Studies on the biosynthesis of paraherquamide. Construction of the amino acid framework

TETRAHEDRON

Pergamon

Tetrahedron 57 72001) 5303±5320

Studies on the biosynthesis of paraherquamide.

Construction of the amino acid framework

Emily M. Stocking,^aJuan F. Sanz-Cervera,^bClifford J. Unkefer^cand Robert M. Williams^a,p

^aDepartment of Chemistry, Colorado State University, Fort Collins, CO 80523, USA

b

Â

Departamento de Quõmica Organica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain

^cNIH Stable Isotope Resource, Los Alamos National Laboratory, Los Alamos, NM 87545, USA

Received 28 February 2001; accepted 23 April 2001

AbstractÐIt has been previously established in this laboratory that the b-methyl-b-hydroxyproline moiety of the potent anthelmintic agent

paraherquamide A, isbiosynthetically derived from l-isoleucine. The downstream events from l-Ile to paraherquamide A have now been

investigated. The synthesis of [1-¹³C]-labeled l-b-methylproline is described by means of a Hoffman±Loef¯er±Freytag reaction sequence

from [1-¹³C]-l-Ile. This amino acid is shown to be a direct biosynthetic precursor to paraherquamide A by feeding and incorporation

experimentsin growing culturesof Penicillium fellutanum. Three tryptophan-containing dipeptidesof l-b-methylproline have been

constructed: [¹³C₂]-2-71,1-dimethyl-2-propenyl)-l-tryptophanyl-37S)-methyl-l-proline; [¹³C₂]-37S)-methyl-l-prolyl-2-71,1-dimethyl-2-pro-

penyl)-l-tryptophan and [¹³C₂]-cyclo-2-71,1-dimethyl-2-propenyl)-l-tryptophan-37S)-methyl-l-proline. [a-¹⁵N, 1-¹³C]-2-71,1-Dimethyl-2-

propenyl)-l-tryptophan was also prepared but none of these substances were found to serve as biosynthetic precursors to paraherquamide A.

1. Introduction

and cyclo-[methylene-¹⁴C]l-tryptophanyl-[5-³H]-l-proline

716) were biosynthetically incorporated into brevianamide

A 717) in signi®cant radiochemical yield 7Scheme 1).⁶

The paraherquamidesare a group of heptacyclic mycotoxins

isolated from various Penicillium sp. that possess potent

anti-parasitic activity^1,2. Membersof the paraherquamide

family include paraherquamide A 71), which displays the

most potent anthelmintic activity of these compounds,

paraherquamidesB-G 7 2±4, 7, 10, 11), VM55595 712),

VM55596 75), VM55597 76), VM55599 713), SB203105

78), and SB200437 79). Although similar in structure, the

paraherquamidesvary with repsect to oxygenation and

substitution of both the proline ring and the prenylated

oxindole ring. Aspart of an ongoing invetsigation into

the biosynthesis of this family of alkaloids, the

construction of the amino acid framework leading to the

bicyclo[2.2.2]ring system of paraherquamide A has been

investigated and the results of this study are disclosed

here 7Fig. 1).^3,4

Later, Williamsand co-workersshowed that [8- ³H₂]deoxy-

brevianamide E 718) wasalos incorporated into brevian-

amide A 717).^5b,cBased on the structural similarities of

these substances, we initially speculated that l-tryptophan

and l-proline might also provide the biosynthetic building

blocksof paraherquamide A with l-tryptophan providing

the oxindole moiety and l-proline forming the framework

of the b-methyl-b-hydroxyproline moiety. The hypothesis

that l-proline could serve as the precursor to the b-methyl-

proline ring could be based on studies conducted by

Arigoni and co-workers, who studied the biosynthesis of

the b-methylproline residue in the cyclic peptide antibiotic

bottromycin 719), a natural substance isolated from

Streptomyces bottropenis 7Fig. 2). These studies demon-

strated that [methyl-¹³C]-l-methionine 720) wasincorpor-

ated into bottromycin giving rise to the 37R)-methyl group

of the b-methyl proline ring of bottromycin with a speci®c

incorporation of 20±25%.⁷In principle, methylation of the

b-position of proline in paraherquamide biosynthesis might

arise via methyl transfer from S-adenosylmethionine to a

2,3-dehydroproline derivative followed by reduction.

However, the ETH research group has demonstrated that

in the instance of bottromycin biosynthesis, the

mechanism for methylation involves a B₁₂-type radical

coupling mechanism that does not implicate loss of the

prolyl a-proton.

To provide some perspective and background concerning

the biogenesis of the paraherquamides, the biosynthesis of

a simpler, structurally related alkaloid, brevianamide A

717), has been the subject of previous study in our

laboratory.⁵In 1974, Birch and coworkersfound that

[5-³H]-l-proline 714), [methylene-³H]-d,l-tryptophan 715)

Keywords: biosynthesis; secondary metabolism; feeding experiments.

Corresponding author. Tel./fax: 11-970-491-3944;

e-mail: rmw@chem.colostate.edu

p

PII: S0040-4020701)00449-5

5304

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

Figure 1.

We previously reported that S-adenosylmethionine only

contributesthe N-methyl group 7C-29) in the para-

herquamide A structure and does not donate the C-17

methyl group. Thisled usto pseculate that the b-methyl

proline moiety of paraherquamide may instead be derived

from l-isoleucine through an oxidative cyclization

process.^3bBoth possible progenitors of the b-methyl-b-

hydroxyproline ring, l-proline and l-isoleucine, were

explored through ¹³C labeling studies and only l-isoleucine

wasfound to provide the

portion of paraherquamide A.

b-methyl-b-hydroxyproline

In addition to the amino acid constituents, paraherquamide

A also contains two ®ve carbon isoprene-derived units: one

comprising the dioxepin ring system and one forming the

unusual bicyclo [2.2.2] diazaoctane ring system. We have

proposed that the latter isoprene group is attached to the

2-position of the indole via an `inverse' prenyl transferase

Scheme 1.

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5305

the temporal sequence of events in building up the complex

polycyclic array of the paraherquamide family.

2. Results and discussion

To determine what the primary amino acid building blocks

of paraherquamide A are, feeding experimentswere

performed on Penicillium fellutanum 7ATCC: 20841) with

[1-¹³C]-l-tryptophan 721), [methyl-¹³C]-l-methionine 722),

[1-¹³C]-l-proline 723) and [1-¹³C]-l-isoleucine 724) 7Fig. 3,

Table 1). The position and the percentage of ¹³C incorpora-

tion in paraherquamide A wasdetermined by meansof ¹³C

²

NMR spectroscopy. The [1-¹³C]-l-tryptophan 721) was

incorporated, into the oxindole ring asexpected, 72.5%

incorporation) with the label at C-12. The [methyl-¹³C]-l-

methionine 722) wasnot incorporated in the b-methyl-

proline ring, but rather, only at C-29, the N-methyl position

Figure 2.

Figure 3.

Table 1. Results for the feeding experiments with compounds 21±27 and 32

Compound

mmol

1 produced

7mmol)

Incorporation at

C-18 7%)

Incorporation at

C-12 7%)

Incorporation at

C-29 7%)

21

22

23

24

25

26

27

32

0.152

0.164

0.149

0.155

0.157

0.081

0.028

0.120

0.091

0.043

0.169

0.041

0.049

0.011

0.053

0. 028

0.019

0.023

0.017

0.014

0.042

0.041

±

0

3.7

3.3

1.8

1.3

1.2

1.7

0

2.5

±

0.4

0.8

0.5

0.9

0

±

0.66

±

14.6

±

that subsequently undergoes a [412] cycloaddition across

an azadiene system derived from the amino acids to form

the bicyclo [2.2.2] ring system.⁸Because of the importance

of this isoprene unit in forming the unusual core structure of

paraherquamide A, several different potential metabolites

were explored containing the `inverse' prenyl group at the

2-position of the indole which might give an indication of

of the monoketopiperazine ring 70.6%) and [1-¹³C]-l-

proline 723) did not show any incorporation. On the other

hand, feeding of [1-¹³C]-l-isoleucine 724) to Penicillium

²

The percentage of incorporation wasalos determined through electro-

spray mass spectrometry. See Section 4 for the incorporation levels and

method of determination.

5306

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

fellutanum followed by harvesting the cells and isolation of

paraherquamide A, revealed that the labeled l-isoleucine

wasincorporated to the extent of 3.3±3.7% with the label

at C-18. It wasthusclear from the feeding experimentswith

the primary amino acidsthat l-isoleucine and not l-proline

incorporation at C-12, respectively. The ¹³C NMR spectra of

the paraherquamide A isolated from these feeding experi-

ments did not provide compelling evidence for site-speci®c

incorporation of both labelsfrom the intact dipeptides. The

observed levels of incorporation are more consistent with

hydrolysis of the dipeptides and re-incorporation of the indi-

vidual amino acidspreusmably coupled with additional

metabolic degradation and reconstitution of ¹³C-enriched

building blocks. Furthermore, incorporation levels deter-

mined from the mass spectra of Paraherquamide A corro-

borated the ®ndingsthat the doubly labeled metabolites

were not incorporated intact 7see Table 3 in Section 4 for

results) 7Fig. 4).

esrvesasthe precurosr to the

b-methyl-b-hydroxyproline

ring. However, it still remained unclear at what point of

the biosynthetic pathway l-isoleucine was converted into

b-methylproline.

We therefore set out to determine if the oxidative cycliza-

tion of the a-amino group of l-Ile on the side chain ethyl

residue occurs before or after condensation with l-trypto-

phan. The doubly labeled dipeptides[ ¹³C₂]-l-isoleucyl-l-

tryptophan 725), [¹³C₂]-l-tryptophanyl-l-isoleucine 726),

and [2,5-¹³C₂]-cyclo-l-tryptophan-l-isoleucine 727) were

synthesized and fed to P. fellutanum to test the latter possi-

bility.^3bAsa result, no incorporation wasdetected by means

of ¹³C NMR for the diketopiperazine 27. However, ¹³C

NMR data showed incorporation for the dipeptides 25 and

26, namely 1.2±1.8% incorporation at C-18 and 0.4±0.9%

Since none of the dipeptideswere incorporated intact, it

therefore seemed logical that oxidative cyclization of

l-isoleucine must precede coupling to l-tryptophan.⁹

Synthesis of [1-¹³C]-37S)-methyl-l-proline 732) from

[1-¹³C]-l-isoleucine 724) wasaccomplihsed according to

the method of Lavergne et al. 7Scheme 2).¹⁰Treatment of

[1-¹³C]-l-isoleucine ethyl ester 728) with t-butylhypochlorite

Figure 4.

Scheme 2.

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5307

afforded the N-chlorinated derivative 729) which, after

exposure to a mercury lamp in 85% H₂SO₄, gave the

Hoffman±Lof¯er±Freytag intermediate 730). Upon

neutralization, this intermediate suffered spontaneous cycli-

zation to give the desired labeled b-methyl-proline ethyl

ester. The reaction mixture was treated with di-tert-butyl-

dicarbonate to facilitate separation of the desired product

from any unreacted [1-¹³C]-l-isoleucine ethyl ester.

Hydrolysis of the puri®ed ethyl ester 31 with LiOH in

aqueousTHF followed by removal of the t-Boc with TFA

and ion exchange chromatography 7Dowex 50WX2-100)

afforded the desired labeled [1-¹³C]-37S)-methyl-l-proline

732) asthe free amino acid.

in the biosynthesis of proteins and other primary and/or

secondary metabolites in the fungus. Thus, 32 serves as a

more ef®cient precursor to paraherquamide.

Once it wasdetermined that l-isoleucine is ®rst converted to

b-methylproline before coupling to l-tryptophan, more

advanced intermediateson the bioysnthetic pathway were

considered. We have recently demonstrated that the addi-

tion of the ®rst equivalent of dimethylallyl pyrophosphate

7DMAPP) occursin formation of the bicyclo [2.2.2] ring

system and that the C₅unit that formspart of the dioxepin

system is installed later.^3cThere are numeroustsagesat

which the addition of the inverted isoprene unit to the indole

system might occur; several of these possibilities are

outlined in Fig. 5. Inverted prenylation of the indole nucleus

might occur after formation of the diketopiperazine 35 in

analogy to what has been established experimentally for the

structurally related brevianamides, to form 36. Alterna-

tively, either l-tryptophan itself or the dipeptides l-trypto-

phanyl-37S)-methyl-l-proline or 37S)-methyl-l-prolyl-l-

tryptophan might be prenylated, to give rise to potential

intermediates 37±39. To explore these possibilities, the

doubly isotopically labeled compounds 35±39 were synthe-

sized and fed to cultures of P. fellutanum.

A feeding experiment on P. fellutanum with [1-¹³C]-37S)-

methyl-l-proline 732) gave paraherquamide A with 14.6%

incorporation of the labeled precursor. A signi®cantly

higher level of incorporation was observed in this case,

when compared with [1-¹³C]-l-isoleucine 724) 714.6 vs.

