Design and synthesis of nucleoproline amino acids for the straightforward preparation of chiral and conformationally constrained nucleopeptides

Article abstract of DOI:10.1002/hlca.201200557

A straightforward synthesis of orthogonally protected nucleoproline (Nup) amino acids and their coupling to oligomers are described. A key step is the attachment of alkynylated nucleobases to Fmoc-protected 4-azidoproline (Fmoc-Azp-OH) by a Cu-catalyzed 1,3-dipolar cycloaddition ('click reaction'). The developed protocol allows preparation of the nucleoprolines in scales of >30g. Solid-phase peptide synthesis proved to be straightforward with these Nup amino acids. The resulting oligonucleoproline peptides adopt defined helices, are very well H₂O soluble, and show comparable cell-penetrating properties as recently reported α-nucleoalanine peptides. Copyright

Full text of DOI:10.1002/hlca.201200557

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Design and Synthesis of Nucleoproline Amino Acids for the Straightforward
Preparation of Chiral and Conformationally Constrained Nucleopeptides
by Rolf A. Kramera)b)1), Konrad H. Bleicher*b), and Helma Wennemers*a)
a) Laboratorium fꢀr Organische Chemie, Departement fꢀr Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich, Hçnggerberg HCI, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(fax: þ 41446321223; e-mail wennemers@org.chem.ethz.ch)
b) Discovery Chemistry, PRCB, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel
(fax: þ 41446886965; e-mail konrad.bleicher@roche.com)
Dedicated to Professor Dieter Seebach on the occasion of his 75th birthday
A straightforward synthesis of orthogonally protected nucleoproline (Nup) amino acids and their
coupling to oligomers are described. A key step is the attachment of alkynylated nucleobases to Fmoc-
protected 4-azidoproline (Fmoc-Azp-OH) by a Cu-catalyzed 1,3-dipolar cycloaddition (ꢁclick reactionꢂ).
The developed protocol allows preparation of the nucleoprolines in scales of > 30 g. Solid-phase peptide
synthesis proved to be straightforward with these Nup amino acids. The resulting oligonucleoproline
peptides adopt defined helices, are very well H2O soluble, and show comparable cell-penetrating
properties as recently reported a-nucleoalanine peptides.
Introduction. – In the last decades, the synthesis and study of synthetic nucleic acids
based on a peptide skeleton has attracted considerable attention [1][2]. As a class of
molecules that contain both nucleobases and amino acids, nucleopeptides are
interesting for therapeutic or diagnostic applications such as the selective binding to
RNA or DNA, or the design of biomaterials [1 – 3]. Since the invention of peptide
nucleic acids (PNA) by Nielsen et al. [4], several different nucleopeptides have been
introduced that differ mainly in the peptidic backbone, and the linker between the
amino acid and the nucleobase [5]. The original PNA consists of an (2-amino-
ethyl)glycin backbone with acyl groups as linkers to the nucleobases (Fig. 1) [4]. Other
nucleopeptides have a- or b-amino acids, for instance, alanine, lysine, serine, or b-
alanine, in their backbone and bear the nucleobases attached to the side chains [5].
Fig. 1. Structure of the original PNA (B ¼ nucleobase) [4a]
Also proline-based nucleopeptides have been investigated as constrained and
preorganized PNA analogs [6]. In these nucleopeptides, the proline derivatives are
1
)
R. A. K. acknowledges funding by the Roche Postdoc Fellowship (RPF) Program.
ꢃ 2012 Verlag Helvetica Chimica Acta AG, Zꢀrich

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connected by different linking units, and the nucleobases are attached at different
positions of the pyrrolidine ring with an alkyl or amide linkage to the proline core.
(Fig. 2). These derivatives are typically synthesized starting from naturally occurring
trans-hydroxy-l-proline. Several of the resulting chiral, constrained, and structurally
preorganized nucleopeptides have improved binding affinities towards DNA or RNA
compared to the original PNA, and discriminate between parallel and antiparallel
binding orientation [6].
Fig. 2. Examples of proline-containing PNA analogs (B ¼ nucleobase) [6]
We have recently introduced azidoproline (Azp)-containing oligoprolines (Fig. 3)
as conformationally well-defined molecular scaffolds that can be easily functionalized
with alkynes using Cu-catalyzed 1,3-dipolar cycloaddition [7] or by amidation with
carboxylic acid derivatives after reduction of the azides to amines [8]. In an aqueous
environment, these oligomers adopt the highly symmetric polyproline II (PPII) helix,
in which every third residue is stacked on top of each other at a distance of ca. 1 nm
(Fig. 3) [8][9]. This conformationally well-defined helix is already adopted at chain
lengths as short as six proline residues. All of these features render functionalizable
oligoprolines intriguing for manifold different applications such as the controlled
formation of silver nanoparticles [10], studies of electron transfer [11], or the
development of functionalized synthetic collagen [12].
Fig. 3. Structure of oligoprolines functionalized in every third position with N3 groups
The synthesis of such functionalizable oligoprolines is straightforward using Fmoc-
Pro-OH and Fmoc-Azp-OH (1) as the functionalizable building block, that can be
easily prepared in large scale and has excellent coupling efficiencies in peptide
synthesis [8].
We envisaged chiral and conformationally constrained oligonucleoprolines, with
yet a different geometry and thereby coordination properties compared to previously
developed nucleopeptides, as useful for the selective binding to RNA or DNA, and the
construction of tailor-made supramolecular assemblies. Towards the synthesis of such
nucleoproline (Nup) peptides by standard solid-phase peptide synthesis (SPPS)
following the Fmoc/tBu protocol, appropriately protected nucleobase proline amino
acids, Fmoc-Nup(X)-OH, are required (Scheme 1). The key step for the synthesis of

