Improved Large-Scale Liquid-Phase Synthesis and High-Temperature NMR Characterization of Short (F-)PNAs

Article abstract of DOI:10.1002/hlca.201100243

We report on a large-scale synthesis of F-PNA trimer 10 and PNA trimer 11. The key improvement is the facile two-step synthesis of (2,4-difluoro-5- methylphenyl)acetic acid (2). Water solubility of the corresponding F-PNA oligomer 10 was achieved by synthesizing solubility enhancer 5a, which is twofold positively charged and only consists of inherent structural elements of PNA. Protected and unpaired PNA n-mers exist in a mixture of 2ⁿ conformers undergoing slow exchange and leading to complicated NMR spectra. Structure analysis was improved by recording ¹H- and ¹³C-NMR spectra at elevated temperatures above the coalescence point. Fully protected backbone derivatives show sharp resonances where expected, and spectra of protected PNAs are remarkably simplified, thereby allowing an interpretation for the first time. Both trimers 10 and 11 are considered as building blocks for a self-replicating system based on PNA. Copyright

Full text of DOI:10.1002/hlca.201100243

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Helvetica Chimica Acta – Vol. 94 (2011)
Improved Large-Scale Liquid-Phase Synthesis and High-Temperature NMR
Characterization of Short (F-)PNAs
by Tobias A. Plçger* and Gꢀnter von Kiedrowski
Lehrstuhl fꢀr Organische Chemie I, Bioorganische Chemie, Ruhr-Universitꢁt Bochum,
Universitꢁtsstraße 150, NC 2/173, D-44780 Bochum
(phone: þ 49-23432-28218; fax: þ 49-23432-14355; tobias@oc1.rub.de)
We report on a large-scale synthesis of F-PNA trimer 10 and PNA trimer 11. The key improvement is
the facile two-step synthesis of (2,4-difluoro-5-methylphenyl)acetic acid (2). Water solubility of the
corresponding F-PNA oligomer 10 was achieved by synthesizing solubility enhancer 5a, which is twofold
positively charged and only consists of inherent structural elements of PNA. Protected and unpaired
PNA n-mers exist in a mixture of 2n conformers undergoing slow exchange and leading to complicated
NMR spectra. Structure analysis was improved by recording 1H- and 13C-NMR spectra at elevated
temperatures above the coalescence point. Fully protected backbone derivatives show sharp resonances
where expected, and spectra of protected PNAs are remarkably simplified, thereby allowing an
interpretation for the first time. Both trimers 10 and 11 are considered as building blocks for a self-
replicating system based on PNA.
1. Introduction. – 19F-NMR Probes have become a powerful tool for studying
secondary-structure phenomena in RNA and DNA [1 – 10]. Yet, this method has not
been utilized for analyzing PNA, a DNA or RNA mimic based on a non-charged,
achiral, and pseudopeptidic backbone consisting of N-(2-aminoethyl)glycine (Aeg; 1)
units [11]. Moreover, there are only five reports on fluorine modified PNA: i) a multi-
step synthesis of (2,4-difluoro-5-methylphenyl)acetic acid (2) and its Boc-protected
monomer [12], ii) a hybridization survey of PNA containing fluoroaromatic residues
[13], iii) two studies on synthesis and hybridization of fluorinated olefinic PNA
[14][15], and iv) one on 18F labeling [16].
PNA is reasoned as a model structure for a primordial genetic material [17 – 23] and
was recently used in the design of ꢂprotocellsꢃ [24 – 27] that are not based on chemistry
occurring in todayꢃs biosphere. While templated reactions involving PNA have been
studied to a good extent [28 – 37], cases for an autocatalytic feedback have not been
identified so far. Thus, we became interested in the potential of PNA to undergo self-
replication and considered a kinetic NMR titration [38] assay as a useful tool for online
monitoring of the system. Among the possible nuclei, 19F appeared to be the most
attractive because of its sensitivity (83% compared to 1H), natural abundance of 100%,
high chemical-shift dispersion and, most notably, its sensitivity to slight changes in its
supramolecular environment. As an example of the latter, alterations in the hybrid-
ization of nucleic acids can induce changes in the chemical shift up to 1.5 ppm [1]. This
encouraged us to explore whether it would be possible to prepare PNAwith reasonable
fluorine probes and good solubility at a scale required for NMR experiments.
ꢄ 2011 Verlag Helvetica Chimica Acta AG, Zꢀrich

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2. Results and Discussion. – 2.1. Fluorine Reporter Group. The introduction of
fluorine probes by means of modified bases is the most common approach, as it places
the F-atom in direct spatial proximity to the supramolecular recognition side. We
considered the fluoroaromatic 2,4-difluorotoluene (3), a nonpolar and almost perfect
isostere of thymine [39][40], as the most promising candidate. To the best of our
knowledge, there is no commercial source for (2,4-difluoro-5-methylphenyl)acetic acid
(2), and only one report on a very laborious six step synthesis which made use of highly
toxic reagents [12].
Here, we present a facile two-step synthesis of 2, starting from commercially
available 2,4-difluorotoluene (3; Scheme 1). First, 3 was treated with paraformalde-
hyde and anhydrous HBr in AcOH as reported previously for the bromomethylation of
aromatic hydrocarbons [41]. To compensate for the deactivating influence of the F-
substituents, we modified the conditions by adding ZnBr2 as Lewis acid catalyst. The
desired benzene derivative 4 was obtained in 66% yield after 4 h of reaction and
subsequent silica-gel chromatography. Conversion to the corresponding Grignard
reagent, followed by treatment with gaseous CO2 gave 2 in 38% overall yield after
chromatography. In addition, these conditions were also suitable to prepare (2,4-
difluorophenyl)acetic acid, the isostere of uracil, from 1,3-difluorobenzene (22%
overall yield; data not shown).
Scheme 1. Synthesis of F-AcOH 2
a) Paraformaldehyde (¼ polyoxymethylene), 33% HBr in AcOH, ZnBr2, 1208, 4 h. b) 1. Mg, Et2O,
reflux; 2. CO2, 08, 1 h; 3. Hþ, H2O.
2.2. Solubility Enhancer. To overcome the drawbacks of the charge neutral
backbone, namely reduced water solubility, pronounced self-organization [42], and
limited cellular uptake [43], terminal positively charged modifiers such as the amino
acid l-lysine [11] and the polyamine spermine [44], as well as backbone modifications
[45 – 47] have been introduced. Furthermore, PNA/DNA chimeras [48][49], PNA-
peptide conjugates [50][51], and PNA conjugates to high-molecular weight PEG [52]
or polyethyleneimine [53] were developed. Typically, these methods involve the
introduction of additional reaction sites and/or stereogenic centers inducing preferred
helical orientations [42]. An improved strategy involved Fmoc-protected monomers
bearing either positively charged or neutral solubility enhancers as side chains instead
of nucleobases [54]. However, for N-terminal modification we envisioned a solubility
enhancer to be as similar to the original PNA structure as possible. This was achieved
by synthesizing a fully N-methylated backbone unit by LeuckartꢀWallach chemistry or
via reductive amination with elemental hydrogen, respectively (Scheme 2). The
resulting achiral solubility enhancer 5a is twofold positively charged at physiological

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pH, while in accordance with the PNA structure. The same advantages also count for its
ethyl analog 5b. Additional charges can, in principle, be introduced by using oligomeric
backbone molecules 6 as starting material for the preparation of polycharged modifiers
7. Concerning the C-terminus, the common l-lysine amide 8 was replaced by N-
(morpholinoethyl)amide 9, thereby maintaining a positive charge and the achiral PNA
structure (Scheme 3).
Scheme 2. Synthesis of the Solubility Enhancer 5a, Structure of the Ethyl Analog 5b, and Approach to
Higher Homologs 7
a) 1. Formalin solution, HCOOH, reflux, overnight; 2. HCl. b) 1. Formalin solution, H2O, 08 ! room
temperature (r.t.), 1 h; 2. H2, Pd/C.
Scheme 3. Widespread C-Terminal l-Lysine Amide 8 and Its Achiral Replacement 9
2.3. Sequence Design. We decided to work with a system of two trimeric building
blocks leading to a hexa-PNA template with palindromic sequence upon condensation
(Scheme 4,a). Thus, we would be able to compare this system with earlier studies from
our laboratory [55 – 57]. The condensation would result in a central amide linkage and,
for instance, be facilitated by the water-soluble EDC.
Starting point of the template design was the crystal structure of a self-
complementary PNA (NcgtacgC) duplex [58]. As described above, natural thymine
was replaced by 2,4-difluorotoluene (3), and the extremities were modified with the
unreactive solubility enhancers 5a and 9. The sequence itself was slightly modified to
place the isostere in the central position of the N-terminal trimer rather than on the
ligation side (NcfgcagC). This position ensures minimal influence of the isostere on the
ligation, while maintaining the fluorine probe in equal spatial proximity to the
recognition side. In summary, we came up with a system consisting of building blocks F-
PNA 10 and PNA 11, which form, upon ligation, template F-PNA 12 (kb; Scheme 4,b),
which is assumed to be able to reversibly preorganize another pair of 10 and 11 (K1) by
H-bonding in a termolecular complex [10 · 11 · 12]. Thus, a pseudo-unimolecular

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Scheme 4. a) Trimeric Building Blocks 10 and 11 Give the Self-Complementary Hexa-PNA 12 upon
Condensation. b) A Model of the Envisioned Self-Replicating System Based on PNA.

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Scheme 4 (cont.)
ligation reaction (ka) should take place leading to template duplex 122. The reversible
dissociation of this duplex affords two template molecules 12, which may both enter
another replication cycle. However, from thermodynamic considerations and data for
published replication systems, it can be concluded that most template molecules 12 will
be present as [12 · 12], a small fraction as [10 · 11 · 12], and another small portion in an
unpaired state. Further information on this topic can be found in [59].
2.4. (F-)PNA Synthesis. PNA typically is synthesized in the solid phase from Fmoc/
Bhoc [60], Boc/Z [61], Fmoc/Mmt [62], or Fmoc/Z [63] monomers according to
peptide-synthesis protocols at a scale between 5 and 20 mmol. Strategies for a more
economical large-scale synthesis of short PNAs in the liquid phase were extensively and
almost exclusively elaborated by Condom and co-workers [64 – 71]. We adopted his
most successful route, the ꢂfully protected backbone approachꢃ (FPBA) [66], to
prepare PNAs 10 and 11 on a large scale (ca. 150 mmol). This strategy requires the
synthesis of a fully protected linear poly[N-(2-(aminoethyl)glycinamide) (poly-AEG)
bearing as many different and orthogonal protecting groups on its secondary amines as
there are different types of nucleic bases in the PNA sequence. Moreover, these
protecting groups must be orthogonal to the protecting groups on the nucleobase acetic
acid units. Additionally, care has to be taken of appropriate protection or modification
of the elongation sites at the C- and N-termini.
The synthesis of PNA 11 is outlined in Scheme 5. It required 2-aminoethylglycine
(1) that was synthesized as reported in [72] and subsequently transferred to its methyl
ester 13. The methyl ester 13 served as starting material for the three orthogonally
protected key synthons 14, 15, and 16. Treatment of 13 with N-[(benzyloxy)carbonyl-
oxy]succinimide (!63% of 17) and subsequent Boc protection of the remaining
secondary amine gave the key synthon 14 in 60% overall yield. Another portion of 13
was protected at its primary amine upon reaction with Boc2O (!63% of 18) and

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Scheme 5. Synthesis of PNA 11
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a) Boc2O, Et3N, CH2Cl2, ꢀ 108 ! r.t., overnight. b) SOCl2, MeOH, reflux, overnight. c) Allyloxycar-
bonyl chloride (Alloc-Cl), Et3N, CH2Cl2, 08 ! r.t., 2 h. d) [(9H-Fluoren-9-yl)methoxy]carbonyl chloride
(Fmoc-Cl), EtNiPr2, CH2Cl2, 08 ! r.t., 4 h. e) N-[(Benzyloxy)carbonyloxy]succinimide (Z-OSu), N-
methylmorpholine (NMM), MeCN, ꢀ 158 ! r.t., 5 h. f) 1m LiOH, THF, 08 ! r.t., 2 h. g) TFA, CH2Cl2,
08 ! r.t., 2 h. h) 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),
EtNiPr2, DMF, r.t., overnight. i) 1. 1m LiOH, THF, 08 ! r.t., 2 h; 2. Fmoc-Cl, 08, 1 h. j) 1. N,N’-
Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (HOSu), CH2Cl2, 08 ! r.t., overnight; 2. 2-
morpholinoethylamine (26), ꢀ 158 ! r.t., 4 h. k) Et2NH, CH2Cl2, r.t., 1.5 h. l) GZCH2COOH (28),
Bromotris(dimethylamino)phosphonium hexafluorophosphate (Brop), Et3N, CH2Cl2, 08 ! r.t., over-
night. m) Pd[P(Ph)3]4, Et2NH, CH2Cl2, 08 ! r.t., 1 h. n) AZCH2COOH (29), Brop, Et3N, DMF, 08 ! r.t.,
overnight. o) TFA, Et3SiH, CH2Cl2, 08 ! r.t., 2 h. p) CZCH2COOH (30), Brop, Et3N, DMF, 08 ! r.t.,
overnight. q) Trifluoromethanesulfonic acid, TFA, m-cresol, thioanisole, r.t., 2 h.

