A Mutasynthesis Approach with a Penicillium chrysogenum ΔroqA Strain Yields New Roquefortine D Analogues

Article abstract of DOI:10.1002/cbic.201402686

Penicillium chrysogenum, which lacks the roqA gene, processes synthetic, exogenously added histidyltryptophanyldiketopiperazine (HTD) to yield a set of roquefortine-based secondary metabolites also produced by the wild-type strain. Feeding a number of synthetic HTD analogues to the ΔroqA strain gives rise to the biosynthesis of a number of new roquefortine D derivatives, depending on the nature of the synthetic HTD added. Besides delivering semisynthetic roquefortine analogues, the mutasynthesis studies presented here also shed light on the substrate preferences and molecular mechanisms employed by the roquefortine C/D biosynthesis gene cluster, knowledge that may be tapped for the future development of more complex semisynthetic roquefortine-based secondary metabolites.

Full text of DOI:10.1002/cbic.201402686

DOI: 10.1002/cbic.201402686
Full Papers
A Mutasynthesis Approach with a Penicillium chrysogenum
DroqA Strain Yields New Roquefortine D Analogues
Kahina Ouchaou,[a] Florian Maire,[b] Oleksandr Salo,[c] Hazrat Ali,[c] Thomas Hankemeier,[b]
Gijsbert A. van der Marel,[a] Dmitri V. Filippov,[a] Roel A. L. Bovenberg,[d, e] Rob J. Vreeken,[b]
Arnold J. M. Driessen,*[c] and Herman S. Overkleeft*[a]
Penicillium chrysogenum, which lacks the roqA gene, processes
synthetic, exogenously added histidyltryptophanyldiketopiper-
azine (HTD) to yield a set of roquefortine-based secondary me-
tabolites also produced by the wild-type strain. Feeding a
number of synthetic HTD analogues to the DroqA strain gives
rise to the biosynthesis of a number of new roquefortine D
derivatives, depending on the nature of the synthetic HTD
added. Besides delivering semisynthetic roquefortine ana-
logues, the mutasynthesis studies presented here also shed
light on the substrate preferences and molecular mechanisms
employed by the roquefortine C/D biosynthesis gene cluster,
knowledge that may be tapped for the future development of
more complex semisynthetic roquefortine-based secondary
metabolites.
Introduction
Roquefortines C and D (Scheme 1) and secondary metabolites
derived from them are produced by many different Penicillium
species, including Penicillium chrysogenum.[1] They are mem-
bers of the broad family of prenylated indole alkaloids[2,3] and
are derived from histidyltryptophanyldiketopiperazine (HTD,
Scheme 2) as the distinguishing precursor. Roquefortines and
their derivatives are of interest because of their structural com-
plexity, their biosynthesis, and their biological activity. Roque-
fortine C was identified to have neurotoxic activity in mice and
is bacteriostatic against a number of Gram-positive bacteria.[4–6]
A downstream product derived from roquefortine C is neoxa-
line, also a compound with attributed antimicrobial activity.[7]
Our work on roquefortines to date has focused on unraveling
their biosynthesis pathway.
We recently reported that roquefortines C and D are pro-
duced in P. chrysogenum by a parallel biosynthesis pathway.[8,9]
In the first step, the common precursor, HTD, is synthesized by
the nonribosomal peptide synthetase RoqA. HTD is subse-
quently transformed in a series of steps into roquefortines and
from these into a number of complex secondary metabolites.
The biosynthesis of the roquefortine indole alkaloids in-
volves two gene clusters.[8,10] RoqA is a nonribosomal peptide
synthetase (NRPS) cluster that accepts and activates l-histidine
and l-tryptophan and condenses these to produce HTD. This
diketopiperazine is accepted as substrate by a further set of
enzymes including the dehydrogenase RoqR and the dimethyl-
allyltryptophan synthase RoqD. These, in concert with a num-
ber of other gene products, are responsible for the biosynthe-
sis of roquefortines C and D and their downstream secondary
metabolites.
[a] Dr. K. Ouchaou, Prof. Dr. G. A. van der Marel, Dr. D. V. Filippov,
Prof. Dr. H. S. Overkleeft
Leiden Institute of Chemistry, Department of Bio-organic Synthesis
Leiden University
Einsteinweg 55, 2333CC Leiden (The Netherlands)
E-mail: h.s.overkleeft@chem.leidenuniv.nl
[b] Dr. F. Maire, Prof. Dr. T. Hankemeier, Dr. R. J. Vreeken
Division of Analytical Biosciences
Leiden Academic Centre for Drug Research
Einsteinweg 55, 2333CC Leiden (The Netherlands)
The parallel action of RoqA and RoqR/RoqD invites a strategy
in which roquefortine analogues are produced through a muta-
tional biosynthesis—or mutasynthesis—approach.[11,12] In muta-
synthesis a gene (or gene cluster) responsible for the produc-
tion of a key secondary metabolite precursor is eliminated, and
the resulting mutant strain is supplemented with a synthetic,
modified version of this precursor in the hope that this will be
taken up and processed to deliver new analogues of the origi-
nal secondary metabolite. Mutasynthesis is considered to have
the advantage over classical precursor-directed biosynthesis
strategies in that competition between endogenous substrate
and added synthetic substrate is eliminated.[11] On the down-
side, engineering NRPS or polyketide synthase (PKS) gene clus-
ters to yield a desired mutant is high-risk in that the secondary
[c] O. Salo, Dr. H. Ali, Prof. Dr. A. J. M. Driessen
Department of Molecular Microbiology
Groningen Biomolecular Sciences and Biotechnology Institute
Zernike Institute for Advanced Materials, University of Groningen
Nijenborgh 7, 9747AG Groningen (The Netherlands)
E-mail: a.j.m.driessen@rug.nl
[d] Prof. Dr. R. A. L. Bovenberg
DSM Biotechnology Center
Alexander Fleminglaan 1, 2613AX Delft (The Netherlands)
[e] Prof. Dr. R. A. L. Bovenberg
Department of Synthetic Biology and Cell Engineering
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen
Nijenborgh 7, 9747AG Groningen (The Netherlands)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402686.
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Scheme 1. Biosynthetic pathway to roquefortines C and D in P. chrysogenum and mutasynthesis strategy reported here (PP=pyrophosphate).
