Enantioselective Synthesis and Application of Small and Environmentally Sensitive Fluorescent Amino Acids for Probing Biological Interactions

Article

Enantioselective Synthesis and Application of Small and

Environmentally Sensitive Fluorescent Amino Acids for Probing

Biological Interactions

Michael Noden and Scott D. Taylor*

Cite This: https://doi.org/10.1021/acs.joc.1c00907

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ABSTRACT: Environmentally sensitive ﬂuorescent amino acids (FlAAs) have been used extensively to probe biological

interactions. However, most of these amino acids are large and do not resemble amino acid side chains. Here, we report the

enantioselective synthesis of two small and environmentally sensitive ﬂuorescent amino acids bearing 7-dialkylaminocoumarin side

chains by alkylation of a Ni(II) glycine Schiﬀ base complex. These amino acids exhibit a large increase in ﬂuorescence as

environment polarity decreases. One of these FLAAs was incorporated into a highly active analog of the cyclic lipopeptide antibiotic

paenibacterin by Fmoc solid-phase peptide synthesis via a new and very eﬃcient route. This peptide was used to probe the

interaction of the antibiotic with model liposomes, lipopolysaccharides, and live bacteria.

INTRODUCTION

acids have been developed and used to study peptide−protein

11−16

■

interactions and peptide−membrane interactions.

Fluorescent labels are often incorporated into peptides and

proteins to probe their structure and function by ﬂuorescence

spectroscopy and to visualize molecular interactions and

intracellular processes by ﬂuorescence microscopy. These

labels are attached to reactive side chains or to the N- or C-

terminus, which has the potential to signiﬁcantly impact the

characteristics of the peptide or protein. Some peptides and

proteins do not require labeling as they contain intrinsically

For our studies on cyclic lipopeptide antibiotics, we had a

need for a solvatochromic FlAA that had the following

properties: (1) displays a large increase in ﬂuorescence upon

its transfer from a polar to an apolar environment; (2) is small

and resembles a natural amino acid, such as tryptophan, as

much as possible; and (3) can be readily incorporated into

peptides using standard Fmoc SPPS. There are a number of

−5

FlAAs, such amino acids 1−5 in Figure 1, that meet the ﬁrst

ﬂuorescent amino acids, such as tryptophan

and kynur-

10,13−16

and third criteria.

Some of them, such as 4-DAPA (3),

enine, which can be used to study interactions with model

biological systems or live cells. However, these naturally

ﬂuorescent amino acids often lack the sensitivity required for

biological studies. As an alternative to labeling, ﬂuorescent

amino acids (FlAAs) may be directly incorporated into

peptides or proteins, minimizing added bulk and potentially

reducing the impact of the structural change on the peptide’s

exhibit very large increases in ﬂuorescence upon transfer from

polar to apolar media, and all are readily incorporated into

peptides using standard SPPS. However, these FLAAs are

either conjugates of diaminopropionic acid to various

Received: April 19, 2021

−10

biological properties.

Solvatochromic FlAAs, which exhibit

a change in ﬂuorescence intensity and/or wavelength as the

environment surrounding the amino acid changes from polar

to apolar or vice versa, are particularly useful for probing

interactions between biomolecules. A number of such amino

XXXX American Chemical Society

The Journal of Organic Chemistry

J. Org. Chem. XXXX, XXX, XXX−XXX

Article

Figure 1. Structures of some known solvatochromic FLAAs (1−6) and proposed solvatochromic FLAAs DMACA (7) and DEACA (8).

ﬂuorophores (1−4) or, like DANA (5), have a ketone moiety

in between the α-carbon and the aromatic group (DANA), or

are very large (DMNA, 4), and therefore, they do not resemble

a natural amino acid such as tryptophan. L-(7-Hydroxycoumar-

in-4-yl)ethylglycine (7-HC, 6) is often the FlAA of choice for

incorporation into peptides and proteins due to its small size

commercially available, while coumarin 9, which is not readily

available, was prepared from 3-(dimethylamino)phenol and

ethyl acetoacetate using a method initially reported by von

Pechmann and Schaal (Scheme 1). Coumarins 9 and 10

Scheme 1. Attempted Synthesis of 7 and 8 Using Williams’

Auxiliary

12,17−19

and high ﬂuorescence quantum yield.

However, the

ﬂuorescence intensity of this amino acid decreases as the

polarity of the environment decreases.

The 7-aminocoumarin family of ﬂuorophores is extremely

useful due to its strong solvatochromism, exhibiting increased

20,21

and blue-shifted ﬂuorescence as solvent polarity decreases.

Compared to hydroxycoumarins, 7-aminocoumarins are

relatively unaﬀected by solvent acidity between pH 4 and

0. Many coumarins of this type have found use as laser dyes,

and their photophysical properties have been studied

2−25

extensively.

This led us to design “minimal” solvatochro-

mic FlAAs 7-dimethylaminocoumarin-4-alanine (7, DMACA)

and 7-diethylaminocoumarin-4-alanine (8, DEACA). Here, we

report the enantioselective syntheses of these two amino acids

suitably protected for Fmoc SPPS. We also demonstrate the

potential of these FlAAs for probing biological interactions by

incorporating DMACA into an analog of paenibacterin, a

broad spectrum cyclic lipodepsipeptide antibiotic, and

monitoring its insertion into model liposomes, lipopolysac-

charides, and live bacteria by ﬂuorescence spectroscopy/

microscopy.

were brominated on the methyl group with LiHMDS and NBS

to give bromomethyl coumarins 11 and 12. Williams’

auxiliary was treated with NaHMDS, and the resulting

metalated auxiliary was reacted with 11 and 12 in THF or

THF/HMPA, which gave 14 in good yield (74%) and

compound 13 in low yield (38%), which was probably due

to the limited solubility of 11 in THF/HMPA. Unfortunately,

compounds 13 and 14 exhibited very limited solubility in most

organic solvents and we were unable to achieve the removal of

the auxiliary and isolate the free amino acids.

RESULTS AND DISCUSSION

■

Chiral coumarin amino acids can be accessed by acid-catalyzed

von Pechmann condensation between substituted phenols and

aspartate- or glutamate-derived β-ketoesters. Although this is

a fairly direct route to these types of compounds, the

puriﬁcation of the coumarin amino acid can be challenging,

sometimes requiring preparative HPLC. Therefore, we

elected to examine a chiral auxiliary approach to the synthesis

of 7 and 8. Since Williams’ auxiliary has been used for the

enantioselective synthesis of coumarin amino acids, we

anticipated that this approach could be used for the

We then decided to prepare 7 and 8 via alkylation of a chiral

Ni(II) glycine Schiﬀ base complex 15 (Scheme 2) as this

auxiliary has been used extensively for the preparation of a

wide variety of unnatural amino acids and the absolute

conﬁguration can be inferred from extensive literature

7,28

32,33

preparation of 7 and 8.

Starting coumarin 10 is

precedent.

Alkylation of the auxiliary with 11 and 12

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

Scheme 2. Synthesis of DMACA and DEACA Using Auxiliary 15

Figure 2. Emission spectra of tripeptides 20 (left) and 21 (right) in solvents of varying polarity. λ_e_x= 380 nm.

proceeded smoothly in DMF with dr values of 86:14 and 84:16

for substrates 11 and 12, respectively. Pleasingly, the major

diastereomers, 16 and 17, readily dissolved in organic solvents,

which were isolated by column chromatography and then

decomposed by acidic hydrolysis to give compounds 7 and 8.