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subsequently treated with Alloc-Cl or Fmoc-Cl to give the synthons 15 and 16,
respectively. Prior to the following condensation steps, 14 and 15 were hydrolyzed with
1m LiOH, and the Boc group of 16 was removed by acidolysis with CF3COOH (TFA)/
CH2Cl2 1:1. Coupling of fragments 19 and 20 was carried out by HBTU activation to
afford the desired bis-AEG 21 in 76% yield after purification by silica-gel
chromatography. Subsequent Boc removal enabled elongation to tris-AEG 22 by
condensation of the resulting amine 23 with carboxylic acid 24. The fully protected
backbone 22 was obtained in 62% yield after silica-gel chromatography. The following
ester hydrolysis resulted in the simultaneous removal of the Fmoc group which was
easily reversed by addition of Fmoc chloride after complete saponification (!69% of
25). Next, the solubility enhancer was introduced via pre-activation of 25 with DCC
and HOSu in CH2Cl2, followed by addition of 2-morpholinoethylamine (26) to afford
compound 27 in 84% yield. The remaining steps consisted of three deprotectionꢀcou-
pling cycles to introduce the nucleobase moieties in form of their Z-protected acetic
acids 28 – 30. These compounds were synthesized according to slightly modified
procedures [60][73]. Selective removal of the Fmoc group on 27 (79% yield), by means
of Et2NH in CH2Cl2, was followed by condensation of guanine unit 28 to 31 with Brop
in CHCl3. The desired compound 32 could be isolated in 86% yield after silica-gel
chromatography. Treatment of 32 with Pd[P(Ph)3]4 and Et2NH as the allyl scavenger
gave amine 33 after 30 min. Subsequent attachment of adenine unit 29 to 33 was also
mediated by Brop reagent, but carried out in DMF instead of CHCl3, as the solubility of
the growing PNA decreased. Purification of the crude product by step-gradient
chromatography on silica gel required highly polar conditions and gave the target
compound 34 in 67% yield. Removal of the remaining Boc group led to the
corresponding TFA salt 35 in 95% yield after precipitation with cold Et2O. Conjugation
of the N-Z cytosine unit 30 with 35 by means of Brop activation afforded the fully
protected tri-PNA 36 in 47% yield after purification by semi-preparative RP-HPLC.
The coupling efficiency dropped by 20% after each introduction of an additional
nucleobase unit, accompanied by reduced solubility of the corresponding oligomers.
The synthesis of the target PNA 11 was achieved by submitting 36 to TFMSAꢀTFA
containing thioanisole and m-cresol as scavengers, which induced simultaneous
deprotection of both the nucleobases and the N-terminal ligation side. The crude
product was precipitated with cold Et2O, and the residue was purified by semi-
preparative RP-HPLC to give 11 in 55% yield after lyophilization. Its purity and
structure was confirmed by RP-HPLC analysis, 1H-NMR spectroscopy, MALDI-TOF,
and high-resolution ESI mass spectrometry.
For circumventing chromatographic problems likely to occur in the presence of the
highly polar, twofold charged solubility enhancer 5a, tri-PNA 10 was synthesized
according to a plan in which this moiety is introduced at an advanced step of the
synthesis. Following the FPBA strategy, this would necessitate the implementation of
another orthogonal protective group. To overcome this, a mixed strategy combining
PNA units as well as fully N-protected N-(2-aminoethyl)glycinamide synthons was
chosen [70] (Scheme 6). To this end, synthon 37 was prepared from 1 by acid-catalyzed
esterification with BnOH under H2O-removing conditions (DeanꢀStark trap; 86%,
38), protection of the primary amine with the Boc group, followed by masking the
secondary amine with the orthogonal Fmoc group. We improved the overall yield for 37

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Scheme 6. Synthesis of PNA 10
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a) BnOH, TsOH, toluene, 1408, Ar, overnight. b) 1. Boc2O, Et3N, CH2Cl2, 08 ! r.t., 5 h; 2. Fmoc-Cl,
EtNiPr2, CH2Cl2, 08 ! r.t., overnight. c) TFA, CH2Cl2, 08 ! r.t., 2 h. d) HBTU, EtNiPr2, DMF, r.t.,
overnight. e) Et2NH, CH2Cl2, r.t., 1.5 h. f) GZCH2COOH (28) or FCH2COOH (2), Brop, Et3N, DMF,
08 ! r.t., overnight. g) Pd[P(Ph)3]4, N,N’-dimethylbarbituric acid (NDMBA), THF, 08 ! r.t., 2 h. h)
TFA, Et3SiH, CH2Cl2, 08 ! r.t., 2 h. i) 1. DCC, HOSu, CH2Cl2, 08 ! r.t., overnight; 2. 5, ꢀ 158 ! r.t., 5 h.
j) TFMSA, TFA, m-cresol, thioanisole, r.t., 2 h.
by introducing both carbamate moieties sequentially in a one-pot reaction (73 vs. 41%
overall yield). N-Deprotection of 37, followed by HBTU-mediated coupling of the
resulting fragment 39 with 19, led to bis-AEG 40. After acidolysis of the Boc group of
40 (!quant. yield of 41), condensation with Z-cytosine PNA fragment 42 was carried
out via another HBTU coupling. Deprotection of the Fmoc group from 43 and
subsequent introduction of GZCH2COOH 28 into 44 were carried out as described for

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compounds 31 and 32, respectively. Unfortunately, Pd-catalyzed Alloc deprotection of
45 in the presence of Et2NH proceeded in low yield. Allylamine formation is
considered as the most prominent side reaction, since the deprotected amine competes
with the scavenger in the trapping of the p-palladium complex [74]. Additionally, it is
known that secondary amines like Et2NH are protonic reversible allyl-group trapping
reagents that accessorily promote allylamine formation through an equilibrium process.
Examples of alternative and irreversible scavengers include CH-acidic compounds like
dimedone or N,N-dimethylbarbituratic acid (NDMBA). When the latter one was used,
the cleavage proceeded selectively and smoothly to give the desired amine 46 in 88%
yield. Subsequent implementation of the fluorine probe was achieved by coupling
FCH2COOH (2) by means of Brop reagent. After TFA treatment of the resulting tri-
PNA 47, the solubility enhancer 5a was introduced via preactivation with DCC and
HOSu. Due to its highly polar character, the crude product was purified by semi-
preparative RP-HPLC to afford 49 in 46% yield. Finally, the water-soluble target PNA
10 was obtained by submitting 49 to cleavage with TFMSA and TFA, as described for
the preparation of PNA 11 (56% yield). After purification by RP-HPLC, its purity and
1
structure could be verified (RP-HPLC, H-NMR, 19F-NMR, MALDI-TOF-MS, HR-
ESI-MS). Analytical RP-HPLC plots and MALDI-TOF mass spectra of tri-PNAs 10
and 11 are depicted in Fig. 1.
Fig. 1. RP-HPLC Traces (left) and MALDI-TOF-MS spectra (right) of 10 and 11
2.5. NMR Spectroscopy. In complexes of PNA with DNA [75][76], RNA [77], and
itself [58], the CO group of the backbone-base linker points exclusively to the C-
terminus (cis-rotamer), whereas another rotameric form (trans-rotamer) coexists in
ssPNA and monomers (Scheme 7). Moreover, this effect also occurs if the secondary
amine of the backbone is protected with a carbamate moiety. The activation energy for
the interconversion has been determined as 19 ꢁ 2 kcal/mol by variable-temperature
1H-NMR spectroscopy on four PNA monomers and one dimer [78]. The rate of
Scheme 7. cis- and trans-Rotamers of a PNA Unit

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exchange was calculated as 0.5 – 2 sꢀ1 at 378. Hence, protected and unpaired PNA n-
mers exist in a mixture of 2n conformers in slow exchange and generate complicated
NMR spectra. This complication can only be avoided by the design of suitable PNA
analogs where conformational constraints exclude the formation of rotational isomers
[79 – 82].
Since we needed a detailed analysis of our poly-AEGs and poly-PNAs, we decided
to accelerate the cis > trans equilibria by elevating the temperature. 1H-NMR Spectra
of tris-AEG 22 at different temperatures revealed a profile typical for the
determination of rotational barriers (Fig. 2). At low temperatures, each H-atom
displayed several resonances due to the putative presence of eight conformers of 22
which could not be differentiated. Then, in an intermediate temperature range (40 –
708), the spectrum consisted of significantly broadened overlapping lines which
coalescenced upon further heating to give single, flat-topped peaks. Finally, in the
regime after coalescence (70 – 1208), the exchange was fast, and sharp lines could be
observed. The data suggest that the barrier heights and, therefore, the coalescence
temperatures (Tc) associated with the given protective groups differ in accordance with
their steric demand (Fmoc ꢂ Alloc and Boc), and depend on their conformational
flexibility that arises from their position within the sequence (Alloc > Boc). However,
1
an improved H resonance assignment of 22 was achieved at 1008. As a single AEG
backbone unit contains four geminal H-atom pairs with nearly isochronous chemical
shifts, a complete assignment in this region was not possible. The 13C-NMR spectra
Fig. 2. Cutouts of 1H-NMR spectra (250 MHz, (D6)DMSO) of Z-Aeg(Boc)-Aeg(Alloc)-Aeg(Fmoc)-
OMe (22) at different temperatures. Resonances assigned to aromatic CH of Fmoc and amide NH groups
(left), AllocCH2CH¼CH2 (middle), and Me group of Boc (right).

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benefit notably from increased signal-to-noise ratio as illustrated in Fig. 3. Given that
conformers are populated according to their relative free energies, one can accomplish
a more than eightfold saving of time or substance.
Fig. 3. Low-field region of 13C-NMR spectra of Z-Aeg(Boc)-Aeg(Alloc)-Aeg(Fmoc)-OMe (22) at 308
(100 MHz, 10835 scans; above) and 1008 (63 MHz, 1024 scans; below). Resonances assigned to 3 CO of
Gly (left) and 4 carbamate-CO (right).
Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(H)-OBn (44) is an example for a class of hybrid
molecules, obtained on the way from the fully protected tris-AEGs via the fully
protected tri-PNAs to the unprotected tri-PNAs. One of its secondary amines is free,
one is protected, and the third one is already modified with a protected nucleobase. As
a consequence of the relative low steric demand of only two substituents, the
1
temperature-dependent H-NMR spectra of 44 showed that rotational barriers were
crossed frequently at 908 to obtain single signals for each H-atom (Fig. 4).
In accordance with the challenges during synthesis and purification, every
additional nucleobase unit complicated the analysis by raising the coalescence
temperature. Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(Fmoc)-OBn (43), for instance, showed
1
a single set of H signals at 1108 and 200 MHz, while the spectrum of the GZ-modified
derivative Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(GZ)-OBn (45) exhibited broader lines and
multiple signals consistent with at least three rotamers (Fig. 5). Nonetheless, compared
with the spectrum at 308, a significant progress in structural analysis could be achieved
by reducing line broadening and the number of resonances for chemical equivalent
nuclei.
The 1H-NMR spectra of the fully protected tri-PNA 49 were recorded in a
temperature range of 30 – 1508 (Fig. 6). The signals assigned to the FCH2 group (2.0 –
2.2 ppm) showed coalescence at 708 and finally gave rise to one defined signal at 908. In
contrast, the resonances assigned to CZ-C(5)H (6.9 ppm) and CZ-C(6)H (7.8 ppm)
groups had a Tc value of 808. Decomposition of the molecule started slowly at 1108 and
could be followed by the appearance of a resonance at 4.5 ppm. It is noteworthy that

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Fig. 4. 1H-NMR Spectra (200 MHz, (D6)DMSO) of Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(H)-OBn (44) at
different temperatures
signals assigned to the N-methyl and the N,N-dimethyl group of the solubility enhancer
revealed different values for Tc. The latter ones (2.8 – 2.9 ppm) showed coalescence at
608 and exhibited a sharp signal at 708, while the broad N-Me signals demonstrated
pronounced shift-shifting (2.7 ! 2.4 ppm), and reached the coalescence point and the
regime of fast exchange after an additional temperature raise of 208. This example
indicates that the distance to the nearest rotation center, as well as the overall
conformal flexibility, have a strong influence on the shift dispersion and on Tc of a given
nucleus. Of course, distance and conformal flexibility are connected with each other.
Anyway, we were able to analyze the spectrum at 1108 by integrating non-overlapping
as well as overlapping signals, and assigning resonances to single H-atoms or classes of
similar ones (Fig. 7).
Spectra of the corresponding unprotected tri-PNA 10, recorded between 30 – 1408,
did not show a single set of resonances until 1008, while narrow line-widths for all C-
bound H-atoms before 1408 (Figs. 8 and 9). A broadened signal at 6.2 ppm was
detected for diverse protonated NH species and the COOH group.
In contrast, tri-PNA 11 (Fig. 10) and its fully protected derivative 36 (not shown)
revealed multiple datasets and line broadening under the above conditions and while
decomposition upon further heating. Both compounds differ from the preceding ones
with respect to their end modifications and their central nucleobase (adenine instead of
2,4-difluorotoluene). Hence, we assume that the higher values for Tc are associated
with the higher steric demand of (protected) adenine compared to the fluoroaromate.

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Fig. 5. Expansions of 1H-NMR spectra (200 MHz, (D6)DMSO, 1108) of Boc-Aeg(CZ)-Aeg(Alloc)-
Aeg(Fmoc)-OBn (43; below) and Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(GZ)-OBn (45; above). Resonances
assigned to the nucleobase(s), the aromatic H-atoms, and the amide NH (left), AllocCH2CH¼CH2
(middle), and the CH2 groups, including CH groups of Fmoc (right).
3. Conclusions and Outlook. – In summary, we designed a system to evaluate
potential PNA self-replication by kinetic 19F-NMR titration and presented an efficient
large-scale synthesis of the building blocks needed. In particular, an improved synthesis
of (2,4-difluoro-5-methylphenyl)acetic acid (2) was presented, reducing the number of
required steps from six to two. Furthermore, the development of an achiral N-terminal
solubility enhancer has been an essential point of our survey. This was addressed with
the synthesis of the fully N-methylated and twofold charged backbone 5a that was
designed to be as similar to the native PNA structure as possible. Moreover, we adopted
and modified the ꢂfully protected backbone approachꢃ (FPBA) of Condom and co-
workers to prepare tri-PNAs 10 and 11. Finally, we presented a solution to a hitherto
ignored problem in the context of liquid-phase PNA synthesis, namely the complexity
of NMR spectra due to the coexistence of 2n conformers in slow exchange. To this end,
1
22 monomers, dimers, and trimers were studied by H-NMR spectroscopy at elevated
temperatures up to 1608. We could obtain spectra in the regime of overall fast exchange,
showing a single set of signals for ten of these compounds. For further nine oligomers,
spectra with a remarkably reduced number of resonances, as well as significantly
narrowed line-widths, were acquired. Thus, a reasonable interpretation and assignment
was able for the first time. Unfortunately, two compounds decomposed under the
conditions studied, and in one case elevated temperatures led to a complication of the
spectrum. Decomposition is especially a concern if the analyte contains unprotected

Helvetica Chimica Acta – Vol. 94 (2011)
1965
Fig. 6. Expansions of 1H-NMR spectra (200 MHz, (D6)DMSO) of 49 at different temperatures.
Resonances assigned to the nucleobases, the aromatic H-atoms of the Ph rings, and amide NH (left),
CH2 (middle), and the Me groups, including one CH2 signal shifting at 908 (right).
Fig. 7. 1H-NMR Spectrum (200 MHz, (D6)DMSO, 1108) of 49. For assignments, see Exper. Part.

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Helvetica Chimica Acta – Vol. 94 (2011)
Fig. 8. Expansions of 1H-NMR spectra (200 MHz, D2O or (D6)DMSO) of tri-PNA 10 at different
temperatures. Resonances assigned to the nucleobases (left) and the remaining H-atoms (right). For
details on calibration, see Exper. Part.
Fig. 9. 1H-NMR Spectrum (200 MHz, (D6)DMSO, 1408) of 10. For assignments, see Exper. Part.