Results and Discussion
metabolite biosynthesis machinery as a whole is compro-
mised.[12]
We reasoned that because of its parallel action of RoqA and
RoqR/D the roquefortine biosynthesis pathway would not be
subject to this caveat. On the basis of this assumption, we set
out to apply our P. chrysogenum strain in which we had geneti-
cally deleted the roqA gene (P. chrysogenum DroqA), as de-
scribed in ref. [8], to reconstitute roquefortine biosynthesis by
external addition to the growth media of HTD. Here we show
our results in this undertaking, as well as the fate of a set of
HTD analogues, 2–10 (Scheme 2), in terms of roquefortine ana-
logue mutasynthesis.
A number of synthesis strategies for the preparation of chiral,
enantiopure diketopiperazines have appeared in the litera-
ture.[13–18] Perusal of these reveals that the preparation of l,l-
or d,d-diketopiperazines—that is, the synthesis of diketopiper-
azines assembled from two l- or d-amino acids—is considera-
bly more complicated than the construction of their l,d-con-
figured counterparts. Thus, as the first research objective we
investigated the synthesis of the natural roquefortine precursor
l,l-HTD (1). The optimized route we arrived at, which is based
on literature[17,19] precedent, is shown in Scheme 3.
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Scheme 2. l,l-HTD (1) and synthetic HTD isomers and analogues 2–10, subjects of this mutasynthesis study.
tities of the desired intermediate to continue our synthesis by
using TMSI. Treatment of in-situ-generated H2N dipeptide 15
with aqueous ammonia yielded monotrityl-protected HTD 16,
which was treated with trifluoroacetic acid in dichloromethane
in the presence of triisopropylsilane (TIS) as a cation scavenger
to yield target compound 1.
An alternative route to diketopiperazines more commonly
applied in the literature[20–22] is depicted in Scheme 4 for the
synthesis of l,d-HTD (3). This route is based on condensation
of an Fmoc-protected a-amino acid (here Fmoc-(Boc)-d-trypto-
Scheme 3. Synthesis of l,l-HTD (1). a) C6F5OH, EDC·HCl, CH2Cl2, RT, 12 h,
74%; b) NEt3, CH2Cl2, RT, 12 h, 87%; c) TMSI, CH3CN, 08C, 2 h; d) NH3·H2O,
MeOH, RT, 16 h, 45%; e) 20% TFA, 2.5% TIS, CH3CN, 08C, 2 h, 99%.
In the first step, bis-Boc-protected tryptophan 11 was trans-
formed into the corresponding pentafluorophenyl ester, which
was condensed with partially protected histidine 13. The re-
sulting fully protected dipeptide 14 was treated with trimethyl-
silyl iodide (TMSI) under conditions reported in the literature[19]
to deblock the tryptophan secondary amine selectively. Al-
though the yield in this step was only moderate, structurally
and enantiomerically pure H2N dipeptide 15 was obtained in
this way. Application of other acidic conditions to obtain 15
proved abortive, and we were able to prepare sufficient quan-
Scheme 4. Synthesis of l,d-HTD (3). a) HATU, Et3N, CH3CN, RT, 12 h, 99%;
b) 50% piperidine in DMF, RT, 2 h, 62%; c) 20% TFA, 2.5% TIS, CH3CN, 08C,
2 h, 96%.
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phan (17)) with a second a-amino acid protected as the
methyl ester (here l-histidine derivative 13). In this way, fully
protected dipeptide 18 is obtained uneventfully. The difference
between the two routes is in the next stage: whereas in the
former example deprotection of the secondary amine proceed-
ed under (Lewis) acidic conditions, in the current case the
Fmoc group is removed under basic (piperidine in DMF) condi-
tions. The advantage in terms of efficiency is that, upon the
Fmoc removal, the liberated basic amine in 19 reacts without
further manipulation with the methyl ester to provide protect-
ed diketopiperazine 20 in good yield. A disadvantage of this
method—at least according to literature reports—is that the
basic conditions applied can give rise to deprotonation of one
of the two a-carbons and thus epimerization and erosion of
enantiomeric purity. Nevertheless, after removal of the Boc/
trityl protective groups in 20 and HPLC purification we were
able to obtain l,d-HTD (3) in good yield and enantiomeric
purity. We thus have two routes at our disposal, and these
combined allowed us to prepare HTD analogues 1, 2, 5, and 6
(by the TMSI route depicted in Scheme 3) as well as 3, 4, 7,
and 8 (by the Fmoc peptide chemistry route depicted in
Scheme 4). HTD analogues 9 and 10 in turn were obtained
from a commercial source.
The outcome of feeding experiments in which the P. chryso-
genum DroqA strain was grown with each of the configuration-
al isomers and structural HTD analogues 2–10 is depicted in
Scheme 5 (see the Supporting Information for LC-MS traces of
these feeding experiments). Growth media were extracted and
analyzed for roquefortine analogue content by LC-MS essen-
tially as done for the feeding experiment with synthetic l,l-
HTD (1).
Exact masses of putative roquefortine analogue metabolites
were calculated and compared against the list of ions high-
lighted during the data processing of the LC-MS chromato-
grams (i.e., ions present in supplemented P. chrysogenum
DroqA cultures and absent in P. chrysogenum DroqA control
culture). It should be noted that no NMR experiment has been
performed on the roquefortine analogue metabolites. Because
the identifications are based on accurate masses only, isomers
of the structures shown in Scheme 5 cannot be ruled out com-
pletely. A first observation we made is that neither of the HTD
analogues made it through the biosynthesis pathways to pro-
duce roquefortine C analogues. At least, if such analogues—or
any of the secondary metabolites derived from roquefortine
C—had been produced, this had occurred in quantities below
our limit of detection. On the positive side, some HTD ana-
logues proved to be acceptable substrates for RoqR (the dehy-
drogenase activity) and some were accepted by RoqD (the di-
methylallyltryptophan synthase activity), whereas configura-
tional isomers 3 and 4 proved metabolically inert.
With l,l-HTD (1) and its configurational isomers (compounds
2–4) and structural analogues (compounds 5–10) to hand, we
set out to study their use in the mutasynthesis of roquefortine
analogues by P. chrysogenum DroqA. Thus, after 4 days of
growth of this strain, l,l-HTD was added at 200 mgmLÀ1
(0.6 mm) final concentration, and growth was continued up to
days 5 or 7. Next, the metabolites produced were analyzed in
samples taken from both 5- and 7-day culture broths after
removal of the cells by filtration, previous work having shown
that roquefortine-related secondary metabolites can be readily
extracted from the medium.[8,9] As shown in Figure 1B, after
7 days of fermentation the P. chrysogenum DroqA strain, previ-
ously prepared by us,[8] had produced neither l,l-HTD nor any
of its derived secondary metabolites. Supplementation with
synthetic l,l-HTD (1), in contrast, restored the roquefortine
metabolic pathway (Figure 1C).
