Tailoring peptide-nucleotide conjugates (PNCs) for nucleotide delivery in bacterial cells

FULL PAPER

DOI: 10.1002/ejoc.201301781

Tailoring Peptide–Nucleotide Conjugates (PNCs) for Nucleotide Delivery in

Bacterial Cells

Swarup De,^[a]Elisabetta Groaz,^[a]and Piet Herdewijn*^[a]

Keywords: Bioorganic chemistry / Peptides / Nucleotides / Peptide–nucleotide conjugates

The design and synthesis of peptide-2Ј-deoxythymidine-5Ј-

O-monophosphate conjugates as potential active delivery

systems for nucleotides into auxotrophic E. coli strains is pre-

sented. A series of oligopeptides were allowed to react with

5Ј-O-(dibenzylphosphate)-2Ј-deoxythymidine or its suitably

3Ј-derivatized analogues to give the relevant peptide–nu-

cleotide adducts, by the formation of a biolabile chemical

connection. Using strategies based on the principles of or-

thogonal protection and activation, rational variations were

made to the linker and the peptide moiety in order to tune

the metabolic stability of the conjugates.

Given these facts, we envisaged that specifically tailored

nucleotide conjugates might act as active delivery systems,

exploiting membrane uptake systems naturally occurring in

bacterial cells for the internalization of essential nutrients,

to eventually facilitate nucleotide delivery across the cyto-

plasmic membrane. In particular, the rationale for our study

hinges on the significance of peptide transporters in pro-

karyotic microorganisms, which are best represented by the

vast family of binding-protein-dependent permeases.^[6]The

derivatization of ligands with peptides is an attractive con-

cept in medicinal chemistry, as it can give cellular access to

synthetic antimicrobial compounds that might be difficult

or impossible to be delivered by direct means.^[7,8]Microbial

permeases are multicomponent ATP (adenosine triphos-

phate) binding cassette (ABC) proteins that mediate the

active transport of peptides, which act as a source of amino

acids, carbon, nitrogen, and energy.^[9,10]In Gram-negative

species, three classes of peptide permeases (Dpp, Tpp, and

Opp) have been found, which have complementary selectivi-

ties for different conformations, sizes, and compositions of

oligopeptide sequences, thus ensuring complete uptake

from the peptide pool. Of the periplasmic proteins required

for initial molecular recognition and binding by permeases,

the oligopeptide binding protein OppA is the most abun-

dant (7–10%) and structurally well-defined.^[11,12]In vitro

binding studies on peptide complexes of E. coli OppA high-

lighted high affinity and broad substrate specificity for

small peptides comprising two up to five or six l-amino

acids, regardless of their sequence.^[13,14]Further investi-

gations have suggested a preference for peptides containing

Ala, Gly, Phe,^[13]and basic residues,^[15]in particular lysines,

which may result from a negatively charged surface in the

vicinity of the initial binding site.

Introduction

The ability to encode uncharted heritable information in

polymers other than DNA and RNA relies upon chemical

diversification and enzymatic proliferation of artificial nu-

cleic acids (XNAs).^[1,2]With the aim of controlling and pre-

venting cross-contamination, new synthetic genetic se-

quences will have to be conceived to satisfy a principle of

orthogonality with respect to wild biodiversity, leading to

the establishment of self-reliant genetic enclaves.^[3]Funda-

mental progress in enzyme evolution has recently been

made with the discovery of engineered polymerase mutants

that are able to recognize and efficiently incorporate back-

bone-modified nucleotide building blocks [hexitol (HNA),

cyclohexene (CeNA), arabino (ANA), 2Ј-deoxy-2Ј-fluoroar-

abino (FANA), threose (TNA), and locked (LNA) nucleic

acids] from a DNA template. XNA sequences up to 72 nt

(nucleotides) in length were generated following canonical

base-pairing selectivity, and reverse-transcribed into

DNA.^[4]Nonetheless, major challenges remain if XNAs are

to reach their true evolutionary potential as genetic repro-

gramming tools in vivo.^[5]The molecular assembly of xeno-

polymers within the host cell requires the intracellular avail-

ability of preactivated unnatural precursors, since most

modified nucleoside substrates are poorly phosphorylated

by host kinases. However, negatively charged nucleotides

suffer from low cell-penetrating ability and a known insta-

bility as a result of their propensity to undergo hydrolysis to

the phosphate-free state of nucleosides by exocytoplasmic

phosphatases before being taken up.

[a] Medicinal Chemistry, Rega Institute for Medical Research, KU

Leuven,

Minderbroedersstraat 10, 3000 Leuven, Belgium

E-mail: piet.herdewijn@rega.kuleuven.be

http://medchem.rega.kuleuven.be/

It has been suggested that the presence of free α-amino

and carboxyl groups at the N- and C-terminals, respectively,

were necessary for binding to E. coli OppA and subsequent

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201301781.

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Tailoring Peptide–Nucleotide Conjugates

transmembrane translocation into the cell to take place, but the amino functionality in side-chain of Lys, were utilized

protected peptides were also transported, albeit less effec- to form the connecting bond. Following internalization of

tively.^[13]However, the presence of a free amino group the conjugate in the cell, the peptide might be hydrolysed

might trigger extracellular metabolism of the peptide conju- by E. coli PepN, the principal cytosolic aminopeptidase en-

gate by peptidases or degradation by chemical means, ow- zyme responsible for the hydrolysis of peptides, whose ac-

ing to its high reactivity.

tivity varies in the following order: ArgϾ AlaϾ Lys Ͼ

With this knowledge in mind, an approach in which Gly.^[18]

oligopeptides are conjugated to nucleotides by a biorevers-

ible linkage, appeared to be logical in an attempt to develop

a new cellular uptake system. It is, however, anticipated that

Results and Discussion

the success of this proposal would strongly depend on the

metabolic stability of the relevant adducts, which should be

Several strategies have been described in the literature to

sufficiently stable to be able to cross the cell wall without prepare peptide–oligonucleotide conjugates connected by

undergoing premature degradation, but still be cleaved at variously functionalized linkers, such as a thioether, a di-

the linker site once in the presence of intracellular enzymes. sulfide, an amide, and an oxime moiety etc.^[19]For our

To assess the viability of our model, we selected 5Ј-thymid- study, we selected five different linkers (Figure 1), which

ylate (dTMP) nucleotide conjugates as initial synthetic tar- were assumed to be liable to cleavage inside the bacterial

gets, for which reliable nutritional selections using E. coli cell, either by a chemical or an enzymatic mechanism, i.e.,

mutants lacking thymidylate synthase are well established. carbamate (OCONH), ester (OCO), oxyamide (ONHCO),

A conjugate derived from a pyridoxal-pyroglutamyl-tri- oxymethyleneoxyamide (OCH₂ONHCO), and oxymethyl-

peptido-nucleotide, which could release the dTMP synthon eneoxy ester (OCH₂OCO). The corresponding peptide–nu-

by an autocatalytic elimination process, was prepared, but cleotide conjugates (PNCs) were synthesized by the reaction

purification and scaling-up of the synthesis of this adduct of the 3Ј-hydroxy group of dTMP with the side-chain func-

was not straightforward.^[16]In this paper, we describe a fur- tionalities of an oligopeptide, for example an amino or a

ther elaboration of this concept with the synthesis of a di- carboxylic acid group.

verse range of peptide–nucleotide conjugate (PNCs) ana-

Firstly, di-, tri-, tetra-, and pentapeptides were conve-

logues, prepared by covalently linking di- to pentapeptidic niently prepared according to the NHS (N-hydroxysuc-

linear chains, through various spacers, to the 3Ј-position of cinimide) activated ester method^[20]starting from commer-

the ribose moiety. Amino acidic residues and chemical con- cially available N-Boc (tert-butoxycarbonyl) protected/for-

nections between dTMP and peptides were systematically mylated amino acids, as shown in Scheme 1. In the first

examined, as summarized in Figure 1. The α-amino group step, NHS-activated carboxy ester derivatives were formed

at the N-terminus was either unmodified or protected as a and subsequently coupled with an incoming amino acid

formylamide (For), which can be hydrolytically cleaved by without the need for protection at the C-terminal. The side-

E. coli peptide deformylase. This enzyme is known to show chain functionalities were orthogonally protected as Boc,

a preference for substrates containing N-formyl methionine, Fmoc (fluorenylmethyloxycarbonyl), Cbz (benzyloxycarb-

followed by N-formyl glycine.^[17]The α- or β-carboxyl onyl), OBn, or tBu during peptide synthesis, and the pro-

groups present in the second or third residue, together with tecting groups were removed prior to coupling to the free

Figure 1. Chemical diversity in the design of novel peptide-nucleotide conjugates (PNCs) as delivery system of natural and modified

nucleotides in prokaryotic cells.

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Scheme 1. Preparation of dipeptides 2a, 2b, and 8, tripeptides 4h, 5a–5g, and 13, tetrapeptide 9, and pentapeptide 10. Reagents and

conditions: (a) (i) NHS, DCC (N,NЈ-dicyclohexylcarbodiimide), THF, 0 °C to room temp., 2–12 h, (ii) amino acid, NaHCO₃, THF/H₂O,

0 °C to room temp., 16 h; (b) Pd/C (10%; Degussa), H₂, EtOH or MeOH, room temp., 6–24 h; (c) TFA, CH₂Cl₂, 0 °C to room temp.,

2 h; (d) Et₃N/CH₂Cl₂(2:1), room temp., 72 h; (e) TFA, thioanisole, CH₂Cl₂, room temp., 4 h.

3Ј-OH group of thymidine. For the acidic deprotection of deoxythymidine was selectively protected with a MMTr (4-

the N-Boc group, thioanisole was used as a radical scaven- monomethoxytrityl) group^[23]to give 5Ј-O-MMTr-2Ј-de-

ger;^[21]an excess of Pd/C was used in order to avoid catalyst oxythymidine 14. This compound was then activated at its

poisoning during removal of the benzyl group of methio- 3Ј-OH group using carbonyldiimidazole (CDI)^[24,25]or 4-

nine-containing peptides. In the case of peptide 4c, the nitrophenyl chloroformate^[26](via intermediate 15), and

Fmoc group was removed using Et₃N in dichloromethane. subsequent reaction with the ε-amino group of the lysine

Ethanol was used as solvent^[22]for the N-Cbz deprotection residue of peptide 5d gave compound 16. After detrityl-

of 12 to give 13, since in the presence of methanol a N- ation, the phosphate group was introduced at the 5Ј-posi-

methylated by-product was formed instead.

tion using dibenzyl-N,N-diisopropyl phosphoramidite and

The construction of PNCs can be accomplished either by hydrogen peroxide as oxidizing agent. The resulting thymid-

linking the peptide at the 3Ј-position of thymidine followed ine-5Ј dibenzyl phosphate product (i.e., 18) was subjected

by 5Ј-O-phosphorylation, or by reversing the order of this to hydrogenation in the presence of Pd/C to produce the

reaction sequence. For the synthesis of our first PNC ana- free phosphate functionality.

logue 19, featuring a carbamoyl linker (OCONH), it was

However, this strategy could not be used for the synthesis

decided to use the first of these two approaches, as shown of N-formyl-l-methionine-containing analogues, presum-

in Scheme 2. In the first step, the 5Ј-hydroxy group of 2Ј- ably due to the instability of the thiomethyl group during

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Tailoring Peptide–Nucleotide Conjugates

Scheme 2. Synthesis of 3Ј-O-carbamoyl peptide conjugate of dTMP 19. Reagents and conditions: (a) MMTrCl, Et₃N, DMAP (4-dimethyl-

aminopyridine), DMF, room temp., 4 h; (b) 4-nitrophenyl chloroformate, Py, CH₂Cl₂, 0 °C to room temp., 24 h; (c) peptide 5d, Et₃N,

CH₂Cl₂, room temp.; (d) AcOH (80%), room temp., 3.5 h; (e) (i) dibenzyl N,N-diisopropylphosphoramidite, tetrazole (0.45 m in MeCN),

CH₂Cl₂, room temp., 12 h, (ii) H₂O₂, –40 °C to room temp., 2 h; (f) Pd/C (10%; Degussa), H₂, Et₃N, MeOH.

the oxidation of P^IIIto P^V, even when milder oxidizing rea- tized with 4-nitrophenyl chloroformate in pyridine to give

gents, such as a dilute solution of I₂in pyridine/THF/H₂O, 4-nitrophenyl carbonate 24 in moderate yield.^[34]The acti-

were used.^[27,28]Therefore, the route to compound 19 was vated carbonate was then treated with amino peptides 5c–

repeated using 3Ј-benzoyl thymidine 21 as starting material, 5g, 8–10, and 13 to generate carbamates 18 and 25–32 in

as shown in Scheme 3.

yields ranging from 64 to 88%, depending on the peptide

The dibenzylphosphate group at the 5Ј-position of 21 used. Finally, hydrogenolysis of the benzyl groups was car-

was introduced first, followed by peptide conjugation. The ried out under mild conditions, in order to prevent re-

same synthetic method was also used to give nine further duction of the double bond of the thymine ring, using palla-

PNCs, 18 and 25–32, built on a carbamoyl linker.

dium on carbon (10%; Degussa) or Pd(OH)₂/C (20%) at

With regard to this second route, it was necessary to pro- atmospheric pressure, smoothly giving phosphate deriva-

tect the secondary OH group to obtain phosphorylated nu- tives 19 and 33–40 in 58–83% yield.

cleoside 23 in good yield.^[29–31]Thus, we elected to protect

Notably, debenzylation by hydrogenation was successful

the free hydroxy functionality of compound 14 using ben- for conjugates containing methionine in the peptide chain.

zoyl chloride.^[32]Standard MMTr deprotection proceeded A larger excess of Pd/C was used during these reactions to

as expected using AcOH (80%) to give 3Ј-O-benzoyl-2Ј-de- avoid poisoning of the catalyst by the thiomethyl group. In

oxythymidine 21, which was in turn phosphorylated with most cases, except for compound 36 where a free amino

dibenzyl N,N-diisopropylphosphoramidite and 1H-tetraz- group is present in the peptidic side-chain, 2 equiv. of Et₃N

ole^[33]at the 5Ј-position. Subsequent oxidation in the pres- was used to neutralize the strong acidity of the phosphoric

ence of hydrogen peroxide gave 5Ј-dibenzylphosphate-3Ј-O- acid group during di-O-benzyl deprotection. The terminal

benzoyl-2Ј-deoxythymidine 22 in 92% yield over two steps. amino group of 37 was deprotected with TFA (trifluoro-

Debenzoylation was effected by treatment of 22 with satu- acetic acid) to give conjugate 41, which was then treated

rated methanolic ammonia to give key intermediate 23 in with LiOH to liberate the terminal carboxyl group and pro-

95% yield. This compound served as a common substrate duce compound 42.

for the subsequent coupling step to explore various 3Ј-O

The synthesis of 3Ј-oxyamide-linked PNC 48 is shown

linkers. As shown in Scheme 3, the 3Ј-OH group was deriva- in Scheme 4. 3Ј-Oxyamine intermediate 46 was synthesized

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Scheme 3. Synthesis of 3Ј-carbamoyl peptide conjugates of dTMP 19 and 33–42. Reagents and conditions: (a) BzCl, Py, 0 °C, 3 h;

(b) AcOH (80%), room temp., 3.5 h; (c) (i) dibenzyl N,N-diisopropylphosphoramidite, tetrazole (0.45 m in MeCN), CH₂Cl₂, room temp.,

12 h, (ii) H₂O₂, –40 °C to room temp., 2 h; (d) NH₃(7 n in MeOH), 0 °C to room temp., 24 h; (e) 4-nitrophenyl chloroformate, Py,

CH₂Cl₂, 0 °C to room temp., 24 h; (f) peptide-NH₂(5c–5g, 8–10 or 13), Et₃N, CH₂Cl₂, room temp.; (g) Pd/C (10%; Degussa), H₂, MeOH

or EtOH, room temp., 24 h; (h) TFA, thioanisole, H₂O, room temp., 3 h; (i) LiOH, THF/MeOH/H₂O (1:1:1), room temp., 3 h.

starting from 2Ј-deoxythymidine according to modified lit- Compound 54 was obtained from 5Ј-O-protected-3Ј-O-

erature procedures.^[35–37]5Ј-O-MMTr-2Ј-deoxy-xylo-thym- methylenethiomethyl-2Ј-deoxythymidine 50, which was syn-

idine 43 was obtained from 2Ј-deoxythymidine in 48% yield thesized by a modified literature procedure.^[41]The acid-

over three steps.^[38]

stable TBDPS (tert-butyldiphenylsilyl) group was selected

The protected hydroxylamine functionality was formed to mask the 5Ј-position in view of potential complications

by Mitsunobu inversion using N-hydroxyphthalimide. Re- at a later stage of the synthetic scheme. The 3Ј-thioacetal

moval of the MMTr group and phosphorylation at the 5Ј- group was converted by treatment with sulfuryl chloride

position, as described above, yielded protected hydroxyl- into the corresponding chloromethyl ether. Without further

amine 45, which was then treated with methylamine (4%) to purification, this compound was treated with N-hydroxy-

restore the 3Ј-ONH₂group. Although stronger nucleophilic phthalimide to give 3Ј-O-aminooxymethylene derivative 51.

reagents like hydrazine hydrate or hydroxylamine are more The 5Ј-TBDPS protection was removed by treatment with

efficient for phthalimide deprotection, our reagent of choice Et₃N·3HF, and the free 5Ј-hydroxy group was phosphor-

was diluted methylamine, to minimize the rate of side-reac- ylated using our general protocol. Phthalimide deprotection

tions at the protected 5Ј-phosphate moiety.

followed by DCC coupling with dipeptides 2a and 2b

Coupling between the 3Ј-oxyamino group of compound yielded conjugates 55 and 56, respectively. Benzyl deprotec-

46 and the free carboxylic acid group of dipeptide 2a in the tion and HPLC purification as described above, yielded the

presence of DCC led to benzyl-protected oxyamide 47, 5Ј-O-monophosphates of the 2Ј-deoxythymidine-3Ј-oxy-

which was subjected to catalytic hydrogenation using Pd/C methyleneoxyamide peptide conjugates as triethylammo-

and Et₃N at atmospheric pressure. Final purification by RP nium salts, i.e., 57 and 58.

(reverse-phase) HPLC yielded 48 as a triethylammonium

Due to its propensity to undergo cleavage by cellular

salt. The common key intermediate in the synthesis of 3Ј- carboxyesterase, the oxymethylene ester moiety has been

oxymethyleneoxyamide PNCs 57 and 58 is compound 54 described in the literature in the context of bioavailable

(Scheme 5), which can be coupled with different peptides antiretroviral prodrugs based on acyclic nucleosides,^[42,43]

with a free carboxylic acid group in their side-chains.^[39,40]and also as a biolabile 2Ј-O-protecting group for the devel-

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Scheme 4. Synthesis of the 3Ј-oxyamide peptide conjugate of dTMP 48. Reagents and conditions: (a) (i) MMTrCl, Et₃N, Py, room temp.,

16 h, (ii) MsCl, Et₃N, room temp., 2 h, (iii) NaOH, EtOH, reflux, 1.5 h; (b) (i) N-hydroxy phthalimide, PPh₃, DIAD, toluene, 0 °C to

room temp., 2 h, (ii) AcOH (80%), room temp., 3 h; (c) (i) dibenzyl N,N-diisopropylphosphoramidite, tetrazole (0.45 m in MeCN),

CH₂Cl₂, room temp., 12 h, (ii) H₂O₂, –40 °C to room temp., 2 h; (d) MeNH₂(4%), EtOH, room temp., 0.5 h; (e) dipeptide 2a, DCC,

DMAP, CH₂Cl₂/DMF, room temp., 24 h; (f) Pd/C (10%; Degussa), H₂, MeOH, Et₃N, room temp., 24 h.

opment of short synthetic double-stranded RNA sequences by bacterial esterases. Analogue 65 was obtained from pre-

with therapeutic applications in vivo.^[44–46]The formation viously prepared protected 2Ј-deoxythymidine-5Ј-O-mono-

of 3Ј-O-methylacyloxy PNC 62 essentially followed known phosphate 23 by standard coupling to the free terminal

procedures for similar transformations; previously synthe- carboxylate of tripeptide 4h in the presence of DCC and

sized intermediate 50 was activated, and the β-carboxyl DMAP as catalyst,^[50]in good yield. Deprotection of the

group of tripeptide 5a was used as

a

nucleophile dibenzyl phosphate by catalytic hydrogenation led to 64,

(Scheme 6).

which, without further purification, underwent removal of

A first attempt using NIS (N-iodosuccinimide)/NCS (N- the NH-Boc protecting groups in the presence of TFA to

chlorosuccinimide) as the activating agent^[47]did not give give final compound 65 as a TFA salt in 62% yield over

satisfactory yields of compound 59. Therefore, we switched two steps (Scheme 7). When Cbz was used in place of Boc,

to the sulfuryl chloride method, which was more success- attempts to remove all the protecting groups in one step by

ful.^[48,49]TBDPS deprotection using Et₃N·3HF and phos- catalytic hydrogenation resulted in a complex mixture of

phorylation at the 5Ј-position was carried out as shown in degradation products.