Helvetica Chimica Acta – Vol. 94 (2011)
1967
Fig. 10. 1H-NMR Spectrum (200 MHz, (D6)DMSO, 1408) of 11. For assignments, see Exper. Part.
reactive groups, e.g., amines or carboxylic acids. Ester 22, for instance, was stable
enough to be analyzed by 13C-NMR spectroscopy at 1008, while the corresponding
carboxylic acid 25 decomposed under Fmoc elimination within a few minutes under
these conditions. Clearly, high-temperature NMR characterization is also limited by the
thermal stability of the spectrometer, especially the probe head. Beside this drawback,
we would like to stress the importance of high-temperature NMR spectra for the
success of our synthetic work presented here, as they provided unprecedented
structural data of intermediate and desired compounds. Most notably, the spectra offer
insight into the dynamic behavior of protected PNAs and AEGs for the first time.
Altogether, the results provide the synthetic base for a large survey aiming at potential
PNA self-replication. The search for optimal conditions for the autocatalytic template-
directed ligation of both trimers is the subject of ongoing experimental studies in our
laboratory. First results reveal that self-replication is detectable and controllable in
novel ways. Full details of self-replication studies will be reported in due course.
Experimental Part
General. Nomenclature. Following the IUPAC nomenclature rules leads to complicated names
(given in parentheses), especially for the larger molecules presented in this work. Therefore, we used a
notation according to the one used for the description of peptide sequences. The diaminoacid N-(2-
aminoethyl)glycine (Aeg; 1) is the constitutional building block which is linked with further Aeg units,
suitably protected or modified on its N-terminus (left end) or C-terminus. The substituents on the

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Helvetica Chimica Acta – Vol. 94 (2011)
internal secondary amine are indicated in parentheses as side-chain protective groups. For instance,
unprotected Aeg 1 is denoted as H-Aeg(H)-OH.
Abbreviations. A: adenine, AcOH: acetic acid, Aeg: N-(2-aminoethyl)glycine, Aem: 2-morpholi-
noethylamine, Alloc: (allyloxy)carbonyl, Bhoc: (benzhydryloxy)carbonyl, Bn: benzyl, Boc: (tert-
butyloxy)carbonyl, Brop: bromotris(dimethylamino)phosphonium hexafluorophosphate, C: cytosine,
DCC: N,N’-dicyclohexylcarbodiimide, EDC: 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, F: 2,4-
difluorotoluene, Fmoc: [(9H-fluoren-9-yl)methoxy]carbonyl, G: guanine, Gly: glycine, HBTU: 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate, HOSu: N-hydroxysuccinimide,
MALDI-TOF: matrix-assisted laser-desorption ionization time of flight, Mmt: 4-monomethoxytrityl,
NDMBA: N,N’-dimethylbarbituric acid, NMM: N-methylmorpholine, TEA: triethylamine, TFA:
trifluoroacetic acid, TFMSA: trifluoromethanesulfonic acid, Z: (benzyloxy)carbonyl.
All chemicals were used as delivered, usually in p.a. quality. All moisture-sensitive reactions were
carried out under Ar. Dry DMF and CH2Cl2 were purchased from Biosolve or Roth. TLC: Merck silica
gel 60 F-254 aluminum plates, visualization by inspection under UV light (254 nm) or by the use of
phosphomolybdic acid stain (4.00 g phosphomolybdic acid hydrate in 100 ml MeOH). Column
chromatography (CC): silica gel (SiO2; ICN silica 32-63 60 ꢅ). Freeze drying: Christ Alpha 1-2. HPLC:
Applied Biosystems Vision Workstation with AFC2000 Roboter and Jetstream 2 Plus Column-
Thermostat; A: 0.1% TFA in deionized and dist. H2O, B: 0.1% TFA in MeCN; Tcolumn , 558. Anal.
HPLC: 250 ꢃ 4.6 mm Supelco Ascentis RP-Amide 5 mm; flow, 1 ml/min. Semi-prep. HPLC: 250 ꢃ 10 mm
Supelco Discovery BIO Wide Pore C18 5 mm; flow, 3 ml/min. NMR: Bruker DPX 200 (200 MHz), DRX
250, or DRX 400 (400 MHz); d in ppm rel. to TMS as internal standard and to external standard TFA for
19F; J in Hz. Temp.-dependent spectra of unprotected PNAs 10 and 11: since the chemical shift of the
remaining undeuterated portions of the solvent is considerably more temp.-dependent in the case of D2O
than (D6)DMSO, calibration was carried out as follows. The spectrum at 308 was calibrated to the solvent
as internal standard. Afterwards, a distinct signal was chosen for calibration of the following spectra (10:
N-Me group; 11: MeCN, remaining from preceding HPLC purification). MS: VG Instruments Autospec
(EI, FAB), Thermo Scientific LTQ-Orbitrap XL (HR-ESI), Bruker daltonics autoflex (MALDI-TOF;
matrices: 2,5-dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, and 2’,4’,6’-trihydroxyacetophe-
none); only characteristic fragments are given with intensities [%] and possible composition in
parentheses.
General Procedure for Removing the Boc Group (GP 1). The protected compound was placed in
CH2Cl2 at 08. In the presence of nucleobases, Et3SiH was added as a scavenger. An equal amount of TFA
was added. After stirring at 08 for 1 h and additional 2 h at r.t., the volatiles were removed by repeated
co-evaporation with toluene. The crude product was used without further purification, or was
precipitated with Et2O and collected by suction.
General Procedure for Saponification of Methyl Esters (GP 2). The ester was placed in THF at 08.
After addition of an equal amount of 1m aq. LiOH (1.5 to 10 equiv.), the mixture was stirred at 08 for 1 h
and at r.t. for another h. The pH was then adjusted to 7 with 1m aq. KHSO4. The org. solvent was removed
by evaporation at reduced pressure, and the resulting mixture was cooled to 08, acidified to pH 2 – 3 with
1m aq. KHSO4, and extracted with AcOEt (3ꢃ). The combined extracts were washed with brine and
dried (MgSO4). Evaporation of the solvent and drying in vacuo yielded the product.
General Procedure for Amide Bond Formation with HBTU (GP 3). HBTU (1 equiv.) was added to a
stirred soln. of the carboxylic acid (1 equiv.) and EtNiPr2 (2 equiv.). After stirring for 30 min, the amine
was added, and the reaction was allowed to proceed overnight under Ar. The mixture was diluted with
the tenfold volume of AcOEt and successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine.
Then, the org. layer was dried (MgSO4) and concentrated under reduced pressure.
General Procedure for Amide Bond Formation with Brop (GP 4). The amine (1 equiv.), the
carboxylic acid (1.12, 1.2, or 1.25 equiv.), and Et3N (2.22 equiv.) were dissolved in CH2Cl2 or DMF at 08.
The reaction was started by the addition of Brop (1.12, 1.2, or 1.25 equiv.) and allowed to proceed
overnight while stirring at r.t. under Ar.
H-Aeg(H)-OH (¼ N-(2-Aminoethyl)glycine; 1). Ethylenediamine (823 g, 13.7 mol) was rapidly
stirred while being cooled in an ice-bath. ClCH2COOH (131 g, 1.39 mol) was added portionwise during
8 h, thereby ensuring that each portion was dissolved before adding further material. The mixture was

Helvetica Chimica Acta – Vol. 94 (2011)
1969
stirred at r.t. for 16 h, concentrated in vacuo, and triturated with DMSO (2 l). After stirring overnight, an
amorphous solid was isolated by suction, washed with DMSO and Et2O, and dried in vacuo: 1 (116 g,
980 mmol, 70%). Colorless solid. 1H-NMR (200 MHz, D2O): 3.24 (s, CH2 of Gly); 3.04 – 2.83 (m,
CH2CH2). 13C-NMR (50 MHz, D2O): 178.26 (CO); 51.56 (CH2 of Gly); 46.53 (NH2CH2CH2); 38.56
(NH2CH2). FAB-MS: 119.1 (100, [M þ H]þ).
(2,4-Difluoro-5-methylphenyl)acetic Acid (2). Mg Turnings (4.87 g, 200 mmol) were placed in dry
Et2O (10 ml). 1-(Bromomethyl)-2,4-difluoro-5-methylbenzene (4; 1.11 g, 5.00 mmol) was added drop-
wise to start the reaction. A soln. of 4 (21.0 g, 95.0 mmol) in dry Et2O (25 ml) was added dropwise over a
period of 10 min, and the mixture was refluxed for 2.5 h. After cooling to ꢀ 108, a stream of dry CO2 gas
was directed through the mixture for 1 h, thereby keeping the temp. below 08. The mixture was
hydrolyzed with ice and 6m aq. HCl. The org. layer was separated, and the aq. phase was extracted with
Et2O. The combined org. phases were washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by CC (SiO2; CH2Cl2 ! CH2Cl2/MeOH 10 :1) to give 2 (10.7 g, 57.5 mmol, 57%).
Colorless solid. Rf (CH2Cl2) 0.20. 1H-NMR (200 MHz, CDCl3): 9.86 (br. s, COOH); 7.06 (dd, t-like, J ¼
8.3, 8.3, arom. HꢀC(6)); 6.78 (dd, t-like, J ¼ 9.6, 9.6, arom. HꢀC(3)); 3.64 (s, CH2); 2.22 (s, Me). 13C-NMR
(50 MHz, CDCl3): 177.12 (CO); 160.56 (dd, 1J(C,F) ¼ 247, 3J(C,F) ¼ 11.5, arom. C(4)); 159.26 (dd,
1J(C,F) ¼ 247, 3J(C,F) ¼ 11.9, arom. C(2)); 133.37 (dd, 3J(C,F) ¼ 6.50, 3J(C,F) ¼ 5.40, arom. CH(6));
2
3
2
4
121.10 (dd, J(C,F) ¼ 17.3, J(C,F) ¼ 3.80, arom. C(5)); 116.33 (dd, J(C,F) ¼ 15.7, J(C,F) ¼ 3.80, arom.
C(1)); 103.54 (dd, t-like, 2J(C,F) ¼ 26.1, 26.1, arom. CH(3)); 33.77 (d, 3J(C,F) ¼ 2.70, CH2); 14.02 (d,
3J(C,F) ¼ 3.50, Me). 19F-NMR (565 MHz, (D6)DMSO): ꢀ 115.57 (m, FꢀC(4)); ꢀ 117.17 (m, FꢀC(2)).
.
.
EI-MS: 186.0 (32, Mþ ), 141.0 (100, [M ꢀ COOH]þ). HR-EI-MS: 186.0502 (30, Mþ , C9H8F2Oþ2 ; calc.
186.0492).
Compound 4. Paraformaldehyde (4.93 g, 156 mmol) was dissolved in 33% HBr/AcOH soln. (80 ml)
followed by the addition of 2,4-difluorotoluene (3; 20.0 g, 156 mmol) and ZnBr2 (15.8 g, 70.0 mmol).
After stirring for 4 h at 1208, the mixture was cooled to r.t. and hydrolyzed with an equal volume of H2O.
The combined org. layers were successively washed with sat. aq. NaHCO3 and brine, dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified by chromatography (SiO2, cyclohexane) to
1
give 4 (22.6 g, 120 mmol, 66%). Colorless oil. Rf (cyclohexane) 0.34. H-NMR (200 MHz, CDCl3): 7.20
(dd, t-like, J ¼ 8.3, 8.3, arom. HꢀC(6)); 6.77 (dd, t-like, J ¼ 9.6, 9.6, arom. HꢀC(3)); 4.46 (s, CH2); 2.23 (s,
Me). 13C-NMR (50 MHz, CDCl3): 161.28 (dd, 1J(C,F) ¼ 250, 3J(C,F) ¼ 11.9, arom. C(4)); 159.01 (dd,
1J(C,F) ¼ 250, 3J(C,F) ¼ 12.3, arom. C(2)); 133.21 (dd, 3J(C,F) ¼ 6.90, 3J(C,F) ¼ 4.20, arom. CH(6));
2
3
2
4
120.91 (dd, J(C,F) ¼ 29.1, J(C,F) ¼ 4.20, arom. C(5)); 121.23 (dd, J(C,F) ¼ 26.1, J(C,F) ¼ 4.20, arom.
C(1)); 103.89 (dd, 2J(C,F) ¼ 24.9, 2J(C,F) ¼ 26.5, arom. CH(3)); 25.39 (d, 3J(C,F) ¼ 3.80, CH2); 14.01 (d,
3J(C,F) ¼ 3.10, Me). 19F-NMR (565 MHz, (D6)DMSO): ꢀ 111.49 (m, FꢀC(4)); ꢀ 115.88 (m, FꢀC(2)).
EI-MS: 268 (72, [M þ 2 Na]þ), 253 (100), 141.0 (78, [M ꢀ Br]þ).
Me2-Aeg(Me)-OH (¼ N-[2-(Dimethylamino)ethyl]-N-methylglycine; 5a). To a soln. of 1 (2.50 g,
21.2 mmol) in H2O (200 ml) at 08, a soln. of formalin (5.68 ml, 73.3 mmol) in H2O (100 ml) was added
dropwise. After stirring for 1 h at r.t., 10% Pd/C (2.00 g) was added, and the mixture was hydrogenated at
3.5 bar. Upon completion of the reaction, the mixture was filtered through a pad of diatomaceous earth
and concentrated in vacuo. All volatiles were removed by repeated co-evaporation with toluene. The
residue was crystallized from MeOH (20 ml) to give 5a (2.54 g, 15.7 mmol, 75%). Colorless solid. For
anal. data, see below.
Me2-Aeg(Me)-OH · 2 HCl (5a · 2 HCl). To 1 (2.50 g, 21.2 mmol) at 08, HCOOH (3.99 ml,
106 mmol), followed by formalin (5.68 ml, 73.3 mmol), was added. The resulting soln. was refluxed
for 12 h, cooled to r.t., and treated with conc. aq. HCl (11.6 ml). The volatiles were evaporated in vacuo,
and the residue was crystallized from MeOH (20 ml) to give 5a · 2 HCl (2.02 g, 8.67 mmol, 41%).
Colorless solid. 1H-NMR (200 MHz, D2O): 4.20 (s, CH2 of Gly); 3.77 (m, CH2CH2); 3.11 (s, MeN); 3.04
(s, Me2N). 13C-NMR (50 MHz, D2O): 168.26 (COOH); 57.31 (CH2 of Gly); 51.29 (CH2); 50.82 (CH2);
43.57 (MeN); 42.17 (Me2N). FAB-MS: 321.2 (10, [2M þ H]þ), 161.1 (100, [M þ H]þ).
Me2-Aeg(Me)-Aeg(C)-Aeg(F)-Aeg(G)-OH · 3 TFA (¼ N-[(2-Amino-6-oxo-1,6-dihydro-9H-purin-
9-yl)acetyl]-N-[2-({N-[2-({N-[(4-amino-2-oxopyrimidin-1(2H)-yl)acetyl]-N-[2-({N-[2-(dimethylami-
no)ethyl]-N-methylglycyl}amino)ethyl]glycyl}amino)ethyl]-N-[(2,4-difluoro-5-methylphenyl)acetyl]gly-
cyl}amino)ethyl]glycine; 10). Me2-Aeg(Me)-Aeg(CZ)-Aeg(F)-Aeg(GZ)-OBn · 2 TFA (49; 305 mg,