Closer perusal of the obtained data allows for some interest-
ing observations. With respect to the configurational isomer
set 1–4, l,d-HTD (3) and d,d-HTD (4) proved to be unaccepta-
ble substrates for both RoqD and RoqR, and from this we can
conclude that the l stereochemistry of the tryptophan residue
is essential. This is underscored by the observation that d,l-
HTD (2) is accepted by RoqD to produce the roquefortine C
analogue 23 (Figure 2). Neither 23 nor 2 is dehydrogenated,
and so the l configuration of the histidine residue is essential
for RoqR as well.
Structural analogues 5–10 all have the appropriate stereo-
chemistry for both RoqD and RoqR, and acceptance by either
of the two therefore relies on their structural and
functional features. RoqR-mediated dehydrogenation
proceeds with 7 (to produce 21, Figure 3) and with 8
(to produce 22), in other words exclusively with
those HTD analogues containing l-histidine as one of
the two a-amino acids incorporated. RoqD in turn is
lenient with regard to the nature of the l-histidine
analogue but accepts, out of the series of HTD ana-
logues screened in this study, only those residues fea-
turing an l-tryptophan moiety. In this way, roqueforti-
ne C analogues 24 (from 5), 25 (from 6), 26 (from 9),
and 27 (from 10) are produced. The fact that none of
these metabolites is further processed to roqueforti-
ne C analogues can also be seen as a positive out-
come in that in this way roquefortine D analogues—
Figure 1. LC-MS chromatograms (total ion current): A) of production of roquefortines by
wild-type P. chrysogenum, and of secondary metabolite production by P. chrysogenum
DroqA B) in the absence and C) in the presence of l,l-HTD at 200 mgmLÀ1 after 7 days of
fermentation (roq. roquefortine, * internal standard).
in themselves interesting compounds in terms of
their structural complexity—can be readily prepared.
Somewhat surprising is the finding that HTD ana-
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Scheme 5. DHTD/roquefortine D analogues produced by mutasynthesis of P. chrysogenum DroqA broth supplemented with HTD isomers and analogues 2–10.
Conclusions
In conclusion, this work demonstrates that the roque-
fortine biosynthesis pathway is amenable to muta-
synthesis studies to deliver new, semisynthetic
DHTD/roquefortine D analogues. From an engineer-
ing point of view, it can be predicted from the
branched nature of the roquefortine biosynthesis
pathway that the deletion of the roqA gene should
yield a strain in which the complete roquefortine bio-
synthesis pathway can be rescued through supple-
mentation with synthetic l,l-HTD. The two enzymes
immediately downstream of HTD biosynthesis, RoqD
and RoqR, appear to be selective for their corre-
sponding amino acids (l-Trp and l-His, respectively),
but not so much for the second amino acid that
completes the diketopiperazine ring. Thus, the small
set of HTD analogues assessed here has already deliv-
ered some interesting semisynthetic secondary me-
tabolites, in particular roquefortine D analogues 23–
27. Further modifications on the HTD core, such as
the benzofuran tryptophan analogue mentioned
above, might yield semisynthetic secondary metabo-
lites with structures resembling those found further
Figure 2. LC-MS analysis of culture broth of P. chrysogenum DroqA 72 h after addition of
2. A) Full chromatogram (* internal standard). B) Extracted ion chromatogram of 2. C) Ex-
tracted ion chromatogram of 23. The analysis was performed with the LTQ-Orbitrap, and
an internal recalibration was performed after acquisition by use of the monoprotonated
ion of 2.
logues 7 and 8, featuring a benzothiophene and a naphthyl
moiety, respectively, as indole analogues, are not acceptable
RoqD substrates. The indole nitrogen appears to be crucial in
the electrophilic aromatic substitution that comprises the first
step in the RoqD-catalyzed indole prenylation process. In view
of this result it would be of interest to investigate the fates of
benzofuran-based HTD analogues or of analogues of 7 and 8
in which the benzothiophene/naphthyl moieties are modified
to bear electron-donating substituents.
downstream in the roquefortine biosynthesis pathway. Here it
should be mentioned that our approach is biased towards the
detection of modified metabolites that are exported into the
medium. We cannot exclude the possibility that additional me-
tabolites are produced from our HTD analogues but are not
detected because they are not exported. Finally, numerous
indole alkaloid secondary metabolites found in nature are de-
rived from diketopiperazines featuring l-tryptophan together
with a-amino acids other than histidine.[23] Generation of the
corresponding diketopiperazine synthase deletion mutants
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EtOAc 100:0!97:3) to give the corresponding com-
1
pound as a white solid (0.522 g, 74%). H NMR (400 MHz,
CDCl3): d=8.21–8.12 (brd, 1H), 7.57 (d, J=7.6 Hz, 1H),
7.52 (s, 1H), 7.35 (m, 1H), 7.27 (m, 1H), 5.05 (m, 1H),
3.45 (m, 1H), 3.35 (m, 1H), 1.67 (s, 9H), 1.44 ppm (s, 9H).
Dipeptide Boc-Trp(Boc)-His(Tr) methyl ester (14): A so-
lution of l-BocTrp(Boc)OC6F5 (0.52 g, 0.9 mmol, 1.0 equiv)
in freshly distilled CH2Cl2 (5 mL) was added dropwise
under argon to
a solution of l-His(Tr)OMe (0.45 g,
1.0 mmol, 1.1 equiv) and NEt3 (0.14 mL, 1.1 equiv) in
freshly distilled CH2Cl2 (5 mL). The reaction mixture was
stirred overnight at room temperature. The solvent was
removed, and the mixture was purified on silica (MeOH
in CH2Cl2, 1%) to afford the expected compound as an
1
amorphous white solid (0.63 g, 87%). H NMR (400 MHz,
CDCl3): d=8.12–8.01 (brs, 1H), 7.60 (d, J=6.8 Hz, 1H),
7.46 (s, 1H), 7.38–7.15 (m, 12H), 7.11–7.03 (m, 6H), 6.43
(s, 1H), 4.78–4.71 (m, 1H), 4.59–4.48 (m, 1H), 3.55 (s, 3H),
3.38–3.10 (m, 2H; AB), 3.05–2.84 (m, 2H; AB), 1.64 (s,
9H), 1.36 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=
171.17–171.09, 165.64, 155.40, 149.55, 141.16, 135.51,
130.53, 129.78, 128.54, 124.37, 122.54, 119.86, 119.26,
116.07, 115.14, 83.43, 79.75, 54.79, 52.77, 52.58, 29.05,
28.31–28.23 ppm; LC-MS: m/z: 798.13 [M++H]+, 1594.73
[2M]+.
