
were observed. For serratin (4a), Luna et al. noticed signals for the CH2 at 2.42 ppm (dd, CDCl3) and
2.62 ppm (dd, CDCl3) and a signal for CHꢀO at 72.4 ppm (CDCl3) in the 13C NMR spectrum.11 These
values seem to be consistent with our observations for diastereomer (RSS)ꢀ21b.
As the 2ꢀtBuꢀoxazolidine is a rather stable oxazolidine, it was decided to synthesize two different
types of oxazolidines as well. The first type was the more labile 2,2ꢀdimethyloxazolidine
(Ser(ΨMe,MePro)), which undergoes rapid deprotection in dilute TFA.36 Unlike Ser(ΨtBuPro) 18b, this
oxazolidine cannot be isolated as such and is commonly introduced via the postꢀinsertion route.36
Therefore, methyl Nꢀ(3ꢀhydroxynonanoyl)serinate 11a was reacted with 2,2ꢀdimethoxypropane
(DMP) with continuous removal of water to yield compound 19c (Scheme 4).37 As 1H NMR analysis
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revealed that this compound, obtained after column chromatography, was less pure than the crude
compound after workꢀup due to degradation during purification, it was decided to proceed with the
reaction sequence without further purification. Hydrolysis of compound 19c with NaOH in a
water:methanol (ratio 1:3) mixture, followed by cyclization gave 2,2ꢀdimethyloxazolidineꢀprotected
oxazepaneꢀ2,5ꢀdione 21c in a total yield of 18% after purification. Once again, the CHꢀO and CH2 of
both diastereomers gave very distinct signals in NMR spectroscopy.
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For the 2ꢀphenylꢀoxazolidine derivative, Lꢀserine methyl ester hydrochloride 10a was neutralized with
Et3N and allowed to react with benzaldehyde 13a to produce oxazolidine derivative 18a (Scheme 4).38
This compound was Nꢀacylated with βꢀhydroxynonanoic acid 9a and the ester functionality was
hydrolyzed under alkaline conditions to furnish compound 20a. The 2ꢀphenyloxazolidineꢀprotected
oxazepanedione 21a was obtained in 53% total isolated yield after the DMAPꢀcatalyzed cyclization
under dilute reaction conditions.
Several reaction conditions were evaluated to deprotect the oxazolidine unit without opening the
sevenꢀmembered lactone (Table 2). A catalytic amount of bismuth(III) bromide was successfully
employed by Cong et al. to deprotect a cyclic N,Oꢀaminal under mild reaction conditions.39 However,
in our case no reaction was observed (Table 2, entry 1AꢀC). This could be explained by the fact that
the reactivity of the oxazolidine ring is dramatically reduced upon amidation.40 When a catalytic
amount of water was added to the reaction mixture, the 2ꢀphenyloxazolidine moiety of the transꢀ
diastereomer (RRS)ꢀ21a got deprotected to the ringꢀopened structure 12a (Table 2, entry 2B). As
traces of the ringꢀopened oxazolidineꢀcontaining product 20a were detected during the course of the
reaction, unlike the deprotected compound 4a, hydrolysis probably preceded deprotection. The other
diastereomer (RSS)ꢀ21a could be recovered from the crude reaction mixture, albeit in a severely
reduced amount. For the two other types of oxazolidines, all stereoisomers seemed to react at a similar
pace and only the deprotected and hydrolyzed product 12a was isolated (Table 2, entries 2A and C). In
an alternative procedure, mild acidic conditions for deprotection were evaluated by employing formic
acid in a THF:H2Oꢀmixture (Table 2, entries 3A and B).41 In the case of the tBuꢀcontaining
oxazolidine 21b, a quick hydrolysis of both diastereomers to 20b was observed. Interestingly, for the
2ꢀphenyloxazolidine 21a the transꢀdiastereomer (RRS)ꢀ21a hydrolyzed significantly faster than the
cisꢀdiastereomer (RSS)ꢀ21a: while the starting compound had a d.r. of 1:1, an alteration to 1:2.7 was
found in the recovered starting material. Treatment with an acidic resin also failed to deliver the
desired compound (Table 2, entries 4A and B).42 5% TFA in dichloromethane left the 2ꢀtBuꢀ
oxazolidine 21b intact, even after a reaction time of 48 h (Table 2, entry 5A).31b Under the same
conditions, the transꢀdiastereomer of the 2.2ꢀdimethyloxazolidine 21c was successfully deprotected
1
(Table 2, entry 5C). The H NMR of the crude reaction mixture, obtained after washing with a
saturated aqueous NaHCO3ꢀsolution to remove the TFA and the ringꢀopened product, revealed the
presence of only the cisꢀdiastereomer. However, the deprotected transꢀisomer (SR)ꢀ4a was not
detected. When the same treatment was applied on the 2ꢀphenyloxazolidine protected sevenꢀ
membered ring 21a a similar observation was made: the transꢀdiastereomer (RRS)ꢀ21a seemed to react
faster, but none of the deprotected serratin (4a) could be isolated (Table 2, entry 5B and SI, Figure
S5). When 4M HCl (g) in dioxane was evaluated for the removal of the oxazolidine moiety,43
a
difference in reactivity between the 2ꢀtBu and the 2ꢀphenyloxazolidine moiety was observed: in the
case of the tBuꢀcompound 21b, the cisꢀdiastereomer (RSS)ꢀ21b got deprotected faster while for the
latter compound 21a the transꢀdiastereomer (RRS)ꢀ21a was deprotected faster. In both cases none of
the deprotected sevenꢀmembered ring 4a could be isolated (Table 2, entry 6A and B). When the
reaction time was prolonged, all of the starting material was converted to the deprotected, hydrolyzed
compound 12a. The protocol with 1,3ꢀpropanedithiol in acidic trifluoroethanol, developed by Corey,44
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also caused a faster deprotection of the transꢀdiastereomer of both the 2ꢀtBu and the 2ꢀ
phenyloxazolidine compared to the cisꢀisomers. Once again, no deprotected serratin (4a) could be
isolated.
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Table 2: Reaction conditions evaluated to achieve the deprotection of the different oxazolidine
moieties of sevenꢀmembered rings 21a, 21b and 21c.39, 41ꢀ44
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Entry Reaction conditions
Oxazolidine
derivative
Ph
Result[a]
1A
1B
1C
2A
0.1 equiv BiBr3, MeCN, r.t., 1 h to o.n.
Idem
Idem
ꢀ[b]
tBu
DiMe
Ph
ꢀ[b]
ꢀ[b]
1 equiv BiBr3, cat. H2O, MeCN, r.t., 1 h to
48 h
Conversion of transꢀdiastereomer
(RRS)ꢀ21a to 12a. No isolation of
(SR)ꢀ4a. No reaction of cisꢀ
diastereomer (RSS)ꢀ21a
.
2B
2C
3A
Idem
Idem
tBu
DiMe
Ph
ꢀ[c]
ꢀ[c]
THF:H2O:HCOOH 3:1:1, r.t., 1 h to 48 h
Ringꢀopening to 20a, trans-
diastereomer (RRS)ꢀ21a reacts
faster than (RSS)ꢀ21a
Ringꢀopening to 20b
ꢀ[b]
3B
4A
Idem
tBu
Amberlyst 15, acetone:H2O 9:1, r.t., 1 h to
Ph
24 h
4B
5A
Idem
tBu
ꢀ[b]
5% TFA in dry CH2Cl2, 0 °C, 3 h, then r.t.,
48 h
Ph
No reaction of cisꢀdiastereomer
(RSS)ꢀ21a, deprotection of transꢀ
diastereomer (RRS)ꢀ21a to 12a
ꢀ[b]
No reaction of cisꢀdiastereomer
(SS)ꢀ21c, deprotection of transꢀ
diastereomer (RS)ꢀ21c to 12a
Formation of 12a, faster reaction
of the cisꢀdiastereomer (RSS)ꢀ21a
Formation of 12a, faster reaction
of the transꢀdiastereomer (RRS)ꢀ
21b
Formation of 12a, faster reaction
of the transꢀdiastereomer (RRS)ꢀ
21a
Formation of 12a, faster reaction
of the transꢀdiastereomer (RRS)ꢀ
5B
5C
Idem
Idem
tBu
DiMe
6A
6B
4M HCl in dioxane, 0 °C to r.t., 1 h to 24 h
Idem
Ph
tBu
7A
7B
3 equiv 1,3ꢀpropanedithiol, 2% HCl in
2,2,2ꢀtrifluoroethanol, r.t., 1 h to 24 h
Ph
Idem
tBu
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21b
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[a] Based on LCꢀMS analysis during the course of the reaction and H NMR analysis of the crude reaction mixture after
workꢀup. [b] No reaction. [c] Deprotection and ringꢀopening or ringꢀopening and deprotection. Only recovery of 12a.