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Helvetica Chimica Acta – Vol. 94 (2011)
214 mmol; see later), m-cresol (0.50 ml, 9.6 mmol), and thioanisole (0.50 ml, 8.5 mmol) were dissolved in
TFA (3 ml). TFMSA (1 ml) was added dropwise, and the mixture was shaken for 2 h at r.t. The reaction
was then quenched by the addition of Et2O (45 ml), and the resulting suspension was cooled to 08. The
precipitate was sedimented by centrifugation and washed with Et2O twice. Purification by RP-HPLC and
subsequent freeze drying gave 10 (157 mg, 120 mmol, 56%). Colorless powder. 1H-NMR (200 MHz,
(D6)DMSO, 1408): 7.63 (d, J ¼ 7.4, HꢀC(6) of C); 7.57 (s, HꢀC(8) of G); under it (br. s, 3 amide NH); 7.16
(t, J ¼ 8.7, 1 arom. H of F); 6.89 (t, J ¼ 9.8, 1 arom. H of F); 6.21 (br. s, various R3NþH, COOH); 5.96 (d,
J ¼ 7.4, HꢀC(5) of C); 4.88 (s, CH2); 4.65 (s, CH2); 4.15 (s, CH2); 4.04 (s, CH2); 4.02 (s, CH2); 3.65 (s,
CH2); 3.58 – 3.17 (m, 14 H of 4 CH2CH2, CH2); 2.85 (s, Me2N); under it (m, 2 H of 4 CH2CH2); 2.37 (s,
Me); 2.17 (s, Me). 19F-NMR (235 MHz, (D6)DMSO, 308; rotamers): ꢀ 78.80 (s, TFA); ꢀ 118.45 (m,
FꢀC(4)); ꢀ 120.99 (m, FꢀC(2)). MALDI-MS: 972.564 (485, [M þ 2 H]þ). HR-ESI-MS: 973.4433 (13,
[M þ 3 H]þ, C41H59F2N16O1þ0 ; calc. 973.4568), 972.4411 (48, [M þ 2 H]þ, C41H58F2N16O1þ0 ; calc. 972.4490),
971.4381 (100, [M þ H]þ, C41H57F2N16O1þ0 ; calc. 971.4411).
H-Aeg(C)-Aeg(A)-Aeg(G)-Aem · 4 TFA (¼2-Amino-N-[12-(2-aminoethyl)-14-(4-amino-2-oxopyri-
midin-1(2H)-yl)-6-[2-(6-amino-9H-purin-9-yl)acetyl]-4,10,13-trioxo-3,6,9,12-tetraazatetradec-1-yl]-1,6-
dihydro-N-(2-{[2-(morpholin-4-yl)ethyl]amino}-2-oxoethyl)-6-oxo-9H-purine-9-acetamide; 11). Z-
Aeg(CZ)-Aeg(AZ)-Aeg(GZ)-Aem · TFA (36; 491 mg, 0.308 mmol; see below), m-cresol (0.50 ml,
9.6 mmol), and thioanisole (0.50 ml, 8.5 mmol) were dissolved in TFA (3 ml). TFMSA (1 ml) was
added dropwise, and the mixture was shaken for 2 h at r.t. The reaction was then quenched by the
addition of Et2O (45 ml), and the resulting suspension was cooled to 08. The precipitate was sedimented
by centrifugation and washed with Et2O twice. Purification by RP-HPLC and subsequent freeze-drying
gave 11 (238 mg, 170 mmol, 55%). Colorless powder. 1H-NMR (200 MHz, (D6)DMSO, 1408; rotamers):
8.56, 8.22, 8.21, 8.02 (4s, HꢀC(2) of A, HꢀC(8) of A); 7.88 (br. m, 3 amide NH); 7.76 (m, HꢀC(C(6) of C,
HꢀC(8) of G); 6.42 (br. s, div. R3NþH); 6.88 (m, HꢀC(5) of C); 5.12 (br. s, base CH2); 4.89 (m, base
CH2); 4.57 (br. s, base CH2); 3.78 – 3.10 (m, 30 H, 2 NCH2CH2O, 4 CH2CH2, 3 CH2 of Gly). MALDI-MS:
949.298 (169, [M þ 2 H]þ). HR-ESI-MS: 950.4438 (13, [M þ 3 H]þ, C38H56N21Oþ9 ; calc. 950.4570),
949.4411 (51, [M þ 2 H]þ, C38H55N21O9þ ; calc. 949.4491), 948.4387 (100, [M þ H]þ, C38H54N21Oþ9 ; calc.
948.4413).
H-Aeg(H)-OMe · 2 HCl (¼ Methyl N-(2-Aminoethyl)glycinate Dihydrochloride; 13 · 2 HCl). To a
suspension of 1 (93.0 g, 787 mmol) in MeOH (1.5 l) at 08, SOCl2 (138 ml, 1.90 mol) was added dropwise.
After refluxing overnight, the volume was reduced to one-third, and Et2O (500 ml) was added. The
suspension was stirred for 30 min in an ice-bath, and the precipitate was collected by suction, washed with
Et2O, and dried in vacuo to give 13 · 2 HCl (130 g, 641 mmol, 81%). Colorless solid. 1H-NMR (200 MHz,
D2O): 4.14 (s, CH2 of Gly); 3.86 (s, MeO); 3.56 – 3.39 (m, CH2CH2). 13C-NMR (50 MHz, D2O): 167.73
(CO); 54.03 (MeO); 48.03 (CH2 of Gly); 39.81 (NH2CH2CH2); 35.79 (NH2CH2). FAB-MS: 154.0 (100,
[M þ Na]þ); 133.1 (70, [M þ H]þ).
Z-Aeg(Boc)-OMe (¼ Methyl N-(2-{[(Benzyloxy)carbonyl]amino}ethyl)-N-[(tert-butoxy)carbo-
nyl]glycinate; 14). A soln. of Boc2O (19.7 g, 93.8 mmol) in CH2Cl2 (90 ml) was added to a soln. of 17
(24.9 g, 93.8 mmol; see below) and Et3N (13.1 ml, 93.8 mmol) in CH2Cl2 (410 ml) at 08. After stirring
overnight at r.t., the mixture was successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine
(500 ml each), dried (MgSO4), filtered, and concentrated in vacuo to give 14 (32.9 g, 89.9 mmol, 96%).
Colorless oil. 1H-NMR (200 MHz, (D6)DMSO; 2 rotamers): 7.34 (s, 5 arom. H of Z); 7.18 (m, NH); 5.01
(s, CH2 of Z); 3.93 (s, 0.90 H of CH2 of Gly); 3.91 (s, 1.10 H of CH2 of Gly); 3.65 (s, 1.30 H of MeO); 3.63
(s, 1.70 H of MeO); 3.07 – 3.29 (m, CH2CH2); 1.37 (s, 4.10 H of t-Bu); 1.32 (s, 4.90 H of t-Bu). 13C-NMR
(50 MHz, (D6)DMSO; 2 rotamers): 170.61 (CO of Gly); 170.64 (CO of Gly); 156.07 (CO of Z); 154.89
(CO of Boc); 154.55 (CO of Boc); 137.21 (arom. C of Z); 137.10 (arom. C of Z); 128.34 (arom. o-CH of
Z); 127.75 (arom. m-CH of Z); 127.66 (arom. p-CH of Z); 79.31 (Me3C); 79.24 (Me3C); 65.27 (CH2 of Z);
65.23 (CH2 of Z); 51.72 (MeO); 49.28, 48.38, 47.24, 47.14 (2 CH2 of Gly, 2 ZNHCH2CH2); the signal for
ZNHCH2 is expected at ca. 40 ppm, and is probably hidden by the DMSO signal; 27.88 (Me of Boc); 27.80
(Me of Boc). FAB-MS: 389.1 (26, [M þ Na]þ), 367.1 (22, [M þ H]þ), 267.0 (100, [M ꢀ Boc]þ).
Boc-Aeg(Alloc)-OMe (¼ Methyl N-[(Allyloxy)carbonyl]-N-(2-{[(tert-butoxy)carbonyl]amino}-
ethyl)glycinate; 15). Boc-Aeg(H)-OMe (18; 24.0 g, 103 mmol; see below) and Et3N (14.4 ml, 103 mmol)
were dissolved in CH2Cl2 (360 ml) at 08. A soln. of Alloc-Cl (14.3 ml, 134 mmol) in CH2Cl2 (30 ml) was

Helvetica Chimica Acta – Vol. 94 (2011)
1971
then added dropwise. After stirring for 2 h at r.t., the solvent was evaporated in vacuo. The residue was
taken up in AcOEt (500 ml) and successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine
(500 ml each). The org. layer was dried (MgSO4), and the solvent was evaporated under reduced
pressure. Compound 15 (27.8 g, 87.9 mmol, 85%) was obtained as a colorless oil and used without further
purification. 1H-NMR (200 MHz, CDCl3 ; 2 rotamers): 5.90 (m, CH2CH¼CH2); 5.25 (m, NH,
CH2CH¼CH2); 4.60 (m, CH2CH¼CH); 4.00 (s, CH2 of Gly); 3.75, 3.74 (2s, MeO); 3.46 (m, BocNHCH2);
3.29 (m, BocNHCH2CH2); 1.43 (s, t-Bu). 13C-NMR (50 MHz, CDCl3; 2 rotamers): 170.83 (CO of Gly);
170.56 (CO of Gly); 156.36 (CO of Boc); 156.21 (CO of Alloc); 156.07 (CO of Alloc); 132.61
(CH2CH¼CH2); 117.88 (CH2CH¼CH2); 117.42 (CH2CH¼CH2); 79.34 (Me3C); 66.66 (CH2CH¼CH2);
66.43 (CH2CH¼CH2); 52.36 (MeO); 49.94 (CH2 of Gly); 49.69 (CH2 of Gly); 49.02 (BocNHCH2CH2);
48.69 (BocNHCH2CH2); 39.19 (BocNHCH2); 28.46 (Me of Boc). FAB-MS: 655.4 (5, [2M þ Na]þ), 633.4
(3, [2M þ H]þ), 339.2 (25, [M þ Na]þ), 317.2 (23, [M þ H]þ), 217.1 (100, [M ꢀ Boc]þ).
Boc-Aeg(Fmoc)-OMe (¼ Methyl N-(2-{[(tert-Butoxy)carbonyl]amino}ethyl)-N-{[(9H-fluoren-9-
yl)methoxy]carbonyl}glycinate; 16). Compound 18 (26.5 g, 114 mmol) and EtNiPr2 (39.7 ml, 228 mmol)
were dissolved in CH2Cl2 (400 ml). After cooling to 08, a soln. of Fmoc-Cl (29.4 g, 144 mmol) in CH2Cl2
(100 ml) was added. The mixture was stirred for 1 h at 08 and for additional 3 h at r.t. After washing with
1m aq. KHSO4, sat. aq. NaHCO3, and brine, the org. phase was dried (MgSO4). Evaporation of the
solvent, followed by CC (SiO2; CH2Cl2/MeOH 50 :1) gave 16 (44.0 g, 96.9 mmol, 86%). Colorless oil. Rf
1
(CH2Cl2/MeOH 50 :1) 0.21. H-NMR (200 MHz, (D6)DMSO, 1108): 7.85 (m, 2 arom. H of Fmoc); 7.62
(m, 2 arom. H of Fmoc); 7.46 – 7.29 (m, 4 arom. H of Fmoc); 6.21 (br. m, NH); 4.39 (m, CH2 of Fmoc);
4.26 (m, CH of Fmoc); 3.97 (s, CH2 of Gly), 3.65 (s, Me), 3.33 (m, 2 H of CH2CH2), 3.09 (m, 2 H of
CH2CH2); 1.39 (s, t-Bu). 13C-NMR (50 MHz, (D6)DMSO, 1108): 169.25 (COOMe); 154.95 (CO of Fmoc,
CO of Boc); 143.32 (arom. C of Fmoc); 140.27 (arom. C of Fmoc); 126.94 (arom. CH of Fmoc); 126.40
(arom. CH of Fmoc); 124.20 (arom. CH of Fmoc); 119.30 (arom. CH of Fmoc); 77.30 (Me3C), 66.56 (CH2
of Fmoc); 50.96 (MeO); 48.47, 47.31, 46.41, 38.26 (CH of Fmoc, CH2 of Gly, CH2CH2); 27.65 (Me of Boc).
FAB-MS: 477 (15, [M þ Na]þ), 455 (5, [M þ H]þ), 355.1 (67, [M ꢀ Boc]þ), 178.0 (100).
Z-Aeg(H)-OMe (¼ Methyl N-(2-{[(Benzyloxy)carbonyl]amino}ethyl)glycinate; 17). To a soln. of Z-
OSu (37.1 g, 150 mmol) in MeCN (600 ml) at ꢀ 158 13 · 2 HCl (30.4 g, 150 mmol), followed by NMM
(41.1 ml, 373 mmol), was added. The mixture was stirred at ꢀ 158 for 3 h, followed at r.t. overnight. After
evaporation of volatiles in vacuo, the residue was taken up in 1m aq. KHSO4 (600 ml) and washed with
AcOEt (600 ml). The pH was adjusted to 8 – 9 with solid NaHCO3, and the aq. soln. was washed with
AcOEt (3 ꢃ 500 ml). The combined org. phases were washed with brine, dried (MgSO4), filtered, and
concentrated to give 17 (25.0 g, 93.9 mmol, 63%). Colorless oil. 1H-NMR (200 MHz, CDCl3; 2 rotamers):
7.38 – 7.13 (m, 5 arom. H of Z); 5.32 (br. s, NH of Z); 5.02 (s, CH2 of Z); 3.64 (s, MeO); 3.32 (s, CH2 of
Gly); 3.20 (dt, J ¼ 5.7, 5.7, ZNHCH2); 2.68 (t, J ¼ 5.7, ZNHCH2CH2). 13C-NMR (50 MHz, CDCl3; 2
rotamers): 172.95 (CO of Gly); 156.55 (CO of Z); 136.63 (arom. C of Z); 128.51 (arom. o-CH of Z); ca.
128.07 (2s (not resolved), arom. p-CH of Z, arom. m-CH of Z); 66.65 (CH2 of Z); 51.86 (MeO); 50.23
(CH2 of Gly); 48.63 (CH2 of Z); 40.63 (ZNHCH2). FAB-MS: 289.0 (11, [M þ Na]þ), 267.1 (100, [M þ
H]þ).
Boc-Aeg(H)-OMe (¼ Methyl N-(2-{[(tert-Butoxy)carbonyl]amino}ethyl)glycinate; 18). Compound
13 · 2 HCl (20.0 g, 98.5 mmol) was suspended in CH2Cl2 (1.2 l) at ꢀ 108, followed by the addition of Et3N
(27.3 ml, 197 mmol). After adding a soln. of Boc2O (21.5 g, 98.5 mmol) in CH2Cl2 (300 ml) dropwise
during 30 min, the mixture was stirred at r.t. overnight. The mixture was successively washed with H2O
and brine, dried (MgSO4), and concentrated in vacuo. The residue was distilled under reduced pressure
to give 18 (14.4 g, 62.0 mmol, 63%). Colorless oil. Rf (CH2Cl2/MeOH 10 :1) 0.55. Rf (AcOEt/MeOH 4 :1)
0.45. 1H-NMR (200 MHz, CDCl3): 3.72 (s, MeO); 3.40 (s, CH2 of Gly); 3.20 (dt, q-like, J ¼ 5.8, 5.8,
BocNHCH2); 2.73 (t, J ¼ 5.8, BocNHCH2CH2); 1.43 (s, t-Bu). 13C-NMR (50 MHz, CDCl3): 173.04 (CO of
Gly); 156.21 (CO of Boc); 79.34 (Me3C); 51.97 (MeO); 50.41 (CH2 of Gly); 48.90, 41.02 (CH2CH2); 28.53
(Me of Boc). FAB-MS: 255.1 (19, [M þ Na]þ), 233.1 (100, [M þ H]þ).
Boc-Aeg(Alloc)-OH (¼ N-[(Allyloxy)carbonyl-]-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)gly-
cine; 19). Compound 15 (27.8 g, 87.8 mmol) was dissolved in THF (180 ml) and treated with an equal
volume of 1m aq. LiOH according to GP 1. After workup, the product 15 (25.1 g, 83.0 mmol, 94%) had
1
sufficient purity to be used in the next synthetic step. H-NMR (200 MHz, (D6)DMSO; 2 rotamers):