3.7%, respectively). This is consistent with the postulate

that 37S)-methyl-l-proline is biosynthesized from l-iso-

leucine and istherefore farther along the pathway to

paraherquamide A. In addition, 37S)-methyl-l-proline isa

non-proteinogenic amino acid that is most likely used

exclusively in the production of paraherquamide A and

related compounds, whereas l-isoleucine must be consumed

The diketopiperazine 35 was synthesized as outlined in

Figure 5.

Scheme 3.

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E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

Scheme 4.

Scheme 3. Coupling of the N-t-Boc protected [1-¹³C]-l-

tryptophan 734) to [1-¹³C]-37S)-methyl-l-proline ethyl

ester 733) wasaccomplihsed in good yield with the BOP

reagent. Removal of the t-Boc group with TFA and

cyclization in re¯uxing toluene containing a catalytic

amount of 2-hydroxypyridine afforded 35 in 48% yield

over the three steps.

¹⁵N-labeled material was chosen to explore the question

whether or not the tryptophanyl moiety wasoxidatively

deaminated at some point in the biosynthesis 7speci®cally

in the formation of the azadiene system for the proposed

intramolecular Diels±Alder cyclization reaction). The

retention or loss of the ¹⁵N-label could therefore provide

signi®cant mechanistic insight into the coupling and cycli-

zation with 37S)-methyl-l-proline. The synthesis of 37 was

accomplished via a Somei-type coupling¹²reaction of 40

with 41 and 0.3 equiv. of PBu₃in re¯uxing acetonitrile

over a period of 8 h 7Scheme 4). The sultam moiety of 42

wasremoved through hydrolysisin a 2:1 mixture of THF

and water with 5 equiv. of LiOH for 24 h. Puri®cation of 43

was accomplished by ¯ash column chromatography which,

Labeled 2-71,1-dimethyl-2-propenyl)-l-tryptophan 737) was

synthesized from the labeled Oppolzer sultam glycinate as

shown in Scheme 4.¹¹Thisfacilitated the preparation of

either the singly labeled ¹³C-tryptophan derivative or the

doubly labeled ¹³C, ¹⁵N-derivative. In the case of 2-71,1-

dimethyl-2-propenyl)-l-tryptophan itself, the doubly ¹³C,

Scheme 5.

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5309

in turn, allowed recovery of the chiral sultam. The N-dithio-

methylmethylene group wasremoved with 10 equiv. of 1N

HCl in THF at room temperature over 24 h. The solvent was

removed in vacuo and the HCl salt was submitted to Dowex

ion exchange chromatography to provide the free amino

acid 37.

Feeding experimentswere performed with the potential

intermediates 35±39 7see Table 2). Somewhat surprisingly,

none of these potential precursors were incorporated intact.

However, ¹³C NMR data indicated that while the dipeptides

38 and 39 showed incorporation at C-18 in 0.44 and 0.92%,

respectively, no incorporation at C-12 was observed. This

again suggests that the dipeptides are catabolized and 37S)-

methyl-l-proline isre-incorporated, but the reveers

prenylated l-tryptophan isnot. The lack of incorporation

doesnot rigorously exclude the intermediacy of 35±39 in

the biosynthesis of paraherquamide A since penetration of

the cell envelope may severely limit uptake of these candi-

date precursors. On the other hand, dipeptides 38 and 39,

presumably release the reverse prenylated tryptophan

derivative 48 inside the cell upon catabolic proteolysis

and the lack of incorporation of thismoiety either intact

from the dipeptidesor asan individual amino acid would

cast doubt on this species as being a biosynthetic building

block.

The doubly labeled diketopiperazine of 2-71,1-dimethyl-2-

propenyl)-l-tryptophan and 37S)-methyl-l-proline was

prepared according to the procedure outlined in Scheme 5.

The intermediate 45 wasprepared in the same manner as 42

in 86% yield. After removal of the sultam with LiOH, the

BOP reagent wasused to carry out itscoupling to 33 in 95%

yield. Removal of the N-dithiomethylmethylene protecting

group followed by cyclization in re¯uxing toluene with a

catalytic amount of 2-hydroxypyridine afforded the diketo-

piperazine 736) in excellent yield. Alternatively, the

dipeptide l-tryptophanyl-37S)-methyl-l-proline 738) was

prepared in essentially quantitative yield through deprotec-

tion of 47 with LiOH, followed by 1 M HCl and Dowex ion

exchange chromatography.

3. Conclusions

The dipeptide [¹³C₂]-37S)-methyl-l-prolyl-l-tryptophan

739) was synthesized according to the procedure outlined

in Scheme 6. [1-¹³C]-2-71,1-Dimethyl-2-propenyl)-l-tryp-

tophan 748) wasprepared in the asme manner as 37 in

77% yield. Protection of the carboxyl group of 48 asthe

methyl ester, followed by coupling to [1-¹³C]-N-Boc-37S)-

methyl-l-proline with the BOP reagent provided the

dipeptide 50. Deprotection of the methyl ester with LiOH

followed by deprotection of the Boc group with TFA and

subsequent ion exchange with Dowex provided the

dipeptide 39 in 69% yield.

Through feeding experimentswith P. fellutanum, the

primary amino acid building blocksof paraherquamide A

have been determined: l-tryptophan, l-methionine and

l-isoleucine. l-Isoleucine appears to undergo an oxidative

cyclization process to be converted into b-methylproline,

which isthen ef®ciently incorporated into paraherquamide

A. The hydroxylation of the b-position of the b-methyl-

proline moiety must therefore occur with net retention of

stereochemistry. However, we have recently shown that the

doubly ¹³C-labeled cycloadduct 51 7Fig. 6) isincorporated

Scheme 6.

Table 2. Results for the feeding experiments with compounds 35±39

Compound

mmol

1 produced 7mmol)

Incorporation at C-18 7%)

Incorporation at C-12 7%)

35

36

37

38

39

0.042

0.059

0.098

0.040

0.031

0.028

0.035

0.026

0.010

0.015

0

0.44

0.92

0

±

0

5310

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

Figure 6.

Scheme 7.

1

intact into paraherquamide A in P. fellutanum,^3cwhich

would dictate that the hydroxylation of b-methylproline

must occur after formation of the bicyclo [2.2.2] diaza-

octane ring system.

phosphate) was purchased from Aldrich Chemical Co. H-

and ¹³C NMR spectra were obtained on a Bruker AC300P

300 MHz NMR spectrometer, a Mercury 300 or Inova 400

Varian NMR spectrometer at Colorado State University.

NMR spectra were taken in CDCl₃7¹H, 7.24 ppm; ¹³C,

77.0 ppm), CD₃OD 7¹H, 4.87 ppm, ¹³C, 49.15 ppm), and

d₆-DMSO 7¹H, 2.50 ppm, ¹³C, 39.51 ppm) obtained from

Cambridge Isotope Labs. Electrospray mass spectra were

obtained with a FisonsVG AutoSpec spectrometer. Exact

masses were obtained with a VG Quattro-SQ spectrometer.

IR spectra were recorded on a Perkin±Elmer 1600 Series

FTIR spectrometer. EM Science Silica Gel 60 was used for

column chromatography and PTLC were performed with

The free amino acid [1-¹³C]-l-tryptophan wasincorporated

and thus constitutes the precursor for the oxindole ring.

Furthermore, [methyl-¹³C]-l-methionine incorporated

exclusively at the N-methyl position 7C29). The results of

feeding experimentswith labeled [ ¹³C₂]-l-isoleucyl-l-tryp-

tophan [¹³C₂]-l-tryptophanyl-l-isoleucine, [2,5-¹³C₂]-

cyclo-l-tryptophan-l-isoleucine and [1-¹³C]-b-methyl-l-

proline suggest that l-isoleucine is ®rst converted to

b-methyl-l-proline before coupling to a tryptophanyl

derivative in the biosynthesis of paraherquamide A. Since

none of the potential reverse prenylated candidates 735±39)

were incorporated intact, the timing of the addition of the

isoprene moiety that culminates in the formation of the

bicyclo [2.2.2] ring system remains unclear. It is thus not

unreasonable to expect that a multi-domain non-ribosomal

peptide synthetase, that would neither charge nor release the

putative dipeptide intermediates, is involved in the early

stages of paraherquamide biosynthesis leading up to 51.¹³

Studiesaimed at clarifying the intermediate metabolitesin

paraherquamide biosynthesis preceding the formation of

metabolite 51 are currently under investigation in these

laboratories.

EM Science precoated TLC plates7Kieslgel 60, F

0.25 mm).

,

254

4.2. Synthesis of [¹³C₂]-l-isoleucyl-l-tryptophan

Prepared according to the route described in Scheme 7.

4.2.1. [1-¹³C]-N-Benzyloxycarbonyl-l-isoleucine. [1-¹³C]-

l-Isoleucine 732.1 mg, 0.244 mmol) was dissolved in dd

H₂O 775 mL) and 5 M NaOH 750 mL). The reaction mixture

wascooled to 0 8C while stirring. Benzyl chloroformate

738.5 mL, 0.270 mmol) and 2 M NaOH 7135 mL) were

added dropwise simultaneously under an argon atmosphere.

After addition wascomplete, the reaction wasbrought to

room temperature slowly and stirred for an additional 2 h.

The reaction mixture was then adjusted to pH 10 with satu-

rated aqueousNa ₂CO₃and extracted four timeswith diethyl

ether. The aqueouslayer wasacidi®ed with 2 M HCl to pH 3

and extracted into diethyl ether four times. The ethereal

layer wasdried over anhydrousmagnesium sulfate, ®ltered,

and the solvent removed in vacuo to provide a colorless oil.

Yield: 57.0 mg, 88%. ¹H NMR 7300 MHz, d₆-DMSO,

1208C): d 0.91 73H, t, J7.3 Hz), 0.92 73H, d, J6.6 Hz),

1.27 71H, m), 1.49 71H, m), 1.84 71H, m), 4.00 71H, dd,

J6.9, 6.9 Hz), 5.08 72H, s), 6.68 71 H, bs), 7.36 75H, s),

11.1 71H, bs). ¹³C NMR 775 MHz, CDCl₃): d 11.8, 15.7,

25.1, 38.0, 58.5 7d, J_C±C55 Hz), 67.8, 128.3, 128.5, 128.6,

136.3, 156.5, 177.3. IR 7NaCl, CH₂Cl₂): 3313, 3047, 2966,

4. Experimental

4.1. General considerations

[1-¹³C]-l-Tryptophan, [methyl-¹³C]-l-methionine and

[1-¹³C]-l-proline were obtained from the LosAlamosNIH

Stable Isotopes Resource. [1-¹³C]-l-isoleucine, 90% enrich-

ment, wasobtained from the LosAlamosNIH Stable

Isotopes Resource; [1-¹³C]-l-isoleucine, 99% enrichment,

was purchased from Cambridge Isotope Labs. All other

reagentswere commercial grade and uesd without puri-

®cation unless otherwise noted. BOP reagent 7benzo-

triazol-1-yloxytris7dimethylamino)phosphonium hexa¯uoro-

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5311

Scheme 8.

1714, 1520, 1455, 1415, 1339, 1092, 739, 697 cm²¹

[a]_D168 7c0.0125, CH₂Cl₂).

m), 1.79 71H, m), 3.27 71H, dd, J5, 15 Hz), 3.34 71H,

dd, J5, 15 Hz), 4.04 71 H, dd, J6, 10 Hz), 4.98 71H,

ddd, J6, 6, 8 Hz), 5.08 74H, s), 5.35 71H, d, J9 Hz,

D₂O exch.), 6.43 71H, d, J8 Hz, D₂O exch.), 6.75 71H,

bs), 7.08 71H, t, J8 Hz), 7.18 71H, ddd, J1, 8, 8 Hz),

7.29 711H, m), 7.50 71H, d, J8 Hz), 7.96 71H, bs, D₂O

exch.). ¹³C NMR 775 MHz, CDCl₃): d 11.6, 15.5, 24.9,

27.9, 37.9, 53.1 7d, J_C±C61 Hz), 59.8 7d, J_C±C52 Hz),

67.2, 67.5, 109.7, 111.5, 118.7, 120.0, 122.5, 123.4, 127.7,

128.3, 128.4, 128.7, 128.7, 128.8, 135.4, 136.3, 156.4,

171.1, 171.6. IR 7NaCl, CH₂Cl₂): 3319, 3045, 2946, 1710,

25

³

4.2.2. [1-¹³C]-l-Tryptophan benzyl ester. [1-¹³C]-l-Tryp-

tophan 744 mg, 0.214 mmol), p-toluenesulfonic acid mono-

hydrate 7102 mg, 0.536 mmol), benzyl alcohol 7250 mL),

and dry toluene 715 mL) were re¯uxed under an argon

atmosphere in a Dean±Stark apparatus half ®lled with

Ê

activated 4 A molecular sieves for 12 h. The toluene was

evaporated under reduced pressure and the residue was

covered with a layer of EtOAc. Saturated aqueoussodium

carbonate was then added to make the solution basic. The

aqueouslayer wasesparated from the organic layer and

extracted three more timeswith EtOAc. After drying with

anhydrous sodium sulfate, the solvent was removed in

vacuo. Yield: 34 mg, 54%. An analytical sample of

unlabeled material wasrecrytsallized from EtOAc and

1655, 1523, 1452, 1348, 1227, 1101, 1029, 738, 694 cm²¹

.