Figure 3. LC-MS analysis of culture broth of P. chrysogenum DroqA 72 h after addition of
7. A) Full chromatogram (* internal standard). B) Extracted ion chromatogram of 7. C) Ex-
tracted ion chromatogram of 21. The analysis was performed with the LTQ-Orbitrap, and
an internal recalibration was performed after acquisition by use of the monoprotonated
ion of 7.
should allow mutasynthesis studies related to those presented
Cyclo-l-Trp-l-His(Tr) (16): TMSI (150 mL, 6.0 equiv) was added at
08C to a solution of compound 15 (0.135 g, 0.17 mmol, 1.0 equiv)
in dry CH3CN (6 mL). The reaction mixture was allowed to warm to
room temperature over 3 h. The reaction was quenched with a so-
lution of aqueous saturated NaHCO3, and the mixture was extract-
ed with CH2Cl2. The organic layer was washed with water and
brine and then dried over MgSO4. After removal of the solvent, the
crude product was dissolved in dry MeOH (6 mL), and aq. NH3.
(0.6 mL) was added. The solution mixture was then stirred over-
night and concentrated under reduced pressure. Purification by
column chromatography (MeOH in CH2Cl2, 4%) yielded the purified
compound as an amorphous beige solid (0.043 g, 45%). 1H NMR
(400 MHz, CDCl3): d=8.78 (s, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.35–7.14
(m, 14H), 7.09–6.80 (m, 9H), 6.15 (s, 1H), 4.30–4.20 (brs, 1H), 4.15–
4.11 (brd, 1H), 3.52–3.46 (brdd, 1H; AB), 3.18–3.12 (brdd, 1H; AB),
2.99–2.95 (brdd, 1H; AB), 1.86–1.82 ppm (brt, 1H; AB); 13C NMR
(100, MHz, CDCl3): d=168.02, 167.30, 142.27, 138.92, 136.28,
136.21, 129.80, 128.23, 127.25, 124.49, 122.24, 120.07, 119.82,
118.97, 111.36, 109.14, 75.44, 55.13, 54.85, 31.67, 30.03 ppm; LC/
MS: 565.93 [M+H]+, 1130.80 [2M]+.
here, especially in the light of the diketopiperazine synthesis
procedures we have developed, and thus enable the synthesis
of both configurational and structural diketopiperazine ana-
logues.
Experimental Section
General: All reagents were commercial grade and were used as re-
ceived unless indicated otherwise. Dichloromethane was distilled
over phosphorus pentoxide. DMF, MeCN, MeOH, piperidine, and
NEt3 were stored over molecular sieves (4 Š). Reactions were moni-
tored by TLC (DC-Alufolien, Merck, Kieselgel 60, F254) with detec-
tion variously by UV absorption (254 nm), by spraying with a
solution of (NH4)6Mo7O24·4H2O (25 gLÀ1) and (NH4)4Ce(SO4)4·2H2O
(10 gLÀ1) in sulfuric acid (10%) followed by charring at ꢀ1508C, or
by spraying with an aqueous solution of KMnO4 (20%) and K2CO3
(10%). Column chromatography was performed on silica gel
(Screening Devices, 0.040–0.063 nm). LC/MS analysis was per-
formed with a LCQ Advantage Max (Thermo Finnegan) instrument
with a Gemini C18 column (Phenomene”). The solvents used were
H2O (A), MeCN (B), and aq. TFA (1.0%, C). 1H and 13C APT-NMR
spectra were recorded with Bruker AV 400 (400/100 MHz) instru-
ments with a pulsed field gradient accessory. Chemical shifts (d)
are given in ppm relative to tetramethylsilane as internal standard.
Coupling constants are given in Hz. All 13C-APT spectra presented
are proton-decoupled.
l,l-HTD (1): A solution of compound 17 (0.043 g, 0.07 mmol) in dry
CH2Cl2 (6 mL) was cooled to 08C. Then TFA (20%) and TIS (2.5%)
were added. The reaction mixture was concentrated in the pres-
ence of toluene and then stirred for 2 h. Purification by column
chromatography (10% MeOH in EtOAc) yielded the compound as
a white solid (0.023 g, 97%). Spectroscopic data were in accordance
with known literature values.[24] [a]D20 =À42 (c=4.06, H2O); 1H NMR
(400 MHz, D2O): d=8.26 (d, J=1.6 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H),
7.49 (d, J=8.0 Hz, 1H), 7.28 (dd, J=8.0, 1.2 Hz, 1H), 7.23–7.17 (m,
2H), 5.90 (d, J=1.6 Hz, 1H), 4.46 (dd, J=3.2, 1.2 Hz, 1H), 4.02 (dd,
J=4.8, 1.2 Hz, 1H), 3.46 (dd, J=14.8, 3.2 Hz, 1H; AB), 3.13 (dd, J=
14.8, 4.8 Hz, 1H; AB), 2.32 (dd, J=14.8, 4.4 Hz, 1H; AB), 1.37 ppm
(dd, J=14.8, 8.8 Hz, 1H; AB).); 13C NMR (100, MHz, D2O): d=169.68,
167.67, 135.85, 133.31, 127.37, 126.71, 125.70, 122.06, 119.72,
119.01, 117.35, 112.06, 107.62, 55.69, 53.09, 28.55, 28.30 ppm;
HRMS: m/z calcd for C17H17N5O2+H+: 324.1461 [M+H]+; found:
324.1452.
(l)-BocTrp(Boc)pentafluorophenyl ester (12): Pentafluorobenzo-
phenol (0.456 g, 2.48, 2.0 equiv) was added under argon to a solu-
tion of N-Boc-N’-Boc-l-tryptophan (0.5 g, 1.24 mmol, 1.0 equiv) in
freshly distilled CH2Cl2 (6.5 mL, 0.2m), followed by EDC·HCl
(0.474 g, 2.48 mmol, 2.0 equiv). The mixture was stirred at room
temperature overnight. The reaction was quenched with a solution
of HCl (1m), and the mixture was extracted with Et2O. The organic
layer was washed with brine and dried over MgSO4, and the sol-
vent was removed. The mixture was purified on silica (pentane/
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Dipeptide Boc-Trp(Boc)-d-His(Tr) methyl ester (28): Applying the
same procedure as described above for compound 15 yielded the
title compound as an amorphous beige solid (0.38 g, 99%).