Scheme 5 to give compound 61. Finally, Pd(OH)₂-mediated

Key intermediate 5Ј-O-(dibenzylphosphate)-2Ј-deoxy-

hydrogenolysis in the presence of Et₃N gave triethylammo- thymidine 23 could also be prepared by an alternative se-

nium salt 62 in moderate yield, due to undesirable side-reac- quence of protection/deprotection steps. The synthesis, as

tions involving cleavage of the linker, giving rise to dTMP shown in Scheme 8, started with the bis-silylation of

and the peptide as by-products.

thymidine to form intermediate 66, which was selectively

The synthesis of PNCs with an ester linker was thought deprotected at the 5Ј-position in good yield (59%) using a

to be particularly promising in view of the known suscep- mixture of TFA and H₂O (10:1).^[51]Compound 67 was then

tibility of this functional group to intracellular hydrolysis subjected to standard phosphorylation followed by

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Scheme 5. Synthesis of 3Ј-oxymethyleneoxyamide peptide conjugates of dTMP 57 and 58. Reagents and conditions: (a) TBDPSCl, imid-

azole, DMF, room temp., 4 h; (b) DMSO, Ac₂O, AcOH, room temp., 48 h; (c) (i) SO₂Cl₂, CH₂Cl₂, 0 °C to room temp., 2 h, (ii) N-

hydroxyphthalimide, DBU (1,8-diazabicycloundec-7-ene), CH₂Cl₂, room temp., 24 h; (d) Et₃NH·3HF, THF, room temp., 36 h; (e) (i) di-

benzyl N,N-diisopropylphosphoramidite, tetrazole (0.45 m in MeCN), CH₂Cl₂, room temp., 12 h, (ii) H₂O₂, –40 °C to room temp., 2 h;

(f) MeNH₂(4%), EtOH, room temp., 0.5 h; (g) dipeptide 2a or 2b, DCC, DMAP, CH₂Cl₂/DMF, room temp., 24 h; (h) Pd/C (10%;

Degussa), H₂, Et₃N, MeOH, room temp., 24 h.

TBDMS (tert-butyldimethylsilyl) cleavage to give the re- gates featuring linker moieties such as carbamate, ester,

quired substrate (i.e., 23) with which to carry out peptide oxyamide, oxymethyleneoxyamide, and oxymethyleneoxy

coupling. In this step, the use of the milder reagent ester, all of which are potentially susceptible to enzymatic

Et₃N·HF was found to give higher yields than TBAF (tetra- or chemical cleavage inside the bacterial cell. Final depro-

butylammonium fluoride). In the presence of the free β- tection of all structures was achieved in high yield by palla-

carboxyl group of tripeptide 5b, DCC, and DMAP, 23 was dium-catalysed hydrogenolysis. Of particular note is the un-

cleanly converted into conjugate 69 and eventually into its expected tolerance of the thiomethyl functionality present

triethylammonium salt 70.

in methionine-containing PNCs of the debenzylation condi-

tions.

Biological studies on the metabolic stability and func-

tional activity of all of the synthesized constructs in culture

medium and bacterial cells (ThyA^–auxotrophic E. coli mu-

Conclusions

The derivatization of ligands with peptides can be used tants) are currently being performed, and the results will be

to achieve a large improvement in the cellular uptake of reported in due course. With a view to the development

hydrophilic structures. We have described the application of of a xenonucleotide delivery system in bacterial cells, the

this methodology to nucleotides, which has led to the devel- introduction of other types of residues is also being ex-

opment of a wide variety of peptide-5Ј-thymidylate conju- plored.

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Scheme 6. Synthesis of 3Ј-oxymethyleneoxy ester–peptide conjugate of dTMP 62. Reagents and conditions: (a) (i) SO₂Cl₂, CH₂Cl₂, 0 °C

to room temp., 2 h, (ii) peptide-CO₂H 5a, DBU, CH₂Cl₂, room temp., 24 h; (b) Et₃NH·3HF, THF, room temp., 36 h; (c) (i) dibenzyl N,N-

diisopropylphosphoramidite, tetrazole (0.45 m in MeCN), CH₂Cl₂, room temp., 12 h, (ii) H₂O₂, –40 °C to room temp., 2 h; (d) Pd(OH)₂/

C (20%), H₂, NaHCO₃, EtOH/H₂O, room temp., 1.5 h.

Scheme 7. Synthesis of 3Ј-carboxy ester–peptide conjugate of dTMP 65. Reagents and conditions: (a) peptide-CO₂H 4h, DCC, DMAP,

CH₂Cl₂/DMF (5:1), room temp., 24 h; (b) Pd/C (10%; Degussa), H₂, MeOH, room temp., 24 h; (c) TFA, thioanisole, H₂O, room temp.,

5 h.

shifts were referenced to residual solvent signals at δ_H/C7.26/77.00

(CDCl₃), 3.31/49.10 ([D₄]methanol), and 2.50/39.50 ([D₆]DMSO)

Experimental Section

General Information: For all reactions, analytical grade solvents ppm relative to tetramethylsilane. Coupling constants are expressed

were used. All moisture-sensitive reactions were carried out under

an argon or nitrogen atmosphere in oven-dried glassware (135 °C).

Reaction temperatures are reported as bath temperatures. Pre-

coated aluminum sheets (254 nm) were used for TLC, and com-

pounds were visualized with UV light (λ = 254 nm). Products were

purified by flash chromatography on ICN silica gel 63–200, 60 Å.

¹H, ¹³C, and ³¹P NMR spectra were recorded with Bruker Avance

300, 500, or 600 MHz spectrometers. For final compounds, ¹H and

¹³C resonance assignments were made using 2D NMR correlation

experiments (COSY, gHSQC and gHMBC). For the sake of clarity,

the NMR signals of protons and carbons for sugar and base moie-

ties are indicated with and without a prime, respectively. Chemical

in Hertz (Hz). Splitting patterns are reported as s (singlet), d (doub-

let), t (triplet), q (quartet), m (multiplet), and br (broad). High-

resolution mass spectra were acquired with a quadrupole orthogo-

nal acceleration time-of-flight mass spectrometer (Synapt G2

HDMS, Waters, Milford, MA). Samples were infused at 3 μL/min,

and spectra were obtained in positive or negative ionization mode

with a resolution of 15000 (FWHM; full width at half maximum),

using leucine enkephalin as lock mass. All the methods

used MeCN/H₂O gradients. Water contained either TFA (0.1%)

or NH₃(0.1%). All final compounds were purified by preparative

RP-HPLC (Xbridge^TMPrep C18 5 μm OBD 19ϫ150 mm

column).

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S. De, E. Groaz, P. Herdewijn

FULL PAPER

Scheme 8. Synthesis of 3Ј-carboxy ester peptide conjugate of dTMP 70. Reagents and conditions: (a) TBDMSCl, Im (imidazole), dry

DMF; (b) TFA/H₂O (10:1), CH₂Cl₂; (c) (i) dibenzyl N,N-diisopropylphosphoramidite, tetrazole (0.45 m in MeCN), CH₂Cl₂, 0 °C to room

temp., 12 h, (ii) H₂O₂, –25 °C to room temp., 2 h; (d) Et₃N·3HF, THF, 24 h; (e) peptide-CO₂H 5b, DCC, DMAP, DMF, CH₂Cl₂, room

temp., 24 h; (f) Pd/C (10%; Degussa), H₂, THF, room temp., 24 h.

General Procedures for Oligopeptides Synthesis

THF (20 mL), and H-l-Glu(OBn)OMe·TFA salt [1.18 g,

3.24 mmol; prepared from Boc-l-Glu(OBn)OMe and TFA in

CH₂Cl₂with thioanisole (1 equiv.)] and NaHCO₃(0.95 g,

11.3 mmol) in 1:1 THF/H₂O (30 mL). ¹H NMR (300 MHz,

CDCl₃): δ = 8.16 (s, 1 H, CHO), 7.35–7.32 (m, 5 H, Ar-H), 7.03

(d, J = 7.7 Hz, 1 H, NH-Glu), 6.47 (d, J = 7.5 Hz, 1 H, NH-Met),

5.12 (s, 2 H, OCH₂Ph), 4.77–4.70 (m, 1 H, αCH-Met), 4.64–4.59

(m, 1 H, αCH-Glu), 3.74 (s, 3 H, OCH₃), 2.64–2.55 (m, 2 H, γCH₂-

Met), 2.52–2.43 (m, 2 H, γCH₂-Glu), 2.29–1.96 (unresolved m, 7

H, βCH₂-Glu, SCH₃, and βCH₂-Met) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 172.7 (δCO-Glu), 171.8 (αCO-Glu), 170.7 (CO-Met),

160.9 (CHO), 135.8 (1C-Ph), 128.7 (Ar-C), 128.5 (Ar-C), 128.4 (Ar-

C), 66.8 (OCH₂Ph), 52.7 (OCH₃), 52.0 (αC-Glu), 50.7 (αC-Met),

31.8 (βC-Met), 30.4 (γC-Glu), 29.9 (γC-Met), 27.0 (βC-Glu), 15.2

(SCH₃) ppm. HRMS: calcd. for C₁₉H₂₆N₂O₆S [M – H]^–411.1584;

found 411.1584.

Method A: DCC (1.3 equiv.) was added to a solution of N-Boc or

N-formyl amino acid (1 equiv.) and N-hydroxysuccinimide

(1.2 equiv.) in dry THF (ca. 4 mL/mmol) at 0 °C. The mixture was

stirred for 0.5 h at 0 °C, then for 2–6 h (monitoring by TLC) at

room temp. under an argon atmosphere. The resulting dicyclohex-

ylurea (DCU) was removed by filtration, and the filtrate was con-

centrated in vacuo to give the corresponding activated ester. This

compound was then dissolved in dry THF (2 mL/mmol), and a

suspension of a second amino acid (1.2 equiv.) and NaHCO₃

(4 equiv.) in water (2 mL/mmol) was then added to this solution at

0 °C under an argon atmosphere. The reaction mixture was stirred

at room temp. for 16 h, then it was cooled to 0 °C and neutralized

by the addition of HCl (1 n). The aqueous layer was extracted with

ethyl acetate (3ϫ), and the combined organic extracts were dried

with Na₂SO₄, filtered, and concentrated under reduced pressure.

The resulting crude residue was purified by column chromatog-

raphy on silica gel (gradient: CH₂Cl₂/MeOH, 98:2, v/v; 96:4, v/v;

94:6, v/v) to give the desired compound.

N-For-L-Met-L-Glu(OBn)-NH₂(1b): Following Method B, dipept-

ide 1b was obtained as a white solid (0.76 g, 68%) in two steps,

starting from N-For-l-Met-OH (0.5 g, 2.82 mmol), N-hydroxysuc-

cinimide (0.39 g, 3.38 mmol), and DCC (0.76 g, 3.67 mmol) in

THF (20 mL), and H-l-Glu(OBn)-NH₂·TFA salt [1.09 g,

3.24 mmol; prepared from Boc-l-Glu(OBn)-NH₂and TFA in

CH₂Cl₂with thioanisole (1 equiv.)] and NaHCO₃(0.95 g,

11.3 mmol) in 1:1 THF/H₂O (30 mL). ¹H NMR (300 MHz, [D₆]-

DMSO): δ = 8.32 (d, J = 8.2 Hz, 1 H, NH-Met), 8.05 (d, J =

8.0 Hz, 1 H, NH-Glu), 8.02 (s, 1 H, CHO), 7.38–7.34 (m, 5 H, Ar-

H), 7.30 (br. s, 1 H, NH-CONH₂), 7.09 (br. s, 1 H, NH-CONH₂),

5.08 (s, 2 H, OCH₂Ph), 4.43–4.36 (m, 1 H, αCH-Met), 4.24–4.16

(m, 1 H, αCH-Glu), 2.44 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.36 (t,

J = 7.8 Hz, 2 H, γCH₂-Glu), 2.02 (s, 3 H, SCH₃), 1.98–1.86 (m, 2

H, βCH₂-Glu), 1.81–1.71 (m, 2 H, βCH₂-Met) ppm. ¹³C NMR

Method B: A minor modification of Method A was made during

the work-up stage when the carboxyl group at the C-terminal in

the peptides was protected as an ester or amide. Thus, after amide

coupling with the activated ester, the reaction mixture was diluted

with NaHCO₃(10% aq.) and then extracted with ethyl acetate.

This made the purification step easier by removing the acidic by-

products.

N-For-L-Met-L-Glu(OBn)OMe (1a): Following Method B, dipept-

ide 1a was obtained as a white solid (1.01 g, 87%) in two steps,

starting from N-For-l-Met-OH (0.5 g, 2.82 mmol), N-hydroxysuc-

cinimide (0.39 g, 3.38 mmol), and DCC (0.76 g, 3.67 mmol) in

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Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

(75 MHz, [D₆]DMSO): δ = 172.7 (αCO-Glu), 172.1 (δCO-Glu), (35 mL), and H-l-Lys(Boc)OH (2.87 g, 11.6 mmol) and NaHCO₃

170.7 (CO-Met), 161.1 (CHO), 136.2 (1C-Ph), 128.4 (Ar-C), 128.0 (2.44 g, 29.1 mmol) in 1:1 THF/H₂O (40 mL). ¹H NMR (300 MHz,

(Ar-C), 127.9 (Ar-C), 65.5 (OCH₂Ph), 51.6 (αC-Glu), 50.6 (αC-

Met), 31.9 (βC-Met), 30.1 (γC-Glu), 29.3 (γC-Met), 27.1 (βC-Glu),

14.6 (SCH₃) ppm. HRMS: calcd. for C₁₈H₂₅N₃O₅S [M + Na]⁺

418.1407; found 418.1407.

[D₆]DMSO): δ = 12.16 (br. s, 1 H, CO₂H), 8.20–8.14 (m, 2 H, 2

NH), 8.06 (s, 1 H, CHO), 6.76 (t, J = 4.9 Hz, 1 H, εNH-Lys), 4.19–

4.12 (m, 1 H, αH-Lys), 3.79 (d, J = 7.5 Hz, 2 H, αH-Gly), 2.91–

2.85 (m, 2 H, εCH₂-Lys), 1.71–1.52 (m, 2 H, βCH₂-Lys), 1.39–1.33

(m, 11 H, tBu and δCH₂-Lys), 1.31–1.22 (m, 2 H, γCH₂-Lys) ppm.

HRMS: calcd. for C₁₄H₂₅N₃O₆[M + Na]⁺354.1636; found

354.1635.

N-For-Gly-L-Glu(OBn)OH (3a): Following Method A, dipeptide 3a

was obtained as a white solid (2.47 g, 79%) in two steps, starting

from N-For-Gly-OH (1.0 g, 9.70 mmol), N-hydroxysuccinimide

(1.34 g, 11.6 mmol), and DCC (2.6 g, 12.6 mmol) in THF (30 mL),

and H-l-Glu(OBn)-OH·TFA salt [3.92 g, 11.2 mmol; prepared

from Boc-l-Glu(OBn)-OH and TFA in CH₂Cl₂with thioanisole (1

equiv.)] and NaHCO₃(3.26 g, 38.8 mmol) in 1:1 THF/H₂O

N-For-L-Met-L-Lys(Boc)OH (3e): Following Method A, dipeptide

3e was obtained as a white solid (1.25 g, 84%), in two steps, starting

from N-For-l-Met-OH (0.65 g, 3.67 mmol), N-hydroxysuccinimide

(0.51 g, 4.40 mmol), and DCC (0.98 g, 4.77 mmol) in THF

(20 mL), and H-l-Lys(Boc)-OH (0.99 g, 4.03 mmol) and NaHCO₃

(1.23 g, 14.7 mmol) in 1:1 THF/H₂O (40 mL). ¹H NMR (300 MHz,

[D₆]DMSO): δ = 12.56 (br. s, 1 H, CO₂H), 8.28 (d, J = 8.3 Hz, 1

H, NH-Met), 8.24 (d, J = 7.6 Hz, 1 H, αNH-Lys), 8.00 (s, 1 H,

CHO), 6.76 (t, J = 5.3 Hz, 1 H, εNH-Lys), 4.50–4.43 (m, 1 H, αH-

Met), 4.15–4.08 (m, 1 H, αH-Lys), 2.91–2.85 (m, 2 H, εCH₂-Lys),

2.44 (t, J = 8.0 Hz, 2 H, γCH₂-Met), 2.03 (s, 3 H, -SCH₃), 1.95–

1.77 (m, 2 H, βCH₂-Met), 1.75–1.56 (m, 2 H, βCH₂-Lys), 1.36–

1.21 (m, 13 H, tBu, δCH₂-Lys, and γCH₂-Lys) ppm. ¹³C NMR

(75 MHz, [D₆]DMSO): δ = 173.4 (CO-Lys), 170.7 (CO-Met), 160.8

(CHO), 155.5 (OCONH), 77.2 (1C of tBu), 51.9 (αC-Lys), 50.1

(αC-Met), 39.6 (εC-Lys), 32.4 (βC-Met), 30.4 (βC-Lys), 29.2 (γC-

Met), 29.1 (δC-Lys), 28.2 (CH₃-tBu), 22.8 (γC-Lys), 14.6 (SCH₃)

ppm. HRMS: calcd. for C₁₇H₃₁N₃O₆S [M – H]^–404.1861; found

404.1861.

1

(60 mL). H NMR (300 MHz, [D₆]DMSO): δ = 12.73 (br. s, 1 H,

CO₂H), 8.23–8.21 (m, 2 H, 2 NH), 8.07 (s, 1 H, CHO), 7.37–7.34

(m, 5 H, Ar-H), 5.09 (s, 2 H, OCH₂Ph), 4.30–4.23 (m, 1 H, αCH-

Glu), 3.78 (app t, J = 5.3 Hz, 2 H, αCH₂-Gly), 2.43 (t, J = 7.7 Hz,

2 H, γCH₂-Glu), 2.10–1.78 (m, 2 H, βCH₂-Glu), 1.81–1.71 (m, 2

H, βCH₂-Met) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 173.0

(αCO-Glu), 172.1 (δCO-Glu), 168.5 (CO-Gly), 161.4 (CHO), 136.2

(1C-Ph), 128.4 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 65.5 (OCH₂Ph),

51.1 (αC-Glu), 40.3 (αC-Gly), 30.0 (γC-Glu), 26.3 (βC-Glu) ppm.

HRMS: calcd. for C₁₅H₁₈N₂O₆[M – H]^–321.1092; found 321.1090.