The amino acids were insoluble in both organic solvents and

water, and so, they could be separated from the residual nickel

salts and the (S)-2-[N-(N′-benzylprolyl)amino]benzophenone

ligand without the use of ion exchange chromatography. The

enantiomeric excess of 7 and 8 was determined using Marfey’s

reagent and found to be 97 and 90%, respectively. For

reference, the racemic amino acids were prepared using the

coupling reagents, and deprotection with concentrated TFA

and typical scavengers. Interestingly, a sample of 20 exhibited a

loss of 14 amu (likely loss of an N-methyl group) after several

weeks in a MeCN/H O solution on the benchtop. Deal-

kylation of 7-dialkyl-4-methylaminocoumarins by singlet-oxy-

gen has been reported by von Trebra and Koch. Regardless,

no decomposition of 18−21 was observed so long as

reasonable precautions were taken, namely, minimizing

prolonged exposure to light and storing long term at low

temperature, preferably as a dry solid.

Both 20 and 21 exhibited absorption maxima near 380 nm

in water or ethanol (Figure S3 ). Excitation at this wavelength

resulted in ﬂuorescence that was highly solvent-dependent. In

aqueous solution, both coumarins exhibited maximum

ﬂuorescence at approximately 480 nm. Decreasing solvent

polarity resulted in a dramatic increase in ﬂuorescence

intensity as well as a distinct blue-shift, as shown in Figure 2.

The spectroscopic properties of these coumarin-containing

tripeptides in water and ethanol are given in Table 1.

27,28

method reported by Leblanc and co-workers (Scheme S1).

Absolute conﬁguration was inferred from extensive literature

2,33

precedent.

Protection of 16 and 17 with Fmoc-OSu gave

8 and 19, suitable building blocks for solid-phase peptide

synthesis (SPPS).

To investigate their properties, compounds 18 and 19 were

used to incorporate 7 and 8 into simple tripeptides bearing Ala

at the N and C-terminal positions (H-Ala-DMACA-Ala-OH

To investigate the potential of these FLAAs for probing

biological interactions, DMACA was incorporated into an

analog of the broad-spectrum peptide antibiotic paenibacterin-

(

20) and H-Ala-DEACA-Ala-OH (21)). The ﬂuorescent

residues proved to be stable to 20% piperidine in DMF,

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

discovered cyclic lipodepsipeptide antibiotics produced by the

Article

Table 1. Spectroscopic Properties of Tripeptides 20 and 21

vancomycin-resistant enterococci. The antibiotic activity of

P-A1 has been attributed to disruption of the outer membrane

λ_A_b_s_.

max

λ_E_m_.

max

(nm)

Stokes

shift

−

of G bacteria and damage of the cytoplasmic membrane of

(cm ¹)

−

(cm⁻¹M⁻¹

)

Φ_F

both G and G bacteria.

−

peptide solvent

(nm)

We have previously reported an Fmoc solid-phase total

H O

EtOH

385

373

393

381

480

457

483

456

5140

4930

4740

4320

1.5 × 10

1.7 × 10

1.8 × 10

2.0 × 10

0.21

0.70

0.12

0.63

synthesis of P-A1. In this synthesis, a linear peptide was

prepared that started with Pro10 attached to the resin and

extended to lipidated D-Orn1. The ester bond was then formed

between Thr3 and Ile13, and the resulting branched peptide

extended to include Lys12 and Val11. The use of a

pseudoproline dipeptide at residues 4 and 5 was found to

signiﬁcantly increase esteriﬁcation yields. Cleavage of the

protected peptide from the resin followed by cyclization and

global deprotection gave P-A1. However, partial epimerization

of the Ile13 residue occurred during the esteriﬁcation reaction.

Although the resulting epimers were separable by semi-

preparative HPLC, we sought a method to further reduce

epimerization. We also wished to determine if the D-Orn

residues at positions 4 and 1 could be replaced with more

economical D-Lys residues. We expected that shortening of the

linear peptide onto which Ile13 was coupled would further

disrupt aggregation, which would result in the esteriﬁcation

reaction proceeding faster, which would reduce epimeriza-

H O

EtOH

Extinction coeﬃcient at λ_A_b_s_.max. Relative quantum yield using

quinine sulfate in 0.5 M H SO as a standard.

A1 (P-A1, Figure 3). P-A1 is a member of a family of recently

tion. Accordingly, we found that formation of the ester bond

on linear peptide 22, which does not contain an acylated

residue at position 1, using DIC/cat. DMAP in the presence of

Triton X-100, resulted in no detectable epimerization

Scheme 3). This approach requires the use of an alloc

(

group for protection of the Ile13 residue since it is orthogonal

to the Fmoc protecting group and allowed for elongation of

the resulting peptide 23 to the acyl tail (peptide 24) before

Figure 3. Structure of paenibacterin-A1.

deprotection of N -Ile and further couplings. The remaining

soil bacterium Paenibacillus thiaminolyticus. It is active against

both Gram negative (G ) and Gram positive (G ) bacteria

including methicillin-resistant Staphylococcus aureus and

amino acid couplings, resin-cleavage, cyclization, and depro-

tection are essentially identical to the originally reported

procedure with only minor deviations. Using this method, an

−

Scheme 3. Synthesis of Peptides 25 and 26

The Journal of Organic Chemistry

J. Org. Chem. XXXX, XXX, XXX−XXX

Article

Figure 4. Interaction of peptide 26 (3 μM) with (A) DOPC/DOPG liposomes (250 μM); (B) DMPC/DMPG liposomes (250 μM); and (C) E.

coli LPS. Studies were conducted in 20 mM HEPES, 150 mM NaCl, pH 7.4 at 37 °C. λ_e_x= 380 nm, λ_e_m= 400−600 nm.

analog of paenibacterin A1 (P-A1) was prepared containing D-

Lys residues in place of D-Orn at positions 1 and 4 (peptide

pronounced with the myristoyl lipids. It stands to reason

that the presence of anionic PG would result in increased

association of the cationic peptide, and this suggests that PG

lipids may have a role in paenibacterin’s mechanism of action

against G⁺bacteria as this lipid is present in the cell

5) in a very good 40% yield. This analog exhibited improved

activity against both Escherichia coli K12 and Bacillus subtilis

046 compared to native P-A1.

We elected to incorporate DMACA into peptide 25 at

membranes of many G bacteria.

position 6. We anticipated that this position would be

amenable to substitution with aromatic residues based on a

family of peptides recently isolated from Paenibacillus alvei that

bear a similarity to paenibacterin and contain Tyr or Phe

residues at that position. The target peptide 26 was prepared

in 41% yield (Scheme 3) and was only two-fold less active than

peptide 25.

Since studies suggest that paenibacterin interacts with

lipopolysaccharides (LPS, also known as endotoxins) in the

−

35,36

outer membrane of G bacteria,

we also examined the

interaction of peptide 26 with LPS. Based on prior studies of a

similar nature conducted by others on the interaction between

dansyl polymyxin and LPS, we expected to observe a large

40,41

increase in ﬂuorescence upon binding of 26 to LPS.

Since it is known that paenibacterin disrupts the bacterial

cytoplasmic membrane, we hypothesized that insertion of the

peptide into the hydrophobic membrane interior would result

in an observable increase in coumarin ﬂuorescence intensity.

As phosphatidylglycerol (PG) is a common component of G⁺

bacterial membranes, we prepared liposomes consisting of

either oleoyl or myristoyl lipids and varying amounts of

phosphatidylglycerol (PG) or phosphatidyl choline (PC). An

increase in ﬂuorescence of peptide 26 was observed in the

presence of these lipids, as shown in Figure 4. We observed

greater ﬂuorescence intensity with liposomes containing 25 or

Instead, at 10 μg/mL LPS, we observed quenching of

ﬂuorescence (Figure 4). The expected increase in ﬂuorescence

was only observed at concentrations of LPS ≥ 30 μg/mL

(Figure 4), which is above the range of typical critical micelle

concentrations (CMC) reported for E. coli LPS (14−22 μg/

42,43

mL).