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Scheme 1. Synthesis Strategy of Nucleobase-Modified Proline Derivatives, Fmoc-Nup(X)-OH, and
Solid-Phase Peptide Synthesis (SPPS) of Oligonucleoprolines
these Fmoc-Nup-OH amino acids is the attachment of alkyne-functionalized nucleo-
bases to azidoproline 1 via a Cu-catalyzed 1,3-dipolar cycloaddition reaction (ꢁclick
reactionꢂ) [7].
Results and Discussion. – To avoid undesired side-reactions in the synthesis of the
a-Fmoc-protected Nup monomers, Fmoc-Nup(X)-OH, the exocyclic amines of
cytosine, adenine, and guanine had to be masked with protecting groups that are
orthogonal to the Fmoc protecting group and are compatible with SPPS. No protection
was necessary for thymine, since it lacks an exocyclic amine group. A general problem
for the synthesis of nucleopeptides is the often low solubility of the nucleobase-
containing building blocks. Porcheddu et al. showed that protection of the exocyclic
amines of the nucleobases with two Boc groups, increases the lipophilicity of the
nucleobases and thereby their solubility in organic solvents [13]. We decided to use the
same protection-group strategy and hence synthesized the bis-Boc-protected nucleo-
bases Boc-cytosine 3 and Boc-adenine 4 in multi-gram scale, according to the known
procedure (Scheme 2) [13b]. This involved tris-Boc protection of cytosine and adenine,
followed by a selective mono-Boc deprotection to yield 3 and 4, respectively.
The alkylation of thymine 2, and the protected cytosine and adenine derivatives, 3
and 4, was achieved using K2CO3 and propargyl bromide in DMF to obtain the
corresponding functionalized nucleobases 5, 6, and 7, respectively, ready for the
attachment to azidoproline 1 (Scheme 2).
The development of an efficient synthesis route to a suitably protected, alkynylated
guanine derivative presented the most challenging part of the Nup synthesis. To
overcome the low reactivity and solubility of guanine, we initially used the more
reactive 6-chloroguanine (8) [14] and increased its solubility with the bis-Boc-
protection strategy. Haloguanine derivative 9 was synthesized according to the same
procedure described for the synthesis of the cytosine and adenine derivatives 3 and 4
[13b], followed by its alkylation with propargyl bromide to yield the alkynylated 6-
haloguanine derivative 10 (Scheme 3). Several known methods for the final conversion
of 10 to the protected guanine 11 were then evaluated, e.g., reaction of the 6-halo group
with O-nucleophiles such as 3-hydroxypropanenitrile in combination with NaH, 2-
cyanoethanol in combination with NaH, Me3N in combination with H2O, or Me3N in
the presence of 3-hydroxypropanenitrile and DBU (¼1,8-diazabicyclo[5.4.0]undec-7-

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Scheme 2. Synthesis of Alkyne-Modified Thymine, Cytosine, and Adenine Derivatives 5, 6, and 7,
Respectively
a) Propargyl bromide, K2CO3, DMF, r.t., 15 h.
Scheme 3. Tentative Synthetis of Bis-Boc-Protected Guanine Derivative 11
ene) [13][15]. Unfortunately, all of these trials resulted either in the decomposition of
10 or in complete recovery of the starting material.
We, therefore, focused our attention on the use of O(6)-benzyl (Bn)-protected
guanine derivatives as starting material, since the Bn protecting group can be cleaved
under the acidic conditions used for cleaving the Boc groups [16]. In a first attempt, we
alkylated 6-chloroguanine (8) with propargyl bromide to yield the haloguanine
derivative 12, which, however, did not react with BnOH to the desired guanine
derivative 13 (Scheme 4) [17].
The successful route to the desired alkynylated guanine building block 17 started by
converting 6-chloroguanine (8) with BnOH to 6-(benzyloxy)guanine (14; Scheme 5)
[18]. Guanine derivative 14 was then reacted to the tris-Boc-protected derivative 15

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Scheme 4. Tentative Synthetis of Benzyl (Bn)-Protected Guanine 13
Scheme 5. Synthesis of Alkynylated Guanine Derivative 17 (DMAP ¼ 4-(dimethylamino)pyridine)
with Boc2O and catalytic amounts of DMAP, followed by selective Boc deprotection at
N(9) with aqueous NaHCO3 in MeOH to yield bis-Boc-protected guanine derivative
16. The final alkylation with propargyl bromide resulted in the desired guanine
derivative 17, ready for the attachment to azidoproline 1. This route allowed
preparation of the bis-Boc-protected and alkynylated building block 17 in a scale of
22 g.
For the conjugation of Fmoc-Azp-OH (1) with the alkyne-functionalized nucleo-
bases 5, 6, 7, and 17, several Cu salts, e.g., CuSO4 · 5 H2O, Cu(OAc)2, CuI, and
[Cu(MeCN)4]PF6, in the presence or absence of sodium ascorbate as reducing agent
and different conditions (solvents, temperatures, etc.) for the 1,3-dipolar cycloaddition
reaction were tested (Scheme 6) [7]. Nearly quantitative conversion in multi-gram
scale was obtained with the very well DMF soluble CuI complex [Cu(MeCN)4]PF6 at
458, whereas the other tested copper salts did either not allow for reaction or provided
the products in poor yields. Using these optimized conditions allowed for the synthesis
of all four nucleoproline derivatives in yields of > 92% and quantities of > 30 g
(Scheme 6).
With all four building blocks in hand, we next studied their coupling efficiencies in
SPPS following the Fmoc/tBu protocol. The monomers Fmoc-Nup(A)-OH, Fmoc-
Nup(T)-OH, and Fmoc-Nup(C)-OH showed comparable coupling yields as standard
Fmoc-protected a-amino acids. Six consecutive coupling steps with the Nup derivatives

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Scheme 6. Synthesis of the a-Fmoc-Protected Nucleoproline Derivatives Fmoc-Nup(X)-OH
a) [Cu(MeCN)4]PF6, DMF, 458, 2 h.
using COMU (¼1-{[(1-cyano-2-ethoxy-2-oxoethylidene)aminooxy](dimethylamino)-
morpholino}uronium hexafluorophosphate) [19] as coupling reagent allowed for
preparing the hexamers Ac-[Nup(A)]6-NH2, Ac-[Nup(T)]6-NH2, and Ac-[Nup(C)]6-
NH2 in purities of > 94%, as judged by HPLC analysis. This corresponds to an average
coupling yield of 99% for each coupling step. The purity of the hexamer, Ac-
[Nup(G)]6-NH2, prepared under identical conditions, was only ca. 65% corresponding
to an average coupling yield of 93% for Fmoc-Nup(G)-OH. The lower coupling
efficiencies of the guanine building block in this all-Nup(G) nucleopeptide might be
due to the steric hindrance of the bulky Nup(G) monomers. Reassuringly, when
Nup(G) was incorporated into mixed nucleopeptides such as Ac-Nup(G)-Nup(C)-
Nup(A)-Nup(T)-Nup(C)-Nup(G)-Nup(A)-NH2 (18), also with Fmoc-Nup(G)-OH,
excellent average coupling efficiencies of ca. 99% for each coupling step were achieved
to yield this peptide in a purity of 94% (Fig. 4).
Similar coupling efficiencies were obtained for mixed proline and Nup peptides, for
example, H-Lys(CF)-[Pro-Nup(T)-Pro]4-Lys-NH2 (19). Conformational analysis of
this peptide by circular dichroism (CD) spectroscopy showed that this nucleobase-