N-For-Gly-L-Glu(OtBu)OH (3b): Following Method A, dipeptide

3b was obtained as a white solid (2.79 g, 84%) in two steps, starting

from N-For-l-Gly-OH (1.00 g, 9.70 mmol), N-hydroxysuccinimide

(1.34 g, 11.6 mmol), and DCC (2.60 g, 12.6 mmol) in THF

(16 mL), and H-l-Glu(OtBu)-OH (2.46 g, 12.1 mmol) and

NaHCO₃(2.24 g, 26.7 mmol, 2.2 equiv.) in 1:1 THF/H₂O (30 mL).

¹H NMR (300 MHz, [D₄]methanol): δ = 8.18 (s, 1 H, CHO), 4.49–

4.45 (m, 1 H, αCH-Glu), 3.99 (br. s, 2 H, αCH₂-Gly), 2.34 (t, J =

7.5 Hz, 2 H, γCH₂-Glu), 2.21–2.09 (m, 1 H, βCH₂-Glu), 1.98–1.88

(m, 1 H, βCH₂-Glu), 1.43 [s, 9 H, C(CH₃)₃] ppm. ¹³C NMR

(75 MHz, [D₄]methanol): δ = 173.0 (CO-Glu), 172.1 (CO-Glu),

169.3 (CO-Gly), 162.6 (CHO), 80.2 (1C-tBu), 51.2 (αC-Glu), 40.2

(αC-Gly), 30.8 (γC-Glu), 26.7 (tBu), 26.2 (βC-Glu) ppm. HRMS:

calcd. for C₁₂H₁₉N₂O₆[M – H]^–287.1248; found 287.1245.

N-For-L-Met-L-Lys(Boc)OMe (3f): Following Method B, dipeptide

3f was obtained as a white solid (1.02 g, 86%), in two steps, starting

from N-For-l-Met-OH (0.5 g, 2.82 mmol), N-hydroxysuccinimide

(0.39 g, 3.39 mmol), and DCC (0.76 g, 3.67 mmol) in THF

(18 mL), and H-l-Lys(Boc)OMe·HCl salt (0.92 g, 3.10 mmol) and

1

NaHCO₃(0.83 g, 9.87 mmol) in 1:1 THF/H₂O (30 mL). H NMR

(300 MHz, [D₆]DMSO): δ = 8.39 (d, J = 7.2 Hz, 1 H, αNH-Lys),

8.29 (d, J = 8.2 Hz, 1 H, NH-Met), 8.01 (s, 1 H, CHO), 6.76 (t, J

= 5.4 Hz, 1 H, εNH-Lys), 4.51–4.44 (m, 1 H, αH-Met), 4.22–4.16

(m, 1 H, αH-Lys), 3.62 (s, 3 H, OCH₃), 2.91–2.85 (m, 2 H, εCH₂-

Lys), 2.44 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.04 (s, 3 H, SCH₃),

1.93–1.77 (m, 2 H, βCH₂-Met), 1.75–1.58 (m, 2 H, βCH₂-Lys), 1.37

(s, 9 H, tBu), 1.35–1.21 (m, 4 H, δCH₂-Lys and γCH₂-Lys) ppm.

¹³C NMR (75 MHz, [D₆]DMSO): δ = 172.4 (CO-Lys), 170.9 (CO-

Met), 160.8 (CHO), 155.5 (OCONH), 77.3 (1C of tBu), 52.0 (αC-

Lys), 51.8 (OCH₃), 50.0 (αC-Met), 39.6 (εC-Lys), 32.3 (βC-Met),

30.3 (βC-Lys), 29.2 (γC-Met), 29.0 (δC-Lys), 28.2 (CH₃-tBu), 22.7

(γC-Lys), 14.6 (SCH₃) ppm. HRMS: calcd. for C₁₈H₃₃N₃O₆S [M –

H]^–418.2017; found 418.2022.

N-For-Gly-L-β-amino-Ala(Fmoc)OH (3c): Following Method A, di-

peptide 3c was obtained as a white solid (1.54 g, 70.3%) in two

steps, starting from N-For-Gly-OH (0.55 g, 5.33 mmol), N-hy-

droxysuccinimide (0.74 g, 6.40 mmol), and DCC (1.43 g,

6.93 mmol) in THF (20 mL) and H-l-β-amino-Ala(Fmoc)-

OH·TFA salt [2.70 g, 6.13 mmol; prepared from Boc-l-β-amino-

Ala(Fmoc)-OH and TFA in CH₂Cl₂with thioanisole (1 equiv.)]

and NaHCO₃(1.79 g, 21.4 mmol) in 1:1 THF/H₂O (50 mL). ¹H

NMR (300 MHz, [D₆]DMSO): δ = 12.19 (br. s, 1 H, CO₂H), 8.24

(t, J = 5.0 Hz, 1 H, NH), 8.17 (d, J = 7.9 Hz, 1 H, NH), 8.08 (s, 1

H, CHO), 7.89 (d, J = 7.4 Hz, 2 H, ArH-Fmoc), 7.69 (d, J =

7.3 Hz, 2 H, ArH-Fmoc), 7.44–7.31 (m, 4 H, ArH-Fmoc), 4.35–

4.22 (unresolved m, 4 H, αH-β-amino-Ala, OCH₂-Fmoc, and CH-

Fmoc), 3.80 (d, J = 5.8 Hz, 2 H, αCH₂-Gly), 3.50–3.30 (m, 2 H,

βCH₂-β-amino-Ala merged with DMSO) ppm. ¹³C NMR

(75 MHz, [D₆]DMSO): δ = 171.5 (CO-β-amino-Ala), 168.5 (CO-

Gly), 161.4 (CHO), 156.3 (OCONH), 143.8 (Ar-C), 140.7 (Ar-C),

127.6 (Ar-C), 127.1 (Ar-C), 125.2 (Ar-C), 120.1 (Ar-C), 65.6

(OCH₂-Fmoc), 52.3 (αC-β-amino-Ala), 46.6 (CH-Fmoc), 41.5 (αC-

Gly), 30.7 (βC-β-amino-Ala) ppm. HRMS: calcd. for C₂₁H₂₁N₃O₆

[M – H]^–410.1357; found 410.1357.

Boc-L-Ala-L-Lys(Cbz)OH (11): Following Method A, dipeptide 11

was obtained as a white solid (1.63 g, 68%), in two steps, starting

from Boc-l-Ala-OH (1.0 g, 5.29 mmol), N-hydroxysuccinimide

(0.73 g, 6.35 mmol), and DCC (1.42 g, 6.88 mmol) in THF

(25 mL), and H-l-Lys(Cbz)OH (1.70 g, 6.08 mmol) and NaHCO₃

(1.78 g, 21.2 mmol) in 1:1 THF/H₂O (50 mL). ¹H NMR (300 MHz,

[D₆]DMSO): δ = 10.49 (br. s, 1 H, CO₂H), 7.80 (d, J = 7.6 Hz, 1

H, αNH-Lys), 7.29–7.23 (m, 5 H, Ar-H), 7.10 (t, J = 5.9 Hz, 1 H,

εNH-Lys), 6.77 (d, J = 7.5 Hz, 1 H, NH-Ala), 4.91 (s, 2 H,

OCH₂Ph), 4.10–4.03 (m, 1 H, αH-Lys), 3.93–3.86 (m, 1 H, αH-

Ala), 2.90–2.85 (m, 2 H, εCH₂-Lys), 1.64–1.41 (m, 2 H, βCH₂-

Lys), 1.35–1.20 (m, 11 H, CH₃-tBu and δCH₂-Lys), 1.16–0.93 (m,

5 H, CH₃-Ala and γCH₂-Lys) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO): δ = 173.6 (CO-Lys), 172.9 (CO-Ala), 156.2 (OCONH),

155.1 (OCONH), 137.4 (1C-Ph), 128.5 (Ar-C), 127.8 (Ar-C), 78.2

N-For-Gly-Lys(Boc)OH (3d): Following Method A, dipeptide 3d

was obtained as a white solid (2.60 g, 81%) in two steps, starting

from N-For-Gly-OH (1.0 g, 9.70 mmol), N-hydroxysuccinimide

(1.34 g, 11.6 mmol), and DCC (2.60 g, 12.6 mmol) in THF

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FULL PAPER

(1C of tBu), 65.2 (-OCH₂Ph), 51.7 (αC-Lys), 49.6 (αC-Ala), 40.2

(εC-Lys), 30.9 (βC-Lys), 29.1 (δC-Lys), 28.3 (CH₃-tBu), 22.6 (γC-

Lys), 18.2 (CH₃-Ala) ppm. HRMS: calcd. for C₂₂H₃₃N₃O₇[M –

H]^–450.2245; found 450.2236.

[D₆]DMSO): δ = 172.8 (δCO-Glu), 172.2 (CO-Ala), 170.8 (αCO-

Glu), 168.3 (CO-Gly), 161.4 (CHO), 136.2 (1C Ph), 128.4 (Ar-C),

128.0 (Ar-C), 127.9 (Ar-C), 65.5 (OCH₂Ph), 51.8 (OCH₃), 51.2

(αC-Glu), 47.6 (αC-Ala), 40.5 (αC-Gly), 29.8 (γC-Glu), 27.5 (βC-

Glu), 16.6 (CH₃-Ala) ppm. HRMS: calcd. for C₁₉H₂₅N₃O₇[M +

H]⁺408.1765; found 408.1765.

N-For-Met-L-Glu-OMe (2a): Pd/C (10%; Degussa; 0.5 g, 50%

w/w) was added to a stirred solution of 1a (1.00 g, 2.44 mmol) in

EtOH, and the mixture was hydrogenated at atmospheric pressure

using a balloon filled with H₂for 16 h. The catalyst was removed

by filtration, and the filtrate was concentrated under reduced pres-

sure. The resulting crude residue was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v;

97:3, v/v; 94:6, v/v) to give compound 2a (0.75 g, 96%) as a white

solid. ¹H NMR (300 MHz, [D₆]DMSO): δ = 12.15 (br. s, 1 H,

CO₂H), 8.43 (t, J = 7.4 Hz, 1 H, NH-Glu), 8.30 (d, J = 7.9 Hz, 1

H, NH-Met), 8.01 (s, 1 H, CHO), 4.89–4.41 (m, 1 H, αH-Met),

4.31–4.23 (m, 1 H, αH-Glu), 3.62 (s, 3 H, OCH₃), 2.44 (t, J =

7.9 Hz, 2 H, γCH₂-Met), 2.29 (t, J = 7.5 Hz, 2 H, γCH₂-Glu), 2.04

(s, 3 H, -SCH₃), 2.01–1.72 (unresolved m, 4 H, βCH₂-Glu and

βCH₂-Met) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 173.6

(δCO-Glu), 172.0 (αCO-Glu), 171.0 (CO-Met), 160.9 (CHO), 51.9

(OCH₃), 51.2 (αC-Glu), 50.1 (αC-Met), 32.1 (βC-Met), 29.8 (γC-

Glu), 29.2 (γC-Met), 25.9 (βC-Glu), 14.6 (SCH₃) ppm. HRMS:

calcd. for C₁₁H₂₀N₂O₆S [M – H]^–319.0969; found 319.0975.

N-For-Gly-L-Glu(OtBu)-L-Phe-OMe (4b): Following Method A,

tripeptide 4b was obtained as a white solid (2.29 g, 69%) in two

steps, starting from 3b (2.11 g, 7.32 mmol), N-hydroxysuccinimide

(1.01 g, 8.78 mmol), and DCC (1.96 g, 9.51 mmol) in THF

(14 mL), and H-l-Phe-OMe·HCl salt (2.00 g, 9.27 mmol) and

NaHCO₃(1.72 g, 20.5 mmol, 2.2 equiv.) in 1:1 THF/H₂O (40 mL).

¹H NMR (300 MHz, [D₄]methanol): δ = 8.18 (s, 1 H, CHO), 7.29–

7.20 (m, 5 H, Ar-H), 4.67 (dd, J = 8.5, 5.6 Hz, 1 H, αCH-Phe),

4.40 (dd, J = 8.3, 5.3 Hz, 1 H, αCH-Glu), 3.92 (br. s, 2 H, αCH₂-

Gly), 3.70 (s, 3 H, OCH₃), 3.21–3.15 (m, 1 H, βCH₂-Phe), 3.06–

2.98 (m, 1 H, βCH₂-Phe), 2.31–2.26 (m, 2 H, γCH₂-Glu), 2.04–2.00

(m, 1 H, βCH₂-Glu), 1.88–1.85 (m, 1 H, βCH₂-Glu), 1.46 [s, 9 H,

C(CH₃)₃] ppm. ¹³C NMR (75 MHz, [D₄]methanol): δ = 172.2

(δCO-Glu), 171.6 (CO), 171.4 (CO), 169.1 (CO), 162.5 (CHO),

136.3 (1C-Ph), 128.5 (Ar-C), 127.8 (Ar-C), 126.2 (Ar-C), 53.5, 52.0,

51.0, 40.3, 36.5, 30.7 (γC-Glu), 26.6 (βC-Glu), 26.5 (tBu) ppm.

HRMS: calcd. for C₂₂H₃₂N₃O₇[M + H]⁺450.2234; found

450.2233.

N-For-Met-L-Glu-NH₂(2b): Following a similar procedure to that

used for the synthesis of compound 2a, 1b (0.76 g, 2.44 mmol) was

hydrogenated in the presence of Pd/C (10%; Degussa; 0.38 g, 50%

w/w) in EtOH, and the resulting product was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:2, v/v;

97:4, v/v; 94:8, v/v) to give 2b (0.51 g, 86%) as a white solid. ¹H

NMR (300 MHz, [D₆]DMSO): δ = 12.09 (br. s, 1 H, CO₂H), 8.33

(d, J = 7.9 Hz, 1 H, NH-Met), 8.04–8.01 (m, NH-Glu and CHO),

7.29 (br. s, 1 H, NH-CONH₂), 7.07 (br. s, 1 H, NH-CONH₂), 4.44–

4.37 (m, 1 H, αH-Met), 4.21–4.14 (m, 1 H, αH-Glu), 2.44 (t, J =

7.9 Hz, 2 H, γCH₂-Met), 2.29 (t, J = 7.6 Hz, 2 H, γCH₂-Glu), 2.03

(s, 3 H, SCH₃), 1.97–1.85 (m, 2 H, βCH₂-Glu), 1.81–1.71 (m, 2 H,

βCH₂-Met) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 174.0

(δCO-Glu), 172.9 (αCO-Glu), 170.6 (CO-Met), 161.2 (CHO), 51.8

(αC-Glu), 50.6 (αC-Met), 31.9 (βC-Met), 30.3 (γC-Glu), 29.3 (γC-

Met), 27.2 (βC-Glu), 14.6 (SCH₃) ppm. HRMS: calcd. for

C₁₁H₁₉N₃O₅S [M – H]^–304.0972; found 304.0975.

N-For-Gly-

L

-β-amino-Ala(Fmoc)-

L

-Phe-OMe

(4c):

Following

Method B, tripeptide 4c was obtained as a white solid (1.42 g,

72.8%) in two steps, starting from 3c (1.4 g, 3.40 mmol), N-hy-

droxysuccinimide (0.47 g, 4.09 mmol), and DCC (0.91 g,

4.42 mmol) in THF (25 mL), and H-l-Phe-OMe·HCl salt (0.84 g,

3.91 mmol) and NaHCO₃(1.14 g, 13.6 mmol) in 1:1 THF/H₂O

(50 mL). ¹H NMR (500 MHz, [D₆]DMSO): δ = 8.41 (d, J = 7.6 Hz,

1 H, NH-Phe), 8.24 (t, J = 4.9 Hz, 1 H, NH-Gly), 8.09 (s, 1 H,

CHO), 8.02 (d, J = 8.1 Hz, 1 H, NH-β-amino-Ala), 7.88 (d, J =

7.6 Hz, 2 H, ArH-Fmoc), 7.69 (d, J = 7.5 Hz, 2 H, ArH-Fmoc),

7.43–7.15 (m, 9 H, ArH-Fmoc and ArH-Phe), 7.17 (t, J = 5.0 Hz,

1 H, βNH-β-amino-Ala), 4.51–4.47 (m, 1 H, αH-Phe), 4.55–4.42

(m, 1 H, αH-β-amino-Ala), 4.30–4.28 (m, 1 H, OCH₂-Fmoc), 4.24–

4.21 (m, 1 H, CH-Fmoc), 3.79 (d, J = 5.7 Hz, 2 H, αCH₂-Gly),

3.56 (s, 3 H, OCH₃), 3.35–3.15 (m, 2 H, βCH₂-β-amino-Ala), 3.06–

2.93 (m, 2 H, βCH₂-Phe) ppm. ¹³C NMR (125 MHz, [D₆]DMSO):

δ = 171.7 (CO-Phe), 169.7 (CO-β-amino-Ala), 168.5 (CO-Gly),

161.6 (CHO), 156.3 (OCONH), 143.9 (1C Fmoc), 140.8 (1C

Fmoc), 137.1 (1C Ph), 129.2 (Ar-C), 129.1 (Ar-C), 128.3 (Ar-C),

127.7 (Ar-C), 127.4 (Ar-C), 127.2 (Ar-C), 126.7 (Ar-C), 125.3 (Ar-

C), 121.5 (Ar-C), 120.1 (Ar-C), 65.7 (OCH₂-Fmoc), 53.8 (αC-Phe),

52.7 (αC-β-amino-Ala), 52.0 (OCH₃), 46.7 (CH-Fmoc), 42.3 (βC-

Phe), 40.7 (αC-Gly), 36.6 (βC-β-amino-Ala) ppm. HRMS: calcd.

for C₃₁H₃₂N₄O₇[M + H]⁺573.2343; found 573.2346.

N-For-L-Met-L-Lys(NH₂·TFA)-OMe (8): Trifluoroacetic acid

(1 mL) was slowly added to a stirred solution of 3f (162.9 mg,

0.388 mmol) and thioanisole (0.046 mL, 0.388 mmol) in CH₂Cl₂

(4 mL) at 0 °C. The mixture was then slowly warmed to room temp.

and stirred for 4 h. All the volatiles were removed under reduced

pressure, and the resulting residue was coevaporated with toluene

(3ϫ) to give compound 8 (168.3 mg, quantitative) as a colourless

foam, which was used directly in the next step without any further

purification.