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As deprotection was observed but isolation of serratin (4a) failed, either due to deprotection followed
by immediate ringꢀopening or by first ringꢀopening and then deprotection, another route for the 2ꢀ
phenyloxazolidine 21a was evaluated. In a first attempt to remove the 2ꢀphenyloxazolidine unit from
21a, hydrogenolysis with Pd/C in ethyl acetate and in MeOH was evaluated, but no reaction (Table 3,
entry 1) or only a limited conversion, combined with degradation (Table 3, entry 2), were obtained.
When Pd(OH)2/C was employed in EtOH, a complete debenzylation was observed, unfortunately
combined with ethanolysis of the deprotected product (Table 3, entry 3). When the reaction was
repeated in ethyl acetate, deprotection without solvolysis was observed (Table 3, entry 4).
Remarkably, both diastereomers behaved differently: only the sevenꢀmembered ring (SS)ꢀ4a with both
substituents in a cisꢀrelationship was obtained and isolated in 22% yield (based on the total amount of
starting material). The diastereomeric ratio of the recovered, oxazolidineꢀcontaining starting material
21a had consequently changed from 1:1 to 1:3, favoring the (RRS)ꢀdiastereomer (SI, Figure S5). This
observed selectivity for (RSS)-21a could be explained by steric factors. The (RSS)-isomer of 21a has
the phenyl and alkyl substituent on the same side of the bicyclic ring system (see Figure 2 for (RSS)-
21b), allowing a relatively unhindered interaction of the opposed side with the palladium catalyst. As
the (RRS)-isomer has the bulky phenyl substituent on one side and the alkyl substituent on the other
side, it is expected that such an interaction with the catalyst proceeds with more difficulty.
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Table 3: Reaction conditions evaluated for the hydrogenolytic removal of the 2ꢀphenyloxazolidine
moiety of 21a
Entry Reaction conditions
Result
ꢀ[a]
1
2
3
4
4 atm H2, 25 wt% Pd/C, EtOAc, r.t., o.n.
4 atm H2, 25 wt% Pd/C, MeOH, r.t., o.n.
4 atm H2, 25 wt% Pd(OH)2/C, EtOH, r.t., o.n.
1 atm H2, 50 wt% Pd(OH)2/C, EtOAc, r.t., 6 h (SS)ꢀ4a (22%). No reaction of the other diaꢀ
stereomer, recovery of starting material 21a
ꢀ[b]
ꢀ[c]
(67%) with a d.r. of 1:3
[a] No reaction. [b] Solvolysis of starting material 21a. [c] Deprotection and solvolysis or solvolysis and deprotection.
The (RSS)ꢀdiastereomer that was deprotected, however, had the correct stereochemistry to deliver
1
serratin (SS)ꢀ4a. In the H NMR spectrum (CDCl3), a signal for NH became visible, consistent with
successful deprotection. Also the CHꢀOꢀsignal at 73.7 ppm (CDCl3) was present, indicating that the
sevenꢀmembered heterocycle was still intact. However, unlike the value around 5.30 ppm (CDCl3)
reported by Luna et al. for CH-O, a multiplet around 4.73ꢀ4.81 ppm (CDCl3) was observed. Also a big
difference for the adjacent CH2 was apparent: while Luna et al. reported two dd at 2.42 and 2.62 ppm
(CDCl3), we detected the corresponding signals at 2.79 and 2.87 ppm (CDCl3).
The values reported by Luna et al. show a lot of similarities with the spectral data of the compound
serratamolide A (22a) (Figure 3 and SI, Table S4). This antimycobacterial cyclodepsipeptide is
produced by Serratia sp. and has the CHꢀO signal at 5.33 ppm (1H NMR, CD3OD), the CHꢀO signal at
73.2 ppm (13C NMR, CD3OD) and the adjacent CH2 at 2.39 and 2.72 ppm (1H NMR, CD3OD).12b That
the actual structure of serratin (4a) could be a serratamolide analogue is also hinted by the observed
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vicinal coupling constants of the OꢀCHCH2C(=O) moiety. While for the compound synthesized in this
study vicinal coupling constants of 11.3 Hz and 3.0 Hz were observed, which corresponds to a
conformation as depicted in figure 2 with one large and one small dihedral angle of the coupling
protons, values of 4.9 Hz and 2.0 Hz were reported by Luna et al.11 These small vicinal coupling
constants were also reported for serratamolide A (22a) (5.0 Hz and 2.6 Hz), which correspond with a
more flexible structure and smaller dihedral angles (SI, Table S4).45 Serratamolide A (22a) is a
symmetrical molecule composed out of two serine units and two times βꢀhydroxydecanoic acid as the
fatty acid moiety. However, the asymmetrical analogue serratamolide F (22b) has a βꢀ
hydroxydecanoic acid moiety but also a βꢀhydroxynonanoic acid moiety in its structure.12b It is known
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that during the biosynthesis of cyclic lipopeptides, a relaxed substrate specificity can give rise to the
46
production of several analogues of one main cyclic lipopeptide compound.12b,
Therefore, if a
symmetrical serratamolide exists with two βꢀhydroxynonanoic acid tails, the corresponding NMR
spectra would contain only a limited number of signals with values nearly identical to those reported
by Luna et al. for serratin (4a) (SI, Table S4).
Figure 3: Structure of secondary metabolites Serratamolide A (22a), Serratamolide F (22b)12b and
serratin (4a)11 produced by Serratia sp.
Conclusion
A method for the synthesis of Nꢀunsubstituted 1,4ꢀoxazepaneꢀ2,5ꢀdiones is presented. The lability of
the lactone moiety excludes the use of many techniques commonly used for the cyclization of
mediumꢀsized heterocycles. Therefore, PMB was applied as a protecting group to force the linear
amino acid precursor in a correct conformation for cyclization to occur. For serine, the oxazolidine or
pseudoꢀproline group was used as a protecting group. Several pseudoꢀprolines and deprotecting
reaction conditions were evaluated but only hydrogenolysis of the 2ꢀphenyloxazlidine moiety with
Pearlman’s catalyst was able to remove the oxazolidine moiety without opening of the lactone. The
application of our methodology led to the identification of the incorrect structural assignment of the
natural product serratin, whose spectral data fit better with a serratamolide structure instead of a 1,4ꢀ
oxazepanꢀ2,5ꢀdione derivative.
Experimental section
General methods. Solvents and chemicals used were bought from commercial suppliers and used as
such, unless stated otherwise. Diethyl ether, toluene and tetrahydrofuran were dried by distillation over
1
sodium/benzophenone ketyl. Dichloromethane was distilled over calcium hydride. H (400 MHz) and
13C (100.6 MHz) NMR spectra were recorded on a Bruker Avance III Nanoꢀbay 400 at room
temperature. IR spectra were recorded in neat form with a PerkinꢀElmer Spectrum One FTIR
spectrometer. Highꢀresolution mass spectra were determined with an Agilent 1100 series HPLC
coupled to an Agilent 6210 TOF mass spectrometer, equipped with an ESI/APCI multimode source.
Melting points were measured with a Kofler bench, type WME Heizbank of Wagner & Munz. The
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reaction mixtures were purified by column chromatography on silica gel (Acros, particle size: 0.035ꢀ
0.070 mm, pore diameter: approximately 6 nm) or by recrystallization. For the structure of (RSS)ꢀ21b,
Xꢀray intensity data were collected at RT on an Agilent Supernova Dual Source (Cu at zero)
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diffractometer equipped with an Atlas CCD detector using
ω scans and CuKα (λ = 1.54184 Å)
radiation. The images were interpreted and integrated with the program CrysAlisPro.47 Using Olex2,48
the structure was solved by direct methods using the ShelXS structure solution program and refined by
fullꢀmatrix leastꢀsquares on F2 using the ShelXL program package.49 Nonꢀhydrogen atoms were
anisotropically refined and the hydrogen atoms in the riding mode and isotropic temperature factors
fixed at 1.2 times U(eq) of the parent atoms (1.5 times for methyl groups).
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Synthesis, hydrolysis and cyclization of
N-(3-hydroxyacyl) amino acids.
General procedure A: -acylation of a primary amine. Triethylamine (1 equiv) was added to a
N
stirred solution of the amino acid methyl ester hydrochloride (1 equiv) in water (5 mL/mmol methyl
ester), followed by the addition of the appropriate carboxylic acid (1 equiv) and 1ꢀethylꢀ3ꢀ[3ꢀ
(dimethylamino)propyl]carbodiimide hydrochloride (1 equiv). After stirring overnight at room
temperature, water (15 mL/mmol methyl ester) was added and the aqueous phase was extracted three
times with ethyl acetate. The combined organic phases were washed with saturated aq. NaHCO3 and
brine. Drying over MgSO4, followed by filtration and evaporation of the solvent in vacuo gave the
crude product. If necessary, a purification step via column chromatography was included.