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Fig. 4. HPLC Chromatogram of crude Ac-Nup(G)-Nup(C)-Nup(A)-Nup(T)-Nup(C)-Nup(G)-Nup-
(A)-NH2 (18)
functionalized peptide adopts a polyproline type-II conformation in aqueous environ-
ments, as indicated by the minima and maxima at wavelengths of 205 and 227 nm,
respectively (Fig. 5) [9][10]. This demonstrates that the triazole nucleobase units do
not influence the conformational preferences of oligoprolines significantly.
Fig. 5. CD Spectrum of mixed proline and nucleoproline peptide H-Lys(CF)-[Pro-Nup(T)-Pro]4-Lys-
NH2 (19; CF ¼ 5(6)-carboxyfluorescein)
We have also investigated the solubility of such artificial Nup analogs. As a
representative, the hexamer [Nup(T)]6 was chosen which showed a remarkably high
solubility of > 3000 ug/ml.
For biological applications of nucleopeptides for, e.g., DNA or RNA targeting, a big
challenge is their transport to the target molecules through the cell membrane into the
cells [1b][20]. Thus, often cell-penetrating peptides such as polyarginines [21] or their
b-amino acid analogs [22] are used as carriers to facilitate the cellular uptake.

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Nucleoalanines Ala(T) conjugated to polyalanines have been reported by Formaggio,
Bianco and co-workers to penetrate the cell membrane without the help of a molecular
transporter [5g]. We compared the cell-penetrating properties of the reported
oligonucleoalanine peptide 20, with those of oligonucleoproline peptide 21, in which
the nucleoalanine Ala(T) moieties of peptide 20 were replaced with nucleoprolines,
Nup(T), and additionally with those of oligonucleoproline peptide 19, in which all
Ala(T) moieties and alanine units of 20 are exchanged with Nup(T) and proline,
respectively. The peptides were labeled with the fluorophore 5(6)-carboxyfluorescein
(CF). Incubation of HeLa cells for 1 h with the different peptides and additionally with
CF as a control at concentrations of 50 mm, followed by fluorescence activated cell
sorting (FACS) studies, allowed determination of the relative cellular uptake of the
peptides into the cells (Fig. 6).
Fig. 6. Cellular uptake of peptides 19 – 21 (MFI ¼ mean fluorescence intensity)
The results show that the uptake efficiencies of oligonucleoprolines are at least
comparable to those of the reference compound 20 (Fig. 6). Nonetheless, these results
have to be taken with caution, since cellular uptake is only observed at very high and
non-physiologically relevant concentrations of 50 mm for all of the peptides.
Conclusions. – We have developed a straightforward synthesis protocol for the
preparation of orthogonally protected nucleoproline monomers. Key step is the
attachment of the alkyne-functionalized nucleobases to Fmoc-Azp-OH by a 1,3-dipolar
cycloaddition reaction, that proceeds in yields of > 92% on a 30-g scale. The resulting
nucleoproline amino acids have excellent coupling efficiencies in SPPS rendering them
attractive for the preparation of chiral, structurally constrained nucleobase-modified
peptides. The CD spectrum of a mixed proline and nucleoproline peptide showed that
the triazole nucleobase units do not influence the polyproline type-II conformation
significantly. These structurally constrained nucleoproline peptides might, therefore,
be useful for applications in targeting RNA or DNA, or for the construction of tailor-
made supramolecular assemblies. In addition, nucleoprolines have good solubility and

