Total Synthesis of Microcystin-LF and Derivatives Thereof

Article abstract of DOI:10.1021/acs.joc.7b00175

Microcystins (MCs) are highly toxic natural products which are produced by cyanobacteria. They can be released to the water during harmful algal blooms and are a serious threat to animals and humans. Described is the total synthesis of the cyanotoxin micr

Full text of DOI:10.1021/acs.joc.7b00175

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Article
Total Synthesis of Microcystin-LF and Derivatives Thereof
Ivan Zemskov, Stefan Altaner, Daniel R. Dietrich, and Valentin Wittmann
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b00175 • Publication Date (Web): 15 Mar 2017
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Total Synthesis of Microcystin-LF and Derivatives
Thereof
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Ivan Zemskov,† Stefan Altaner,‡ Daniel R. Dietrich,‡ and Valentin Wittmann†,*
†Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB),
University of Konstanz, 78457 Konstanz (Germany)
‡Department of Biology and Graduate School Biological Sciences (GBS), University of
Konstanz, 78457 Konstanz (Germany)
E-mail: mail@valentin-wittmann.de
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ABSTRACT: Microcystins (MCs) are highly toxic natural products which are produced by
cyanobacteria. They can be released to the water during harmful algal blooms and are a serious
threat to animals and humans. Described is the total synthesis of the cyanotoxin microcystin-
LF (MC-LF, 1a) and two derivatives thereof. Deuterated derivative 1b is of interest as an
internal standard during MC quantification in biological samples by mass spectrometry and
alkyne-labeled 1c can be employed for toxin derivatization by click chemistry with an azide-
containing reporter molecule or as an activity-based probe to identify interaction partners.
Application of tert-butyl ester protecting groups for erythro--D-methylaspartic acid and -D-
glutamic acid were key for an isomerization-free synthesis. The analytical data of synthetic
MC-LF were identical to those of an authentic sample of the natural product. All derivatives
1a-c were determined to be potent inhibitors of protein phosphatase-1 with similar activity.
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Introduction
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Microcystins (MCs) are highly toxic natural products which are produced by cyanobacteria and,
according to the WHO, are amongst the most dangerous water pollutants.1 Widespread harmful
algal blooms in densely populated areas, which are favored by global warming along with
eutrophication of surface waters,2 resulted in a shutdown of the public water supply for millions
of people, for example in Toledo, Ohio, USA, (August 2014) and around lake Taihu in China
(July 2007).2-3 The sole analytical method for congener-specific MC detection enabling the
identification and quantification of MCs is liquid chromatography-mass spectrometry/mass
spectrometry (LC-MS/MS).2b,4 However, the lack of certified reference compounds limits the
applicability of this method.2b Although research on MCs has been conducted since 1878,5 their
physiological function in cyanobacteria is still under scientific debate.6 Consequently, the total
synthesis of MCs would provide access to a yet missing variety of reference compounds as well
as microcystin derivatives for structure-activity relationship studies enabling future studies into
the biological roles of MCs. Furthermore, the high cytotoxicity of MCs makes this scaffold an
attractive lead structure for the development of novel potent anticancer drugs.
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Figure 1. Microcystin-LF (MC-LF) and some amino acid variations (grey) of naturally
occurring congeners. The two amino acids indicated in the name (LF in the current example)
denote the natural L-amino acids in positions 2 and 4. Adda = (2S,3S,4E,6E,8S,9S)-3-amino-9-
methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, D-Ala = D-alanine, -D-Glu = -D-
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glutamic acid, Mdha = N-methyldehydroalanine, -D-MeAsp = erythro--D-methylaspartic
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acid.
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As shown in Figure 1, MCs consist of three D-amino acids in positions 1, 3, and 6, the -amino
acid Adda5, and N-methyldehydroalanine (Mdha7) which is involved in covalent binding of
MCs to protein phosphatases by Michael addition of a proximate cysteine residue.7 Two L-
amino acids in positions 2 and 4 are variable and count for the major differences between the
more than 100 individual congeners, that have been reported so far.8 Despite numerous
attempts9 the total synthesis of a natural microcystin (MC-LA) was accomplished only once.10
This landmark achievement featured protection of -D-MeAsp3 and -D-Glu6 by methyl esters
that were cleaved under basic conditions in the last synthetic step. Unfortunately, this treatment
led to the formation of several uncharacterized isomers of the final product.10 In our
interpretation the isomerization can be attributed to a cyclization involving -D-MeAsp3 (I,
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Scheme 1) leading to aspartimide II which is prone to isomerization at the chiral centers in -
and -positions. Ring opening by basic hydrolysis can lead to the stereoisomers III of desired
MC-LA as well as constitutional isomers IV. Despite extensive investigation of different
methyl ester cleavage conditions, the isomerization could not be avoided and resulted in
significant loss of material.10 To identify natural MC-LA, the isomeric mixture was separated
by HPLC and the isomers were compared to an authentic sample of the cyanotoxin.10 The
identification of the desired isomers was particularly intricate during the synthesis of unnatural
derivatives for which no standard was available.11
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Scheme 1. Proposed formation of aspartimide II and subsequent isomerization during the
saponification of methyl ester protected MC-LA I
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Here, we present an isomerization-free synthetic strategy and its application to the first total
synthesis of MC-LF (1a) and its deuterated derivative 1b (Scheme 2) which is of interest as an
internal standard during MC quantification by mass spectrometry. Since this approach delivers
only one isomer, the synthesis of unnatural MC derivatives becomes feasible which we
demonstrate by the preparation of derivative 1c. The alkyne-labeled 1c can be employed for
toxin derivatization by click chemistry with an azide-containing reporter molecule, e.g. biotin
or a fluorescent dye and, furthermore, be used as an activity-based probe to identify interaction
partners.12 According to crystal structures of MCs in complex with protein phosphatases13 the
side chain at position 4 of the MC backbone is not involved in binding and thus represents an
optimal position for synthetic modifications.
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Results and Discussion
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Scheme 2. Retrosynthetic analysis of MC derivatives 1a-c
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Retrosynthetic Analysis. For the synthesis of 1a-c we followed a fragment-based strategy
using tert-butyl esters as protecting groups for -D-MeAsp3 and -D-Glu6 in order to suppress
aspartimide formation.14 In contrast to the previous approach,10 in which N-
methylphosphonylsarcosine was incorporated and subsequently converted into Mdha7 by a
Horner-Wadsworth-Emmons reaction, we incorporated
N-methylphenylselenocysteine
(NMeSecPh) as Mdha precursor that was transformed into Mdha in the final step of the
synthesis. For the macrocyclization, we decided to use the peptide bond between residues 4 and
5 which is also the cyclization site during biosynthesis of MCs.15 In addition, this disconnection
has proven reliable in the former synthetic approach.10
The linear precursors 2a-c were synthesized in a convergent manner from fragments 3, 4 and
5a-c. Several syntheses of Boc-Adda-OH 3 have been reported.9a,16 We followed the route
developed by Pearson et al. that delivers 3 in 13 steps with the best overall yield.16a Fmoc groups
served as N-terminal protection of fragments 4 and 5a-c. The presence of Fmoc, tert-butyl ester
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and the phenylselenocysteine moiety in fragments 4 and 5a-c required a C-terminal protection
which can be cleaved in presence of these three groups and that withstands the conditions of
Fmoc deprotection. In our approach we used the phenacyl (Pac) protecting group which is
removed under mild reductive conditions.17 Tetrapeptide 4 and dipeptides 5a-c were obtained
from NMeSecPh derivative 6,18 -D-MeAsp derivative 7 and the building blocks 8a,17 8b, and
8c.
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Scheme 3. Synthesis of tetrapeptide fragment 4a
aHATU represents 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate.
Synthesis of Tetrapeptide 4. We started our synthesis with the preparation of tetrapeptide
fragment 4 (Scheme 3). Boc-NMeSecPh-OH 6 was synthesized starting from Boc-N-methyl-
L-serine 9 using the strategy developed by van der Donk and coworkers for the synthesis of
Boc-SecPh-OH19 and coupled with dipeptide 11 to give 12.18 Tripeptide 12 was then
deprotected with TFA and coupled with Fmoc-D-Glu-Ot-Bu to give fragment 4.
