Syntheses of 5′-Nucleoside Monophosphate Derivatives with Unique Aminal, Hemiaminal, and Hemithioaminal Functionalities: A New Class of 5′-Peptidyl Nucleotides

Article abstract of DOI:10.1002/chem.201600721

A number of synthetically useful transformations have been developed to generate novel 5′-peptidyl nucleoside monophosphate analogues that incorporate sensitive phosphoaminal, -hemiaminal or -hemithioaminal functionalities. The strategies adopted entailed

Full text of DOI:10.1002/chem.201600721

DOI: 10.1002/chem.201600721
Full Paper
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Synthetic Biology |Hot Paper|
Syntheses of 5’-Nucleoside Monophosphate Derivatives with
Unique Aminal, Hemiaminal, and Hemithioaminal Functionalities:
A New Class of 5’-Peptidyl Nucleotides
Swarup De, Elisabetta Groaz, Lia Margamuljana, and Piet Herdewijn*[a]
Abstract: A number of synthetically useful transformations
have been developed to generate novel 5’-peptidyl nucleo-
side monophosphate analogues that incorporate sensitive
phosphoaminal, -hemiaminal or -hemithioaminal functionali-
ties. The strategies adopted entailed the coupling between
dipeptides, which enclose a reactive Ca-functionalized gly-
cine residue and phosphate or phosphorothioate moieties.
These developments led to potentially powerful and general
methodologies for the preparation of a-phosphorylated
pseudopeptides as well as nucleoside monophosphate
mimics. The resulting conjugates are of interest for a variety
of important applications, which range from drug develop-
ment to synthetic biology, as pronucleotides or artificial
building blocks for the enzymatic synthesis of xenobiotic in-
formation systems. The potential of all dipeptide-TMP conju-
gates as pyrophosphate mimics in the DNA polymerization
reaction was tested, and the influence of the nature of the
linker was evaluated by in vitro chain elongation assay in
the presence of wild-type microbial DNA polymerases.
Introduction
tion of the exceptional role that is displayed by this class of
compounds is relevant to both the fields of medicinal chemis-
try and synthetic biology, which allows for the design of new
drugs that are able to bypass the multistep anabolic cascade,
which is required for nucleoside activation,[7] or unnaturally ac-
tivated building blocks for the biosynthesis of artificial genetic
polymers (XNAs).[8]
Amino acids are a common, non-toxic structural motif in nu-
merous prodrug compounds, some of which have shown inter-
esting biological activities.[1] For example, peptidomimetic con-
jugates were designed to increase the solubility of polar nu-
cleoside analogues (i.e., acyclovir, ganciclovir, didanosine),
which enhances their cellular permeability and bioavailability
following oral administration, by targeting intestinal oligopep-
tide transporters.[2] The phosphoramidate family of nucleotide
prodrugs contains an amino acid ester linked to the 5’-phos-
phate group through a PÀN bond, which is a key characteristic
element.[3] This species is sufficiently stable during transport,
but the nucleotide adduct is readily cleaved by intracellular en-
zymes with phosphoramidase activity.[4] As a result, this pro-
cess releases the promoiety as a biodegradable intermediate,
which then re-enters the cellular metabolism and concomitant-
ly leads to the delivery of the parent active nucleoside mono-
phosphate.
A surprising discovery was made by Richert and co-workers,
who recently showed that it is possible to form peptidyl chains
that are linked to RNA through a 5’-phosphoramidic bond
from the spontaneous oligomerization between ribonucleoside
phosphates and amino acids under plausible prebiotic condi-
tions and in the absence of cellular machinery.[9] The resulting
peptidyl RNA, which is produced alongside simple genetic ma-
terial, thus emerged as an early protein precursor in primitive
metabolic pathways.
However, from a synthetic perspective, the N-terminus of an
amino acid/peptide chain is the most common site for conju-
gation to the 5’-phosphate moiety of NMPs.[4a,6,10] Aminoacyl
adenylates, which can be considered as molecular isomers of
amino acid phosphoramidate nucleosides, are important acti-
vated intermediates in protein biosynthesis.[11] The connecting
phosphoric–carboxylic mixed anhydride bond makes those
molecules too unstable to be synthetically useful.[12] Naturally
occurring[13] and relatively less reactive peptidyl nucleotide an-
alogues that contain active carboxyl groups were synthe-
sized.[14]
We have previously reported that analogous amidate mono-
ester derivatives that comprise amino acids[5] (i.e., l-Asp, l-His)
as well as small peptides[6] represent synthetic alternatives to
natural nucleoside triphosphates and act as direct substrates in
the DNA-polymerase-catalyzed synthesis of DNA. The recogni-
[a] Dr. S. De, Dr. E. Groaz, L. Margamuljana, Prof. Dr. P. Herdewijn
KU Leuven
We wished to explore whether the reaction between nucleo-
side monophosphates and the alpha carbon of a suitable func-
tionalized amino acid residue would allow us to produce
novel, synthetically and biologically useful molecules. To this
end, we designed a range of l-Ala-Gly nucleotide conjugates
Rega Institute for Medical Research, Medicinal Chemistry
Minderbroedersstraat 10, 3000 Leuven (Belgium)
E-mail: Piet.Herdewijn@rega.kuleuven.be
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201600721.
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peptides but also with the increased nucleophilicity of the unit
that originates from the phosphorus-containing nucleophile in
the final products.
Results and Discussion
Figure 1. General design of molecular targets: Ca-X-peptide–nucleotide con-
jugates (X=O, N, S).
Synthetic routes need to be cautiously designed and executed
by using suitable orthogonal protection/deprotection methods
as well as reaction conditions that are compatible with the
sensitive phosphodiester and mixed phosphoester units of
newly designed conjugates. Our retrosynthetic analysis for the
proposed target compounds is shown in Figure 2. It became
apparent that our target conjugates could be obtained by nu-
cleophilic substitution either at the phosphorus group itself
(Route 1) or at the a-position of glycine (Route 2).
that were connected through a phosphodiester or a mixed
amido/thioate-phosphoester bond, as illustrated in Figure 1.
The formation of a-heterosubstituted peptides from electro-
philic precursors typically requires the presence of a good leav-
ing group at the a-carbon center of a glycine residue, which is
commonly in the form of an alkoxide,[15] a carboxylate,[16] or
a halide group.[17] Although a-substituted glycines can rapidly
decompose by elimination of the a-group, their stabilization
can be accomplished by masking the lone pair of the glycyl ni-
trogen through N-acylation (i.e., formation of a peptide
bond).[16a,18] Small heteroatom-centered (i.e., N, O, and S) alkyl
and aryl nucleophiles were shown to be suitable reaction part-
ners.[16c,19] Most notably, glycine-containing peptides that were
modified at the a-carbon with a toxophoric agent[16a] or
a phenyl derivative[20] were found not only to be accepted and
actively transported by peptide permeases into bacterial cells
but also to undergo hydrolysis in the presence of intracellular
peptidases, which thus liberates the attached cargo.
Among the various options that are available for the forma-
tion of a phosphodiester linkage, we selected the approach
that is based on the use of a dialkylphosphoramidite inter-
mediate followed by in situ oxidation in our initial efforts to
access phosphohemiaminal diester target 1 (Scheme 1). a-Hy-
droxy glycine derivative 5 was obtained as a diastereoisomeric
mixture in good yield by coupling z-l-AlaÀNH2 with benzyl
glyoxalate in accordance with an improved protocol,[16a] which
comprised the addition of a catalytic amount of p-TSA to in-
crease the reaction rate. As all attempts to generate bis(alkyl)-
N,N-diisopropyl phosphoramidite derivative 6 met with failure,
we turned to an alternative strategy that was based on the
conversion of 3’-O-benzyl thymidine 8 into the corresponding
pro-moiety 9[21] followed by phosphoramidite coupling with
the a-OH of glycine 5 and in situ oxidation. Although thymi-
dine phosphoramidite 9 could be successfully isolated, the
subsequent coupling with glycine 5 in the presence of 1-H tet-
To the best of our knowledge, the synthetic utility of this
transformation has not been demonstrated for highly charged
compounds. It was anticipated that efficient NÀCaÀXÀP bond
formation involved overcoming challenges that were associat-
ed not only with the inherent chemical instability of the parent
Figure 2. Retrosynthetic analysis of 5’-peptidyl nucleoside monophosphate targets.
Scheme 1. Attempted routes towards the synthesis of the phosphoester of (l-alanyl-d,l-2-aminoglycine)-TMP 1. Reagents and conditions: (a) Benzyl glyoxa-
late, p-TSA, CH2Cl2, 168 h; (b) Benzyloxy-bis(N,N-diisopropylphosphoramino) phosphine, 0.45m 1H-tetrazole in CH3CN, CH2Cl2, 08C to RT, 4 h.
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razole and oxidation with H2O2 did not produce the desired
compound 7, but 3’-O-benzyl-(benzyloxy)-TMP was formed in-
stead. Despite screening of various coupling agents with differ-
ent pKa values, which included 4,5-dicyanoimidazole, 2-ethyl-
thio tetrazole, and 2-benzylthio tetrazole, none were found to
be beneficial to the outcome of the above reaction.
glycine derivative 5 was activated towards nucleophilic substi-
tution at the a-position by conversion to its a-acetoxy deriva-
tive 12, which was then reacted with the triethylammonium
salt of TMP (Scheme 3). Although at first a single product was
thought to have formed on the basis of mass spectroscopic
analysis of the crude residue, further RP-HPLC purification re-
vealed a mixture of products with the same mass. After in-
depth two-dimensional NMR analysis of the separated prod-
ucts, it could be concluded that the desired phosphodiester
derivative 13 was formed as a minor product (8% yield) along
with thymidylate N3-peptide adduct 14 as the major
product (40% yield). Various dimeric products and
a 3’-TMP-peptide adduct were also produced in trace
amounts in the course of the reaction. This result can
be rationalized on the basis of the hard soft acid
base principle (HSAB): a soft center, such as an a-
carbon, prefers a soft donor; in the present case, the
electrons will preferentially be donated from the N3-
atom rather than from the oxygen atom of the phos-
Therefore, we proceeded to investigate a method that was
based on a more reactive chlorophosphate intermediate, as
depicted in Scheme 2. 3’-O-benzyl thymidine 8 was converted
into its activated form, 5’-O-dichlorophospho-3’-O-benzyl-thy-
Scheme 2. Route towards the synthesis of the phosphoester of (l-alanyl-d,l-2-aminogly-
cine)-TMP 1. Reagents and conditions: (a) POCl3, trimethyl phosphate, À208C to RT, 8 h;
(b) Compound 5, base, CH2Cl2, 08C to RT, 16 h.
phate group. Despite numerous attempts to improve
the reaction yields, longer times and higher tempera-
tures only led to the formation of additional unde-
sired side products. As the polarity similarities be-
tween the 3’-TMP-peptide adduct and compound 13
midine 10, by using POCl3 in trimethyl phosphate, which was
then treated in situ with glycine 5 in the presence of N-methyl
imidazole. Surprisingly, the reaction did not afford the desired
product, but it resulted in the formation of 3-O-benzyl-TMP
along with other uncharacterized byproducts. A number of
bases with different pKa’s, such as Et3N, DIPEA, and pyridine,
were employed but yielded the same results. This may be due
to the low nucleophilicity of the a-OH group, a steric hin-
drance at the a-position, the instability of the mono-chloro-
phosphate intermediate during quenching of the reaction, or
a base-promoted[22] elimination reaction at the a-position of
the desired compound.
did not allow their separation by preparative HPLC, the two re-
gioisomers were separated in the subsequent step. Finally, re-
moval of all protecting groups of compound 13 by reductive
hydrogenation with 10% Pd/C Degussa-type followed by prep-
arative HPLC purification yielded the desired conjugate 1 in
moderate yield (42%). During the conversion of compound 13
into target 1, we observed the formation of TMP as an impuri-
ty, whereas when the same reaction conditions were applied
to compound 14, we isolated the desired product 15 in excel-
lent yield (92%).
In principle, the synthesis of phosphoaminal analogue 2
could be accessed through either Route 1 or 2, as shown in
the retrosynthetic analysis (Figure 2). Above, we reported the
competition for nucleophilic attack in Route 1 between the N3-
atom of thymine and the thymidine-5’-O-phosphoramidate ni-
Owing to the perceived difficulties in achieving the synthesis
of compound 1, we next switched to Route 2 of the proposed
retrosynthetic analysis (Figure 2). Accordingly, the a-hydroxy
Scheme 3. Synthesis of the phosphoester of (l-alanyl-d,l-2-aminoglycine)-TMP 1. Reagents and conditions: (a) Ac2O, pyridine, 08C, 8 h; (b) TMP triethylammo-
nium salt, Et3N, DMF, 408C, 24 h; (c) 10% Pd/C (Degussa-type), EtOH/H2O 5:1, RT, 4 h.