1972
Helvetica Chimica Acta – Vol. 94 (2011)
12.67 (br. s, COOH); 6.75 (br. m, NH); 6.01 – 5.73 (m, CH2CH¼CH2); 5.34 – 5.11 (m, CH2CH¼CH2); 4.51
(m, CH2CH¼CH); 3.92, 3.89 (2s, CH2 of Gly); 3.33 – 3.05 (m, CH2CH2); 1.36 (s, t-Bu). 13C-NMR
(50 MHz, (D6)DMSO; 2 rotamers): 171.18 (COOH); 171.07 (COOH); 155.59, 155.46, 155.20 (CO of
Boc, CO of Alloc); 133.22 (CH2CH¼CH2); 116.70 (CH2CH¼CH2); 116.34 (CH2CH¼CH2); 77.67
(Me3C); 65.34 (CH2CH¼CH2); 65.13 (CH2CH¼CH2); 48.97 (CH2 of Gly); 48.81 (CH2 of Gly); 47.78
(BocNHCH2CH2); 47.13 (BocNHCH2CH2); 38.36 (BocNHCH2); 38.06 (BocNHCH2); 28.18 (Me of
Boc). FAB-MS: 325.1 (100, [M þ Na]þ), 303 (19, [M þ H]þ), 203.1 (73, [M ꢀ Boc]þ).
H-Aeg(Fmoc)-OMe · TFA (¼ Methyl N-(2-Aminoethyl)-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-
glycinate; 20). Compound 16 (44.0 g, 96.6 mmol) was treated with CH2Cl2 (100 ml) and TFA (100 ml)
according to in GP 1. After precipitation with Et2O, an amorphous solid was isolated by suction. Crude 20
(41.2 g, 87.8 mmol, 91%) was directly used in the next step. FAB-MS: 709.1 (5, [2M þ H]þ), 377.0 (11,
[M þ Na]þ), 355.1 (100, [M þ H]þ).
Boc-Aeg(Alloc)-Aeg(Fmoc)-OMe (¼ Methyl N-(2-{[N-(2-{[(tert-Butoxy)carbonyl]amino}ethyl)-N-
[(prop-2-en-1-yloxy)carbonyl]glycyl]amino}ethyl)-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycinate;
21). Compounds 19 (13.9 g, 45.8 mmol) and 20 (21.5 g, 45.8 mmol) were treated according to GP 3. The
crude product was purified by CC (SiO2; AcOEt/MeOH 9 :1) to give 21 (22.4 g, 35.0 mmol, 76%).
Colorless foam. Rf (AcOEt/MeOH 9 :1) 0.6. 1H-NMR (250 MHz, (D6)DMSO, 1008; 2 rotamers, the
Fmoc group is not in the regime of fast exchange): 7.87 (m, 2 arom. H of Fmoc); 7.61 (d, J ¼ 7.4, 2 arom. H
of Fmoc); 7.5 (br. m, NH); 7.45 – 7.29 (m, 4 arom. H of Fmoc); 6.35 (br. t, NH); 5.90 (ddt, J ¼ 17, 11, 5.2,
CH2CH¼CH2); 5.27 (ddt, dq-like, J ¼ 17, 1.6, 1.6, CH2CH¼CHZHE); 5.15 (ddt, dq-like, J ¼ 11, 1.6, 1.6,
CH2CH¼CHZHE); 4.51 (dt, J ¼ 5.2, 1.6, 2 H, CH2CH¼CH2); 4.39 (m, CH2 of Fmoc); 4.26 (m, CH of
Fmoc); 3.97 (s, N(Alloc)CH2CONH); 3.82 (s, 1.6 H of N(Fmoc)CH2COOMe); 3.74 (s, 0.4 H of
N(Fmoc)CH2COOMe); 3.65, 3.64 (2s, MeO); 3.36 – 3.08 (m, 2 CH2CH2); 1.39 (s, t-Bu). 13C-NMR
(50 MHz, (D6)DMSO; 2 rotamers, the Fmoc group is not in the regime of fast exchange): 169.39
(COMe); 168.32 (CO of Gly); 155.03, 155.01 (2 CO); 143.34 (arom. C of Fmoc); 140.31 (arom. C of
Fmoc); 132.79 (CH2CH¼CH2); 127.01 (arom. CH of Fmoc); 126.47 (arom. CH of Fmoc); 124.26 (arom.
CH of Fmoc); 119.38 (arom. CH of Fmoc); 116.08 (CH2CH¼CH2); 77.24 (Me3C); 66.64 (CH2CH¼CH2);
64.79 (CH2 of Fmoc); 51.07 (Me); 50.02 (CH2); 49.32 (CH2); 48.58 (CH2); 48.62 (CH2); 47.65 (CH2);
47.08 (CH2); 46.40 (CH of Fmoc); 38.18 (CH2); 36.90 (CH2); 36.91 (CH2); 27.71 (Me of Boc). FAB-MS:
678.3 (47, [M þ K]þ), 661.3 (97, [M þ Na]þ), 539.3 (33, [M ꢀ Boc]þ), 178.1 (100).
Z-Aeg(Boc)-Aeg(Alloc)-Aeg(Fmoc)-OMe (¼ Methyl N-[2-({[({N-[(Allyloxy)carbonyl]-N-(2-{[N-
(2-{[(benzyloxy)carbonyl]amino}ethyl)-N-{[((tert-butoxy)carbonyl]glycyl}amino)ethyl]glycyl}ami-
no)ethyl]-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycinate; 22). Z-Aeg(Boc)-OH (24; 12.3 g,
34.9 mmol; see below) and H-Aeg(Alloc)-Aeg(Fmoc)-OMe (23; 22.8 g, 34.9 mmol; see below) were
coupled according to GP 3. Purification by CC (SiO2; AcOEt/MeOH 9 :1) afforded 22 (19.0 g,
21.7 mmol, 62%). Colorless foam. Rf (AcOEt/MeOH 9 :1) 0.28. 1H-NMR (250 MHz, (D6)DMSO, 1008):
7.85 (d, J ¼ 7.0, 2 arom. H of Fmoc); 7.61 (d, J ¼ 7.5, 2 arom. H of Fmoc); under it (br. s, NH); 7.57 (br. s,
NH); 7.44 – 7.28 (m, 4 arom. H of Fmoc, 5 arom. H of Z); 6.81 (br. t, NH); 5.90 (ddt, J ¼ 17, 11, 5.2,
CH2CH¼CH2); 5.28 (ddt, dq-like, J ¼ 17, 1.6, 1.6, CH2CH¼CHZHE); 5.15 (ddt, dq-like, J ¼ 11, 1.6, 1.6,
CH2CH¼CHZHE); 5.04 (s, CH2 of Z); 4.52 (dt, J ¼ 5.2, 1.6, CH2CH¼CH2); 4.39 (m, CH2 of Fmoc); 4.26
(br. t, J ¼ 6.2, CH of Fmoc); 3.97 (s, CH2 of Gly); 3.84 (s, CH2 of Gly); 3.74 (s, CH2 of Gly); 3.64 (s, Me);
3.35 – 3.15 (m, 3 CH2CH2); 1.38 (s, t-Bu). 13C-NMR (63 MHz, (D6)DMSO, 1008): 169.37 (COOMe);
168.65 (CO of Gly); 168.38 (CO of Gly); 155.51, 155.04, 155.00, 154.48 (CO of Z, CO of Boc, CO of Alloc,
CO of Fmoc); 143.34 (arom. C of Fmoc); 140.32 (arom. C of Fmoc); 136.78 (arom. C of Z); 132.75
(CH2CH¼CH2); 127.66 (arom. CH of Fmoc); 127.01 (arom. o-CH of Z); 126.90 (arom. m-CH of Z);
126.47 (arom. p-CH of Z); 124.26 (arom. CH of Fmoc); 119.38 (arom. CH of Fmoc); 116.16
(CH2CH¼CH2); 78.59 (Me3C); 66.63 (CH2CH¼CH2); 64.84 (CH2 of Fmoc); 51.07 (Me); 46.40 (CH of
Fmoc); signals of the remaining CH2 groups are located in the range of 51.13 – 37.74 ppm, but cannot be
separated unambiguously from the baseline under the given conditions (76 mm sample, 1024 scans); 27.50
(Me of Boc). FAB-MS: 895.3 (35, [M þ Na]þ), 873.3 (15, [M þ H]þ), 795.3 (3, [M ꢀ Boc þ Na]þ), 773.3
(36, [M ꢀ Boc]þ), 91.0 (100).
H-Aeg(Alloc)-Aeg(Fmoc)-OMe · TFA (¼ Methyl N-[2-({N-[(Allyloxy)carbonyl]-N-(2-aminoethyl)-
glycyl}amino)ethyl]-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycinate; 23). Compound 21 (22.4 g,

Helvetica Chimica Acta – Vol. 94 (2011)
1973
35.0 mmol) was treated with CH2Cl2 (40 ml) and TFA (40 ml) according to GP 1. The resulting oil
(22.8 g, 34.9 mmol, 99%) was directly used in the next step. FAB-MS: 561.2 (17, [M þ Na]þ), 539.2 (100,
[M þ H]þ).
Z-Aeg(Boc)-OH (¼ N-(2-{[(Benzyloxy)carbonyl]amino}ethyl)-N-[(tert-butoxy)carbonyl]glycine;
24). The product was prepared from a soln. of 14 (32.9 g, 89.9 mmol) in THF (180 ml) according to
GP 2. After adjusting the pH to 2 – 3, the product precipitated as an amorphous solid. The precipitate was
collected by suction, washed with H2O and Et2O, and dried in vacuo to give 24 (29.4 g, 83.4 mmol, 93%).
Colorless solid. 1H-NMR (200 MHz, (D6)DMSO; 2 rotamers): 12.61 (br. s, COOH); 7.34 (m, 5 arom. H
of Z); 7.17 (br. m, NH of Z); 5.01 (s, CH2 of Z); 3.86 (s, 1 H of CH2 of Gly); 3.82 (s, 1 H of CH2 of Gly);
3.35 – 3.11 (m, CH2CH2); 1.37 (s, 4 H of t-Bu); 1.34 (s, 5 H of t-Bu). 13C-NMR (50 MHz, (D6)DMSO; 2
rotamers): 171.61 (CO of Gly); 171.46 (CO of Gly); 156.16 (CO of Z); 156.09 (CO of Z); 154.93 (CO of
Boc); 154.78 (CO of Boc); 137.24 (arom. C of Z); 137.13 (arom. C of Z); 128.36 (arom. o-CH of Z); 127.75
(arom. m-CH of Z); 127.65 (arom. p-CH of Z); 79.09 (Me3C); 79.07; (Me3C); 65.28 (CH2 of Z); 65.22
(CH2 of Z); 49.30 (CH2 of Gly); 48.44 (CH2 of Gly); 47.26 (ZNHCH2CH2); 47.16 (ZNHCH2CH2); the
signal for ZNHCH2 is expected at ca. 40 ppm, and is probably hidden by the DMSO signal; 27.93 (Me of
Boc), 27.87 (Me of Boc). FAB-MS: 375.1 (15, [M þ Na]þ), 353.1 (24, [M þ H]þ), 297.1 (25, [M ꢀ tBu]þ),
253.1 (81, [M ꢀ Boc]þ), 91.0 (100).
Z-Aeg(Boc)-Aeg(Alloc)-Aeg(Fmoc)-OH (¼ N-[2-({[{N-[(Allyloxy)carbonyl]-N-(2-{[N-(2-{[(ben-
zyloxy)carbonyl]amino}ethyl)-N-{[(tert-butoxy)carbonyl]glycyl}amino)ethyl]glycyl}amino)ethyl]-N-
{[(9H-fluoren-9-yl)methoxy]carbonyl}glycine; 25). Compound 22 (19.0 g, 21.7 mmol) was dissolved in
THF (43.4 ml) at 08; 1m aq. LiOH (43.4 ml) was added, and the mixture was stirred at 08 for 1 h and an
additional h at r.t. Fmoc Elimination, which had taken place to some extent, was reversed by addition of
Fmoc-Cl (7.31 g, 28.2 mmol, 1.3 equiv.) and stirring for 1 h at 08. The mixture was then adjusted to pH 3
with a 1m aq. KHSO4 and extracted with AcOEt (3 ꢃ 150 ml). The combined org. phases were washed
with brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by CC (SiO2, AcOEt/
MeOH 9 :1 ! 1:1) to give 25 (12.9 g, 15.0 mmol, 69%). Colorless foam. Rf (AcOEt/MeOH 1:1) 0.46.
1H-NMR (250 MHz, (D6)DMSO; rotamers): 12.74 (br. s, COOH); 8.00 (br. s, 2 NH); 7.89 (br. d, J ¼ 7.4,
2 arom. H of Fmoc); 7.67 (d, J ¼ 7.3, 1 arom. H of Fmoc); 7.62 (d, J ¼ 7.5, 1 arom. H of Fmoc); 7.45 – 7.23
(m, 4 arom. H of Fmoc, 5 arom. H of Z, NH); 6.01 – 5.76 (m, CH2CH¼CH2); 5.20 (m, CH2CH¼CH2); 5.00
(s, CH2 of Z); 4.50 (m, CH2CH¼CH2); 4.27 (m, CH2 of Fmoc, CH of Fmoc); 4.04 – 3.68 (m, 3 CH2 of
Gly); 3.39 – 3.13 (m, 3 CH2CH2); 1.36, 1.31 (2s, Me3C). Elevated temps. led to fast Fmoc elimination,
probably due to formation of a cyclic anhydride with the carboxy. Therefore, the characterization was not
applicable in the overall fast regime. FAB-MS: 881.2 (45, [M þ Na]þ), 859.2 (13, [M þ H]þ), 759.2 (28,
[M ꢀ Boc]þ), 91.0 (100).
Z-Aeg(Boc)-Aeg(Alloc)-Aeg(Fmoc)-Aem (¼ N-[15-[(Allyloxy)carbonyl]-9-[(tert-butoxy)carbon-
yl]-3-{[(9H-fluoren-9-yl)methoxy]carbonyl}-20-(morpholin-4-yl)-5,11,17-trioxo-3,6,9,12,15,18-hexaaza-
icos-1-yl]carbamic Acid Phenylmethyl Ester; 27). Compound 25 (12.8 g, 14.9 mmol) and HOSu (2.56 g,
22.3 mmol, 1.5 equiv.) were dissolved in CH2Cl2 (60 ml) at 08 under Ar, followed by the addition of DCC
(3.38 g, 16.4 mmol, 1.1 equiv.). The mixture was allowed to warm to r.t. while stirring overnight. After
cooling to ꢀ 158, 4-(2-aminoethyl)morpholine (26; 1.96 ml, 14.9 mmol, 1 equiv.) was added, and stirring
was continued for 1 h at ꢀ 158 and additional 3 h at r.t. The dicyclohexylurea was filtered off, washed with
CH2Cl2, and the filtrate was concentrated under reduced pressure. The residue was taken up in AcOEt
(500 ml), successively washed with sat. aq. NaHCO3 and brine (252 ml each), dried (MgSO4), and
concentrated in vacuo. The crude product was purified by CC (SiO2; AcOEt/MeOH 2 :1) to give 27
(12.1 g, 12.5 mmol, 84%). Colorless foam. Rf (AcOEt/MeOH 2 :1) 0.32. 1H-NMR (250 MHz,
(D6)DMSO, 1008): 7.85 (d, J ¼ 7.4, 2 arom. H of Fmoc); 7.72 (br. t, NH); 7.64 (d, J ¼ 7.3, 2 arom. H of
Fmoc); under it (NH); 7.48 – 7.26 (m, 4 arom. H of Fmoc, 5 arom. H of Z, NH); 6.81 (br. t, NH); 5.90 (ddt,
J ¼ 17, 11, 5.2, CH2CH¼CH2); 5.27 (ddt, dq-like, J ¼ 17, 1.6, 1.6, CH2CH¼CHZHE); 5.15 (ddt, dq-like, J ¼
11, 1.6, 1.6, CH2CH¼CHZHE); 5.04 (s, CH2 of Z); 4.52 (br. dt, CH2CH¼CH2); 4.30 (m, CH2 of Fmoc, CH
of Fmoc); 3.88 (s, CH2 of Gly); 3.85 (s, CH2 of Gly); 3.74 (s, CH2 of Gly); 3.54 (m, 2 NCH2CH2O); 3.37 –
3.11 (m, 3.5 CH2CH2); 2.38 (m, 2 NCH2CH2O, 0.5 CH2CH2); 1.38 (s, t-Bu). Elevated temps. led to efficient
Fmoc elimination, probably due to the presence of the tertiary amine. Therefore, the characterization
by 13C-NMR spectroscopy was not possible. MALDI-MS: 971.917 (68, [M þ H]þ), 871.771 (248,