The decrease in ﬂuorescence at 10 μg/mL LPS

suggests that in the absence of LPS micelles or aggregates into

which the peptide can insert, we instead see association with

LPS that suppresses ﬂuorescence emission. This can also be

seen if LPS is titrated into a solution of peptide 26 (Figure 5).

This results in a steady reduction in ﬂuorescence until an LPS

concentration of 12 μg/mL, at which point no further decrease

0% phosphatidylglycerol (PG). This eﬀect was more

The Journal of Organic Chemistry

J. Org. Chem. XXXX, XXX, XXX−XXX

DMACA exhibits a large increase in quantum yield upon

Article

Figure 7. Micrographs of bacteria stained with 30 μg/mL peptide 26

in 1× PBS for 30 min. (A) Confocal laser scanning micrograph (405

nm excitation). (B) Diﬀerential interference contrast (DIC) micro-

graph. (C) Merged image. Bacteria were immobilized on 1% agarose

pads. The red circle highlights a bright spot found at the poles of E.

coli cells.

Figure 5. Titration of peptide 26 (3 μM) with LPS in buﬀer (pH 7.2,

mM HEPES). λ_e_x= 380 nm, λ_e_m= 400−600 nm.

occurs at 15 μg/mL LPS. The most probable explanation for

this decrease in ﬂuorescence is concentration-dependent self-

quenching, which is indicative of aggregation of the peptide−

LPS complex. This explanation is supported by the ﬁnding that

the extent of quenching is reduced when nonﬂuorescent

peptide 25 is mixed with an excess of ﬂuorescent peptide 26

before addition of LPS (Figure 6). Within the hybrid

aggregates formed from these mixtures, the ﬂuorescent

peptides are separated from one another, and less quenching

is observed (Figure 6B,C). These results suggest that

paenibacterin binds to free LPS, and the resulting paenibacter-

in−LPS complex readily aggregates.

insertion into the cell. Because of this increase in brightness,

the background ﬂuorescence was negligible, and the stained

bacteria were clearly visible. For B. subtilis, the peptide appears

to be capable of entering the cell. For E. coli, a modest increase

in brightness was observed at the membrane, particularly at the

polar regions of the cell. The cause of this eﬀect has not yet

been determined but may indicate that paenibacterin

accumulates at the cell poles. We anticipate that this

ﬂuorescent analog will facilitate the further study of

paenibacterin’s mechanism of action, allowing us to observe

its eﬀects on bacterial cells over time. We also anticipate that

DMACA or DEACA could be incorporated into other peptide

antibiotics to give ﬂuorescent analogs of suﬃcient brightness to

be easily visualized by ﬂuorescence microscopy.

To demonstrate that peptides containing these ﬂuorescent

−

amino acids can be used to image G and G bacteria by

confocal microscopy, live E. coli or B. subtilis were suspended in

a solution of 26 and then the suspension was added directly to

an agarose pad (Figure 7). The staining procedure was very

convenient, and no rinsing steps were necessary. As expected,

CONCLUSIONS

■

We have developed a facile method for the enantioselective

synthesis of two 7-dialkylamino coumarin amino acids,

DMACA and DEACA, suitable for use in Fmoc-SPPS. These

Figure 6. Eﬀect of LPS on the ﬂuorescence of peptide 26 in the presence and absence of peptide 25 in 5 mM HEPES, pH 7.2. λ = 380 nm, λ_e_m

00−600 nm. (A) Peptide 26 (3 μM) alone. (B) 3:1 mixture of peptide 26 and peptide 25 (3 μM total). (C) 1:1 mixture of peptide 26 and peptide

5 (3 μM total). (D) Peptide 26 (1.5 μM) alone.

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

amino acids function as environmental probes, exhibiting an

increase in ﬂuorescence as environment polarity decreases. We

also report a synthesis of a highly active P-A1 analog using an

improved route employing mainly Fmoc chemistry that

minimizes epimerization. This route was used to prepare

peptide 26, a P-A1 analog containing DMACA. Peptide 26

exhibited increased ﬂuorescence in the presence of liposomes,

and a greater increase if PG was present, suggesting that the

presence of PG in bacterial membranes may promote

membrane binding. Above the CMC of LPS, peptide 26

showed an increase in ﬂuorescence consistent with the peptide

inserting into a hydrophobic micelle or aggregate. In the

presence of sub-CMCs of LPS, peptide 26 showed

concentration-dependent self-quenching, which is indicative

of binding to free LPS followed by aggregation of the peptide−

LPS complex. LPS or endotoxins are powerful activators of

septic shock. Inappropriate antibiotic treatment can induce the

liberation of LPS into the circulation and cause septic

allowed to warm to −30 °C at which point the ﬂask was transferred to

an acetone-dry ice bath and cooled to −78 °C. N-Bromosuccinimide

(

1.47 g, 8.28 mmol, 1.2 equiv) was added as a solution in dry THF

20 mL). After stirring for 30 min at −78 °C the reaction was

quenched with 0.1 N HCl (100 mL). The mixture was extracted

several times with DCM. The organic fractions contained an insoluble

yellow solid. The combined organic extracts were concentrated by

rotary evaporation, then resuspended in EtOAc, and concentrated

again. The resulting orange-yellow solid was suspended in minimal

EtOAc and ﬁltered, rinsing with cold EtOAc, which gave 11 as a

yellow solid (1.50 g, 5.3 mmol, 77% yield). H NMR (CD

, 300

MHz): δ 7.54 (d, 1H, J = 8.9 Hz), 6.72 (dd, 1H, J = 8.9, 2.5 Hz), 6.57

(

d, 1H, J = 2.4 Hz), 6.14 (s, 1H), 4.44 (s, 2H), 3.06 (s, 6H); C{ H}

NMR (CD Cl , 75 MHz): δ 161.1, 156.3, 152.9, 150.3, 125.2, 110.0,

09.1, 106.9, 98.5, 40.1, 27.3; HRMS (+ESI) m/z: [M + H] calcd for

C H BrNO 282.0124; found 282.0149.

-(Bromomethyl)-7-(diethylamino)-2H-chromen-2-one (12).

Compound 12 was prepared in 59−66% yield from 4 according to

the procedure reported by Li et al. The NMR spectra of the product

matched what was reported in the literature. H NMR (CDCl , 300

4−46

shock.

Huang and Yousef have suggested that paenibac-

MHz): δ 7.47 (d, 1H, J = 9 Hz), 6.60 (dd, 1H, J = 9, 2 Hz), 6.49 (d,

H, J = 2 Hz), 6.12 (s, 1H), 4.37 (s, 2H), 3.40 (q, 4H, J = 7 Hz), 1.20

terin may neutralize released LPS and thus may be capable of

reducing sepsis during antibiotic treatment. Our results lend

support to this hypothesis as they indicate that paenibacterin is

capable of binding to free LPS. Peptide 26 allowed for direct

visualization of the bacteria by ﬂuorescence microscopy. We

anticipate that this ﬂuorescent analog will facilitate further

mechanism of action studies, allowing us to observe the eﬀects

of paenibacterin on bacterial cells over time.

50.9, 150.3, 125.3, 109.2, 108.6, 106.1, 97.8, 44.8, 27.1, 12.4; HRMS

(+ESI) m/z: [M + H] calcd for C H BrNO 310.0437; found

14 17 2

10.0466.