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trogen atom; therefore, the more selective approach,
Route 1, was employed. The synthesis of a-amino
glycine derivative 17 from its corresponding hydroxyl
derivative 5 was performed via an azide intermediate
(Scheme 4). Azido compound 16 was obtained in
good yield from glycine derivative 5 by using the
DPPA/DBU method,[23] and it was then converted into
its amino form 17 by classical reduction in the pres-
ence of PPh3. The ensuing phosphoramidate coupling
was carried out under standard DCC coupling condi-
tions[24] to afford a diastereoisomeric mixture of com-
pounds 18a and 18b in good yield. Our initial efforts
to separate isomers 18a,b after the final debenzyla-
tion step turned out to be problematic; however, the
protected diastereoisomers could be successfully sep-
arated by preparative RP-HPLC to yield pure 18a and
18b in an approximate 1:1 ratio. Finally, catalytic hy-
Scheme 5. Synthesis of the phosphoaminal of (l-alanyl-d,l-2-aminoglycine methyl ester)-
TMP 23. Reagents and conditions: (a) Pb(OAc)4, dry EtOAc, 4 Š MS, reflux, 2 h; (b) 4-me-
thoxybenzylamine, DIPEA, dry DMF, 408C, 24 h; (c) 10% Pd/C, EtOH, RT, 5 h (d) TMP tri-
ethylammonium salt, DCC, Et3N, tBuOH/H2O 4:1, 858C, 2.5 h; (e) TFA, thioanisole, CH2Cl2,
08C to RT, 4 h.
drogenation effected the cleavage of the two protecting
groups, which gave rise to the corresponding isomers 2a and
2b in moderate yields.
protecting group with TFA in the presence of the radical scav-
enger thioanisole produced the desired methyl ester derivative
23; in this case, no TMP formation was observed. As for previ-
ously described compounds 18a,b, a late stage effort was
made to separate the diastereoisomers of 22 and 23 by prepa-
rative RP-HPLC, but it was not found to be effective in this
case.
As it is not unreasonable to speculate that the free a-carbox-
yl group of glycine might play a role in the formation of TMP
as a side product during the isolation of nucleotides 1, 2a and
2b, we were interested to synthesize phosphoaminal ester an-
alogue 23, in which the carboxyl group of glycine is protected
as a methyl ester. A suitable choice of orthogonal protecting
groups was crucial for the synthesis of compound 23
(Scheme 5), and the main synthetic strategy resembled that
which was employed for nucleotides 2ab. Unlike benzyl-pro-
tected a-acetoxy glycine derivative 12, which was synthesized
in a convergent manner, the synthesis of a-acetoxy derivative
19 followed a more practical degradative approach, which
started from N-Boc-l-Ala-l-Ser-OMe in the presence of
Pb(OAc)4,[22] because the related protecting groups were well
tolerated during the reaction. The acetoxy derivative 19 was
then converted to its protected amino derivative 20 by nucleo-
philic substitution in the presence of 4-methoxybenzyl amine.
Subsequent deprotection by Pd/C-catalyzed hydrogenation
yielded the desired amine 21 in excellent yield (96%). Phos-
phoaminal ester 22 was obtained in good yield by using the
standard coupling protocol, and successive removal of the Boc
Although both proposed retrosynthetic routes are feasible
for the synthesis of thioethyl phosphate derivative 4, it was an-
ticipated that the instability of the thioethyl-TMP synthon
could be problematic. As described in the literature, this unit
can be readily synthesized,[25] but when it is formed it immedi-
ately decomposes through nucleophilic attack of the naked
thiol group to give TMP and thiirane.[26] According to Route 2,
thioethanol analogue 24 was formed by selective S-alkylation
of the corresponding acetoxy compound 12 with 2-mercapto-
ethanol in good yield (70%), as shown in Scheme 6. The phos-
phoramidite approach could in theory be utilized for the syn-
thesis of phosphoester 25; however, selective oxidation of PIII
to PV in the presence of a sulfur atom posed a real chal-
lenge.[27] Therefore, the POCl3 method was adopted instead,
which involved the formation of intermediate 10 from 3’-O-
benzyl thymidine and its in situ treatment with thioethanol an-
alogue 24 in the presence of N-methyl imidazole. Quenching
Scheme 4. Synthesis of the phosphoaminals of (l-alanyl-d,l-2-aminoglycine)-TMP 2a and 2b. Reagents and conditions: (a) DPPA, DBU, dry toluene, 08C to RT,
16 h; (b) PPh3, THF, reflux, 2 h; (c) TMP triethylammonium salt, Et3N, DCC, tBuOH/H2O 4:1, 858C, 2.5 h; (d) 10% Pd/C (Degussa-type), EtOH/H2O 5:1, RT, 4 h.
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controlled reaction conditions. Finally, the removal of the Boc
group was achieved by using the TFA/thioanisole system fol-
lowed by the simultaneous hydrolysis of the glycine ester and
the 3’-benzoyl group with LiOH, which furnished nucleotide 4
in good yield (72%) over two steps.
As separation of diastereoisomers by preparative RP-HPLC
represents a limitation with regards to the quantities of prod-
ucts that can be purified, we tried to perform this step at the
later stages of the synthetic pathway for all the conjugates.
However, effective separation could only be achieved in some
cases. Therefore, it was thought that the enzymatic separation
of the starting diastereoisomeric peptides could help to obtain
larger quantities of pure final compounds. Accordingly, the dia-
stereoisomeric mixture of N-Boc-l-Val-d,l-benzyloxy-Gly-OMe
was successfully resolved by enzyme-catalyzed ester hydrolysis
by using subtilisin Carlsberg (Scheme 8).[22] Conversely, the ap-
plication of similar conditions with N-Boc-l-Ala-d,l-benzyloxy-
Gly-OMe resulted in a poor separation, which may be due to
a low substrate specificity.
Scheme 6. Attempted route towards the synthesis of the phosphoester of
(l-alanyl-d,l-2-mercaptoethanolglycine)-TMP 4. Reagents and conditions:
(a) 2-mercaptoethanol, Et3N, dry DMF, 08C to RT,16 h; (b) POCl3, trimethyl
phosphate, À208C to RT, 8 h (c) N-methyl imidazole, CH2Cl2, 08C to RT,16 h.
followed by preparative RP-HPLC purification furnished phos-
phoester 25 as a diastereoisomeric mixture in 66% yield over
two steps. For the conversion of protected derivative 25 into
nucleotide 4, we attempted to remove the Cbz and benzyl
groups by hydrogenation with a range of palladium com-
pounds, which included 10% Pd/C, 10% Pd/C (Degussa), 20%
Pd(OH)2/C, Pd-black, and Pd(OAc)2. Here, the use of an excess
of catalyst was necessary because of catalyst poisoning that
can occur in the presence of sulfur atoms. However, all cata-
lysts failed to provide nucleotide 4; harsh reaction conditions
led to undesired over-reduced side products, whereas mild
conditions resulted in the recovery of unreacted starting mate-
rial.
For our last targets, phosphohemithioaminal ester deriva-
tives 38a and 38b, the initially followed retrosynthetic Route 2
was quickly replaced by the approach described in Scheme 9
as it was not possible to isolate the N-Boc-l-alanyl-d,l-2-thio-
glycine methyl ester synthon in a satisfactorily pure form,
which is likely due to the instability of the free thiol group.
The synthesis of thymidine-5’-O-phosphorothioate 36 is report-
ed in the literature by using a solid-phase methodology;[29] de-
spite being effective for small quantities, this protocol was not
reproducible upon scale-up.
Therefore, an alternative protecting group strategy for the
synthesis of target
4 was considered, as illustrated in
Scheme 7. Thioethanol methyl ester 26 was isolated in good
yield (77%) from its acetoxy derivative 19. In analogy to the
synthesis of compound 25, the corresponding phosphodiester
product 29 was obtained in low yield (12%) over two
steps. It was found that the low conversion rate of
nucleoside 27 into dichlorophosphoester intermedi-
ate 28 was the main cause for the formation of com-
plex byproducts in the next base-promoted step.
Other methods were explored to improve the yield
of intermediate 28, which included the use of POCl3
in combination with different bases and solvents,[28]
Scheme 8. Route towards the enzymatic separation of diastereoisomeric peptides. Re-
agents and conditions: (a) DABCO, dry THF, BnOH, À788C to RT, 24 h; (b) subtilisin Carls-
but none of them led to improved conversions, and
the formation of dimers was detected even under berg, 1m NaOH, DMF/H2O 1:1, pH 7.2, 558C.
Scheme 7. Synthesis of the phosphoester of (l-alanyl-d,l-2-mercaptoethanolglycine)-TMP 4. Reagents and conditions: (a) 2-mercaptoethanol, Et3N, dry DMF,
08C to RT,16 h; (b) POCl3, trimethyl phosphate, À208C to RT, 8 h (c) N-methyl imidazole, CH2Cl2, 08C to RT,16 h; (d) TFA, CH2Cl2, thioanisole, 08C to RT, 4 h;
(e) LiOH, MeOH/H2O 1:1, 08C to RT, 3 h.
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Scheme 9. Synthesis of the phosphohemithioaminals of (l-alanyl-d,l-2-aminoglycine methyl ester)]-TMP 38a and 38b. Reagents and conditions: (a) 2-mercap-
to ethanol, DIPEA, diethyl ether, À208C to RT, 2.5 h, (b) (i) 3’-O-benzoyl thymidine, S-ethylthio tetrazole, CH2Cl2, 08C to RT, 3 h; (ii) S8, RT, overnight; (c) 3-hy-
droxy propionitrile, DBU, RT, 8 h; (d) 25% NH3, 558C, sealed tube, 16 h; (e) Compound 19, Et3N, DMF, 378C, 4 h; (f) TFA, thioanisole, CH2Cl2, 08C to RT, 3 h.
[30]
Thus, monothiophosphorylation with PSCl3 was next ex-
plored to access compound 36 in one step from thymidine,
but the reaction rates were found to be sluggish and the pro-
cess was non-selective in the presence of an excess of reagent.
However, the protection of 3’-OH of thymidine and the use of
an improved oxathiaphospholane approach that was adapted
from the literature could overcome this issue.[31] As depicted in
Scheme 9, compound 34 was obtained from N,N-diisopropyl
phosphoramidous dichloride and 2-mercapto ethanol in the
presence of DIPEA, and without further purification, it was re-
acted with 3’-O-benzoyl thymidine and S-ethylthio tetrazole. In
situ sulfurization with elemental S8 furnished oxathiaphospho-
lane 35 in excellent yield (93%) over three steps. Opening of
the oxathiaphospholane ring of compound 35 with 3-hydroxy-
propionitrile and simultaneous removal of 2-cyanoethyl and 3’-
benzoyl groups by using 25% aqueous ammonia in a sealed
tube produced compound 36 in good yield. Selective S-alkyla-
tion over N3-alkylation of compound 36 with fragment 19
gave compound 37 in only 4 h as a diastereoisomeric mixture.
Ultimately, Boc cleavage and preparative RP-HPLC purification
allowed the isolation of diastereoisomers 38a and 38b.
merases in directed enzyme evolution as well as in medicinal-
chemistry-focused programs. As dipeptides are comparable in
size to the pyrophosphate group,[6] which is realeased during
DNA biosynthesis, it was envisioned that l-Ala-Gly could also
act as an alternative leaving group in the enzyme-catalyzed
DNA polymerization reaction. In this study, the activities of dif-
ferent thermophilic and mesophilic microbial DNA polymerases
were compared, which included Therminator, Vent (exo-), and
the Klenow fragment (exo-) of E. coli DNA Polymerase I. To in-
vestigate the strand elongation capacity of all the final ana-
logues that exhibit a-substituted glycines connected through
various phosphoheteroamic linkers (1, 2a, 2b, 23, 4, 38a and
38b), we carried out a polyacrylamide gel-based template-de-
pendent incorporation assay of multiple nucleotides by using
a tagged 5’-radiolabelled g-33P or a fluorescent DNA primer
(P1) that was annealed to templates T1–3 (Table 1).
Amongst all the compounds screened as potential triphos-
phate mimics, phosphoaminal ester analogues 2a and 2b ex-
hibited interesting incorporation efficiencies with all tested
polymerases (Figure 3A). In contrast, compound 1 showed in-
corporation with only the Therminator polymerase and with
a poor level of efficiency. On the basis of these observations, it
can be deduced that the free carboxyl group along with an ap-
propriate chain length are both crucial factors for incorpora-
tion. Vent (exo-) showed poorer efficiency than Therminator
and the Klenow fragment; it only produced 4% (P+4) strand
formation for 2a and 11 % (P+3) for 2b at 1 mm concentration
after 60 min. Therminator gave 76% yield of the full length
product (P+5) and up to 4% (P+4) strand formation for 2a
and 2b, respectively. The Klenow fragment showed the best
incorporation efficiency, which led to 27% formation of the full
length product (P+5) and up to 11 % yield of the (P+4) strand
at 1 mm concentration after only 15 min. Notably, compound
When the two diastereoisomers were separately subjected
to ester hydrolysis with LiOH, we isolated TMP as the major
product. The in cellulo mode of breakdown of a-substituted
glycine-based peptide conjugates after peptidase cleavage was
postulated by Gilverg et al.[16a] In this context, the formation of
TMP as a side product during the synthesis of compounds 1–3
could be explained by the anchimeric assistance of the a-car-
boxyl group of glycine towards the phosphorus atom, which
led to the formation of a 5-membered-ring intermediate. This
phenomenon has previously been noticed at different rates in
the presence of phosphate, phosphoramidate, and phosphor-
othiate moieties.[32] By masking the free a-carboxyl group of
glycine as a methyl ester, it was possible to resolve the meta-
stability of the corresponding compounds.