25

[a]_D112.78 7c0.01, CH₂Cl₂).

4.2.4. [¹³C₂]-l-Isoleucyl-l-tryptophan. [¹³C₂]-Z-l-Iso-

leucine-l-tryptophan benzyl ester 715.4 mg, 0.028 mmol)

was suspended with 20% Pd7OH)₂/C 710 mg) in 2 mL of

95% ethanol. After bubbling ®rst argon and then H_27g)

through the solution, it was placed under H_27g)atmosphere

with an H_27g)balloon. The reaction wasdetermined to be

complete by meansof TLC after 16 h. The reaction mixture

was®ltered through a pad of Celite and evaporated to

dryness. Yield: 9 mg, 100%. A pinkish solid was obtained

by dissolving the dipeptide in deionized, distilled water and

lyophilizing, mp 908C 7decomp). ¹H NMR 7300 MHz,

CD₃OD): d 0.85 73H, t, J7 Hz), 0.91 73H, d, J7 Hz),

1.10 71H, m), 1.42 71H, m), 1.81 71H, m), 3.19 71H, dd,

J7.3, 14.7 Hz), 3.36 71H, dd, J5, 16 Hz), 3.52 71 H, d,

J5 Hz), 4.61 71H, dd, J5, 7 Hz), 6.97 71H, ddd, J1, 8,

8 Hz), 7.04 71H, ddd, J1, 8, 8 Hz), 7.11 71H, bs), 7.28 71H,

d, J8 Hz), 7.62 71H, d, J7 Hz). ¹³C NMR 775 MHz,

CD₃OD): d 11.9, 15.5, 25.3, 29.3, 38.5, 57.0 7d,

J_C±C56 Hz), 59.8 7d, J_C±C47 Hz), 112.2, 112.3, 119.7,

119.8, 124.5, 129.4, 138.1, 170.4, 177.6. IR 7NaCl,

CH₃OH): 3404, 3025, 2966, 1652, 1593, 1456, 1393,

1

hexane to provide a cream colored solid, mp 74±758C. H

NMR 7300 MHz, CDCl₃): d 1.71 72H, bs, D₂O exch.), 3.08

71H, dd, J7.3, 14.4 Hz), 3.27 71H, dd, J5, 14.4 Hz), 3.87

71H, bs), 5.12 72H, s), 6.91 71 H, d, J2 Hz), 7.10 71H, ddd,

J1, 7, 7 Hz), 7.18 71H, ddd, J1, 7, 7 Hz), 7.25 71H, d

J6 Hz), 7.26, 71H, m), 7.32 72H, d, J6 Hz), 7.32 72H, t,

J4 Hz), 7.60 71H, d, J7 Hz), 8.21 71H, bs, D₂O exch.).

¹³C NMR 775 MHz, CDCl₃): d 31.0, 55.4 7d, J_C±C60 Hz),,

66.9, 77.4, 111.3, 111.4, 119.0, 122.4, 123.1, 127.8, 128.5,

128.5, 128.8, 135.9, 136.5, 175.6. IR 7NaCl, CH₂Cl₂): 3389,

3161, 3047, 1731, 1586, 1451, 1177, 742, 695 cm²¹

.

[a]_D130.88 7c0.0065, CH₂Cl₂).²

25

4.2.3. [¹³C₂]-N-Benzyloxycarbonyl-l-isoleucyl-l-trypto-

phan benzyl ester. [1-¹³C]-l-Tryptophan benzyl ester

734.0 mg, 0.115 mmol) wasadded to [1- ¹³C]-l-N-

benzyloxycarbonyl-isoleucine 735.0 mg, 0.131 mmol)

dissolved in acetonitrile 75 mL). Triethylamine 724 mL,

0.172 mmol) and BOP reagent 750.92 mg, 0.115 mmol)

were added to the ¯ask. After stirring for 2 h at room

temperature, brine 710 mL) wasadded to the reaction

mixture. The solution was extracted three times with

EtOAc. The combined EtOAc layerswere extracted with

2 M HCl, water, 0.5 M NaHCO₃, and brine successively.

742 cm²¹. [a]_D12.28 7c0.005, CH₃OH).

25

4.3. Synthesis of [¹³C₂]-l-tryptophanyl-l-isoleucine 126)

Prepared according to the route described in Scheme 8.

4.3.1. [1-¹³C]-l-Isoleucine benzyl ester. [1-¹³C]-l-Iso-

leucine 732.2 mg, 0.244 mmol), p-toluenesulfonic acid

monohydrate 751 mg, 0.268 mmol), benzyl alcohol

7100 mL), and dry toluene 715 mL) were re¯uxed for 24 h

under an argon atmosphere through an addition funnel half

The EtOAc fraction wasdried over MgSO , ®ltered and

4

evaporated to dryness. The crude product was puri®ed by

meansof radial chromatography with a 3% mixture of

methanol in CH₂Cl₂. Yield: 51.8 mg, 86%. An analytical

sample of unlabeled material was recrystallized from

EtOAc and hexane to provide a white solid, mp 152±

1538C. ¹H NMR 7300 MHz, CDCl₃): d 0.81 73H, t

J7 Hz), 0.86 73H, d, J7 Hz), 1.04 71H, m), 1.27 71H,

Ê

®lled with activated 4 A molecular sieves. The toluene was

evaporated under reduced pressure and the residue was

covered with a layer of CH₂Cl₂. Saturated aqueoussodium

carbonate was then added to make the solution basic. The

aqueouslayer wasesparated from the organic layer and

extracted three more timeswith CH Cl₂. After drying over

2

anhydrous sodium sulfate, the solvent was removed in

³

The reported variable temperature ¹H- and ¹³C NMR data were measured

in the unlabeled compound. ¹³C-labeled compound wasidentical by TLC

mobility and ¹H- and ¹³C NMR at 228C.

5312

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

vacuo to give a colorless oil. Yield: 34.4 mg, 63%. ¹H NMR

7300 MHz, CDCl₃): d 0.88 73H, t, J7.3 Hz), 0.93 73H, d,

J6.7 Hz), 1.19 71H, m), 1.42 71H, m), 1.55 72H, bs, D₂O

exch.), 1.78 71H, m), 3.40 71H, d, J5 Hz), 5.14 71H, d,

J12 Hz), 5.18 71H, d, J12 Hz), 7.36 75H, m). ¹³C NMR

775 MHz, CDCl₃): d 11.8, 16.0, 24.8, 39.3, 59.4 7d, J_C±

reaction mixture, it wasplaced under H _27g)atmosphere with

an H_{2 7g)}balloon. After 16 h TLC analysis showed the reac-

tion to be complete. The reaction mixture was®ltered

through a pad of Celite and evaporated in vacuo. Yield:

30 mg, 100%. A white solid was obtained by dissolving

the dipeptide in distilled deionized water and lyophilizing,

mp 1718C 7decomp). ¹H NMR 7300 MHz, CD₃OD): d 0.90

73H, t, J7 Hz), 0.92 73H, d, J7 Hz), 1.14 71H, m), 1.55

71H, m), 1.88 71H, m), 3.17 71H, dd, J8, 15 Hz), 3.44 71H,

dd, J5, 15 Hz), 4.15 71H, dd, J6, 6 Hz), 4.27 71H, d,

J5 Hz), 7.04 71H, ddd, J1, 8, 8 Hz), 7.11 71H, ddd,

J1, 7, 7 Hz), 7.21 71H, bs), 7.35 71H, d, J8 Hz), 7.70

71H, d, J8 Hz). ¹³C NMR 775 MHz, CD₃OD): d 12.3,

16.5, 18.2, 26.3, 29.3, 39.2, 55.4 7d, J_C±C53 Hz), 60.0 7d,

J_C±C60 Hz), 108.7, 112.6, 119.4, 120.3, 122.9, 125.8,

128.6, 138.4, 170.2, 175.9. IR 7NaCl, CH₃OH): 3272,

57 Hz), 66.7, 128.5, 128.7, 136.0, 175.7. IR 7NaCl,

C

25

CH₂Cl₂): 3385, 2962, 1732, 1456, 1167 cm²¹. [a]_D

116.28 7c0.015, CH₂Cl₂).

4.3.2. N-Benzyloxycarbonyl-[1-¹³C]-l-tryptophan. [1-¹³C]-

l-Tryptophan 750.0 mg, 0.244 mmol) wasprotected in the

same manner as described for N-benzyloxycarbonyl-[1-¹³C]-

1

l-isoleucine. Yield: 83.0 mg, 100%. H NMR 7300 MHz,

CDCl₃): d 3.33 72H, m), 4.75 71H, dd, J5, 12 Hz), 5.08

71H, d, J12 Hz), 5.14 71H, d, J12 Hz), 5.37 71H, d,

J8 Hz, D₂O exch.), 6.90 71H, s), 7.08 71H, t, J7 Hz),

7.19 71H, t, J7 Hz), 7.33, 76H, s), 7.57 71H, d, J8 Hz),

8.09 71H, bs, D₂O exch.) 10.22 71H, bs, D₂O exch.). ¹³C

NMR 7100 MHz, CDCl₃): d 27.6, 54.5 7d, J_C±C60 Hz),

67.1, 77.4, 109.9, 111.3, 118.6, 119.8, 122.2, 123.1, 127.6,

128.2, 128.3, 128.5, 136.0, 156.0, 176.5. IR 7NaCl,

CH₃OH): 3414, 2962, 1704, 1515, 1455, 1260, 1020,

3075, 2962, 1665, 1592, 1457, 1404, 742 cm²¹

.

25

[a]_D16.68 7c0.005, CH₃OH).

4.4. Synthesis of cyclo-l-isoleucine-l-tryptophan 127)

Prepared according to the route described in Scheme 9.

795 cm²¹. [a]_D24.08 7c0.01, CH₃OH).

4.4.1. [1-¹³C]-N-t-Butoxycarbonyl-l-isoleucine. NaOH

70.5 mL of a 1.0 M solution) was added to [1-¹³C]-l-iso-

leucine 732.2 mg, 0.244 mmol, 90% ¹³C) dissolved in

dioxane 71.0 mL) and dd H₂O 70.5 mL). The solution was

cooled in an ice bath while stirring and di-tert-butyl

dicarbonate 762 mL, 0.270 mmol) wasadded. The reaction

mixture wasbrought to room temperature and stirred for one

hour. The dioxane wasthen evaporated under reduced pres-

sure and the residue was covered with a layer of EtOAc and

acidi®ed to pH 2±3 with KHSO₄. The aqueouslayer was

separated from the organic layer and extracted two more

timeswith EtOAc. The EtOAc fractionswere pooled,

dried over MgSO₄, and evaporated under reduced pressure

25

4.3.3. [¹³C₂]-N-Benzyloxycarbonyl-l-tryptophanyl-l-iso-

leucine benzyl ester. The coupling of [1-¹³C]-N-benzyl-

oxycarbonyl-l-tryptophan 753.8 mg, 0.242 mmol) and

[1-¹³C]-l-isoleucine benzyl ester 781.4 mg, 0.239 mmol)

wasperformed asdescribed for [ ¹³C₂]-Z-l-isoleucyl-l-tryp-

tophan-benzyl ester. Yield: 123.0 mg, 99%. An analytical

sample of unlabeled material was recrystallized from

EtOAc and hexane to provide a white solid, mp 128±

1298C. ¹H NMR 7300 MHz, CDCl₃): d 0.71 73H, d,

J7.1 Hz), 0.79 73H, t, J7.3 Hz), 0.94 71H, m), 1.23

71H, m), 1.75 71H, m), 3.15 71H, dd, J7.8, 14.5 Hz),

3.33 71H, dd, J5, 14.5 Hz), 4.51 72H, m), 5.08 72H, d,

J3 Hz), 5.13 72H, s), 5.6 71H, d, J6 Hz, D₂O exch.),

6.22 71H, d, J8 Hz, D₂O exch.), 6.96 71H, bs), 7.10 71H,

t, J7 Hz), 7.18 71H, ddd, J1, 7, 7 Hz), 7.35 711H, m),

7.69 71H, d, J7 Hz), 8.05 71H, bs, D₂O exch.). ¹³C NMR

775 MHz, CDCl₃): d 11.7, 15.4, 25.2, 28.8, 38.0, 55.7 7d,

J_C±C55 Hz), 56.9 7d, J_C±C61 Hz), 67.1, 67.2, 110.6,

111.4, 119.0, 120.0, 122.5, 123.6, 127.5, 128.3, 128.4,

128.6, 128.7, 128.8, 135.6, 136.5, 156.2, 171.3, 171.3. IR

7NaCl, CH₂Cl₂): 3308, 3044, 2946, 1710, 1656, 1518, 1452,

1

to leave a colorless oil. Yield: 51.6 mg, 92%. H NMR

7300 MHz, d₆-DMSO, 1208C): d 0.88 73H, t, J7.7 Hz),

0.91 73H, d, J6.6 Hz), 1.22 71H, m), 1.42 79H, s), 1.46

71H, m), 1.80 71H, m), 3.92 71H, dd, J7.7, 7.7 Hz), 6.04

71H, bs), 10.29 71H, bs). ¹³C NMR 775 MHz, CDCl₃): d

11.6, 15.5, 24.4, 28.3, 37.8, 57.8 7d, J_C±C58 Hz), 80.0,

155.7, 177.2. IR 7NaCl, CH₂Cl₂): 3326, 3110, 2971, 2587,

1715, 1663, 1509, 1454, 1401,1365, 1248, 1165 cm²¹

[a]_D15.48 7c0.012, CH₂Cl₂).