Benzyl (3S,5S,6R)-3-((7-(Dimethylamino)-2-oxo-2H-chromen-4-

yl)methyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (13). Bro-

mocoumarin 11 (423 mg, 1.50 mmol, 1.06 equiv) and (5S,6R)-4-

benzyloxycarbonyl-5,6-diphenylmorpholin-2-one (552 mg, 1.42

mmol, 1 equiv) were dispersed in dry THF (27 mL) and HMPA

(

3 mL) before cooling to −78 °C, and then 1 M NaHMDS in THF

EXPERIMENTAL SECTION

■

(1.5 mL, 1.5 mmol, 1.06 equiv) was added dropwise. After 2 h, the

solution was warmed to 0 °C and stirred for a further 30 min before

warming to room temperature. The reaction mixture was diluted in

DCM (200 mL) and washed with water (3× 50 mL) and brine before

condensing. The residue was dissolved in a minimal volume of DCM

by heating and then precipitated by the addition of heptane. The

resulting suspension was decanted to separate the product from a red

residue. The yellow solid was then collected by centrifugation, giving

General. Commercially available reagents were used without

further puriﬁcation. The Gly/Ni(II)/BPB auxiliary (15) was prepared

7−49

as described in the literature.

Alloc-Ile was prepared following

literature procedures. DMF, 4-methylpiperidine (4-MP), 2-methyl-

piperidine (2-MP), and TFA were purchased from commercial

suppliers and used without further puriﬁcation. Tetrahydrofuran

(

THF) was dried over sodium metal and benzophenone. Dichloro-

methane (DCM) was dried over calcium hydride. High-resolution

mass spectra were acquired using an Orbitrap instrument in positive

electrospray ionization mode (+ESI). Minimum inhibitory concen-

trations were determined by the broth dilution method as previously

13 (316 mg, 0.54 mmol, 38% yield). H NMR ((CD ) SO, 300

MHz): rotameric mixture δ 8.03 (d, 1H, J = 9 Hz), 7.50−6.98 (m,

12H), 6.84−6.44 (m, 6H), 6.18−6.05 (m, 1H), 5.42−5.31 (m, 1H),

5.21−4.86 (m, 3H), 3.65 (dt , 1H, J = 12, 5 Hz), 3.42 (t, 1H, J = 12

app

7,51

described.

ﬂask was immersed in a hot oil bath.

-(Dimethylamino)-4-methyl-2H-chromen-2-one (9). Compound

was prepared according to the procedure reported by von

For all reactions that required heating, the reaction

Hz), 2.99 (m, 6H); C{ H} NMR ((CD ) SO, 75 MHz): rotameric

3 2

mixture δ 166.9, 161.1, 161.0, 156.0, 155.8, 154.4, 153.4, 153.1, 151.6,

151.4, 136.7, 136.4, 136.2, 136.0, 135.2, 135.1, 129.3, 129.1, 128.9,

128.7, 128.5, 128.4, 128.3, 128.0, 127.9, 127.7, 127.6, 126.9, 126.8,

126.0, 125.5, 110.6, 110.4, 109.9, 109.4, 108.3, 108.0, 98.2, 78.4, 68.2,

67.2, 60.5, 60.4, 57.0, 40.2, 35.3; ESI-MS m/z: [M + H] calcd for

C H N O 589.2333; found 589. 2310.

Pechmann and Schaal. To a solution of zinc chloride (2.50 g,

8.3 mmol, 0.5 equiv) in ethanol (10 mL) were added ethyl

acetoacetate (5.35 mL, 42.3 mmol, 1.16 equiv) and 3-

dimethylamino)phenol (5.00 g, 36.4 mmol, 1.0 equiv). The solution

(

Benzyl (3S,5S,6R)-3-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)-

was heated to reﬂux under nitrogen for 20 h. After cooling to room

temperature, the mixture was poured into 75 mL of 0.5% HCl (aq.)

and slowly stirred for 2 h. The resulting green solid was ﬁltered and

rinsed with water. The solid was dissolved in minimal concentrated

HCl (aq.) and then precipitated with 200 mL of water. The solid was

collected by suction ﬁltration and then recrystallized from 90% EtOH

to give compound 9 as purple-red crystals (4.52 g, 22.1 mmol, 61%

yield). The NMR spectra of the product closely matched what had

methyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (14). Bromo-

coumarin 12 (310 mg, 1 mmol, 1 equiv) and (5S,6R)-4-

benzyloxycarbonyl-5,6-diphenylmorpholin-2-one (387 mg, 1 mmol,

1 equiv) were dispersed in dry THF (20 mL) before cooling to −78

°C, and then 1 M NaHMDS in THF (1.1 mL, 1.1 mmol, 1.1 equiv)

was added dropwise. After 2 h, the solution was warmed to room

temperature. The reaction mixture was diluted in DCM (100 mL),

washed with water (3× 20 mL) and brine, dried over Na SO , and

52,53

previously been reported.

H NMR (CDCl , 300 MHz): δ 7.38

condensed. The product (R = 0.26, 10:0.5:0.1 DCM/EtOAc/

(

d, 1 H, J = 9 Hz), 6.60 (dd, 1H, J = 9 Hz, 2 Hz), 6.50 (s, 1H), 5.95

s, 1H), 3.03 (s, 6H), 2.33 (s, 3H); HRMS (+ESI) m/z: [M + H]

triethylamine) was isolated by ﬂash chromatography, giving 14 (455

mg, 0.74 mmol, 74% yield). H NMR (CDCl , 300 MHz): rotameric

calcd for C H NO 204.1019; found 204.1037.

mixture δ 7.78 (d, 1H, J = 9 Hz), 7.46−6.39 (m, 17H), 6.00−5.62 (m,

-(Bromomethyl)-7-(dimethylamino)-2H-chromen-2-one (11).

2H), 5.50−4.90 (m, 5H), 3.78−3.48 (m, 6H), 1.19 (m, 6H); C{ H}

Compound 11 was prepared following a literature procedure for the

NMR ((CD ) SO, 75 MHz): rotameric mixture δ 161.1, 161.0, 156.5,

bromination of coumarin 10. Compound 9 (1.4 g, 6.9 mmol, 1

equiv) was dispersed in dry THF (70 mL) and then cooled to −41 °C

in an acetonitrile-dry ice bath. LiHMDS was added as a 1 M solution

in THF (17.3 mL, 17.3 mmol, 2.5 equiv), and then the bath was

156.3, 154.4, 153.4, 151.5, 151.3, 150.9, 150.6, 136.7, 136.4, 136.2,

136.0, 135.2, 135.1, 129.5, 129.1, 128.9, 128.7, 128.5, 128.4, 128.0,

127.7, 127.6, 127.1, 126.9, 126.8, 126.6, 126.3, 125.8, 110.1, 109.9,

109.4, 108.8, 107.8, 107.4, 97.5, 79.3, 78.4, 68.3, 67.2, 66.9, 60.4, 57.1,

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

6.0, 44.5, 36.2, 35.4, 12.8; ESI-MS m/z: [M + H]⁺calcd for

C H N O 617.2646; found 617.2644.

0.304, 1 N HCl). Enantiomeric purity was determined by

derivatization with Marfey’s reagent.