Table 1. Overview of primer and templates sequences used in the incor-
poration experiments.
Chain elongation experiments
In our laboratory, we are currently investigating the non-ca-
nonical mimics of nucleoside triphosphates as valuable targets
for a range of studies into the substrate scope of DNA poly-
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Figure 3. (A) Chain elongation efficiencies of compounds 2a and 2b by using Vent (exo-) (0.01 UmLÀ1), Therminator (0.01 UmLÀ1) and Klenow fragment
(0.05 UmLÀ1) polymerases at 1 mm after 60 min. (B) Chain elongation profile of compounds 2a and 2b as substrates into the P1:T1 complex by Klenow poly-
merase (0.05 UmLÀ1).
2a exhibited better substrate properties than its diastereoiso-
mer 2b; this was probably because of different binding effica-
cy in the enzyme pocket, which might be approached with the
aid of a modeling study. As the best incorporation results were
obtained with compound 2a and the Klenow fragment (exo-)
polymerase, we compared the kinetic parameters of 2a with
its natural substrate counterpart TTP based on the Single Com-
plete Hot model.
cedures for the synthesis of a-phosphorylated pseudopeptides
and nucleoside monophosphate mimics.
In a template-based multiple incorporation assay, analogues
2a and 2b featuring a phosphoaminal ester linker and the
Klenow fragment (exonuclease-free) of DNA polymerase I were
found to be the best substrate and the most effective poly-
merase, respectively, and resulted in the best strand elongation
rates. We found that a free carboxyl group at the C-terminal of
the dipeptidic chain was necessary for binding in the active
site of the enzyme, but it was also accountable for the destabi-
lization of the conjugates and led to hydrolytic cleavage
through anchimeric assistance. The further exploration of such
dipeptides as leaving groups for DNA polymerization can be
a valuable tool for synthetic biology, nucleotide delivery, poly-
merase chain reaction (PCR), and as potential prodrugs for an-
tiviral therapy.
Steady-state kinetic analysis (Table 2) indicated a comparable
Vmax value, a KM that was 991-fold higher, and a final Vmax/KM
ratio for compound 2a that was 994-fold lower than the natu-
ral substrate. This result falls within the range of kinetic values
that have been previously reported for other leaving groups in
our laboratory.[24]
Conclusion
A number of useful strategies were developed and effectively
refined to generate different phosphodiester and mixed (N/O,
S/O) phosphoester linker moieties in the preparation of
a novel class of 5’-modified conjugates of TMP with the dipep-
tide l-Ala-Gly. The considerable challenges posed by the sensi-
tive nature of the derived phosphoaminal, -hemiaminal or
-hemithioaminal functionalities demanded a systematic elabo-
ration of specifically tailored synthetic technologies. The intro-
duction of an amino group at the a-position of a glycine resi-
due successfully activated the corresponding dipeptide for
attack at the phosphorus atom of TMP, which led to the requi-
site phosphoaminal ester conjugates. 5’-Peptidyl nucleotides
that contain either an OÀCaÀOÀP or a SÀCaÀOÀP bond were
otherwise assembled by a nucleophilic substitution reaction of
the a-acetoxy group of glycine. Overall, we feel that the exten-
sive synthetic optimization that was needed during the execu-
tion of this work could develop into new general reaction pro-
Experimental Section
General Information
For all reactions, analytical grade solvents were used. TMP-disodi-
um salt was transformed into its triethylammonium salt by treat-
ment with Amberlite IRA-120B ion exchange resin (H+ form) and
then with triethylamine. All moisture-sensitive reactions were car-
ried out in oven-dried glassware (1358C) under a nitrogen or
argon atmosphere. Reaction temperatures are reported as bath
temperatures. Pre-coated aluminum sheets (254 nm) were used for
TLC. Compounds were visualized with UV light (l=254 nm). Prod-
ucts were purified by flash chromatography on ICN silica gel 63–
200 mm, 60 Š. All final compounds were purified by preparative RP-
HPLC (XbridgeTM Prep C18 5 mm OBD 19”150 mm column) by
using an eluent gradient of H2O/CH3CN with 50 mmol TEAB as
eluent buffer. 1H, 13C, and 31P NMR spectra were recorded on
Bruker Avance 300, 500, or 600 MHz spectrometers. Final com-
pounds were characterized by using 2D NMR (H-COSY, HSQC,
HMBC, TOCSY, and NOESY) techniques. For the sake of clarity, NMR
signals of protons and carbons for sugar and base moieties are in-
dicated with and without a prime, respectively. Chemical shifts
were referenced to residual solvent signals at dH/C =7.26/77.00
(CDCl3), 3.31/49.10 (CD3OD), 1.94/118.7 (CD3CN), and 2.50/39.50
([D6]DMSO) relative to TMS as an internal standard wherever ap-
plied. Coupling constants are expressed in hertz (Hz) and were di-
Table 2. Steady-state kinetics of single nucleotide incorporation into
P1:T3 by Klenow fragment (0.005 UmLÀ1).
Substrate
TTP
Compound 2a 30.48Æ1.04
Vmax [nmminÀ1
30.59Æ0.74
]
KM [mm]
Vmax/KM [”10À3 minÀ1
]
0.088Æ0.006 347.61
87.21Æ9.21 0.3495
Chem. Eur. J. 2016, 22, 8167 – 8180
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8173
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Full Paper
rectly obtained from the spectra. Splitting patterns are designated
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br
(broad), and apparent (app). High-resolution mass spectra (HRMS)
were obtained on a quadruple orthogonal acceleration time-of-
flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA).
Samples were infused at 3 mLminÀ1 and spectra were obtained in
positive (or negative) ionization mode with a resolution of 15000
(FWHM) by using leucine enkephalin as lock mass.
The reaction mixture was stirred at the same temperature for 8 h
and monitored by TLC. Upon completion, the mixture was concen-
trated in vacuo (bath temp. ꢀ158C). The residue was diluted with
EtOAc (150 mL) and washed with H2O (2”50 mL), 1n HCl (2”
50 mL), 5% NaHCO3 (2”50 mL), and brine (100 mL). The organic
phase was dried over Na2SO4, filtered, and concentrated in vacuo.
The resulting crude residue was triturated with cold Et2O to give
compound 12 (2.02 g, 76%) as a white solid. 1H NMR (300 MHz,
CDCl3) d=7.59, 7.54 (2”d, J=8.5 Hz, 1H, NH-Gly), 7.35–7.26 (m,
10H, 2”OBn), 6.43, 6.39 (2”d, J=8.9 Hz, 1H, aH-Gly), 5.31 (app t,
1H, NH-Ala), 5.20 (s, 2H, CH2-CO2Bn), 5.09 (s, 2H, CH2-Cbz), 4.35–
4.30 (m, 1H, aH-Ala), 2.05 (s, 3H, CH3-OAc), 1.38 and 1.36 ppm (2”
d, J=7.0 Hz, 3H, CH3-Ala); 13C NMR (75 MHz, CDCl3) d=172.4 and
172.4 (CO-Ala), 170.3 and 170.3 (CO-OAc), 166.4 (CO-Gly), 156.0
(OCONH), 136.1 (1C of OCH2Ph), 134.8 (1C of OCH2Ph), 128.8 (Ar-C),
128.7 (Ar-C), 128.4 (Ar-C), 128.2 (Ar-C), 72.3 and 72.3 (aC-Gly), 68.3
(CH2-CO2Bn), 67.4 and 67.4 (CH2-Cbz), 50.5 (aC-Ala), 20.7 (CH3-OAc),
18.5 and 18.2 ppm (CH3-Ala); HRMS (ESI+): m/z calcd for
C22H24N2O7: 451.1476 [M+Na]+; found: 451.1474.
N-Carbobenzyloxy-l-alanyl-d,l-2-hydroxyglycine benzyl
ester (5)
Benzyl glyoxalate (1.85 g, 11.25 mmol) and a catalytic amount of p-
toluene sulfonic acid (0.03 g, 0.18 mmol) were added to a stirred
suspension of N-carbobenzoxy-l-alanine amide (2.0 g, 9.0 mmol) in
CH2Cl2 (40 mL). The reaction mixture was stirred at room tempera-
ture for 7 days and monitored by TLC. Upon completion, the reac-
tion mixture was concentrated in vacuo and the resulting crude
residue was purified by column chromatography on silica gel
(EtOAc/hexane, 1:4–2:3–3:2, v/v) to give compound 5 (2.44 g,
1
70%) as a white solid. H NMR (300 MHz, [D6]DMSO) d=8.79 and
5’-O-(N-Carbobenzyloxy-l-alanyl-d,l-2-aminoglycine benzyl
ester)-TMP (13) and 3-N-(N-carbobenzyloxy-l-alanyl-d,l-2-
aminoglycine benzyl ester)-TMP triethylammonium salt (14)
8.77 (2”d, J=8.5 Hz, 1H, NHÀGly), 7.44 (d, J=7.9 Hz, 1H, NHÀAla),
7.37–7.33 (m, 10H, 2”OBn), 6.74–6.65 (m, 1H, aHÀGly), 5.53 (br s,
1H, aOHÀGly), 5.16–5.14 (m, 2H, CH2ÀCO2Bn), 5.02 (s, 2H, CH2À
Cbz), 4.17–4.07 (m, 1H, aHÀAla), 1.19 ppm (app t, J=7.7 Hz, 3H,
CH3ÀAla); 13C NMR (75 MHz, [D6]DMSO) d=172.5 and 172.5 (CO-
Ala), 169.6 and 169.6 (CO-Gly), 155.6 (NHCONH), 137.0 and 137.0
(1C of OCH2Ph), 135.7 and 135.7 (1C of OCH2Ph), 128.4 and 128.4
(Ar-C), 128.2 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 127.8 and 127.8 (Ar-
C), 127.7 and 127.7 (Ar-C), 71.4 and 71.4 (aC-Gly), 66.1 and 66.1
(CH2-CO2Bn), 65.4 and 65.4 (CH2-Cbz), 49.9 and 49.8 (aC-Ala), 18.1
and 18.0 ppm (CH3-Ala); HRMS (ESI): m/z calcd for C20H22N2O6:
385.1405 [MÀH]À; found: 385.1402.
Et3N (0.45 mL, 3.234 mmol) was added to a stirred solution of TMP-
triethylammonium salt (344 mg, 0.809 mmol) and compound 12
(476 mg, 1.496 mmol) in dry DMF (6 mL). The reaction mixture was
heated at 408C for 24 h. It was allowed to cool to room tempera-
ture, the volatiles were removed in vacuo, and the resulting resi-
due was dissolved in water and lyophilized. The crude product was
purified by preparative RP-HPLC (98% H2O+2% CH3CN and 98%
CH3CN+2% H2O). The collected fractions were freeze-dried repeat-
edly until the mass remained constant to afford compound 13
(49.6 mg, 8%) as a minor product and compound 14 (270 mg,
40%) as the major product, both as white solids. Data for com-
3’-O-Benzyl-5’-O-(O-benzyl-N,N-diisopropyl phosphoramidi-
te)thymidine (9)
1
pound 13: H NMR (600 MHz, CD3OD) d=7.77 and 7.76 (2”s, 1H,
H-6), 7.36–7.26 (m, 10H, 2”OBn), 6.32–6.29 (m, 1H, H-1’), 6.06 and
6.01 (2”d, J=10.0 Hz, 1H, aH-Gly), 5.22–5.15 (m, 2H, CH2-CO2Bn),
5.09–5.03 (m, 2H, CH2-Cbz), 4.49–4.46 (m, 1H, H-3’), 4.19–4.16 (m,
1H, aH-Ala), 4.07–4.02 (m, 2H, H-5’ and H-5’’), 3.99–3.97 (m, 1H, H-
4’), 2.22–2.14 (m, 2H, H-2’ and H-2’’), 1.90 and 1.89 (2”s, 3H, CH3-
Thy), 1.30–1.27 ppm (m, 3H, CH3-Ala, merged with Et3N); 13C NMR
(125 MHz, CD3OD) d=175.1 and 175.0 (CO-Ala), 169.2 (CO-Gly),
166.6 and 166.5 (C-4), 158.2 and 158.1 (OCONH), 152.5 (C-2), 138.1
(C-6 and 1C OBn), 136.9 (1C OBn), 129.6 (Ar-C), 129.5 (Ar-C), 129.4
A
solution of benzyloxy-bis(N,N-diisopropylamino)phosphine
(1.68 mL, 0.92 mmol, 0.549m in anhydrous CH2Cl2) was added to
a stirred solution of compound 8 (300 mg, 0.903 mmol) in anhy-
drous CH2Cl2 (18 mL) followed by a solution of 1H-tetrazole
(2.04 mL, 0.92 mmol, 0.45m in anhydrous AcCN) at 08C. The reac-
tion mixture was stirred at room temperature for 4 h, and it was
then quenched with saturated aq. NaHCO3 and extracted with
CH2Cl2. The organic phase was dried over Na2SO4 and concentrated
in vacuo. The crude residue was purified by flash chromatography
on silica gel (Et3N/EtOAc/hexane, 0.2:20:79.8–0.2:40:59.8–
0.2:80:19.8). The fractions that contained the phosphoramidite
were combined and concentrated to afford compound 9 (497 mg,
72% over two steps) as a white solid. 1H NMR (300 MHz, CDCl3) d=
7.67 and 7.56 (2”s, 1H, H-6), 7.36–7.24 (m, 10H, 2”OBn), 6.43–
6.34 (m, 1H, H-1’), 4.80–4.63 (m, 2H, CH2PÀOBn), 4.58–4.34 (m, 2H,
H-5’ and H-5’’), 4.27 and 4.23 (2”s, 2H, CH2À3’-OBn), 3.79–3.78 (m,
1H, H-3’), 3,64–3.60 (m, 3H, H-4’ and 2”H-iPr), 2.54–2.38 (m, 1H,
H-2’), 2.01–1.95 (m, 1H, H-2’’), 1.86 and 1.80 (2”s, 3H, CH3ÀThy),
1.23–1.15 ppm (m, 12H, CH3, 4”iPr); 31P NMR (121 MHz, CDCl3) d=
148.6 and 148.1 ppm; HRMS (ESI+): m/z calcd for C30H40N3O6P:
570.2727 [M+H]+; found: 570.2732.