.

25

4.4.2. [1-¹³C]-l-Tryptophan methyl ester. [1-¹³C]-l-Tryp-

tophan 750 mg, 0.244 mmol, 98% ¹³C) and d, l-10-

camphorsulfonic acid 7115 mg, 0.495 mmol) were dissolved

in 20 mL of absolute methanol. The solution was re¯uxed

25

1337, 1221, 1139, 738, 694 cm²¹. [a]_D11.08 7c0.01,

CH₂Cl₂).

4.3.4. [¹³C₂]-l-Tryptophanyl-l-isoleucine 126). [¹³C₂]-Z-

Ê

l-Tryptophanyl-l-isoleucine

benzyl

ester

748 mg,

through an addition funnel containing 3 A molecular sieves

for 24 h under an argon atmosphere. The methanol was

evaporated off under reduced pressure and the residue was

0.0881 mmol) and 20% Pd7OH)₂/C 715 mg) were suspended

in 2 mL of 95% ethanol. After bubbling argon through the

Scheme 9.

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5313

covered with a layer of EtOAc. Saturated aqueoussodium

carbonate was then added to make the solution basic. The

aqueouslayer wasesparated from the organic layer and

extracted 3 more timeswith EtOAc. After drying over

anhydrous sodium sulfate, the solvent was removed in

vacuo to leave a yellow oil. Yield: 41.3 mg, 78%. ¹H

NMR 7300 MHz, CDCl₃): d 1.83 72H, bs, D₂O exch.),

3.07 71H, dd, J7.6, 14.4 Hz), 3.31 71H, dd, J4.7,

14.4 Hz), 3.73 73H, s), 3.86 71H, dd, J5, 7 Hz), 7.01

71H, d, J2 Hz), 7.14 71H, ddd, J1, 7, 7 Hz), 7.20 71H,

ddd, J1, 7, 7 Hz), 7.34 71H, d, J8 Hz), 7.63 71H, d,

J8 Hz), 8.52 71H, bs, D₂O exch.). ¹³C NMR 775 MHz,

CDCl₃): d 30.9, 52.2, 55.1 7d, J_C±C59 Hz), 111.0, 111.4,

118.8, 119.6, 122.2, 123.2, 127.6, 136.4, 175.9. IR 7NaCl,

CH₂Cl₂): 3387, 3162, 2936, 1732, 1586, 1448, 1201, 1099,

71H, ddd, J1, 9, 13 Hz), 3.50 71H, ddd, J3, 3, 14 Hz),

3.80 71H, m), 4.21 71H, m), 7.04 71H, s), 7.06 71H, t,

J7 Hz), 7.13 71H, t, J7 Hz), 7.32 71H, d, J8 Hz),

7.56 71H, d, J8 Hz). ¹³C NMR 7100 MHz, CDCl₃15

dropsCD OD): d 11.5, 14.9, 23.4, 30.4, 38.1, 54.9 7d,

3

J_C±C51 Hz), 59.8 7d, J_C±C52 Hz), 108.8, 111.3, 118.4,

119.6, 122.2, 132.8, 126.8, 136.4, 166.9, 167.9. IR 7NaCl,

CH₃OH): 3319, 3198, 3045, 2957, 1666, 1457, 1326, 1095,

25

733 cm²¹. [a]_D231.58 7c0.0065, CH₃OH).

4.4.5. Synthesis of l-[1-¹³C]-31S)-methyl-proline 132). For

the synthesis of 32, essentially the procedure of Lavergne et

al.¹¹wasused with the exception that a Hg 8 lamp wasused

in lieu of a Rayonnet lamp for the Hoffman±Lof¯er±

Freytag reaction. In addition, N-Boc protection followed

by ¯ash column chromatography was used to purify the

7S)-b-methyl-proline instead of PTLC of the free amino

acid.

1007, 742 cm²¹. [a]_D120.48 7c0.01, CH₂Cl₂).

25

4.4.3. [¹³C₂]-l-N-t-Butoxycarbonyl-isoleucyl-l-trypto-

phan methyl ester. A mixture of [¹³C]-l-tryptophan methyl

ester 741.3 mg, 0.180 mmol) and 41.4 mg 70.179 mmol) of

[1-¹³C]-N-t-butoxycarbonyl-l-isoleucine was dissolved in

acetonitrile 72.75 mL). One equivalent of triethylamine

725 mL, 0.180 mmol) and BOP reagent 779.5 mg,

0.180 mmol) were added to the ¯ask. After stirring for 2 h

at room temperature, brine 710 mL) wasadded to the reac-

tion mixture. The solution was extracted three times with

EtOAc and the combined EtOAc layerswere extracted with

2 M HCl, water, 0.5 M NaHCO₃, and brine successively.

The organic layer wasdried over MgSO ₄, ®ltered, and

evaporated to dryness. The crude product was puri®ed by

Chromatotron with a 9:1 mixture of CH₂Cl₂/CH₃OH. Yield:

60.0 mg, 80%. An analytical sample of unlabeled material

wasrecrytsallized from EtOAc and hexane to provide a

white crystalline solid, mp 142±1438C. ¹H NMR

7300 MHz, d₆-DMSO, 1208C): d 0.82 73H, t, J7.3 Hz),

0.85 73H, d, J7.7 Hz), 1.10 71H, m), 1.41 79H, s), 1.44

71H, m), 1.72 71H, m), 2.89 71H, bs), 3.17 71H, dddd, J6.6,

15.0, 15.0, 15.0 Hz), 3.59 73H, s), 3.90 71H, dd, J6.6,

8.8 Hz), 4.66 71H, dd, J7.3, 14.3 Hz), 6.03 71H, d,

J9.5 Hz), 6.99 71H, ddd, J1.1, 7.0 Hz), 6.08 71H, ddd,

J1.1, 7.0, 7.0 Hz), 7.12 71H, d, J2.2 Hz), 7.35 71H, d,

J7.7 Hz), 7.51 71H, d, J7.7 Hz), 7.70 71H, d, J7.0 Hz,

10.47 71H, bs). ¹³C NMR 775 MHz, CDCl₃): d 11.6, 15.5,

24.8, 27.9, 28.5, 37.7, 52.7 7d, J_C±C62 Hz), 59.3 7d,

J_C±C60 Hz), 80.0, 109.8, 111.5, 118.6, 119.8, 122.4,

123.3, 127.7, 136.4, 155.9, 171.5, 172.2. IR 7NaCl,

CH₂Cl₂): 3305, 2967, 1657, 1514, 1442, 1360, 1248,

4.4.6. Synthesis of [1-¹³C]-l-isoleucine ethyl ester 128).

7 equiv. of thionyl chloride 71.93 mL, 26.5 mmol) were

added dropwise to a suspension of [1-¹³C]-l-isoleucine

7500 mg, 3.8 mmol) in 23 mL of absolute ethanol at 08C.

The mixture wasbrought to room temperature and stirred

until all the solids dissolved and then re¯uxed for an addi-

tional 10 h. The reaction mixture wasconcentrated under

reduced pressure, re-dissolved in water and made basic

7pH9±10) by the addition of 20% aqueousammonia.

The aqueouslayer wasextracted with EtOAc four time.s

The combined organic layerswere dried over anhydrous

Na₂SO₄and concentrated under reduced pressure to afford

a colorless oil. To remove residual EtOAc, the product was

re-dissolved in CH₂Cl₂and again concentrated under

reduced pressure. Due to the volatility of the product, it

should not be placed on a high vacuum line. Yield:

1

545 mg, 3.4 mmol, 90%. H NMR 7300 MHz, CDCl₃): d

0.87 73H, t, J7.3 Hz), 0.91 73H, d, J6.6 Hz), 1.15 71H,

m), 1.25 73H, t, J7.0 Hz), 1.40 71H, m), 1.45 72H, bs, D₂O

exch.), 1.71 71H, m), 3.30 71H, d, J5.1 Hz), 4.15 72H, m).

¹³C NMR 775 MHz, CDCl₃): d 11.6, 14.2, 15.6, 24.6, 39.2,

59.0 7d, J_C±C57 Hz), 60.4, 175.5. HRMS 7FAB¹) calcd for

C₇¹³C₁H₁₈O₂N₁7M1H) 161.1371, found 161.1373.

4.4.7. N-Cl-[1-¹³C]-l-Isoleucine ethyl ester. To a solution

of [1-¹³C]-l-isoleucine ethyl ester 7360.5 mg, 2.2 5 mmol)

in benzene 72.8 mL) at 08C wasadded one equivalent of

freshly

prepared

t-butyl

hypochlorite

7228 mL,

25

1165 cm²¹. [a]_D135.38 7c0.01, CH₂Cl₂).

2.25 mmol). The reaction mixture waskept below 5 8C and

stirred in the dark for 1.5 h. The mixture was washed with

1 mL of 0.1N HCl, and water 7three times, 1 mL aliquots)

4.4.4. [¹³C]₂-cyclo-l-Isoleucine-l-tryptophan 127). [¹³C₂]-

N-Boc-l-isoleucine-l-tryptophan-methyl ester 759 mg,

0.141 mmol) was dissolved in CH₂Cl₂710 mL). The solu-

tion wascooled to 0 8C and tri¯uoroacetic acid 71 mL) was

added. The reaction wastsirred at 0 8C under an argon

atmosphere for 5 h. The solvent and most of the TFA

were removed in vacuo. The residue was dissolved in dry

toluene and re¯uxed under an argon atmosphere for 1 h. The

toluene wasevaporated in vacuo and the crude product was

puri®ed by meansof ¯ash column chromatography with a

9:1 mixture of CH₂Cl₂/CH₃OH to provide a white solid, mp

before drying over anhydrousNa SO₄and concentrating

2

under reduced pressure at room temperature to afford a

yellow oil. The product wascarried on to the next tsep

without further puri®cation. Yield: 424 mg, 2.18 mmol,

97%. ¹H NMR 7300 MHz, CDCl₃): d 0.89 73H, t,

J7.3 Hz), 0.92 73H, d, J5.1 Hz), 1.21 71H, m), 1.30

73H, t, J7.7 Hz), 1.53 71H, m), 1.72 71H, m), 3.45 71H,

dd, J6.6, 11.4 Hz), 4.25 72H, q, J7.0 Hz), 4.61 71H, d,

J11.4 Hz). ¹³C NMR 775 MHz, CDCl₃): d 11.2, 14.3,

15.4, 5.8, 38.2, 61.2, 72.3 7d, J_C±C57 Hz), 172.7.

1

1638C 7decomp). Yield: 37 mg, 87%. H NMR 7400 MHz,

CDCl₃15 dropsCD ₃OD): d 0.76 73H, t, J7 Hz), 0.87 73H,

4.4.8. N-Boc-[1-¹³C]-31S)-methyl-l-proline ethyl ester. A

d, J7 Hz), 0.91 71H, m), 1.09 71H, m), 1.83 71H, m), 3.03

solution of [1-¹³C]-N-Cl-l-isoleucine ethyl ester 7424 mg,

5314

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

2.18 mmol) in 85% H₂SO₄714.5 mL) at 08C wasprepared in

a photochemical reaction vessel ®tted with a quartz

immersion well and Hg8 lamp. After purging the system

with argon for 15 min, the reaction mixture wasthen

irradiated with a mercury lamp for 40 h under an

atmosphere of argon while maintaining the temperature

between 0±58C. The reaction wastetsed for completion

by taking an aliquot 75 drops) of the reaction mixture and

treating with 5 mL of a 5% KI solution 7in 1:1 water,

acetone). When a clear pale yellow color wasobtained the

reaction wascomplete. After addition of 100 mL of an ice/

water mixture, the reaction wascarefully neutralized with

aqueous10 M NaOH at 0 8C. The reaction mixture was

concentrated under reduced pressure, the residue was

taken up in absolute ethanol and the solids ®ltered off.