N-For-Gly-L-Lys(Boc)-L-Phe-OMe (4d): Following Method B, tri-

N-For-Gly-

L

-Glu(OBn)-

L

-Ala-OMe (4a): Following Method B, tri-

peptide 4d was obtained as a white solid (1.82 g, 72%) in two steps,

starting from 3d (1.7 g, 5.13 mmol), N-hydroxysuccinimide (0.71 g,

6.16 mmol), and DCC (1.38 g, 6.67 mmol) in THF (35 mL), and

H-l-Phe-OMe·HCl salt (1.33 g, 6.16 mmol) and NaHCO₃(1.72 g,

20.52 mmol) in 1:1 THF/H₂O (50 mL). H NMR (500 MHz, [D₆]-

DMSO): δ = 8.37 (d, J = 7.4 Hz, 1 H, NH-Phe), 8.19 (t, J = 5.2 Hz,

1 H, NH-Gly), 8.06 (s, 1 H, CHO), 7.97 (d, J = 8.1 Hz, 1 H, NH-

peptide 4a was obtained as a white solid (2.04 g, 79%) in two steps,

starting from 3a (2.04 g, 6.33 mmol), N-hydroxysuccinimide

(0.87 g, 7.59 mmol), and DCC (1.70 g, 8.23 mmol) in THF

(36 mL), and H-l-Ala-OMe·HCl salt (1.02 g, 7.28 mmol) and

NaHCO₃(2.13 g, 25.32 mmol) in 1:1 THF/H₂O (40 mL). ¹H NMR

(300 MHz, [D₆]DMSO): δ = 8.43 (d, J = 6.8 Hz, 1 H, NH-Ala),

1

8.21 (t, J = 5.2 Hz, 1 H, NH-Gly), 8.10 (d, J = 8.1 Hz, 1 H, NH- Lys), 7.30–7.19 (m, 5 H, Ar-H), 6.73 (t, J = 5.1 Hz, 1 H, εNH-

Glu), 8.06 (s, 1 H, CHO), 7.38–7.32 (m, 5 H, Ar-H), 5.09 (s, 2 H,

OCH₂Ph), 4.40–4.33 (m, 1 H, αCH-Glu), 4.30–4.21 (m, 1 H, αCH-

Ala), 3.77 (d, J = 7.3 Hz, 2 H, αCH₂-Gly), 3.59 (s, 3 H, OCH₃),

2.41 (t, J = 8.1 Hz, 2 H, γCH₂-Glu), 2.02–1.75 (m, 2 H, βCH₂-

Lys), 4.49–4.41 (m, 1 H, αH-Phe), 4.31–4.24 (m, 1 H, αH-Lys),

3.76 (d, J = 5.7 Hz, 2 H, αH-Gly), 3.57 (s, 3 H, OCH₃), 3.06–2.92

(m, 2 H, βCH₂-Phe), 2.89–2.83 (m, 2 H, εCH₂-Lys), 1.61–1.43 (m,

2 H, βCH₂-Lys), 1.37–1.29 (m, 11 H, tBu and δCH₂-Lys), 1.26–

Glu), 1.29 (d, J = 7.3 Hz, 3 H, CH₃-Ala) ppm. ¹³C NMR (75 MHz, 1.13 (m, 2 H, γCH₂-Lys) ppm. ¹³C NMR (125 MHz, [D₆]DMSO):

2332

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Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

δ = 171.7 (CO-Phe), 171.6 (CO-Lys), 168.0 (CO-Gly), 161.4

βCH₂-Phe), 2.47 (t, J = 6.8 Hz, 2 H, γCH₂-Met), 2.04 (s, 3 H,

(CHO), 155.5 (OCONH), 137.1 (1C-Ph), 129.0 (Ar-C), 128.2 (Ar- SCH₃), 2.00–1.90 (m, 2 H, βCH₂-Met), 1.79–1.63 (m, 2 H, βCH₂-

C), 126.5 (Ar-C), 77.3 (1C of tBu), 53.6 (αC-Phe), 52.1 (αC-Lys),

51.8 (OCH₃), 40.4 (αC-Gly), 39.6 (εC-Lys), 36.5 (βC-Phe), 31.9

(βC-Lys), 29.3 (δC-Lys), 28.3 (CH₃-tBu), 22.4 (γC-Lys) ppm.

HRMS: calcd. for C₂₄H₃₆N₄O₇[M + H]⁺493.2656; found

493.2659.

Lys), 1.47–1.40 (m, 11 H, tBu and δCH₂-Lys), 1.35–1.28 (m, 2 H,

γCH₂-Lys) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 172.0 (CO-Phe),

171.5 (CO-Lys), 171.1 (CO-Met), 161.4 (CHO), 156.2 (OCONH),

136.0 (1C of Phe), 129.2 (Ar-C), 128.6 (Ar-C), 127.2 (Ar-C), 79.1

(1C of tBu), 53.7 (αC-Phe), 52.9 (αC-Lys), 52.4 (OCH₃), 50.6 (αC-

Met), 40.2 (εC-Lys), 37.9 (βC-Phe), 32.8 (βC-Met and βC-Lys),

30.0 (γC-Met), 29.6 (δC-Lys), 28.5 (CH₃-tBu), 22.6 (γC-Lys), 15.4

(SCH₃) ppm. HRMS: calcd. for C₂₇H₄₂N₄O₇S [M – H]^–565.2701;

found 565.2700.

N-For-L-Met-L-Lys(Boc)-L-Ala-OMe (4e): Following Method B,

tripeptide 4e was obtained as a white solid (1.50 g, 88.5%) in two

steps, starting from 3e (1.4 g, 3.45 mmol), N-hydroxysuccinimide

(0.48 g, 4.14 mmol), and DCC (0.93 g, 4.49 mmol) in THF

(30 mL), and H-l-Ala-OMe·HCl salt (0.53 g, 3.80 mmol) and

NaHCO₃(0.87 g, 10.36 mmol) in 1:1 THF/H₂O (40 mL). ¹H NMR

N-For-L-Met-L-Lys(Boc)-L-Lys(Boc)-OH (4h): Following Method

A, tripeptide 4h was obtained as a white solid (0.54 g, 62%) in two

(300 MHz, [D₆]DMSO): δ = 8.30 (t, J = 6.9 Hz, 1 H, NH), 8.27 steps, starting from 3e (0.56 g, 1.38 mmol), N-hydroxysuccinimide

(d, J = 9.4 Hz, 1 H, NH), 8.02–8.00 (m, 2 H, CHO and NH), 6.73 (0.19 g, 1.66 mmol), and DCC (0.37 g, 1.79 mmol) in THF

(t, J = 5.3 Hz, 1 H, εNH-Lys), 4.47–4.40 (m, 1 H, αH-Met), 4.29– (15 mL), and H-l-Lys(Boc)-OH (0.39 g, 1.59 mmol) and NaHCO₃

4.20 (m, 2 H, αH-Lys and αH-Ala), 3.61 (s, 3 H, OCH₃), 2.91–2.84 (0.46 g, 5.52 mmol) in 1:1 THF/H₂O (20 mL). ¹H NMR (300 MHz,

(m, 2 H, εCH₂-Lys), 2.42 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.02 (s,

3 H, SCH₃), 1.94–1.72 (m, 2 H, βCH₂-Met), 1.69–1.49 (m, 2 H,

βCH₂-Lys), 1.40–1.31 (m, 11 H, CH₃-tBu and δCH₂-Lys), 1.29–

[D₆]DMSO): δ = 12.51 (s, 1 H, CO₂H), 8.28 (d, J = 8.0 Hz, 1 H,

NH-Met), 8.04–8.02 (m, 3 H, 2 NH-Lys and CHO), 6.77–6.71 (m,

2 H, 2 εNH-Lys), 4.47–4.40 (m, 1 H, αH-Met), 4.28–4.24 (m, 1 H,

1.20 (m, 5 H, CH₃-Ala and γCH₂-Lys) ppm. ¹³C NMR (75 MHz, αH-Lys_N-ter), 4.23–4.21 (m, 1 H, αH-Lys_C-ter), 2.91–2.83 (m, 4 H,

[D₆]DMSO): δ = 172.9 (CO-Ala), 171.4 (CO-Lys), 170.5 (CO-Met), 2 εCH₂-Lys), 2.42 (t, J = 7.8 Hz, 2 H, γCH₂-Met), 2.02 (s, 3 H,

160.9 (CHO), 155.5 (OCONH), 77.3 (1C of tBu), 52.2 (αC-Lys),

51.8 (OCH₃), 50.3 (αC-Met), 47.5 (αC-Ala), 39.8 (εC-Lys), 32.2

(βC-Met), 31.7 (βC-Lys), 29.3 (γC-Met), 29.2 (δC-Lys), 28.2 (CH₃-

SCH₃), 1.90–1.70 (m, 2 H, βCH₂-Met), 1.69–1.48 (m, 4 H, 2 βCH₂-

Lys), 1.40–1.15 (m, 26 H, 2 CH₃-tBu, 2 δCH₂-Lys, and 2 γCH₂-

Lys) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 173.4 (CO-

tBu), 22.5 (γC-Lys), 16.8 (CH₃-Ala), 14.6 (SCH₃) ppm. HRMS: Lys_C-ter), 171.5 (CO-Met), 170.5 (CO-Lys_N-ter), 160.9 (CHO), 155.5

calcd. for C₂₁H₃₈N₄O₇S [M + Na]⁺513.2353; found 513.2352.

(2 OCONH), 77.3 (1C of tBu), 52.3 (αC-Lys_N-ter), 51.8 (αC-

Lys_C-ter), 50.3 (αC-Met), 39.6 (2 εC-Lys), 32.2 (βC-Met), 31.6 (βC-

Lys), 30.6 (βC-Lys), 29.3 (γC-Met), 29.1 (2 δC-Lys), 28.2 (CH₃-

tBu), 22.6 (2 γC-Lys), 14.6 (SCH₃) ppm. HRMS: calcd. for

C₂₈H₅₁N₅O₉S [M – H]^–632.3334; found 632.3334.

N-For- -Met- -Lys(Boc)- -Lys(Cbz)-OMe (4f): Following Method

L

B, tripeptide 4f was obtained as a white solid (1.07 g, 67%) in two

steps, starting from 3e (0.92 g, 2.27 mmol), N-hydroxysuccinimide

(0.31 g, 2.72 mmol), and DCC (0.61 g, 2.95 mmol) in THF

(20 mL), and H-l-Lys(Cbz)-OMe·HCl salt (0.86 g, 2.61 mmol) and N-For-L-Met-L-Lys(Boc)-L-Ala-OH (4i): Following Method A, tri-

1

NaHCO₃(0.76 g, 9.08 mmol) in 1:1 THF/H₂O (30 mL). H NMR

peptide 4i was obtained as a white solid (2.04 g, 85%) in two steps,

starting from 3e (2.04 g, 5.03 mmol), N-hydroxysuccinimide

(300 MHz, [D₆]DMSO): δ = 8.31–8.25 (m, 2 H, 2 NH), 8.02–8.00

(m, 2 H, CHO and NH), 7.36–7.30 (m, 5 H, Ar-H), 7.22 (t, J = (0.70 g, 6.04 mmol), and DCC (1.35 g, 6.54 mmol) in THF

5.1 Hz, 1 H, εNH-Lys_C-ter), 6.73 (t, J = 5.0 Hz, 1 H, εNH- (40 mL), and H-l-Ala-OH (0.49 g, 5.53 mmol) and NaHCO₃

Lys_N-ter), 5.00 (s, 2 H, OCH₂Ph), 4.47–4.40 (m, 1 H, αH-Met), (1.69 g, 20.12 mmol) in 1:1 THF/H₂O (50 mL). ¹H NMR

4.32–4.25 (m, 1 H, αH-Lys_N-ter), 4.22–4.15 (m, 1 H, αH-Lys_C-ter),

3.61 (s, 3 H, OCH₃), 2.99–2.93 (m, 2 H, εCH₂-Lys), 2.89–2.83 (m,

(300 MHz, [D₆]DMSO): δ = 12.52 (br. s, 1 H, CO₂H), 8.28 (d, J =

8.0 Hz, 1 H, αNH-Met), 8.13 (d, J = 7.1 Hz, 1 H, αNH-Ala), 8.03–

2 H, εCH₂-Lys), 2.41 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.02 (s, 3 H, 8.01 (m, 2 H, CHO and αNH-Lys), 6.73 (t, J = 4.4 Hz, 1 H, εNH-

SCH₃), 1.94–1.70 (m, 2 H, βCH₂-Met), 1.69–1.44 (m, 4 H, 2 βCH₂-

Lys), 1.40–1.17 (m, 17 H, CH₃-tBu, 2 δCH₂-Lys, and 2 γCH₂-Lys)

ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 172.4 (CO-Lys_C-ter),

171.6 (CO-Met), 170.4 (CO-Lys_N-ter), 161.0 (CHO), 156.0

(OCONH), 155.5 (OCONH), 137.3 (1C-Ph), 128.3 (Ar-C), 127.7

Lys), 4.47–4.40 (m, 1 H, αH-Met), 4.26–4.15 (m, 2 H, αH-Ala and

αH-Lys), 2.89–2.84 (m, 2 H, εCH₂-Lys), 2.43 (t, J = 7.8 Hz, 2 H,

γCH₂-Met), 2.03 (s, 3 H, -SCH₃), 1.93–1.70 (m, 2 H, βCH₂-Met),

1.63–1.48 (m, 2 H, βCH₂-Lys), 1.40–1.33 (m, 11 H, tBu and δCH₂-

Lys), 1.30–1.19 (m, 5 H, CH₃-Ala and γCH₂-Lys) ppm. ¹³C NMR

(Ar-C), 77.3 (1C of tBu), 65.1 (OCH₂Ph), 52.3 (αC-Lys_N-ter), 51.7 (75 MHz, [D₆]DMSO): δ = 172.0 (CO₂H), 171.2 (CO-Lys), 170.5

(OCH₃and αC-Lys_C-ter), 50.4 (αC-Met), 40.0 (εC-Lys), 32.1 (βC-

Met), 31.9 (βC-Lys), 30.5 (βC-Lys), 29.3 (γC-Met), 29.2 (δC-Lys),

28.8 (δC-Lys), 28.2 (CH₃-tBu), 22.6 (γC-Lys), 22.5 (γC-Lys), 14.5

(CO-Met), 161.0 (CHO), 155.5 (OCONH), 77.3 (1C of tBu), 52.2

(αC-Lys), 50.4 (αC-Met), 47.4 (αC-Ala), 40.2 (εC-Lys), 32.2 (βC-

Met), 31.7 (βC-Lys), 29.3 (γC-Met), 29.2 (δC-Lys), 28.3 (CH₃-tBu),

(SCH₃) ppm. HRMS: calcd. for C₃₂H₅₁N₅O₉S [M – H]^–680.3334; 22.6 (γC-Lys), 17.1 (CH₃-Ala), 14.6 (SCH₃) ppm. HRMS: calcd.

found 680.3336.

for C₂₀H₃₆N₄O₇S [M – H]^–475.2232; found 475.2231.

N-For- -Met- -Lys(Boc)-

L

L-Phe-OMe (4g): Following Method B,

Boc- -Ala- -Lys(Cbz)- -Ala-OMe (12): Following Method B, tri-

L

tripeptide 4g was obtained as a white solid (1.02 g, 86%) in two

steps, starting from 3e (0.5 g, 1.23 mmol), N-hydroxysuccinimide

(0.17 g, 1.48 mmol), and DCC (0.33 g, 1.60 mmol) in THF

(18 mL), and H-l-Phe-OMe·HCl salt (0.29 g, 1.36 mmol) and

NaHCO₃(0.41 g, 4.93 mmol) in 1:1 THF/H₂O (30 mL). H NMR

(300 MHz, CDCl₃): δ = 8.14 (s, 1 H, CHO), 7.69 (d, J = 8.1 Hz, 1

H, αNH-Lys), 7.60–7.57 (m, 2 H, NH-Met and NH-Phe), 7.27–

peptide 12 was obtained as a white solid (1.10 g, 75%) in two steps,

starting from 11 (1.23 g, 2.72 mmol), N-hydroxysuccinimide

(0.38 g, 3.27 mmol), and DCC (0.73 g, 3.54 mmol) in THF

(20 mL), and H-l-Ala-OMe·HCl (0.44 g, 3.13 mmol) and NaHCO₃

(0.92 g, 10.92 mmol) in 1:1 THF/H₂O (30 mL). ¹H NMR

(300 MHz, CDCl₃): δ = 7.33–7.29 (m, 5 H, Ar-H), 6.99–6.94 (m, 2

H, 2 NH), 5.28–5.22 (m, 2 H, 2 NH), 5.08 (s, 2 H, OCH₂Ph), 4.57–

1

7.11 (m, 5 H, Ar-H Phe), 5.03–4.98 (m, 1 H, εNH-Lys), 4.96–4.88 4.44 (m, 2 H, αH-Lys and αH-Ala_C-ter), 4.20–4.16 (m, 1 H, αH-

(m, 1 H, αH-Met), 4.84–4.77 (m, 1 H, αH-Phe), 4.74–4.69 (m, 1 H, Ala_N-ter), 3.69 (s, 3 H, OCH₃), 3.21–3.14 (m, 2 H, εCH₂-Lys), 1.89–

αH-Lys), 3.69 (s, 3 H, OCH₃), 3.11–3.01 (m, 4 H, εCH₂-Lys and 1.61 (m, 2 H, βCH₂-Lys), 1.55–1.47 (m, 2 H, δCH₂-Lys), 1.42 (s, 9

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2333

S. De, E. Groaz, P. Herdewijn

H, CH₃-tBu), 1.39–1.36 (m, 5 H, CH₃-Ala_C-terand γCH₂-Lys), 1.32 quantitative) as a colourless foam, which was used directly in the

FULL PAPER

(d, J = 7.0 Hz, 3 H, CH₃-Ala_N-ter) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 173.3 (CO-Ala_C-ter), 172.9 (CO-Ala_N-ter), 173.3 (CO-

Lys), 156.8 (OCONH), 155.7 (OCONH), 136.7 (1C-Ph), 128.6 (Ar-

C), 128.3 (Ar-C), 128.2 (Ar-C), 80.3 (1C of tBu), 66.7 (OCH₂Ph),

52.8 (αC-Lys), 52.5 (OCH₃), 50.3 (αC-Ala_N-ter), 48.2 (αC-Ala_C-ter),

40.4 (εC-Lys), 31.9 (βC-Lys), 29.3 (δC-Lys), 28.4 (CH₃-tBu), 22.2

(γC-Lys), 18.4 (CH₃-Ala_N-ter), 18.0 (CH₃-Ala_C-ter) ppm. HRMS:

calcd. for C₂₆H₄₀N₄O₈[M + H]⁺537.2919; found 537.2916.

next step without any further purification.

N-For- -Met- -Lys(NH₂·TFA)- -Ala-OMe (5e): Following a sim-

L

ilar procedure to that used for the synthesis of 8, compound 4e

(200 mg, 0.407 mmol), thioanisole (0.048 mL, 0.407 mmol), and

trifluoroacetic acid (2 mL) in CH₂Cl₂(6 mL) gave 5e (205.5 mg,

quantitative) as a colourless foam, which was used directly in the

next step without any further purification.

N-For-L-Met-L-Lys(NH₂·TFA)-L-Lys(Cbz)-OMe (5f): Following a

N-For-Gly-L-Glu-L-Ala-OMe (5a): Following a similar procedure

similar procedure to that used for the synthesis of 8, compound

4f (400 mg, 0.587 mmol), thioanisole (0.07 mL, 0.587 mmol), and

trifluoroacetic acid (3 mL) in CH₂Cl₂(9 mL) gave 5f (408.1 mg,

quantitative) as a colourless foam, which was used directly in the

next step without any further purification.

to that used for the synthesis of 2a, compound 4a (2.00 g,

4.91 mmol) was hydrogenated in the presence of Pd/C (10%; De-

gussa; 0.20 g, 10% w/w) in MeOH (60 mL) to give 5a (1.42 g, 91%)

as a white solid after purification. ¹H NMR (300 MHz, [D₆]-

DMSO): δ = 12.09 (br. s, 1 H, γCO₂H-Glu), 8.41 (d, J = 6.8 Hz, 1

H, NH-Ala), 8.21 (t, J = 5.2 Hz, 1 H, NH-Gly), 8.09–8.06 (m, 2

H, NH-Glu and CHO), 4.36–4.29 (m, 1 H, αCH-Glu), 4.27–4.20

(m, 1 H, αCH-Ala), 3.77 (d, J = 5.8 Hz, 2 H, αCH₂-Gly), 3.61 (s,

3 H, OCH₃), 2.25 (t, J = 8.1 Hz, 2 H, γCH₂-Glu), 1.95–1.67 (m, 2

H, βCH₂-Glu), 1.28 (d, J = 7.0 Hz, 3 H, CH₃-Ala) ppm. ¹³C NMR

(75 MHz, [D₆]DMSO): δ = 174.0 (δCO-Glu), 172.9 (CO-Ala),

171.0 (αCO-Glu), 168.3 (CO-Gly), 161.5 (CHO), 51.9 (OCH₃), 51.4

(αC-Glu), 47.6 (αC-Ala), 40.5 (αC-Gly), 29.9 (γC-Glu), 27.6 (βC-

Glu), 16.7 (CH₃-Ala) ppm. HRMS: calcd. for C₁₂H₁₉N₃O₇[M –

H]^–316.1150; found 316.1152.