(Ar-C), 129.3 (Ar-C), 129.0 (Ar-C), 128.9 (Ar-C), 112.0 (C-5), 87.5 and
2
87.4 (d, 3JC,P =9.4 Hz, C-4’), 86.2 and 86.1 (C-1’), 75.3 (d, JC,P
=
12.2 Hz, aC-Gly), 72.9 and 72.8 (C-3’), 68.5 (CH2-CO2Bn), 67.7 (CH2-
2
Cbz), 66.6 and 66.5 (d, JC,P =5.4 Hz, C-5’), 51.8 (aC-Ala), 40.9 (C-2’),
18.4 and 18.2 (CH3-Ala), 12.6 ppm (CH3-Thy); 31P NMR (202 MHz,
CD3OD) d=À2.1 and À2.2 ppm; HRMS (ESI): m/z calcd for
C30H35N4O13P: 689.1865 [MÀH]À; found: 689.1879.
1
Data for compound 14: H NMR (500 MHz, CD3OD) d=7.93 (s, 1H,
H-6), 7.37–7.29 (m, 11 H, aH-Gly and 2”OBn), 6.34–6.30 (m, 1H, H-
1’), 5.28–5.08 (m, 2H, CH2-CO2Bn), 5.06–5.04 (m, 2H, CH2-Cbz), 4.50
(br s, 1H, H-3’), 4.26–4.19 (m, 1H, aH-Ala), 4.08–4.04 (m, 3H, H-4’,
H-5’ and H-5’’), 2.26–2.19 (m, 2H, H-2’ and H-2’’), 1.95 (s, 3H, CH3-
Thy), 1.34 and 1.32 ppm (2”d, J=7.1 Hz, 3H, CH3-Ala, merged with
Et3N); 13C NMR (125 MHz, CD3OD) d=175.4 and 175.3 (CO-Ala),
168.3 and 168.2 (CO-Gly), 164.3 (C-4), 158.3 and 158.2 (OCONH),
151.7 (C-2), 138.1 and 138.0 (1C OBn), 137.5 and 137.4 (C-6), 136.7
(1C OBn), 129.6 (Ar C), 129.5 (Ar C), 129.4 (Ar C), 129.2 (Ar C), 129.1
(Ar C), 129.0 (Ar C), 128.9 (Ar C), 128.8 (Ar C), 111.3 and 111.2 (C-5),
N-Carbobenzyloxy-l-alanyl-d,l-2-acetoxyglycine benzyl ester
(12)
Pyridine (21.6 mL) was added dropwise to a stirred suspension of
compound 5 (2.4 g, 6.21 mmol) in acetic anhydride (30 mL) at 08C.
Chem. Eur. J. 2016, 22, 8167 – 8180
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
3
87.9 and 87.8 (d, JC,P =8.8 Hz, C-4’), 87.1 and 87.0 (C-1’), 72.7 and
dried over Na2SO4, filtered, and concentrated in vacuo, and the re-
sultant crude residue was purified by column chromatography on
silica gel (EtOAc/hexane, 1:9–1:4–2:3, v/v) to give compound 16
(1.51 g, 71%) as a colorless foam. 1H NMR (300 MHz, CDCl3) d=
7.81–7.77 (2”d, J=7.1 Hz, 1H, NH-Gly), 7.34–7.30 (m, 10H, 2”
OBn), 5.79 and 5.78 (2”d, J=8.2 Hz, 1H, aH-Gly), 5.65 (d, J=
6.3 Hz, 1H, NH-Ala), 5.21 and 5.20 (2”s, 2H, CH2-CO2Bn), 5.08 and
5.07 (2”s, 2H, CH2-Cbz), 4.43–4.38 (m, 1H, aH-Ala), 1.36 ppm (app
t, J=6.3 Hz, 3H, CH3-Ala); 13C NMR (75 MHz, CDCl3) d=173.3 (CO-
Ala), 166.4 (CO-Gly), 156.1 (OCONH), 136.2 (1C of OCH2Ph), 134.4
(1C of OCH2Ph), 128.9 (Ar-C), 128.7 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C),
128.3 (Ar-C), 128.1 (Ar-C), 68.5 (aC-Gly), 67.3 (CH2-CO2Bn), 64.7 and
64.6 (CH2-Cbz), 50.4 (aC-Ala), 18.6, 18.3 ppm (CH3-Ala); HRMS (ESI+
): m/z calcd for C20H21N5O5 : 434.1435 [M+Na]+; found: 434.1432.
72.6 (C-3’), 68.9 (CH2-CO2Bn), 67.8 and 67.7 (CH2-Cbz), 65.9 (d,
2JC,P =4.8 Hz, C-5’), 58.2 and 58.1 (aC-Gly), 52.0 and 51.6 (aC-Ala),
41.1 and 41.0 (C-2’), 18.0 and 17.9 (CH3-Ala), 13.0 ppm (CH3-Thy);
31P NMR (202 MHz, CD3OD) d=0.7 ppm; HRMS (ESI): m/z calcd for
C30H35N4O13P : 689.1865 [MÀH]À; found: 689.1862.
5’-O-(l-Alanyl-d,l-2-aminoglycine)-TMP (1)
10% Pd/C, Degussa-type (6 mg, 0.2 equiv w/w) was added to
a stirred solution of compound 13 (30 mg, 0.0434 mmol, 1 equiv)
in EtOH/H2O (5:1, 5 mL), and evacuation was carried out with hy-
drogen atmosphere replacements (3”). The reaction mixture was
stirred at room temperature for 4 h under an atmospheric pressure
of hydrogen. Upon completion, the catalyst was removed by filtra-
tion through a cellulose filter (0.45 mm), and the ethanol was re-
moved in vacuo (bath temp. ꢀ108C). The residue was lyophilized
to obtain a crude product that was purified by preparative RP-
HPLC (98% H2O+2% CH3CN and 98% CH3CN+2% H2O). The col-
lected fractions were freeze-dried repeatedly until the mass re-
mained constant to afford compound 1 as a white solid (8.5 mg,
42%). 1H NMR (300 MHz, D2O) d=7.73 and 7.70 (2”s, 1H, H-6),
6.37–6.30 (m, 1H, H-1’), 5.73 (d, J=9.0 Hz, 1H, aH-Gly), 4.60–4.54
(m, 1H, H-3’), 4.18–4.02 (m, 4H, H-4’, aH-Ala, H-5’ and H-5’’), 2.37–
2.33 (m, 2H, H-2’ and H-2’’), 1.91 (s, 3H, CH3-Thy), 1.53 (d, J=
7.1 Hz, 3H, CH3-Ala); 13C NMR (125 MHz, D2O) d=172.8 (CO-Gly),
169.9 and 169.8 (CO-Ala), 166.4 (C-4), 151.5 (C-2), 137.1 and 137.0
(C-6), 111.5 and 111.5 (C-5), 85.1 and 85.0 (d, 3JC,P =9.2 Hz, C-4’),
84.7 and 84.6 (C-1’), 74.8 (C-3’), 70.9 and 70.8 (aC-Gly), 64.9 and
N-Carbobenzyloxy-l-alanyl-d,l-2-aminoglycine benzyl ester
(17)
Triphenylphospine (0.92 g, 3.50 mmol) was added to a stirred solu-
tion of compound 16 (1.2 g, 2.92 mmol) and water (0.26 mL,
14.6 mmol) in THF (15 mL). The reaction mixture was heated at
reflux for 2 h, cooled to room temperature, and concentrated in
vacuo. Water (100 mL) was added to the residue, and the solution
was extracted with CH2Cl2 (3”100 mL). The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo. The resul-
tant crude residue was purified by column chromatography on
silica gel (MeOH/CH2Cl2, 1:99–1:49–1:19, v/v) to give compound 17
(0.79 g, 70%) as a colorless foam and triphenylphosphine oxide as
1
a minor impurity. H NMR (300 MHz, CDCl3) d=7.77 and 7.73 (2”d,
2
64.8 (d, JC,P =5.2 Hz, C-5’), 48.8 and 48.7 (aC-Ala), 38.4 and 38.3 (C-
J=8.3 Hz, 1H, NH-Gly), 7.31–7.29 (m, 10H, 2”OBn), 5.74 (app t, J=
9.0 Hz, 1H, NH-Ala), 5.17–5.07 (m, 3H, aH-Gly and CH2-CO2Bn), 5.06
and 5.04 (2”s, 2H, CH2-Cbz), 4.30–4.20 (m, 1H, aH-Ala), 2.84 (br s,
2H, aNH2-Gly), 1.31 and 1.30 ppm (2”d, J=7.0 Hz, 3H, CH3-Ala);
13C NMR (75 MHz, CDCl3) d=172.7 (CO-Ala), 170.5 (CO-Gly), 156.0
(OCONH), 136.3 (1C of OCH2Ph), 135.1 (1C of OCH2Ph), 128.6 (Ar-C),
128.5 (Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 67.6 (aC-Gly),
67.0 (CH2-CO2Bn), 60.3 and 60.2 (CH2-Cbz), 50.5 (aC-Ala), 18.5 ppm
(CH3-Ala); HRMS (ESI+): m/z calcd for C20H23N3O5: 408.1530
[M+Na]+; found: 408.1523.
2’), 15.9 and 15.8 (CH3-Ala), 11.4 ppm (CH3-Thy); 31P NMR (121 MHz,
D2O) d=À2.0 ppm; HRMS (ESI): m/z calcd for C15H23N4O11P:
465.1028 [MÀH]À; found: 465.1029.
3-N-(l-Alanyl-d,l-2-aminoglycine)-TMP triethylammonium
salt (15)
Following a similar procedure as the one used for the synthesis of
compound 1, compound 15 was obtained as a white solid
(179.1 mg, 92%) starting from 14 (260 mg, 0.343 mmol), 10% Pd/C,
Degussa-type (52 mg, 0.2 eq w/w) in a 5:1 EtOH:H2O mixture
Thymidine-5’-O-(N-carbobenzyloxy-l-alanyl-2-aminoglycine
benzyl ester) phosphoramidates (18a) and (18b)
1
(15 mL). H NMR (500 MHz, D2O) d=7.76 and 7.74 (2”s, 1H, H-6),
6.90 and 6.86 (2”s, 1H, aH-Gly), 6.32 (app t, J=6.8 Hz, 1H, H-1’),
4.55–4.52 (m, 1H, H-3’), 4.21–4.16 (m, 1H, aH-Ala), 4.15–4.14 (m,
1H, H-4’), 4.11–4.01 (m, 2H, H-5’ and H-5’’), 2.36–2.33 (m, 2H, H-2’
and H-2’’), 1.91 (s, 3H, CH3-Thy), 1.58 and 1.41 ppm (2”d, J=
7.1 Hz, 3H, CH3-Ala); 13C NMR (150 MHz, D2O) d=171.0 and 170.9
(CO-Gly), 170.5 and 170.4 (CO-Ala), 164.1 (C-4), 150.7 (C-2), 135.9
Et3N (0.1 mL, 0.737 mmol) was added to a stirring suspension of
TMP-triehtylammonium salt (400 mg, 0.737 mmol) and compound
17 (640 mg, 1.659 mmol) in tBuOH/H2O (4:1, 15 mL) to facilitate
dissolution, followed by DCC (532 mg, 2.579 mmol). The reaction
mixture was heated at 858C for 2.5 h, and the reaction progress
was monitored by TLC (CH2Cl2/MeOH/H2O, 17:7:1). Upon comple-
tion, the mixture was cooled to room temperature and concentrat-
ed in vacuo. The resulting residue was resuspended in water and
lyophilized to give a crude material, which was purified by column
chromatography on silica gel (CH2Cl2/MeOH/H2O 19:1:0–9:1:0–
17:7:1) to provide the desired nucleoside phosphoramidates as
salts. Semi-preparative RP-HPLC (98% H2O+2% CH3CN and 98%
CH3CN+2% H2O) was employed for further separation to obtain
the pure diastereoisomers in a ꢀ1:1 ratio as white powders
(131 mg of compound 18a, 129 mg of compound 18b, overall
yield 18a+18b: 260 mg, 51%). The isolated products were freeze-
dried repeatedly until the mass remained constant. Data for com-
3
(C-6), 110.6 (C-5), 85.6 (C-1’), 85.3 (d, JC,P =9.3 Hz, C-4’), 70.7 (C-3’),
64.4 (C-5’), 58.3 and 58.2 (aC-Gly), 48.7 and 48.6 (aC-Ala), 38.6 (C-
2’), 16.1 and 16.0 (CH3-Ala), 11.9 ppm (CH3-Thy); 31P NMR (202 MHz,
D2O) d=0.04 ppm; HRMS (ESI): m/z calcd for C15H23N4O11P
465.1028 [MÀH]À; found: 465.1025.