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comparable cellular-uptake efficiencies in HeLa cells as previously reported a-
nucleoalanines.
Experimental Part
General. Materials and reagents were of the highest commercially available grade and used without
further purification. Compounds 3, 4, and 14 were synthesized according to the procedures reported by
Porcheddu et al. [13b]. Reactions were monitored by TLC using Merck silica gel (SiO2) 60 F 254 plates;
visualization by UV and ninhydrin. Anal. HPLC: Reprosil Gold 120 C18 RP 5 mm (150 mm ꢀ 4 mm)
column from Dr. Maisch GmbH. Prep. HPLC: Reprosil Gold 120 C18 RP 5 mm (150 mm ꢀ 16 mm)
column from Dr. Maisch GmbH. Flash column chromatography (CC): Merck SiO2 60 or Fluka SiO2 60,
particle size 40 – 63 mm. CD Spectra: Chirascan spectrometer (Applied Biophysics Ltd, Leatherhead,
UK). 1H- and 13C-NMR spectra: Bruker DPX 400 spectrometer; chemical shifts in ppm, with the solvent
signal as a reference. ESI-MS: Bruker solariX 94 instrument.
tert-Butyl 6-(Benzyloxy)-2-{[bis(tert-butoxy)carbonyl]amino}-9H-purine-9-carboxylate (15). To a
suspension of 6-(benzyloxy)-9H-purin-2-amine (14; 50.0 g, 207 mmol) and DMAP (2.30 g, 20.7 mmol,
0.1 equiv.) in THF was added Boc2O (130 g, 745 mmol, 3.6 equiv.). The mixture was stirred overnight at
r.t. The excess amount of THF was removed at reduced pressure, and the crude product was dissolved in
CH2Cl2 (1500 ml), washed with HCl aq. (1n, 300 ml) and brine (2 ꢀ 300 ml), dried (Na2SO4), and all
volatiles were removed at reduced pressure. The remaining residue was purified by CC (SiO2; CH2Cl2 !
CH2Cl2/MeOH 99 :1) to give 15 (67.1 g, 124 mmol, 60%). Colorless solid. Rf (CH2Cl2/MeOH 98 :2) 0.53.
1H-NMR (400 MHz, (D6)DMSO): 8.76 (s, 1 H); 7.57 – 7.52 (m, 2 H); 7.47 – 7.37 (m, 3 H); 5.64 (s, 2 H);
1.66 (s, 9 H); 1.46 (s, 18 H). 13C-NMR (101 MHz, (D6)DMSO): 153.2; 152.7; 151.2; 150.8; 148.0; 145.9;
131.2; 129.4; 129.4; 87.6; 84.5; 84.1; 83.9; 31.9; 28.3; 28.3. HR-ESI-MS: 564.2422 ([M þ Na]þ,
C27H35N5NaOþ7 ; calc. 564.2434).
Di(tert-butyl) [6-(Benzyloxy)-9H-purin-2-yl]imidodicarbonate (16). To a soln. of 15 (50 g,
92.4 mmol) in MeOH (1000 ml) was added sat. aq. NaHCO3 (450 ml). The turbid soln. was stirred at
508 for 1 h. After removal of MeOH at reduced pressure, H2O (500 ml) was added, and the aq.
suspension was extracted with CH2Cl2 (3 ꢀ 500 ml). The combined org. layers were dried (Na2SO4),
filtered, and all volatiles were removed at reduced pressure to give pure 16 (40.4 g, 91.3 mmol, 99%).
Colorless solid. Rf (CH2Cl2/MeOH 98 :2) 0.10. 1H-NMR (400 MHz, (D6)DMSO): 14.11 (s, 1 H); 8.49 (s,
1 H); 7.57 – 7.52 (m, 2 H); 7.46 – 7.34 (m, 3 H); 5.63 (s, 2 H); 1.44 (s, 18 H). 13C-NMR (101 MHz,
(D6)DMSO): 153.1; 152.2; 151.6; 151.4; 145;2; 129.4; 129.3; 84.1; 83.5; 55.8; 31.9; 28.3. HR-ESI-MS:
464.1901 ([M þ H]þ, C22H27N5NaOþ5 ; calc. 464.1910).
Di(tert-Butyl) [6-(Benzyloxy)-9-(prop-2-yn-1-yl)-9H-purin-2-yl]imidodicarbonate (17). To a sus-
pension of 16 (59.6 g, 135 mmol) and anh. K2CO3 (18.7 g, 135 mmol, 1.0 equiv.) in DMF (400 ml), was
added propargyl bromide (80 wt-% in toluene, 54.5 ml, 405 mmol, 3.0 equiv.). The mixture was stirred at
r.t. for 15 h. After removal of the solvent at reduced pressure, the residue was suspended in H2O
(500 ml). The aq. soln. was extracted with CH2Cl2 (3 ꢀ 500 ml), the combined org. layers were dried
(Na2SO4), and all volatiles were removed at reduced pressure. The remaining residue was purified by CC
(SiO2; AcOEt/hexane 2 :3 ! 3 :2) to give 17 (22.1 g, 46.1 mmol, 34%). Colorless solid. Rf (AcOEt/
1
hexane 3 :2) 0.35. H-NMR (400 MHz, (D6)DMSO): 8.55 (s, 1 H); 7.52 – 7.57 (m, 2 H); 7.37 – 7.46 (m,
3 H); 5.66 (s, 2 H); 5.17 (d, J ¼ 2.6, 2 H); 3.55 (t, J ¼ 2.5, 1 H); 1.42 (s, 18 H). 13C-NMR (101 MHz,
(D6)DMSO): 161.0; 153.4; 152.0; 151.3; 145.3; 136.8; 129.4; 129.2; 129.2; 119.9; 83.7; 78.7; 77.1; 69.1; 33.8;
28.3. HR-ESI-MS: 502.2067 ([M þ Na]þ, C25H29N5NaO5þ ; calc. 502.2066).
5-Methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (5). To a suspension of thymine (2; 15.0 g,
119 mmol) and anh. K2CO3 (16.5 g, 119 mmol, 1.0 equiv.) in DMF (400 ml) was added propargyl bromide
(80 wt-% in toluene, 48.0 ml, 357 mmol, 3.0 equiv.). The mixture was stirred at r.t. for 15 h. After removal
of the solvent at reduced pressure, the residue was suspended in H2O (500 ml). The aq. soln. was
extracted with CH2Cl2 (3 ꢀ 500 ml), and the combined org. layers were dried (Na2SO4), and all volatiles
were removed at reduced pressure. The remaining residue was purified by CC (SiO2; AcOEt/hexane