General procedure B:
N-acylation of a secondary amine. The Nꢀacylation was executed according
to the procedure of Falorni et al. with minor adaptations.33 Briefly, the appropriate fatty acid (1 equiv)
was dissolved in ethyl acetate (2 mL/mmol fatty acid) and cooled to 0 °C whereafter Nꢀ
methylmorpholine (1 equiv) was added, followed by the dropwise addition of isobutyl chloroformate
(1 equiv). The resulting turbid suspension was stirred for 20 min at 0 °C. The secondary amine (1.05
equiv), dissolved in a minimal amount of ethyl acetate, was added at the same temperature and after 2
h the reaction mixture was allowed to warm to room temperature and stirred overnight. Water was
added and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases
were washed with a saturated solution of aqueous NaHCO3 and brine, dried (MgSO4) and the solvent
was removed via rotary evaporation to yield the Nꢀacylated product.
General procedure C: hydrolysis. 5 Equiv of NaOH, as a 2M aqueous solution, were added to the
methyl ester dissolved in methanol (ratio MeOH:H2O 3:1). The reaction mixture was left to stir at
room temperature for 3 h to overnight, followed by an extraction step with hexane. The aqueous phase
was acidified with 2M aqueous HCl and extracted three times with ethyl acetate. After washing with
brine, followed by drying (MgSO4) and removal of the drying agent by filtration, the solvent was
removed by rotary evaporation to yield the crude hydrolyzed product, which was purified, if
applicable, via recrystallization in diethyl ether:hexane.
General procedure D: cyclization. The free carboxylic acid was dissolved in dry CH2Cl2 to obtain a
20 mM solution whereafter 1.1 equiv of EDC.HCl and 0.2 equiv of DMAP were added. The resulting
reaction mixture was stirred overnight at room temperature, after which the solvent was removed in
vacuo and the residue redissolved in ethyl acetate:water 1:1. The aqueous phase was extracted twice
with ethyl acetate, the organic phases were combined and subsequently washed with a saturated
solution of aqueous NaHCO3 and brine. Drying with MgSO4, filtration and rotary evaporation of the
solvent yielded the crude sevenꢀmembered ringꢀcontaining product, which was purified via column
chromatography.
Methyl
N-(3-hydroxynonanoyl)-L-serinate 11a. This compound was synthesized by reacting Lꢀ
serine methyl ester hydrochloride 10a (1 equiv, 3.1 g, 20 mmol) with βꢀhydroxynonanoic acid 9a (1
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equiv, 3.48 g, 20 mmol), following general procedure A to yield 2.91 g (10.6 mmol, 53% yield) of
compound 11a. Diastereomers (ratio 1:1) could not be separated via flash chromatography (ethyl
acetate:petroleum ether 4:1). Colorless powder. Melting point: 65ꢀ67 °C. Yield: 53%. Rf = 0.24 (ethyl
1
acetate:petroleum ether 4:1). H NMR (400 MHz, CDCl3): δ = 0.89 (3H, t, J = 6.8 Hz), 1.22ꢀ1.62
(10H, m), 2.35 (0.5H, dd, J = 14.8 Hz, 9.4 Hz), 2.36 (0.5H, dd, J = 14.7 Hz, 9.3 Hz), 2.467 (0.5H, d, J
= 14.7 Hz), 2.474 (0.5H, d, J = 14.7 Hz), 3.54 (0.5H, br s), 3.80 (3H, s), 3.70ꢀ4.10 (4.5H, m), 4.64ꢀ
4.72 (1H, m), 7.02 (0.5H, d, J = 7.7 Hz), 7.08 (0.5H, d, J = 7.7 Hz) ppm. 13C NMR (100 MHz,
CDCl3): δ = 14.1, 22.6, 25.50, 25.53, 31.8, 37.0, 37.1, 43.15, 43.21, 52.77, 52.84, 54.7, 62.7, 68.8,
69.0, 171.1, 171.3, 172.6, 172.9 ppm. MS (ESI): m/z (%): 276 (M+H+, 100). HRMS Calcd. for
C13H25NO5H+ 276.1805; found: 276.1804. IR (neat, cmꢀ1) νmax 1061, 1546, 1622, 1652, 1722, 1742,
2854, 2924, 2952, 3290.
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N-(3-Hydroxynonanoyl)-
L-serine 12a. This compound was synthesized by hydrolyzing methyl Nꢀ(3ꢀ
hydroxynonanoyl)ꢀ ꢀserinate 11a (1 equiv, 100 mg, 0.36 mmol) according to general procedure C to
L
yield 86 mg (0.33 mmol, 91% yield) of compound 12a. Diastereomers (ratio 1:1) could not be
separated. Colorless powder. Melting point: 112ꢀ114 °C. Yield: 91%. 1H NMR (400 MHz, CD3OD): δ
= 0.81 (3H, t, J = 6.8 Hz), 1.10ꢀ1.45 (10H, m), 2.25ꢀ2.35 (2H, m), 3.73 (1H, dd, J = 11.2 Hz), 3.81
(1H, ddd, J = 11.2 Hz, 4.8 Hz, 1.9 Hz), 3.83ꢀ3.92 (1H, m), 4.41 (1H, dd, J = 7.8 Hz, 4.0 Hz) ppm. 13C
NMR (100 MHz, CD3OD): δ = 13.0, 22.3, 25.21, 25.22, 29.0, 31.6, 36.8, 43.1, 43.2, 54.7, 61.48,
61.55, 68.3, 68.4, 172.0, 172.96, 173.02 ppm. MS (ESI): m/z (%): 262 (M+H+, 100), 284 (M+Na+, 40).
HRMS Calcd. for C12H23NO5H+ 262.1649; found: 262.1653. IR (neat, cmꢀ1) νmax 1055, 1204, 1414,
1531, 1614, 1657, 2851, 2920, 3341.
Methyl
N-(3-hydroxynonanoyl)-L-prolinate 11b. This compound was synthesized by reacting Lꢀ
proline methyl ester hydrochloride 10b (1 equiv, 1.66 g, 10 mmol) with βꢀhydroxynonanoic acid 9a (1
equiv, 1.74 g, 10 mmol), following general procedure A. After purification via flash chromatography
(ethyl acetate:petroleum ether 1:1), 1.67 g (6.6 mmol, 66% yield) of compound 11b was obtained.
Diastereomers (ratio 1:1) could not be separated via flash chromatography. Both diastereomers existed
as a mixture of two rotamers in a 5:1 ratio. Colorless oil. Yield: 66%. Rf = 0.12 (ethyl
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acetate:petroleum ether 1:1). H NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.5 Hz), 1.21ꢀ1.62
(10H, m), 1.88ꢀ2.26 (4H, m), 2.26ꢀ2.55 (2H, m), 3.45ꢀ3.75 (2H, m), 3.74, 3.75 (2.5H, 2 x s), 3.766,
3.772 (0.5H, 2 x s), 3.95ꢀ4.10 (1H, m), 4.37 (0.1H, dd, J = 8.6 Hz, 2.5 Hz), 4.41 (0.1H, dd, J = 8.4 Hz,
2.6 Hz), 4.49 (0.4H, dd, J = 8.6 Hz, 3.7 Hz), 4.53 (0.4H, dd, J = 8.4 Hz, 3.5 Hz) ppm. 13C NMR (100
MHz, CDCl3): δ = 14.0, 22.5, 24.5, 24.6, 25.4, 25.5, 29.10, 29.14, 29.2, 29.6, 31.7, 36.4, 36.5, 40.2,
40.6, 40.69, 40.7, 46.1, 46.2, 47.0, 47.1, 52.18, 52.20, 52.5, 52.6, 58.4, 58.5, 59.2, 59.4, 67.7, 68.0,
68.1, 68.3, 171.7, 171.8, 171.9, 172.0, 172.2, 172.3, 172.6 ppm. MS (ESI): m/z (%): 286 (M+H+, 100).
HRMS Calcd. for C15H27NO4H+ 286.2013; found: 286.2012. IR (neat, cmꢀ1) νmax 1038, 1173, 1196,
1300, 1373, 1393, 1435, 1626, 1744, 2859, 2928, 2953, 3438.