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Scheme 4. Synthesis of Fmoc-D-MeAsp-Ot-Bu 7
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Synthesis of Dipeptides 5a-c. In position 3 of the vast majority of the MCs erythro--methyl
D-aspartic acid (D-MeAsp) is found. Up to date several synthetic routes towards derivatives of
this amino acid have been published.10,16e-g,20 However, all these published approaches feature
methyl or ethyl ester protecting groups, which are crucial for the generation of the stereocenter
at the -carbon atom. Since the literature-known building blocks cannot be easily transformed
into 7, we synthesized Fmoc-D-MeAsp-Ot-Bu 7 starting from D-aspartic acid 13 as shown in
Scheme 4. The amino group of 13 was benzylated by reductive amination and the -carboxy
group was regioselectively esterified under acidic conditions to give benzyl ester 14. The free
-carboxy group of 14 was tert-butylated and subsequently the secondary amine was protected
with the phenylfluorenyl (PhFl) group to give 15. This sterically hindered group suppresses
deprotonation at the -carbon and allows enolate formation by selective deprotonation at the -
position of 15 in the subsequent step. Thus, treatment of 15 with lithium
bis(trimethylsilyl)amide (LHMDS) followed by addition of methyl iodide led to exclusive
methylation of the -position to give erythro-16 and threo-16 in a ratio of 5:1. The
stereoselectivity of this step can be explained by preferred formation of the (Z)-lithium enolate
which adopts a hydrogen-in-plane conformation that is attacked opposite to the bulky nitrogen
protecting groups.21 The isomers erythro-16 and threo-16 were readily separated by column
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chromatography and the configuration of erythro-16 was verified by complete deprotection and
subsequent NMR analysis22 (see Supporting Information) as well as optical rotation
measurement.20 Hydrogenolysis of erythro-16 followed by Fmoc protection of the free amino
group gave building block 7.
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Scheme 5. A) Synthesis of alkyne labeled building block 8c. B) Synthesis of dipeptide
fragments 5a-cb
bFor definitions of a-c, see Scheme 2
The phenacyl protected propargyl tyrosine building block Boc-Tyr(Prg)-OPac 8c (Prg =
propargyl) was synthesized in three steps starting from Boc-Tyr-OH 17 (Scheme 5A). Double
propargylation of 17 followed by saponification of the propargyl ester gave 18 that was
alkylated with phenacyl bromide yielding 8c in a high yield. 8c as well as literature known 8a17
and analogously prepared, isotopically labeled 8b were N-terminally deprotected and coupled
with D-MeAsp derivative 7 to give dipeptides 5a-c (Scheme 5B).
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Scheme 6. Synthesis of linear heptapeptides 22a-c by fragment couplingsb
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bFor definitions of a-c, see Scheme 2
Synthesis of Linear Heptapeptides. With all fragments in hand, heptapeptides 2a-c were
assembled (Scheme 6). The C-terminal phenacyl ester of tetrapeptide 4 was reductively cleaved
to give 19. Fmoc deprotection of dipeptides 5a-c was performed with diluted (5%) piperidine
in DMF to avoid possible cleavage of the Pac ester giving 20a-c. The subsequent fragment
coupling of 19 and a slight excess of 20a-c was performed with HATU/collidine leading to 21a-
c. Under these conditions isomerization at the C-terminal Leu residue of fragment 19 is
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minimized as it was also reported by others.10,23 The hexapeptides 21a-c were N-terminally
deprotected and Boc-Adda-OH 3 was coupled to give heptapeptides 2a-c. Removal of the Pac
group led to macrocyclization precursors 22a-c. We found that it was essential to purify
compounds 22a-c by RP-HPLC to prevent by-product formation during the subsequent
macrocyclization step.
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Scheme 7. Deprotection and macrocyclization
Macrocyclization and Final Steps. The C-termini of 22a-c were activated as
pentafluorophenyl (Pfp) ester and the t-butyl esters along with the Boc group were removed by
TFA treatment (Scheme 7). Macrocyclization was induced under basic conditions applying a
two-phase system of chloroform and phosphate buffer (pH = 9.5) to give cyclopeptides 23a-c
which were purified by RP-HPLC. According to HPLC approximately 20% (in case of 23a and
23b) and 10% (in case of 23c), respectively, of an isomeric product, possibly due to
epimerization at C-terminal amino acid during macrocyclization, were observed. Subsequent
selenoxide elimination under mild oxidative conditions gave the desired MC derivatives 1a-c.
All final products were fully characterized by one- and two-dimensional NMR spectroscopy
and HRMS. The analytical data of synthetic MC-LF (1a) were identical to those of a
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commercial MC-LF sample confirming the structure of the synthetic material. In addition, co-
injection of both a mixture of natural and synthetic 1a as well as a mixture of 1a and 1b led to
single peaks in the LC-MS chromatograms (Supporting Information).
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Figure 2. PP1 inhibition assay with natural MC-LF and synthetic compounds 1a, 1c, and 23c
(values from three independent experiments). APP1 = activity of PP1
Inhibition of Protein Phosphatase-1. In order to confirm that the biological activities of
synthetic and natural MC-LF are identical, we determined the potency of both compounds to
inhibit the hydrolysis of p-nitrophenyl phosphate catalyzed by protein phosphatase-1 (PP1,
Figure 2). As expected, the IC50 values obtained with this robust assay24 are very similar within
the accuracy of this assay (synthetic 1a: IC50 = 870 pM, natural 1a: IC50 = 1.2 nM) further
verifying the authenticity of the synthetic material. We also evaluated the inhibitory potency of
the propargylated derivative 1c and its precursor 23c lacking the Michael acceptor Mdha. The
similar IC50 value of 1c (IC50 = 1.7 nM) shows that the modification at position 4 has only a
minor influence on phosphatase binding and inhibition as expected from the crystal structure
of PP1 in complex with MC-LR.13b Phenylselenocystein derivative 23c, lacking the capability
to covalently bind to PP1, has a similar inhibitory potency (IC50 = 2.1 nM). Our results show
that neither the modification at position 4 nor the one at position 7 of the scaffold disrupt PP1
inhibition. The fact that the absence of the Michael acceptor in compound 23c does not result
in reduced inhibition potency is in line with previous findings that have shown that covalent
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binding of the toxin to PP1 is a slow process and not required for phosphatase inhibition which
is rather achieved by the initial fast non-covalent interaction. However, it is remarkable that the
large side chain of NMeSecPh is tolerated during this binding process, thereby dramatically
expanding the application domain of derivatized MCs.
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Conclusion
In summary, we developed a novel strategy for the synthesis of MCs. Application of the Fmoc/t-
Bu protecting group strategy enabled the isomerization-free synthesis of MC-LF as well as a
deuterated and an alkyne-labeled derivative thereof. The incorporation of
a
phenylselenocysteine moiety as precursor for the Mdha residue gave access to a novel potent
and reversible phosphatase inhibitor that cannot undergo covalent binding to the protein. The
synthesized MC derivatives can be broadly applied for improved MC detection and
quantification and pave the way for future approaches to understanding the biological roles of
MCs in various organisms.
Experimental Section
General Experimental Methods. Technical solvents (petroleum ether and EtOAc) were
distilled prior to use. THF and CH2Cl2 were distilled from Na/K or CaH2 under inert atmosphere
immediately prior to use. Peptide grade DMF was used for peptide couplings. A sample of
natural microcystin-LF was obtained from Enzo Life Sciences (ALX-350-081-C100, 100 g)
and used as received. The compounds 3,16a 6,18 8a,17 and 10,18 were synthesized according to
the literature. For the reactions performed under inert gas conditions (nitrogen) Schlenk
technique and oven dried glassware were used. Analytical thin layer chromatography (TLC)
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was performed using TLC silica gel 60 F254 coated aluminum sheets (Merck). Spots were
visualized either by UV light (= 254 nm) or by dipping and heating using ethanolic ninhydrin
solution (3 % w/v), aqueous potassium permanganate (1 % w/v), anisaldehyde solution (135
mL EtOH, 5 mL conc. H2SO4, 15 mL glacial acetic acid, and 3.7 mL p-anisaldehyde), or
Seebach’s stain (25 g phosphomolybdic acid, 10 g Ce(SO4)2 · 4H2O, and 60 mL conc. H2SO4
in 1 L H2O). Preparative flash column chromatography (FC) was performed using Geduran 60
silica gel (40-60 m, Merck). NMR spectra were recorded on Bruker Avance III 400 or Bruker
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Avance III 600 instruments. Chemical shifts  are reported in ppm relative to solvent signals
(CDCl3 H = 7.26 ppm, C = 77.2 ppm; CD3OD H = 3.34 ppm; C = 49.0 ppm; DMSO-d6 H =
2.50 ppm, C = 39.5 ppm). For all new compounds two-dimensional NMR experiments (COSY,
HSQC, and HMBC) were used for signal assignments. For numbering of carbons see supporting
information.