Evaporation of the ethanolic solution under reduced

pressure left crude l-[1-¹³C]-37S)-methyl-proline ethyl

ester 7298 mg, 1.87 mmol, 86%). The crude product was

dissolved in 7.48 mL of a 1:1 mixture of dioxane and

water and cooled to 08C. Di-tert-butyl dicarbonate

7449 mg, 2.06 mmol, 1.1 equiv.) and solid K₂CO₃

7258 mg, 1.87 mmol, 1 equiv.) were added. The reaction

was slowly brought to room temperature and stirred for a

total of 8 h. The dioxane wasremoved under reduced

pressure, the reaction was lowered to a pH of 2 with 10%

aqueousKHSO ₄, and extracted four timeswith EtOAc. The

organic layer wasdried over anhydrousNa ₂SO₄and the

solvent was removed in vacuo to afford [1-¹³C]-N-Boc-

37S)-methyl-l-proline ethyl ester 731) asan oil. Yield:

255 mg, 0.99 mmol 745% from of [1-¹³C]-N-Cl-l-isoleucine

ethyl ester). [1-¹³C]-N-Boc-l-isoleucine ethyl ester was also

recovered 750.3 mg, 0.19 mmol, 9% from of [1-¹³C]-N-Cl-

[1-¹³C]-N-Boc-37S)-methyl-l-proline wastaken up in

0.5 mL of CH₂Cl₂and placed under argon while stirring at

08C. TFA 7348 mL, 4.34 mmol, 20 equiv.) wasadded slowly

to this solution. The resulting solution was allowed to come

to room temperature while continuing to stir over 3 h. The

CH₂Cl₂and excess TFA were removed in vacuo. The crude

reaction mixture wasloaded onto a Dowex 50X2-100 ion

exchange column and washed with 500 mL of water until

the pH of the eluate wasneutral. The free amino acid was

then eluted with 2% NH₄OH. The NH₄OH solution was

evaporated under reduced pressure. The residue was then

taken up in a small amount of distilled deionized water and

lyophilized to leave [1-¹³C]-37S)-methyl-l-proline 732) as

an off-white amorphoussolid. Yield: 28 mg, 0.215 mmol,

99%. ¹H NMR 7300 MHz, D₂O): d 1.23 71H, d J7.0 Hz),

1.68 71H, m), 2.20 71H, m), 2.39 71H, m), 3.39 72H, m), 3.60

71H, dd, J4.0, 6.7 Hz). ¹³C NMR 775 MHz, D₂O): d 13.1,

27.7, 33.7, 40.5, 62.6 7d, J_C±C53 Hz), 169.8. Enhanced ¹³

C

peak. IR 7NaCl, CH₃OH): 3389±2284, 2958, 1556, 1434,

.

1378, 1299, 1282, 1212, 1122, 1030, 961 cm²¹

25

[a]_D2278 7c0.1, H₂O). 7Literature: [a]_D2298^28

7c0.1, H₂O)) HRMS 7FAB¹) calcd for C₅¹³C₁H₁₂O₂N₁

7M1H) 131.0902, found 131.0907.

4.5. Synthesis of [¹³C₂]-cyclo-l-tryptophan-31S)-methyl-

l-proline 135)

4.5.1. [¹³C₂]-N-Boc-l-tryptophan-31S)-methyl-l-proline

ethyl ester. [1-¹³C]-l-Tryptophan 760 mg, 0.292 mmol)

was dissolved in 1.17 mL of a 1:1 mixture of dioxane and

water and cooled to 08C under an inert atmosphere. Di-tert-

butyl dicarbonate 776.1 mL, 0.331 mmol, 1.1 equiv.) and

1

l-isoleucine ethyl ester). H NMR 7300 MHz, d₆-DMSO,

anhydrousK CO₃740.3 mg, 0.292 mmol, 1 equiv.) were

2

1208C): d 1.13 73H, d, J7.0 Hz), 1.24 73H, t, J7.0 Hz),

1.40 79H, s), 1.52 71H, m), 2.01 71H, m), 2.27 71H, m), 3.33

71H, m), 3.49 71H, m), 3.72 71H, d, J5.5 Hz), 4.15 72H,

m). ¹³C NMR 775 MHz, d₆-DMSO, 1208C): d 13.2, 17.4,

27.4, 31.0, 38.7, 44.9, 59.4, 65.4, 78.2, 152.6, 171.3. IR

7NaCl, CH₂Cl₂): 2974, 2933, 2877, 1747, 1704, 14769,

1455, 1397, 1366, 1278, 1249, 1174, 1148, 1115,

added to the stirring solution. The reaction mixture was

brought to room temperature and stirred for 3 h. The

dioxane was removed under reduced pressure, the reaction

mixture waslowered to pH 2 with 10% aqueousKHSO ,

4

and extracted four timeswith EtOAc. The organic layer was

dried over anhydrousNa ₂SO₄and the solvent was removed

in vacuo to afford [1-¹³C]-N-Boc-l-tryptophan 734). In a

separate reaction vessel, [1-¹³C]-N-Boc-37S)-methyl-l-

proline ethyl-ester 731, 75.4 mg, 0.292 mmol) wastaken

up in 0.5 mL of CH₂Cl₂, placed under argon and stirred at

08C. To thissolution, TFA 7469 mL, 5.84 mmol, 20 equiv.)

was added slowly. The solution was allowed to come to

room temperature while being stirred for 3 h. The CH₂Cl₂

and excess TFA were removed in vacuo to leave the [1-¹³C]-

37S)-methyl-l-proline ethyl ester TFA salt as an oil 733).

The crude protected amino acidswere combined with one

equivalent of BOP reagent 7129 mg, 0.292 mmol) and

2 equiv. of triethylamine 781.4 mL, 0.584 mmol) in

438 mL of dry acetonitrile. The reaction wastsirred for

3.5 h at room temperature under an argon atmosphere. A

saturated aqueous solution of NaCl was added and the

mixture wasextracted three timeswith EtOAc. The

combined organic extractswere dried over anhydrous

NaSO₄and concentrated under reduced pressure. The

crude product waspuri®ed by meansof ¯ash column chro-

matography 7eluted with 50% EtOAc/hexane) to provide

25

1032 cm²¹. [a]_D244.18 7c1.24, CH₃OH).

4.4.9. l-[1-¹³C]-31S)-Methylproline. Compound 31

762 mg, 0.24 mmol) wasdioslved in 5 mL of anhydrous

methanol. NaOH 748 mg, 1.2 mmol, 5 equiv.) wasadded

and the solution was re¯uxed for 2 h. The methanol was

removed in vacuo and 0.1 M HCl wasadded to achieve a

pH2. Thismixture wasextracted three timeswith EtOAc

and the combined organic layerswere dried over anhydrous

Na₂SO₄and evaporated under reduced pressure to afford

[1-¹³C]-N-Boc-37S)-methyl-l-proline asa oslid. Yield:

50 mg, 0.217 mmol, 90%. An analytical sample of

unlabeled material wasrecrystallized from EtOAc and hex-

ane to provide a solid with mp 147±1488C. ¹H NMR

7300 MHz, d₆-DMSO, 1208C): d 1.12 73H, d, J7.0 Hz),

1.40 79H, s) 1.48 71H, m), 1.99 71H, m), 2.28 71H, m), 3.32

71H, m), 3.47 71H, m), 3.66 71H, d, J5.5 Hz), 11.77 71H,

bs). ¹³C NMR 775 MHz, d₆-DMSO, 1208C): d 17.7, 27.5,

31.0, 38.7, 44.8, 65.4, 152.8, 172.6. IR 7NaCl, CH₂Cl₂):

3438 7broad), 3106 7broad), 2965, 2604 7broad), 1694,

[¹³C₂]-N-Boc-l-tryptophan-l-37S)-methyl-proline

ethyl

25

1413, 1252, 1157 cm²¹. [a]_D21358 7c0.1, CH₂Cl₂).

ester as a foam. An analytical sample of unlabeled material

wasrecrytsallized from EtOAc and hexane to provide a

white solid, melting point 152.58C. Yield: 101.8 mg,

HRMS 7FAB¹) calcd for C₁₀¹³C₁H₂₀O₄N₁7M1H)

231.1426, found 231.1420.

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5315

0.229 mmol, 78% 72 steps). ¹H NMR 7300 MHz, d₆-DMSO,

1208C):^§d 1.12 73H, br s), 1.21 73H, t, J7.3 Hz), 1.34

710H, s), 1.57 71H, m), 2.08 71H, br s), 2.23 71H, br s),

2.87±3.37 74H, m), 3.86 71H, m), 4.13 72H, dd, J7.0,

14.3 Hz), 4.54 71H, br s), 6.11 71H, br s), 7.00 71H, dd,

J1.1, 7.0 Hz), 7.08 71H, dd, J1.1, 7.0 Hz), 7.35 71H, d,

J8.0 Hz), 7.56 71H, d, J8.0 Hz), 10.48 71H, br s). ¹³C

NMR 775 MHz, d₆-DMSO, 1208C): d 13.2, 17.1, 26.8, 32.1,

36.4, 45.2, 52.4 7d, J_CC55 Hz), 59.5, 65.3 7d, J_CC60 Hz),

77.7, 109.2, 110.6, 117.3, 117.7, 120.2, 123.1, 127.0, 135.8,

154.1, 170.0, 170.5. IR 7NaCl, CH₂Cl₂): 3323, 3057, 2966,

2926, 2873, 1738, 1711, 1648, 1510, 1455, 1391, 1366,

under reduced pressure and the crude product was puri®ed

by means of ¯ash silica gel column chromatography 720%

EtOAc/hex.) to give 361 mg, 0.630 mmol of compound 42

1

772% yield). H NMR 7400 MHz, CDCl₃): d 0.76 73H, s),

0.86 73H, s), 1.2772H, m), 1.55 71H, m), 1.56 73H, s), 1.61

73H, s), 1.65 71H, dd, J3.9, 3.9 Hz), 1.80 72H, m), 1.89

71H, dd, J7.8, 13.7 Hz), 2.25 73H, s), 2.36 73H, s), 3.32

71H, d, J13.7 Hz), 3.36 71H, m), 3.39 71H, d, J14.0 Hz),

3.48 71H, m), 3.83 71H, dd, J5.0, 7.0 Hz), 5.11 71H, dd,

J1, 10.6 Hz), 5.15 71H, dd, J0.8, 17.6 Hz), 5.54 71H, m),

6.19 71H, dd, J10.6, 17.6 Hz), 7.0171H, ddd, J1.2, 7.0,

7.0 Hz), 7.05 71H, ddd, J1.6, 7.0, 7.0 Hz), 7.16 71H, dd,

J1.6, 6.6 Hz), 7.74 71H, dd, J2.0, 7.0 Hz), 7.79 71H, br

s). ¹³C NMR 7100 MHz, CDCl₃): d 14.8 7d, J_N±C5 Hz),

15.9, 19.8, 20.4, 26.4, 27.6, 27.7, 29.8, 32.8, 38.2, 39.5,

44.6, 47.5, 48.1, 53.2, 65.3, 66.7 7d, J_C±C58 Hz), 105.7

7d, J_N±C3 Hz), 109.6, 111.6, 118.9, 120.1, 121.2, 129.9,

134.1, 140.6, 146.5, 161.7, 171.9 7d, J_N±C3 Hz). IR 7NaCl,

CH₂Cl₂): 3411, 3081, 3055, 2961, 2926, 2883, 1653, 1558,

1351, 1297, 1255, 1170, 1098, 1030, 955, 863, 742 cm²¹

.

[a]_D218.98 7c1.2, CH₂Cl₂). HRMS 7FAB¹) calcd for

25

C₂₂¹³C₂H₃₄O₅N₃7M1H) 446.2566, found 446.2574.

4.5.2. [¹³C₂]-cyclo-l-Tryptophan-l-31S)-methylproline

135). To a stirred solution of [¹³C₂]-N-Boc-l-tryptophan-

37S)-methyl-l-proline ethyl ester 7102 mg, 0.229 mmol) at

08C under an inert atmosphere, TFA 7353 mL, 4.58 mmol,

20 equiv.) was slowly added. The solution was brought to

room temperature and wasstirred for 3 h. The CH ₂Cl₂and

excess TFA were removed in vacuo. The residue was taken

1541, 1457, 1335, 1207, 1133, 1049, 1009, 912, 733 cm²¹

.