N-For-L-Met-L-Lys(NH₂·TFA)-L-Phe-OMe (5g): Following a sim-

ilar procedure to that used for the synthesis of 8, compound 4g

(220 mg, 0.388 mmol), thioanisole (0.046 mL, 0.388 mmol), and

trifluoroacetic acid (1 mL) in CH₂Cl₂(4 mL) gave 5g (225.4 mg,

quantitative) as a colourless foam, which was used directly in the

next step without any further purification.

Boc-L-Ala-L-Lys-L-Ala-OMe (13): Following a similar procedure to

that used for the synthesis of 2a, compound 12 (1.10 g, 2.05 mmol)

was hydrogenated in the presence of Pd/C (10%; Degussa; 0.11 g,

10% w/w) in EtOH (40 mL) to give 13 (0.82 g, quantitative) as a

colourless foam, which was used directly in the next step without

any further purification. ¹H NMR (500 MHz, [D₆]DMSO): δ =

8.39 (d, J = 6.4 Hz, 1 H, NH-Ala_C-ter), 7.73 (d, J = 8.0 Hz, 1 H,

NH-Lys), 6.96 (d, J = 7.4 Hz, 1 H, NH-Ala_N-ter), 4.44 (br. s, 2 H,

εNH₂-Lys), 4.29–4.204 (m, 2 H, αH-Lys and αH-Ala_C-ter), 4.00–

3.92 (m, 1 H, αH-Ala_N-ter), 3.60 (s, 3 H, OCH₃), 2.57 (t, J = 6.9 Hz,

2 H, εCH₂-Lys), 1.69–1.45 (m, 2 H, βCH₂-Lys), 1.45–1.36 (m, 11

H, δCH₂-Lys and CH₃-tBu), 1.28–1.21 (m, 5 H, CH₃-Ala_C-terand

γCH₂-Lys), 1.15 (d, J = 7.1 Hz, 3 H, CH₃-Ala_N-ter) ppm. ¹³C NMR

(125 MHz, CDCl₃): δ = 172.9 (CO-Ala_C-ter), 172.4 (CO-Ala_N-ter),

171.4 (CO-Lys), 155.0 (OCONH), 78.1 (1C of tBu), 51.8 (αC-Lys

and OCH₃), 49.7 (αC-Ala_N-ter), 47.5 (αC-Ala_C-ter), 40.4 (εC-Lys),

32.1 (βC-Lys), 30.7 (δC-Lys), 28.1 (CH₃-tBu), 22.1 (γC-Lys), 18.0

N-For-Gly-L-Glu-L-Phe-OMe (5b): A solution of 4b (0.13 g,

0.29 mmol) in CH₂Cl₂(4 mL) was cooled to 0 °C, and trifluoro-

acetic acid (1.33 mL) was then added. The reaction mixture was

stirred for 15 min at 0 °C, and then for 2 h at room temp. Toluene

(6 mL) was then added, and the volatiles were removed in vacuo.

The crude residue was purified by column chromatography on sil-

ica gel (gradient CH₂Cl₂/MeOH, 98:1, v/v; 96:5, v/v; 94:7, v/v,

94:10, v/v) to give 5b (0.11 g, quant.) as a white foam. ¹H NMR

(300 MHz, [D₄]methanol): δ = 8.08 (s, 1 H, CHO), 7.21–7.10 (m,

5 H, Ar-H), 4.61–4.56 (m, 1 H, αCH-Phe), 4.35 (dd, J = 8.3, 5.6 Hz,

1 H, αCH-Glu), 3.83 (br. s, 2 H, αCH₂-Gly), 3.59 (s, 3 H, OCH₃),

3.11–3.04 (m, 1 H, βCH₂-Phe), 2.96–2.88 (m, 1 H, βCH₂-Phe), 2.26

(t, J = 8.0 Hz, 2 H, γCH₂-Glu), 1.97–1.93 (m, 1 H, βCH₂-Glu),

1.83–1.79 (m, 1 H, βCH₂-Glu) ppm. ¹³C NMR (75 MHz, [D₄]-

methanol): δ = 173.5 (δCO-Glu), 170.2 (CO), 169.9 (CO), 167.6

(CO), 161.1 (CHO), 134.8 (1C-Ph), 127.0 (Ar-C), 126.3 (Ar-C),

124.6 (Ar-C), 50.6, 49.5, 46.6, 38.8, 34.9, 27.8 (γC-Glu), 25.1 (βC-

Glu) ppm. HRMS: calcd. for C₁₈H₂₂N₃O₇[M – H]^–392.1463;

found 392.1462.

(CH₃-Ala_N-ter), 16.8 (CH₃-Ala_C-ter

) ppm. HRMS: calcd. for

C₁₈H₃₄N₄O₆[M + H]⁺403.2551; found 403.2543.

N-For- -Met- -Lys(Boc)- -Ala- -Ala-OH (6a): Following Method

L

A, tetrapeptide 6a was obtained as a white solid (0.99 g, 86%) in

one pot without filtration of the DCU, starting from 4i (1.0 g,

2.10 mmol), N-hydroxysuccinimide (0.29 g, 2.52 mmol), and DCC

(0.56 g, 2.73 mmol) in THF (18 mL), and H-l-Ala-OH (0.21 g,

2.31 mmol) and NaHCO₃(0.70 g, 8.39 mmol) in 1:1 THF/H₂O

N-For-Gly-L-β-amino-Ala-L-Phe-OMe (5c): A solution of 4c

(0.92 g, 10.92 mmol) in Et₃N/CH₂Cl₂(2:1; 21 mL) was stirred at

room temp. for 72 h. After the reaction was complete, the mixture

was concentrated under reduced pressure, and the crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 98:2, v/v; 96:4, v/v; 94:6, v/v) to give 5c (0.57 g,

1

(40 mL). H NMR (300 MHz, [D₆]DMSO): δ = 12.55 (br. s, 1 H,

CO₂H), 8.28 (d, J = 8.0 Hz, 1 H, αNH-Met), 8.10–8.03 (m, 2 H,

αNH-Lys and αNH-Ala_N-ter), 8.02 (s, 1 H, CHO), 7.94 (d, J =

7.3 Hz, 1 H, αNH- Ala_C-ter), 6.73 (t, J = 5.3 Hz, 1 H, εNH-Lys),

4.47–4.40 (m, 1 H, αH-Met), 4.31–4.27 (m, 1 H, αH-Ala_C-ter), 4.24–

4.16 (m, 2 H, αH-Lys and αH-Ala_N-ter), 2.89–2.84 (m, 2 H, εCH₂-

Lys), 2.43 (t, J = 7.8 Hz, 2 H, γCH₂-Met), 2.03 (s, 3 H, SCH₃),

1.95–1.73 (m, 2 H, βCH₂-Met), 1.68–1.44 (m, 2 H, βCH₂-Lys),

1.40–1.33 (m, 11 H, tBu and δCH₂-Lys), 1.32–1.17 (m, 8 H, 2 CH₃-

Ala and γCH₂-Lys) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ =

173.9 (CO-Ala_C-ter), 171.8 (CO-Lys), 171.0 (CO-Ala_N-ter), 170.6

(CO-Met), 160.9 (CHO), 155.5 (OCONH), 77.3 (1C of tBu), 52.5

(αC-Lys), 50.4 (αC-Met), 47.7 (αC-Ala_C-ter), 47.4 (αC-Ala_N-ter),

40.2 (εC-Lys), 32.2 (βC-Met), 31.5 (βC-Lys), 29.3 (γC-Met), 29.2

(δC-Lys), 28.3 (CH₃-tBu), 22.7 (γC-Lys), 18.1 (CH₃-Ala_C-ter), 17.2

(CH₃-Ala_N-ter), 14.6 (SCH₃) ppm. HRMS: calcd. for C₂₃H₄₁N₅O₈S

1

68%) as a colourless foam. H NMR (300 MHz, [D₆]DMSO): δ =

8.45–8.43 (m, 1 H, NH-Phe), 8.20–8.19 (m, 1 H, NH-Gly), 8.07–

7.99 (m, 2 H, CHO and NH-β-amino-Ala), 7.29–7.15 (m, 5 H,

ArH-Phe), 4.50–4.42 (m, 1 H, αH-Phe), 4.31–4.29 (m, 1 H, αH-β-

amino-Ala), 3.69 (d, J = 5.7 Hz, 2 H, αCH₂-Gly), 3.58 (s, 3 H,

OCH₃), 3.41 (br. s, 2 H, βNH₂-β-amino-Ala), 3.33–3.08 (m, 2 H,

βCH₂-β-amino-Ala), 3.03–2.88 (m, 2 H, βCH₂-Phe) ppm. HRMS:

calcd. for C₁₆H₂₂N₄O₅[M + H]⁺351.1663; found 351.1667.

N-For-Gly-L-Lys(NH₂·TFA)-L-Phe-OMe (5d): Following a similar

procedure to that used for the synthesis of 8, compound 4d

(265.6 mg, 0.539 mmol), thioanisole (0.063 mL, 0.539 mmol), and

trifluoroacetic acid (3 mL) in CH₂Cl₂(9 mL) gave 5d (272.9 mg, [M + Na]⁺570.2568; found 570.2571.

2334

www.eurjoc.org

Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

N-For- -Met- -Lys(Boc)- -Ala- -Phe-OMe

Method B, tetrapeptide 6b was obtained as a white solid (0.55 g,

82%) in one pot without filtration of the DCU, starting from 4i

(0.5 g, 1.05 mmol), N-hydroxysuccinimide (0.15 g, 1.26 mmol), and

L

(6b):

Following

5Ј-O-(4-Monomethoxytrityl)-2Ј-deoxythymidine (14):^[23]Triethyl-

amine (3.61 mL, 25.8 mmol) was added to a stirred solution of thy-

midine (2.50 g, 10.32 mmol) in dry DMF (26 mL). DMAP (0.10 g,

0.825 mmol) and 4-monomethoxytrityl chloride (6.37 g,

DCC (0.28 g, 1.36 mmol) in THF (10 mL), and H-l-Phe-OMe·HCl 20.64 mmol) were then added, and the resulting mixture was stirred

(0.25 g, 1.15 mmol) and NaHCO₃(0.26 g, 3.15 mmol) in 1:1 THF/

at room temp. for 4 h. The reaction mixture was quenched with

H₂O (30 mL). ¹H NMR (300 MHz, [D₆]DMSO): δ = 8.29–8.24 (m, water (260 mL), and the aqueous layer was extracted with ethyl

2 H, αNH-Met and αNH-Phe), 8.05–8.02 (m, 2 H, CHO and αNH- acetate (3ϫ 150 mL). The combined organic extracts were dried

Lys), 7.90 (d, J = 7.4 Hz, 1 H, αNH-Ala), 7.30–7.19 (m, 5 H, Ar-

with Na₂SO₄, filtered, and concentrated under reduced pressure.

H Phe), 6.73 (t, J = 5.7 Hz, 1 H, εNH-Lys), 4.49–4.44 (m, 1 H, The crude residue was purified by column chromatography on sil-

αH-Met), 4.32–4.27 (m, 1 H, αH-Ala), 4.25–4.19 (m, 1 H, αH-Lys), ica gel (gradient hexane/EtOAc, 2:1, v/v; 1:1, v/v; 1:2, v/v) to give 14

3.57 (s, 3 H, OCH₃), 3.05–2.94 (m, 2 H, βCH₂-Phe), 2.92–2.82 (m, (5.14 g, 97%) as a colourless foam. ¹H NMR (300 MHz, CDCl₃): δ

2 H, εCH₂-Lys), 2.43 (t, J = 7.8 Hz, 2 H, γCH₂-Met), 2.02 (s, 3

= 8.79 (br. s, 1 H, NH), 7.57 (d, J = 1.1 Hz, 1 H, 6-H), 7.42–7.39

H, -SCH₃), 1.95–1.73 (m, 2 H, βCH₂-Met), 1.65–1.44 (m, 2 H, (m, 4 H, Ar-H), 7.33–7.22 (m, 8 H, Ar-H), 6.86–6.83 (m, 2 H, Ar-

βCH₂-Lys), 1.40–1.24 (m, 11 H, tBu and δCH₂-Lys), 1.24–1.13 (m,

5 H, CH₃-Ala and γCH₂-Lys) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO): δ = 172.2 (CO-Ala), 171.7 (CO-Phe), 171.0 (CO-Lys),

170.6 (CO-Met), 160.9 (CHO), 155.5 (OCONH), 136.9 (1C of Phe),

129.0 (Ar-C), 128.2 (Ar-C), 126.5 (Ar-C), 77.3 (1C of tBu), 53.5

(αC-Phe), 52.4 (αC-Lys), 51.8 (OCH₃), 50.4 (αC-Met), 47.8 (αC-

Ala), 40.2 (εC-Lys), 36.6 (βC-Phe), 32.2 (βC-Met), 31.5 (βC-Lys),

29.3 (γC-Met), 29.2 (δC-Lys), 28.2 (CH₃-tBu), 22.7 (γC-Lys), 18.2

(CH₃-Ala), 14.6 (SCH₃) ppm. HRMS: calcd. for C₃₀H₄₇N₅O₈S [M

+ Na]⁺660.3038; found 660.3049.

H), 6.42 (dd, J = 7.6, 6.1 Hz, 1 H, 1Ј-H), 4.59–4.57 (m, 1 H, 3Ј-

H), 4.08–4.05 (m, 1 H, 4Ј-H), 3.79 (s, 3 H, OCH₃), 3.49–3.35 (m,

2 H, 5Ј-H and 5ЈЈ-H), 2.44–2.29 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.47 (d,

J = 1.1 Hz, 3 H, CH₃) ppm. HRMS: calcd. for C₃₀H₃₀N₂O₆[M +

Na]⁺537.1996; found 537.1998.

5Ј-O-(4-Monomethoxytrityl)-3Ј-O-(4-nitrophenyl carbonate)-2Ј-de-

oxythymidine (15): Compound 14 (1.00 g, 1.95 mmol) was coevapo-

rated with dry pyridine (2ϫ 5 mL), then it was redissolved in dry

pyridine (5 mL). A suspension of 4-nitrophenyl chloroformate

(0.432 g, 2.14 mmol) in a mixture of CH₂Cl₂/pyridine (70:1; 5 mL)

was slowly added to this solution at 0 °C. The reaction mixture was

then stirred overnight at room temp. The volatiles were removed

under reduced pressure, and the resulting residue was redissolved

in CH₂Cl₂(4 mL). This solution was slowly added into vigorously

stirring diethyl ether (30 mL). The resulting precipitate was filtered

off, and the filtrate was poured into stirring hexane (100 mL) to

give a white precipitate, which was filtered and dried to give 15

N-For-L-Met-L-Lys(NH₂·TFA)-L-Ala-L-Phe-OMe (9): Following a

similar procedure to that used for the synthesis of 8, compound

6b (248 mg, 0.388 mmol), thioanisole (0.046 mL, 0.388 mmol), and

trifluoroacetic acid (1 mL) in CH₂Cl₂(4 mL) gave 9 (253 mg, quan-

titative) as a colourless foam, which was used directly in the next

step without any further purification.

1

N-For-L-Met-L-Lys(Boc)-L-Ala-L-Ala-L-Phe-OMe (7): Following

(0.87 g, 66%) as a white solid. H NMR (300 MHz, CDCl₃): δ =

Method B, pentapeptide 7 was obtained as a white solid (0.87 g,

81%) in two steps, starting from 6a (0.83 g, 1.51 mmol), N-hydroxy-

succinimide (0.21 g, 1.82 mmol), and DCC (0.41 g, 1.97 mmol) in

THF (20 mL), and H-l-Phe-OMe·HCl (0.36 g, 1.67 mmol) and

8.81 (br. s, 1 H, NH), 8.31–8.27 (m, 2 H, Ar-H), 7.61 (s, 1 H, 6-

H), 7.39–7.26 (m, 14 H, Ar-H), 6.87–6.84 (m, 2 H, Ar-H), 6.39 (dd,

J = 8.8, 5.6 Hz, 1 H, 1Ј-H), 5.51–5.45 (m, 1 H, 3Ј-H), 4.32 (br. s,

1 H, 4Ј-H), 3.79 (s, 3 H, OCH₃), 3.60–3.47 (m, 2 H, 5Ј-H and 5ЈЈ-

H), 2.79–2.47 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.43 (s, 3 H, CH₃) ppm.

¹³C NMR (75 MHz, CDCl₃): δ = 163.6, 159.1, 155.2, 152.0, 150.5,

145.7, 143.7, 135.2, 134.6, 130.5, 128.5, 128.4, 128.3, 127.6, 125.5,

122.2, 121.8, 113.5 112.0, 84.4, 83.6, 80.5, 63.9, 55.4, 38.0,

11.8 ppm. HRMS: calcd. for C₃₇H₃₃N₃O₁₀[M + Na]⁺702.2058;

found 702.2081.

1

NaHCO₃(0.38 g, 4.54 mmol) in 1:1 THF/H₂O (38 mL). H NMR

(300 MHz, [D₆]DMSO): δ = 8.28 (d, J = 8.3 Hz, 1 H, αNH-Met),

8.23 (d, J = 7.6 Hz, 1 H, αNH-Phe), 8.06 (d, J = 7.7 Hz, 1 H,

αNH-Lys), 8.02 (s, 1 H, CHO), 7.96 (d, J = 7.2 Hz, 1 H, αNH-

Ala), 7.85 (d, J = 7.7 Hz, 1 H, αNH-Ala), 7.30–7.19 (m, 5 H, Ar-

H Phe), 6.73 (t, J = 5.4 Hz, 1 H, εNH-Lys), 4.49–4.40 (m, 2 H,

αH-Met and αH-Phe), 4.30–4.20 (unresolved m, 3 H, 2 αH-Ala and

αH-Lys), 3.58 (s, 3 H, OCH₃), 3.06–2.93 (m, 2 H, βCH₂-Phe), 2.90–

2.84 (m, 2 H, εCH₂-Lys), 2.43 (t, J = 7.7 Hz, 2 H, γCH₂-Met), 2.03

(s, 3 H, SCH₃), 1.95–1.73 (m, 2 H, βCH₂-Met), 1.72–1.47 (m, 2 H,

βCH₂-Lys), 1.40–1.31 (m, 11 H, tBu and δCH₂-Lys), 1.24–1.12 (m,

8 H, 2 CH₃-Ala and γCH₂-Lys) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO): δ = 172.2 (CO-Ala_C-ter), 171.7 (CO-Phe), 171.5 (CO-

Ala_N-ter), 171.2 (CO-Lys), 170.6 (CO-Met), 161.0 (CHO), 155.5

(OCONH), 137.0 (1C of Phe), 129.0 (Ar-C), 128.2 (Ar-C), 126.5

(Ar-C), 77.3 (1C of tBu), 53.5 (αC-Phe), 52.5 (αC-Lys), 51.8

(OCH₃), 50.4 (αC-Met), 47.9 (αC-Ala), 47.9 (αC- Ala_C-ter), 47.7

(αC- Ala_N-ter), 40.2 (εC-Lys), 36.6 (βC-Phe), 32.2 (βC-Met), 31.5

(βC-Lys), 29.3 (γC-Met), 29.2 (δC-Lys), 28.3 (CH₃-tBu), 22.7 (γC-

Lys), 18.2 (CH₃-Ala), 17.9 (CH₃-Ala), 14.6 (SCH₃) ppm. HRMS:

calcd. for C₃₃H₅₂N₆O₉S [M – H]^–707.3443; found 707.3451.

5Ј-O-(4-Monomethoxytrityl)-3Ј-O-[N-For-Gly-

L-Lys(ε-carb-

amate)- -Phe-OMe]-2Ј-deoxythymidine (16): Compound 15

L

(132 mg, 0.195 mmol) in dry CH₂Cl₂was added to a stirred solu-

tion of tripeptide 5d (110 mg, 0.217 mmol) and Et₃N (0.105 mL,

0.868 mmol) in dry CH₂Cl₂(5 mL) at 0 °C, and the stirring was

continued at room temp. for 16 h. The reaction mixture was then

diluted with CH₂Cl₂and washed twice with NaHCO₃(10% aq.).