N-(3-Hydroxynonanoyl)-
L-proline 12b. This compound was synthesized by hydrolyzing methyl Nꢀ
(3ꢀhydroxynonanoyl)ꢀ ꢀprolinate 11b (1 equiv, 1.43 g, 5 mmol) according to general procedure C to
L
give 1.21 g (4.45 mmol) of compound 12b in 89% yield. Spectral data were obtained from a mixture
of two diastereomers in a 1:1 ratio. Both diastereomers existed as a mixture of two rotamers in a 5:1
ratio. Colorless powder. Melting point: 51ꢀ53 °C. Yield: 89%. 1H NMR (400 MHz, CDCl3): δ = 0.88
(3H, t, J = 6.4 Hz), 1.23ꢀ1.61 (10H, m), 1.88ꢀ2.38 (4H, m), 2.38ꢀ2.56 (2H, m), 3.45ꢀ3.76 (2H, m),
4.03ꢀ4.11 (1H, m), 4.37 (0.1H, dd, J = 6.9 Hz, 4.0 Hz), 4.46 (0.1H, dd, J = 7.5 Hz, 3.3 Hz), 4.50ꢀ4.58
(0.8H, m), 7.50 (1H, br s) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.1, 21.0, 24.5, 24.6, 25.49, 25.51,
28.7, 28.8, 29.2, 31.8, 36.1, 36.4, 36.5, 40.6, 40.7, 40.9, 41.1, 46.4, 47.5, 47.6, 59.02, 58.98, 59.4,
59.5, 68.0, 68.5, 172.2, 172.4, 172.7, 172.8, 174.3 ppm. MS (ESI): m/z (%): 272 (M+H+, 100). HRMS
Calcd. for C14H25NO4H+ 272.1856; found: 272.1865. IR (neat, cmꢀ1) νmax 1045, 1173, 1198, 1240,
1373, 1392, 1628, 1703, 1740, 2889, 2930, 2972, 2982, 3448, 3651.
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synthesized by ring closing acid 12b (1 equiv, 0.54 g, 2 mmol) according to general procedure D to
give 0.43 g of cyclic compound 4b in 84% yield after flash chromatography (ethyl acetate:petroleum
ether 1:1). Diastereomers (ratio 1:1) could not be separated via flash chromatography. Colorless
powder. Melting point: 87ꢀ89 °C. Yield: 84%. Rf = 0.21 (ethyl acetate:petroleum ether 1:1). 1H NMR
(400 MHz, CDCl3): δ = 0.89 (3H, t, J = 6.5 Hz), 1.24ꢀ1.55 (8H, m), 1.56ꢀ1.80 (2H, m), 1.80ꢀ2.02 (2H,
m), 2.15ꢀ2.24 (1H, m), 2.55ꢀ2.64 (1H, m), 2.76 (1H, dd, J = 18.5 Hz, 11.5 Hz), 2.86 (1H, dd, J = 18.5
Hz, 2.4 Hz), 3.58ꢀ3.69 (2H, m), 4.67 (1H, dd, J = 7.0 Hz, 7.0 Hz), 4.72ꢀ4.82 (1H, m) ppm. 13C NMR
(100 MHz, CDCl3): δ = 14.0, 22.5, 25.2, 28.9, 29.6, 31.6, 35.0, 42.4, 48.3, 55.7, 73.5, 166.9, 169.6
ppm. MS (ESI): m/z (%): 254 (M+H+, 100). HRMS Calcd. for C14H23NO3H+ 254.1751; found:
254.1763. IR (neat, cmꢀ1) νmax 1223, 1383, 1438, 1616, 1748, 2859, 2902.
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Methyl
N-(3-hydroxydodecanoyl)sarcosinate 11c. This compound was synthesized by reacting
sarcosine methyl ester hydrochloride 10c (1 equiv, 1.40 g, 10 mmol) with βꢀhydroxydodecanoic acid
9b (1 equiv, 1.74 g, 10 mmol), following general procedure A. After flash chromatography (ethyl
acetate:petroleum ether 3:1), 1.87 g (6.2 mmol, 62% yield) of compound 11c was obtained. Spectral
data were obtained from a mixture of two rotamers in a 4:1 ratio. Colorless powder. Melting point: 52ꢀ
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54 °C. Yield: 62%. Rf = 0.29 (ethyl acetate:petroleum ether 3:1). H NMR (400 MHz, CDCl3): δ
rotamer 1 (major): 0.88 (3H, t, J = 6.8 Hz), 1.19ꢀ1.61 (16H, m), 2.40 (1H, dd, J = 16.5 Hz, 9.4 Hz),
2.55 (1H, dd, J = 16.5 Hz, 2.4 Hz), 3.07 (3H, s), 3.75 (3H, s), 3.93 (1H, br d, J = 2.8 Hz), 3.98ꢀ4.07
(1H, m), 4.09 (1H, d, J = 17.3 Hz), 4.19 (1H, d, J = 17.4 Hz) ppm; rotamer 2 (minor): 0.88 (3H, t, J =
6.8 Hz), 1.19ꢀ1.61 (16H, m), 2.25 (1H, dd, J = 16.2 Hz, 9.2 Hz), 2.35ꢀ2.41 (1H, m), 2.99 (3H, s), 3.79
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(3H, s), 3.98ꢀ4.07 (2H, m), 3.99 (1H, d, J = 18.2 Hz), 4.10 (1H, d, J = 18.2 Hz) ppm. C NMR (100
MHz, CDCl3): δ rotamer 1 (major): 14.1, 22.6, 29.3, 29.5, 29.6, 31.9, 36.4, 36.5, 39.5, 49.1, 52.2, 68.7,
169.6, 173.6 ppm; rotamer 2 (minor): 14.1, 22.6, 29.3, 29.5, 29.6, 31.9, 34.7, 36.4, 39.2, 51.3, 52.5,
68.7, 169.1, 173.3 ppm. MS (ESI): m/z (%): 302 (M+H+, 100). HRMS Calcd. for C16H31NO4H+
302.2326; found: 302.2337. IR (neat, cmꢀ1) νmax 1210, 1418, 1471, 1489, 1634, 1746, 2852, 2922,
2954, 3478.
N-(3-Hydroxydodecanoyl)sarcosine 12c. This compound was synthesized by hydrolyzing 1.56 g of
methyl ester 11c (1 equiv, 5.2 mmol) according to general procedure C to yield 1.33 g (4.6 mmol, 89%
yield) of compound 12c. Spectral data were obtained from a mixture of two rotamers in a 3:1 ratio.
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Colorless powder. Melting point: 71ꢀ73 °C. Yield: 83%. H NMR (400 MHz, CDCl3): δ rotamer 1
(major): 0.89 (3H, t, J = 6.8 Hz), 1.19ꢀ1.65 (16H, m), 2.49 (1H, dd, J = 16.3 Hz, 9.2 Hz), 2.57 (1H, dd,
J = 16.2 Hz, 2.7 Hz), 3.10 (3H, s), 3.97ꢀ4.21 (3H, m), 7.85 (1H, br s) ppm; rotamer 2 (minor): 0.89
(3H, t, J = 6.8 Hz), 1.19ꢀ1.65 (16H, m), 2.40ꢀ2.50 (2H, m), 3.01 (3H, s), 3.97ꢀ4.21 (3H, m), 7.85 (1H,
br s) ppm. 13C NMR (100 MHz, CDCl3): δ rotamer 1 (major): 14.1, 22.7, 25.6, 25.7, 29.3, 29.5, 29.6,
31.9, 35.9, 36.3, 36.8, 39.6, 49.5, 68.2, 172.2, 174.1 ppm; rotamer 2 (minor): 14.1, 22.7, 25.6, 25.7,
29.3, 29.5, 29.6, 31.9, 35.9, 36.3, 35.0, 39.2, 51.3, 68.7, 171.3, 173.6 ppm. MS (ESI): m/z (%): 288
(M+H+, 100). HRMS Calcd. for C15H29NO4H+ 288.2169; found: 288.2172. IR (neat, cmꢀ1) νmax 1258,
142, 1418, 1497, 1638, 1726, 2849, 2918, 3353.
4-Methyl-7-nonyl-1,4-oxazepane-2,5-dione 4c. This compound was synthesized by ring closing acid
12c (1 equiv, 0.14 g, 0.50 mmol) according to general procedure D. After purification via flash
chromatography (ethyl acetate:petroleum ether 2:1), 84 mg (0.31 mmol, 62% yield) of cyclic
compound 4c was obtained. Colorless powder. Melting point: 81ꢀ83 °C. Yield: 62%. Rf = 0.23 (ethyl
acetate:petroleum ether 2:1). 1H NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.8 H), 1.20ꢀ1.80 (16H,
m), 2.85 (1H, dd, J = 17.5 Hz, 9.5 Hz), 2.91 (1H, dd, J = 17.5 Hz, 3.7 Hz), 3.08 (3H, s), 3.96 (1H, d, J
= 15.8 Hz), 4.53 (1H, d, J = 15.8 Hz), 4.63ꢀ4.71 (1H, m) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.3,
22.8, 25.2, 29.35, 29.42, 29.57, 29.62, 32.0, 35.7, 37.2, 42.3, 52.7, 76.1, 167.8, 168.9 ppm. MS (ESI):
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m/z (%): 270 (M+H+, 100). HRMS Calcd. for C15H27NO3H+ 270.2064; found: 270.2058. IR (neat, cmꢀ
1) νmax 1213, 1339, 1493, 1624, 1736, 2857; 2924.