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2 :3 ! 3 :2) to give 5 (14.4 g, 88.1 mmol, 74%). Colorless solid. Rf (AcOEt/hexane 3 :2) 0.35. The
spectroscopic data are in accordance to those reported in [23].
Di(tert-butyl) [2-(Prop-2-ynoyl)pyrimidin-4-yl]imidodicarbonate (6). To a suspension of 3 (30.0 g,
96.4 mmol) and anh. K2CO3 (13.3 g, 96.4 mmol, 1.0 equiv.) in DMF (300 ml) was added propargyl
bromide (80 wt-% in toluene, 38.8 ml, 289 mmol, 3.0 equiv.). The mixture was stirred at r.t. for 15 h.
After removal of the solvent at reduced pressure, the residue was suspended in H2O (500 ml). The aq.
soln. was extracted with CH2Cl2 (3 ꢀ 500 ml), the combined org. layers were dried (Na2SO4), and all
volatiles were removed at reduced pressure. The remaining residue was purified by CC (SiO2; AcOEt/
hexane 2 :3 ! 1:1) to give 6 (21.9 g, 62.7 mmol, 65%). Colorless solid. Rf (AcOEt) 0.54. 1H-NMR
(400 MHz, (D6)DMSO): 8.29 (d, J ¼ 7.3, 1 H); 6.89 (d, J ¼ 7.3, 1 H); 4.70 (d, J ¼ 2.5, 2 H); 3.52 (t, J ¼ 2.5,
1 H); 1.53 (s, 18 H). 13C-NMR (101 MHz, (D6)DMSO): 162.8; 154.3; 150.5; 150.1; 96.9; 85.5; 78.9; 77.6;
39.7; 28.1. HR-ESI-MS: 372.1543 ([M þ Na]þ, C17H23N3NaO5þ ; calc. 372.1535).
Di(tert-butyl) [9-(Prop-2-yn-1-yl)-9H-purin-6-yl]imidodicarbonate (7). To a suspension of
4
(30.0 g, 89,5 mmol) and anh. K2CO3 (12.3 g, 89.5 mmol, 1.0 equiv.) in DMF (300 ml) was added
propargyl bromide (80 wt-% in toluene, 36.1 ml, 269 mmol, 3.0 equiv.). The mixture was stirred at r.t. for
15 h. After removal of the solvent at reduced pressure, the residue was suspended in H2O (500 ml). The
aq. soln. was extracted with CH2Cl2 (3 ꢀ 500 ml), the combined org. layers were dried (Na2SO4), and all
volatiles were removed at reduced pressure. The remaining residue was purified by CC (SiO2; AcOEt/
hexane 2 :3 ! 1:1) to give 7 (19.7 g, 52.8 mmol, 59%). Colorless solid. Rf (AcOEt) 0.59. 1H-NMR
(400 MHz, (D6)DMSO): 8.93 (s, 1 H); 8.74 (s, 1 H); 5.26 (d, J ¼ 2.5, 2 H); 3.58 (t, J ¼ 2.5, 1 H); 1.42 (s,
18 H). 13C-NMR (101 MHz, (D6)DMSO): 153.5; 152.6; 150.9; 150.1; 147.1; 128.5; 84.3; 78.5; 77.4; 34.0;
28.2. HR-ESI-MS: 396.1634 ([M þ Na]þ, C18H23N5NaO4þ ; calc. 396.1648).
(4S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-proline (Fmoc-Nup(T)-OH). A soln. of 5 (10.9 g, 66.7 mmol)
and Fmoc-Azp-OH (1; 25.2 g, 66.7 mmol, 1.0 equiv.) in DMF (500 ml) was degassed in an ultrasonic bath
for 20 min, followed by the addition of [Cu(MeCN)4]PF6 (1.24 g, 3.34 mmol, 5 mol-%). The soln. was
stirred at 458 for 2 h. After removal of the solvent at reduced pressure, the residue was suspended in a sat.
soln. of EDTA (¼ethylenediaminetetraacetic acid) in H2O (0.5m, 500 ml).The aq. soln. was extracted
with CH2Cl2 (3 ꢀ 1000 ml), and the combined org. layers were washed with aq. sat. NaCl (1000 ml), dried
(Na2SO4), and all volatiles were removed at reduced pressure. The remaining residue was purified by CC
(SiO2; AcOEt/hexane 2 :3 ! 3 :2) to give Fmoc-Nup(T)-OH (35.5 g, 65.5 mmol, 98%). Colorless solid.
Rf (AcOEt/hexane 3 :2) 0.35. The NMR spectra indicated two conformers (cis and trans conformation
1
around the carbamate bound) in a ratio of 1:2.1. H-NMR (400 MHz, (D6)DMSO): major conformer:
11.35 (s, NH); 8.28 (s, HꢁC(6) thymine); 7.95 – 7.89 (m, 2 H of Fmoc); 7.75 – 7.69 (m, 2 H of Fmoc); 7.63 (s,
1 H of triazole); 7.48 – 7.41 (m, 2 H of Fmoc); 7.40 – 7.32 (m, 2 H of Fmoc); 5.33 – 5.22 (m, HꢁCg(Pro));
4.94 (s, CH2 of thymine); 4.12 – 4.38 (m, HꢁCa(Pro), CH2 of Fmoc, CH of Fmoc, HꢁCb(Pro)); 3.73 – 3.84
(m, HꢁCb(Pro)); 2.90 – 3.01 (m, HꢁCd(Pro)); 2.41 – 2.53 (m, HꢁCd(Pro)); 1.79 (s, Me of thymine); minor
conformer: 11.35 (s, NH); 8.28 (s, HꢁC(6) thymine); 7.95 – 7.89 (m, 2 H of Fmoc); 7.75 – 7.69 (m, 2 H of
Fmoc); 7.63 (s, 1 H of triazole); 7.48 – 7.41 (m, 2 H of Fmoc); 7.40 – 7.37 (m, 2 H of Fmoc); 5.33 – 5.28 (m,
HꢁCg(Pro)); 4.94 (s, CH2 of thymine); 4.54 (dd, J ¼ 7.4, HꢁCa(Pro)); 4.12 – 4.38 (m, CH2 of Fmoc, CH of
Fmoc, HꢁCb(Pro)); 3.73 – 3.84 (m, HꢁCb(Pro)); 3.01 – 3.11 (m, HꢁCd(Pro)); 2.52 – 2.63 (m, HꢁCd(Pro));
1.79 (s, Me of thymine). 13C-NMR (101 MHz, (D6)DMSO): major conformer: 165.3; 151.8; 144.8; 143.7;
142.1; 141.7; 128.8; 128.2; 126.4; 126.2; 124.1; 121.2; 109.9; 68.3; 68.0; 59.1; 58.8; 57.6; 53.8; 52.4; 51.8; 47.7;
47.6; 43.1; 35.6; 35.6; minor conformer: 165.3; 151.8; 144.8; 143.7; 142.1; 141.7; 128.8; 128.2; 126.4; 126.2;
124.1; 121.2; 109.9; 68.3; 68.0; 59.1; 58.4; 57.6; 53.8; 52.4; 51.8; 47.7; 47.6; 43.1; 36.8; 36.7. HR-ESI-MS:
543.1990 ([M þ H]þ, C28H27N6Oþ6 ; calc. 543.1992).
(4S)-4-{4-[(4-{[Bis(tert-butoxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-
1-yl}-1-{[(9H-fluoren-9-yl)methoxy]carbonyl}-l-proline (Fmoc-Nup(C)-OH). A soln. of 6 (17.4 g,
49.9 mmol) and 1 (18.9 g, 49.9 mmol, 1.0 equiv.) in DMF (500 ml) was degassed in an ultrasonic bath
for 20 min, followed by the addition of [Cu(MeCN)4]PF6 (0.928 g, 2.50 mmol, 5 mol-%). The soln. was
stirred at 458 for 2 h. After removal of the solvent, the residue was suspended in a sat. soln. of EDTA in
H2O (0.5m, 500 ml). The aq. soln. was extracted with CH2Cl2 (3 ꢀ 1000 ml), the combined org. layers
were washed with aq. sat NaCl (1000 ml), dried (Na2SO4), and all volatiles were removed at reduced