The organic layer was dried with Na₂SO₄, filtered, and concen-

trated under reduced pressure. The crude residue was purified by

column chromatography on silica gel (gradient CH₂Cl₂/EtOH,

99:1, v/v; 97:3, v/v; 94:6, v/v) to give 16 (160 mg, 79%) as a white

solid. ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.39 (br. s, 1 H, NH),

8.40 (d, J = 7.4 Hz, 1 H, NH-Phe), 8.22 (t, J = 5.4 Hz, 1 H, NH-

Gly), 8.06 (s, 1 H, CHO), 7.99 (d, J = 8.2 Hz, 1 H, NH-Lys), 7.53

(s, 1 H, 6-H), 7.53–7.19 (m, 18 H, Ar-H and εNH-Lys), 6.23 (dd,

J = 7.8, 6.5 Hz, 1 H, 1Ј-H), 5.24–5.23 (m, 1 H, 3Ј-H), 4.48–4.41

(m, 1 H, αH-Phe), 4.31–4.25 (m, 1 H, αH-Lys), 4.05 (br. s, 1 H, 4Ј-

H), 3.75 (s, 2 H, CH₂-Gly), 3.56 (s, 3 H, OCH₃), 3.39–3.20 (m, 2

N-For-

lowing a similar procedure to that used for the synthesis of 8, com-

pound (275 mg, 0.388 mmol), thioanisole (0.046 mL,

L-Met-L-Lys(NH₂·TFA)-L-Ala-L-Ala-L-Phe-OMe (10): Fol-

7

0.388 mmol), and trifluoroacetic acid (1 mL) in CH₂Cl₂(4 mL) H, 5Ј-H and 5ЈЈ-H), 2.99–2.94 (m, 4 H, εCH₂-Lys and βCH₂-Phe),

gave 10 (281 mg, quantitative) as a colourless foam, which was used

directly in the next step without any further purification.

2.47–2.25 (m, 1 H, 2Ј-H and 2ЈЈ-H), 1.77 (s, 3 H, CH₃-Thy), 1.62–

1.55 (m, 2 H, βCH₂-Lys), 1.54–1.45 (m, 5 H, δCH₂-Lys and CH₃-

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2335

S. De, E. Groaz, P. Herdewijn

FULL PAPER

Thy), 1.37–1.24 (m, 2 H, γCH₂-Lys) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO): δ = 171.7, 171.6, 168.1, 163.6, 161.4, 158.3, 155.2, 150.4,

144.1, 143.8, 137.1, 135.4, 134.6, 130.1, 129.0, 128.2, 127.9, 127.1,

126.5, 113.3, 109.9, 83.6, 83.2, 74.2, 63.9, 55.1, 63.6, 52.1, 51.7,

40.4, 36.8, 36.5, 31.9, 29.1, 22.4, 11.6 ppm. HRMS: calcd. for

C₅₀H₅₆N₆O₁₂[M + Na]⁺955.3848; found 955.3813.

84.5, 84.2, 75.9, 63.9, 55.3, 38.2, 11.8 ppm. HRMS: calcd. for

C₃₇H₃₄N₂O₇[M + Na]⁺641.2258; found 641.2257.

5Ј-Hydroxy-3Ј-O-benzoyl-2Ј-deoxythymidine (21):^[52]A solution of

20 (3.35 g, 5.41 mmol) in AcOH (80%; 40 mL) was stirred at room

temp. for 3.5 h. The volatiles were then removed under reduced

pressure, and the residue was coevaporated twice with toluene. The

residue was purified by column chromatography on silica gel (gra-

dient CH₂Cl₂/MeOH, 100:0, v/v; 98:2, v/v; 96:4, v/v) to give 21

5Ј-Hydroxy-3Ј-O-[N-For-Gly-L-Lys(ε-carbamate)-L-Phe-OMe]-

2Ј-deoxythymidine (17): A solution of 16 (147 mg, 0.157 mmol) in

AcOH (80%; 2 mL) was stirred at room temp. for 3.5 h. The vola-

tiles were then removed under reduced pressure, and the residue

was coevaporated twice with toluene. The crude residue was puri-

fied by column chromatography on silica gel (gradient CH₂Cl₂/

MeOH, 100:0, v/v; 98:2, v/v; 96:5, v/v) to give 17 (85 mg, 82%) as

1

(1.7 g, 91%) as a white solid. H NMR (300 MHz, [D₆]DMSO): δ

= 11.35 (br. s, 1 H, NH), 8.03–8.00 (m, 2 H, o-H of Ph), 7.79 (d,

J = 1.1 Hz, 1 H, 6-H), 7.71–7.66 (m, 1 H, p-H of Ph), 7.58–7.53

(m, 2 H, m-H of Ph), 6.30 (app t, J = 7.1 Hz, 1 H, 1Ј-H), 5.49–

5.48 (m, 1 H, 3Ј-H), 5.26 (br. s, 1 H, OH), 4.18–4.15 (m, 1 H, 4Ј-

a white solid. ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.32 (br. s, 1 H), 3.71 (br. s, 2 H, 5Ј-H and 5ЈЈ-H), 2.43–2.39 (m, 2 H, 2Ј-H and

H, NH), 8.38 (d, J = 7.3 Hz, 1 H, NH-Phe), 8.19 (t, J = 5.1 Hz, 1 2ЈЈ-H), 1.80 (d, J = 1.0 Hz, 3 H, CH₃) ppm. ¹³C NMR (75 MHz,

H, NH-Gly), 8.06 (s, 1 H, CHO), 7.96 (d, J = 7.9 Hz, 1 H, NH-

Lys), 7.73 (s, 1 H, 6-H), 7.31–7.19 (m, 5 H, Ar-H), 6.17 (dd, J =

8.5, 5.9 Hz, 1 H, 1Ј-H), 5.19 (t, J = 4.7 Hz, 1 H, εNH-Lys), 5.11–

5.09 (m, 1 H, 3Ј-H), 4.48–4.40 (m, 1 H, αH-Phe), 4.31–4.23 (m, 1

H, αH-Lys), 3.93 (br. s, 1 H, 4Ј-H), 3.75 (d, J = 5.6 Hz, 2 H, αCH₂-

Gly), 3.63–3.60 (m, 2 H, 5Ј-H and 5ЈЈ-H), 3.57 (s, 3 H, OCH₃),

3.05–2.89 (m, 4 H, εCH₂-Lys and βCH₂-Phe), 2.33–2.17 (m, 2 H,

2Ј-H and 2ЈЈ-H), 1.78 (s, 3 H, CH₃-Thy), 1.59–1.47 (m, 2 H, βCH₂-

Lys), 1.46–1.34 (m, 2 H, δCH₂-Lys), 1.28–1.15 (m, 2 H, γCH₂-Lys)

ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 171.8, 171.6, 168.1,

163.7, 161.4, 155.4, 150.5, 137.1, 135.8, 129.1, 128.3, 126.6, 109.7,

85.5, 83.7, 74.6, 61.5, 40.4, 36.8, 36.5, 31.9, 30.7, 29.1, 22.4,

12.3 ppm. HRMS: calcd. for C₃₀H₄₀N₆O₁₁[M + H]⁺661.2827;

found 661.2828.

[D₆]DMSO): δ = 165.2 (COPh), 163.7 (C-4), 150.5 (C-2), 135.9 (C-

6), 133.6 (p-C of Ph), 129.4 (1C of Ph), 129.3 (o-C of Ph), 128.8

(m-C of Ph), 109.8 (C-5), 84.6 (C-1Ј), 83.8 (C-4Ј), 75.7 (C-3Ј), 61.4

(C-5Ј), 36.7 (C-2Ј), 12.3 (CH₃) ppm. HRMS: calcd. for

C₃₇H₃₄N₂O₇[M + H]⁺347.1237; found 347.1238.

5Ј-O-(Dibenzylphosphate)-3Ј-O-benzoyl-2Ј-deoxythymidine (22):

Tetrazole (0.45 m in MeCN; 51.33 mL, 23.10 mmol) and then di-

benzyldiisopropyl phosphoramidite (3.47 mL, 10.16 mmol) were

added to a stirred suspension of 21 (1.6 g, 4.62 mmol) in dry

CH₂Cl₂(20 mL) at 0 °C, and the reaction mixture was stirred at

room temp. for 12 h. The mixture was cooled to –40 °C, H₂O₂

(35%; 1.97 mL, 23.10 mmol) was slowly added, and the resulting

mixture was stirred at 0 °C for 1 h and for an additional 1 h at

room temp. Then the reaction mixture was again cooled to 0 °C

and finally quenched with NaHSO₃(10% w/v aq.; 30 mL). The

aqueous layer was extracted with CH₂Cl₂(3ϫ 100 mL), and the

combined organic extracts were dried with Na₂SO₄and concen-

trated under reduced pressure. The crude residue was purified by

column chromatography on silica gel (gradient hexane/EtOAc, 3:1,

v/v; 2:1, v/v; 1:1, v/v) to give 22 (2.59 g, 92%) as a colourless foam.

¹H NMR (300 MHz, CDCl₃): δ = 9.38 (br. s, 1 H, NH), 8.05–8.02

(m, 2 H, Ar-H), 7.63–7.58 (m, 1 H, Ar-H), 7.49–7.44 (m, 3 H, 6-

H and Ar-H), 7.35 (br. s, 10 H, Ar-H), 6.46 (dd, J = 9.0, 5.4 Hz, 1

H, 1Ј-H), 5.37–5.35 (m, 1 H, 3Ј-H), 5.15–5.03 (m, 4 H, OCH₂Ph),

4.38–4.34 (m, 1 H, 4Ј-H), 4.25 (br. s, 2 H, 5Ј-H and 5ЈЈ-H), 2.48–

2.42 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.86 (s, 3 H, CH₃) ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 166.0 (COPh), 163.8 (C-4), 150.7 (C-2),

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-Gly-L-Lys(ε-carbamate)-L-

Phe-OMe]-2Ј-deoxythymidine (18): Tetrazole (0.45 m in MeCN;

3.53 mL, 1.59 mmol) and then dibenzyldiisopropyl phosphoramid-

ite (0.24 mL, 0.697 mmol) were added to a stirred suspension of 17

(210 mg, 0.317 mmol) in dry CH₂Cl₂(5 mL) at 0 °C, and the reac-

tion mixture was stirred at room temp. for 12 h. The mixture was

cooled to –40 °C, then H₂O₂(35 %; 0.14 mL, 1.59 mmol) was

slowly added, and the resulting mixture was stirred first at 0 °C for

1 h and then at room temp. for 1 h. The mixture was cooled and

quenched with NaHSO₃(10% w/v aq.; 6 mL). The aqueous layer

was extracted with CH₂Cl₂(3ϫ 30 mL), and the combined organic

extracts were dried with Na₂SO₄, filtered and concentrated under

reduced pressure. The crude residue was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v;

98:2, v/v; 96:4, v/v) to give 18 (230 mg, 79%) as a colourless foam.

3

135.5 (d, J_C,P= 6.0 Hz, 1C of OCH₂Ph), 135.0 (C-6), 133.7 (Ar-

C), 129.8 (Ar-C), 129.1 (Ar-C), 129.0 (Ar-C), 128.8 (Ar-C), 128.6

3

(Ar-C), 128.2 (Ar-C), 111.9 (C-5), 84.6 (C-1Ј), 83.0 (d, J_C,P

=

5Ј-O-(4-Monomethoxytrityl)-3Ј-O-benzoyl-2Ј-deoxythymidine (20):

Benzoyl chloride (0.80 mL, 6.91 mmol) was slowly added to a

stirred solution of 14 (3.09 g, 6.01 mmol) in pyridine (25 mL) at

0 °C, and the resulting mixture was stirred for 3 h at 0 °C. The

reaction mixture was then diluted with CH₂Cl₂(200 mL), and the

mixture was washed with saturated aq. NaHCO₃and brine. The

organic layer was dried with Na₂SO₄, filtered, evaporated and co-

evaporated with toluene. The crude residue was purified by column

chromatography on silica gel (gradient hexane/EtOAc, 3:1, v/v; 2:1,

v/v; 1:1, v/v) to give 20 (3.53 g, 95%) as a colourless foam. ¹H

2

8.3 Hz, C-4Ј), 75.3 (C-3Ј), 69.9 (app t, J_C,P= 5.3 Hz, OCH₂), 67.3

(d, J_C,P= 5.7 Hz_,C-5Ј), 37.3 (C-2Ј), 12.4 (CH₃) ppm. ³¹P NMR

2

(121 MHz, CDCl₃): δ = –0.6 ppm. HRMS: calcd. for C₃₇H₃₄N₂O₇

[M + H]⁺607.1840; found 607.1840.

5Ј-O-(Dibenzylphosphate)-2Ј-deoxythymidine (23): A solution of 22

(2.52 g, 4.15 mmol) in NH₃(7 n in MeOH; 30 mL) was stirred at

0 °C for 3 h and then at room temp. for 24 h. The volatiles were

removed under reduced pressure, and the crude residue was puri-

fied by column chromatography on silica gel (gradient CH₂Cl₂/

NMR (300 MHz, CDCl₃): δ = 9.56 (br. s, 1 H, NH), 8.06–8.03 (m, MeOH, 99:1, v/v; 98:2, v/v; 97:3, v/v) to give 23 (1.98 g, 95%) as a

2 H, Ar-H), 7.66 (s, 1 H, 6-H), 7.60–7.25 (m, 15 H, Ar-H), 6.87–

colourless foam. ¹H NMR (300 MHz, CDCl₃): δ = 9.41 (br. s, 1 H,

6.84 (m, 2 H, Ar-H), 6.56 (dd, J = 8.7, 5.7 Hz, 1 H, 1Ј-H), 5.74– NH), 7.34–7.33 (m, 11 H, 6-H and Ar-H), 6.29 (app t, J = 6.7 Hz,

5.72 (m, 1 H, 3Ј-H), 4.30–4.29 (m, 1 H, 4Ј-H), 3.78 (s, 3 H, OCH₃), 1 H, 1Ј-H), 5.11–4.99 (m, 4 H, OCH₂Ph), 4.37–4.33 (m, 1 H, 3Ј-

3.61–3.51 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.69–2.51 (m, 2 H, 2Ј-H and

H), 4.22–4.14 (m, 2 H, 5Ј-H and 5ЈЈ-H), 4.04–4.00 (m, 2 H, 4Ј-H),

2ЈЈ-H), 1.43 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 2.35–2.27 (m, 1 H, 2Ј-H), 2.03–1.96 (m, 1 H, 2ЈЈ-H), 1.81 (d, J =

166.0, 164.0, 158.9, 150.8, 143.9, 143.8, 135.4, 134.8, 133.6, 130.5,

129.8, 129.4, 128.6, 128.5, 128.4, 128.1, 127.4, 113.4, 111.8, 87.5,

1.0 Hz, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 164.0

(C-4), 150.7 (C-2), 149.8 (1C of PhNO₂), 145.36 (p-C of PhNO₂),

2336

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Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

3

135.7 (C-6), 135.4 (d, J_C,P= 7.3 Hz, 1C of OCH₂Ph), 129.1 (Ar- Phe), 2.38–2.14 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.80 (s, 3 H, CH₃-Thy)

C), 128.9 (Ar-C), 128.2 (Ar-C), 111.5 (C-5), 85.0 (C-1Ј), 84.8 (d,

ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 172.0 (CO-Phe), 169.7 (CO-

2

³J_C,P= 7.5 Hz, C-4Ј), 71.1 (C-3Ј), 70.1–70.0 (2 d, J_C,P= 5.6 Hz, 2

β-amino-Ala), 169.4 (CO-Gly), 164.0 (C-4), 162.4 (CHO), 156.5

2

3

OCH₂Ph), 66.9 (d, J_C,P= 5.7 Hz_,C-5Ј), 40.2 (C-2Ј), 12.5 (CH₃) (OCONH), 151.1 (C-2), 136.1 (C-6), 135.5 (d, J_C,P= 5.5 Hz, 1C

ppm. ³¹P NMR (121 MHz, CDCl₃): δ = –0.5 ppm. HRMS: calcd.

of OCH₂Ph), 135.1 (1C Ph-Phe), 129.3 (Ar-C), 129.0 (Ar-C), 128.9

(Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 127.2 (Ar-C), 112.0 (C-5), 84.6

for C₂₄H₂₇N₂O₈P [M + H]⁺503.1578; found 503.1580.

3

2

(C-1Ј), 82.8 (d, J_C,P= 7.7 Hz, C-4Ј), 75.5 (C-3Ј), 69.9 (app t, J_C,P

= 5.0 Hz, 2 OCH₂Ph), 67.4 (d, ²J_C,P= 4.9 Hz_,C-5Ј), 53.9 (αC-Phe),

53.6 (αC-β-amino-Ala), 52.6 (OCH₃), 43.3 (βC-β-amino-Ala), 41.7

(αC-Gly), 37.8 (C-2Ј), 37.2 (βC-Phe), 12.5 (CH₃) ppm. ³¹P NMR

(121 MHz, CDCl ₃) : δ = – 0 . 7 p p m . H R M S : c a l c d . for

C₄₁H₄₇N₆O₁₄P [M + H]⁺879.2960; found 879.2966.

5Ј-O-(Dibenzylphosphate)-3Ј-O-(4-nitrophenyl Carbonate)-2Ј-deoxy-

thymidine (24): Compound 23 (1.12 g, 2.23 mmol) was coevapo-

rated with dry pyridine (2ϫ 5 mL), then it was redissolved in dry

pyridine (7 mL), and a suspension of 4-nitrophenyl chloroformate

(0.539 g, 2.67 mmol) in a mixture of CH₂Cl₂/pyridine (70:1;

7.1 mL) was slowly added at 0 °C. The reaction mixture was stirred

at 0 °C for 2 h, and at room temp. for 24 h. The volatiles were

removed under reduced pressure, the residue was redissolved in

CH₂Cl₂(150 mL), and this solution was washed with saturated aq.