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Methyl
N-(3-hydroxydodecanoyl)glycinate 11d. This compound was synthesized by reacting
glycine methyl ester hydrochloride 10d (1 equiv, 1.89 g, 15 mmol) with βꢀhydroxydodecanoic acid 9b
(1 equiv, 3.24 g, 15 mmol), following general procedure A. After purification via flash
chromatography (ethyl acetate:petroleum ether 3:1), 2.83 g (9.9 mmol, 66% yield) of compound 11d
was obtained. Colorless powder. Melting point: 77ꢀ79 °C. Yield: 66%. Rf = 0.27 (ethyl
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acetate:petroleum ether 3:1). H NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.9 Hz), 1.19ꢀ1.59
(16H, m), 2.32 (1H, dd, J = 15.1 Hz, 9.1 Hz), 2.44 (1H, dd, J = 15.1 Hz, 2.7 Hz), 3.34 (1H, br d, J =
3.6 Hz), 3.77 (3H, s), 3.97ꢀ4.05 (1H, m), 4.06ꢀ4.14 (2H, m), 6.34 (1H, br s) ppm. 13C NMR (100 MHz,
CDCl3): δ = 14.1, 22.7, 25.4, 25.5, 29.3, 29.55, 29.59, 31.9, 36.9, 41.1, 42.7, 52.5, 68.7, 170.6, 172.7
ppm. MS (ESI): m/z (%): 288 (M+H+, 100). HRMS Calcd. for C15H29NO4H+ 288.2169; found:
288.2168. IR (neat, cmꢀ1) νmax = 1221, 1379, 1392, 1434, 1442, 1550, 1643, 1742, 2851, 2919, 2956,
3312.
N-(3-Hydroxydodecanoyl)glycine 12d. This compound was synthesized by hydrolyzing methyl ester
11d (1 equiv, 1.75 g, 6.1 mmol) according to general procedure C to yield 1.35 g (4.9 mmol, 81%
yield) of compound 12d. Colorless powder. Melting point: 92ꢀ94 °C. Yield: 94%. 1H NMR (400 MHz,
CD3OD): δ = 0.80 (3H, t, J = 6.9 Hz), 1.14ꢀ1.43 (16H, m), 2.25 (1H, dd, J = 14.3 Hz, 7.6 Hz), 2.30
(1H, dd, J = 14.3 Hz, 5.3 Hz), 3.78 (1H, d, J = 17.8 Hz), 3.85 (1H, d, J = 17.8 Hz), 3.84ꢀ3.90 (1H, m)
ppm. 13C NMR (100 MHz, CD3OD): δ = 13.1, 22.3, 25.3, 29.1, 29.3, 31.7, 36.7, 40.4, 43.2, 68.3,
171.7, 173.3 ppm. MS (ESI): m/z (%): 274 (M+H+, 100), 296 (M+Na+, 28). HRMS Calcd. for
C14H27NO4H+ 274.2013; found: 274.2009. IR (neat, cmꢀ1) νmax 1246, 1262, 1421, 1447, 1556, 1640,
1708, 2849, 2922, 3262, 3326.
Methyl
N-benzyl-N-(3-hydroxynonanoyl)glycinate 15a. This compound was synthesized by
reacting the methyl ester of Nꢀbenzyl glycine 14a (1 equiv, 0.59 g, 3.0 mmol) with βꢀhydroxynonanoic
acid 9a, (1 equiv, 0.52 g, 3.0 mmol) following general procedure B to yield 0.55 g (1.6 mmol, 55%
yield) of ester 15a. Spectral data were obtained from a mixture of two rotamers in a 7:3 ratio. Yellow
oil. Yield: 55%. 1H NMR (400 MHz, CDCl3): δ rotamer 1 (major): 0.87 (3H, t, J = 6.6 Hz), 1.26ꢀ1.61
(10H, m), 2.49 (1H, dd, J = 16.2 Hz, 9.4 Hz), 2.62 (1H, dd, J = 16.2 Hz, 2.4 Hz), 3.73 (3H, s), 3.85ꢀ
4.15 (4H, m), 4.55ꢀ4.77 (2H, m), 7.16ꢀ7.41 (5H, m) ppm; rotamer 2 (minor): 0.87 (3H, t, J = 6.6 Hz),
1.26ꢀ1.61 (10H, m), 2.32 (1H, dd, J = 16.3 Hz, 9.3 Hz), 2.45 (1H, dd, J = 16.3 Hz, 2.3 Hz), 3.72 (3H,
s), 3.85ꢀ4.15 (4H, m), 4.55ꢀ4.77 (2H, m), 7.16ꢀ7.41 (5H, m) ppm. 13C NMR (100 MHz, CDCl3): δ
rotamer 1 (major): 14.1, 22.59, 25.5, 29.3, 31.8, 36.39, 39.4, 46.9, 52.1, 52.2, 68.3, 126.7, 128.4,
129.1, 135.6, 169.7, 173.9 ppm; rotamer 2 (minor): 14.1, 22.61, 25.5, 29.2, 31.8, 36.43, 39.5, 48.2,
49.5, 52.5, 68.2, 127.8, 128.0, 128.7, 136.2, 169.3, 173.4 ppm. MS (ESI): m/z (%): 336 (M+H+, 100).
HRMS Calcd. for C19H29NO4H+ 336.2169; found: 336.2176. IR (neat, cmꢀ1) νmax 1001, 1175, 1200,
1368, 1406, 1435, 1452, 1634, 1709, 1748, 2857, 2928, 2953, 3451.
N-Benzyl-N-(3-hydroxynonanoyl)glycine 16a. This compound was synthesized by hydrolyzing
methyl ester 15a (1 equiv, 0.50 g, 1.5 mmol) according to general procedure C to give 0.41 g (1.3
mmol) of compound 16a in 86% yield. Spectral data were obtained from a mixture of two rotamers in
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a 1.9:1 ratio. Colorless powder. Melting point 91ꢀ93 °C. Yield: 86%. H NMR (400 MHz, CDCl3): δ
rotamer 1 (major): 0.86ꢀ0.91 (3H, m), 1.19ꢀ1.68 (10H, m), 2.45ꢀ2.65 (2H, m), 3.90ꢀ4.21 (3H, m), 4.58
(1H, d, J = 16.8 Hz), 4.69 (1H, d, J = 16.7 Hz), 7.10ꢀ7.41 (5H, m), 7.60 (1H, br s) ppm; rotamer 2
(minor): 0.86ꢀ0.91 (3H, m), 1.19ꢀ1.68 (10H, m), 2.45ꢀ2.65 (2H, m), 3.90ꢀ4.21 (3H, m), 4.45ꢀ4.55 (1H,
m), 4.78ꢀ4.86 (1H, m), 7.10ꢀ7.41 (5H, m), 7.60 (1H, br s) ppm. 13C NMR (100 MHz, CDCl3): δ
rotamer 1 (major): 14.1, 22.61, 25.5, 29.2, 31.8, 36.3, 39.5, 47.1, 52.3, 68.5, 126.8, 128.4, 129.1,
135.2, 172.4, 174.4 ppm; rotamer 2 (minor): 14.1, 22.64, 25.6, 29.2, 31.8, 35.9, 39.5, 48.0, 49.6, 68.7,
127.8, 128.1, 128.8, 136.2, 171.5, 173.6 ppm. MS (ESI): m/z (%): 322 (M+H+, 100). HRMS Calcd. for
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C18H27NO4H+ 322.2013; found: 322.2009. IR (neat, cmꢀ1) νmax 1188, 1213, 1420, 1476, 1626, 1730,
2855, 2927, 3310.