Optical rotation: [a]_D17.58 7CH₃OH, c0.53). HRMS

7FAB¹) calcd for C₂₈¹³C₁H₄₀N₁¹⁵N₁O₃S₃: 576.2236. Found

576.2243 7M1H).

up in aqueous10% NaCO and extracted three timeswith

3

4.6.2. [a-¹⁵N, 1-¹³C]-N-bismethylsulfanyl-ethene-2-11,1-

dimethyl-2-propenyl)-l-tryptophan 143). Solid LiOH

714 mg, 0.59 mmol, 5.0 equiv.) wasadded to a THF/water

72:1) solution of 42 767.5 mg, 0.118 mmol, 0.03 M). The

mixture wasstirred at ambient temperature for 24 h under

an atmosphere of argon. The THF was removed under

reduced pressure and the remaining aqueous layer was

acidi®ed to pH2 with 10% aqueousKHSO ₄before extract-

ing with dichloromethane three times. The product was

puri®ed by meansof ¯ash column chromatography 7eluted

with 2% CH₃OH/CH₂Cl₂) to provide 37.5 mg of compound

43 784% yield). ¹H NMR 7400 MHz, CDCl₃): d 1.55 73H, s),

1.56 73H, s), 2.18 73H, s), 2.40 73H, s), 3.29 71H, m), 3.59

71H, dddd, J2.7, 3.5, 3.5, 14.8 Hz), 4.78 71H, m), 5.16

71H, d, J10.5 Hz), 5.18 71H d, J17.6 Hz), 6.12 71H,

dd, J10.5, 17.2 Hz), 7.03 71H, ddd, J0, 7.8, 7.8 Hz),

7.09 71H, ddd, J1.2, 7.0, 7.0 Hz), 7.23 71H, d,

J7.0 Hz), 7.52 71H, d, J7.8 Hz), 7.88 71H, br s), 10.02

71H, very br s). ¹³C NMR 7300 MHz, CDCl₃): d 14.9

7J_N±C5 Hz), 15.4, 27.6, 27.7, 29.6, 39.2, 66.4 7d,

J_C±C58 Hz), 106.4, 110.1, 112.1, 118.6, 119.1, 121.3,

130.0, 134.1, 140.4, 145.8, 170.0, 173.0 7J_N±C3 Hz). IR

7NaCl, CH₂Cl₂): 3374, 3054, 2923, 2852, 1667, 1547, 1461,

EtOAc. The combined organic extractswere dried over

anhydrousNaSO ₄and concentrated under reduced pressure.

The crude product wasdiosslved in 1.5 mL of toluene,

2-hydroxypyridine 74.4 mg, 0.046 mmol, 0.2 equiv.) was

added, and the solution was re¯uxed under an argon

atmosphere for 4 h. The toluene was removed under reduced

pressure and the product puri®ed by means of ¯ash column

chromatography 7eluted with 2% CH₃OH/CH₂Cl₂) to

provide [¹³C₂]-cyclo-l-tryptophan-37S)-methyl-l-proline

735) asa white solid. Yield: 42 mg, 0.140 mmol, 61%. ¹H

NMR 7400 MHz, CDCl₃): d 1.27 73H, d J6.6 Hz), 1.53

71H, m), 2.13 71H, m), 2.34 71H, m), 2.94 71H, ddd, J2.3,

10.9, 14.8 Hz), 3.55 73H, m), 3.73 71H. d, J14.7 Hz), 4.32

71H, m), 5.63 71H d, J4.3 Hz), 7.09 71H, d, J2.0 Hz),

7.12 71H, ddd, J0.8, 7.8, 7.8 Hz), 7.22 71H, ddd, J1.2,

8.2, 8.2 Hz), 7.38 71H, d, J8.2 Hz), 7.57 71H, d,

J8.5 Hz), 8.19 71H, br s). ¹³C NMR 7100 MHz, CDCl₃):

d 18.2, 26.8, 31.6, 37.1, 44.1, 54.2 7d, J_CC52 Hz), 64.0 7d,

J_CC52 Hz), 109.97d, J_CC29 Hz), 111.5, 118.5, 120.0,

122.8, 123.2, 126.7, 136.6, 165.6, 169.4. IR 7NaCl,

CH₂Cl₂): 3389, 3054, 2922, 2851, 1632, 1612, 1454,

1415, 1301, 1102, 737, 693, 673 cm²¹. [a]_D292.98

25

7c1.2, 5% CH₃OH/CH₂Cl₂). HRMS 7FAB¹) calcd for

C₁₅¹³C₂H₂₀O₂N₃7M1H) 300.1623, found 300.1617.

25

1428, 1307, 1242, 1009, 913, 742 cm²¹. [a]_D116.48

7c0.495, CH₂Cl₂). HRMS 7FAB¹) calcd for

C₁₈¹³C₁H₂₅N₁¹⁵N₁O₂S₂: 379.1361. Found 379.1353 7M1H).

4.6. Synthesis of [a-¹⁵N, 1-¹³C]-2-11,1-dimethyl-2-

propenyl)-l-tryptophan 137)

4.6.3. [a-¹⁵N, 1-¹³C]-2-11,1-Dimethyl-2-propenyl)-l-tryp-

tophan 137). Compound 43 737.5 mg, 0.099 mmol) was

stirred with 1 M HCl 70.99 mL, 10 equiv.) in THF

71.8 mL, 0.05 M) at room temperature for 24 h under an

argon atmosphere. The THF was removed under reduced

pressure and the remaining aqueous layer was extracted

with diethyl ether. The diethyl ether layer waswahsed

twice with a small amount of water and the combined

aqueouslayerswere extracted twice with diethyl ether.

The aqueous layer was evaporated under reduced pressure,

re-dissolved in a small amount of distilled water and loaded

onto a Dowex H¹column. The column waswashed several

4.6.1. [2-¹⁵N, 1-¹³C]-2-Amino-N-bismethylsulfanyl-ethene-

3-[2-11,1-dimethyl-allyl)-1H-indol-3-yl]-1-110,10-dimethyl-

3,3-dioxo-3-thia-4-aza-tricyclo[5.2.1.0^0,0]dec-4-yl)-propan-

1-one 142). The [¹³C, ¹⁵N]-labeled Oppolzer sultam

glycinate 740, 330 mg, 0.887 mmol), the gramine derivative

76, 255 mg, 1.05 mmol, 1.2 equiv.), and tri-n-butylpho-

sphine 766 mL, 0.263 mmol, 0.3 equiv.) were re¯uxed in

acetonitrile 77.9 mL, 0.11 M) under argon for 8 h. After

cooling to room temperature, the solvent was concentrated

§

The observed peaks were still broad at 1208C in DMSO-d₆.

5316

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

timeswith distilled water before eluting with approximately

200 mL of 3% aqueousNH ₄OH. The eluate wasevaporated

in vacuo and then re-suspended in a small amount of de-

ionized water. The resulting solution was lyophilized to give

compound 37 asan off-white oslid in 59% yield 716 mg,

0.058 mmol). ¹H NMR 7400 MHz, CD₃OD): d 1.55 73H, s),

1.57 73H, s), 3.12 71H, m), 3.58 71H, m), 3.88 71H, m), 4.11

71H, dd, J1.2, 10.5 Hz), 5.14 71H, dd, J0.8, 17.6 Hz),

6.19 71H, dd, J10.9, 17.6 Hz), 6.99 71H, ddd, J0, 7.0,

7.8 Hz), 7.05 71H, ddd, J0.8, 7.0, 7.8 Hz), 7.32 71H, d,

J8.2 Hz), 7.59 71H, d, J7.8 Hz). ¹³C NMR 775 MHz,

CD₃OD): 28.7, 29.0, 40.4, 58.0 7dd, J_N±C5 Hz,

J_C±C54 Hz), 105.7 7J_N±C3 Hz), 112.1, 112.4, 118.9,

120.3, 122.3, 130.7, 136.8, 143.1, 147.9, 175.4. IR 7neat,

NaCl): 3689±1738 7broad), 3349, 2966, 2924, 1631, 1538,

1461, 1381, 1334, 1302, 919, 745 cm²¹. [a]_D17.58

7CH₃OH, c0.53). HRMS 7FAB1): Calcd for

C₁₅¹³C₁H₂₁N₁¹⁵N₁O₂, 275.1607. Found 275.1594 7M1H).

1547, 1461, 1428, 1307, 1242, 1009, 913, 742 cm²¹

.

25

[a]_D116.48 7c0.495, CH₂Cl₂). HRMS 7FAB¹) calcd

for C₁₈¹³C₁H₂₅N₂O₂S₂378.1391. Found 378.1378 7M1H).

4.7.3. [1-¹³C]-N-Bismethylsulfanyl-ethene-2-11,1-dimethyl-

2-propenyl)-l-tryptophanyl-31S)-methyl-l-proline ethyl

ester 147). N-Boc-37S)-methyl-l-proline ethyl ester 731,

57.4 mg, 0.222 mmol) wastaken up in 0.5 mL of CH Cl₂,

2

placed under argon and stirred at 08C. TFA 7469 mL,

5.84 mmol, 20 equiv.) was then added slowly. The solution

wasbrought to room temperature and wastsirred an

additional 3 h. The CH₂Cl₂and excess TFA were removed

in vacuo to leave the [1-¹³C]-37S)-methyl-l-proline ethyl

ester TFA salt 733) asan oil. To 33 wasadmixed 46

784 mg, 0.222 mmol) with 1 equiv. of BOP reagent

798 mg, 0.222 mmol) and 2 equiv. of triethylamine

768 mL, 0.488 mmol) in 3.4 mL of dry acetonitrile. The

reaction wastsirred for 3.5 h at room temperature under

an argon atmosphere. A saturated aqueous solution of

NaCl wasadded and the mixture wasextracted three

timeswith EtOAc. The combined organic extractswere

dried over anhydrousNa ₂SO₄and concentrated under

reduced pressure. The crude product was puri®ed via ¯ash

column chromatography 7eluted with 4% CH₃OH/CH₂Cl₂)

to afford 47. Yield: 106.8 mg, 0.206 mmol, 93% 72 steps).

¹H NMR 7300 MHz, d₆-DMSO, 1208C): d 1.10 73H, br s),

1.21 73H, t, J7.3 Hz), 1.56 76H, s), 2.65 71H, br s), 2.28

72H, m), 2.88 76H, s), 3.38 72H, br s), 3.56 71H, br s), 3.76

71H, br s), 3.8871H, dd, J4.8, 4.8 Hz), 4.12 72H, dddd,

J2.9, 7.0, 7.0, 7.0 Hz), 4.90 71H, br s), 5.05 71H, dd,

J1.1, 10.6 Hz), 5.08 71H dd, J1.1, 17.6 Hz), 6.26 71H,

dd, J10.3, 17.2 Hz), 6.91 71H, ddd, J1.1, 7.3, 7.3 Hz),

6.99 71H, ddd, J1.1, 7.0, 7.0 Hz), 7.29 71H, d, J7.7 Hz),

7.46 71H, d, J7.3 Hz), 9.98 71H, br s). ¹³C NMR 775 MHz,

d₆-DMSO, 1208C): d 13.2, 13.9, 17.4, 27.3, 27.4, 28.1, 32.0,

38.4, 45.2, 59.5, 67.0 7d, J_CC57.7 Hz), 109.9, 110.1 117.3,

117.6, 119.6, 129.1, 134.4, 140.2, 146.1, 167.0, 169.0,

170.7. IR 7NaCl, CH₂Cl₂): 3319, 3052, 2966, 2927, 2874,

1699, 1598, 1577, 1460, 1429, 1312, 1235, 1174, 1154,

4.7. Synthesis of [¹³C₂]-cyclo-2-11,1-dimethyl-2-

propenyl)-l-tryptophan-31S)-methyl-l-proline 136)

4.7.1. [1-¹³C]-2-Amino-N-bismethylsulfanyl-ethene-3-[2-

11,1-dimethyl-allyl)-1H-indol-3-yl]-1-110,10-dimethyl-

3,3-dioxo-3-thia-4-aza-tricyclo[5.2.1.0^0,0]dec-4-yl)-

propan-1-one 145). Preparation of 45 from 44 7190 mg,

0.508 mmol) wascarried out in the asme manner as 42

from 40. Yield: 249 mg, 0.435 mmol, 86%. ¹H NMR

7400 MHz, CDCl₃): d 0.78 73H, s), 0.85 73H, s), 1.25 72H,

m), 1.55 71H, m), 1.57 73H, s), 1.61 73H, s), 1.65 71H, dd,

J3.5, 3.5 Hz), 1.78 72H, m), 1.89 71H, dd, J7.8,

13.7 Hz), 2.24 73H, s), 2.36 73H, s), 3.30 71H, d,

J13.7 Hz), 3.37 71H, m), 3.38 71H, d, J13.7 Hz), 3.49

71H, ddd, J1.6, 6.5, 14.0 Hz), 3.83 71H, dd, J4.7,

7.4 Hz), 5.11 71H, d, J10.9 Hz), 5.15 71H, d,

J18.8 Hz), 5.56 71H, dd, J7.8, 7.8 Hz), 6.20 71H, dd,

J10.6, 17.6 Hz), 7.03 72H, m), 7.17 71H, d, J8.2 Hz),

7.75 71H, d, J7.0 Hz), 7.89 71H, br s). ¹³C NMR

7100 MHz, CDCl₃): d 14.7, 15.7, 19.6, 20.3, 26.2, 27.5,

27.6, 29.7, 32.6, 38.0, 39.4, 44.5, 47.4, 48.0, 53.0, 65.1,

66.6 7d, J_CC57 Hz), 105.5, 109.5, 111.4, 118.7, 119.9,

121.0, 129.7, 134.1, 140.5, 146.4, 161.4 7d, J_CC6 Hz),

173.5. IR 7NaCl, CH₂Cl₂): 3411, 3081, 3055, 2961, 2926,

2883, 1653, 1558, 1541, 1457, 1335, 1207, 1133, 1049,

1032, 1008, 912, 742 cm²¹. [a]_D274.98 7c0.595,

25

CH₂Cl₂). HRMS 7FAB¹) calcd for C₂₅¹³C₂H₃₈N₃O₃S₂

518.2422. Found 518.2422 7M1H).

4.7.4. [¹³C₂]-Cyclo-2-11,1-dimethyl-2-propenyl)-l-trypto-

phan-31S)-methyl-l-proline 136). Compound 47 757 mg,

0.113 mmol) watsisrred with 1 M HCl 71.13 mL,

10 equiv.) in THF 72.26 mL, 0.05 M) at room temperature

1009, 912, 733 cm²¹

. HRMS 7FAB1): Calcd for

C₂₈¹³C₁H₄₀N₃O₃S₃575.2265. Found 575.2264 7M1H).