Semi-preparative and analytical RP-HPLC was performed using a Shimadzu LC-20A
prominence system (LC-20AT pumps, SIL-20A auto sampler, CTO-20AC column oven, SPD-
M2OA PDA detector, CBM-20A communication bus module and LC-Solution software). A
Kinetex 5u C18 100A, AXIA (250 x 21.2 mm, Phenomenex) column was used as a stationary
phase at a flow rate of 10 mL min–1 unless mentioned otherwise. LC-MS measurements were
performed on a Shimadzu LCMS-2020 system (LC-20 AD high pressure pumps, SIL-20AT
HAT autosampler, CTO-20AC column oven, SPD-20A UV-Vis detector, CBM-20A
communication bus module, LCMS-2020 ESI-detector and LC-MS-Solution software) using a
Nucleodur 100-3 C18ec (4 x 125 mm, Macherey-Nagel) reversed phase column as stationary
phase at a flow rate of 0.4 mL min–1. A gradient of water with 0.1 % formic acid (mobile phase
A) and acetonitrile with 0.1 % formic acid (mobile phase B) was used for the HPLC and LC-
MS separations. Elemental analyses were performed by the microanalytical laboratory of the
University of Konstanz using an Elementar vario EL instrument. HRMS measurements were
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General Procedures
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GP1: Phenacyl protection.17 The amino acid derivative (1.0 equiv) is dissolved in EtOAc.
Then Et3N (1.1 equiv) and phenacyl bromide (1.1 equiv) are added and the reaction mixture is
stirred at rt for 4h. During the reaction, the formation of a white precipitate can be observed.
After 4h the mixture is diluted with EtOAc and washed with brine (1x), sat. NaHCO3 solution
(1x), and brine (2x). The organic phase is dried over Na2SO4, filtered, and concentrated under
reduced pressure.
GP2: Phenacyl deprotection. The protected peptide is dissolved in MeOH/DMF (8:2, 8 L
mg-1 peptide). Then Mg-turnings (0.16 mg/mg peptide) and acetic acid (0.8 L mg-1 peptide)
are added and the reaction mixture is cooled to 0 °C. After 20 min cooling is removed and the
reaction mixture is stirred at rt for 30 min. The reaction mixture is filtered, the volatiles are
removed under reduced pressure, and the residue is taken up in CH2Cl2/MeOH (9:1). The
mixture is filtered through 3 cm3 silica column which is extensively washed with
CH2Cl2/MeOH (9:1). The solvents are removed under reduced pressure and the residue is
lyophilized. The obtained deprotected peptide is used without further purification.
GP3: Fmoc deprotection. The peptide is dissolved in 5% piperidine in DMF (v/v). After 3
min, the solution is diluted with DMF and the volatiles are removed under reduced pressure.
The residue is coevaporated with toluene (3x) and used without further purification.
GP4: Boc/t-Bu deprotection. The starting material is dissolved in neat TFA and stirred at rt
for 30 min. Subsequently the TFA is removed and the residue is coevaporated with toluene,
EtOAc and petroleum ether (each 1x) and used without further purification.
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GP5: Fmoc protection. The amino acid (1.0 equiv) is suspended in Na2CO3 (10% w/v, 1.25
mL mmol-1 amino acid). Then Fmoc-OSu (1.3 equiv) in dioxane (1.875 mL mmol-1 amino acid)
is added, and the mixture is stirred at rt overnight. The reaction mixture is diluted with H2O and
EtOAc, acidified with aqueous NaHSO4 (1M) to pH < 3, and extracted with EtOAc (3x). The
combined organic phases are washed with brine (1x), dried over Na2SO4, filtered, and the
volatiles removed under reduced pressure. The crude product is purified with FC.
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GP6: Peptide coupling. The amine and carboxylic acid are dissolved in DMF. The resulting
solution is cooled to 0 °C. Then base (DIPEA or collidine) and after 5 min the coupling reagent
(HATU or HBTU) are added. The resulting yellowish solution is stirred at 0 °C for 45 min and
at rt overnight. The reaction mixture is diluted with EtOAc and washed subsequently with H2O,
1:1 diluted saturated citric acid solution, 1:1 diluted saturated NaHCO3 solution, and brine (each
1x). The organic phase is dried over Na2SO4, filtered, and purified with FC.
GP7: Macrocylization. The HPLC-purified C-terminally deprotected heptapeptide (1.0 equiv)
is placed in a 4 mL glass vial. Then pentafluorophenol (1.95 equiv), dissolved in freshly distilled
EtOAc (24.2 mL mmol-1 peptide) and solid DCC are added. The reaction mixture is cooled to
0 °C, stirred for 90 min and then at rt for 8-10h. Afterwards, the solvent is removed and the
activated peptide is treated with TFA according to GP4. The resulting C-terminally activated
unprotected heptapeptide is dissolved in CHCl3 (515 mL mmol-1 peptide). The resulting
solution is added dropwise over a period of 10 min to a vigorously mechanically stirred mixture
of CHCl3 (875 mL mmol-1 peptide) and pH = 9.5 phosphate buffer (1M, 875 mL mmol-1
peptide). After 30 min, the reaction mixture is diluted with a small amount of CHCl3 and H2O.
The phases are separated, the aqueous phase is acidified with NaHSO4 (1M) to pH < 3 and
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extracted with EtOAc (3x). The organic phases are combined, washed with brine, and dried
over Na2SO4. The crude macrocyclic heptapeptide is lyophilized and purified using HPLC.
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GP8: Selenoxide elimination. The macrocyclic peptide is dissolved in MeCN/H2O (3:2, 1 mL)
and 30% aqueous H2O2 (4 L) is added. After 1h at rt, the reaction mixture is quenched with
Me2S (50 L) and purified by HPLC.
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Synthesized Compounds (Sorted According to Compound Number)
Microcystin-LF (1a). The C-terminally unprotected heptapeptide 22a (15 mg, 10.9 mol)
was macrocyclized using pentafluorophenol (4.0 mg, 21.3 mol) and DCC (2.8 mg, 13.3 mol)
according to GP7. The crude macrocycle 23a was purified by semi-preparative RP-HPLC
(gradient: 60-90% B in 30 min, tR = 13.3 min). The cyclic peptide 23a was transformed to 1a
according to GP8 and the reaction mixture was separated by semi-preparative RP-HPLC
(gradient: 50-70% B in 20 min, tR = 13.2 min). MC-LF 1a was obtained as a white amorphous
solid (2.0 mg, 19%). LC-MS analysis of synthetic 1a coinjected with natural MC-LF resulted
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in a single peak with the expected mass (Figure S3). H NMR (600 MHz, CD3OD, 300 K)
 8.89 (d, J = 9.6 Hz, 1H, NH MeAsp), 8.17 – 8.08 (m, 2H, NH Leu, NH Phe), 7.46 (d, J = 8.2
Hz, 1H, NH Ala), 7.31 (d, J = 9.2 Hz, 1H, NH Adda), 7.27 – 7.25 (m, 2H, Ar), 7.23 – 7.14 (m,
8H, Ar), 6.35 (d, J = 15.5 Hz, 1H, H-5 Adda), 5.89 (s, 1H, C=CH2), 5.51 (d, J = 9.8 Hz, 1H, H-
7 Adda), 5.45 (s, 1H, C=CH2), 5.43 (dd, J = 15.5, 8.6 Hz 1H, H-4 Adda), 4.74 – 4.69 (m, H-3
Adda), 4.58 – 4.49 (m, 3H, H- MeAsp, H- Phe, H- Ala), 4.45 (t, J = 7.4 Hz, 1H, H- Glu),
4.22 – 4.18 (m, 1H, H- Leu), 3.50 (dd, J = 14.1, 3.2 Hz, 1H, H- Phe), 3.38 (s, 3H, -NCH3),
3.30 – 3.27 (m, 1H, H-9 Adda), 3.26 (s, 3H, -OCH3), 2.93 (dq, J = 7.2, 3.3 Hz, 1H, H- MeAsp),
2.84 (dd, J = 14.0, 4.8 Hz, 1H, H-10 Adda), 2.74 – 2.68 (m, 2H, H-10 Adda, H-2 Adda), 2.66
– 2.55 (m, 4H, H-8 Adda, 2x H- Glu, H- Phe), 2.19 – 2.11 (m, 1H, H- Glu), 1.91 – 1.83 (m,
1H, H- Leu), 1.80 – 1.68 (m, 2H, H- Glu, H- Leu), 1.65 (s, 3H, 3xH-6’ Adda), 1.53 (ddd, J
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= 13.9, 9.8, 4.2 Hz, 1H, H- Leu), 1.11 (d, J = 6.9 Hz, 3H, 3xH-2’ Adda), 1.04 (d, J = 6.7 Hz,
3H, 3xH-8’ Adda), 0.99 (d, J = 7.4 Hz, 3H, CH3 Ala), 0.88 (d, J = 6.6 Hz, 3H, 3xH- Leu),
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0.86 (d, J = 6.5 Hz, 3H, 3xH- Leu), 0.76 (d, J = 7.2 Hz, 3H, CH3 MeAsp); C NMR (151
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MHz, CD3OD, 300 K)  178.4 (C=O MeAsp), 176.5 (C=O), 176.4 (C=O), 176.0 (C=O), 175.7
(C=O), 175.2 (C=O), 175.2 (C=O), 171.5 (C=O Phe), 166.0 (C=O Mdha), 146.2 (-C=CH2),
140.6 (C Ar), 139.4 (C-5 Adda), 139.2 (C Ar) , 137.6 (C-7 Adda), 133.7 (C-6 Adda), 130.5 (C
Ar), 130.1 (2xC Ar), 129.5 (2xC Ar), 129.2 (C Ar), 127.9 (C Ar), 127.1 (C Ar), 125.9 (C-4
Adda), 114.4 (-C=CH2), 88.4 (C-9 Adda), 58.8 (-OCH3), 56.1 (C-7 Adda), 55.3 (C- MeAsp),
55.3 (C- Phe), 55.2 (C- Leu), 53.3 (C- Glu), 49.6 (C- Ala), 46.0 (C-2 Adda), 40.8 (C-
Leu), 40.6 (C- MeAsp), 39.0 (C-10 Adda), 38.5 (-NCH3), 38.1 (C- Phe), 37.8 (C-8 Adda),
33.4 (C- Glu), 29.5 (C- Glu), 25.7 (C- Leu), 23.5 (C- Leu), 21.2 (C- Leu), 17.4 (CH3 Ala),
16.5 (C-8’ Adda), 16.4 (C-2’ Adda), 15.0 (CH3 MeAsp), 12.9 (C-6’ Adda). HRMS (ESI-
Orbitrap) m/z: [M+H]+ Calcd for C52H72N7O12 986.52335; Found 986. 52418.