Helvetica Chimica Acta – Vol. 95 (2012)
2631
pressure. The remaining residue was purified by CC (SiO2; AcOEt/hexane 2 :3 ! 3 :2) to give Fmoc-
Nup(C)-OH (34,8 g, 46.9 mmol, 94%). Colorless solid. Rf (AcOEt/hexane 3 :2) 0.35. The NMR spectra
indicated two conformers (cis and trans conformation around the carbamate bound) in a ratio of 1:1.8.
1H-NMR (400 MHz, (D6)DMSO): major conformer: 8.33 – 8.29 (m, 1 H of cytosine); 7.98 (s, 1 H of
triazole); 7.91 – 7.89 (m, 2 H of Fmoc); 7.73 – 7.68 (m, 2 H of Fmoc); 7.47 – 7.42 (m, 2 H of Fmoc); 7.40 –
7.33 (m, 2 H of Fmoc); 6.90 – 6.86 (m, 1 H of cytosine); 5.35 – 5.23 (m, HꢁCg(Pro)); 5.14 (s, CH2 of
triazole); 4.13 – 4.40 (m, HꢁCa(Pro), CH2 of Fmoc, CH of Fmoc, HꢁCb(Pro)); 3.76 – 3.85 (m,
HꢁCb(Pro)); 2.94 – 3.01 (m, HꢁCd(Pro)); 2.45 – 2.56 (m, HꢁCd(Pro)); 1.51 (s, 2 Me3C); minor con-
former: 8.33 – 8.29 (m, 1 H of cytosine); 7.98 (s, 1 H of triazole); 7.91 – 7.89 (m, 2 H of Fmoc); 7.73 – 7.68
(m, 2 H of Fmoc); 7.47 – 7.42 (m, 2 H of Fmoc); 7.40 – 7.33 (m, 2 H of Fmoc); 6.90 – 6.86 (m, 1 H of
cytosine); 5.33 – 5.23 (m, HꢁCg(Pro)); 5.14 (s, CH2 of triazole); 4.59 – 4.51 (m, HꢁCa(Pro)); 4.13 – 4.40
(m, CH2 of Fmoc, CH of Fmoc, HꢁCb(Pro)); 3.76 – 3.85 (m, HꢁCb(Pro)); 3.01 – 3.11 (m, HꢁCd(Pro));
2.57 – 2.66 (m, HꢁCd(Pro)); 1.51 (s, 2 Me3C). 13C-NMR (101 MHz, (D6)DMSO): major conformer:
163.3; 162.8; 154.9; 154.8; 151.6; 150.3; 144.7; 142.9; 141.7; 128.7; 128.2; 126.3; 126.2; 124.5; 121.2; 96.7;
85.6; 68.3; 68.0; 58.8; 58.4; 52.4; 51.8; 47.7; 47.6; 45.5; 35.49; 35.2; 38.3; 28.3; minor conformer: 163.3;
162.8; 154.9; 154.8; 151.6; 150.3; 144.7; 142.9; 141.7; 128.8; 128.2; 126.3; 126.6; 124.5; 121.1; 96.7; 85.6;
68.3; 68.0; 58.8; 57.6; 52.4; 51.8; 47.7; 47.6; 45.5; 36.6; 36.4; 28.3. HR-ESI-MS: 750.2858 ([M þ Na]þ,
C37H41N7NaOþ9 ; calc. 750.2863).
(4S)-4-[4-({6-[Bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}methyl)-1H-1,2,3-triazol-1-yl]-1-
{[(9H-fluoren-9-yl)methoxy]carbonyl}-l-proline (Fmoc-Nup(A)-OH). A soln. of 7 (16.6 g, 44.5 mmol)
and 1 (16.8 g, 44.5 mmol, 1.0 equiv.) in DMF (500 ml) was degassed in an ultrasonic bath for 20 min,
followed by the addition of [Cu(MeCN)4]PF6 (0.828 g, 2.23 mmol, 5 mol-%). The soln. was stirred at 458
for 2 h. After removal of the solvent, the residue was suspended in a sat. soln. of EDTA in H2O (0.5m,
500 ml). The aq. soln. was extracted with CH2Cl2 (3 ꢀ 1000 ml), the combined org. layers were washed
with aq. sat. NaCl (1000 ml), dried (Na2SO4), and all volatiles were removed at reduced pressure. The
remaining residue was purified by CC (SiO2; AcOEt/hexane 2 :3 ! 3 :2) to give Fmoc-Nup(A)-OH
(31.7 g, 42.3 mmol, 95%). Colorless solid. Rf (AcOEt/hexane 3 :2) 0.35. The NMR spectra indicated two
conformers (cis and trans conformation around the carbamate bound) in a ratio of 1:1.9. 1H-NMR
(400 MHz, (D6)DMSO): major conformer: 8.74 (s, 1 H of adenine); 8.44 (s, 1 H of adenine); 7.99 (s, 1 H
of triazole); 7.94 – 7.89 (m, 2 H of Fmoc); 7.75 – 7.67 (m, 2 H of Fmoc); 7.47 – 7.41 (m, 2 H of Fmoc); 7.39 –
7.32 (m, 2 H of Fmoc); 5.65 (s, CH2 of triazole); 5.34 – 5.23 (m, HꢁCg(Pro)); 4.12 – 4.38 (m, HꢁCa(Pro),
CH2 of Fmoc, CH of Fmoc, HꢁCb(Pro)); 3.73 – 3.84 (m, HꢁCb(Pro)); 2.89 – 2.99 (m, HꢁCd(Pro)); 2.41 –
2.52 (m, HꢁCd(Pro)); 1.42 (s, 2 Me3C); minor conformer: 8.74 (s, 1 H of adenine); 8.44 (s, 1 H of
adenine); 7.99 (s, 1 H of triazole); 7.94 – 7.89 (m, 2 H of Fmoc); 7.75 – 7.67 (m, 2 H of Fmoc); 7.47 – 7.41 (m,
2 H of Fmoc); 7.39 – 7.32 (m, 2 H of Fmoc); 5.65 (s, CH2 of triazole); 5.34 – 5.23 (m, HꢁCg(Pro)); 4.56 –
4.50 (m, HꢁCa(Pro)); 4.12 – 4.38 (m, CH2 of Fmoc, CH of Fmoc, HꢁCb(Pro)); 3.73 – 3.84 (m,
HꢁCb(Pro)); 3.01 – 2.99 (m, HꢁCd(Pro)); 2.56 – 2.63 (m, HꢁCd(Pro)); 1.42 (s, 2 Me3C). 13C-NMR
(101 MHz, (D6)DMSO): major conformer: 163.4; 153.9; 152.6; 151.2; 150.1; 147.8; 144.8; 142.8; 141.7;
128.8; 128.6; 128.2; 126.4; 126.2; 124.3; 121.2; 68.3; 67.9; 59.3; 59.0; 58.5; 57.8; 52.5; 51.9; 47.7; 47.6; 35.7;
35.2; 35.0; 28.3; minor conformer: 163.4; 153.9; 152.6; 151.2; 150.1; 147.8; 144.8; 142.8; 141.7; 128.8; 128.6;
128.2; 126.4; 126.2; 124.3; 121.2; 68.3; 67.9; 59.3; 59.0; 58.5; 57.8; 52.5; 51.9; 47.7; 47.6; 36.1; 36.0; 28.3. HR-
ESI-MS: 774.2980 ([M þ Na]þ, C38H41N9NaO8þ ; calc. 774.2976).
(4S)-4-[4-({6-(Benzyloxy)-2-[bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}methyl)-1H-1,2,3-triazol-
1-yl]-1-{[(9H-fluoren-9-yl)methoxy]carbonyl}-l-proline (Fmoc-Nup(G)-OH). A soln. of 17 (23,4 g,
48.8 mmol) and 1 (18.5 g, 48.8 mmol, 1.0 equiv.) in DMF (500 ml) was degassed in an ultrasonic bath for
20 min, followed by the addition of [Cu(MeCN)4]PF6 (0.906 g, 2.44 mmol, 5 mol-%). The soln. was
stirred at 458 for 2 h. After removal of the solvent, the residue was suspended in a sat. soln. of EDTA in
H2O (0.5m, 500 ml). The aq. soln. was extracted with CH2Cl2 (3 ꢀ 1000 ml), and the combined org. layers
were washed with aq. sat. NaCl (1000 ml), dried (Na2SO4), and all volatiles were removed at reduced
pressure. The remaining residue was purified by CC (SiO2; AcOEt/hexane 2 :3 ! 3 :2) to give Fmoc-
Nup(G)-OH (38.5 g, 44.9 mmol, 92%). Colorless solid. Rf (AcOEt/hexane 3 :2) 0.35. The NMR spectra
indicated two conformers (cis and trans conformation around the carbamate bound) in a ratio of 1:2.1.
1H-NMR (400 MHz, (D6)DMSO): major conformer: 8.71 (s, 1 H of guanine); 7.99 (s, 1 H of triazole);