NaHCO₃(2ϫ 70 mL). The organic layer was dried with Na₂SO₄,

filtered, and concentrated under reduced pressure. The crude resi-

due was purified by column chromatography on silica gel (gradient

hexane/EtOAc, 2:1, v/v; 1:1, v/v; 1:3, v/v) to give 24 (0.99 g, 67%)

as a colourless foam. ¹H NMR (300 MHz, CDCl₃): δ = 8.43 (br. s,

1 H, NH), 8.32–8.29 (m, 2 H, Ar-H), 7.41–7.26 (m, 13 H, 6-H and

Ar-H), 6.39 (dd, J = 9.3, 5.3 Hz, 1 H, 1Ј-H), 5.17–5.15 (m, 1 H,

3Ј-H), 5.12–5.00 (m, 4 H, OCH₂Ph), 4.28–4.27 (m, 1 H, 4Ј-H),

4.26–4.20 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.48–2.41 (m, 1 H, 2Ј-H), 2.04–

1.93 (m, 1 H, 2ЈЈ-H), 1.86 (d, J = 1.0 Hz, 3 H, CH₃) ppm. ¹³C

NMR (75 MHz, CDCl₃): δ = 162.9 (C-4), 154.7 (COPhNO₂), 151.5

(C-2), 149.8 (1C of PhNO₂), 145.36 (p-C of PhNO₂), 135.0 (d, ³J_C,P

= 5.8 Hz, 1C of OCH₂Ph), 134.4 (C-6), 128.7 (Ar-C), 128.6 (Ar-

C), 128.5 (Ar-C), 128.4 (Ar-C), 127.8 (Ar-C), 125.1 (Ar-C), 121.3

(Ar-C), 111.6 (C-5), 84.1 (C-1Ј), 81.9 (d, ³J_C,P= 8.2 Hz, C-4Ј), 79.1

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-Gly-L-Lys(ε-carbamate)-L-

Phe-OMe]-2Ј-deoxythymidine (18): Compound 18 was prepared ac-

cording to the general procedure starting from 24 (300 mg,

0.449 mmol), tripeptide 5d (273 mg, 0.539 mmol), and Et₃N

(0.25 mL, 1.80 mmol) in dry CH₂Cl₂(15 mL). The crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:2, v/v; 92:4, v/v) to give conjugate 18

(322 mg, 78%) as a colourless foam. ¹H NMR (300 MHz, [D₄]-

methanol): δ = 8.15 (s, 1 H, CHO), 7.46 (s, 1 H, 6-H), 7.36 (br. s,

10 H, Ar-H of OBn), 7.29–7.17 (m, 5 H, Ar-H of Phe), 6.23 (dd,

J = 8.6, 5.8 Hz, 1 H, 1Ј-H), 5.12–5.08 (m, 5 H, 3Ј-H and 2

OCH₂Ph), 4.67–4.63 (m, 1 H, αH-Phe), 4.38–4.33 (m, 1 H, αH-

Lys), 4.30–4.26 (m, 2 H, 5Ј-H and 5ЈЈ-H), 4.17–4.16 (m, 1 H, 4Ј-

H), 3.90 (s, 2 H, CH₂-Gly), 3.67 (s, 3 H, OCH₃), 3.16–2.96 (m, 4

H, εCH₂-Lys and βCH₂-Phe), 2.33–2.27 (m, 1 H, 2Ј-H), 2.12–2.02

(m, 1 H, 2ЈЈ-H), 1.77 (s, 3 H, CH₃-Thy), 1.71–1.58 (m, 2 H, βCH₂-

Lys), 1.51–1.47 (m, 2 H, δCH₂-Lys), 1.33–1.29 (m, 2 H, γCH₂-

Lys) ppm. ¹³C NMR (75 MHz, [D₄]methanol): δ = 173.9 (CO-Phe),

173.3 (CO-Lys), 170.7 (CO-Gly), 166.1 (C-4), 164.2 (CHO), 157.8

(OCONH), 152.2 (C-2), 138.1 (1C Ph-Phe), 137.1 (C-6), 137.0 (d,

³J_C,P= 6.1 Hz, 1C of OCH₂Ph), 130.3 (Ar-C), 129.9 (Ar-C), 129.8

(Ar-C), 129.5 (Ar-C), 129.2 (Ar-C), 127.9 (Ar-C), 112.1 (C-5), 86.2

2

(C-3Ј), 69.6 (app t, J_C,P= 4.9 Hz, OCH₂Ph), 66.6 (d, J_C,P

=

5.5 Hz_,C-5Ј), 36.7 (C-2Ј), 12.0 (CH₃) ppm. ³¹P NMR (121 MHz,

CDCl₃): δ = –0.5 ppm. HRMS: calcd. for C₃₁H₃₀N₃O₁₂

P

[M + H]⁺668.1640; found 668.1638.

3

(C-1Ј), 84.4 (d, J_C,P= 7.7 Hz, C-4Ј), 75.7 (C-3Ј), 71.2–71.1 (2 d,

2

²J_C,P= 5.7 Hz, 2 OCH₂Ph), 68.7 (d, J_C,P= 5.8 Hz_,C-5Ј), 55.2

General Procedure for the Synthesis of 3Ј-Carbamoyl Peptide Conju-

gates of dTMP (18 and 25–32): A solution of compound 24 in dry

CH₂Cl₂was added to a stirred solution of peptide-NH₂·TFA salt

(1 equiv.) and Et₃N (4 equiv.) in dry CH₂Cl₂at 0 °C, and the stir-

ring was continued at room temp. for 24 h. The reaction mixture

was then diluted with CH₂Cl₂and washed twice with NaHCO₃

(20% aq.). The organic layer was dried with Na₂SO₄, filtered, and

concentrated under reduced pressure. The crude residue was puri-

fied by column chromatography on silica gel (gradient CH₂Cl₂/

MeOH, 99:1, v/v; 98:2, v/v; 96:4, v/v) to give the desired 3Ј-carbam-

oyl PNCs.

(αC-Phe), 54.4 (αC-Lys), 52.7 (OCH₃), 42.0 (αC-Gly), 41.5 (εC-

Lys), 38.3 (C-2Ј), 38.1 (βC-Phe), 32.8 (βC-Lys), 30.3 (δC-Lys), 23.8

(γC-Lys), 12.5 (CH₃) ppm. ³¹P NMR (121 MHz, [D₄]methanol): δ

= –1.2 ppm. HRMS: calcd. for C₄₄H₅₃N₆O₁₄P [M – H]^–919.3284;

found 919.3287.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-

L-Phe-OMe]-2Ј-deoxythymidine (26): Compound 26 was prepared

according to the general procedure starting from 24 (246.2 mg,

0.367 mmol), tripeptide 5g (225.4 mg, 0.3882 mmol), and Et₃N

(0.22 mL, 1.55 mmol) in dry CH₂Cl₂(20 mL). The crude residue

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-Gly-

L-β-amino-Ala-(β- was purified by column chromatography on silica gel (gradient

carbamate)- -Phe-OMe]-2Ј-deoxythymidine (25): Compound 25

L

CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:5, v/v) to give conjugate 26

(330 mg, 85%) as a colourless foam. ¹H NMR (300 MHz, [D₆]-

DMSO): δ = 11.36 (br. s, 1 H, NH-Thy), 8.33 (d, J = 7.5 Hz, 1 H,

NH-Phe), 8.27 (d, J = 8.4 Hz, 1 H, NH-Met), 8.01–8.00 (m, 2 H,

CHO and αNH-Lys), 7.50 (s, 1 H, 6-H), 7.36–7.34 (m, 11 H, Ar-

H of OBn and εNH-Lys), 7.26–7.19 (m, 5 H, Ar-H of Phe), 6.19

(app t, J = 7.3 Hz, 1 H, 1Ј-H), 5.07–5.04 (m, 5 H, 3Ј-H and 2

was prepared according to the general procedure starting from 24

(240 mg, 0.365 mmol), tripeptide 5c (179 mg, 0.510 mmol), and

Et₃N (0.05 mL, 0.539 mmol) in dry CH₂Cl₂(15 mL). The crude

residue was purified by column chromatography on silica gel (gra-

dient CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:5, v/v) to give conju-

1

gate 25 (259 mg, 82%) as a colourless foam. H NMR (300 MHz,

CDCl₃): δ = 9.93 (br. s, 1 H, NH-Thy), 8.18 (s, 1 H, CHO), 7.65 OCH₂Ph), 4.50–4.39 (m, 2 H, αH-Met and αH-Phe), 4.26–4.20 (un-

(d, J = 7.2 Hz, 1 H, NH-Phe), 7.65 (d, J = 7.2 Hz, 1 H, NH-β- resolved m, 3 H, αH-Lys, 5Ј-H, and 5ЈЈ-H), 4.10 (br. s, 1 H, 4Ј-H),

amino-Ala), 7.39 (s, 1 H, 6-H), 7.32 (br. s, 10 H, Ar-H of OBn),

7.26–7.09 (m, 5 H, Ar-H of Phe), 6.39 (t, J = 6.3 Hz, 1 H, NH-

3.56 (s, 3 H, OCH₃), 3.05–2.89 (m, 4 H, εCH₂-Lys and βCH₂-Phe),

2.39 (t, J = 7.8 Hz, 2 H, γCH₂-Met), 2.23–2.18 (m, 2 H, 2Ј-H and

Gly), 6.25 (dd, J = 8.3, 5.2 Hz, 1 H, 1Ј-H), 5.07–4.98 (m, 5 H, 3Ј- 2ЈЈ-H), 2.00 (s, 3 H, SCH₃), 1.91–1.72 (m, 2 H, βCH₂-Met), 1.69

H and 2 OCH₂Ph), 4.83–4.76 (m, 1 H, αH-Phe), 4.75–4.68 (m, 1

H, αH-β-amino-Ala), 4.28–4.17 (m, 2 H, 5Ј-H and 5ЈЈ-H), 4.14 (br.

s, 1 H, 4Ј-H), 4.04–3.88 (m, 2 H, CH₂-Gly), 3.67 (s, 3 H, OCH₃),

(s, 3 H, CH₃-Thy), 1.63–1.45 (m, 2 H, βCH₂-Lys), 1.40–1.36 (m, 2

H, δCH₂-Lys), 1.28–1.19 (m, 2 H, γCH₂-Lys) ppm. ¹³C NMR

(75 MHz, [D₆]DMSO): δ = 171.7 (CO-Phe), 171.5 (CO-Lys), 170.4

3.58–3.33 (m, 2 H, βCH₂-β-amino-Ala), 3.16–2.99 (m, 2 H, βCH₂- (CO-Met), 163.5 (C-4), 160.9 (CHO), 155.2 (OCONH), 150.4 (C-

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2337

S. De, E. Groaz, P. Herdewijn

FULL PAPER

2), 137.0 (1C of Phe), 135.9 (d, J_C,P= 6.8 Hz, 1C of OCH₂Ph),

135.4 (C-6), 129.0 (Ar-C), 128.5 (Ar-C), 128.4 (Ar-C), 128.2 (Ar-

C), 127.8 (Ar-C), 126.5 (Ar-C), 110.0 (C-5), 83.9 (C-1Ј), 82.2 (d,

3

(Ar-C), 127.7 (Ar-C), 127.6 (Ar-C), 110.0 (C-5), 83.9 (C-1Ј), 82.2

(d, J_C,P= 8.2 Hz, C-4Ј), 73.6 (C-3Ј), 68.8–68.7 (3 OCH₂Ph), 65.1

3

2

(d, J_C,P= 3.7 Hz_,C-5Ј), 52.3 (αC-Lys), 52.6 (αC-Lys and OCH₃),

³J_C,P= 7.4 Hz, C-4Ј), 73.6 (C-3Ј), 68.7 (2 d, J_C,P= 5.5 Hz, 2 50.4 (αC-Met), 39.5 (2 εC-Lys merged with [D₆]DMSO), 36.0

2

OCH₂Ph), 67.0 (d, J_C,P= 5.7 Hz_,C-5Ј), 53.4 (αC-Phe), 52.2 (αC- (C-2Ј), 32.1 (βC-Met), 31.8 (2 βC-Lys), 30.5 (γC-Met), 29.3

Lys), 51.8 (OCH₃), 50.3 (αC-Met), 40.2 (εC-Lys), 36.5 (βC-Phe),

36.0 (C-2Ј), 32.2 (βC-Met), 31.7 (βC-Lys), 29.3 (γC-Met), 29.0 (δC- Thy) ppm. ³¹P NMR (121 MHz, [D₆]DMSO): δ = –0.9 ppm.

Lys), 22.5 (γC-Lys), 14.6 (-SCH₃), 12.0 (CH₃-Thy) ppm. ³¹P NMR HRMS: calcd. for C₅₂H₆₈N₇O₁₆PS [M – H]^–1108.4108; found

(δC-Lys), 28.9 (δC-Lys), 22.5 (2 γC-Lys), 14.5 (SCH₃), 12.0 (CH₃-

(121 MHz, [D₆]DMSO): δ = –0.9 ppm. HRMS: calcd. for 1108.4100.

C₄₇H₅₉N₆O₁₄PS [M – H]^–993.3474; found 993.3478.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-Boc-

L-Ala-L-Lys(ε-carbamate)-

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-

L

-Met-

L

-Lys(ε-carbamate)-

L-Ala-OMe]-2Ј-deoxythymidine (29): Compound 29 was prepared

L

-Ala-OMe]-2Ј-deoxythymidine (27): Compound 27 was prepared

according to the general procedure starting from 24 (505 mg,

0.756 mmol), tripeptide 13 (350 mg, 0.89 mmol), and Et₃N

(0.16 mL, 1.13 mmol) in dry CH₂Cl₂(20 mL). The crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:2, v/v; 92:4, v/v) to give conjugate 29

(619 mg, 88%) as a colourless foam. ¹H NMR (500 MHz, CDCl₃):

δ = 9.41 (br. s, 1 H, NH-Thy), 7.44 (s, 1 H, 6-H), 7.33 (br. s, 10 H,

Ar-H of OBn), 7.06 (br. s, 2 H, NH-Ala_C-Terand NH-Lys), 6.29

(dd, J = 9.1, 5.3 Hz, 1 H, 1Ј-H), 5.62 (br. s, 1 H, ε-NH-Lys), 5.39

according to the general procedure starting from 24 (226.6 mg,

0.339 mmol), tripeptide 5e (205.5 mg, 0.407 mmol), and Et₃N

(0.19 mL, 1.36 mmol) in dry CH₂Cl₂(15 mL). The crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:5, v/v) to give conjugate 27

(224.5 mg, 72%) as a colourless foam. H NMR (500 MHz, [D₆]-

DMSO): δ = 11.3 (br. s, 1 H, NH-Thy), 8.31 (d, J = 6.8 Hz, 1 H,

NH-Ala), 8.27 (d, J = 8.1 Hz, 1 H, NH-Met), 8.03–8.01 (m, 2 H,

1

CHO and αNH-Lys), 7.50 (s, 1 H, 6-H), 7.36–7.35 (m, 11 H, Ar- (br. s, 1 H, NH-Ala_N-Ter), 5.10–5.01 (m, 5 H, 3Ј-H and 2 OCH₂Ph),

H of OBn and εNH-Lys), 6.19 (app t, J = 7.3 Hz, 1 H, H-1Ј), 5.08–

5.05 (m, 5 H, H-3Ј and 2 OCH₂Ph), 4.46–4.41 (m, 1 H, αH-Met),

4.55–4.48 (m, 2 H, αH-Ala_C-Terand αH-Lys), 4.29–4.26 (m, 2 H,

5Ј-H), 4.21–4.19 (m, 2 H, αH-Ala_N-Terand 5ЈЈ-H), 4.16 (br. s, 1 H,

4.27–4.22 (unresolved m, 4 H, αH-Lys, αH-Ala, H-5Ј, and H-5ЈЈ), 4Ј-H), 3.74 (s, 3 H, OCH₃), 3.58–3.33 (m, 2 H, εCH₂-Lys), 2.31–

4.11 (br. s, 1 H, H-4Ј), 3.61 (s, 3 H, OCH₃), 2.99–2.95 (m, 2 H, 2.25 (m, 2 H, 2Ј-H), 1.93–1.86 (m, 2 H, 2ЈЈ-H and βCH_2Ј-Lys), 1.84

εCH₂-Lys), 2.42 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.22–2.19 (m, 2

(s, 3 H, CH₃-Thy), 1.71–1.63 (m, 1 H, βCH_2ЈЈ-Lys), 1.57–1.48 (m,

H, H-2Ј and H-2ЈЈ), 2.02 (s, 3 H, SCH₃), 1.91–1.72 (m, 2 H, βCH₂- 1 H, δCH₂-Lys), 1.42 (s, 9 H, tBu), 1.40–1.34 (m, 8 H, γCH₂-Lys,

Met), 1.69 (s, 3 H, CH₃-Thy), 1.67–1.48 (m, 2 H, βCH₂-Lys), 1.43–

1.37 (m, 2 H , δ CH₂-Lys), 1.33–1.22 (m, 5 H, γCH₂-Lys and CH₃-

Ala). ¹³C NMR (125 MHz, [D₆]DMSO): δ = 172.9 (CO-Ala), 171.4

2 CH₃-Ala) ppm. ¹³C NMR (125 MHz, CDCl₃): δ = 173.4 (CO-

Ala_C-Ter), 173.2 (CO-Ala_N-Ter), 171.3 (CO-Lys), 163.9 (C-4), 155.7

(2 OCONH), 150.8 (C-2), 135.5 (d, ³J_C,P= 5.6 Hz, 1C of OCH₂Ph),

(CO-Lys), 170.5 (CO-Met), 163.6 (C-4), 161.0 (CHO), 155.2 135.2 (C-6), 129.0 (Ar-C), 128.8 (Ar-C), 128.2 (Ar-C), 111.9 (C-5),

3

(OCONH), 150.4 (C-2), 135.9 (d, J_C,P= 6.9 Hz, 1C of OCH₂Ph), 84.6 (C-1Ј), 83.1 (d, J_C,P= 8.0 Hz, C-4Ј), 80.2 (1C tBu), 75.0 (C-

2

135.4 (C-6), 128.5 (Ar-C), 128.4 (Ar-C), 127.8 (Ar-C), 110.0 (C-5), 3Ј), 69.9–69.8 (2 d, J_C,P= 5.4 Hz, 2 OCH₂Ph), 67.4 (d, J_C,P

=

83.9 (C-1Ј), 82.2 (d, ³J_C,P= 7.2 Hz, C-4Ј), 73.6 (C-3Ј), 68.7 (d, ²J_C,P

= 5.4 Hz, 2 OCH₂Ph), 67.1 (d, ²J_C,P= 5.9 Hz_,C-5Ј), 52.1 (αC-Lys),

51.8 (OCH₃), 50.4 (αC-Met), 47.5 (αC-Ala), 40.2 (εC-Lys), 36.0

5.8 Hz_,C-5Ј), 52.8 (αC- Ala_C-Ter), 52.6 (OCH₃), 48.2 (αC-Lys and

αC-Ala_N-Ter), 40.6 (εC-Lys), 37.4 (C-2Ј), 29.1 (βC-Lys), 28.4 (δC-

Lys and CH₃-tBu), 22.4 (γC-Lys), 18.0 (2 CH₃-Ala), 12.4 (CH₃-

(C-2Ј), 32.2 (βC-Met), 31.6 (βC-Lys), 29.3 (γC-Met), 29.1 (δC-Lys), Thy) ppm. ³¹P NMR (202 MHz, CDCl₃): δ = –0.7 ppm. HRMS:

22.5 (γC-Lys), 16.8 (CH₃-Ala), 14.6 (-SCH₃), 12.0 (CH₃-Thy) ppm. calcd. for C₄₃H₅₉N₆O₁₅P [M – H]^–929.3703; found 929.3715.