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[Phe-d5 ]-Microcystin-LF (1b). The macrocyclic derivative 23b (5 mg, 4.35 mol) was
transformed to 1a according to GP8, and the reaction mixture was separated by semi-
preparative RP-HPLC (gradient: 50-70% B in 20 min, tR = 13.6 min). The microcystin 1b was
obtained as a white amorphous solid (3.3 mg, 76%). LC-MS analysis of synthetic 1b coinjected
with MC-LF resulted in a single peak with the expected masses of both deuterated 1b and
undeuterated MC-LF (Figure S4). 1H NMR (600 MHz, CD3OD, 284 K)  8.16 (d, J = 6.8 Hz,
1H, NH Leu), 8.07 (d, J = 9.1 Hz, 1H, NH MeAsp), 7.56 (d, J = 8.4 Hz, 1H, NH Ala), 7.27 –
7.25 (m, 2H, Ar), 7.23 – 7.15 (m, 3H, Ar), 6.35 (d, J = 15.5 Hz, 1H, H-5 Adda), 5.89 (s, 1H, -
C=CH2), 5.51 (d, J = 9.9 Hz, 1H, H-7 Adda), 5.46 (s, 1H, -C=CH2), 5.45 (dd, J = 15.6, 8.6 Hz,
1H, H-4 Adda), 4.69 (dd, J = 11.0, 8.7 Hz, 1H, H-3 Adda), 4.58 – 4.49 (m, 3H, H- MeAsp,
H- Phe-d5, H- Ala), 4.40 (t, J = 7.5 Hz, 1H, H- Glu), 4.23 – 4.13 (m, 1H, H- Leu), 3.49
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(dd, J = 14.1, 3.4 Hz, 1H, H- Phe-d5), 3.37 (s, 3H, -NCH3), 3.30 – 3.28 (m, 1H, H-9 Adda),
3.26 (s, 3H, -OCH3), 2.95 (dq, J = 7.2, 3.5 Hz, 1H, H- MeAsp), 2.85 (dd, J = 14.0, 4.7 Hz,
1H, H-10 Adda), 2.80 (dd, J = 10.9, 6.9 Hz, 1H, H-2 Adda), 2.69 (dd, J = 13.9, 7.3 Hz, 1H, H-
10 Adda), 2.65 – 2.52 (m, 4H, H-8 Adda, H- Phe-d5, 2xH- Glu), 2.17 – 2.07 (m, 1H, H-
Glu), 1.90 (ddd, J = 15.5, 12.8, 4.1 Hz, 1H, H- Leu), 1.80 – 1.69 (m, 2H, H- Glu, H- Leu),
1.65 (s, 3H, 3xH-6’ Adda), 1.52 (ddd, J = 13.8, 10.2, 3.9 Hz, 1H, H- Leu), 1.10 (d, J = 6.9 Hz,
3H, 3xH-2’ Adda), 1.04 (d, J = 6.7 Hz, 3H, 3xH-8’ Adda), 0.97 (d, J = 7.4 Hz, 3H, -CH3 Ala),
0.88 (d, J = 6.6 Hz, 3H, 3xH- Leu), 0.85 (d, J = 6.6 Hz, 3H, 3xH- Leu), 0.75 (d, J = 7.2 Hz,
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3H, CH3 MeAsp); 13C NMR (151 MHz, CD3OD, 284 K):  = 178.5 (C=O MeAsp), 176.6
(C=O), 176.5 (C=O), 176.2 (C=O), 175.6 (C=O), 175.3 (C=O), 175.2 (C=O), 171.6 (C=O Phe),
166.0 (C=O Mdha), 146.2 (-C=CH2), 140.5 (C Ar), 139.3 (C-5 Adda), 139.0 (C Ar), 137.5 (C-
7 Adda), 133.7 (C-6 Adda), 130.6 (C Ar), 129.2 (C Ar), 127.1 (C Ar), 126.0 (C-4 Adda), 114.5
(-C=CH2), 88.3 (C-9 Adda), 58.7 (-OCH3) , 56.2 (C-7 Adda), 55.6 (C- MeAsp), 55.2 (C-
Phe-d5), 55.1 (C- Leu), 53.6 (C- Glu), 49.6 (C- Ala), 45.7 (C-2 Adda), 40.8 (C- Leu),
38.9 (C-10 Adda), 38.5 (-NCH3), 38.0 (C- Phe-d5), 37.7 (C-8 Adda), 33.4 (C- Glu), 29.2 (C-
 Glu), 25.7 (C- Leu), 23.6 (C- Leu), 21.2 (C- Leu), 17.3 (-CH3 Ala), 16.6 (C-8’ Adda),
16.3 (C-2’ Adda), 15.0 (CH3 MeAsp), 12.9 (C-6’ Adda); HRMS (ESI-TOF) m/z: [M+H]+ Calcd
for C52H67D5N7O12 991.5547; Found 991.5553.
Microcystin-LY(Prg) (1c). The macrocyclic derivative 23c (6.2 mg, 5.18 mol) was
transformed to 1c according to GP8 and the reaction mixture was separated by semi-preparative
RP-HPLC (gradient: 50-70% B in 20 min, tR = 14.1 min). The microcystin 1c was obtained as
a white amorphous solid (2.3 mg, 43 %). 1H NMR (600 MHz, CD3OD, 300 K)  8.89 (d, J =
9.6 Hz, 1H, NH Tyr(Prg)), 8.21 (d, J = 8.7 Hz, 1H, NH MeAsp), 8.11 (d, J = 6.7 Hz, 1H, NH
Leu), 7.43 (d, J = 8.0 Hz, 1H, NH Ala), 7.28 – 7.26 (m, 2H, Ar), 7.22 – 7.16 (m, 3H, Ar), 7.09
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(d, J = 8.6 Hz, 2H, Ar), 6.84 (d, J = 8.7 Hz, 2H, Ar), 6.35 (d, J = 15.5 Hz, 1H, H-5 Adda), 5.90
(s, 1H, -C=CH2), 5.51 (d, J = 9.8 Hz, 1H, H-7 Adda), 5.46 (s, 1H, -C=CH2), 5.42 (dd, J = 15.5,
8.7 Hz, 1H, H-4 Adda), 4.71 (dd, J = 11.0, 8.6 Hz, 1H, H-3 Adda), 4.65 (d, J = 2.3 Hz, 2H, -
OCH2- Tyr(Prg)), 4.57 – 4.50 (m, 3H, H- MeAsp, H- Tyr(Prg), H- Ala), 4.46 (dd, J = 8.8,
6.1 Hz, 1H, H- Glu), 4.22 – 4.15 (m, 1H, H- Leu), 3.44 (dd, J = 14.1, 3.1 Hz, 1H, H-
Tyr(Prg)), 3.37 (s, 3H, -NCH3), 3.30 – 3.26 (m, 1H, H-9 Adda), 3.26 (s, 3H, -OCH3), 2.96 –
2.91 (m, 1H, H- MeAsp), 2.91 (t, J = 2.4 Hz, 1H, -C≡CH), 2.84 (dd, J = 14.0, 4.7 Hz, 1H, H-
10 Adda), 2.73 – 2.66 (m, 2H, H-2 Adda, H-10 Adda), 2.63 (dq, J = 9.9, 6.6 Hz, 1H, H-8 Adda),
2.61 – 2.56 (m, 1H, 2xH- Glu), 2.51 (dd, J = 14.1, 11.9 Hz, 1H, H- Tyr(Prg)), 2.19 – 2.11
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(m, 1H, H- Glu), 1.89 – 1.83 (m, 1H, H- Leu), 1.79 – 1.73 (m, 1H, H- Leu), 1.73 – 1.65 (m,
1H, H- Glu), 1.65 (d, J = 1 Hz, 3H, 3xH-6’ Adda), 1.52 (ddd, J = 13.7, 9.8, 4.1 Hz, 1H, H-
Leu), 1.11 (d, J = 6.9 Hz, 3xH-2’ Adda), 1.04 (d, J = 6.7 Hz, 3xH-8’ Adda), 1.01 (d, J = 7.4
Hz, 3H, CH3 Ala), 0.89 (d, J = 6.6 Hz, 3H, 3xH- Leu), 0.86 (d, J = 6.6 Hz, 3H, 3xH- Leu),
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0.81 (d, J = 7.2 Hz, 3H, CH3 MeAsp); C NMR (151 MHz, CD3OD, 300 K)  178.4 (C=O
MeAsp), 176.4 (C=O), 175.9 (C=O), 175.8 (C=O), 175.2 (C=O), 175.0 (C=O), 171.5 (C=O),
166.0 (C=O), 165.9 (C=O), 158.0 (-C-O-CH2-), 146.2 (-C=CH2), 140.5 (C Ar Adda), 139.4 (C-
5 Adda), 137.7 (C-7 Adda), 133.7 (C-6 Adda), 131.8 (C Ar Tyr(Prg)), 131.1 (2xC Ar Tyr(Prg)),
130.5 (2xC Ar Adda), 129.2 (2xC Ar Adda), 127.1 (CH Ar Adda), 125.8 (C-4 Adda), 116.0
(2xC Ar Tyr(Prg)), 114.4 (-C=CH2), 88.4 (C-9 Adda), 79.8 (-C≡CH), 76.8 (-C≡CH), 58.7 (-
OCH3), 56.5 (-OCH2-), 56.0 (C-3 Adda), 55.4, 55.3, 55.2 (m, 3C, C- Leu, C- MeAsp, C-
Tyr(Prg)), 53.2 (C- Glu), 49.6 (C- Ala), 46.0 (C-2 Adda), 40.8 (C- Leu), 40.5 (C-
MeAsp), 39.0 (C-10 Adda), 38.5 (-NCH3), 37.7 (C-8 Adda), 37.2 (C- Tyr(Prg)), 33.4 (C-
Glu), 29.6 (C- Glu), 25.7 (C- Leu), 23.5 (CH3 Leu), 21.2 (CH3 Leu), 17.4 (CH3 Ala), 16.5
(C-8’ Adda), 16.4 (C-2’ Adda), 15.2 (CH3 MeAsp), 12.9 (C-6’ Adda); HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C55H74N7O13 1040.5339; Found 1040.5353.