1974
Helvetica Chimica Acta – Vol. 94 (2011)
[M ꢀ Boc]þ). HR-ESI-MS: 973.4910 (17, [M þ 3 H]þ, C50H69N8O1þ2 ; calc. 973.5034), 972.4885 (58, [M þ
2 H]þ, C50H68N8O1þ2 ; calc. 972.4956), 971.4853 (100, [M þ H]þ, C50H67N8O1þ2 ; calc. 971.4878).
Z-Aeg(Boc)-Aeg(Alloc)-Aeg(H)-Aem (¼ N-[9-[(Allyloxy)carbonyl]-3-[(tert-butoxy)carbonyl]-20-
(morpholin-4-yl)-5,11,17-trioxo-3,6,9,12,15,18-hexaazaeicos-1-yl]carbamic Acid Phenylmethyl Ester; 31).
Compound 27 (11.1 g, 11.4 mmol) was dissolved in CH2Cl2 at r.t. and treated with Et2NH (17.8 ml,
171 mmol). After stirring for 1.5 h, the solvent was evaporated in vacuo, and the residue was purified by
CC (SiO2; AcOEt/MeOH 1:1 ! 0 :1) to give 31 (6.78 g, 9.05 mmol, 79%). Colorless foam. Rf (AcOEt/
MeOH 1:1) 0.15. 1H-NMR (200 MHz, (D6)DMSO, 1108): 7.73 (br. t, NH); 7.66 (br. t, NH); 7.54 (br. m,
NH); 7.34 (m, 5 arom. H of Z); 6.88 (br. t, NH); 5.89 (ddt, J ¼ 17, 11, 5.2, CH2CH¼CHZHE); 5.28 (ddt, dq-
like, J ¼ 17, 1.6, 1.6, CH2CH¼CHZHE); 5.16 (ddt, dq-like, J ¼ 11, 1.6, 1.6, CH2CH¼CHZHE); 5.04 (s, CH2 of
Z); 4.52 (dt, J ¼ 5.2, 1.6, CH2CH¼CH2); 3.86 (s, CH2 of Gly); 3.73 (s, CH2 of Gly); 3.74 (s, CH2 of Gly);
3.57 (m, 2 NCH2CH2O); 3.38 – 3.14 (m, 3.5 CH2CH2); 3.10 (s, CH2 of Gly); 2.40 (m, 2 NCH2CH2O,
0.5 CH2CH2); 1.38 (s, t-Bu). FAB-MS: 771.3 (21, [M þ Na]þ), 749.4 (59, [M þ H]þ), 649.3 (13, [M ꢀ
Boc]þ), 91 (100). HR-ESI-MS: 751.4239 (10, [M þ 3 H]þ, C35H59N8O1þ0 ; calc. 751.4354), 750.4212 (40,
[M þ 2 H]þ, C35H58N8O1þ0 ; calc. 750.4276), 749.4178 (100, [M þ H]þ, C35H57N8O1þ0 ; calc. 749.4197).
Z-Aeg(Boc)-Aeg(Alloc)-Aeg(GZ)-Aem (¼ 9-[(Allyloxy)carbonyl]-N-{15-[2-(1,6-dihydro-6-oxo-2-
{[(phenylmethoxy)carbonyl]amino}-9H-purin-9-yl)acetyl]-3-[(tert-butoxy)carbonyl]-20-(morpholin-4-
yl)-5,11,17-trioxo-3,6,9,12,15,18-hexaazaicos-1-yl}carbamic Acid Phenylmethyl Ester; 32). Compound 31
(6.78 g, 9.05 mmol) and GZ-AcOH (28; 3.88 g, 11.3 mmol) were coupled in CH2Cl2 (35 ml) according to
GP 4. The mixture was diluted with CHCl3 and successively washed with sat. aq. NaHCO3 and brine,
dried (MgSO4), and concentrated in vacuo. The crude product was purified by CC (SiO2 ; AcOEt/MeOH
1:1 ! 0 :1) to give 32 (8.15 g, 7.78 mmol, 86%). Colorless foam. Rf (AcOEt/MeOH 1:1) 0.10. 1H-NMR
(200 MHz, (D6)DMSO, 1108): 7.78 (s, HꢀC(8) of G); 7.73 (br. t, NH); 7.66 (br. t, NH); 7.54 (br. m, NH);
7.40 – 7.27 (m, 10 arom. H of Z); 6.77 (br. t, NH); 5.89 (ddt, J ¼ 17, 11, 5.2, CH2CH¼CHZHE); 5.28 (s, CH2
of Z of G); 5.27 (ddt, dq-like, J ¼ 17, 1.6, 1.6, CH2CH¼CHZHE); 5.15 (ddt, dq-like, J ¼ 11, 1.6, 1.6,
CH2CH¼CHZHE); 5.04 (s, CH2 of Z); 4.98 (br. s, CH2ꢀN(9) of G); 4.51 (dt, J ¼ 5.2, 1.6, CH2CH¼CH2);
4.06, 3.95 (2 br. s, CH2 of Gly); 3.87 (br. s, CH2 of Gly); 3.74 (s, CH2 of Gly); 3.55 (m, 2 NCH2CH2O);
3.35 – 3.14 (m, 3.5 CH2CH2); 2.41 (m, 2 NCH2CH2O, 0.5 CH2CH2); 1.38 (s, t-Bu). FAB-MS: 1096 (42,
[M þ Na]þ), 1074.6 (31, [M þ H]þ), 154.0 (100). HR-ESI-MS: 1077.5062 (5, [M þ 4 H]þ, C50H71N13Oþ14
;
calc. 1077.5243), 1076.5035 (20, [M þ 3 H]þ, C50H70N13O1þ4 ; calc. 1076.5165), 1075.5010 (57, [M þ 2 H]þ,
C50H69N13O1þ4 ; calc. 1075.5087), 1074.4983 (100, [M þ H]þ, C50H68N13O1þ4 ; calc. 1074.5008).
Z-Aeg(Boc)-Aeg(H)-Aeg(GZ)-Aem (¼ N-{6,9-Dihydro-9-[15-[(tert-butoxy)carbonyl]-3-(2-{[2-
(morpholin-4-yl)ethyl]amino}-2-oxoethyl)-2,7,13,19-tetraoxo-21-phenyl-20-oxa-3,6,9,12,15,18-hexaaza-
heneicos-1-yl]-6-oxo-1H-purin-2-yl}carbamic Acid Phenylmethyl Ester; 33). Compound 32 (8.98 g,
8.58 mmol) and Et2NH (26.7 ml, 257 mmol, 30 equiv.) were dissolved in CH2Cl2 (60 ml) at 08. After the
addition of Pd[P(Ph)3]4 (991 mg, 0.858 mmol), the mixture was stirred at r.t. for 1 h. The volatiles were
evaporated in vacuo, and the residue was purified by CC (SiO2; AcOEt/MeOH 1:1 ! 0 :1) to give 33
(5.21 g, 5.27 mmol, 61%). Colorless foam. Rf (MeOH) 0.19. 1H-NMR (400 MHz, (D6)DMSO;
rotamers): 8.17 (br. s, NH); 7.86 (br. s, 2 NH); 7.79, 7.78 (2s, HꢀC(8) of G); 7.42 – 7.28 (m, 10 arom. H
of Z); 7.24 (br. t, NH); 5.233, 5.226 (2s, CH2 of Z of G); 5.00 (s, PhCH2); 5.05, 4.93 (2s, CH2ꢀN(9) of G);
4.15, 3.92 (2s, CH2 of Gly); 3.75, 3.70 (2s, CH2 of Gly); 3.56 – 3.06 (m, CH2 of Gly, 3.5 CH2CH2,
2 NCH2CH2O); 2.35 (m, 2 NCH2CH2O, 0.5 CH2CH2); 1.36, 1.31 (2s, Me3C). Elevated temps. led to line
broadening that complicated the analysis. MALDI-MS: 991.208 (120, [M þ H]þ), 1013.249 (210, [M þ
Na]þ).
Z-Aeg(Boc)-Aeg(AZ)-Aeg(GZ)-Aem (¼ N-(6,9-Dihydro-9-{15-[(tert-butoxy)carbonyl]-3-(2-{[2-
(morpholin-1-yl)ethyl]amino}-2-oxoethyl)-2,7,13,19-tetraoxo-21-phenyl-9-[2-(6-{[(phenylmethoxy)car-
bonyl]amino}-9H-purin-9-yl)acetyl]-20-oxa-3,6,9,12,15,18-hexaazaheneicos-1-yl}-6-oxo-1H-purin-2-yl)-
carbamic Acid Phenylmethyl Ester; 34). Compound 33 (5.21 g, 5.26 mmol) and AZ-AcOH (29; 2.15 g,
6.58 mmol) were coupled in DMF according to GP 4. The mixture was concentrated in vacuo with a dry-
ice evaporator, and the residue was purified by CC (SiO2; MeOH) to give 34 (4.62 g, 3.56 mmol, 67%).
Colorless foam. Rf (MeOH) 0.14. 1H-NMR (200 MHz, (D6)DMSO, 1108; rotamers): 8.57, 8.23, 8.14, 7.96
(4m, HꢀC(2) of A, HꢀC(8) of A); 7.92 – 7.17 (m, 15 arom. H of Z, HꢀC(8) of G, NH); 6.79 (br. t, NH);
6.66 (br. s, NH); 6.09 (br. s, NH); 5.25 (s, CH2 of Z); 5.16 (br. s, CH2ꢀN(9)); 5.03 (s, CH2 of Z); 4.87 (br. s,