(

S)-BPB/Ni(II)/(S)-2-Amino-3-(7-(dimethylamino)-2-oxo-2H-chro-

men-4-yl)propanoic Acid Complex (16). Bromocoumarin 11 (2.82 g,

0.0 mmol, 1 equiv) and Gly/Ni(II)/BPB auxiliary, 15 (4.98 g, 10.0

(S)-2-Amino-3-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)-

propanoic Acid (DEACA, 8). A solution of compound 17 (1.5 g, 2.06

mmol) in 2:1 MeOH/1 M HCl (50 mL) was heated to 50−60 °C for

2 h. After cooling to room temperature, the mixture was neutralized

with ammonium hydroxide. The solution was partially concentrated in

vacuo and ﬁltered, and the solid was rinsed several times with water,

mmol, 1 equiv), were dissolved in dry DMF (20 mL), and the

solution was degassed by bubbling Ar through the solution. Ground

NaOH (1 g, 25 mmol, 2.5 equiv) was added, and the solution was

stirred at room temperature for 30 min. The solution was then poured

into 0.5 M AcOH (aq., 100 mL), which was then extracted with DCM

3×). The organic extracts were concentrated by rotary evaporation,

then re-dissolved in EtOAc (200 mL), and washed with 6× 70 mL of

dried, and then rinsed with EtOAc, which gave compound 8 as a

yellow solid, (478 mg, 76% yield, 90% ee). H NMR (D O with 5%

DCl, 500 MHz): δ 7.44 (d, 1H, J = 8.2 Hz), 7.08 (s, 1H), 6.99 (d, 1H,

J = 8.1 Hz), 6.04 (s, 1H), 3.88 (br t, 1H, J = 6 Hz), 3.18−2.99 (m,

(

H), 2.81 (dd, 1H, J = 14.4 Hz, 7.7 Hz), 0.48 (s, 6H); C{ H} NMR

H O and once with brine. The major diastereomer (R = 0.27, 2:1

(

D O with 5% DCl, 75 MHz): δ 169.7, 161.6, 153.4, 149.5, 139.3,

DCM/acetone) was isolated by ﬂash chromatography (4:1 to 7:3

DCM/acetone) to give 16 as a red solid (3.84 g, 5.49 mmol, 55%

yield). H NMR (CDCl , 500 MHz): δ 8.22 (d, 1H, J = 8.7 Hz), 8.03

26.9, 119.7, 118.7, 117.8, 111.8, 53.8, 51.1, 31.4, 9.5; HRMS (+ESI)

m/z: [M + H] calcd for C H N O 305.1496; found 305.1508.

16 21 2 4

[α] = 12.1 (c 0.105, 1 N HCl). Enantiomeric purity was determined

(

d, 2H, J = 7.7 Hz), 7.53−7.43 (m, 2H), 7.40−7.27 (m, 4H), 7.19−

by derivatization with Marfey’s reagent.

.12 (m, 2H), 6.90 (d, 1H, J = 8.7 Hz), 6.79 (d, 1H, J = 7.6 Hz),

.68−6.59 (m, 2H), 6.46 (d, 1H, J = 2.3 Hz), 6.29 (dd, 1H, J = 8.8,

.5 Hz), 5.87 (s, 1H), 4.40−4.31 (m, 2H), 3.52 (d, 1H, J = 12.8 Hz),

.45−3.31 (m, 5H), 3.00 (s, 6H), 2.68−2.59 (m, 1H), 2.57−2.44 (m,

Fmoc-DMACA (18). Compound 7 (50 mg, 0.18 mmol, 1 equiv)

was dispersed in 10% Na CO (aq., 3 mL) and dioxane (2 mL) and

then cooled to 0 °C. Fmoc-OSu (73 mg, 0.22 mmol, 1.2 equiv) was

added dropwise as a solution in dioxane (3 mL). The solution was

allowed to warm to rt, then stirred for 16 h, then acidiﬁed with 1 M

HCl, and extracted with EtOAc (3× 10 mL). The combined organic

extracts were washed with brine, dried over Na SO , ﬁltered, and

H), 2.20−1.99 (m, 3H); C{ H} NMR (CDCl , 75 MHz): δ 180.2,

77.4, 171.3, 161.3, 156.0, 152.9, 150.3, 142.9, 133.6, 133.3, 133.2,

32.7, 131.5, 129.9, 129.0, 128.9, 128.2, 127.6, 125.9, 125.2, 123.6,

20.6, 111.0, 108.8, 108.7, 98.2, 70.4, 69.9, 63.2, 57.3, 40.1, 38.1, 30.8,

concentrated in vacuo. Puriﬁcation by ﬂash chromatography (toluene/

EtOAc/AcOH, 8:2:0.5) gave 18 as a green solid (67 mg, 72% yield).

4.0; HRMS (+ESI) m/z: [M + H] calcd for C H N NiO

99.2112; found 699.2097. The minor diastereomer (626 mg, R =

.38, 2:1 DCM:acetone) was isolated as a mixture with an

unidentiﬁed impurity and was not further puriﬁed. The diastereomeric

H NMR ((CD ) SO, 300 MHz): δ 7.88−7.77 (m, 3H), 7.70−7.52

(

m, 3H), 7.41−7.20 (m, 4H), 6.71 (dd, 1H, J = 9.0, 2.0 Hz), 6.52 (d,

H, J = 2.0 Hz), 5.99 (s, 1H), 4.33−4.04 (m, 4H), 3.30−3.10 (m,

ratio was thus estimated to be >86:14.

13 1

H), 2.96 (s, 6H); C{ H} NMR ((CD ) SO, 75 MHz): δ 173.1,

(

S)-BPB/Ni(II)/(S)-2-Amino-3-(7-(diethylamino)-2-oxo-2H-chro-

61.0, 156.4, 156.0, 153.3, 153.2, 144.2, 144.1, 141.1, 128.1, 27.5,

25.7, 125.6, 120.5, 109.7, 109.3, 108.1, 98.2, 66.2, 53.5, 47.0, 40.1,

men-4-yl)propanoic Acid (17). The Gly/Ni(II)/BPB auxiliary 15

(

2.14 g, 5.00 mmol, 1 equiv) was alkylated with bromocoumarin 12

1.55 g, 5.00 mmol) and NaOH (0.500 g, 12.5 mmol, 2.5 equiv) using

the same procedure described above for compound 16. The major

diastereomer (R = 0.39, 2:1 DCM/acetone) was isolated by ﬂash

chromatography (3:1 DCM/acetone) to give 17 as a red solid (1.94 g,

.66 mmol, 53% yield). H NMR (CDCl , 300 MHz): δ 8.22 (d, 1H,

3.1; HRMS (+ESI) m/z: [M + H] calcd for C H N O 499.1863;

found 499.1845. Alternatively, following acidiﬁcation, the precipitate

could be ﬁltered and then rinsed with water and ether. The resulting

product is suﬃciently pure for use in Fmoc-SPPS.

Fmoc-DEACA (19). Compound 19 was prepared from 8 (20 mg,

.066 mmol, 1 equiv) using the same procedure described above for

J = 8.8 Hz), 8.02 (d, 2H, J = 7.4 Hz), 7.52−7.28 (m, 6H), 7.19−7.09

the preparation of compound 18. Puriﬁcation by ﬂash chromatog-

(

m, 2H), 6.89 (d, 1H, J = 9.1 Hz), 6.76 (d, 2H, J = 7.5 Hz), 6.69−

raphy (toluene/EtOAc/AcOH, 8:2:0.5) gave 19 as a yellow solid (23

.57 (m, 2H), 6.43 (d, 1H, J = 2.5 Hz), 6.25 (dd, 1H, J = 9.2, 2.6 Hz),

mg, 67% yield). H NMR (CDCl , 300 MHz): δ 7.70 (d, 2H, J = 7.3

.84 (s, 1H), 4.42−4.31 (m, 2H), 3.52 (d, 1H, J = 12.8 Hz), 3.48−

Hz), 7.59 (d, 1H, J = 8.5 Hz), 7.49 (dd, 2H, J = 16.8, 7.4 Hz), 7.39−

.24 (m, 9H), 2.72−2.42 (m, 2H), 2.20−1.96 (m, 2H), 1.16 (t, 6H, J

.17 (m, 4H), 6.52 (d, 1H, J = 8.6), 6.45 (s, 1H), 6.07 (s, 1H), 5.86

7.1); ¹³C{ H} NMR (CDCl , 75 MHz): δ 180.1, 177.5, 171.3,

(d, 1H, J = 6.9 Hz), 4.72 (m, 1H), 4.37 (t, 1H, J = 8.0 Hz), 4.27 (t,

61.4, 156.4, 150.6, 150.3, 142.9, 133.6, 133.3, 133.2, 132.7, 131.5,

29.8, 129.0, 128.9, 128.2, 127.6, 125.9, 125.5, 123.6, 120.6, 110.4,

08.5, 108.1, 97.6, 70.4, 69.9, 63.1, 57.2, 44.7, 38.2, 30.8, 23.9, 12.4;