2632
Helvetica Chimica Acta – Vol. 95 (2012)
7.94 – 7.88 (m, 2 H of Fmoc); 7.77 – 7.65 (m, 2 H of Fmoc); 7.50 – 7.29 (m, 4 H of Fmoc, 5 arom. H); 5.61 (s,
CH2 of triazole); 5.24 – 5.14 (m, HꢁCg(Pro)); 4.36 – 4.08 (m, HꢁCa(Pro), CH2 of Fmoc, CH of Fmoc,
HꢁCb(Pro)); 3.76 – 3.64 (m, HꢁCb(Pro)); 2.92 – 2.81 (m, HꢁCd(Pro)); 2.42 – 2.32 (m, HꢁCd(Pro)); 1.42
(s, 2 Me3C); minor conformer: 8.32 (s, 1 H of guanine); 7.98 (s, 1 H of triazole); 7.94 – 7.88 (m, 2 H of
Fmoc); 7.77 – 7.65 (m, 2 H of Fmoc); 7.50 – 7.29 (m, 4 H of Fmoc, 5 arom. H); 5.69 (s, CH2 of triazole);
5.24 – 5.14 (m, HꢁCg(Pro)); 4.55 – 4.47 (m, HꢁCa(Pro)); 4.13 – 4.40 (m, CH2 of Fmoc, CH of Fmoc,
HꢁCb(Pro)); 3.76 – 3.85 (m, HꢁCb(Pro)); 2.95 – 3.04 (m, HꢁCd(Pro)); 2.46 – 2.53 (m, HꢁCd(Pro)); 1.51
(s, 2 Me3C). 13C-NMR (101 MHz, (D6)DMSO): major conformer: 163.3; 157.6; 151.8; 151.7; 149.4; 143.5;
141.7; 136.8; 129.5; 129.2; 128.9; 128.7; 128.2; 126.2; 123.8; 121.2; 111.9; 69.0; 67.8; 59.6; 57.7; 52.4; 51.8;
47.7; 35.2; 35.1; 28.4; minor conformer: 163.3; 157.6; 151.8; 151.7; 149.4; 143.5; 141.7; 136.8; 129.5; 129.2;
128.9; 128.7; 128.2; 126.18; 123.8; 121.2; 111.9; 69.0; 67.8; 58.4; 57.7; 52.4; 51.8; 47.6; 36.2; 36.1; 28.4. HR-
ESI-MS: 858.3569 ([M þ H]þ, C45H48N9O9þ ; calc. 858.3575).
Solid-Phase Peptide Synthesis (SPPS). For SPPS, Rink Amide-ChemMatrixꢄ resin from Biotage was
used. For coupling of Nup monomers to solid support, a soln. of the appropriate Nup monomer (4 equiv.)
and COMU [19] (4 equiv.) were each dissolved in DMF (0.6m), and EtNiPr2 (8 equiv., dissolved in NMP,
3m) was added to the suspension of the amino functionalized resin in DMF after 3 min of preactivation.
The mixture was shaken for 60 min and washed with DMF (5ꢀ) and CH2Cl2 (5ꢀ). Couplings were
monitored by the qual. Kaiser [24] and Chloranil [25] tests.
For Fmoc deprotection, a soln. of 40% piperidine in DMF was added to the resin, and the suspension
was agitated for 3 min. After filtration, the procedure was repeated for 10 min. The resin was then
washed with DMF (7ꢀ). For the final acetylation step of peptides Ac-[Nup(T)]6-NH2, Ac-[Nup(C)]6-
NH2, Ac-[Nup(A)]6-NH2, Ac-[Nup(G)]6-NH2, and 18, Ac2O/Et3N/CH2Cl2 8 :1:1 (v/v/v) was added to
the resin. The mixture was agitated for 15 min and washed with CH2Cl2 (5ꢀ). For cleavage and
deprotection of the peptides from solid support, the resin was agitated for 1 h in TFA/H2O/TIS
(¼triisopropylsilane) 95 :2.5 :2.5 before collecting the soln. by filtration. This procedure was repeated
twice, and the combined solns. were concentrated under reduced pressure. After addition of cold Et2O, a
white solid precipitated which was centrifuged, and the supernatant was decanted. The solid was
suspended in Et2O, sonicated, centrifuged, and the supernatant was decanted. The residual white solid
was dissolved in H2O and lyophilized to obtain white foam. For HPLC analyses and semiprep.
purification, MeCN (A), and H2O containing 1% of MeCN and 0.1% TFA (B) were used as eluents. The
flow used for semiprep. HPLC was 6 and 1 ml/min for anal. HPLC. After semiprep. purification, all
collected fractions were analyzed by anal. HPLC again, and only pure fractions were combined.
Data of Ac-[Nup(T)]6-NH2 (¼(4S)-1-Acetyl-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-proline). HPLC: tR 9.46 min; gradient: 90 to 40% B over
20 min at 508. HR-ESI-MS: 1872.7261 ([M þ H]þ, C80H90N37O1þ9 ; calc. 1872.7214).
Data of Ac-[Nup(A)]6-NH2 (¼(4S)-1-Acetyl-4-{4-[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-tria-
zol-1-yl}-l-prolyl-(4S)-4-{4-[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-
[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(6-amino-9H-purin-9-yl)-
methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-
yl}-l-prolyl-(4S)-4-{4-[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-proline). HPLC: tR
5.59 min; gradient: 90 to 40% B over 20 min at 508. HR-ESI-MS: 1926.7876 ([M þ H]þ, C80H84N55O7þ ;
calc. 1926.7908).
Data of Ac-[Nup(C)]6-NH2 (¼(4S)-1-Acetyl-4-{4-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-1H-
1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-
l-prolyl-(4S)-4-{4-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-
{4-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(4-amino-2-
oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(4-amino-2-oxopyrimidin-