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4-Benzyl-7-hexyl-1,4-oxazepane-2,5-dione 17a. This compound was synthesized by ring closing acid
16a (1 equiv, 0.28 g, 0.86 mmol) according to general procedure D. After purification via flash
chromatography (ethyl acetate:petroleum ether 4:1), 0.20 g (0.65 mmol, 75% yield) of cyclic
compound 17a was obtained. Colorless powder. Melting point 53ꢀ55 °C. Yield: 75%. Rf = 0.33 (ethyl
acetate:petroleum ether 4:1). 1H NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.8 Hz), 1.21ꢀ1.55 (8H,
m), 1.58ꢀ1.81 (2H, m), 2.90ꢀ3.02 (2H, m), 3.94 (1H, d, J = 15.9 Hz), 4.31 (1H, d, J = 15.9 Hz), 4.49
(1H, d, J = 14.7 Hz), 4.67 (1H, tt, J = 12.6 Hz, 4.6 Hz), 4.89 (1H, d, J = 14.7 Hz), 7.23ꢀ7.38 (5H, m)
ppm. 13C NMR (100 MHz, CDCl3): δ = 14.0, 22.5, 25.0, 28.9, 31.6, 35.6, 42.2, 50.2, 52.1, 76.0, 128.1,
128.2, 129.0, 135.9, 167.5, 168.8 ppm. MS (ESI): m/z (%): 304 (M+H+, 100). HRMS Calcd. for
C18H25NO3H+ 304.1907; found: 304.1904. IR (neat, cmꢀ1) νmax 1134, 1161, 1233, 1296, 1344, 1423,
1439, 1454, 1638, 1749, 2849, 2920, 2951.
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Methyl
N-(3-hydroxynonanoyl)-N-(4-methoxybenzyl)glycine 15b. This compound was synthesized
by reacting the methyl ester of NꢀPMB glycine 14b (1 equiv, 5.23 g, 25 mmol) with βꢀ
hydroxynonanoic acid 9a (1 equiv, 4.36 g, 25 mmol), following general procedure B to yield 7.1 g
(19.5 mmol, 78% yield) of ester 15a. Spectral data were obtained from a mixture of two rotamers in a
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2:1 ratio. Yellow oil. Yield: 78%. H NMR (400 MHz, CDCl3): δ rotamer 1 (major): 0.85ꢀ0.90 (3H,
m), 1.21ꢀ1.63 (10H, m), 2.50 (1H, dd, J = 16.1 Hz, 9.4 Hz), 2.63 (1H, dd, J = 16.2 Hz, 2.3 Hz), 3.72
(3H, s), 3.83 (3H, s), 3.84ꢀ4.03 (2H, m), 4.01ꢀ4.11 (1H, m), 4.49ꢀ4.69 (2H, m), 6.89 (2H, d, J = 8.6
Hz), 7.11 (2H, d, J = 8.6 Hz) ppm; rotamer 2 (minor): 0.85ꢀ0.90 (3H, m), 1.21ꢀ1.63 (10H, m), 2.30
(1H, dd, J = 16.3 Hz, 9.1 Hz), 2.43 (1H, dd, J = 16.3 Hz, 2.2 Hz), 3.71 (3H, s), 3.79 (3H, s), 3.84ꢀ4.03
(2H, m), 4.01ꢀ4.11 (1H, m), 4.49ꢀ4.69 (2H, m), 6.85 (2H, d, J = 8.6 Hz), 7.15 (2H, d, J = 8.6 Hz) ppm.
13C NMR (100 MHz, CDCl3): δ rotamer 1 (major): 14.1, 22.6, 25.5, 29.2, 29.3, 31.8, 36.4, 39.4, 46.6,
51.6, 52.2, 55.33, 68.3, 114.4, 128.2, 129.9, 159.4, 169.7, 173.8 ppm; rotamer 2 (minor): 14.1, 22.6,
25.5, 29.2, 29.3, 31.8, 36.4, 39.5, 47.9, 48.9, 52.5, 55.29, 68.1, 114.1, 127.3, 128.2, 128.6, 159.3,
169.3, 173.4 ppm. MS (ESI): m/z (%): 366 (M+H+, 100). HRMS Calcd. for C20H31NO5H+ 366.2275;
found: 366.2268. IR (neat, cmꢀ1) νmax 1032, 1173, 1200, 1246, 1422, 1437, 1512, 1612, 1632, 1748,
2857, 2928, 2953, 3478.
N-(3-Hydroxynonanoyl)-N-(4-methoxybenzyl)glycine 16b. Methyl ester 15b (1 equiv, 7.0 g, 19.2
mmol) was hydrolyzed following general procedure C to yield 6.4 g (18.2 mmol) of carboxylic acid
16b in 95% yield. Spectral data were obtained from a mixture of two rotamers in a 2.3:1 ratio.
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Colorless powder. Melting point: 70ꢀ72 °C. Yield: 95%. H NMR (400 MHz, CDCl3): δ rotamer 1
(major): 0.87ꢀ0.93 (3H, m), 1.24ꢀ1.71 (10H, m), 2.58 (1H, dd, J = 16.0 Hz, 9.0 Hz), 2.65 (1H, dd, J =
16.0 Hz, 2.5 Hz), 3.83 (3H, s), 4.03 (1H, d, J = 17.4 Hz), 4.11 (1H, d, J = 17.3 Hz), 4.01ꢀ4.21 (1H, m),
4.54 (1H, d, J = 16.3 Hz), 4.63 (1H, d, J = 16.3 Hz), 6.92 (2H, d, J = 8.5 Hz), 7.13 (2H, d, J = 8.5 Hz)
ppm; rotamer 2 (minor): 0.87ꢀ0.93 (3H, m), 1.24ꢀ1.71 (10H, m), 2.45ꢀ2.54 (2H, m), 3.82 (3H, s), 3.89
(1H, d, J = 18.7 Hz), 3.95 (1H, d, J = 18.7 Hz), 4.01ꢀ4.21 (1H, m), 4.54 (1H, d, J = 16.3 Hz), 4.63
(1H, d, J = 16.3 Hz), 4.83 (1H, d, J = 14.8 Hz), 6.87 (2H, d, J = 8.5 Hz), 7.18 (2H, d, J = 8.5 Hz) ppm.
13C NMR (100 MHz, CDCl3): δ rotamer 1 (major): 14.1, 22.6, 25.5, 29.23, 31.8, 36.4, 39.6, 46.9, 51.8,
55.35, 68.4, 114.5, 127.0, 128.3, 159.5, 172.4, 174.3 ppm; rotamer 2 (minor): 14.1, 22.6, 25.6, 29.19,
31.8, 35.8, 39.5, 47.6, 48.9, 55.29, 68.7, 114.2, 128.2, 129.9, 159.3, 171.7, 173.4 ppm. MS (ESI): m/z
(%): 352 (M+H+, 100). HRMS Calcd. for C19H29NO5H+ 352.2118; found: 352.2110. IR (neat, cmꢀ1)
νmax 1036, 1175, 1225, 1246, 1398, 1483, 1512, 1620, 1724, 2857, 2920, 3380.
7-Hexyl-4-(4-methoxybenzyl)-1,4-oxazepane-2,5-dione 17b. This compound was synthesized by
ring closing acid 16b (1 equiv, 6.17 g, 17.5 mmol) according to general procedure D. After
purification via flash chromatography (ethyl acetate:petroleum ether 1:1), 2.33 g (7 mmol, 40% yield)
of compound 17b was obtained. Colorless powder. Melting point: 54ꢀ56 °C. Yield: 40%. Rf = 0.28
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(ethyl acetate:petroleum ether 1:1). H NMR (400 MHz, CDCl3): δ = 0.88 (3H, t, J = 6.7 Hz), 1.21ꢀ
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1.55 (8H, m), 1.56ꢀ1.80 (2H, m), 2.88ꢀ3.00 (2H, m), 3.80 (3H, s), 3.94 (1H, d, J = 15.9 Hz), 4.28 (1H,
d, J = 15.9 Hz), 4.43 (1H, d, J = 14.6 Hz), 4.64 (1H, tt, J = 12.7 Hz, 4.5 Hz), 4.81 (1H, d, J = 14.6 Hz),
6.87 (2H, d, J = 8.6 Hz), 7.19 (2H, d, J = 8.6 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.0, 22.5,
25.0, 28.8, 31.6, 35.6, 42.1, 50.0, 51.4, 55.3, 76.0, 114.3, 127.9, 129.6, 159.4, 167.6, 168.7 ppm. MS
(ESI): m/z (%): 334 (M+H+, 100). HRMS Calcd. for C19H27NO4H+ 334.2013; found: 334.2001. IR
(neat, cmꢀ1) νmax 1032, 1175, 1182, 1204, 1223, 1244, 1306, 1325, 1352, 1512, 1628, 1730, 2855,
2926, 2954.