HRMS (+ESI) m/z: [M + H] calcd for C H N NiO 727.2425;

H, J = 7.8 Hz), 4.14 (t, 1H, J = 6.7 Hz), 3.40−3.12 (m, 6H), 1.10 (t,

H, J = 6.3 Hz); C{ H} NMR (CDCl ,75 MHz): δ 173.2, 163.5,

56.2, 156.1, 152.6, 150.9, 143.6, 141.2, 127.7, 127.1, 125.6, 125.2,

125.1, 119.9, 109.3, 108.7, 108.3, 97.7, 67.4, 53.6, 47.0, 44.7, 34.3,

12.4; HRMS (+ESI) m/z: [M + H] calcd for C H N O 527.2177;

found 727.2419. The minor diastereomer (370 mg, R = 0.5, 2:1

DCM/acetone) was also isolated as a mixture with an unidentiﬁed

impurity and was not further puriﬁed. The diastereomeric ratio was

thus estimated to be >84:16.

found 527.2170. As with compound 18, following acidiﬁcation, the

precipitate could be ﬁltered and then rinsed with water and ether,

giving a product suﬃciently pure for use in Fmoc-SPPS.

(

S)-2-Amino-3-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)-

propanoic Acid (DMACA, 7). A solution of compound 16 (3.74 g,

.35 mmol) in MeOH (64 mL) and 1 M HCl (aq., 32 mL) was

Ala-DMACA-Ala (20) and Ala-DEACA-Ala (21). 2′-Cl-TrtCl resin

(

0.25 mmol, 1 equiv, 167 mg, 1.5 mmol/g) was swelled in dry DCM

and then ﬁltered. Fmoc-Ala-OH (234 mg, 0.75 mmol, 3 equiv) and

DIPEA (349 μL, 2 mmol, 8 equiv) in dry DCM (2 mL) were added

to the resin and agitated for 16 h. After ﬁltration, the resin was capped

by rinsing three times with DCM/MeOH/DIPEA (17:2:1) followed

by rinsing with DCM and DMF. The N-terminus was deprotected

with 4-methylpiperidine (20% in DMF, 5 min and then 20 min)

followed by ﬁltration and washing with DMF. 18 (187 mg, 0. 375

mmol, 1.5 equiv) or 19 (198 mg, 0.375 mmol, 1.5 equiv) was coupled

using HOAt (51 mg, 0.375 mmol, 1.5 equiv) and DIC (59 μL, 0.375

mmol, 1.5 equiv) in 3 mL of DMF for 16 h. The resin was ﬁltered and

washed with DMF, and the N-terminus was deprotected with 4-

methylpiperidine, then ﬁltered, and washed. Fmoc-Ala-OH (311 mg,

1 mmol, 4 equiv) was coupled using HOAt (136 mg, 1 mmol, 4

equiv) and DIC (155 μL, 1 mmol, 4 equiv) in 3 mL of DMF for 3 h.

The resin was rinsed with DMF and the N-terminus deprotected with

heated to 55−60 °C for 2 h. The solution was allowed to cool to

room temperature and then neutralized with ammonium hydroxide.

The solution was partially concentrated in vacuo, and then the

aqueous suspension was washed 3× with 75 mL of EtOAc to remove

the BPB ligand. The aqueous fraction was lyophilized to give a green

solid that was triturated with water several times, collecting by

centrifugation, to give compound 7 as a yellow-green solid (1.41 g,

5% yield, 97% ee). H NMR (D O with 5% DCl, 500 MHz): δ 7.59

(

d, 1H, J = 8.8 Hz), 7.31 (s, 1H), 7.24 (d, 1H, J = 8.3 Hz), 6.21 (s,

H), 4.05 (t, 1H, J = 6.7 Hz), 3.23 (dd, 1H, J = 15.2, 6.6 Hz), 3.00

(

dd, 1H, J = 14.9, 8.2 Hz), 2.90 (s, 3H); C{ H} NMR (D O with

% DCl, 75 MHz): δ 169.7, 161.7, 153.2, 149.5, 144.2, 126.9, 119.4,

17.8, 117.0, 110.1, 51.1, 46.2, 31.4; HRMS (+ESI) m/z: [M + H]

calcd for C H N O 277.1183; found 277.1189. [α] = 10.3 (c

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

-methylpiperidine. The tripeptides were cleaved from the resin with

and collected by centrifugation. The peptide was deprotected with

mL of TFA/TIPS/H O (95:2.5:2.5) for 90 min. The cleavage

TFA/TIPS/H O (90:5:5) for 90 min followed by concentration

solution was ﬁltered, the ﬁltrate was concentrated under an N stream,

under an N stream and precipitation with cold MTBE. The crude

and then the peptide was precipitated with cold ether. The crude

peptide was collected by centrifugation and puriﬁed by semi-

peptide was puriﬁed by semi-preparative RP-HPLC (C8, H O/

MeCN + 0.1% TFA, linear gradient: 70/30 to 50/50 over 60 min,

ﬂow rate = 10 mL/min, λ = 220/280 nm). Collected fractions were

pooled and lyophilized to give the TFA salt of peptide 25 as a white

powder (17.8 mg, 40% based on estimated resin loading). Purity of

preparative RP-HPLC (C18, H O/MeCN + 0.1% TFA, linear

gradient: 90/10 to 60/40 over 40 min, ﬂow rate = 10 mL/min, λ =

20/280 nm).

Ala-DMACA-Ala (20) was obtained as a yellow powder (18.9 mg,

8% yield). The purity of the peptide was veriﬁed by analytical RP-

the peptide was veriﬁed by analytical RP-HPLC (C18, MeCN/H O +

0.1% TFA, linear gradient: 10/90 to 90/10 over 40 min, ﬂow rate = 1

HPLC(C18, MeCN/H O + 0.1% TFA, linear gradient: 10/90 to 90/

mL/min, λ = 220 nm), t = 23.6 min (Figure S35); HRMS (+ESI) m/

0 over 40 min, ﬂow rate = 1 mL/min, λ = 220 nm), t = 11.1 min

z: [M + 2H] calcd for C H N O 817.0732; found 817.0749.

81 151 17 17

Paenibacterin A1-(D-Lys1, D-Lys4, DMACA6) (26). Peptide 26 was

prepared using the same method described above for peptide 25.

Fmoc-DMACA-OH (18) (2.5 equiv) was installed at position 6 using

COMU (2.4 equiv), oxyma (2.4 equiv), and NMM (8 equiv) in DMF

(2 mL) for 2 h. Lyophilization of RP-HPLC fractions gave the TFA

salt of peptide 26 as a yellow powder (19.5 mg, 41% based on

estimated resin loading). Purity of the peptide was veriﬁed by

19.1925; found 419.1929.