1H NMR (400 MHz, CDCl3): d=8.10–8.05 (brs, 1H), 7.85–7.80 (brs,
1H; NH), 7.58 (d, J=7.6 Hz, 1H), 7.47–7.17 (m, 13H), 7.09–7.07 (m,
6H), 6.59 (s, 1H), 5.40–5.30 (brs, 1H; NH), 4.72–4.62 (brs, 1H),
4.50–4.40 (brs, 1H), 3.56 (s, 3H), 3.21–2.90 (m, 4H; AB), 1.61 (s,
9H), 1.35 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=171.17–
171.09, 165.64, 155.40, 149.54, 141.61, 135.03, 130.57, 128.68,
128.20, 124.33, 122.47, 119.87, 119.07, 115.58, 115.11, 83.39, 79.88,
54.56, 52.46, 52.24, 28.77, 28.23, 28.13 ppm; LC-MS: m/z: 798.13
[M+H]+, 1594.73 [2M]+.
111.37, 108.82, 55.64, 55.28, 39.95, 39.73 ppm; HRMS: m/z calcd for
C20H19O2N3 +H+: 334.1556 >[M+H]+; found: 334.1548.
Dipeptide Boc-Trp(Boc)-Tyr(OBn) methyl ester (31): Applying the
same procedure as described above for compound 15 yielded the
title compound as an amorphous white solid (0.38 g, 79%);
1H NMR (400 MHz, CDCl3): d=8.14 (d, J=8.0 Hz, 1H), 7.59 (d, J=
8.0 Hz, 1H), 7.46–7.22 (m, 8H), 6.78–6.74 (m, 4H), 6.25 (d, J=
6.0 Hz, 1H; NH), 5.12–5.05 (brs, 1H; NH), 4.98 (s, 2H), 4.72–4.67 (m,
1H), 4.44–4.38 (m, 1H), 3.62 (s, 3H), 3.23–3.06 (m, 2H; AB), 2.93 (d,
J=6.0 Hz, 2H; AB), 1.62 (s, 9H), 1.41 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3): d=171.37, 170.87, 158.04, 137.10, 130.33, 128.71,
128.10, 127.90, 127.65, 124.79, 124.57, 122.88, 122, 84, 119.23,
118.32, 115.48, 115.42, 114.99, 83.78, 70.09, 54.70, 53.44, 52.37,
37.23, 28.42, 28.33, 28.24 ppm.
Cyclo-l-Trp-d-His(Tr) (29): Applying the same procedure as de-
scribed above for compound 17 yielded the title compound as an
amorphous beige solid (0.15 g, 52%). 1H NMR (400 MHz, CDCl3):
d=8.91–8.40 (brs, 1H; NHindole), 7.54 (d, J=8.0 Hz, 1H), 7.43–7.40
(brs, 1H; NH), 7.32–7.20 (m, 11 H), 7.13–6.96 (m, 9H), 6.86–6.76
(brs, 1H; NH), 6.56 (s, 1H), 4.08–4.02 (brs, 1H), 3.75–3.69 (brs, 1H),
3.40–3.35 (brdd, 1H; AB), 3.18–3.02 (m, 2H; AB), 2.78–2.68 ppm
(brdd, 1H; AB); 13C NMR (100 MHz, CDCl3): d=168.20, 167.91,
142.24, 138.76, 136.45, 136.25, 129.74, 128.21, 127.06, 124.37,
122.25, 119.74, 118.76, 111.47, 109.10, 55.61, 54.75, 31.20,
30.45 ppm; LC-MS: m/z: 565.93 [M+H]+, 1130.80 [2M]+.
Cyclo-l-Trp-l-Tyr(OBn) (32): Applying the same procedure as de-
scribed above for compound 17 yielded the title compound as an
amorphous beige solid (0.10 g, 43%). 1H NMR (400 MHz,
[D6]DMSO): d=10.89 (s, 1H; NH), 7.87 (brs, 1H; NH), 7.66 (brs, 1H;
NH), 7.48 (d, J=8.0 Hz), 7.53–7.22 (m, 6H), 7.07 (t, J=8.0 Hz, 1H),
7.01–6.93 (m, 2H), 6.82 (d, J=8.4 Hz, 2H), 6.61 (d, J=8.4 Hz, 2H),
5.01 (s, 2H), 4.01–3.96 (brs, 1H), 3.78–3.82 (brs, 1H), 2.80 (dd, J=
14.4, 4.4 Hz, 1H; AB), 2.54–2.50 (m, 1H; AB), 2.42 (dd, J=13.6 Hz,
4.4 Hz, 1H; AB), 1.80 ppm (dd, J=13.6 Hz, 7.2 Hz, 1H; AB); 13C NMR
(100 MHz, [D6]DMSO): d=166.82, 166.24, 157.09, 137.18, 136.07,
130.74, 128.62, 128.38, 127.71, 127.53, 127.44, 124.42, 120.91,
118.72, 118.42, 114.42, 111.34, 108.88, 69.12, 55.75, 55.23, 38.89,
29.88 ppm.
l,d-HTD (2): The procedure was the same as described above for
compound 1. Purification on silica followed by HPLC gave the title
compound as a white solid (0.06 g, 73%). Spectroscopic data were
in accordance with known literature values.[24] [a]D20 =+18 (c=3.28,
H2O); 1H NMR (400 MHz, CD3OD): d=8.00 (s, 1H), 7.58 (d, J=
8.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.11–7.05 (m, 2H), 7.00 (t, J=
8.0 Hz, 1H), 6.79 (s, 1H,), 4.02 (t, J=4.0 Hz, 1H), 3.40 (dd, J=4.0,
14.4 Hz, 1H; AB), 3.20 (dd, J=4.0, 14.4 Hz, 1H; AB), 3.03 (t, J=
4.4 Hz, 1H), 2.94 (dd, J=4.4, 15.2 Hz, 1H; AB), 2.80 ppm (dd, J=
4.4, 15.2 Hz, 1H; AB); 13C NMR (100 MHz, CD3OD): d=169.46,
169.35, 135.67, 131.79, 128.84, 126.05, 125.98, 122.60, 120.18,
119.75, 118.89, 112.25, 109.04, 57.41, 54.67, 30.94, 29.57 ppm; LC-
MS: m/z: 324.07 [M+H]+; HRMS: m/z calcd for C17H17N5O2+H+:
324.1461 [M+H]+; found: 324.1450.
Cyclo-l-Trp-l-Tyr (6): Pd/C (0.08 g) was added to a solution of com-
pound 34 (0.081 g, 0.18 mmol) in EtOH (15 mL). After flushing with
H2 (balloon, three times), the reaction mixture was stirred over-
night under H2. After having been filtered on celite and concentrat-
ed, the crude product was purified on silica (MeOH in CH2Cl2, 5%)
to afford the purified compound as a white solid (0.028 g, 45%).