Helvetica Chimica Acta – Vol. 94 (2011)
1975
CH2ꢀN(9)); 4.52 (s, CH2 of Z); 4.23 (br. s, 2 CH2 of Gly); 3.81, 3.76 (2 br. s, CH2 of Gly); 3.57 – 3.18 (m,
2 NCH2CH2O, 3.5 CH2CH2); 2.41 (m, 2 NCH2CH2O, 0.5 CH2CH2); 1.38 (s, t-Bu). MALDI-MS: 1322.861
(127, [M þ Na]þ), 1300.814 (171, [M þ 2 H]þ). HR-ESI-MS: 1302.5703 (8, [M þ 4 H]þ, C61H78N18Oþ15
;
calc. 1302.5894), 1301.5671 (26, [M þ 3 H]þ, C61H77N18O1þ5 ; calc. 1301.5816), 1300.5652 (75, [M þ 2 H]þ,
C61H76N18O1þ5 ; calc. 1300.5737), 1299.5621 (100, [M þ H]þ, C61H75N18O1þ5 ; calc. 1299.5659).
Z-Aeg(H)-Aeg(AZ)-Aeg(GZ)-Aem · TFA (¼ N-(6,9-Dihydro-9-{3-(2-{[2-(morpholin-4-yl)ethyl]-
amino}-2-oxoethyl)-2,7,13,19-tetraoxo-21-phenyl-9-[2-(6-{[(phenylmethoxy)carbonyl]amino}-9H-purin-
9-yl)acetyl]-20-oxa-3,6,9,12,15,18-hexaazaheneicos-1-yl}-6-oxo-1H-purin-2-yl)carbamic Acid Phenyl-
methyl Ester; 35). Compound 34 (1.38 g, 1.06 mmol) was treated with TFA (5 ml) according to GP 1
in the presence of Et3SiH (1.00 ml, 6.26 mmol). The crude product was taken up in AcOEt (10 ml),
triturated with Et2O, filtered off with suction, washed with Et2O, and dried to give 35 (1.44 g, 1.01 mmol,
95%). Amorphous solid. As the product decomposed at 1108, no HT-NMR characterization was possible.
MALDI-MS: 1223.223 (355, [M þ Na]þ), 1200.790 (877, [M þ 2 H]þ). HR-ESI-MS: 1202.5178 (6, [M þ
4 H]þ, C56H70N18Oþ13 ; calc. 1202.5369), 1201.5152 (24, [M þ 3 H]þ, C56H69N18O1þ3 ; calc. 1201.5291),
1200.5123 (68, [M þ 2 H]þ, C56H68N18O1þ3 ; calc. 1200.5213), 1199.5062 (100, [M þ H]þ, C56H67N18Oþ13
;
calc. 1199.5135).
Z-Aeg(CZ)-Aeg(AZ)-Aeg(GZ)-Aem · TFA (¼ N-(6,9-Dihydro-9-{3-(2-{[2-(morpholin-4-yl)ethyl]-
amino}-2-oxoethyl)-2,7,13,19-tetraoxo-15-[2-(2-oxo-4-{[(phenylmethoxy)carbonyl]amino}pyrimidin-
1(2H)-yl)acetyl]-21-phenyl-9-[2-(6-{[(phenylmethoxy)carbonyl]amino}-9H-purin-9-yl)acetyl]-20-oxa-
3,6,9,12,15,18-hexaazaheneicos-1-yl}-6-oxo-1H-purin-2-yl)carbamic Acid Phenylmethyl Ester; 36). Com-
pounds 35 (1.00 g, 0.701 mmol) and CZ-AcOH (30; 266 mg, 0.876 mmol) were placed in DMF (5 ml) and
coupled according to GP 4. After completion of the reaction, the mixture was concentrated in vacuo with
i
a dry-ice evaporator. The residue was triturated with PrOH, purified by sonication, and the resulting
precipitate was collected by suction. The crude product (1.08 g) was purified by semi-prep. RP-HPLC.
Freeze drying gave 36 (531 mg, 0.332 mmol, 47%). Colorless powder. 1H-NMR (200 MHz, (D6)DMSO,
1108; rotamers): 8.56, 8.22, 8.21, 8.02 (4s, HꢀC(2) of A, HꢀC(8) of A); 7.76 (m, HꢀC(6) of C, HꢀC(8) of
G); 7.92 – 7.17 (m, 20 arom. H of Z); 6.88 (m, HꢀC(5) of C); 5.27 (s, CH2 of Z); 5.24 (s, CH2 of Z); 5.20 (s,
CH2 of Z); 5.19 (s, CH2 of Z); 5.05 (br. s, base CH2); 5.01 (br. s, base CH2); 4.70 (br. s, base CH2); 3.78 –
3.10 (m, 2 NCH2CH2O, 4 CH2CH2, 3 CH2 of Gly). The occurrence of very broad s between 8.10 – 7.60 and
at 6.42 was a result of the diverse NH species and complicated integration. MALDI-MS: 1508.376 (34,
[M þ Na]þ), 1486.173 (156, [M þ 3 H]þ). HR-ESI-MS: 1487.5917 (12, [M þ 4 H]þ, C70H81N21O1þ7 ; calc.
1487.6119), 1486.5889 (37, [M þ 3 H]þ, C70H80N21O1þ7 ; calc. 1486.6041), 1485.5863 (87, [M þ 2 H]þ,
C70H79N21O1þ7 ; calc. 1485.5962), 1484.5843 (100, [M þ H]þ, C70H78N21O1þ7 ; calc. 1484.5884).
Boc-Aeg(Fmoc)-OBn (¼ Benzyl N-(2-{[(tert-Butoxy)carbonyl]amino}ethyl)-N-{[(9H-fluoren-9-
yl)methoxy]carbonyl}glycinate; 37). A suspension of 38 (12.0 g, 20.4 mmol; see below) in (420 ml)
was cooled to 08 in an ice bath under Ar. EtNiPr2 (7.11 ml, 40.8 ml) was then added, followed by the
dropwise addition of Boc2O (4.45 g, 20.4 mmol) in 120 ml of CH2Cl2 during 20 min. The mixture was
stirred for 1 h at 08 and for a further 4 h at r.t. After cooling to 08, EtNiPr2 (3.55 ml, 20.4 mmol) was
added, followed by the addition of FmocꢀCl (5.28 g, 20.4 mmol). The mixture was stirred overnight and
then successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine (250 ml each), dried (MgSO4),
and concentrated in vacuo. The resulting oil was purified by CC (SiO2; CH2Cl2/MeOH 50 :1) to give 37
(7.89 g, 14.9 mmol, 73%). Colorless oil. Rf (DCM/MeOH 50 :1) 0.41. 1H-NMR (200 MHz, (D6)DMSO; 2
rotamers): 7.90 (d, J ¼ 7.2, 1 arom. H of Fmoc); 7.86 (d, J ¼ 7.2, 1 arom. H of Fmoc); 7.67 (d, J ¼ 7.2, 1 arom.
H of Fmoc); 7.57 (d, J ¼ 7.2, 1 arom. H of Fmoc); 7.33 (m, 4 arom. H of Fmoc, 5 arom. H of Bn); 6.71 (br. s,
NH); 5.15 (s, 1 H, PhCH2); 5.10 (s, 1 H, PhCH2); 4.12 (m, CH of Fmoc, CH2 of Fmoc, CH2 of Gly); 3.26,
3.06 (2m, CH2CH2); 1.35 (s, t-Bu). FAB-MS: 553.3 (16, [M þ Na]þ), 531.3 (6, [M þ H]þ), 431.2 (57, [M ꢀ
Boc]þ), 178.1 (100).
H-Aeg(H)-OBn · 2 TsOH (¼ Benzyl N-(2-Aminoethyl)glycinate; 38). BnOH (60 ml) and TsOH
(29.4 g, 154 mmol) were added to a suspension of 1 (7.56 g, 64.0 mmol) in toluene (500 ml). The mixture
was refluxed (oil bath, 1408) for 6 h and then concentrated in vacuo to ca. 150 ml. Et2O (500 ml) was
added, and the mixture was kept at ꢀ 208 overnight. The precipitate was collected by suction, washed
with three portions of Et2O, and dried in vacuo to give 38 (32.2 g, 54.7 mmol, 86%). Amorphous solid.
1H-NMR (200 MHz, D2O): 7.71 (m, 4 arom. H of TsOH), 7.39 (s, 5 arom. H of Bn); 7.29 (m, 4 arom. H of

1976
Helvetica Chimica Acta – Vol. 94 (2011)
TsOH); 5.20 (s, PhCH2); 4.06 (s, CH2 of Gly); 3.52 (m, CH2CH2); 2.32 (s, 2 Me, TsOH). 13C-NMR
(50 MHz, D2O): 166.61 (CO); 142.32, 139.64 (2 arom. C of TsOH); 134.43 (arom. C, Bn); 129.45, 128.93,
128.85, 128.49, 125.40 (3 arom. CH of Bn, 2 arom. CH of TsOH); 68.52 (PhCH2); 47.71, 44.18
(H3NþCH2CH2, CH2 of Gly); 35.43 (H3NþCH2); 20.54 (Me of TsOH). FAB-MS: 231 (8, [M þ Na]þ),
209.1 (100, [M þ H]þ), 119.1 (20, [M ꢀ Bn]þ).
H-Aeg(Fmoc)-OBn · TFA (¼ Benzyl N-(2-Aminoethyl)-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-
glycinate; 39). The reaction of 37 (63.6 g, 120 mmol) with TFA (100 ml) in CH2Cl2 (100 ml) was
performed according to GP 1. The crude product was precipitated with Et2O and collected by suction to
give 39 (56.5 g, 104 mmol, 86%). Amorphous solid. 1H-NMR (200 MHz, (D6)DMSO; 2 rotamers): 7.87
(m, 2 arom. H of Fmoc, H3Nþ); 7.61 (m, 2 arom. H of Fmoc); 7.35 (m, 4 ꢃ arom. H of Fmoc, 5 arom. H of
Bn); 5.16 (s, 0.78 H, PhCH2); 5.12 (s, 1.22 H, PhCH2); 4.20 (m, CH of Fmoc, CH2 of Fmoc, CH2 of Gly);
3.49, 2.90 (2m, CH2CH2). 13C-NMR (50 MHz, (D6)DMSO; 2 rotamers): 169.65 (COOBn); 169.62
(COOBn); 155.68 (CO of Fmoc); 155.48 (CO of Fmoc); 143.72 (arom. C of Fmoc); 143.62; (arom. C of
Fmoc); 140.72 (arom. C of Fmoc); 135.68 (arom. C of Bn); 135.60 (arom. C of Bn); 128.43 (arom. CH of
Bn); 128.16 (arom. CH of Bn); 127.92 (arom. CH of Bn); 127.74 (arom. CH of Fmoc); 127.68 (arom. CH
of Fmoc); 127.19 (arom. CH of Fmoc); 127.06 (arom. CH of Fmoc); 125.02 (arom. CH of Fmoc); 124.87
(arom. CH of Fmoc); 120.19 (arom. CH of Fmoc); 120.12 (arom. CH of Fmoc); 67.62, 67.29, 66.23, 66.15
(CH2 of Fmoc, PhCH2); 49.41, 48.73, 46.40, 45.84, 37.47, 37.13 (CH of Fmoc, CH2 of Gly, CH2CH2). The
signal of one CH2 of CH2CH2 was expected at ca. 40 ppm and was supposed to be hidden by the signal of
DMSO. FAB-MS: 453.2 (10, [M þ Na]þ), 431.2 (100, [M þ H]þ).
Boc-Aeg(Alloc)-Aeg(Fmoc)-OBn (¼ Benzyl N-(2-{[N-(2-{(tert-Butoxy)carbonyl]amino}ethyl)-N-
[(prop-2-en-1-yloxy)carbonyl]glycyl]amino}ethyl)-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycinate;
40). The coupling of 19 (13.7 g, 45.3 mmol) with 39 (24.7 g, 45.3 mmol) was performed according to GP 3.
The crude product was purified by CC (SiO2; cyclohexane/AcOEt 1:2 ! 1:5) to yield pure 40 (23.7 g,
32.7 mmol, 73%). Colorless foam. 1H-NMR (250 MHz, (D6)DMSO, 1008): 7.85 (d, J ¼ 7.3, 2 arom. H of
Fmoc); 7.61 (d, J ¼ 7.5, 2 arom. H of Fmoc); 7.56 (br. t, NH); 7.43 – 7.27 (m, 4 arom. C of Fmoc, 5 arom. H
of Bn); 6.35 (br. t, NH); 5.89 (ddt, J ¼ 17, 11, 5.2, CH2CH¼CHZHE); 5.26 (ddt, dq-like, J ¼ 17, 1.6, 1.6,
CH2CH¼CHZHE); 5.14 (ddt, dq-like, J ¼ 11, 1.6, 1.6, CH2CH¼CHZHE); 5.14 (s, PhCH2); 4.52 (dt, J ¼ 5.2,
1.6, CH2CH¼CH2); 4.36 (d, J ¼ 6.3, CH2 of Fmoc); 4.23 (br. t, J ¼ 6.3, CH of Fmoc); 4.06 (s, CH2 of Gly);
3.82 (s, CH2 of Gly); 3.42 – 3.06 (m, 2 CH2CH2); 1.38 (s, t-Bu). 13C-NMR (63 MHz, (D6)DMSO, 1008):
168.86 (CO of Gly); 168.33 (CO of Gly); 155.03 (CO of Fmoc, CO of Alloc); 143.32 (arom. C of Fmoc);
140.29 (arom. C of Fmoc); 135.30 (arom. C of Bn); 132.78 (CH2CH¼CH2); 127.78 (arom. CH of Fmoc);
127.42 (arom. CH of Fmoc); 127.17 (arom. o-CH of Bn); 127.02 (arom. m-CH of Bn); 126.48 (arom. p-CH
of Bn); 124.30 (arom. CH of Fmoc); 119.38 (arom. CH of Fmoc); 116.08 (CH2CH¼CH2); 77.24 (Me3C);
66.71, 65.57, 64.79 (CH2CH¼CH2, BnCH2, CH2 of Fmoc); 50.03, 48.89, 47.66, 46.40, 38.19 (CH of Fmoc,
2 CH2 of Gly, 2 CH2CH2); signals of the remaining CH2 groups were located between 50.84 and 35.43 ppm,
but could not be separated unambiguously from the baseline under the given conditions (93 mm sample,
734 scans); 27.72 (Me of Boc). FAB-MS: 737 (100, [M þ Na]þ), 715.4 (13, [M þ H]þ), 615.3 (78, [M ꢀ
Boc]þ).
H-Aeg(Alloc)-Aeg(Fmoc)-OBn · TFA (¼ Benzyl N-[2-({N-(2-Aminoethyl)-N-[(prop-2-en-1-yloxy)-
carbonyl]glycyl}amino)ethyl]-N-{[(9 H-fluoren-9-yl)methoxy]carbonyl}glycinate; 41). The reaction of
40 (12.8 g, 17.9 mmol) with TFA (30 ml) in CH2Cl2 (30 ml) was performed according to GP 1. The crude
product (13.0 g, 17.9 mmol, 100%) was directly used for the next step. 1H-NMR (200 MHz, (D6)DMSO,
1008; rotamers): 7.85 (m, 2 arom. H of Fmoc, NH); 7.60 (m, 2 arom. H of Fmoc); 7.35 (m, 4 arom. H of
Fmoc, 5 arom. H of Bn); 5.88 (m, CH2CH¼CHZHE, NH); 5.22 (m, CH2CH¼CH2); 5.14 (s, PhCH2); 4.54
(m, CH2CH¼CH2); 4.37 (m, CH2 of Fmoc); 4.24 (m, CH of Fmoc); 4.06, 3.96 (2s, CH2 of Gly); 3.91, 3.90
(2s, CH2 of Gly); 3.55 (br. t, J ¼ 6.1, 2 H of 2 CH2CH2), 3.30 (m, 4 H of 2 CH2CH2), 3.05 (br. t, J ¼ 6.1,
2 H of 2 CH2CH2). FAB-MS: 637.3 (20, [M þ Na]þ), 615.3 (90, [M þ H]þ), 154 (100).
Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(Fmoc)-OBn (¼ Benzyl N-{2-[(N-[(Allyloxy)carbonyl]-N-{2-[(N-
{[(4-{[(benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl]acetyl}-N-(2-{[(tert-butoxy)carbonyl]-
amino}ethyl)glycyl]amino}ethyl)glycyl]amino}ethyl)-N-{[(9H-fluoren-9-yl)methoxy)carbonyl}glycinate;
43). The coupling of Boc-Aeg(CZ)-OH (42; 9.01 g, 17.9 mmol) with 41 (13.0 g, 17.9 mmol) was performed
according to GP 3. The crude product was purified by CC (SiO2; AcOEt/MeOH 9 :1) to yield 43 (12.8 g,