Ala-DEACA-Ala (21) was obtained as a yellow powder (35.5 mg,

2% yield). The purity of the peptide was veriﬁed by analytical RP-

HPLC (C18, MeCN/H O + 0.1% TFA, linear gradient: 10/90 to 90/

0 over 40 min, ﬂow rate = 1 mL/min, λ = 220 nm), t = 12.2 min

22 31 4 6

47.2238; found 447.2242.

Paenibacterin A1-(D-Lys1, D-Lys4) (25). 2′-Cl-TrtCl TentaGel

analytical RP-HPLC (C18, MeCN/H O + 0.1% TFA, linear gradient:

10/90 to 90/10 over 40 min, ﬂow rate = 1 mL/min, λ = 220 nm), t =

24.2 min (Figure S37); HRMS (+ESI) m/z: [M + 2H]⁺calcd for

resin (theoretical substitution = 0.23 mmol/g, 217 mg, 50 μmol, 1

equiv) was loaded using Fmoc-Pro (4 equiv) and disopropylethyl-

amine (DIPEA, 8 equiv) in dry DCM (2 mL) for 16 h followed by

ﬁltration/rinsing with DCM/MeOH/DIPEA (17:2:1, 3× 3 min)

followed by DCM, DMF, and DCM again (2 mL, 3× 3 min each).

The resin loading was estimated to be 42% using the method of Gude

C H N O 896.5892; found 896.5913.

156 18 19

Fmoc-(D)-Lys(Boc)-Ser(Ψ^M^e^,^M^ePro)-OH. This dipeptide was pre-

pared from Fmoc-(D)-Lys(Boc)-OH following a procedure previously

reported for the synthesis of the analogous (D)-Orn-Ser dipeptide.

The (D,L)-dipeptide was obtained as a white solid (2.07 g, 54%).

NMR spectra closely resemble that of the (D,D) or (L,L)-dipeptides

et al. The Fmoc group was removed using 20% 4-methylpiperidine

4-MP) in DMF (2× 2 mL, 5 min and then 15 min). The resin was

rinsed between each coupling/deprotection step with DMF (2 mL,

55,56

(

reported in the literature.

H NMR ((CD ) SO, 300 MHz): δ

3 2

7.90−7.75 (m, 3H), 7.67 (d, 2H, J = 7.3 Hz), 7.38 (t, 2H, J = 7.5 Hz),

7.28 (t, 2H, J = 7.5 Hz), 6.69 (t br, 1H, J = 5.1 Hz), 4.90 (d, 1H, J =

5.7 Hz), 4.36−3.98 (m, 5H), 3.86 (q, 1H, J = 6.9 Hz), 2.95−2.75 (m,

× 1 min). Fmoc-Ile-OH (4 equiv) was installed using 1-[(1-(cyano-

-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino-

methylene)]methanaminium hexaﬂuoro-phosphate (COMU, 3.9

equiv), ethyl (hydroxyimino)cyanoacetate (oxyma, 3.9 equiv), and

N-methylmorpholine (NMM, 8 equiv) in DMF (2 mL) for 4 h. The

Fmoc group was removed with 20% 2-methylpiperidine (2-MP) in

DMF containing 0.1% formic acid (2× 2 mL, 5 min and then 7 min)

followed by rinsing and coupling of Fmoc-D-Ser(OtBu)-OH and

2H), 1.64−0.98 (m, 21H); C{ H} NMR ((CD ) SO, 75 MHz): δ

172.8, 169.7, 156.8, 156.0, 144.2, 144.2, 141.2, 128.1, 127.5, 125.7,

120.5, 95.6, 77.8, 67.0, 66.1, 59.2, 53.8, 47.1, 30.8, 29.8, 28.7, 25.3,

23.5, 23.2; HRMS (+ESI) m/z: [M + H] Calcd for C H N O

596.2966; found 596.3013.

Synthesis of Racemic DMACA and DEACA and Derivatiza-

7,28

Fmoc-D-Lys(Boc)-OH with COMU/oxyma/NMM for 2 h until

completed by a ninhydrin test. Fmoc-D-Lys-Ser(Ψ

tion with Marfey’s Reagent.

To prepare (±)-DMACA,

Me,Me

Pro)-OH (2.5

bromocoumarin 11 (500 mg, 1.77 mmol, 1.1 equiv), diethyl

acetamidomalonate (349 mg, 1.61 mmol, 1 equiv), and sodium

ethoxide (225 mg, 3.3 mmol, 2.05 equiv) were dissolved in EtOH (20

mL) and heated to reﬂux for 12 h. The solution was then cooled and

concentrated. When attempting to isolate the desired product by ﬂash

equiv) was coupled with COMU (2.4 equiv), oxyma (2.4 equiv), and

NMM (5 equiv) for 2 h. Fmoc-Thr(OH) (4 equiv) and then Fmoc-

Val (4 equiv) were each coupled with DIC (4 equiv) and HOAt (4

equiv) for 3 h. The Fmoc group was removed after each coupling

using 4-MP as described above. Prior to esteriﬁcation, the resin was

rinsed with DCM and dried in vacuo over KOH. Alloc-Ile (10 equiv)

was activated with DIC (10 equiv) in dry DCM (2 mL) at 0 °C and

allowed to warm to room temperature over 30 min. The solution was

added to the dry resin, and then 1% triton X-100 and DMAP (0.1

equiv) were added before mixing for 20 h. Fmoc-D-Lys(Boc)-OH and

pentadecanoic acid were coupled with COMU/oxyma/NMM, and

the Fmoc group was removed using 20% 2-MP in DMF (5 min and

then 20 min). The resin was treated with Pd(PPh ) (0.2 equiv) and

chromatography (SiO , 1−3% DCM in MeOH), it was found to elute

along with residual diethyl acetamidomalonate. Fractions containing

mostly the product were pooled and concentrated before dissolving in

6 M HCl (aq.) and heating to reﬂux for 16 h. After cooling, the

solution was neutralized with ammonium hydroxide and then

lyophilized. The resulting solid was dispersed in water, ﬁltered, and

rinsed with water and ether to give racemic DMACA (37 mg). The

proton NMR spectra matched that of the pure enantiomer. H NMR

(D O with 5% DCl, 300 MHz): δ 7.57 (d, 1H, J = 8.7 Hz), 7.29 (d,

dimedone (10 equiv) as an allyl scavenger, mixing for 90 min by

1H, J = 2.4 Hz), 7.21 (dd, 1H, J = 8.6, 2.3 Hz), 6.19 (s, 1H), 4.03 (t,

1H, J = 7.0 Hz), 3.20 (dd, 1H, J = 15.1, 6.6 Hz), 2.97 (dd, 1H, J =

15.1, 8.1 Hz), 2.88 (s, 6H) (Figure S21); HRMS (+ESI) m/z: [M +

bubbling N . The resin was then rinsed with 0.5% DIPEA in DMF

(

3× 2 mL) followed by 0.5% sodium diethyldithiocarbamate

trihydrate in DMF (3× 2 mL). Fmoc-Lys and Fmoc-Val were

coupled with COMU/oxyma/NMM, and 20% 2-MP in DMF

containing 0.1% formic acid (2× 2 mL, 5 min and then 7 min) was

used for Fmoc deprotection. After the ﬁnal Fmoc deprotection, the

resin was rinsed with DCM, iPrOH, and DCM again before drying in

vacuo over KOH. The peptide was cleaved from the resin with 30%

HFIP in DCM (2 mL, 3× 30 min), and the resin was rinsed with

DCM, iPrOH, and DCM. Residual peptide was isolated from the

resin by further rinsing with DMF. The cleavage solution was

concentrated by rotary evaporation, then the peptide was dissolved in

DMF (50 mL), and DIPEA (8 equiv), HOAt (4 equiv), and PyAOP

H] Calcd for C H N O 277.1183; found 277.1189.