Spectroscopic data were in accordance with known literature
values.[28] 1H NMR (CD3OD): d=7.59 (d, J=8.0 Hz, 1H), 7.34 (d, J=
8.0 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 7.08–7.03 (m, 2H), 6.62 (d, J=
8.4 Hz, 2H), 6.46 (d, J=8.4 Hz, 2H), 4.20–4.15 (brs?, 1H), 3.85 (dd,
J=8.4, 3.6 Hz, 1H), 3.04 (dd, J=14.8, 4.4 Hz, 1H; AB), 2.78 (dd, J=
14.8, 4.4 Hz, 1H; AB), 2.56 (dd, J=13.6, 3.6 Hz, 1H; AB), 1.45 ppm
(dd, J=13.6, 8.4 Hz, 1H; AB); 13C NMR (CD3OD): d=169.66, 169.32,
157.54, 138.02, 131.97, 128.90, 127.70, 125.80, 122.66, 120.22,
119.95, 116.16, 112.49, 109.62, 57.87, 57.06, 40.57, 31.21 ppm;
HRMS: m/z calcd for C20H19O3N3 +H+: 350.1505 [M+H]+; found:
350.1498.
Dipeptide Boc-Trp(Boc)-Phe methyl ester (30): Applying the same
procedure as described above for compound 15 yielded the title
compound as an amorphous white solid (0.29 g, 69%). Spectro-
scopic data were in accordance with known literature values.[25]
1H NMR (400 MHz, CDCl3): d=8.13 (d, J=6.8 Hz, 1H), 7.59 (d, J=
7.6 Hz, 1H), 7.44 (s, 1H), 7.32 (t, J=7.2 Hz, 1H), 7.23 (t, J=7.2 Hz,
1H), 7.16 (m, 3H), 6.88 (m, 2H), 6.27 (d, J=6.4 Hz, 1H; NH), 5.09
(brs, 1H; NH), 4.72 (m, 1H), 4.43 (m, 1H), 3.62 (s, 3H), 3.13 (m, 2H),
2.98 (m, 2H; AB), 1.64 (s, 9H), 1.41 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3): d=171.27, 170.91, 135.65, 130.34, 129.27, 128.61, 127.22,
124.77, 124.52, 122.86, 119.19, 115.40, 83.76, 54.70, 53.14, 52.37,
38.05, 28.38, 28.31, 28.18 ppm.
Dipeptide Fmoc-d-Trp(Boc)-l-His(Tr)methyl ester (18): HATU
(0.216 g, 0.57 mmol, 1.0 equiv) was added to a solution of Fmoc-d-
Trp(Boc)COOH (0.300 g, 0.57 mmol, 1.0 equiv) in freshly distilled
CH2Cl2 (3 mL). After 15–30 min, a solution of l-His(Tr)OMe (0.255 g,
0.57 mmol, 1.0 equiv) and NEt3 (0.16 mL, 2.0 equiv) in freshly dis-
tilled CH2Cl2 (3 mL) was added dropwise. The reaction mixture was
stirred overnight. After removal of the solvent, the mixture was
purified on silica (MeOH in CH2Cl2, 1%) to afford the expected
compound as an amorphous white solid (0.51 g, 99%). 1H NMR
(400 MHz, CDCl3): d=8.10–8.05 (brs, 1H), 8.02–7.90 (brs, 1H; NH),
7.72 (m, 2H), 7.66–7.62 (brd, 1H), 7.56–7.48 (m, 3H), 7.40–7.16 (m,
16H), 7.08–7.04 (m, 6H), 6.53 (s, 1H), 5.80–5.70 (brs, 1H; NH), 4.72–
4.66 (brs, 1H), 4.60–4.52 (brs, 1H), 4.32–4.21 (m, 2H), 4.18–4.11
(brs, 1H), 3.55 (s, 3H), 3.21–3.18 (m, 2H; AB), 3.10–2.95 (brs, 1H;
AB), 2.79–2.75 (brs, 1H; AB), 1.58 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3): d=171.11–170.75, 156.03, 149.57, 143.87, 143.76, 141.79,
141.22, 129.69, 128.26, 128.19, 127.72, 127.11, 125.23, 124.60,
Cyclo-l-Trp-l-Phe (5): Applying the same procedure as described
above for compound 17 yielded the title compound as an amor-
phous beige solid (0.06 g, 36%). Spectroscopic data were in
accordance with known literature values.[26,27] 1H NMR (400 MHz,
[D6]DMSO): d=10.89 (s, 1H; NH), 7.91 (brs, 1H; NH), 7.70 (brs, 1H;
NH), 7.48 (d, J=8.0 Hz, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.19–7.15 (m,
3H), 7.07 (t, J=6.8 Hz, 1H), 7.00–6.95 (m, 2H), 6.71–6.69 (m, 2H),
3.97 (brs, 1H), 3.86 (brs, 1H), 2.80 (dd, J=14.4 Hz, 4.0 Hz, 1H; AB),
2.54–2.43 (m, 2H; AB), 1.84 ppm (dd, J=13.2 Hz, 6.8 Hz, 1H; AB);
13C NMR (100 MHz, [D6]DMSO): d=166.85, 166.23, 136.54, 136.07,
129.73, 128.07, 127.55, 126.41, 124.45, 120.93, 118.79, 118.47,
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124.47, 122.65, 119.95, 119.71, 119.06, 115.40, 115.26, 83.46, 67.28,
55.22, 52.63, 52.27, 47.05, 28.76–28.41, 28.18 ppm.
(100 MHz, CD3OD): d=170.89, 169.86, 137.95, 136.53, 129.21,
126.15, 122.66, 120.28, 120.21, 112.51, 109.54, 57.28, 55.95,
30.80 ppm; LC-MS: m/z: 324.07 [M+H]+; HRMS: m/z calcd for
C17H17N5O2 +H+: 324.1461 [M+H]+; found: 324.1449.
Cyclo-d-Trp(Boc)-l-His(Tr) (20): Dry piperidine (3 mL) was added to
a solution of compound 18 (0.51 g, 0.56 mmol) in dry DMF (3 mL).