:
N-Carbobenzyloxy-l-alanyl-d,l-2-azidoglycine benzyl ester
(16)
DPPA (1.56 mL, 7.26 mmol) was added dropwise to a stirred sus-
pension of compound 5 (2.0 g, 5.18 mmol) and DBU (1.08 mL,
7.26 mmol) in dry toluene (50 mL) at 08C. The reaction mixture
was slowly warmed to room temperature and left stirring for 16 h.
The resulting mixture was concentrated in vacuo (bath temp.
ꢀ158C). The residue was diluted with EtOAc (150 mL) and washed
with brine (50 mL) and 5% HCl (2”50 mL). The organic phase was
1
pound 18a: H NMR (500 MHz, CD3CN+D2O, 508C) d=7.55 (s, 1H,
H-6), 7.32–7.28 (m, 10H, 2”OBn), 6.14 (app t, J=6.8 Hz, 1H, H-1’),
5.21 and 5.16 (2”d, J=10.9 Hz, 1H, aH-Gly), 5.14–4.99 (m, 4H,
CH2-CO2Bn and CH2-Cbz), 4.38–4.36 (m, 1H, H-3’), 4.11–4.06 (m, 1H,
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3
aH-Ala), 3.93–3.91 (m, 1H, H-4’), 3.87–3.82 (m, 2H, H-5’ and H-5’’),
2.17–2.13 (m, 2H, H-2’ and H-2’’), 1.80 and 1.79 (2”s, 3H, CH3-Thy),
1.20 ppm (app t, J=7.0 Hz, 3H, CH3-Ala); 13C NMR (150 MHz,
CD3CN+D2O, 108C) d=174.6 (CO-Ala), 171.3 (CO-Gly), 166.3 (C-4),
157.5 (OCONH), 151.5 (C-2), 137.9 (C-6), 137.5 (1C of OCH2Ph),
136.2 (1C of OCH2Ph), 129.5 (Ar-C), 129.4 (Ar-C), 129.2 (Ar-C), 129.0
(150 MHz, D2O) d=172.2 (d, JC,P =9.2 Hz, CO-Gly), 169.9 (CO-Ala),
3
166.4 (C-4), 151.6 (C-2), 137.3 (C-6), 111.5 (C-5), 85.3 (d, JC,P
=
2
8.4 Hz, C-4’), 84.9 (C-1’), 71.0 (C-3’), 64.3 (d, JC,P =4.7 Hz, C-5’), 61.1
(d, 2JC,P =9.1 Hz, aC-Gly), 48.7 (aC-Ala), 38.5 (C-2’), 16.0 (CH3-Ala),
11.5 ppm (CH3-Thy); 31P NMR (121 MHz, D2O) d=4.2 ppm; HRMS
(ESI): m/z calcd for C15H24N5O10P: 464.1188 [MÀH]À; found:
464.1187.
3
(Ar-C), 128.7 (Ar-C), 111.9 (C-5), 86.4 (d, JC,P =8.9 Hz, C-4’), 84.8 (C-
2
1’), 71.7 (C-3’), 68.3 (CH2-CO2Bn), 67.4 (CH2-Cbz), 64.9 (d, JC,P
=
2
3.9 Hz, C-5’), 61.1 (d, JC,P =9.8 Hz, aC-Gly), 51.1 (aC-Ala), 39.8 (C-2’),
18.1 (CH3-Ala), 12.6 ppm (CH3-Thy); 31P NMR (202 MHz,
CD3CN+D2O, 508C) d=4.4 ppm; [a]25809 =À0.1478 (c=1 in CH3OH);
HRMS (ESI): m/z calcd for C30H36N5O12P: 688.2025 [MÀH]À; found:
688.2040.
N-Boc-l-Alanyl-d,l-2-acetoxyglycine methyl ester (19)
Molecular sieves (4 Š, 5 g) and Pb(OAc)4 (17.89 g, 40.30 mmol)
were added to a stirring solution of Boc-l-Ala-l-Ser-OMe (3.9 g,
13.43 mmol) in dry ethyl acetate (220 mL)under an inert atmos-
phere. The reaction mixture was heated at reflux for 2 h and
cooled to room temperature. The solid was removed by filtration
through a pad of Celite, and the organic phase was stirred with
10% aq. citric acid until it became nearly colourless. The organic
phase was separated, washed with 10% aq. citric acid, water, and
brine, dried over Na2SO4, filtered, and concentrated in vacuo to
give a crude diastereoisomeric mixture of compound 19 (4.27 g,
quant.) as a colourless foam, which was used in the next step with-
Data for compound 18b: 1H NMR (300 MHz, CD3CN+D2O, 258C)
d=7.54 (s, 1H, H-6), 7.32–7.27 (m, 10H, 2”OBn), 6.14 (app t, J=
6.8 Hz, 1H, H-1’), 5.21 and 5.16 (2”d, J=10.9 Hz, 1H, aH-Gly),
5.11–4.93 (m, 4H, CH2-CO2Bn and CH2-Cbz), 4.37–4.32 (m, 1H, H-3’),
4.11–4.01 (m, 1H, aH-Ala), 3.95–3.90 (m, 1H, H-4’), 3.89–3.85 (m,
2H, H-5’ and H-5’’), 2.17–2.11 (m, 2H, H-2’ and H-2’’), 1.79 and 1.77
(2”s, 3H, CH3-Thy), 1.19 ppm (app t, J=7.0 Hz, 3H, CH3-Ala);
13C NMR (150 MHz, CD3CN+D2O, 108C) d=174.7 (CO-Ala), 171.3
(CO-Gly), 166.3 (C-4), 157.5 (OCONH), 152.0 (C-2), 137.9 (C-6), 137.5
(1C of OCH2Ph), 136.3 (1C of OCH2Ph), 129.5 (Ar-C), 129.4 (Ar-C),
129.0 (Ar-C), 128.7 (Ar-C), 112.0 (C-5), 86.3 (d, 3JC,P =8.7 Hz, C-4’),
85.5 (C-1’), 71.6 (C-3’), 68.4 (CH2-CO2Bn), 67.4 (CH2-Cbz), 64.9 (d,
1
out any further purification. H NMR (300 MHz, CDCl3) d=7.70–7.52
(m, 1H, NH-Gly), 6.40 and 6.38 (2”d, J=9.1 Hz, 1H, aH-Gly), 5.31
(app t, J=6.9 Hz, 1H, NH-Ala), 4.27–4.13 (m, 1H, aH-Ala), 3.81 and
3.80 (2”s, 3H, OCH3-Gly), 2.11 (s, 3H, CH3-OAc), 1.45 (s, 9H, tBu),
1.38 and 1.37 ppm (2”d, J=7.1 Hz, 3H, CH3-Ala); 13C NMR (75 MHz,
CDCl3) d=172.8 (CO-Ala), 170.3 and 170.2 (CO-Gly), 167.2 and
167.1 (CO-OAc), 155.6 (OCONH), 88.7 (1C-tBu), 72.3 and 72.2 (aC-
Gly), 53.4 and 53.3 (OCH3-Gly), 50.3 and 50.1 (aC-Ala), 28.4 (tBu),
20.7 (CH3-OAc), 17.8 and 17.6 ppm (CH3-Ala); HRMS (ESI+): m/z
calcd for C13H22N2O7: 341.1319 [M+Na]+; found: 341.1318.
2
2JC,P =5.1 Hz, C-5’), 61.1 (d, JC,P =9.1 Hz, aC-Gly), 51.1 (aC-Ala), 39.7
(C-2’), 18.1 (CH3-Ala), 12.6 ppm (CH3-Thy); 31P NMR (121 MHz,
CD3CN+D2O, 258C) d=3.2 ppm; [a]25809 =À0.0528 (c=1 in CH3OH);
HRMS (ESI): m/z calcd for C30H36N5O12P: 688.2025 [MÀH]À; found:
688.2019.
Thymidine-5’-O-(l-alanyl-2-aminoglycine) phosphoramidate
(2a)
N-Boc-l-Alanyl-d,l-2-p-anisylaminoglycine methyl ester (20)
DIPEA (3.01 mL, 17.28 mmol) was added to a stirring solution of
compound 19 (2.2 g, 6.91 mmol) and 4-methoxybenzylamine
(1.0 mL, 7.60 mmol) in dry DMF (20 mL) at room temperature. The
reaction mixture was heated at 408C for 24 h and then concentrat-
ed in vacuo. Water (200 mL) was added to the residue and the or-
ganics were extracted with ethyl acetate (3”120 mL). The com-
bined organic phases were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude material
was purified by column chromatography on silica gel (EtOAc/
hexane, 2:3–3:2–4:1) to give a diastereoisomeric mixture of com-
pound 20 (2.07 g, 76%) as a colorless foam. 1H NMR (300 MHz,
CDCl3) d=7.22 (d, J=8.5 Hz, 1H, oH-PMB), 7.04–6.93 (m, 1H, NH-
Gly), 6.84 (d, J=8.5 Hz, 1H, mH-PMB), 5.27 (d, J=7.8 Hz, 1H, aH-
Gly), 5.08–4.99 (m, 1H, NH-Ala), 4.20–4.18 (m, 1H, aH-Ala), 3.78 (s,
3H, OCH3-PMB), 3.73–3.70 (m, 5H, OCH3-Gly and CH2-PMB), 2.37 (br
s, 1H, NH-PMB), 1.46 and 1.45 (2”s, 9H, tBu), 1.37 ppm (app t, J=
7.0 Hz, 3H, CH3-Ala); 13C NMR (75 MHz, CDCl3) d=173.1 (CO-Ala),
170.6 and 170.5 (CO-Gly), 159.0 (Ar-C), 155.6 (OCONH), 131.3 and
131.2 (1C of PMB), 129.7 (Ar-C), 129.3 (Ar-C), 114.3 (Ar-C), 113.9 (Ar-
C), 80.4 (1C-tBu), 64.4 and 64.3 (aC-Gly), 55.4 (OCH3-PMB), 52.9
(OCH3-Gly), 50.3 (aC-Ala), 48.5 (CH2-PMB), 28.4 (tBu), 18.3 and
18.1 ppm (CH3-Ala); HRMS (ESI): m/z calcd for C19H29N3O6: 394.1983
[MÀH]À; found: 394.1973.
Following a similar procedure as the one used for the synthesis of
compound 1, compound 2a was obtained as a white solid
(45.5 mg, 54%) starting from 18a (125 mg, 0.136 mmol, 1 equiv),
10% Pd/C, Degussa-type (25 mg, 0.2 eq w/w) in EtOH/H2O (5:1,
10 mL). 1H NMR (300 MHz, D2O) d=7.63 (d, J=1.1 Hz, 1H, H-6),
6.14 (app t, J=6.8 Hz, 1H, H-1’), 5.23 (d, J=11.3 Hz, 1H, aH-Gly),
4.50–4.45 (m, 1H, H-3’), 4.06–4.02 (m, 1H, H-4’), 4.00–3.88 (m, 3H,
aH-Ala, H-5’ and H-5’’), 2.31–2.26 (m, 2H, H-2’ and H-2’’), 1.82 (d,
J=1.1 Hz, 3H, CH3-Thy), 1.44 ppm (d, J=7.1 Hz, 3H, CH3-Ala);
13C NMR (150 MHz, D2O) d=172.0 (d, 3JC,P =9.1 Hz, CO-Gly), 169.9
(CO-Ala), 166.3 (C-4), 151.5 (C-2), 137.3 (C-6), 111.5 (C-5), 85.2 (d,
2
3JC,P =9.0 Hz, C-4’), 84.8 (C-1’), 70.8 (C-3’), 64.2 (d, JC,P =4.6 Hz, C-5’),
2
61.1 (d, JC,P =6.5 Hz, aC-Gly), 48.7 (aC-Ala), 38.4 (C-2’), 16.0 (CH3-
Ala), 11.5 ppm (CH3-Thy); 31P NMR (121 MHz, D2O) d=4.4 ppm;
HRMS (ESI): m/z calcd for C15H24N5O10P: 464.1188 [MÀH]À; found:
464.1185.