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Removal of the N-PMB group of 17b. NꢀPMBꢀprotected 1,4ꢀoxazepaneꢀ2,5ꢀdione 17b (1 equiv, 2.3
g, 6.9 mmol) was dissolved in 250 mL of a 4:1 ethyl acetateꢀwaterꢀmixture and cooled to 0 °C. Cerium
ammonium nitrate (CAN, 5 equiv, 18.9 g, 34.5 mmol) was added and after 2 h, 150 mL of a saturated
solution of aqueous NaHCO3 was added. After a slow phase separation, the aqueous phase was
extracted twice with 150 mL of ethyl acetate. The combined organic phases were washed once with
brine. Drying with MgSO4, filtration and removal of the solvent in vacuo yielded the crude sevenꢀ
membered ring, which was purified immediately via column chromatography (ethyl acetate:petroleum
ether 1:1), to yield 200 mg of compound 4e as a colorless powder (14% yield).
7-Hexyl-1,4-oxazepane-2,5-dione 4e. Colorless powder. Melting point: 62ꢀ64 °C. Yield: 14%. Rf =
1
0.15 (ethyl acetate:petroleum ether 1:1). H NMR (400 MHz, CDCl3): δ = 0.89 (3H, t, J = 6.5 Hz),
1.22ꢀ1.61 (8H, m), 1.61ꢀ1.83 (2H, m), 2.83 (2H, d, J = 6.8 Hz), 3.85 (1H, dd, J = 15.3 Hz, 7.4 Hz),
4.40 (1H, d, J = 15.3 Hz), 4.72 (1H, quint., J = 6.5 Hz), 6.18 (1H, d, J = 7.6 Hz) ppm. 13C NMR (100
MHz, CDCl3): δ = 14.0, 22.5, 25.1, 28.8, 31.6, 35.1, 41.7, 44.5, 74.7, 167.9, 170.5 ppm. MS (ESI): m/z
(%): 214 (M+H+, 100). HRMS Calcd. for C11H19NO3H+ 214.1438; found: 214.1438. IR (neat, cmꢀ1)
νmax 1090, 1113, 1348, 1422, 1447, 1489, 1551, 1638, 1707, 1738, 2849, 2924, 2953, 3256.
Synthesis of 2-tbutyl-oxazolidine-protected oxazepane-2,5-dione 21b. 2ꢀtBuꢀOxazolidine 18b (1.05
equiv, 0.75 g, 4.0 mmol) was Nꢀacylated according to general procedure B. The Nꢀacylated 2ꢀtBuꢀ
oxazolidine 19b was immediately subjected to a hydrolysis and cyclization reaction according to
general procedures C and D. The crude oxazepaneꢀ2,5ꢀdione 21b, present as a 1:1ꢀmixture of
diastereomers, was purified via column chromatography (Reveleris X2 automated flash
chromatography instrument: gradient increase over 5 column volumes (CV) from 100% hexane to
90% hexane 10% ethyl acetate, hold 5 CV, gradient increase over 10 CV to 50% hexane ꢀ 50% ethyl
acetate, hold 2 CV, then 2 CV 100% ethyl acetate) followed by recrystallization in diethyl
ether:hexane to successfully separate both diastereomers and to give 0.28 g (0.91 mmol, 24% yield) of
(RRS)-21b, 0.33 g (1.1 mmol, 28% yield) of (RSS)-21b and 0.13 g (0.42 mmol, 11% yield) of a
mixture of (RRS)/(RSS)ꢀ21b.
(3R,7R,9aS)-3-(tButyl)-7-hexyltetrahydro-3H,5H,9H-oxazolo[4,3-c][1,4]oxazepine-5,9-dione
(RRS)-21b (trans-diastereomer). Colorless powder. Melting point: 94ꢀ96 °C. Yield: 24%. Rf = 0.29
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(ethyl acetate:petroleum ether 3:1). H NMR (400 MHz, CDCl3): δ = 0.89 (3H, t, J = 6.8 Hz), 0.95
(9H, s), 1.19ꢀ1.55 (8H, m), 1.63ꢀ1.84 (2H, m), 2.72 (1H, d, J = 16.6 Hz), 3.14 (1H, dd, J = 16.5 Hz,
11.2 Hz), 4.44 (1H, dd, J = 9.1 Hz, 9.1 Hz), 4.52 (1H, dd, J = 9.3 Hz, 8.3 Hz), 4.68ꢀ4.79 (2H, m), 5.36
(1H, s) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.0, 22.5, 24.5, 25.9, 28.9, 31.6, 37.2, 38.7, 42.6,
59.4, 69.2, 79.7, 96.2, 166.3, 171.7 ppm. MS (ESI): m/z (%): 312 (M+H+, 100). HRMS Calcd. for
ꢀ
C17H28NO4 310.2024; found: 310.2016. IR (neat, cmꢀ1) νmax 1119, 1179, 1215, 1231, 1362, 1369,
1381, 1680, 1703, 1711, 2870, 2930, 2970, 2980.
(3R,7S,9aS)-3-(tButyl)-7-hexyltetrahydro-3H,5H,9H-oxazolo[4,3-c][1,4]oxazepine-5,9-dione
(RSS)-21b (cis-diastereomer). Colorless needleꢀlike crystals. Melting point: 96ꢀ98 °C. Yield: 28%. Rf
= 0.28 (ethyl acetate:petroleum ether 3:1). 1H NMR (400 MHz, CDCl3): δ = 0.89 (3H, t, J = 6.8 Hz),
0.95 (9H, s), 1.21ꢀ1.58 (8H, m), 1.61ꢀ1.89 (2H, m), 2.85 (1H, dd, J = 17.7 Hz, 9.8 Hz), 2.97 (1H, dd, J
= 17.6 Hz, 2.6 Hz), 4.36 (1H, dd, J = 9.2 Hz, 8.0 Hz), 4.62 (1H, dd, J = 9.2 Hz, 9.2 Hz), 4.71ꢀ4.83
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(2H, m), 5.44 (1H, s) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.0, 22.5, 24.5, 26.4, 28.8, 31.5, 35.5,
38.9, 42.5, 55.1, 68.6, 75.2, 97.0, 167.6, 168.5 ppm. MS (ESI): m/z (%): 312 (M+H+, 100). HRMS
ꢀ
Calcd. for C17H28NO4 310.2024; found: 310.2028. IR (neat, cmꢀ1) νmax 1076, 1125, 1175, 1211, 1233,
1354, 1364, 1375, 1396, 1649, 1738, 2889, 2924, 2970, 2982.
Synthesis of 2,2-dimethyloxazolidine protected oxazepane-2,5-dione 21c. To methyl Nꢀ(3ꢀ
hydroxynonanoyl)serinate 11a (1 equiv, 0.55 g, 2 mmol), dissolved in dry toluene (20 mL) under a
nitrogen atmosphere, were sequentially added 2,2ꢀdimethoxypropane (DMP) (5 equiv, 1.2 mL, 10
mmol) and pꢀtoluenesulfonic acid monohydrate (0.1 equiv, 40 mg, 0.2 mmol). The mixture was heated
to reflux with a DeanꢀStark apparatus for 3 h to remove water. After cooling down, water (20 mL) was
added. The aqueous phase was extracted twice with ethyl acetate (2 x 20 mL), the organic phases were
combined and washed with brine (20 mL), dried (MgSO4), filtered and the solvent was removed in
vacuo. The crude oxazolidine 19c was hydrolyzed and cyclized according to the general procedures C
and D to yield compound 21c (0.10 g, 0.36 mmol) in 18% total yield after column chromatography
(Reveleris X2 automated flash chromatography instrument: 5 column volumes (CV) 100% hexane,
gradient increase over 15 CV to 50% hexane ꢀ 50% ethyl acetate, hold 2 CV, then 2 CV 100% ethyl
acetate). Diastereomers (ratio 1:1.3) could not be separated via flash chromatography.
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(9aS)-7-Hexyl-3,3-dimethyltetrahydro-3
H,5
H,9H-oxazolo[4,3-
c][1,4]oxazepine-5,9-dione
21c.