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Fmoc--D-Glu(Ot-Bu)-N-MeSecPh-D-Ala-Leu-OPac (4). Tripeptide 12 (297 mg, 449.5
mol) was deprotected according to GP4. The N-terminally deprotected tripeptide (91 mg,
107.5 mol) and Fmoc-D-Glu-Ot-Bu (249 mg, 584.4 mol) were dissolved in DMF (2 mL).
Peptide coupling was performed according to GP6 using HATU (222 mg, 584.4 mol) and
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DIPEA (313 L, 1.8 mmol). The crude product was purified by FC to give tetrapeptide 4 (390
mg, 90%) as a white amorphous solid: Rf = 0.77 (EtOAc); 1H NMR (400 MHz, CDCl3, 300 K)
 7.80 – 7.70 (m, 4H, 4x H-Ar), 7.68 – 7.54 (m, 3H, 3x H-Ar), 7.53 – 7.47 (m, 1H, 2x H-Ar),
7.45 – 7.35 (m, 4H, 4x H-Ar), 7.33 – 7.25 (m, 2H, 2x H-Ar), 7.24 – 7.19 (m, 4H, 3x H-Ar, NH
Ala), 6.84 (d, J = 8.3 Hz, 1H, NH Leu), 5.57 (d, J = 8.0 Hz, 1H, NH Glu), 5.35 (dd, J = 10.2,
5.3 Hz, 1H, H- NMeSecPh), 5.19 (d, J = 16.5 Hz, 1H, -C(O)CH2-O), 4.94 (d, J = 16.5 Hz,
1H, -C(O)CH2-O), 4.65 – 4.53 (m, 2H, H- Leu, H- Ala), 4.44 – 4.38 (m, 1H, -OCH2-CH-
Fmoc), 4.35 – 4.25 (m, 2H, -OCH2-CH- Fmoc, H- Glu), 4.17 (t, J = 6.9 Hz, 1H, -OCH2-CH-
Fmoc), 3.63 (dd, J = 13.0, 5.2 Hz, 1H, H- NMeSecPh), 3.19 (dd, J = 12.9, 10.5 Hz, 1H, H-
NMeSecPh), 2.76 (s, 3H, -NCH3), 2.47 – 2.32 (m, 2H, 2x H- Glu), 2.18 (dd, J = 15.6, 4.4 Hz,
1H, H- Glu), 1.82 – 1.73 (m, J = 3.1 Hz, 2H, H- Leu, H- Leu), 1.70 – 1.60 (m, 2H, H- Leu,
H- Glu), 1.48 (s, 9H, tBu), 1.36 (d, J = 7.1 Hz, 3H, CH3 Ala), 0.97 (d, J = 6.3 Hz, 3H, 3xH-
Leu), 0.95 (d, J = 6.3 Hz, 3H, 3xH- Leu); 13C NMR (101 MHz, CDCl3, 300 K)  173.4 (C=O),
172.6 (C=O), 172.1 (C=O), 171.7 (C=O), 171.3 (C=O), 170.5 (C=O), 156.5 (C=O), 144.3 (C
Ar), 143.8 (C Ar), 141.5 (C Ar), 141.4 (C Ar), 134.1 (C Ar), 134.1 (C Ar), 133.2 (C Ar), 129.8
(C Ar), 129.3 (2C Ar), 129.0 (C Ar), 127.8 (C Ar), 127.8 (C Ar), 127.4 (C Ar), 127.3 (C Ar),
127.3 (C Ar), 125.5 (C Ar) 125.3 (C Ar), 120.0 (2C Ar), 82.7 (-C(CH3)3), 67.0 (-OCH2-CH-
Fmoc), 66.3 (CH2 Pac), 58.0 (C- NMeSecPh), 53.4 (-OCH2-CH- Fmoc), 50.7 (C-Leu), 48.6
(C-Ala), 47.2 (C-Glu), 41.2 (C-Leu), 32.2 (-NCH3), 28.84 (C-Glu), 28.75 (C-Glu),
28.2 (3C, -C(CH3)3), 26.4 (C- NMeSecPh), 25.0 (C-Leu), 23.1 (C- Leu), 21.8 (C- Leu)
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16.1 (CH3 Ala); HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C51H61N4O10Se 969.35474; Found
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969.35351.
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Fmoc--D-MeAsp(Ot-Bu)-Phe-OH (5a). Boc-Phe-OPac17 8a (120 mg, 313 mol) was
deprotected according to GP4. The obtained H-Phe-OPac and Fmoc-D-MeAsp-Ot-Bu 7 (140
mg, 329 mol) were dissolved in DMF (3 mL). Peptide coupling was performed according to
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GP6 using HATU (125 mg, 329 mol) and DIPEA (165 L, 329 mol). The crude product was
purified by FC (petroleum ether/EtOAc 2:1) to give dipeptide 5a (153 mg, 71%) as a white
amorphous solid: Rf = 0.77 (petroleum ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3, 300 K)
 7.95 – 7.87 (m, 2H, Ar), 7.76 (d, J = 7.6 Hz, 2H, Ar), 7.65 – 7.60 (m, 2H, Ar), 7.52 – 7.48
(m, 2H, Ar), 7.40 – 7.37 (m, 2H, Ar), 7.34 – 7.20 (m, 8H, Ar), 6.13 – 6.05 (m, 1H, NH MeAsp,
NH Phe), 5.50 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.34 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.01 (ddd, J
= 13.0, 7.4, 5.5 Hz, 1H, H- Phe), 4.41 (dd, J = 10.2, 7.2 Hz, 1H, -OCH2-CH- Fmoc), 4.36 –
4.28 (m, 2H, -OCH2-CH- Fmoc, H- MeAsp), 4.24 (t, J = 7.2 Hz, 1H, -OCH2-CH- Fmoc), 3.42
(dd, J = 14.2, 5.6 Hz, 1H, H-Phe), 3.19 (dd, J = 14.2, 7.1 Hz, 1H, H- Phe), 3.04 (qd, J = 7.3,
3.8 Hz, 1H, H- MeAsp), 1.41 (s, 9H, t-Bu), 1.15 (d, J = 7.3 Hz, 3H, CH3 MeAsp); 13C NMR
(101 MHz, CDCl3, 300 K)  191.4 (C=O), 173.6 (C=O), 171.2 (C=O), 170.1 (C=O), 157.1
(C=O), 144.2 (C Ar), 144.0 (C Ar), 141.4 (C Ar), 135.9 (C Ar), 134.3 (C Ar), 134.1 (C Ar),
129.6 (2C Ar), 129.1 (2C, Ar), 128.8 (2C Ar), 127.9 (2C Ar), 127.8 (2C Ar), 127.4 (C Ar),
127.2 (2C Ar), 127.2 (C Ar), 125.5 (C Ar), 125.4 (C Ar), 120.1 (C Ar) 120.0 (C Ar), 82.5 (-
C(CH3)3), 67.3 (-OCH2-CH- Fmoc), 66.7 (CH2 Pac), 57.1 (-OCH2-CH- Fmoc), 53.0 (C- Phe),
47.3 (C- MeAsp), 41.6 (C- MeAsp), 37.8 (C- Phe), 28.0 (3C, -C(CH3)3), 15.2 (CH3
MeAsp); HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C41H43N2O8 691.30139; Found 691.30123.