4.7.2. [1-¹³C]-N-Bismethylsulfanyl-ethene-2-11,1-dimethyl-

2-propenyl)-l-tryptophan 146). The preparation of 46

from 45 740 mg, 0.070 mmol) wasachieved in the asme

manner as 43 from 42. Yield: 23 mg, 0.061 mmol, 87%.

¹H NMR 7400 MHz, CDCl₃): d 1.55 73H, s), 1.56 73H, s),

2.18 73H, s), 2.40 73H, s), 3.29 71H, ddd, J2.7, 10.1,

14.4 Hz), 3.37 71H, ddd, J2.7, 2.7, 14.8 Hz), 4.79 71H,

ddd, J3.9, 3.9, 8.2 Hz), 5.16 71H, dd, J1.2, 10.5 Hz),

5.19 71H dd, J0, 17.6 Hz), 6.12 71H, dd, J10.5,

17.1 Hz) 7.03 71H, ddd, J1.2, 8.2, 8.2 Hz), 7.09 71H,

ddd, J1.2, 7.1, 7.1 Hz), 7.23 71H, d, J8.2 Hz), 7.52

71H, d, J7.4 Hz), 7.88 71H, br s), 10.02 71H, very br s).

¹³C NMR 7100 MHz, CDCl₃): d 14.9, 15.4, 27.6, 27.7, 29.6,

39.2, 66.4 7d, J_CC56 Hz), 66.4, 106.4, 110.1, 112.1, 118.6,

119.1, 121.3, 130.0, 134.1, 140.4, 145.8, 173.5 7enhanced

¹³C peak). IR 7NaCl, CH₂Cl₂): 3374, 3054, 2923, 2852, 1667,

for 24 h. Enough aqueousoslution of 10% NaCO

was

3

added to turn the pH basic and the reaction was extracted

three timeswith EtOAc. The combined organic extracts

were dried over anhydrousNaSO and concentrated under

4

reduced pressure. The crude product was dissolved in 2 mL

of toluene, 2-hydroxypyridine 72.0 mg, 0.023 mmol,

0.2 equiv.) wasadded, and the solution re¯uxed under an

argon atmosphere for 4 h. The toluene was removed under

reduced pressure and the product puri®ed by means of ¯ash

column chromatography 7eluted with 4% CH₃OH/CH₂Cl₂)

to provide 36 as a white solid which was recrystallized from

ethyl acetate and hexane. Yield: 40 mg, 0.109 mmol, 97%.

¹H NMR 7400 MHz, CDCl₃): d 1.28 73H, d, J6.24 Hz),

1.54 76H, s), 2.16 71H, m), 2.39 71H, m), 3.15 71H, ddd,

J1.6, 11.3, 14.4 Hz), 3.54 72H, m), 3.61 71H, ddd, J1.9,

9.4, 9.4 Hz), 3.68 71H, dd, J3.9, 15.2 Hz), 4.38 71H, d,

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

5317

J11.7 Hz), 5.16 71H, dd, J0, 17.9 Hz), 5.17 71H dd, J0,

10.9 Hz), 5.6 71 H, br s), 6.26 71H, dd, J10.9, 17.9 Hz),

7.08 71H, t, J7.4 Hz), 6.99 71H, t, J7.0 Hz), 7.30 71H, d,

J8.2 Hz), 7.46 71H, d, J8.2 Hz), 8.02 71H, br s). ¹³C

NMR 7100 MHz, CDCl₃): d 18.2, 26.0, 27.9, 28.0, 31.6,

37.2, 39.0, 44.1, 54.8 7d, J_CC52.2 Hz), 64.0 7d,

J_CC53.7 Hz), 104.7, 110.8, 112.8, 117.9, 120.1, 122.2,

129.1, 134.3, 141.4, 145.6, 165.9, 169.3. IR 7NaCl,

CH₂Cl₂): 3364, 3051, 2969, 2867, 1633, 1461, 1403,

4.7.7. [1-¹³C]-2-11,1-Dimethyl-2-propenyl)-l-tryptophan

methyl ester 149). [1-¹³C]-2-71,1-Dimethyl-2-propenyl)-l-

tryptophan 748, 51 mg, 0.187 mmol) and d,l-camphor-

sulfonic acid 7115 mg, 0.497 mmol, 2.0 equiv.) were

dissolved in 2.5 mL of anhydrous methanol. Under an

argon atmosphere, the resulting solution was re¯uxed for

Ê

24 h through an addition funnel containing activated 3 A

molecular sieves. The methanol was removed under reduced

pressure and the residue was covered with a layer of ethyl

acetate. The solution was made basic by the addition of

aqueous saturated sodium carbonate. The aqueous layer

wasesparated from the organic and then extracted three

timeswith ethyl acetate. The organic extractswere pooled

together and dried over anhydrous sodium sulfate. Finally,

the solvent was removed in vacuo to afford 49 753.5 mg,

1321, 1305, 1252, 1223, 1009, 921, 743 cm²¹

.

[a]_D2308 7c0.07, CH₂Cl₂). HRMS 7FAB¹) calcd for

25

C₂₀¹³C₂H₂₇N₃O₂367.2170. Found 367.2185 7M1H).

4.7.5. [¹³C₂]-2-11,1-Dimethyl-2-propenyl)-l-tryptophanyl-

31S)-methyl-l-proline 138). Five equivalentsof LiOH

76.8 mg, 0.285 mmol) were added to a 0.03 M solution of

47 729.5 mg, 0.057 mmol) in THF/H₂O 72:1). The mixture

wastsirred at room temperature for 24 h. The THF was

removed in vacuo and the residue was acidi®ed to pH2

0.186 mmol, 99% yield) asa yellowihs oil.

¹H NMR

7300 MHz, CDCl₃): d 1.62 76 H, s), 1.72 72H, br s), 3.12

71H, dd, J9.5, 14.3 Hz), 3.39 71H, dd, J5.1, 14.7 Hz),

3.73 73H, s), 3.94 71H, dd, J4.8, 9.5 Hz), 5.22 71H, dd,

J1.1, 10.6 Hz), 5.23 71H, dd, J1.1, 17.6 Hz), 6.19 71H,

dd, J10.3, 17.6 Hz), 7.12 71H, ddd, J1.1, 7.0 Hz), 7.18

71H, dd, J1.1, 7.0 Hz), 7.33 71H, dd, J0.7, 7.0 Hz), 7.6

71H, dd, J0.7, 7.7 Hz), 7.98 71H, br s). ¹³C NMR

with 10% aqueousKHSO before extracting three times

4

with EtOAc. The combined organic layerswere dried over

anhydrousNa SO₄, ®ltered and evaporated to dryness. The

2

residue was re-suspended in THF 71.14 mL), 1N HCl was

added 70.57 mL), and the mixture wastsirred for 24 h at

room temperature. The THF and excess HCl were removed

under reduced pressure. The residue was loaded onto a

suitably prepared Dowex 50WX2-100 ion exchange

column, then desalted with de-ionized water and eluted

7100 MHz, CD₃OD):

d

27.8, 27.9, 31.2, 51.9 7d,

J_C±C3 Hz), 55.4 7d, J_C±C54 Hz), 56.1, 106.7, 110.4,

112.0, 118.4, 119.2, 121.4, 129.6, 134.1, 140.4, 145.9,

180.0. IR 7neat, NaCl): 3396, 3284, 3081, 3056, 2962,

2924, 2853, 1699, 1653, 1457, 1436, 1260, 1199, 1154,

1095, 1018, 919, 862, 799, 742 cm²¹. [a]_D216.58

25

with 2% aqueousNH OH. The eluate wasevaporated in

4

vacuo and then re-suspended in a small amount of de-

ionized water lyophilized to give a white solid 722 mg,

0.057 mmol, 100% yield). H NMR 7400 MHz, D₂O): d

7CH₂Cl₂, c0.085). HRMS 7FAB1): Calcd for

C₁₆¹³C₁H₂₃N₂O₂: 288.1793. Found 288.1797 7M1H).

1

0.12 73H, d, J6.6 Hz), 0.49 71H, m), 1.53 71H, m), 1.56

73H, s), 1.60 73H, s), 2.00 71H, m), 2.63 71H, dd, J0,

3.0 Hz), 3.08 71H, m), 3.14 71H, dd, J0, 14.4 Hz), 3.30

72H, m), 3.84 71H, dd, J0, 9.4 Hz), 5.21 71H dd, J0,

10.9 Hz), 5.27 71H, dd, J0.8, 17.2 Hz), 6.28 71H, dd,

J10.9, 17.2 Hz), 7.10 71H, t, J7.4 Hz), 7.18 71H, t,

J7.0 Hz), 7.40 71H, d, J8.2 Hz), 7.42 71H, d,

J9.0 Hz). ¹³C NMR 7100 MHz, D₂O): d 17.8, 26.9, 27.1,

29.3, 29.4, 38.9, 39.0, 46.4, 52.8 7d, J_C±C52 Hz), 68.9 7d,

J_C±C54 Hz), 103.8, 111.4, 111.9, 117.6, 119.4, 121.6,

129.0, 134.5, 142.2, 146.0, 173.3, 178.7. IR 7NaCl,

CH₂Cl₂): 3566±1600 7broad), 3421, 3355, 3056, 2965,

4.7.8. [¹³C₂]-N-Boc-31S)-methyl-l-proline-2-11,1-dimethyl-

2-propenyl)-l-tryptophan methyl ester 150). N-Boc-37S)-

methyl-l-proline 742.5 mg, 0.185 mmol, 1.0 equiv.) was

mixed with 49 753 mg, 0.185 mmol, 1.0 equiv.), BOP

reagent 782 mg, 0.185 mmol, 1.0 equiv.), and Et₃N

728 mL, 0.2035 mmol, 1.1 equiv.) in dry acetonitrile

72.8 mL) and stirred at room temperature under an inert

atmosphere for 4 h. A saturated aqueous solution of NaCl

wasadded and the reaction wasextracted four timeswith

EtOAc. The combined organic layerswere washed with 2 M

HCl, water, 10% NaHCO₃7aq.), water and brine succes-

sively. The organic phase was dried over anhydrous

Na₂SO₄and evaporated to dryness under reduced pressure.

The product was puri®ed by means of ¯ash silica gel column

chromatography with 40% EtOAc/Hex to give 750)

2930, 2873, 1558, 1458, 1362, 921, 743 cm²¹

.

[a]_D1328 7c0.1, H₂O). HRMS 7FAB¹) calcd for

25

C₂₀¹³C₂H₃₀N₃O₃386.2354. Found 386.2347 7M1H).

1

769.5 mg, 75%) as a colorless glass. H NMR 7300 MHz,

4.7.6. [1-¹³C]-2-11,1-dimethyl-2-propenyl)-l-tryptophan

148). Compound 48 was synthesized from compound 44 in

66% yield with the procedure described for the preparation

of 37 from 43. ¹H NMR 7400 MHz, CD₃OD): d 1.57 73H, s),

1.59 73H, s), 2.99 71H, dd, J0, 12.5 Hz), 3.37 71H, m),

3.6871H, m), 5.08 71H, d, J0, 10.5 Hz), 5.09 71H, dd, J0,

17.5 Hz), 6.20 71H, dd, J10.5, 17.5 Hz), 7.03 71H, t,

J7.0 Hz), 7.03 71H, t, J7.0 Hz), 7.30 71H, d,

J7.8 Hz), 7.65 71H, d, J7.7 Hz). ¹³C NMR 7100 MHz,

CD₃OD): d 28.9, 28.0, 33.1, 40.5, 58.8 7J_C±C55 Hz) 108.2,

111.8, 111.9, 119.7, 119.8, 122.0, 131.1, 136.7, 142.5,

148.3, 182.3. IR 7neat, NaCl): 3349, 2966, 2924, 1631,

d₆-DMSO, 1208C): d 1.00 73H, d, J7.0 Hz), 1.33 71H, m),

1.38 79H, s), 1.54 73H, s), 1.55 73H, s), 1.72 71H, m), 1.97

71H, m), 2.89 71H, br s), 3.12 71H, m), 3.18 71H, dd, J0,

8.4 Hz), 3.35 72H, m), 3.45 73H, s), 3.64 71H, d, J5.5 Hz),

4.73 71H, dd, J7.3, 14.6 Hz), 5.03 71H, d, J10.3 Hz),

5.10 71H, d, J17.6 Hz), 6.21 71H, ddd, J1.5, 10.6,

17.6 Hz), 6.94 71H, t, J7.3 Hz), 7.01 71H, t, J7.0 Hz),

7.33 71H, d, J8.0 Hz), 7.37 71H, br s), 7.47 71H, d,

J8.0 Hz). ¹³C NMR 7100 MHz, CDCl₃): d 18.4, 27.5,

27.6, 28.1, 29.67, 31.5, 39.1, 45.7, 52.1, 52.9 7d, J_C±

63 Hz), 67.9 7d, J_C±C53 Hz), 80.5, 105.2, 110.4,

C

112.4, 117.6, 119.9, 121.7, 130.1, 134.0, 140.6, 145.6,

154.6, 172.2, 172.7. IR 7neat, NaCl): 3337, 2968, 2929,

2875, 1703, 1625, 1504, 1462, 1435, 1392, 1365, 1248,

1538, 1461, 1381, 1334, 1302, 919, 745 cm²¹

.