White powder. Melting point: 88ꢀ90 °C. Yield: 18%. Rf = 0.21 (ethyl acetate:petroleum ether 3:1). 1H
NMR (400 MHz, CDCl3): δ diastereomer 1 (major): 0.91 (3H, t, J = 6.8 Hz), 1.25ꢀ1.82 (10H, m), 1.61
(3H, s), 1.64 (3H, s), 2.63 (1H, dd, J = 15.1 Hz, 2.2 Hz), 3.06 (1H, dd, J = 15.1 Hz, 11.7 Hz), 4.23ꢀ
4.33 (1H, m), 4.59ꢀ4.67 (1H, m), 4.70ꢀ4.81 (2H, m) ppm; diastereomer 2 (minor): 0.91 (3H, t, J = 6.8
Hz), 1.25ꢀ1.82 (10H, m), 1.62 (3H, s), 1.69 (3H, s), 2.80 (1H, dd, J = 18.5 Hz, 10.8 Hz), 2.88 (1H, dd,
J = 18.5 Hz, 2.8 Hz), 4.23ꢀ4.33 (1H, m), 4.51 (1H, dd, J = 9.6 Hz, 7.6 Hz), 4.59ꢀ4.67 (1H, m), 4.70ꢀ
4.81 (1H, m) ppm. 13C NMR (100 MHz, CDCl3): δ diastereomer 1 (major): 14.0, 22.5, 23.5, 24.5,
24.9, 25.1, 28.8, 28.9, 31.6, 34.8, 37.5, 43.1, 59.7, 65.8, 79.8, 96.6, 164.8, 167.9 ppm; diastereomer 2
(minor): 14.0, 22.5, 23.8, 24.5, 25.1, 25.3, 28.8, 28.9, 31.6, 34.8, 37.5, 43.4, 54.6, 65.7, 73.8, 98.3,
166.9, 167.1 ppm. MS (ESI): m/z (%): 284 (M+H+, 100). HRMS Calcd. for C15H25NO4H+ 284.1856;
found: 284.1860. IR (neat, cmꢀ1) νmax 1072, 1085, 1155, 1209, 1233, 1252, 1335, 1381, 1418, 1632,
1667, 1703, 1742, 2889, 2916, 2932, 2972, 2982.
Synthesis of 2-phenyloxazolidine protected oxazepane-2,5-dione 21a. To a stirred solution of Lꢀ
serine methyl ester hydrochloride 10a (1 equiv, 3.1 g, 20 mmol) in 60 mL of anhydrous CH2Cl2 and 15
mL Et3N at room temperature was added 4.8 g of anhydrous MgSO4 (2 equiv, 40 mmol), followed by
freshly distilled benzaldehyde 13a (1.1 equiv, 2.24 mL, 22 mmol) under a nitrogen atmosphere. The
reaction mixture was stirred at room temperature overnight, filtered and the filtrate was concentrated
under reduced pressure. The resulting 2ꢀphenyloxazolidine 18a was Nꢀacylated analogously to the
tBuꢀderivative 18b, followed by hydrolysis and cyclization (procedures C and D) to give the
oxazepaneꢀ2,5ꢀdione 21a, as a 1:1ꢀmixture of diastereomers in 53% overall yield (3.5 g, 10.6 mmol).
Diastereomers (ratio 1:1) could not be separated via flash chromatography (ethyl acetate:petroleum
ether 3:1).
(3R,9aS)-7-Hexyl-3-phenyltetrahydro-3H,5H,9H-oxazolo[4,3-
c][1,4]oxazepine-5,9-dione
21a.
Colorless powder. Melting point: 96ꢀ98 °C. Yield: 53% Rf = 0.14 (ethyl acetate:petroleum ether 3:1).
1H NMR (400 MHz, CDCl3): δ diastereomer 1: 0.88ꢀ0.95 (3H, m), 1.28ꢀ1.90 (10H, m), 2.75 (1H, d, J
= 15.8 Hz), 3.17 (1H, dd, J = 15.8 Hz, 11.2 Hz), 4.35ꢀ4.44 (1H, m), 4.47ꢀ4.54 (1H, m), 4.75ꢀ4.83 (1H,
m), 4.85ꢀ4.97 (1H, m), 6.50 (1H, s), 7.27ꢀ7.31 (1H, m), 7.35ꢀ7.42 (4H, m) ppm; diastereomer 2: 0.88ꢀ
0.95 (3H, m), 1.28ꢀ1.90 (10H, m), 2.91 (2H, d, J = 6.8 Hz), 4.35ꢀ4.44 (1H, m), 4.47ꢀ4.54 (1H, m),
4.75ꢀ4.83 (1H, m), 4.85ꢀ4.97 (1H, m), 6.52 (1H, s), 7.27ꢀ7.31 (1H, m), 7.35ꢀ7.42 (4H, m) ppm. 13C
NMR (100 MHz, CDCl3): δ diastereomer 1: 14.0, 22.5, 24.5, 28.8, 28.9, 31.57, 31.58, 35.1, 37.4, 41.9,
59.0, 68.1, 79.9, 89.9, 126.1, 128.7, 129.2, 137.2, 165.4, 169.3 ppm; diastereomer 2: 14.0, 22.5, 24.5,
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28.8, 28.9, 31.57, 31.58, 35.1, 37.4, 42.1, 54.2, 67.1, 74.2, 91.7, 126.3, 126.8, 129.1, 137.0, 166.0,
167.5 ppm. MS (ESI): m/z (%): 332 (M+H+, 100). HRMS Calcd. for C19H25NO4H+ 332.1856; found:
332.1868. IR (neat, cmꢀ1) νmax 1061, 1117, 1184, 1221, 1389, 1425, 1655, 1676, 1703, 1724, 2857,
2926, 2953.
Debenzylation of 21a. 500 mg (1 equiv, 1.5 mmol) of 2ꢀphenyloxazolidine 21a (d.r. 1:1) was
dissolved in 30 mL of ethyl acetate whereafter 250 mg of Pd(OH)2/C (20 wt% loading) was added and
the reaction mixture was stirred under a H2 atmosphere at room temperature for 6 h. Subsequently, the
reaction mixture was filtered through celite and the solvent removed in vacuo. The crude mixture was
purified via column chromatography (ethyl acetate: petroleum ether 1:1 to 100% ethyl acetate) to yield
82 mg (0.32 mmol, 22% yield) of deprotected compound 4a, alongside with 334 mg (1 mmol) of
recovered 2ꢀphenyloxazolidine 21a (d.r. 1:3).
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(3S,7S)-7-Hexyl-3-(hydroxymethyl)-1,4-oxazepane-2,5-dione (SS)-4a Colorless powder. Melting
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point: 81ꢀ83 °C. Yield: 22%. Rf = 0.08 (ethyl acetate:petroleum ether 1:1). H NMR (400 MHz,
CDCl3): δ = 0.89 (3H, t, J = 6.7 Hz), 1.23ꢀ1.57 (8H, m), 1.58ꢀ1.83 (2H, m), 2.60ꢀ2.66 (1H, m), 2.79
(1H, dd, J = 18.7 Hz, 3.0 Hz), 2.87 (1H, dd, J = 18.6 Hz, 11.3 Hz), 3.89 (1H, ddd, J = 12.3 Hz, 8.3 Hz,
4.3 Hz), 4.06 (1H, ddd, J = 12.0 Hz, 5.0 Hz, 5.0 Hz), 4.46ꢀ4.51 (1H, m), 4.73ꢀ4.81 (1H, m), 6.45 (1H,
br s) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.0, 22.5, 25.1, 28.8, 31.5, 34.9, 41.7, 52.8, 61.1, 73.7,
169.5, 171.6 ppm. MS (ESI): m/z (%): 244 (M+H+, 100). HRMS Calcd. for C12H21NO4H+ 244.1543;
found: 244.1543. IR (neat, cmꢀ1) νmax 1013, 1028, 1072, 1084, 1134, 1186, 1231, 1329, 1391, 1429,
1624, 1742, 2853, 2916, 2953, 3098, 3206.
Associated content
Supporting information. This material is available free of charge on the ACS publications website at
DOI: x.
Xꢀray data of compound (RSS)-21b (CIF)
2D NOESY spectrum of 11a.
Zoomꢀin of the1H and 13C NMR spectra of compound 12c and 4c.
Reaction conditions evaluated for the Nꢀdebenzylation of compound 17a.
Reaction conditions evaluated for the Nꢀacylation of 2ꢀtBuꢀoxazolidine 18b.
Spectral data of 1,4ꢀoxazepaneꢀ2,5ꢀdiones reported in literature.
Overlay of the 1H NMR spectra of 2ꢀphenyloxazolidine protected compound 21a and compound 21a
recovered after treatment with 5% TFA and treatment with H2 gas and Pd(OH)2/C.
Spectral data of Serratamolide A (22a).
Crystal data for compound (RSS)ꢀ21b.
1
Copies of H NMR and 13C NMR spectra of compounds 4a-c, 4e, 11a-d, 12a-d, 15a-b, 16a-b, 17a-b
and 21a-c.
Acknowledgements
The authors thank Ghent University (BOF 14/DOC_V/285) for financial support of this research.
KVH thanks the Hercules Foundation (project AUGE/11/029 “3DꢀSPACE: 3D Structural Platform
Aiming for Chemical Excellence”) and the Research Foundation – Flanders (FWO) for funding. Elena
Semina is thanked for the translation of the Cyrillic article.
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