Fmoc--D-MeAsp(Ot-Bu)-Phe-d5-OH (5b). Boc-Phe-d5-OPac 8b (43 mg, 109.7 mol) was
deprotected according to GP4. The obtained H-Phe-d5-OPac and Fmoc-D-MeAsp-Ot-Bu 7 (49
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mg, 115.0 mol) were dissolved in DMF (1 mL). Peptide coupling was performed according to
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GP6 using HATU (44 mg, 115.0 mol) and DIPEA (57.2 L, 329.0 mol). The crude product
was purified by FC (petroleum ether/EtOAc 2:1) to give dipeptide 5b (66 mg, 86%) as a white
amorphous solid: Rf = 0.77 (petroleum ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3, 300 K)
 7.93 – 7.87 (m, 2H, Ar), 7.76 (d, J = 7.6 Hz, 2H, Ar), 7.66 – 7.59 (m, 3H, Ar), 7.52 – 7.48
(m, 2H, Ar), 7.40 – 7.48 (m, 2H, Ar), 7.32 – 7.27 (m, 2H, Ar), 6.12 – 6.03 (m, 2H, NH MeAsp,
NH Phe-d5), 5.50 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.34 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.01 (ddd,
J = 13.0, 7.2, 5.5 Hz, 1H, H- Phe), 4.41 (dd, J = 10.2, 7.2 Hz, 1H, -OCH2-CH- Fmoc), 4.36 –
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4.28 (m, 2H, -OCH2-CH- Fmoc, H- MeAsp), 4.24 (t, J = 7.3 Hz, 1H, -OCH2-CH- Fmoc), 3.42
(dd, J = 14.2, 5.6 Hz, 1H, H- Phe), 3.19 (dd, J = 14.2, 7.1 Hz, 1H, H- Phe), 3.04 (qd, J = 7.1,
3.9 Hz, 1H, H- MeAsp), 1.41 (s, 9H, t-Bu), 1.15 (d, J = 7.2 Hz, 3H, CH3 MeAsp); 13C NMR
(101 MHz, CDCl3, 300 K)  191.4 (C=O), 173.6 (C=O), 171.2 (C=O), 170.1 (C=O), 157.1
(C=O), 141.4 (C Ar), 134.3 (C Ar), 134.1 (C Ar), 129.1 (2C Ar), 127.9 (2C Ar), 127.8 (2C Ar),
127.2 (2C Ar), 125.5 (C Ar), 125.4 (C Ar), 120.1 (2C Ar), 82.5 (-C(CH3)3), 67.4 (-OCH2-CH-
Fmoc), 66.7 (CH2 Pac), 56.1 (-OCH2-CH- Fmoc), 53.0 (C- Phe-d5), 47.4 (C- MeAsp), 41.6
(C- MeAsp), 37.7 (C- Phe-d5), 28.0 (3C, -C(CH3)3), 15.2 (CH3 MeAsp); HRMS (ESI-TOF)
m/z: [M+H]+ Calcd for C41H38D5N2O8 696.3328; Found 696.3313.
Fmoc--D-MeAsp(Ot-Bu)-Tyr(Prg)-OH (5c). Boc-Tyr(Prg)-OPac 8c (137 mg, 313 mol)
was deprotected according to GP4. The obtained H-Tyr(Prg)-OPac and Fmoc-D-MeAsp-Ot-
Bu 7 (140 mg, 329 mol) were dissolved in DMF (3 mL). Peptide coupling was performed
according to GP6 using HATU (125 mg, 329 mol) and DIPEA (165 L, 329 mol). The crude
product was purified by FC to give dipeptide 5c (181 mg, 84%) as a white amorphous solid: Rf
= 0.3 (petroleum ether/EtOAc 2:1); 1H NMR (400 MHz, CDCl3, 300 K)  7.94 – 7.86 (m, 2H,
Ar), 7.75 (d, J = 7.5 Hz, 2H, Ar), 7.67 – 7.58 (m, 3H, Ar), 7.52 – 7.48 (m, 2H, Ar), 7.40 – 7.36
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(m, 2H, Ar), 7.31 – 7.27 (m, 2H, Ar), 7.17 (d, J = 8.6 Hz, 2H, Ar), 6.94 – 6.90 (m, 2H, Ar),
6.11 – 6.03 (m, 2H, NH MeAsp, NH Phe), 5.50 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.33 (d, J = 16.3
Hz, 1H, CH2 Pac), 4.97 (ddd, J = 13.3, 6.8, 6.0 Hz, 1H, H- Tyr(Prg)), 4.65 (d, J = 2.3 Hz, 2H,
-OCH2- Tyr(Prg)), 4.42 (dd, J = 10.1, 7.2 Hz, 1H, -OCH2-CH- Fmoc), 4.37 – 4.30 (m, 2H, H-
 MeAsp, -OCH2-CH- Fmoc), 4.29 – 4.21 (m, 1H, -OCH2-CH- Fmoc), 3.36 (dd, J = 14.3, 5.8
Hz, 1H, H- Tyr(Prg)), 3.16 (dd, J = 14.3, 6.8 Hz, 1H, H- Tyr(Prg)), 3.05 (qd, J = 7.3, 3.5 Hz,
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1H, H- MeAsp), 2.49 (t, J = 2.2 Hz, 1H, -C≡CH), 1.41 (s, 9H, t-Bu), 1.17 (d, J = 7.2 Hz, 3H,
CH3 MeAsp); 13C NMR (101 MHz, CDCl3, 300 K)  191.4 (C=O), 173.6 (C=O), 171.2 (C=O),
170.1 (C=O), 157.1 (C=O) 156.9 (C Ar), 141.4 (C Ar) , 134.3 (C Ar), 134.1 (C Ar), 130.7 (2C,
Ar), 129.1 (2C, Ar), 128.8 (C Ar), 127.9 (2C, Ar), 127.8 (2C, Ar), 127.2 (C Ar), 127.2 (C Ar),
125.5 (C Ar), 125.4 (C Ar), 120.1 (C Ar), 115.2 (2C, Ar), 82.5 (-C(CH3)3), 78.7 (-C≡CH), 75.7
(-C≡CH), 67.4 (-OCH2-CH- Fmoc), 66.7 (CH2 Pac), 57.1 (C- MeAsp), 56.0 (-OCH2-
Tyr(Prg)), 53.1 (C- Tyr(Prg)), 47.3 (m, 1H, -OCH2-CH- Fmoc), 41.6 (C- MeAsp), 36.9 (C-
 Tyr(Prg)), 28.0 (3C, -C(CH3)3), 15.3 (CH3 MeAsp); HRMS (ESI-TOF) m/z: [M+H]+ Calcd
for C44H45N2O9 745.31196; Found 745.31092.
Fmoc-D-MeAsp-Ot-Bu (7). The amino acid erythro-16 (1.00 g, 1.6 mmol) was suspended in
methanol (13 mL) and 10% Pd/C catalyst (410 mg, wet, 53.7% water) was added. The reaction
mixture was hydrogenated overnight at slightly positive hydrogen pressure and filtered through
a Celite pad. The residue was further treated according to GP5 using Fmoc-OSu (703 mg, 2.084
mmol), dioxane (3 mL) and 10% w/v aqueous Na2CO3 (2 mL). The crude product was purified
with FC (petroleum ether/EtOAc/AcOH 74:25:1) to give 7 (572 mg, 84%) as a white
amorphous solid: Rf = 0.5 (petroleum ether/EtOAc/AcOH 49:50:1); mp 55-57 °C; []D27 +11.8
(c 1.0, MeCN); 1H NMR (400 MHz, CDCl3, 300 K)  7.76 (d, J = 7.5 Hz, 2H, Ar), 7.62 (d, J =
7.4 Hz, 2H, Ar), 7.42 – 7.38 (m, 2H, Ar), 7.34 – 7.29 (m, 2H, Ar), 5.75 (d, J = 8.9 Hz, 1H, NH),
4.59 (dd, J = 8.9, 3.6 Hz, 1H, H-), 4.49 – 4.34 (m, 2H, CH2 Fmoc), 4.25 (t, J = 7.1 Hz, 1H,
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CH Fmoc), 3.30 (qd, J = 7.3, 3.6 Hz, 1H, H-) 1.46 (s, 9H, tBu), 1.27 (d, J = 7.3 Hz, 3H, CH3);
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13C NMR (101 MHz, CDCl3, 300 K)  178.7 (C=O), 169.7 (C=O), 156.7 (C=O Fmoc), 144.1
(C Ar), 143.9 (C Ar), 141.5 (2C Ar), 127.9 (2C Ar), 127.2 (2C Ar), 125.3 (2C Ar), 120.1 (C
Ar), 120.1 (C Ar), 83.2 (-C(CH3)3), 67.4 (CH2 Fmoc), 56.2 (C-), 47.3 (CH Fmoc), 41.5 (C-),
28.0 (3C, -C(CH3)3), 13.0 (CH3); HRMS (ESI-TOF) m/z: [2M+H]+ Calcd for C48H55N2O12
851.3750; Found 851.3708; Anal. Calcd for C24H27NO6: C, 67.75; H, 6.40; N, 3.29. Found: C
67.64; H, 6.50; N, 3.38.