The reaction mixture was stirred for 2 h, then quenched with
water, and extracted with EtOAc. The organic layers were com-
bined, washed with brine, and dried over MgSO4. After removal of
the solvent, the crude product was purified on silica (MeOH in
CH2Cl2, 5%) to afford the expected compound as an amorphous
white solid (0.23 g, 62%). 1H NMR (400 MHz, CDCl3): d=8.19–8.11
(brs, 1H), 7.57 (s, 1H; NH), 7.52–7.48 (m, 2H), 7.43–7.22 (m, 12H),
7.19 (t, J=7.6 Hz, 1H), 7.11–7.06 (m, 6H), 6.61 (s, 1H), 6.10 (s, 1H;
NH), 4.17–4.02 (m, 2H), 3.49 (dd, J=2.8, 14.0 Hz, 1H; AB), 3.14 (dd,
J=3.2, 15.2 Hz, 1H; AB), 3.97 (dd, J=2.8, 14.0 Hz, 1H; AB), 2.86
(dd, J=3.2, 15.2 Hz, 1H; AB), 1.66 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3): d=167.67, 167.11, 142.25, 138.85, 136.17, 129.78, 129.58,
128.28, 125.05, 122.99, 119.84, 119.01, 115.56, 114.52, 84.08, 55.12,
54.82, 31.40, 30.34, 28.30 ppm; LC-MS: m/z: 666.00 [M+H]+.
Dipeptide Fmoc-l-benzothiophenylalanine-l-His(Tr) methyl ester
(35): Applying the procedure as described above for compound 20
afforded the title compound as an amorphous white solid (0.30 g,
1
65%). H NMR (400 MHz, CDCl3): d=7.90 (s, 1H), 7.82–7.66 (m, 6H),
7.49–7.18 (m, 20H), 7.11–7.02 (m, 6H), 6.78 (s, 1H), 5.84 (d, J=
6.4 Hz, 1H; NH), 4.78–4.70 (m, 1H), 4.59–4.51 (m, 1H), 4.24–4.02
(m, 3H; FmocCHÀCH2), 3.55 (s, 3H), 3.39–3.31 (m, 1H; AB), 3.27–
3.05 ppm (m, 3H; AB); 13C NMR (100 MHz, CDCl3): d=171.59,
169.88, 165.68, 156.44, 143.64, 143.60, 141.13, 140.33, 139.98,
138.65, 136.08, 131.17, 130.50, 128.57, 128.94, 128.71, 128.64,
127.97, 127.85, 127.73, 127.14, 126.92, 125.27, 125.19, 124.68,
124.40, 124.27, 122.78, 121.56, 119.89, 78.33, 67.48, 55.07, 52.70,
52.04, 46.77, 30.51, 27.19 ppm.
Cyclo-l-benzothiophenylalanine-l-His(Tr) (36): Applying the pro-
cedure as described above for compound 22 afforded the title
compound as an amorphous white solid (0.19 g, 89%); 1H NMR
(400 MHz, CDCl3): d=7.80 (d, J=8.0 Hz, 1H), 7.67 (d, J=8.0 Hz,
1H), 7.60 (s, 1H; NH), 7.38–7.01 (m, 19H), 6.16 (s, 1H), 4.38–4.31
(m, 1H), 4.29–4.10 (m, 1H), 3.51–3.41 (m, 1H; AB), 3.37–3.25 (m,
1H; AB), 2.95–2.88 (m, 1H; AB), 1.82–1.71 ppm (m, 1H; AB);
13C NMR (100 MHz, CDCl3): d=168.11, 166.75, 142.12, 140.18,
138.78, 138.63, 135.81, 130.34, 129.69, 128.11, 125.71, 124.42,
124.35, 122.63, 122.06, 119.95, 75.39, 54.71–54.64, 32.52,
31.80 ppm.
d,l-HTD (3): The same procedure as described for l,l-HTD (1), fol-
lowed by purification by HPLC, yielded the title compound as
a white solid (0.10 g, 96%). Spectroscopic data were in accordance
1
with known literature values.[24] [a]D20 =À17 (c=4.84, H2O); H NMR
(400 MHz, CD3OD): d=7.57 (d, J=8.0 Hz, 1H), 7.51 (s, 1H), 7.32 (d,
J=8.0 Hz, 1H), 7.11–7.05 (m, 2H), 7.01 (t, J=8.0 Hz, 1H), 6.65 (s,
1H), 3.95 (t, J=4.0 Hz, 1H), 3.40–3.35 (m, 1H; AB), 3.15–3.08 (m,
2H; AB), 2.98–2.68 ppm (m, 2H; AB); 13C NMR (100 MHz, CD3OD):
d=170.89, 169.86, 137.95, 136.25, 128.78, 125.93, 122.56, 120.13,
119.72, 112.24, 109.14, 57.20, 55.23, 30.80 ppm; LC-MS: m/z: 324.07
[M+H]+; HRMS: m/z calcd for C17H17N5O2 +H+: 324.1461 [M+H]+;
found: 324.1449.
Cyclo-l-benzothiophenylalanine-l-His (7): Applying the procedure
as described for l,l-HTD (1), followed by HPLC purification, yielded
the title compound as a white solid (0.066 g, 62%). [a]2D0 =À33 (c=
11.8, H2O); 1H NMR (400 MHz, D2O): d=8.30 (d, J=1.2 Hz, 1H),
8.00–7.94 (m, 1H), 7.84–7.79 (m, 1H), 7.52–7.44 (m, 2H), 7.37 (s,
1H), 6.13 (d, J=1.2 Hz, 1H), 4.52 (dd, J=1.2, 4.8 Hz, 1H), 4.07 (dd,
J=1.2, 4.8 Hz, 1H), 3.52 (dd, J=4.8, 15.2 Hz, 1H; AB), 3.26 (dd, J=
4.8, 15.2 Hz, 1H; AB), 2.39 (dd, J=4.8, 15.2 Hz, 1H; AB), 1.67 ppm
(dd, J=8.0, 15.2 Hz, 1H; AB); 13C NMR (100 MHz, D2O): d=169.11,
167.46, 139.84, 138.75, 133.40, 129.63, 127.10, 126.64, 124.76–
124.67, 123.18, 122.18, 177.33, 55.13, 53.16, 31.09, 28.28 ppm;
HRMS: m/z calcd for C17H16N4O2S+H+: 341.1072 [M+H]+; found:
341.1076.