25

[a]_D111.58 7H₂O, c0.23). HRMS 7FAB1): Calcd

for C₁₅¹³C₁H₂₁N₂O₂: 274.1637. Found 274.1643 7M1H).

1167, 1151, 1120 cm²¹. [a]_D229.18 7H₂O, c0.34).

25

5318

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

HRMS 7FAB1): Calcd for C₂₆¹³C₂H₄₀N₃O₅: 500.3035.

Found 500.3040 7M1H).

wasplaced into each of two ¯askscontaining the fungus.

Two control ¯asks were also set up, containing only sterile

trace element solution, for each feeding experiment. The

¯asks were put into the incubator at 258C for 10 daysand

swirled daily to ensure even distribution of the precursor.

4.7.9. [¹³C₂]-2-11,1-Dimethyl-2-propenyl)-l-tryptophanyl-

31S)-methyl-l-proline 139). Five equivalentsof 1N LiOH

7230 mL, 0.23 mmol) were added to a 0.03 M THF/H₂O

72:1) solution of 50 723 mg, 0.046 mmol). The mixture

wastsirred at room temperature for 24 h. The THF was

removed in vacuo and the residue was acidi®ed to pH2

A detergent had to be used to dissolve the water-insoluble

proposed precursors 735 and 36). The water-insoluble

compounds were dissolved in a 10% solution of absolute

ethanol/chloroform. Tween 80 70.2 mL) wasadded to the

dissolved precursor. The samples were then sonicated at

408C under a sterile stream of argon until the solvent was

completely removed. Sterile trace element solution was

added to the each precursor/Tween 80 residue 7see Table

2 for the volume and molarity of each solution) and the

mixture wassonicated for ,15 min to aid in micelle forma-

tion. Each of the solutions was then placed into two ¯asks

containing the fungus. Two control ¯asks were also set up,

each containing only sterile trace element solution. The

¯asks were put into the incubator at 258C for 10 daysand

swirled daily to ensure even distribution of the precursor.

with 10% aqueousKHSO before extracting three times

4

with EtOAc. The combined organic layerswere dried over

anhydrousNa SO₄, ®ltered and evaporated to dryness. The

2

residue was re-suspended in CH₂Cl₂70.5 mL), TFA was

added 774 mL, 0.92 mmol, 20 equiv.), and the mixture was

stirred for 24 h at room temperature. The CH₂Cl₂and excess

TFA were removed under reduced pressure. The residue

was dissolved in water and extracted with EtOAc three

times. The aqueous layer was then evaporated to dryness.

The residue was re-suspended in water, loaded onto a suit-

ably prepared Dowex ion exchange column, desalted with

de-ionized water, and eluted with 2% aqueousNH ₄OH. The

eluate was evaporated in vacuo and then re-suspended in a

small amount of de-ionized water. Finally, the resulting

solution was lyophilized to give compound 39 asa white

solid 712.3 mg, 0.032 mmol, 69% yield). ¹H NMR

7400 MHz, D₂O): d 1.00 73H, d, J6.2 Hz), 1.46 71H, m),

1.58 73H, s), 1.58 73H, s), 1.88 72H, m), 2.94 71H, m), 3.19

72H, m), 3.35 71H, m), 3.35 71H, dd, J5.5, 14.8 Hz), 4.59

71H, m), 5.20 71H, dd, J0.8, 10.5 Hz), 5.23 71H, dd, J0,

17.5 Hz), 6.27 71H, dd, J10.5, 17.5 Hz), 7.13 71H, t,

J7.0 Hz), 7.19 71H, t, J7.0 Hz), 7.42 71H, d,

J8.2 Hz), 7.65 71H, d, J7.8 Hz). ¹³C NMR 7100 MHz,

D₂O): d 17.0, 27.2, 27.3, 32.5, 38.9, 39.0, 45.6, 56.9 7d,

J_C±C54 Hz), 66.19 7d, J_C±C52 Hz), 106.1 7d,

J_C±C5 Hz), 111.1, 111.8, 118.4, 119.4, 121.4, 129.6,

134.6, 142.0, 146.3, 170.6, 178.4. IR 7NaCl, CH₂Cl₂):

3630±1700 7broad), 3292, 3056, 2964, 2964, 2929, 2872,

The aqueousmedia wasdecanted off and stored at 4 8C with

1±2 mL of chloroform. The mycelial cellsfrom each ¯ask

were harvested, combined with the cells from the duplicate

experiment and homogenized with 500 mL of methanol in

an Oster blender. The methanol suspensions of mycelial

cellswere placed in a hsaker at room temperature for

24 h. Celite 710 g) was added to each suspension before

®ltering through Whatman #2 paper. The ®ltrate wasstored

at 48C. The residual mycelia and Celite were re-suspended

in methanol, placed in the shaker for an additional 42 h and

re-®ltered.

The methanol solutions from both ®ltrations were combined

and evaporated in vacuo. The aqueoussolution from each

feeding experiment wasadded to the corresponding metha-

nolic residue and the mixture was acidi®ed to pH 4 with

12 mL of glacial acetic acid. The acidic solution was

extracted four timeswith 150 mL portionsof ethyl acetate.

The organic layer wasdicsarded. The aqueouslayer was

brought to pH 9±10 by the addition of 50 mL of 5 M

NaOH. The aqueouslayer wasthen extracted four times

with ethyl acetate. The combined organic layerswere

washed with brine, dried over anhydrous sodium carbonate

and evaporated to dryness. The paraherquamide from each

feeding experiment waspuri®ed via radial chromatography

and thin layer chromatography with a gradient elution of

4±10% methanol in methylene chloride 7Table 3).

1558, 1540, 1457, 1362, 921, 743 cm²¹. [a]_D18.08

25

7c0.1, H₂O). HRMS 7FAB¹) calcd for C₂₀¹³C₂H₃₀N₃O₃

386.2354. Found 386.2359 7M1H).

4.8. Feeding experiments with P. fellutanum

Sporesfrom P. fellutanum suspended in a 15% aqueous

glycerol solution were spread onto malt extract agar slants

720 g malt extract, 20 g glucose, 1 g peptone and 20 g agar

per liter of distilled, de-ionized water); 50 mL of the suspen-

sion was used per slant. The slants were placed in an

incubator at 258C for 7±10 days. The spores from two slants

were scraped into each 4-L ¯ask containing 400 mL of

sterile glucose corn steep liquor 740 g glucose and 22 g

corn steep liquor per one liter of distilled de-ionized

water). The inoculated ¯asks were placed in an incubator

at 258C for 5±7 days. The glucose corn steep liquor was

removed leaving a disk of the fungus. The undersides of the

disks, the mycelial cells, were rinsed with 100 mL of sterile

water.

4.9. Calculation of the percentage incorporation from

¹³C NMR data

To compensate for the effect of variable nuclear Overhauser

enhancementsand different relaxation timesthat caues

carbon signal integrals to differ within the same spectrum,

the relative peak heightsof labeled and unlabeled para-

herquamide A were compared. First, the ratio of the integral

of the labeled peak to the added integralsof the rest of the

peakswasobtained. Then, the ratio of the integral of the

corresponding peak in the unlabeled spectrum to the added

integrals in the rest of the spectrum was found. The total

percentage of ¹³C at the labeled position was found by

Sterile trace element solution 735 mM NaNO₃, 5.7 mM

K₂HPO₄, 4.2 mM MgSO₄, 1.3 mM KCl, 36 mM

FeSO₄´7H₂O, 25 mM MnSO₄´H₂O, 7 mM ZnSO₄´7H₂O,

1.5 mM CuCl₂´2H₂O) containing the biosynthetic precursor

7see Table 2 for the volume and molarity of each solution)

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

Table 3. Paraherquamide A isolated from feeding experiments

5319

Proposed precursor

Amt. precursor 7mmol)

Molarity 7M)

Volume 7mL)

Amt. 1 produced 7mmol)

21

22

24

27

Control

24

Control

25

26

Control

25

26

Control

23

Control

32

Control

35

36

0.152

0.161

0.155

0.043

±

0.157

±

0.081

0.120

±

0.028

0.0914

±

0.149

±

0.169

±

0.0424

0.0594

±

7.60£10²⁴

8.04£10²⁴

7.75£10²⁴

4.30£10²⁴

±

100

50

100

75

100

50

75

100

0.0405

0.0486

0.0527

0.0420

0.0222

0.0283

0.0193

0.0194

0.0170

0.0182

0.0231

0.0141

0.0277

0.0109

0.0182

0.0405

0.0101

0.0284

0.0347

0.0217

0.0263

0.0103

0.0146

0.0123

7.85£10²⁴

±

5.45£10²⁴

6.00£10²⁴

±

7.85£10²⁴

±

7.45£10²⁴

±

8.45£10²⁴

±

2.12£10²⁴

2.97£10²⁴

±

Control

37

38

39

Control

0.126

0.040

0.031

±

6.30£10²⁴

2.00£10²⁴

1.56£10²⁴

±

Table 4. Incorporationscalculated from electrospray MS data

Compound

mmol

1 produced 7mmol)

Single-label incorporation 7%)

Double-label incorporation 7%)

21

22

23

24

25

26

27

32

35

36

37

38^a

39^a

0.152

0.164

0.149

0.155

0.157

0.081

0.028

0.120

0.091

0.043

0.169

0.042

0.059

0.098

0.040

0.031

0.041

0.049

0.011

0.053

0. 028

0.019

0.023

0.017

0.014

0.042

0.041

0.028

0.035

0.026

0.010

0.015

1.2

0.4

0

±

0.3

0.25

0.08

0.3

0

±

0

±

N/A

3.0

2.2

1.6

0.77

0.5

1.5

0.61

10.1

0

N/A

a

After several attempts, we were unable to obtain reliable mass spectral data for the paraherquamide A isolated from the feeding experiments performed with

thiscompound.

dividing the ratio of integralsobtained from the labeled

paraherquamide A by the ratio from the unlabeled para-

herquamide A and multiplying by 1.1%, the natural

abundance of ¹³C. The percentage of ¹³C enrichment at

the labeled position was obtained by subtracting 1.1%

from the total percentage of ¹³C. The percentage of pre-

cursor incorporated was determined by multiplying the

percentage of ¹³C enrichment by the mmol of para-

herquamide A produced and then dividing thisnumber by

the mmol of isotopically enriched precursor.

determined according to the method outlined by Lambert et

al.¹⁴The percentage of precursor incorporated was deter-

mined by multiplying the percentage of ¹³C enrichment by

the mmol of paraherquamide A produced and then dividing

this number by the mmol of isotopically enriched precursor

7Table 4).

Acknowledgements

Thiswork wassupported by the NIH 7Grant CA70375 to

R. M. W.), the NIH Division of Research Resources Grant

7RR-02231 to C. J. U.) and the DGICYT of Spain 7Proyecto

PB98-1438 to J. F. S.-C.). We also wish to acknowledge the

ACS Division of Organic Chemistry Fellowship 7sponsored

by SmithKline Beecham) and the Pharmacia-Upjohn

Company for Fellowship support 7to E. M. S.). Mass spectra

were obtained on instruments supported by the NIH Shared

4.10. Calculation of percentage incorporation from mass

spectra data

Results were calculated by means of electrospray mass

spectroscopy, thus the base peak was the M1H peak and

wasset to 100%. Percentage of [ ¹³C] enrichment in para-

herquamide A from ¹³C-labeled biosynthetic precursors was

5320

E. M. Stocking et al. / Tetrahedron 57 ;2001) 5303±5320

Chem. Soc. 1989, 111, 3064±3065. 7b) Sanz-Cervera, J. F.;

Glinka, T.; Williams, R. M. J. Am. Chem. Soc. 1993, 115,

347±348. 7c) Sanz-Cervera, J. F.; Glinka, T.; Williams,

R. M. Tetrahedron 1993, 49, 8471. 7d) Domingo, L. R.;

Sanz-Cervera, J. F.; Williams, R. M.; Picher, M. T.; Marco,

J. A. J. Org. Chem. 1997, 62, 1662±1667.

Instrumentation Grant GM49631. Special thanks to R. M.

Lemieux for translation of Ref. 10.

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Studies on the biosynthesis of paraherquamide. Construction of the amino acid framework

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