Thymidine-5’-O-(l-alanyl-2-aminoglycine) phosphoramidate
(2b)
Following a similar procedure as the one used for the synthesis of
1, compound 2b was obtained as a white solid (43.0 mg, 51%)
starting from compound 18b (125 mg, 0.136 mmol, 1 equiv) and
10% Pd/C, Degussa-type (25 mg, 0.2 eq w/w) in EtOH/H2O (5:1,
10 mL). 1H NMR (300 MHz, D2O) d=7.68 (s, 1H, H-6), 6.25 (app t,
J=6.9 Hz, 1H, H-1’), 5.24 and 5.19 (2”d, J=11.0 Hz, 1H, aH-Gly),
4.52–4.47 (m, 1H, H-3’), 4.11–4.07 (m, 1H, H-4’), 4.06–3.90 (m, 3H,
aH-Ala, H-5’ and H-5’’), 2.34–2.29 (m, 2H, H-2’ and H-2’’), 1.86 (s,
3H, CH3-Thy), 1.47 and 1.46 (2”d, J=7.0 Hz, 3H, CH3-Ala); 13C NMR
N-Boc-l-Alanyl-d,l-2-aminoglycine methyl ester (21)
10% Pd/C (0.18 g, 0.1 equiv w/w) was added to a stirring solution
of compound 20 (1.8 g, 2.44 mmol) in EtOH (80 mL), and the mix-
ture was hydrogenated at atmospheric pressure by using a balloon
filled with H2 for 5 h at room temperature. The catalyst was re-
Chem. Eur. J. 2016, 22, 8167 – 8180
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moved by filtration through a pad of Celite, and the filtrate was
concentrated in vacuo. The resulting crude residue was purified by
column chromatography on silica gel (MeOH/CH2Cl2, 1:99–1:49–
1:24) to give a diastereoisomeric mixture of compound 21 (1.2 g,
Thy); 31P NMR (202 MHz, D2O) d=À0.1 ppm; HRMS (ESI): m/z calcd
for C16H26N5O10P: 478.1344 [MÀH]À; found: 478.1346.
N-Carbobenzyloxy-l-alanyl-d,l-2-mercaptoethanolglycine
benzyl ester (24)
1
96%) as a colorless foam. H NMR (300 MHz, CDCl3) d=7.41 (br s,
1H, NH-Gly), 5.28–5.24 (m, 2H, aH-Gly and NH-Ala), 4.20–4.18 (m,
1H, aH-Ala), 3.79 (s, 3H, OCH3-Gly), 2.24 (br s, 1H, aNH2-Gly), 1.45
(s, 9H, tBu), 1.36 ppm (d, J=7.1 Hz, 3H, CH3-Ala); 13C NMR (75 MHz,
CDCl3) d=173.1 and 173.0 (CO-Ala), 171.2 and 171.1 (CO-Gly),
155.5 (OCONH), 80.3 (1C-tBu), 60.2 and 60.1 (aC-Gly), 53.0 (OCH3-
Gly), 50.1 (aC-Ala), 28.3 (tBu), 18.3 and 18.2 ppm (CH3-Ala); HRMS
(ESI+): m/z calcd for C19H29N3O6: 298.1377 [M+Na]+; found:
298.1390.
Et3N (0.55 mL, 3.92 mmol) was added dropwise to a stirring solu-
tion of compound 12 (1.4 g, 3.27 mmol) and 2-mercaptoethanol
(0.25 mL, 3.59 mmol) in dry DMF (12 mL) at 08C. The reaction mix-
ture was slowly warmed to room temperature and left stirring for
16 h. The resulting mixture was concentrated in vacuo. Water
(100 mL) was added to the residue and the mixture was extracted
with ethyl acetate (3”100 mL). The combined organic phases were
washed with brine, dried over Na2SO4, filtered, and concentrated.
The resultant crude material was purified by column chromatogra-
phy on silica gel (EtOAc/hexane, 1:4–2:3–3:2) to give a diastereoiso-
meric mixture of compound 24 (1.2 g, 70%) as a colorless foam.
1H NMR (300 MHz, [D6]DMSO) d=8.83 and 8.79 (2”d, J=8.2 Hz,
1H, NH-Gly), 7.47 (d, J=7.3 Hz, 1H, NH-Ala), 7.39–7.32 (m, 10H, 2”
OBn), 5.46 and 5.44 (2”d, J=10.0 Hz, 1H, aH-Gly), 5.19–5.16 (m,
2H, CH2-CO2Bn), 5.00 (s, 2H, CH2-Cbz), 4.96–4.93 (m, 1H, OH), 4.19–
4.12 (m, 1H, aH-Ala), 3.54–3.48 (m, 2H, OCH2CH2S), 2.72–2.65 (m,
2H, OCH2CH2S), 1.19 ppm (app t, J=7.8 Hz, 3H, CH3-Ala); 13C NMR
(75 MHz, [D6]DMSO) d=172.7 and 172.6 (CO-Ala), 168.5 and 168.4
(CO-Gly), 155.7 and 155.6 (OCONH), 137.0 (1C of OCH2Ph), 135.6
and 135.5 (1C of OCH2Ph), 128.5 (Ar-C), 128.4 (Ar-C), 128.2 (Ar-C),
127.9 (Ar-C), 127.7 (Ar-C), 66.8 and 66.7 (CH2-CO2Bn), 65.5 (CH2-
Cbz), 60.4 and 60.3 (OCH2CH2S), 53.2 and 53.1 (aC-Gly), 49.9 and
49.8 (aC-Ala), 32.7 and 32.6 (OCH2CH2S), 18.1 and 18.0 ppm (CH3-
Ala); HRMS (ESI): m/z calcd for C22H26N2O6S: 445.1439 [MÀH]À;
found: 445.1443.
Thymidine-5’-O-(N-Boc-l-alanyl-d,l-2-aminoglycine methyl
ester) phosphoramidate triethylammonium salt (22)
Following a similar procedure as the one used for the synthesis of
compounds 18a,b, the triethylammonium salt of compound 22
was obtained as a diastereoisomeric mixture (538 mg, 74%, white
solid) starting from TMP-triehtylammonium salt (560 mg,
1.07 mmol), compound 21 (647 mg, 2.35 mmol), triethylamine
(163 mL, 1.17 mmol), and DCC (881 mg, 4.27 mmol) in tBuOH/H2O
1
(4:1, 15 mL). H NMR (500 MHz, CD3OD) d=7.68 (d, J=1.1 Hz, 1H,
H-6), 6.35–6.31 (m, 1H, H-1’), 5.30 and 5.22 (2”d, J=11.1 Hz, 1H,
aH-Gly) 4.48–4.45 (m, 1H, H-3’), 4.10–4.05 (m, 1H, aH-Ala), 4.02–
4.00 (m, 1H, H-4’), 3.99–3.95 (m, 2H, H-5’ and H-5’’), 3.71 and 3.69
(2”s, 3H, OCH3-Gly), 2.29–2.17 (m, 2H, H-2’ and H-2’’), 1.94 (d, J=
1.1 Hz, 3H, CH3-Thy), 1.44 and 1.43 (2”s, 9H, tBu), 1.29 ppm (app t,
J=7.1 Hz, 3H, CH3-Ala, merged with Et3N); 13C NMR (125 MHz,
CD3OD) d=175.2 (CO-Ala), 172.1 and 172.0 (CO-Gly), 166.5 (C-4),
157.5 (OCONH), 152.5 (C-2), 138.3 (C-6), 111.9 (C-5), 87.6 (app t,
3JC,P =9.5 Hz, C-4’), 86.2 and 86.0 (C-1’), 80.6 (1C-tBu), 73.0 and 72.9
3’-O-Benzyl-5’-O-(N-carbobenzyloxy-l-alanyl-d,l-2-mercap-
toethanolglycine benzyl ester) TMP triethylammonium salt
(25)
2
(C-3’), 65.7 (app t, JC,P =6.9 Hz, C-5’), 61.7 (aC-Gly), 53.1 and 53.0
(OCH3-Gly), 51.3 (aC-Ala), 40.8 (C-2’), 28.7 (tBu), 18.4 (CH3-Ala),
12.7 ppm (CH3-Thy); 31P NMR (202 MHz, CD3OD) d=3.4 ppm; HRMS
(ESI): m/z calcd for C21H34N5O12P: 578.1869 [MÀH]À; found:
578.1873.
POCl3 (169 mL, 3.92 mmol) was added dropwise to a stirring solu-
tion of compound 8 (0.5 g, 1.50 mmol) in dry trimethyl phosphate
(5 mL) at À208C. The reaction mixture was slowly warmed to room
temperature and was left stirring for 8 h until reaction completion
(monitored by TLC). The solution was cooled to 08C, and a solution
of compound 24 (0.672 g, 1.50 mmol) and N-methyl imidazole
(0.36 mL, 4.51 mmol) in dry CH2Cl2 (14 mL) was added. The result-
ing mixture was stirred at room temperature for 16 h. It was then
quenched with water at 08C, the volatiles were removed in vacuo,
water was added to the residue, and the mixture was lyophilized
(3”). The resultant crude residue was purified by column chroma-
tography on silica gel (MeOH/CH2Cl2, 1:99–3:97–8:92) and by prep-
arative RP-HPLC (25 mmol TEAB in 98% H2O+2% CH3CN and
25 mmol TEAB in 98% CH3CN+2% H2O). The collected fractions
were freeze-dried repeatedly until the mass remained constant to
obtain a diastereoisomeric mixture of compound 25 (935 mg,
Thymidine-5’-(l-alanyl-d,l-2-aminoglycine methyl ester)
phosphoramidate TFA salt (23)
TFA (1.5 mL) was added to a stirring solution of compound 22
(220 mg, 0.323 mmol) and thioanisole (42 mL, 0.356 mmol) in
CH2Cl2 (4.5 mL) at 08C. The reaction mixture was stirred at 08C for
1 h and then at room temperature for 3 h. After reaction comple-
tion, the volatiles were removed in vacuo and coevaporated three
times with toluene to remove residual TFA. The residue was redis-
solved in H2O and lyophilized (2”) to obtain a crude residue,
which was purified by preparative RP-HPLC (98% H2O+2% CH3CN
and 98% CH3CN+2% H2O). The collected fractions were freeze-
dried repeatedly until the mass remained constant to obtain a dia-
stereoisomeric mixture of compound 23 (158 mg, 85%, white
1
66%) as a white solid. H NMR (300 MHz, CD3OD) d=7.76 (s, 1H,
H-6), 7.35–7.25 (m, 15H, 3”OBn), 6.45 (dd, J=8.7, 5.8 Hz, 1H, H-1’),
5.51 (d, J=13.4 Hz, 1H, aH-Gly), 5.24–5.13 (m, 2H, CH2-CO2Bn), 5.06
(s, 2H, CH2-Cbz), 4.57–4.55 (m, 2H, CH2-3’OBn), 4.34–4.31 (m, 1H,
aH-Ala), 4.25–4.18 (m, 1H, H-3’ and H-4’), 4.07–4.01 (m, 2H, H-5’
and H-5’’), 4.00–3.90 (m, 2H, OCH2CH2S), 2.93–2.78 (m, 2H,
OCH2CH2S), 2.40–2.17 (m, 2H, H-2’ and H-2’’), 1.91 (s, 3H, CH3-Thy),
1.34 and 1.32 ppm (2”d, J=7.2 Hz, 3H, CH3-Ala); 13C NMR (75 MHz,
CD3OD) d=175.2 (CO-Ala), 169.7 and 169.6 (CO-Gly), 166.4 and
166.3 (C-4), 158.1 (OCONH), 152.5 and 152.4 (C-2), 139.4 (1C Ar-C),
138.2 (1C Ar-C), 137.9 (C-6), 136.8 (1C Ar-C), 129.6 (Ar-C), 129.5 (Ar-
C), 129.4 (Ar-C), 129.3 (Ar-C), 129.2 (Ar-C), 129.0 (Ar-C), 128.9 (Ar-C),
1
solid). H NMR (600 MHz, D2O) d=7.72 (s, 1H, H-6), 6.29 (app t, J=
7.0 Hz, 1H, H-1’), 5.64 (d, J=9.5 Hz, 1H, aH-Gly), 4.54–4.52 (m, 1H,
H-3’), 4.18–4.15 (m, 1H, aH-Ala), 4.14–4.12 (m, 1H, H-4’), 4.07–4.03
(m, 2H, H-5’ and H-5’’), 3.84 (s, 3H, OCH3-Gly), 2.32–2.30 (m, 2H, H-
2’ and H-2’’), 1.86 (s, 3H, CH3-Thy), 1.53 and 1.52 ppm (2”d, J=
7.2 Hz, 3H, CH3-Ala); 13C NMR (150 MHz, D2O) d=171.6 and 171.4
(CO-Ala), 166.3 (CO-Gly), 166.2 (C-4), 151.5 (C-2), 137.1 (C-6), 111.4
3
(C-5), 85.2 (app t, JC,P =9.0 Hz, C-4’), 84.8 (C-1’), 70.9 (C-3’), 64.7 (d,
2JC,P =4.5 Hz, C-5’), 56.8 (d, 2JC,P =7.5 Hz, aC-Gly), 54.1 (OCH3-Gly),
48.5 (aC-Ala), 38.6 (C-2’), 15.9 and 15.8 (CH3-Ala), 11.4 ppm (CH3-
Chem. Eur. J. 2016, 22, 8167 – 8180
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128.8 (Ar-C), 128.7 (Ar-C), 112.1 and 112.0 (C-5), 86.2 and 86.1 (C-1’),
5’-O-(l-Alanyl-d,l-2-mercaptoethanolglycine) TMP (4)
3
85.1 (d, JC,P =8.7 Hz, C-4’), 81.1 and 81.0 (C-3’), 72.2 and 72.1 (CH2-
Following a similar procedure as the one used for the synthesis of
compound 23, the TFA salt of the crude diastereoisomeric amine
30 was obtained as a sticky mass (ꢀ100 mg, quant.) starting from
compound 29 (100 mg, 0.134 mmol), thioanisole (16 mL,
0.134 mmol), and TFA (0.5 mL) in CH2Cl2 (1.5 mL). HRMS (ESI): calcd
for C25H33N4O12PS [M-H]À calcd.: 643.1480, found: 643.1472. The ob-
tained residue 30 was treated, without further purification, with
LiOH (19.4 mg, 0.809 mmol) in MeOH/H2O (1:1, 2 mL) at 08C, and
the solution was stirred at room temperature for 3 h. After reaction
completion, the mixture was neutralized with 5% acetic acid and
concentrated in vacuo. The crude residue was purified by prepara-
tive RP-HPLC (98% H2O+2% CH3CN and 98% CH3CN+2% H2O).