(±)-DEACA was prepared from bromocoumarin 12 (200 mg, 0.64

mmol) in the manner described above. The product (R = 0.12, 1:1

EtOAc/hexane) was isolated by ﬂash chromatography (SiO , 3:7 and

then 1:1 EtOAc/hexane) though apparent instability that resulted in

partial decomposition. It was then dissolved in 6 M HCl and reﬂuxed

for 12 h before cooling, neutralizing with ammonium hydroxide and

lyophilizing. The resulting solid was dispersed in water and rinsed

with both water and ether to give a yellow powder (79 mg) consisting

of racemic DEACA contaminated with 7-diethylamino-4-

(hydroxymethyl)coumarin, conﬁrmed by H NMR (Figure S22 )

(

4 equiv) were added. After stirring for 72 h at room temperature, the

and ESI-MS; HRMS (+ESI) m/z: [M + H] Calcd for C H N O

solution was concentrated by rotary evaporation and the protected

305.1496; found 305.1509.; HRMS (+ESI) m/z: [M + H] + Calcd for

peptide was suspended in MeOH, then precipitated with cold H O,

C H N O 305.1496; found 248.1266.

16 21

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

https://pubs.acs.org/doi/10.1021/acs.joc.1c00907.

Article

For determination of enantiomeric excess, coumarin amino acids

2.5 μmol) were treated with Marfey’s reagent (3.5 μmol) in acetone

aliquots. Emission spectra were collected using a PTI Quantamaster

ﬂuorimeter, with an excitation at 380 nm and an emission from 400 to

600 nm and a step size of 3 nm. This was performed in duplicate, and

representative results from a single trial are shown in Figure 5. In

Figure 6, the peak area is the average of two repeated measurements

at each concentration, with error bars depicting the deviation.

Staining of Bacteria with Peptide 26. LB media were

inoculated with either B. subtilis 1046 or E. coli K12 from an

overnight culture and then incubated at 37 °C. When the OD600

reached ca. 0.1, bacteria were collected by centrifugation and then

rinsed three times with phosphate buﬀered saline (1× PBS). The

pellet was then resuspended in a 30 μg/mL solution of peptide 20 in

1× PBS (50−100 μL) for 30 min at room temperature. Then, 2 μL of

this suspension was added to a 1% agarose pad on a microscope slide.

Once the droplet had been absorbed, a coverslip was added. The

edges were sealed with beeswax before imaging on a Zeiss LSM 700

confocal microscope. Images were collected with a ×100 oil

immersion objective with additional zoom as needed. Coumarin

ﬂuorescence was recorded by exciting at 405 nm with the pinhole size

set to 1 AU.

(

100 μL) and 1 M NaHCO (aq. 200 μL), stirring at 40 °C for 1 h

before centrifuging. Samples of the supernatant were analyzed by RP-

HPLC (C8, Water/MeCN + 0.1% TFA, linear gradient: 80/20 to 60/

0 over 40 min, ﬂow rate = 1 mL/min, λ = 340 nm, Figures S1 and

S2 ). The major enantiomer elutes before the minor, as expected since

most L-amino acids elute before their respective D-enantiomers when

reacted with Marfey’s reagent.

Determination of Quantum Yields and Extinction Coef-

ﬁcients for Peptides 20 and 21. Absorbance and ﬂuorescence

measurements were performed on a Tecan M1000 instrument, with

bandwidths set to 5 nm for all measurements. Corrected ﬂuorescence

spectra were acquired using identical settings for all samples. The

excitation wavelength was the wavelength of maximum absorption.

The extinction coeﬃcients in Table 1 were determined by measuring

the absorbance of two independently prepared stock solutions of

known concentration. Each solution was measured in duplicate for a

total of four separate measurements (error is estimated to be ±10%).

The concentration of tripeptide stock solutions was determined by

weight, accounting for 1 equiv of residual TFA. Relative ﬂuorescence

quantum yields (Φ ) were measured using quinine sulfate in 0.5 M

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

■

H SO (Φ = 0.546) as a standard. Solutions of 20 or 21 were

prepared in either ethanol or water and diluted so that the maximum

absorbance was less than 0.10. For each peptide/solvent combination,

four solutions were prepared with maximum absorbance values

ranging from 0.02 to 0.08. The solutions were brieﬂy sonicated

following dilution. The absorbance of each sample was plotted against

the ﬂuorescence peak area from 400 to 600 nm, and the slope of this

line (R²> 0.99) was used to calculate quantum yield with the

NMR spectra and HRMS for all new compounds, HPLC

chromatograms of compounds 7 and 8 modiﬁed with

Marfey’s reagent, absorbance spectra of tripeptides 20

and 21, and analytical HPLC chromatograms and

HRMS analyses of peptides 20, 21, 25, and 26 (PDF)

8,59

following equation:

Φ = Φ × (m /m ) × (η /η ) , where x

refers to the sample and st refers to the standard, m is the slope, and η

is the refractive index of the solvent. As a control, the quantum yield

of 7-diethylamino-4-methylcoumarin (10) in ethanol was found to be

AUTHOR INFORMATION

Corresponding Author

■

.61, which falls well within the range reported in the literature (Φ =

23,61

.5−0.7,

depending on extent of quenching by O ).

Scott D. Taylor − Department of Chemistry, University of

Waterloo, Waterloo, Ontario N2L 3G1, Canada;

orcid.org/0000-0001-5449-5940; Email: s5taylor@

uwaterloo.ca

Interaction of Peptide 26 with Liposomes. Stock solutions of

,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristo-

yl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DMPG), 1,2-

dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-

glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG) were

prepared in chloroform and then mixed in the following PC/PG

molar ratios: 1:0, 3:1, and 1:1. The chloroform was removed under an

Author

Michael Noden − Department of Chemistry, University of

Waterloo, Waterloo, Ontario N2L 3G1, Canada

N stream and then high vacuum, leaving a thin ﬁlm of lipid, which

was re-suspended in buﬀer (20 mM HEPES, 150 mM NaCl, pH 7.4)

to give a total lipid concentration of 5 mM. The solution was

sonicated above 35 °C for 30 min (or until mostly transparent) to

generate small unilamellar vesicles (SUVs). A 100 μM stock solution

of peptide 26 was prepared in buﬀer. A corning black 96-well plate

was preloaded with buﬀer and preheated to 37 °C before aliquots of

the lipid and peptide solutions were added to give ﬁnal concentrations

of 250 μM lipid and 3 μM peptide and a volume of 200 μL. Emission

spectra (λ_e_x= 380 nm, λ_e_m= 400−600 nm) were collected using a

Tecan M1000 plate reader. This was performed in triplicate for each

mixture with a single representative emission spectrum being depicted

in Figure 4.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.joc.1c00907

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

Interaction of Peptide 26 with LPS. Stock solutions containing

■

00 μg/mL LPS from E. coli O127:B8 (obtained from Millipore-

Sigma) were prepared in buﬀer (20 mM HEPES, 150 mM NaCl, pH

.4). Similar to the procedure described above, a corning black 96-

well plate was preloaded with HEPES buﬀered saline and preheated to

7 °C before aliquots of the LPS and peptide solutions were added to

This work was supported by the Natural Sciences and

Engineering Research Council of Canada (NSERC) Discovery

grant to S.D.T. (RGPIN-2017-04233). M.N. is grateful to the

Government of Ontario for a post-graduate scholarship.

give ﬁnal concentrations of 10, 30, 60, or 100 μg/mL LPS and 3 μM

peptide and a volume of 200 μL. Emission spectra (λ = 380 nm, λ

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