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Boc-Phe-d5-OPac (8b) Boc-Phe-d5-OH (390 mg, 1.44 mmol) was dissolved in EtOAc (6 mL)
and reacted according to GP1. The product 8b was obtained as a white amorphous solid (260
mg, 46%): Rf = 0.55 (petroleum ether/EtOAc 7:3); 1H NMR (400 MHz, CDCl3, 300 K)  7.91
(d, J = 7.7 Hz, 2H, Ar), 7.62 (t, J = 7.4 Hz, 1H, Ar), 7.50 (t, J = 7.7 Hz, 2H, Ar), 5.49 (d, J =
16.3 Hz, 1H, CH2 Pac), 5.31 (d, J = 16.4 Hz, 1H, CH2 Pac), 4.97 (d, J = 7.7 Hz, 1H, NH), 4.77
– 4.72 (m, 1H, H-), 3.35 (dd, J = 14.1, 5.3 Hz, 1H, H-), 3.15 (dd, J = 14.0, 7.0 Hz, 1H, H-
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), 1.40 (s, 9H, Bu); C NMR (101 MHz, CDCl3, 300 K):  = 191.7 (C=O), 171.8 (C=O),
155.3 (C Ar), 134.2 (C Ar), 129.1 (2C, Ar), 127.9 (2C, Ar), 80.1 (-C(CH3)3), 66.6 (CH2 Pac),
54.5 (C-), 38.2 (C-), 28.4 (3C, -C(CH3)3); HRMS (ESI-TOF) m/z: [2M+H]+ Calcd for
C44H41D10N2O10 777.4166; Found 777.4151.
Boc-Tyr(Prg)-OPac (8c). The carboxylic acid 18 (350 mg, 1.10 mmol) was dissolved in
EtOAc (5.5 mL) and reacted according to GP1. The product 8c was obtained as a white
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amorphous solid (421 mg, 90%): Rf = 0.55 (petroleum ether/EtOAc 7:3); mp 94-95 °C; []D
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-21.2 (c 0.5, MeCN); H NMR (400 MHz, CDCl3, 300 K)  7.95 – 7.88 (m, 2H, Ar), 7.65 –
7.60 (m, 1H, Ar), 7.50 (t, J = 7.7 Hz, 2H, Ar), 7.19 (d, J = 8.5 Hz, 2H, Ar), 6.94 – 6.90 (m, 2H
Ar), 5.50 (d, J = 16.4 Hz, 1H, CH2 Pac), 5.30 (d, J = 16.3 Hz, 1H, CH2 Pac), 4.95 (d, J = 7.9
Hz, 1H, NH), 4.74 – 4.69 (m, 1H, H-), 4.67 (d, J = 2.4 Hz, 2H, -OCH2- Tyr(Prg)), 3.29 (dd, J
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= 14.2, 5.4 Hz, 1H, H-), 3.11 (dd, J = 14.0, 6.7 Hz, 1H, H-), 2.51 (t, J = 2.4 Hz, 1H, -C≡CH),
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1.41 (s, 9H, tBu); 13C NMR (101 MHz, CDCl3, 300 K)  191.7 (C=O), 171.8 (C=O), 156.8 (-
C-O-CH2-), 155.3 (C=O), 134.2 (C Ar), 130.7 (2C, Ar), 129.2 (C Ar), 129.1 (2C, Ar), 127.9
(2C, Ar), 115.1 (C Ar), 80.1 (-C(CH3)3), 78.8 (-C≡CH), 75.6 (-C≡CH), 66.5 (CH2 Pac), 56.0 (-
OCH2- Tyr(Prg)), 54.5 (C-), 37.5 (C-), 28.4 (3C, -C(CH3)3); HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C25H28NO6: 438.19111; Found 438.19216; Anal. Calcd for C25H27NO6: C, 68.64; H,
6.22; N, 3.20. Found: C, 68.56; H, 6.11; N, 3.32.
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Boc-N-MeSecPh-D-Ala-Leu-OPac (12).18 Boc-Leu-OPac17 (500 mg, 1.43 mmol) was
deprotected according to GP4. Then H-Leu-OPac, Boc-D-Ala-OH (325 mg, 1.72 mmol) and
HOBt (232 mg, 1.72 mmol) were dissolved in CH2Cl2 (5 mL) and coupled according to GP6
using HBTU (651 mg, 1.72 mmol) and DIPEA (980 L, 5.72 mmol). The crude product was
purified by FC to give Boc-D-Ala-Leu-OPac18 (500 mg, 83%) as a white solid: Rf = 0.66
(PE/EtOAc 1:1); The analytical data for Boc-D-Ala-Leu-OPac were in agreement with the
published ones.18 The dipeptide Boc-D-Ala-Leu-OPac (502 mg, 1.195 mmol) was deprotected
according to GP4 to give H-D-Ala-Leu-OPac 11 that was immediately used in the next step.
Crude deprotected dipeptide 11 (1.195 mmol) and Boc-NMeSecPh-OH 6 (471 mg, 1.314
mmol) were dissolved in DMF (5 mL) and coupled according to GP6 using HATU (500 mg,
1.314 mmol) and DIPEA (832 L, 4.778 mmol). The crude product was purified by FC
(petroleum ether/EtOAc 1:1) to give title compound 12 (683 mg, 90%) as a white amorphous
solid. The analytical data for 12 were in agreement with the published ones.18 1H NMR (400
MHz, CDCl3)  7.92 – 7.81 (dd, J = 8.5, 1.3 Hz, 2H, Ar), 7.62 (dt, J = 7.5, 1.3 Hz, 1H, Ar),
7.54 – 7.45 (m, 4H, Ar), 7.27 – 7.23 (m, 3H, Ar), 6.79 (b, 1H, NH Leu), 6.53 (b, 1H, NH Ala),
5.47 (d, J = 16.3 Hz, 1H, CH2 Pac), 5.22 (d, J = 16.3 Hz, 1H, CH2 Pac), 4.69 (b, 1H, H-Leu),
4.50 (b, 2H, H-Ala, H- NMeSecPh), 3.60 (dd, J = 13.0, 5.9 Hz, 1H, H- NMeSecPh), 3.16
(b, 1H, H- NMeSecPh), 2.80 (s, 3H, -NMe), 1.90 – 1.66 (m, 3H, 2xH-Leu, H-Leu), 1.45
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Hz, 3H, CH3 Leu).
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Bn-D-Asp(OBn)-OH (14). Starting from Bn-D-Asp-OH (8.61 g, 38.6 mmol), compound 14
(10.16 g, 84%) was prepared according to a procedure published for the synthesis of Bn-L-
Asp(OBn)-OH.25 The analytical data of 14 were in agreement with the data published for its
enantiomer.25 1H NMR (400 MHz, DMSO-d6, 300 K)  7.43 – 7.18 (m, 10H, Ar), 5.10 (s, 2H,
1H, -COOCH2-), 3.92 (d, J = 13.4 Hz, 1H, N-CH2-), 3.80 (d, J = 13.4 Hz, 1H, N-CH2-), 3.55
(t, J = 6.6 Hz, 1H, H-), 2.80 (dd, J = 16.0, 6.2 Hz, 1H, H-), 2.70 (dd, J = 16.0, 7.1 Hz, 1H,
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H-); C NMR (101 MHz, DMSO-d6, 300 K)  170.9 (C=O), 170.1 (C=O), 150.0 (C Ar),
136.8 (C Ar), 136.2 (C Ar), 129.9 (C Ar), 129.0 (C Ar), 128.9 (2C, Ar), 128.6 (C Ar), 128.4
(2C, Ar), 128.0 (C Ar), 124.4 (C Ar), 66.5 (-COOCH2-), 56.1 (C-), 50.4 (N-CH2-), 35.4 (C-
).