The collected fractions were freeze-dried repeatedly until the mass
remained constant to afford a diastereoisomeric mixture of com-
pound 4 (51 mg, 72% over two steps) as a white solid. 1H NMR
(500 MHz, D2O) d=7.60 (s, 1H, H-6), 6.39 (app t, J=6.9 Hz, 1H, H-
1’), 5.22 (d, J=20.5 Hz, 1H, aH-Gly), 4.56–4.53 (m, 1H, H-3’), 4.12–
4.10 (m, 1H, H-4’), 4.07–4.03 (m, 2H, H-5’ and H-5’’), 4.00–3.94 (m,
2H, OCH2CH2S), 3.51–3.46 (m, 1H, aH-Ala), 2.83–2.79 (m, 2H,
OCH2CH2S), 2.31–2.26 (m, 2H, H-2’ and H-2’’), 1.85 (s, 3H, CH3-Thy),
1.21 and 1.19 (2”d, J=7.0 Hz, 3H, CH3-Ala); 13C NMR (125 MHz,
D2O) d=177.0 (CO-Gly), 173.0 (CO-Ala), 165.5 (C-4), 156.7 (C-2),
2
3’OBn), 68.6 and 68.5 (CH2-CO2Bn), 67.6 (CH2-Cbz), 66.9 (d, JC,P
=
5.5 Hz, C-5’), 65.8 and 65.7 (d, 2JC,P =5.4 Hz, OCH2CH2S), 54.8 and
54.7 (aC-Gly), 52.0 and 51.9 (aC-Ala), 38.1 (C-2’), 32.1 and 32.0
(OCH2CH2S), 18.3 and 18.2 (CH3-Ala), 12.7 ppm (s, CH3-Thy);
31P NMR (121 MHz, CD3OD) d=À0.5 ppm; HRMS (ESI): m/z calcd
for C39H45N4O13PS: 839.2368 [MÀH]À; found: 839.2376.
N-Boc-l-Alanyl-d,l-2-mercaptoethanolglycine methyl ester
(26)
Following a similar procedure as the one used for the synthesis of
compound 24, a diastereoisomeric mixture of compound 26 was
obtained as a colorless foam (3.5 g, 77%) starting from compound
19 (4.28 g, 13.43 mmol), 2-mercaptoethanol (1.04 mL, 14.77 mmol),
and Et3N (2.24 mL, 16.12 mmol) in dry DMF (12 mL). 1H NMR
(300 MHz, CDCl3) d=7.63 (br s, 1H, NH-Gly), 5.56–5.50 (m, 1H, aH-
Gly), 5.10 and 5.06 (2”d, J=7.0 Hz, 1H, NH-Ala), 4.28–4.13 (m, 1H,
aH-Ala), 3.92–3.77 (m, 5H, OCH2CH2S and OCH3-Gly), 2.90–2.85 (m,
2H, OCH2CH2S), 1.46 and 1.45 (2”s, tBu), 1.38 and 1.37 ppm (2”d,
J=7.0 Hz, 3H, CH3-Ala); 13C NMR (75 MHz, CDCl3) d=172.5 (CO-
Ala), 169.8 and 169.6 (CO-Gly), 155.6 (OCONH), 80.7 (1C-tBu), 62.1
(OCH2CH2S), 53.2 (OCH3-Gly), 52.9 (aC-Gly), 50.4 (aC-Ala), 34.2 and
34.1 (OCH2CH2S), 28.4 (tBu), 18.0 and 17.9 ppm (CH3-Ala); HRMS
(ESI+): m/z calcd for C13H24N2O6S: 359.1247 [M+Na]+; found:
359.1240.
3
135.9 and 135.8 (C-6), 111.3 (C-5), 84.4 (d, JC,P =8.4 Hz, C-4’), 84.3
2
2
(C-1’), 70.5 and 70.4 (C-3’), 64.5 (d, JC,P =5.1 Hz, C-5’), 64.1 (d, JC,P
=
5.5 Hz, OCH2CH2S), 55.6 (aC-Gly), 49.2 (aC-Ala), 38.2 (C-2’), 29.6 (d,
3JC,P =7.8 Hz, OCH2CH2S), 19.1 (CH3-Ala), 12.0 and 11.9 ppm (CH3-
Thy); 31P NMR (202 MHz, D2O) d=À0.3 ppm; HRMS (ESI): m/z calcd
for C17H27N4O11PS: 525.1062 [MÀH]À; found: 525.1058.
N-Boc-l-Alanyl-d,l-2-O-benzylglycine methyl ester (31)
3’-O-Benzoyl-5’-O-(N-Boc-l-alanyl-d,l-2-mercaptoethanolgly-
cine methyl ester)TMP (29)
A solution of DABCO (1.35 g, 12.0 mmol) in dry THF (15 mL) was
added to a stirring solution of compound 19 (1.59 g, 5.0 mmol) in
dry THF (100 mL) at À788C. After stirring at the same temperature
for 10 min, benzyl alcohol (0.62 mL, 6 mmol) was added and the
solution was stirred for an additional 6 h at À788C. The reaction
mixture was slowly warmed to room temperature and the stirring
was continued for 24 h. The reaction was cooled to 08C, quenched
with 10% aq. citric acid (100 mL), and the volatiles were removed
in vacuo. The residue was extracted with ethyl acetate (3”150 mL)
and the combined organic phases were washed with 10% aq.
citric acid, saturated aq. NaHCO3, water, and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The resulting crude
product was purified by column chromatography on silica gel (ace-
tone/petroleum ether, 1:99–1:19–1:9) to give a diastereoisomeric
mixture of 31 (0.82 g, 45%) as a colorless oil. 1H NMR (300 MHz,
[D6]acetone) d=8.01 (d, J=8.6 Hz, NH-Gly), 7.39–7.28 (m, Ar-H
OBn), 6.26 (br s, 1H, NH-Ala), 5.69 and 5.67 (2”d, J=9.3 Hz, 1H,
aH-Gly), 4.71–4.56 (m, 2H, CH2-OBn), 4.24–4.19 (m, 1H, aH-Ala),
3.74 and 3.73 (2”s, 3H, OCH3-Gly), 1.42 and 1.41 (2”s, 9H, tBu),
1.36 and 1.35 ppm (2”d, J=7.2 Hz, 3H, CH3-Ala); 13C NMR (75 MHz,
[D6]acetone) d=174.5 (CO-Ala), 168.9 and 168.8 (CO-Gly), 156.4
(OCONH), 138.7 and 138.6 (1C of OBn), 129.1 (Ar-C), 129.0 (Ar-C),
128.9 (Ar-C), 128.8 (Ar-C), 128.5 (Ar-C), 79.5 (1C-tBu), 77.7 and 77.6
(aC-Gly), 70.7 and 70.6 (CH2-OBn), 52.8 (OCH3-Gly), 51.2 (aC-Ala),
28.5 (tBu), 18.2 and 18.0 ppm (CH3-Ala); HRMS (ESI+): m/z calcd for
C18H26N2O6: 367.1863 [M+H]+; found: 367.1860.
Following a similar procedure as the one used for the synthesis of
compound 25, a diastereoisomeric mixture of compound 29 was
obtained as a white solid (101.5 mg, 12%) starting from 27
(394 mg, 1.138 mmol), POCl3 (128 mL, 1.366 mmol) in dry trimethyl
phosphate (4 mL) and compound 26 (383 mg, 1.138 mmol), N-
methyl imidazole (272 mL, 1.366 mmol) in dry CH2Cl2 (10 mL). The
resultant crude residue was purified by column chromatography
on silica gel (MeOH/CH2Cl2, 1:99–3:97–8:92) and preparative RP-
HPLC (98% H2O+2% CH3CN and 98% CH3CN+2% H2O). The col-
lected fractions were freeze-dried repeatedly until the mass re-
1
mained constant. H NMR (300 MHz, CD3OD) d=8.06 (d, J=7.5 Hz,
2H, oH-OBz), 7.78 (s, 1H, H-6), 7.64 (t, J=7.4 Hz, 1H, pH-OBz), 7.64
(t, J=7.6 Hz, 2H, mH-OBz), 6.45 (app t, J=7.5 Hz, 1H, H-1’), 5.67–
5.62 (m, 1H, H-3’), 5.59–5.56 (m, 1H, aH-Gly), 4.67–4.37 (m, 1H, H-
4’), 4.35–4.23 (m, 2H, H-5’ and H-5’’), 4.18–4.04 (m, 3H, OCH2CH2S
and aH-Ala), 3.74 (s, 3H, OCH3-Gly), 3.02–2.92 (m, 2H, OCH2CH2S),
2.58–2.50 (m, 2H, H-2’ and H-2’’), 1.95 (s, 3H, CH3-Thy), 1.42 (s, 9H,
tBu), 1.30 and 1.29 ppm (2”d, J=7.0 Hz, 3H, CH3-Ala); 13C NMR
(150 MHz, CD3OD) d=175.4 and 175.3 (CO-Ala), 170.4 and 170.3
(CO-Gly), 167.3 (CO-OBz), 166.3 and 166.2 (C-4), 157.6 (OCONH),
152.5 and 152.4 (C-2), 137.8 (C-6), 134.6 and 134.5 (1C Ar-C), 130.9
(Ar-C), 130.8 (Ar-C), 130.7 (Ar-C), 130.6 (Ar-C), 129.7 (Ar-C), 129.6
3
(Ar-C), 112.4 and 112.3 (C-5), 86.3 and 86.1 (C-1’), 85.0 (d, JC,P
=
=
2
8.5 Hz, C-4’), 80.6 (1C-tBu), 77.5 and 77.2 (C-3’), 67.0 (d, JC,P
3.0 Hz, C-5’), 66.1 (d, 2JC,P =6.0 Hz, OCH2CH2S), 54.7 and 54.6 (aC-
Gly), 53.5 (OCH3-Gly), 51.5 (aC-Ala), 38.2 and 38.1 (C-2’), 32.0
(OCH2CH2S), 28.7 (tBu), 18.2 and 18.1 (CH3-Ala), 12.9 and 12.8 ppm
(CH3-Thy); 31P NMR (202 MHz, D2O) d=À0.5 ppm; HRMS (ESI): m/z
calcd for C30H41N4O14PS: 743.2005 [MÀH]À; found: 743.2014.
N-Boc-l-Alanyl-l-2-O-benzylglycine (32)
subtilisin Carlsberg (20 mg) was added to a stirring solution of the
diastereoisomeric mixture 31 (400 mg, 1.092 mmol) in DMF/H2O
Chem. Eur. J. 2016, 22, 8167 – 8180
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8178
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(1:1, 16 mL) at 558C. NaOH (0.61 mL, 1m) was added dropwise to
maintain the pH of the reaction at 7.2 (monitored by a pH-meter).
Upon completion, the volatiles were removed in vacuo. The resi-
due was dissolved in 5% NaHCO3 and extracted with ethyl acetate
(3”50 mL), and the combined organic phases were washed with
5% NaHCO3, 10% aq. citric acid, water, and brine, dried over
Na2SO4, filtered, and concentrated in vacuo to give the diastereo-
isomeric mixture (320 mg, recovered). The aqueous phase was
acidified with 10% citric acid and extracted with ethyl acetate (3”
50 mL). The combined organic phases were washed with 10% aq.
citric acid, water, and brine, dried over Na2SO4, filtered, and con-
centrated in vacuo to get the pure diastereoisomer (L,L) 32
Thymidine-5’-O-phosphorothioate triethylammonium salt
(36)
DBU (0.22 mL, 1.476 mmol) was added dropwise to a stirring solu-
tion of compound 35 (650 mg, 1.342 mmol) and 3-hydroxypropio-
nitrile (0.46 mL, 6.708 mmol) in dry CH2Cl2 (18 mL), and the reaction
mixture was stirred at room temperature for 8 h. It was concentrat-
ed in vacuo and the crude residue was purified by flash chroma-
tography on silica gel (MeOH/CHCl3, 1:49–1:9–1:4). The fractions
that contained the phosphorothioate monoester [31P NMR
(121 MHz, CD3OD) d=56.6 ppm; HRMS (ESI): m/z calcd for
C20H22N3O8PS: 494.0792 [MÀH]À; found: 494.0789] were combined
and concentrated in vacuo. The resulting residue was treated with
25% NH3 in a sealed tube at 558C for 16 h. Ammonia was removed
in vacuo, and the residue was diluted with water and lyophilized.