Helvetica Chimica Acta – Vol. 95 (2012)
2633
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-proline). HPLC: tR 10.22 min; gradient: 98 to 70% B over
20 min at 508. HR-ESI-MS: 1782.7246 ([M þ H]þ, C74H84N43O1þ3 ; calc. 1782.7234).
Data of Ac-[Nup(G)]6-NH2 (¼(4S)-1-Acetyl-4-{4-[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-1H-
1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-
l-prolyl-(4S)-4-{4-[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-
[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(2-amino-6-hy-
droxy-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(2-amino-6-hydroxy-9H-purin-9-
yl)methyl]-1H-1,2,3-triazol-1-yl}-l-proline). HPLC: tR 11.58 min; gradient: 98 to 70% B over 20 min at
508. HR-ESI-MS: 2022.7598 ([M þ H]þ, C80H84N55O1þ3 ; calc. 2022.7603).
Data of (4S)-1-Acetyl-4-{4-[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-
prolyl-(4S)-4-{4-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-
[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(4-amino-2-oxopyrimidin-
1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(2-amino-6-hydroxy-9H-purin-9-yl)methyl]-
1H-1,2,3-triazol-1-yl}-l-prolyl-(4S)-4-{4-[(6-amino-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-pro-
line (18). HPLC: tR 11.17 min; gradient: 98 to 60% B over 20 min at 508. HR-ESI-MS: 2203.8601 ([M þ
H]þ, C91H99N54O1þ6 ; calc. 2203.8593).
Data of N6-[4-Carboxy-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-l-lysyl-l-prolyl-(4S)-4-{4-
[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-l-prolyl-l-
prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-
prolyl-l-prolyl-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-
1,2,3-triazol-1-yl}-l-prolyl-l-prolyl-l-prolyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-l-prolyl-l-lysinamide (19). HPLC: tR 13.70 min; gradi-
ent: 90 to 55% B over 20 min at 508. HR-ESI-MS: 2617.1451 ([M þ H]þ, C125H150N37O2þ8 ; calc. 2617.1446).
Data of N6-[4-Carboxy-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-l-lysyl-l-alanyl-3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-l-alanyl-l-alanyl-l-alanyl-3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)-l-alanyl-l-alanyl-l-alanyl-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
l-alanyl-l-alanyl-l-alanyl-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-l-alanyl-l-alanyl-l-ly-
sinamide (20). HPLC: tR 12.75 min; gradient: 90 to 55% B over 20 min at 508. HR-ESI-MS: 1980.8254
([M þ H]þ, C89H114N25O2þ8 ; calc. 1980.8260).
Data of N6-[4-Carboxy-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-l-lysyl-l-alanyl-(4S)-4-{4-
[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-l-alanyl-l-
alanyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-
prolyl-l-alanyl-l-alanyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]-1H-
1,2,3-triazol-1-yl}-l-prolyl-l-alanyl-l-alanyl-(4S)-4-{4-[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}-l-prolyl-l-alanyl-l-lysinamide (21). HPLC: tR 13.15 min; gradi-
ent: 90 to 55% B over 20 min at 508. HR-ESI-MS: (C109H134N37Oþ28 ; calc. [M þ H]þ, 2409.0194).
2409.0202.
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Received October 5, 2012