Bn-D-Asp(OBn)-Ot-Bu. Bn-D-Asp(OBn)-OH 14 (4.0 g, 12.8 mmol) was suspended in
t-BuOAc (72 mL) and 70% aqueous perchloric acid (1.840 mL, 15.2 mmol) was added
dropwise. After stirring for 18h at rt, water (40 mL) was added and the phases were separated.
The aqueous phase was extracted with EtOAc (1x 30 mL). The organic phases were combined
and washed with saturated NaHCO3 solution (3x). During the workup, formation of a white
precipitate could be observed. The organic phase was filtered, washed with brine and dried with
Na2SO4. The volatiles were removed and Bn-D-Asp(OBn)-Ot-Bu was obtained as a pale yellow
oil (3.168 g, 67%). The analytical data for Bn-D-Asp(OBn)-Ot-Bu were in agreement with the
data published for its enantiomer.25 []D26 +17.5 (c 1.0, MeCN); 1H NMR (400 MHz, CDCl3,
300 K)  7.38 – 7.21 (m, 10H, Ar), 5.15 (d, J = 12.3 Hz, 1H, -COOCH2-) 5.11 (d, J = 12.3 Hz,
1H, -COOCH2-), 3.87 (d, J = 12.9 Hz, 1H, N-CH2-), 3.71 (d, J = 12.9 Hz, 1H, N-CH2-), 3.59
(dd, J = 7.1, 5.9 Hz, 1H, H-), 2.75 (dd, J = 15.6, 5.9 Hz, 1H, H-), 2.67 (dd, J = 15.6, 7.2 Hz,
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1H, H-), 1.45 (s, 9H, t-Bu); 13C NMR (101 MHz, CDCl3, 300 K) 172.8 (C=O), 171.0 (C=O),
139.9 (C Ar), 135.9 (C Ar), 128.7 (C Ar), 128.5 (2C Ar), 128.4 (2C Ar), 128.4 (C Ar), 128.4
(C Ar), 127.2 (C Ar), 81.8 (-C(CH3)3), 66.6 (-COOCH2-), 58.0 (C-), 52.2 (N-CH2-), 38.6 (C-
), 28.2 (3C, -C(CH3)3).
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(2R)-4-Benzyl-1-tert-butyl-N-benzyl-N-(9-phenylfluoren-9-yl)-aspartate (15). To the
solution of Bn-D-Asp(OBn)-Ot-Bu (1.587 g, 4.274 mmol) in dry acetonitrile (47 mL) anhydrous
K3PO4 (1.088 g, 5.192 mmol) and phenylfluorenyl bromide (1.324 g, 4.122 equiv) were added.
The resulting heterogenic mixture was mechanically stirred for 24h at rt, filtered, and the
solvent was removed. The crude product was purified by FC to give 15 (1.870 g, 72%) as a
white solid. The analytical data for 15 were in agreement with the data published for its
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enantiomer.25 Rf = 0.25 (petroleum ether/EtOAc 15:1); []D29 -25.0 (c 1.0, MeCN); H NMR
(400 MHz, CDCl3, 300 K)  7.82 (d, J = 7.1 Hz, 2H, Ar), 7.74 (d, J = 7.5 Hz, 1H, Ar), 7.66 (d,
J = 7.4 Hz, 1H, Ar), 7.60 (d, J = 7.5 Hz, 1H, Ar), 7.56 (d, J = 7.5 Hz, 1H, Ar), 7.47 (d, J = 7.0
Hz, 2H, Ar), 7.37 (td, J = 7.5, 1.0 Hz, 1H, Ar), 7.34 – 7.16 (m, 11H, Ar), 7.11 – 7.05 (m, 2H,
Ar), 4.87 (d, J = 12.5 Hz, 1H, -COOCH2-), 4.80 (d, J = 12.5 Hz, 1H, -COOCH2-), 4.22 (d, J =
13.8 Hz, 1H, N-CH2-), 3.93 (dd, J = 10.9, 2.6 Hz, 1H, H-), 3.85 (d, J = 13.9 Hz, 1H, N-CH2-
), 2.61 (dd, J = 15.8, 10.9 Hz, 1H, H-), 1.98 (dd, J = 15.9, 2.7 Hz, 1H, H-), 1.08 (s, 9H, t-
Bu); 13C NMR (101 MHz, CDCl3, 300 K)  171.3 (C=O), 171.2 (C=O), 147.7 (C Ar), 146.4 (C
Ar), 143.8 (C Ar), 141.0 (C Ar), 140.3 (C Ar), 139.0 (C Ar), 136.0 (C Ar), 129.7 (2C Ar), 128.7
(2C Ar), 128.5 (2C Ar), 128.5 (2C Ar), 128.2 (2C Ar), 128.1 (C Ar), 128.0 (2C Ar), 127.7 (C
Ar), 127.7 (C Ar), 127.4 (2C Ar), 127.3 (C Ar), 127.1 (C Ar), 126.7 (C Ar), 120.6 (C Ar), 120.0
(2C Ar), 80.8 (-C(CH3)3), 79.7 (C-Ph), 66.0 (-COOCH2-), 57.6 (C-), 51.8 (N-CH2-), 34.4 (C-
), 27.8 (3C, -C(CH3)3); Anal. Calcd for C41H39NO4: C, 80.76; H, 6.45; N, 2.30. Found: C,
80.58; H, 6.53; N, 2.46.
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(2R,3S)-4-Benzyl-1-tert-butyl-N-benzyl-N-(9-phenylfluoren-9-y1)-3-methylaspartate
(eryhtro-16). A solution of LHMDS in THF (1.0 M, 8.25 mL, 8.25 mmol) was placed in a
Schlenk flask under nitrogen atmosphere and cooled to < -20 °C. First a solution of aspartic
acid derivative 15 (2.0 g, 3.28 mmol in dry THF (15 mL) and then a solution of methyl iodide
(707 L, 11.35 mmol) in dry THF (11 mL) were added slowly. The mixture was stirred for 3h
at < -20 °C and 30 min at room temperature and quenched with sat. aqueous NH4Cl (10 mL)
and water (10 mL). The phases were separated and the aqueous phase was extracted with EtOAc
(100 mL). The combined organic phases were washed with brine (1x), dried over Na2SO4,
filtered and the solvents were removed. The crude product was purified by FC to give eryhtro-
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16 (1.17 g, 57%) as a white solid: Rf = 0.4 (petroleum ether/EtOAc 15:1) mp 115.5 °C; []D
+250.0 (c 1.0, MeCN); 1H NMR (400 MHz, CDCl3, 300 K)  7.83 (d, J = 7.4 Hz, 1H, Ar), 7.73
(d, J = 7.4 Hz, 1H, Ar), 7.62 (d, J = 6.9 Hz, 3H, Ar), 7.56 (d, J = 7.7 Hz, 1H, Ar), 7.47 (d, J =
7.0 Hz, 2H, Ar), 7.44 – 7.08 (m, 15H, Ar), 5.14 (d, J = 12.4 Hz, 1H, -COOCH2-), 5.02 (d, J =
12.4 Hz, 1H, -COOCH2-), 4.67 (d, J = 14.3 Hz, 1H, CH2 N-Bn), 4.26 (d, J = 14.3 Hz, 1H, CH2
N-Bn), 3.97 (d, J = 9.9 Hz, 1H, H-), 2.75 (dq, J = 9.9, 7.0 Hz, 1H, H-), 1.06 (s, 9H, t-Bu),
0.79 (d, J = 7.1 Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3, 300 K)  174.2 (-COOBn), 170.3
(-COOt-Bu), 147.1 (C Ar), 146.0 (C Ar), 145.3 (C Ar), 142.2 (C Ar), 142.0 (2C, Ar), 139.6 (C
Ar), 136.2 (C Ar), 129.2 (2C, Ar), 128.7 (C Ar), 128.6 (2C, Ar), 128.4 (2C, Ar), 128.3 (C Ar),
128.3 (C Ar), 128.2 (2C, Ar), 128.2 (2C, Ar), 128.0 (2C, Ar), 127.7 (C Ar), 127.6 (C Ar), 127.5
(2C, Ar), 127.4 (C Ar), 127.0 (C Ar), 126.7 (C Ar), 80.9 (-C(CH3)3), 80.5 (C-Ph), 66.1 (CH2
Bn), 64.0 (C-), 51.7 (CH2 Bn), 42.5 (C-), 27.8 (3C, -C(CH3)3), 15.4 (CH3); Anal. Calcd for
C42H41NO4: C, 80.87; H, 6.63; N, 2.25. Found: C, 80.84; H, 6.67; N, 2.42.
H--D-MeAsp-OH, ((3S)--D-methylaspartic acid). The amino acid erythro-16 (200 mg,
1.6 mmol) was suspended in methanol (2.84 mL) and 10% Pd/C catalyst (82 mg, wet, 53.7%
water) was added. The reaction mixture was hydrogenated overnight at a slightly positive
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