Helvetica Chimica Acta – Vol. 94 (2011)
1977
11.6 mmol, 65%). Colorless foam. Rf (AcOEt/MeOH 9 :1) 0.15. 1H-NMR (200 MHz, (D6)DMSO, 1108):
7.78 (m, HꢀC(6) of C, 2 arom. CH of Fmoc, NH); 7.60 (d, J ¼ 7.4, 2 arom. H of Fmoc), 7.40 (br. t, J ¼ 5.1,
NH); 7.42 – 7.25 (m, 5 arom. H of Z, 5 arom. H of Bn, 4 arom. CH of Fmoc); 6.92 (d, J ¼ 7.3, HꢀC(5) of
C); 6.37 (br. s, NH); 5.89 (ddt, J ¼ 17, 11, 5.2, CH2CH¼CHZHE); 5.27 (ddt, dq-like, J ¼ 17, 1.6, 1.6,
CH2CH¼CHZHE); 5.14 (ddt, dq-like, J ¼ 11, 1.6, 1.6, CH2CH¼CHZHE); 5.21 (s, PhCH2); 5.14 (s, PhCH2);
4.70 (s, CH2ꢀN(1)); 4.51 (dt, J ¼ 5.2, 1.6, CH2CH¼CH2); 4.36 (d, J ¼ 6.0, CH2 of Fmoc); 4.22 (br. t, J ¼ 6.0,
CH of Fmoc); 4.06 (s, CH2 of Gly); 3.98 (br. s, CH2 of Gly); 3.84 (s, CH2 of Gly); 3.46 – 3.16 (m,
3 CH2CH2); 1.40 (s, t-Bu). FAB-MS: 1122.6 (40, [M þ Na]þ), 91.1 (100).
Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(H)-OBn (¼ Benzyl N-({[2-[(N-[(Allyloxy)carbonyl]-N-(2-{[(N-(4-
{[(benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl)acetyl]-N-(2-{[(tert-butoxy)carbonyl]ami-
no}ethyl)glycyl]amino}ethyl)glycyl]amino}ethyl)glycinate; 44). Compound 43 (7.40 g, 6.72 mmol) was
dissolved in CH2Cl2 at r.t. and treated with Et2NH (10.4 ml, 101 mmol). After stirring for 1.5 h, the
solvent was evaporated in vacuo, and the residue was triturated with AcOEt (50 ml) and Et2O (200 ml).
The resulting suspension was stirred for 10 min; then, the precipitate was collected by filtration and
rinsed with Et2O to give 44 (5.06 g, 5.77 mmol, 86%). Amorphous solid. 1H-NMR (200 MHz,
(D6)DMSO, 1108): 7.82 (d, J ¼ 7.3, HꢀC(6) of C); under it (br. m, NH); 7.52 (br. t, NH); 7.40 (m, 5
arom. H of Z, 5 arom. H of Bn); 6.93 (d, J ¼ 7.3, HꢀC(5) of C); 6.39 (br. s, NH); 5.90 (ddt, J ¼ 17, 11, 5.2,
CH2CH¼CHZHE); 5.33 – 5.12 (m, CH2CH¼CH2); 5.21 (s, PhCH2); 5.15 (s, PhCH2); 4.71 (s, CH2ꢀN(1));
4.52 (dt, J ¼ 5.2, 1.6, CH2CH¼CH2); 3.98 (s, CH2 of Gly); 3.86 (s, CH2 of Gly); 3.51 – 3.15 (m,
2.5 CH2CH2); 3.41 (s, CH2 of Gly); 2.68 (t, J ¼ 6.3, 0.5 CH2CH2); 1.41 (s, t-Bu). MALDI-MS: 878.940
(128, [M þ H]þ). HR-ESI-MS: 880.4082 (13, [M þ 3 H]þ, C42H58N9O1þ2 ; calc. 880.4205), 879.4054 (48,
[M þ 2 H]þ, C42H57N9O1þ2 ; calc. 879.4126), 878.4023 (100, [M þ H]þ, C42H56N9O1þ2 ; calc. 878.4048).
Boc-Aeg(CZ)-Aeg(Alloc)-Aeg(GZ)-OBn (¼ Benzyl N-[(Allyloxy)carbonyl]-N-[(2-{[(benzyloxy)-
carbonyl]amino}-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl]-N-(2-{[N-({2-[(N-{[4-{[(benzyloxy)carbonyl]-
amino}-2-oxopyrimidin-1(2H)-yl]acetyl}-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)glycyl]amino}ethyl)-
glycyl]amino}ethyl)glycinate; 45). The reaction of 44 (4.84 g, 5.51 mmol) with 28 (2.12 g, 6.17 mmol) in
DMF (30 ml) was performed according to GP 4. The resulting mixture was diluted with AcOEt (300 ml),
successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine (250 ml each), dried (MgSO4),
filtered, and concentrated in vacuo. The resulting oil was purified by CC (SiO2; AcOEt/MeOH 4 :1) to
1
give 45 (4.80 g, 3.99 mmol, 72%). Colorless foam. Rf (AcOEt/MeOH 4 :1) 0.08. H-NMR (200 MHz,
(D6)DMSO, 1108; rotamers): 10.76 (br. s, 2 NH of Z); 7.80 (m, HꢀC(6) of C, HꢀC(8) of G, 2 NH); 7.37
(m, 10 arom. H of Z, 5 arom. H of Bn); 6.93 (m, HꢀC(5) of C); 6.39 (br. s, NH); 5.88 (m,
CH2CH¼CHZHE); 5.31 – 5.03 (m, CH2CH¼CH2, PhCH2, CH2ꢀN(9) of G); 5.27 (s, PhCH2); 5.21 (s,
PhCH2); 4.71 (s, CH2ꢀN(1) of C); 4.50 (m, CH2CH¼CH2); 4.24 (s, CH2 of Gly); 3.98 (s, CH2 of Gly); 3.89
(s, CH2 of Gly); 3.55 – 3.19 (m, 3 CH2CH2); 1.40 (s, t-Bu). FAB-MS: 1203.5 (8, [M þ Hþ), 91.1 (100).
Boc-Aeg(CZ)-Aeg(H)-Aeg(GZ)-OBn (¼ Benzyl N-[(2-{[(Benzyloxy)carbonyl]amino}-6-oxo-1,6-
dihydro-9H-purin-9-yl)acetyl]-N-(2-{[N-(2-[(N-{[4-{[(benzyloxy)carbonyl]amino}-2-oxopyrimidin-1-
(2H)-yl]acetyl}-N-{2-{[(tert-butoxy)carbony]amino}ethyl)glycyl]amino}ethyl)glycyl]amino}ethyl)glyci-
nate; 46). Compound 45 (5.17 g, 4.30 mmol) was dissolved in dry THF, followed by the addition of
NDMBA (15.4 g, 98.8 mmol) and Pd[P(Ph)3]4 (991 mg, 0.858 mmol) one after another. After stirring at
r.t. for 2 h, the solvent was evaporated in vacuo, and the residue was purified by CC (SiO2; AcOEt/
MeOH 2 :1 ! 1:1) to give 46 (4.21 g, 3.76 mmol, 88%). Colorless foam. Rf (AcOEt/MeOH) 0.28.
1H-NMR (200 MHz, (D6)DMSO, 1108; rotamers): 7.77 (m, HꢀC(6) of C, HꢀC(8) of G, 2 NH); 7.33 (m,
10 arom. of Z, 5 arom. H of Bn); 6.91 (m, HꢀC(5) of C); 6.39 (br. s, NH); 5.88 (m, CH2CH¼CHZHE);
5.31 – 5.03 (m, CH2CH¼CH2, PhCH2, CH2ꢀN(9) of G); 5.27 (s, PhCH2); 5.21 (s, PhCH2); 4.71 (s,
CH2ꢀN(1) of C); 4.50 (m, CH2CH¼CH2); 4.24 (s, CH2 of Gly); 3.98 (s, CH2 of Gly); 3.89 (s, CH2 of Gly);
3.55 – 3.19 (m, 3 CH2CH2); 1.40 (s, t-Bu). MALDI-MS: 1143.260 (317, [M þ Na]þ), 1120.603 (1053, [M þ
2 H]þ). HR-ESI-MS: 1122.4695 (5, [M þ 4 H]þ, C53H66N14O1þ4 ; calc. 1122.4883), 1121.46703 (22, [M þ
3 H]þ, C53H65N14Oþ14 ; calc. 1121.4804), 1120.4646 (61, [M þ 2 H]þ, C53H64N14O1þ4 ; calc. 1120.4726),
1119.4620 (100, [M þ H]þ, C53H63N14O1þ4 ; calc. 1119.4648).
Boc-Aeg(CZ)-Aeg(F)-Aeg(GZ)-OBn (¼ Benzyl N-[(2-{[(Benzyloxy)carbonyl]amino}-6-oxo-1,6-di-
hydro-9H-purin-9-yl)acetyl]-N-[2-({[(N-{2-[(N-{[4-{[(benzyloxy)carbonyl]amino}-2-oxopyrimidin-
1(2H)-yl]acetyl}-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)glycyl]amino}ethyl]-N-[(2,4-difluoro-5-

1978
Helvetica Chimica Acta – Vol. 94 (2011)
methylphenyl)acetyl]glycyl}amino)ethyl]glycinate; 47). The coupling of 2 (300 mg, 1.61 mmol) with 46
(1.50 g, 1.34 mmol) was performed according to GP 3. The resulting mixture was diluted with AcOEt
(200 ml), successively washed with 1m aq. KHSO4, sat. aq. NaHCO3, and brine (200 ml each), dried
(MgSO4), filtered, and concentrated in vacuo. The resulting oil was purified by CC (SiO2; AcOEt/MeOH
2 :1) to give 47 (1.29 g, 1.01 mmol, 75%). Colorless foam. Rf (AcOEt/MeOH 2 :1) 0.24. 1H-NMR
(200 MHz, (D6)DMSO, 1108; rotamers): 7.97 – 7.72 (m, 5 H), 7.47 – 7.10 (m, 15 H), 6.92 – 6.81 (m, 2 H)
(HꢀC(6) of C, HꢀC(5) of C, HꢀC(8) of G, HꢀC(6) of F, HꢀC(3) of F, 10 arom. H of Z, 5 arom. H of Bn,
2 NH); 6.42 (br. s, NH); 5.25, 5.20, 5.17, 5.03, 4.92 (5s, 3 PhCH2); 4.73 (m, base CH2); 4.10 (m, 2 base
CH2); 3.65 – 2.76 (m, 3 CH2 of Gly, 3 CH2CH2) 1.98 (br. s, Me of F); 1.39 (m, t-Bu). 19F-NMR (235 MHz,
(D6)DMSO; rotamers): ꢀ 119.63 (m, FꢀC(4)); ꢀ 120.14 (m, FꢀC(2)). FAB-MS: 1309.3 (100, [M þ
Na]þ).
H-Aeg(CZ)-Aeg(F)-Aeg(GZ)-OBn · TFA (¼ Benzyl N-[2-({N-(2-{[N-(2-Aminoethyl)-N-{[4-{[(ben-
zyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl]acetyl}glycyl]amino}ethyl)-N-[(2,4-difluoro-5-meth-
ylphenyl)acetyl]glycyl}amino)ethyl]-N-[(2-{[(benzyloxy)carbonyl]amino}-6-oxo-1,6-dihydro-9H-purin-
9-yl)acetyl]glycinate; 48). Compound 47 (1.43 g, 1.11 mmol) was treated with TFA (5 ml) and Et3SiH
(2.5 ml, 15.7 mmol) according to GP 1. The crude product was precipitated with Et2O and collected by
suction to give 48 (1.27 g, 0.975 mmol, 88%). Amorphous solid. 1H-NMR (200 MHz, (D6)DMSO, 1108;
rotamers): 8.08 (br. s, NH); 7.95 – 7.68 (m, 4 H), 7.40 – 7.34 (m, 15 H), 7.11 (br. s, 1 H), 6.93 – 6.82 (m, 2 H)
(HꢀC(6) of C, HꢀC(5) of C, HꢀC(8) of G, HꢀC(6) of F, HꢀC(3) of F, 10 arom. H of Z, 5 arom. H of Bn,
NH); 6.42 (br. s, NH); 5.27, 5.21, 5.18, 5.02 (4s, 3 PhCH2); 4.80 – 4.60 (m, base CH2); 4.26 (br. s, 2 H), 4.08
(br. m, 4 H); 3.64 – 3.09 (br. m, 14 H) (2 base CH2, 3 CH2 of Gly, 3 CH2CH2); 1.98 (br. s, Me of F). 19F-
NMR (235 MHz, (D6)DMSO; rotamers): ꢀ 77.01 (s, F3CCOOH); ꢀ 119.40, ꢀ 120.12 (2m, FꢀC(2),
FꢀC(4)). MALDI-MS: 1188.331 (70, [M þ 2 H]þ). HR-ESI-MS: 1190.4525 (8, [M þ 4 H]þ,
C57H64F2N14O1þ3 ; calc. 1190.4745), 1189.4532 (24, [M þ 3 H]þ, C57H63F2N14O1þ3 ; calc. 1189.4667),
1188.4504 (67, [M þ 2 H]þ, C57H62F2N14Oþ13 ; calc. 1188.4589), 1187.4474 (100, [M þ H]þ, C57H61F2N14Oþ13
;
calc. 1187.4510).
Me2-Aeg(Me)-Aeg(CZ)-Aeg(F)-Aeg(GZ)-OBn · 2 TFA (¼ Benzyl N-[(2-{[(Benzyloxy)carbonyl]-
amino}-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl]-N-{2-[(N-{2-[(N-{[4-{[(benzyloxy)carbonyl]amino}-2-
oxopyrimidin-1(2H)-yl]acetyl}-N-[2-({N-[2-(dimethylamino)ethyl]-N-methylglycyl}amino)ethyl]glycyl)-
N-[(2,4-difluoro-5-methylphenyl)acetyl]glycyl)amino]ethyl}glycinate; 49). Compounds 5a and 48 were
coupled as described for the preparation of 27. The crude product was purified by RP-HPLC to give 49
(305 mg, 214 mmol, 46%). Colorless powder. 1H-NMR (200 MHz, (D6)DMSO, 1108): 7.76 (m, HꢀC(6)
of C, arom. H of F, HꢀC(8) of G, 2 NH); 7.41 – 7.34 (m, 10 arom. H of Z, 5 arom. H of Bn); 7.12 (br. t,
NH); 6.89 (m, HꢀC(5) of C, arom. H of F); 5.27 (s, PhCH2); 5.21 (s, PhCH2); 5.18 (br. s, PhCH2); 5.02
(br. s, base CH2); 4.69 (br. s, base CH2); 4.24 (br. s, base CH2); 4.02 (br. s, 2 CH2 of Gly); 3.63 – 3.20 (br.
m, 2 CH2 of Gly, 3.5 CH2CH2); 2.90 (t, J ¼ 6.0, 0.5 CH2CH2); 2.84 (s, Me2N); 2.40 (s, MeN); 2.12 (br. s, Me
of F). 19F-NMR (235 MHz, (D6)DMSO; rotamers): ꢀ 77.83 (s, 2 F3CCOOH); ꢀ 119.39, ꢀ 120.09 (2m,
FꢀC(2), FꢀC(4)). MALDI-MS: 1330.842 (577, [M þ 2 H]þ. HR-ESI-MS: 1332.5663 (8, [M þ 4 H]þ,
C64H78F2N16O1þ4 ; calc. 1332.5851), 1331.5630 (24, [M þ 3 H]þ, C64H77F2N16O1þ4 ; calc. 1331.5773),
1330.5610 (73, [M þ 2 H]þ, C64H76F2N16O1þ4
;
calc. 1330.5695), 1329.5583 (100, [M þ H]þ,
C64H75F2N16Oþ14 ; calc. 1329.5616).
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Received June 9, 2011