Dipeptide Fmoc-d-Trp(Boc)-d-His(Tr) methyl ester (33): The same
procedure as described above for compound 20 afforded the title
compound as an amorphous white solid (0.40 g, 99%); 1H NMR
(400 MHz, CDCl3): d=8.10–8.05 (m, 1H), 7.74–7.12 (m, 23H), 7.08–
7.04 (m, 6H), 6.59 (s, 1H), 6.05–5.97 (brs, 1H; NH), 4.80–4.71 (m,
1H), 4.64–4.55 (m, 1H), 4.29–4.04 (m, 3H; FmocCHÀCH2), 3.55 (s,
3H), 3.28–3.15 (m, 2H; AB), 3.05–3.02 (m, 2H; AB), 1.61 ppm (s,
9H); 13C NMR (100 MHz, CDCl3): d=171.10–170.63, 156.09, 149.56,
143.87, 143.76, 141.79, 141.22, 129.69, 128.26, 128.19, 127.72,
127.11, 125.23, 124.60, 124.47, 122.65, 119.95, 119.71, 119.06,
115.40, 115.26, 83.51, 67.24, 55.22, 52.63, 52.27, 47.05, 28.76–28.41,
28.18 ppm.
Dipeptide Fmoc-l-naphthylalanine-l-His(Tr) methyl ester (37):
Applying the procedure as described above for compound 20 af-
forded the title compound as an amorphous white solid (0.17 g,
58%). 1H NMR (400 MHz, CDCl3): d=7.81–7.68 (m, 7H), 7.60–7.49
(brs, 1H; NH), 7.47–7.18 (m, 23H), 7.09–7.03 (m, 6H), 6.61 (s, 1H),
5.61–5.59 (brs, 1H; NH), 4.82–4.76 (m, 1H), 4.67–4.59 (m, 1H),
4.31–4.24 (m, 1H; FmocCHÀCH2), 4.19–4.07 (m, 2H; FmocCHÀCH2),
3.52 (s, 3H), 3.49–3.42 (m, 1H; AB), 3.21–3.17 (m, 1H; AB), 3.08–
3.01 ppm (m, 2H; AB); 13C NMR (100 MHz, CDCl3): d=171.10,
170.59, 156.25, 143.85–143.81, 141.31–141.28, 133.86, 133.62,
132.59, 129.76, 128.85–127.02, 126.29–125.23, 120.63, 120.00, 67.47,
56.15, 52.56, 52.41, 47.06, 38.47, 28.05 ppm; LC-MS: m/z: 831.13
[M+H]+, 1661.80 [2M]+.
Cyclo-d-Trp(Boc)-d-His(Tr) (34): Applying the procedure as de-
scribed above for compound 22 afforded the title compound as an
amorphous white solid (0.14 g, 57%). 1H NMR (400 MHz, CDCl3):
d=8.11 (d, J=8.0 Hz, 1H), 7.55–7.46 (m, 3H), 7.40–7.30 (m, 10H),
7.23 (t, J=8.0 Hz, 1H), 7.16–7.08 (m, 7H), 6.48 (s, 1H), 5.95 (s, 1H;
NH), 4.32–4.29 (m, 1H), 4.23–4.20 (m, 1H), 3.59–3.52 (m, 1H; AB),
3.22–3.15 (m, 1H; AB), 3.02–2.96 (m, 1H; AB), 2.50–2.40 (m, 1H;
AB), 1.66 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=167.80,
166.99, 142.22, 138.95, 136.17, 129.83, 129.58, 128.28, 125.07,
123.03, 119.76, 119.01, 115.58, 114.59, 84.08, 55.05, 54.15, 31.03,
29.47, 28.31 ppm.
d,d-HTD (4): Applying the procedure as described for l,l-HTD (1),
followed by HPLC purification, yielded the title compound as
a white solid (0.14 g, 31%). Spectroscopic data were in accordance
Cyclo-l-naphthylalanine-l-His(Tr) (38): Applying the procedure as
described above for compound 22 afforded the title compound as
an amorphous white solid (0.07 g, 58%). 1H NMR (400 MHz,
CD3OD): d=7.62–7.50 (m, 3H), 7.42–7.32 (m, 9H), 7.28–7.21 (m,
3H), 7.19–7.14 (m, 1H), 7.11–7.03 (m, 6H), 6.96–6.91 (m, 1H), 5.59
(s, 1H), 4.39–4.33 (brs, 1H), 3.99–3.95 (m, 1H), 3.47–3.30 (m, 1H;
AB), 3.12–3.04 (m, 1H; AB), 2.16 (dd, J=4.4, 14.0 Hz, 1H; AB), 0.64–
1
with known literature values.[24] [a]D20 =+35 (c=2.56, H2O); H NMR
(400 MHz, CD3OD): d=7.63 (d, J=7.6 Hz, 1H), 7.48 (s, 1H), 7.37 (d,
J=8.0 Hz, 1H), 7.17–7.04 (m, 3H), 5.79 (s, 1H), 4.26–4.20 (m, 1H),
3.92–3.86 (m, 1H), 3.34–3.22 (m, 1H; AB), 3.18–3.09 (m, 1H; AB),
2.48–2.41 (m, 1H; AB), 1.12–1.06 ppm (m, 1H; AB); 13C NMR
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0.56 ppm (m, 1H; AB); LC-MS: m/z: [M+H]+ =577.00; [2M]+
1152.93.
=
[7] D. P. Overy, K. F. Nielsen, J. Smedsgaard, J. Chem. Ecol. 2005, 31, 2373–
2390.
[8] H. Ali, M. Ries, J. Nijland, P. Lankhorst, T. Hankemeier, R. Bovenberg, A.
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Cyclo-l-naphthylalanine-l-His (8): Applying the procedure as de-
scribed for l,l-HTD (1), followed by HPLC purification, yielded the
title compound as a white solid (0.019 g, 49%)); [a]2D0 =À39.7 (c=
1
2.82, H2O); H NMR (400 MHz, D2O): d=7.95–7.85 (m, 3H), 7.74 (d,
J=0.8 Hz, 1H), 7.64–7.53 (m, 3H), 7.28 (dd, J=2.0, 8.4 Hz, 1H), 6.38
(s, 1H), 4.55 (dd, J=3.6, 4.8 Hz, 1H), 4.13 (dd, J=4.8, 6.8 Hz, 1H),
3.37 (dd, J=3.6, 14.0 Hz, 1H; AB), 3.10 (dd, J=4.8, 14.0 Hz, 1H;
AB), 2.31 (dd, J=4.8, 15.6 Hz, 1H; AB), 1.81 ppm (dd, J=6.8,
15.6 Hz, 1H; AB); 13C NMR (100 MHz, D2O): d=169.06, 167.59,
133.02, 132.98, 132.41, 132.23, 129.07, 128.76, 128.06, 127.72,
127.70, 126.78, 126.58, 126.50, 117.29, 55.66, 53.10, 37.98,
27.76 ppm; HRMS: m/z calcd for C19H18N4O2 +H+: 335.1508
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Acknowledgements
This work was supported by the Perspective Genbiotics program
subsidized by STW (Stichting Technische Wetenschappen).
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