The crude product was purified by preparative RP-HPLC (50 mmol
TEAB in 98% H2O+2% CH3CN and 50 mmol TEAB in 50%
CH3CN+50% H2O). The collected fractions were freeze-dried re-
peatedly until the mass remained constant to afford compound 36
1
(54 mg, 14%) as a white solid. H NMR (300 MHz, CD3CN) d=7.56
(d, J=8.6 Hz, NH-Gly), 7.36–7.30 (m, Ar-H OBn), 5.67 (br s, 1H, NH-
Ala), 5.55–5.50 (m, 1H, aH-Gly), 4.67–4.53 (m, 2H, CH2-OBn), 4.10–
4.02 (m, 1H, aH-Ala), 1.41 (s, 9H, tBu), 1.28 ppm (d, J=7.2 Hz, 3H,
CH3-Ala); 13C NMR (75 MHz, CD3CN) d=175.1 (CO-Gly), 169.2 (CO-
Ala), 156.6 (OCONH), 138.4 (1C of OBn), 129.3 (Ar-C), 129.1 (Ar-C),
128.8 (Ar-C), 80.2 (1C-tBu), 77.6 (aC-Gly), 70.8 (CH2-OBn), 51.4 (aC-
Ala), 28.5 (tBu), 17.9 ppm (CH3-Ala); HRMS (ESI+): m/z calcd for
C17H24N2O6: 351.1561 [M+H]+, found: 351.1563.
1
(497 mg, 72% over two steps) as a white solid. H NMR (300 MHz,
D2O) d=7.80 (d, J=1.0 Hz, 1H, H-6), 6.34 (app t, J=7.0 Hz, 1H, H-
1’), 4.60–4.56 (m, 1H, H-3’), 4.20–4.18 (m, 1H, H-4’), 4.12–4.08 (m,
2H, H-5’ and H-5’’), 2.36–2.32 (m, 2H, H-2’ and H-2’’), 1.92 ppm (d,
J=1.0 Hz, 3H, CH3); 13C NMR (75 MHz, D2O) d=166.2 (C-4), 151.4
2-N,N-Diisopropylamino-1,3,2-oxathiaphospholane (34)
3
(C-2), 137.1 (C-6), 111.4 (C-5), 85.2 (d, JC,P =9.4 Hz, C-4’), 84.7 (C-1’),
71.0 (C-3’), 64.5 (d, 2JC,P =5.1 Hz, C-5’), 38.4 (C-2’), 11.3 ppm (CH3-
Thy); 31P NMR (121 MHz, D2O) d=50.2 ppm; HRMS (ESI): m/z calcd
for C10H15N2O7PS: 337.0265 [MÀH]À; found: 337.0262.
DIPEA (664 mL, 3.811 mmol) was slowly added to a stirring solution
of
N,N-diisopropyl
phosphoramidous
dichloride
(293 mL,
1.588 mmol) and 2-mercapto ethanol (112 mL, 1.588 mmol) in dry
diethyl ether (8 mL) at À208C. The reaction mixture was then
stirred at the same temperature for 30 min and slowly warmed to
room temperature over 2 h. The resultant white precipitate was
isolated by filtration under an inert atmosphere, and the filtrate
was concentrated (bath temp. ꢀ158C) and dried in vacuo to give
crude compound 34 (301 mg, quant.) as a colorless oil. The prod-
uct was checked for purity by using 31P NMR and was immediately
used in the next step without any further purification.
Thymidine-5’-O-(N-Boc-l-alanyl-d,l-2-aminoglycine methyl
ester)-phosphorothioate (37)
Et3N (0.45 mL, 3.234 mmol) was added to a stirred solution of com-
pound 19 (344 mg, 0.809 mmol) and compound 36 (309 mg,
0.970 mmol) in dry DMF (6 mL). The reaction mixture was heated
at 378C for 4 h. After cooling to RT, DMF was removed in vacuo,
and the resulting residue was dissolved in water and lyophilized.
The crude product was purified by preparative RP-HPLC (98%
H2O+2% CH3CN and 98% CH3CN+2% H2O). The collected frac-
tions were freeze-dried repeatedly until the mass remained con-
stant to afford a diastereoisomeric mixture of compound 37
3’-O-Benzoyl-5’-(2-thio-1,3,2-oxathiaphospholane)-thymidine
(35)
1
(329 mg, 68%) as a white solid. H NMR (500 MHz, CD3OD) d=7.74
A solution of S-ethylthiotetrazole (273 mg, 2.094 mmol) in dry
CH2Cl2 (5 mL) was added slowly to a stirring suspension of crude
34 (301 mg, 1.588 mmol) and compound 27 (500 mg, 1.444 mmol)
in dry CH2Cl2 (18 mL) at 08C, and the reaction mixture was left stir-
ring at room temperature for 3 h. When the solution became ho-
mogeneous, dry elemental sulfur (153 mg) was added and it was
left stirring overnight. The mixture was filtered, and the filtrate was
concentrated in vacuo. The resulting crude residue was purified by
column chromatography on silica gel (MeOH/CHCl3, 0:100–
0.5:95.5–1:99) to give product 35 (650 mg, 93%) as a pale yellow
(s, 1H, H-6), 6.35 (q, J=6.9 Hz, 1H, H-1’), 5.76–5.69 (m, 1H, aH-Gly),
4.50–4.46 (m, 1H, H-3’), 4.16–4.08 (m, 4H, H-4’, aH-Ala, H-5’ and H-
5’’), 3.79 and 3.78 (2”s, 3H, OCH3-Gly), 2.29–2.25 (m, 2H, H-2’ and
H-2’’), 1.96 and 1.94 (2”s, 3H, CH3-Thy), 1.47 and 1.46 (2”s, 9H,
tBu), 1.35 and 1.34 ppm (2d, J=7.1 Hz, 3H, CH3-Ala; merged with
Et3N); 13C NMR (125 MHz, CD3OD) d=175.4 and 175.3 (CO-Ala),
170.8 and 170.7 (CO-Gly), 166.4 (C-4), 157.6 (OCONH), 152.4 (C-2),
3
138.0 (C-6), 112.0 and 111.9 (C-5), 87.0 (d, JC,P =7.6 Hz, C-4’), 86.2
(C-1’), 80.8 and 80.8 (1C-tBu), 72.7 and 72.6 (C-3’), 67.0 (C-5’), 55.0
and 54.9 (aC-Gly), 53.6 (OCH3-Gly), 51.7 and 51.6 (aC-Ala), 40.8 and
40.7 (C-2’), 28.7 (tBu), 18.2 and 18.0 (CH3-Ala), 12.7 and 12.6 ppm
(CH3-Thy); 31P NMR (202 MHz, CD3OD) d=13.1 ppm; HRMS (ESI): m/
z calcd for C21H33N4O12PS: 595.1480 [MÀH]À, found: 595.1486.
1
foam. H NMR (300 MHz, CDCl3) d=9.09 (br s, 1H, NH-Thy), 8.07 (d,
J=1.0 Hz, 1H, H-6), 8.05–7.46 (m, 5H, OBz), 6.53–6.46 (m, 1H, H-1’),
4.58–4.54 (m, 1H, H-3’), 4.64–4.44 (m, 4H, H-5’, H-5’’ and
OCH2CH2S), 4.42–4.39 (m, 1H, H-4’), 3.61–3.52 (m, 2H, OCH2CH2S),
2.67–2.60 (m, 1H, H-2’), 2.36–2.23 (m, 1H, H-2’’), 2.01 ppm (d, J=
1.0 Hz, 3H, CH3); 13C NMR (75 MHz, CDCl3) d=166.3 (CO-OBz),
164.0 (C-4), 150.6 (C-2), 135.4 and 135.3 (C-6), 133.9 (1C-OBz), 129.9
(Ar-C), 128.7 (Ar-C), 112.1 and 112.0 (C-5), 85.1 and 85.0 (C-1’), 83.2
(d, 3JC,P =8.7 Hz, C-4’), 75.5 and 75.4 (C-3’), 70.9 (d, 3JC,P =11.3 Hz,
Thymidine-5’-O-(l-alanyl-(À)-2-aminoglycine methyl ester)-
phosphorothioate (38a) and thymidine-5’-O-(l-alanyl-(+)-2-
aminoglycine methyl ester)-phosphorothioate (38b)
2
OCH2CH2S), 68.1 (d, JC,P =6.6 Hz, C-5’), 37.7 and 37.6 (C-2’), 37.1 (d,
A similar synthetic protocol as the one used for the synthesis of
compound 23 was employed for the synthesis of 38a,b starting
from compound 37 (160 mg, 0.2293 mmol), thioanisole (33 mL,
0.356 mmol), and TFA (1.5 mL) in CH2Cl2 (4.5 mL). Semi-preparative
3JC,P =16.3 Hz, OCH2CH2S), 12.7 and 12.6 ppm (CH3-Thy); 31P NMR
(121 MHz, CDCl3) d=104.3 and 104.1 ppm; HRMS (ESI+): m/z calcd
for C19H21N2O6PS2: 507.0420 [M+Na]+; found: 507.0421.
Chem. Eur. J. 2016, 22, 8167 – 8180
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8179
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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345–358; b) P. Schimmel, Annu. Rev. Biochem. 1987, 56, 125–158.
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RP-HPLC (98% H2O+2% CH3CN and 98% CH3CN+2% H2O) was
employed for further separation to obtain the TFA salts of pure
diastereoisomers as a ꢀ1:1 ratio (isomer 38a: 59.6 mg; isomer
38b: 60 mg) as white powders (overall yield 38a+38b: 119.6 mg,
84%). The isolated products were freeze-dried repeatedly until the
mass remained constant. Data for compound 38a: 1H NMR
(300 MHz, D2O) d=7.71 (d, J=1.0 Hz, 1H, H-6), 6.35 (app t, J=
7.0 Hz, 1H, H-1’), 5.60 (d, J=15.7 Hz, 1H, aH-Gly) 4.61–4.56 (m, 1H,
H-3’), 4.20–4.18 (m, 1H, H-4’), 4.16–4.09 (m, 3H, aH-Ala, H-5’ and H-
5’’), 3.81 (s, 3H, OCH3-Gly), 2.42–2.37 (m, 2H, H-2’ and H-2’’), 1.94
(d, J=1.0 Hz, 3H, CH3-Thy), 1.55 ppm (d, J=7.1 Hz, 3H, CH3-Ala);
13C NMR (150 MHz, D2O) d=170.0 (d, 3JC,P =6.0 Hz, CO-Gly), 169.7
(CO-Ala), 166.3 (C-4), 151.5 (C-2), 137.1 (C-6), 111.5 (C-5), 84.8 (d,
[13] a) G. Harris, J. W. Davies, R. Parsons, Nature 1958, 182, 1565–1567; b) G.
Harris, J. W. Davies, Nature 1959, 184, 788–789.
2
3JC,P =9.2 Hz, C-4’), 84.7 (C-1’), 70.8 (C-3’), 65.3 (d, JC,P =5.4 Hz, C-5’),
53.6 (aC-Gly and OCH3-Gly), 48.7 (aC-Ala), 38.4 (C-2’), 15.9 (CH3-
Ala), 11.5 ppm (CH3-Thy); 31P NMR (202 MHz, D2O) d=15.1 ppm;
[a]25089 =À0.5188 (c=1 in CH3OH); HRMS (ESI): m/z calcd for
C16H25N4O10PS: 495.0956 [MÀH]À; found: 495.0956. Data for com-
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1
pound 38b: H NMR (500 MHz, D2O) d=7.69 (s, 1H, H-6), 6.36 (app
t, J=7.0 Hz, 1H, H-1’), 5.64 (d, J=14.5 Hz, 1H, aH-Gly) 4.57–4.54
(m, 1H, H-3’), 4.19–4.16 (m, 1H, H-4’), 4.15–4.04 (m, 3H, aH-Ala, H-
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2’’), 1.88 (s, 3H, CH3-Thy), 1.53 ppm (d, J=7.1 Hz, 3H, CH3-Ala);
13C NMR (125 MHz, D2O) d=169.8 (d, 3JC,P =6.8 Hz, CO-Gly), 169.7
(CO-Ala), 166.2 (C-4), 151.5 (C-2), 137.3 (C-6), 111.4 (C-5), 84.9 (d,
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2
3JC,P =8.9 Hz, C-4’), 84.8 (C-1’), 70.6 (C-3’), 65.4 (d, JC,P =5.3 Hz, C-5’),
53.6 (OCH3-Gly), 53.3 (d, 2JC,P =3.0 Hz, aC-Gly), 48.8 (aC-Ala), 38.1
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20
589
d=15.1 ppm; [a] = +0.7308 (c=1 in CH3OH); HRMS (ESI): m/z
calcd for C16H25N4O10PS: 495.0956 [MÀH]À; found: 495.0959.
Acknowledgements
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We thank FWO (Vlaanderen) (G.078014N and G.08816N) for fi-
nancial support. The research leading to these results has re-
ceived funding from the European Research Council under the
European Union’s Seventh Framework Programme (FP7/2007-
2013)/ERC Grant agreement no ERC-2012-ADG 20120216/
320683. We thank Chantal Biernaux for final typesetting.
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Keywords: phosphoaminals
polymerase chain reaction · synthetic biology
·
nucleosides
·
peptides
·
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Published online on May 2, 2016
Chem. Eur. J. 2016, 22, 8167 – 8180
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