³¹P NMR (202 MHz, [D₆]DMSO): δ = –0.9 ppm. HRMS: calcd.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-

for C₄₁H₅₅N₆O₁₄PS [M – H]^–917.3161; found 917.3145.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For- -Met- -Lys(ε-carbamate)-

-Lys(Cbz)-OMe]-2Ј-deoxythymidine (28): Compound 28 was pre-

OMe]-2Ј-deoxythymidine (30): Compound 30 was prepared accord-

ing to the general procedure starting from 24 (246.2 mg,

0.367 mmol), dipeptide 8 (168.3 mg, 0.3882 mmol), and Et₃N

(0.22 mL, 1.55 mmol) in dry CH₂Cl₂(15 mL). The crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:4, v/v) to give conjugate 30

(276.5 mg, 84 %) as a colourless foam. ¹H NMR (300 MHz,

CDCl₃): δ = 10.30 (br. s, 1 H, NH-Thy), 8.20 (s, 1 H, CHO), 7.56

(d, J = 7.9 Hz, 1 H, NH-Met), 7.46 (s, 1 H, 6-H), 7.37–7.34 (m, 11

H, Ar-H of OBn and αNH-Lys), 6.30 (dd, J = 9.0, 5.3 Hz, 1 H,

1Ј-H), 5.95 (t, J = 5.5 Hz, εNH-Lys), 5.12–5.04 (m, 5 H, 3Ј-H and

2 OCH₂Ph), 4.76–4.69 (m, 1 H, αH-Met), 4.61–4.54 (m, 1 H, αH-

Lys), 4.27–4.22 (unresolved m, 3 H, 4Ј-H, 5Ј-H, and 5ЈЈ-H), 3.73

(s, 3 H, OCH₃), 3.18–3.11 (m, 2 H, εCH₂-Lys), 2.57 (t, J = 7.2 Hz,

L

pared according to the general procedure starting from 24

(326.3 mg, 0.489 mmol), tripeptide 5f (408.1 mg, 0.587 mmol), and

Et₃N (0.33 mL, 2.35 mmol) in dry CH₂Cl₂(15 mL). The crude resi-

due was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:2, v/v; 92:4, v/v) to give conjugate 28

(347 mg, 64%) as a colourless foam. ¹H NMR (300 MHz, [D₆]-

DMSO): δ = 11.53 (br. s, 1 H, NH-Thy), 8.31–8.25 (m, 2 H, 2 NH),

8.03–8.01 (m, 2 H, CHO and NH), 7.50 (s, 1 H, 6-H), 7.36–7.19

(m, 15 H, Ar-H of OBn), 7.23–7.19 (m, 1 H, NH), 6.18 (app t, J

= 7.2 Hz, 1 H, 1Ј-H), 5.07–5.04 (m, 5 H, 3Ј-H and 2 POCH₂Ph-

OBn), 4.99 (s, 2 H, OCH₂-Cbz), 4.47–4.39 (m, 1 H, αH-Met), 4.30–

4.15 (m, 4 H, 2 αH-Lys, 5Ј-H, and 5ЈЈ-H), 4.09 (br. s, 1 H, 4Ј-H), 2 H, γCH₂-Met), 2.30–2.24 (m, 1 H, 2Ј-H), 2.17–1.98 (m, 5 H,

3.61 (s, 3 H, OCH₃), 2.99–2.92 (m, 4 H, 2 εCH₂-Lys), 2.41 (t, J = SCH₃and βCH₂-Met), 1.92–1.65 (m, 6 H, CH₃-Thy, 2ЈЈ-H, and

7.8 Hz, 2 H, γCH₂-Met), 2.22–2.16 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.01 βCH₂-Lys), 1.58–1.48 (m, 2 H, δCH₂-Lys), 1.42–1.32 (m, 2 H,

(s, 3 H, SCH₃), 1.90–1.76 (m, 2 H, βCH₂-Met), 1.69 (s, 3 H, CH₃- γCH₂-Lys) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 172.5 (CO-Lys),

Thy), 1.67–1.49 (m, 4 H, 2 βCH₂-Lys), 1.42–1.35 (m, 4 H, 2 δCH₂- 171.2 (CO-Met), 164.1 (C-4), 161.7 (CHO), 155.6 (OCONH), 151.1

Lys), 1.33–1.17 (m, 4 H, 2 γCH₂-Lys) ppm. ¹³C NMR (75 MHz,

(C-2), 135.4 (d, ³J_C,P= 6.2 Hz, 1C of OCH₂Ph), 135.1 (C-6), 128.8

[D₆]DMSO): δ = 172.4 (CO-Lys_C-Ter), 171.6 (CO-Met), 170.4 (CO- (Ar-C), 128.7 (Ar-C), 128.0 (Ar-C), 111.9 (C-5), 84.4 (C-1Ј), 83.0

3

2

Lys_N-Ter), 163.5 (C-4), 161.0 (CHO), 155.2 (2 OCONH), 150.4 (C-

2), 137.2 (C-6), 135.9 (d, J_C,P= 6.7 Hz, 1C of OCH₂Ph), 135.4

(d, J_C,P= 8.0 Hz, C-4Ј), 74.7 (C-3Ј), 69.7 (2 d, J_C,P= 5.5 Hz, 2

3

2

OCH₂Ph), 67.3 (d, J_C,P= 5.4 Hz_,C-5Ј), 52.4 (OCH₃), 52.0 (αC-

(1C of Ph-Cbz), 128.5 (Ar-C), 128.4 (Ar-C), 128.3 (Ar-C), 127.8

Lys), 51.0 (αC-Met), 40.4 (εC-Lys), 37.3 (C-2Ј), 31.5 (βC-Met),

2338

www.eurjoc.org

Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

29.9 (βC-Lys and γC-Met), 28.8 (δC-Lys), 22.4 (γC-Lys), 15.2 of Phe), 135.9 (d, J_C,P= 6.9 Hz, 1C of OCH₂Ph), 135.4 (C-6),

3

(-SCH₃), 12.3 (CH₃-Thy) ppm. ³¹P NMR (121 MHz, CDCl₃): δ =

129.0 (Ar-C), 128.5 (Ar-C), 128.4 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-

3

–0.8 ppm. HRMS: calcd. for C₃₁H₄₄N₅O₁₃PS [M – H]^–756.2320; C), 126.5 (Ar-C), 110.0 (C-5), 83.9 (C-1Ј), 82.2 (d, J_C,P= 7.3 Hz,

2

found 756.2347.

C-4Ј), 73.6 (C-3Ј), 68.7 (2 d, J_C,P= 5.4 Hz, 2 OCH₂Ph), 67.0 (d,

²J_C,P= 5.3 Hz_,C-5Ј), 53.5 (αC-Phe), 52.5 (αC-Lys), 51.8 (OCH₃),

50.4 (αC-Met), 47.9 (αC-Ala), 47.7 (αC-Ala), 40.2 (εC-Lys), 36.6

(βC-Phe), 36.0 (C-2Ј), 32.2 (βC-Met), 31.4 (βC-Lys), 29.3 (γC-

Met), 29.0 (δC-Lys), 22.7 (γC-Lys), 18.2 (CH₃-Ala), 17.9 (CH₃-

Ala), 14.6 (-SCH₃), 12.0 (CH₃-Thy) ppm. ³¹P NMR (121 MHz,

[D₆]DMSO): δ = –0.9 ppm. HRMS: calcd. for C₅₃H₆₉N₈O₁₆PS

[M – H]^–1135.4217; found 1135.4203.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-

L-Ala-L-Phe-OMe]-2Ј-deoxythymidine (31): Compound 31 was pre-

pared according to the general procedure starting from 24

(246.2 mg, 0.367 mmol), tetrapeptide 9 (253 mg, 0.3882 mmol), and

Et₃N (0.22 mL, 1.55 mmol) in dry CH₂Cl₂(20 mL). The crude resi-

due was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:5, v/v) to give conjugate 31

(339 mg, 82%) as a colourless foam. ¹H NMR (300 MHz, [D₆]-

DMSO): δ = 11.36 (br. s, 1 H, NH-Thy), 8.30–8.25 (m, 2 H, NH-

Phe and NH-Met), 8.05 (d, J = 8.0 Hz, 1 H, αNH-Lys), 8.02 (s, 1

H, CHO), 7.91 (d, J = 7.5 Hz, 1 H, αNH-Ala), 7.50 (s, 1 H, 6-H),

7.36–7.32 (m, 11 H, Ar-H of OBn and εNH-Lys), 7.29–7.19 (m, 5

H, Ar-H of Phe), 6.19 (app t, J = 7.3 Hz, 1 H, 1Ј-H), 5.07–5.04

(m, 5 H, 3Ј-H and 2 OCH₂Ph), 4.49–4.40 (m, 2 H, αH-Met and

αH-Phe), 4.33–4.18 (unresolved m, 4 H, αH-Ala, αH-Lys, 5Ј-H,

and 5ЈЈ-H), 4.12–4.08 (m, 1 H, 4Ј-H), 3.57 (s, 3 H, OCH₃), 3.05–

2.90 (m, 4 H, εCH₂-Lys and βCH₂-Phe), 2.43 (t, J = 7.8 Hz, 2 H,

γCH₂-Met), 2.23–2.17 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.02 (s, 3 H,

SCH₃), 1.95–1.75 (m, 2 H, βCH₂-Met), 1.70 (s, 3 H, CH₃-Thy),

1.64–1.45 (m, 2 H, βCH₂-Lys), 1.41–1.34 (m, 2 H, δCH₂-Lys),

1.29–1.22 (m, 2 H, γCH₂-Lys), 1.17 (d, J = 6.9 Hz, 3 H, CH₃-Ala)

ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ = 172.2 (CO-Ala), 171.7

(CO-Phe), 171.0 (CO-Lys), 170.6 (CO-Met), 163.6 (C-4), 161.0

(CHO), 155.2 (OCONH), 150.4 (C-2), 136.9 (1C of Phe), 135.9 (d,

³J_C,P= 6.9 Hz, 1C of OCH₂Ph), 135.4 (C-6), 129.0 (Ar-C), 128.5

(Ar-C), 128.4 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 126.5 (Ar-C),

General Procedure for the Synthesis of 3Ј-O-(Peptide carbamate)-2Ј-

deoxythymidine-5Ј-monophosphate Salts (19 and 33–40): Pd/C

(10%; Degussa; 10–100% w/w) was added to a stirred solution of

5Ј-O-(dibenzylphosphate)-3Ј-O-(peptide carbamate)-2Јdeoxythymi-

dine (1 equiv.) in EtOH or MeOH, and the mixture was hydroge-

nated at atmospheric pressure using a balloon filled with H₂for

24 h. The catalyst was removed by filtration, and the filtrate was

concentrated under reduced pressure. The resulting crude residue

was purified RP-HPLC [TEAB (triethylammonium bicarbonate;

50 mmol) in H₂O/MeCN, 98:2; and TEAB (50 mmol) in H₂O/

MeCN, 50:50]. The collected eluate was freeze-dried repeatedly un-

til constant mass to give the desired 3Ј-O-(peptide carbamate)-2Ј-

deoxythymidine-5Ј-monophosphate triethylammonium salts.

3Ј-O-[N-For-Gly-L-β-amino-Ala-(β-carbamate)-L-Phe-OMe]-2Ј-

deoxythymidine-5Ј-monophosphate Triethylammonium Salt (33):

Compound 33 was obtained as a white solid (149 mg, 81%) accord-

ing to the general procedure, starting from a stirred solution of

25 (180 mg, 0.205 mmol), Et₃N (0.057 mL, 0.410 mmol), and Pd/C

(10%; Degussa; 18 mg, 10% w/w) in MeOH (15 mL). ¹H NMR

(500 MHz, D₂O): δ = 8.16 (s, 1 H, CHO), 7.79 (s, 1 H, 6-H), 7.31–

7.19 (m, 5 H, Ar-H of Phe), 6.32 (dd, J = 8.4, 6.6 Hz, 1 H, 1Ј-H),

5.22–5.21 (m, 1 H, 3Ј-H), 4.72–4.69 (m, 1 H, αH-Phe), 4.48–4.45

(m, 1 H, αH-β-amino-Ala), 4.30–4.29 (m, 1 H, 4Ј-H), 4.05–4.03 (m,

2 H, 5Ј-H and 5ЈЈ-H), 3.91 (d, J = 3.4 Hz, 2 H, CH₂-Gly), 3.70 (s,

3 H, OCH₃), 3.44–3.24 (m, 2 H, βCH₂-β-amino-Ala), 3.22–2.95 (m,

2 H, βCH₂-Phe), 2.39–2.36 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.88 (s, 3 H,

CH₃-Thy) ppm. ¹³C NMR (125 MHz, D₂O): δ = 172.4 (CO-Phe),

170.3 (CO-β-amino-Ala), 170.1 (CO-Gly), 165.9 (C-4), 164.2

(CHO), 156.9 (OCONH), 151.1 (C-2), 136.6 (C-6), 135.6 (1C of

Ph), 128.6 (Ar-C), 128.0 (Ar-C), 126.5 (Ar-C), 111.3 (C-5), 84.1 (C-

3

110.0 (C-5), 83.9 (C-1Ј), 82.2 (d, J_C,P= 7.4 Hz, C-4Ј), 73.6 (C-3Ј),

2

68.7 (2 d, J_C,P= 5.4 Hz, 2 OCH₂Ph), 67.0 (d, J_C,P= 4.9 Hz_,C-

5Ј), 53.5 (αC-Phe), 52.4 (αC-Lys), 51.8 (OCH₃), 50.4 (αC-Met),

47.8 (αC-Ala), 40.2 (εC-Lys), 36.6 (βC-Phe), 36.0 (C-2Ј), 32.2 (βC-

Met), 31.5 (βC-Lys), 29.3 (γC-Met), 29.0 (δC-Lys), 22.6 (γC-Lys),

18.2 (CH₃-Ala), 14.6 (-SCH₃), 12.0 (CH₃-Thy) ppm. ³¹P NMR

(121 MHz, [D₆]DMSO): δ = –0.9 ppm. HRMS: calcd. for

C₅₀H₆₄N₇O₁₅PS [M – H]^–1064.3846; found 1064.3867.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-

L

-Ala- -Ala- -Phe-OMe]-2Ј-deoxythymidine (32): Compound 32

L

was prepared according to the general procedure starting from 24

(246.2 mg, 0.367 mmol), pentapeptide 10 (281 mg, 0.3882 mmol),

and Et₃N (0.22 mL, 1.55 mmol) in dry CH₂Cl₂(20 mL). The crude

residue was purified by column chromatography on silica gel (gra-

dient CH₂Cl₂/MeOH, 99:1, v/v; 97:3, v/v; 92:6, v/v) to give conju-

3

2

1Ј), 83.0 (d, J_C,P= 8.7 Hz, C-4Ј), 75.8 (C-3Ј), 64.3 (d, J_C,P

=

5.5 Hz_,C-5Ј), 53.4 (αC-Phe), 52.7 (αC-β-amino-Ala), 52.3 (OCH₃),

40.6 (βC-β-amino-Ala), 40.3 (αC-Gly), 36.0 (βC-Phe), 35.9 (C-2Ј),

11.0 (CH₃-Thy) ppm. ³¹P NMR (202 MHz, D₂O): δ = 0.3 ppm.

HRMS: calcd. for C₂₇H₃₅N₆O₁₄P [M – H]^–697.1876; found

697.1884.

1

gate 32 (380 mg, 86%) as a colourless foam. H NMR (300 MHz,

[D₆]DMSO): δ = 11.36 (br. s, 1 H, NH-Thy), 8.29 (d, J = 8.6 Hz,

1 H, NH-Met), 8.23 (d, J = 7.5 Hz, 1 H, NH-Phe), 8.07 (d, J =

7.6 Hz, 1 H, αNH-Lys), 8.02 (s, 1 H, CHO), 7.96 (d, J = 7.3 Hz, 1

H, αNH-Ala), 7.86 (d, J = 7.6 Hz, 1 H, αNH-Ala), 7.50 (s, 1 H, 6-

H), 7.36–7.32 (m, 11 H, Ar-H of OBn and εNH-Lys), 7.29–7.19

3Ј-O-[N-For-Gly-L-Lys-(ε-carbamate)-L-Phe-OMe]-2Ј-deoxythym-

idine-5Ј-monophosphate Triethylammonium Salt (19): Compound 19

was obtained as a white solid (255 mg, 83%) according to the gene-

(m, 5 H, Ar-H of Phe), 6.19 (app t, J = 7.3 Hz, 1 H, 1Ј-H), 5.07– ral procedure, starting from a stirred solution of 18 (300 mg,

5.04 (m, 5 H, 3Ј-H and 2 OCH₂Ph), 4.49–4.40 (m, 2 H, αH-Met 0.326 mmol), Et₃N (0.091 mL, 0.651 mmol), and Pd/C (10%; De-

1

and αH-Phe), 4.29–4.19 (unresolved m, 5 H, 2 αH-Ala, αH-Lys, 5Ј- gussa; 30 mg, 10% w/w) in MeOH (25 mL). H NMR (500 MHz,

H, and 5ЈЈ-H), 4.12–4.08 (m, 1 H, 4Ј-H), 3.57 (s, 3 H, OCH₃), 3.05– D₂O): δ = 8.17 (s, 1 H, CHO), 7.85 (s, 1 H, 6-H), 7.35–7.19 (m, 5

2.89 (m, 4 H, εCH₂-Lys and βCH₂-Phe), 2.43 (t, J = 7.9 Hz, 2 H,

γCH₂-Met), 2.25–2.17 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.03 (s, 3 H, (m, 1 H, 3Ј-H), 4.73–4.70 (m, 1 H, αH-Phe), 4.31 (br. s, 1 H, 4Ј-

SCH₃), 1.96–1.75 (m, 2 H, βCH₂-Met), 1.69 (s, 3 H, CH₃-Thy), H), 4.27–4.21 (m, 1 H, αH-Lys), 4.04–4.00 (m, 2 H, 5Ј-H and 5ЈЈ-

H, Ar-H of Phe), 6.32 (dd, J = 7.5, 6.1 Hz, 1 H, 1Ј-H), 5.26–5.23

1.67–1.46 (m, 2 H, βCH₂-Lys), 1.43–1.34 (m, 2 H, δCH₂-Lys), H), 3.96 (s, 2 H, CH₂-Gly), 3.73 (s, 3 H, OCH₃), 3.12–3.10 (m, 2

1.31–1.22 (m, 2 H, γCH₂-Lys), 1.20–1.13 (m, 6 H, 2 CH₃-Ala) ppm. H, εCH₂-Lys), 3.08–3.02 (m, 2 H, βCH₂-Phe), 2.45–2.40 (m, 2 H,

¹³C NMR (75 MHz, [D₆]DMSO): δ = 172.1 (CO-Ala_C-ter), 171.7

2Ј-H and 2ЈЈ-H), 1.93 (s, 3 H, CH₃-Thy), 1.69–1.58 (m, 2 H, βCH₂-

(CO-Phe), 171.5 (CO-Ala_N-ter), 171.1 (CO-Lys), 170.6 (CO-Met), Lys), 1.51–1.44 (m, 2 H, δCH₂-Lys), 1.29–1.26 (m, 2 H, γCH₂-Lys

163.5 (C-4), 161.0 (CHO), 155.4 (OCONH), 150.4 (C-2), 137.0 (1C merged with Et₃N) ppm. ¹³C NMR (125 MHz, D₂O): δ = 177.2

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2339

S. De, E. Groaz, P. Herdewijn

FULL PAPER

(CO-Phe), 175.3 (CO-Lys), 172.7 (CO-Gly), 172.5 (C-4), 170.5 Salt (36): Compound 36 was prepared according to the general

(CHO), 164.4 (OCONH), 157.3 (C-2), 137.2 (C-6), 136.6 (1C of procedure starting from a stirred solution of 28 (300 mg,

Ph), 129.1 (Ar-C), 128.2 (Ar-C), 126.5 (Ar-C), 111.8 (C-5), 84.6 (C-

0.270 mmol) and Pd(OH)₂/C (20%; 150 mg, 50% w/w) in EtOH/

H₂O, 10:1 (25 mL). After HPLC purification, the resulting trieth-

3

2

1Ј), 83.0 (d, J_C,P= 7.5 Hz, C-4Ј), 75.8 (C-3Ј), 63.7 (d, J_C,P

=

4.0 Hz_,C-5Ј), 55.7 (αC-Phe), 53.6 (αC-Lys), 53.5 (OCH₃), 43.3 (αC-

ylammonium salt was further acidified with HCl to stabilize the

1

Gly), 40.7 (εC-Lys), 37.5 (C-2Ј), 36.2 (βC-Phe), 30.3 (βC-Lys), 28.0 free amino group to give 36 (173.5 mg, 58%) as a white solid. H

(δC-Lys), 22.0 (γC-Lys), 12.1 (CH₃-Thy) ppm. ³¹P NMR

(202 MHz, D₂O): δ = 3.8 ppm. HRMS: calcd. for C₃₀H₄₁N₆O₁₄P

[M – H]^–739.2345; found 739.2336.

NMR (600 MHz, D₂O): δ = 8.11 (s, 1 H, CHO), 7.78 (s, 1 H, 6-

H), 6.34 (dd, J = 8.6, 6.2 Hz, 1 H, 1Ј-H), 5.23–5.22 (m, 1 H, 3Ј-

H), 4.49–4.47 (m, 1 H, αH-Met), 4.38–4.28 (m, 4 H, 2 αH-Lys and

4Ј-H), 4.09 (br. s, 1 H, 5Ј-H and 5ЈЈ-H), 3.73 (s, 3 H, OCH₃), 3.13–

3.10 (m, 2 H, εCH₂-Lys), 2.98–2.94 (m, 2 H, εCH₂-Lys), 2.57–2.50

(m, 2 H, γCH₂-Met), 2.43–2.36 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.07 (s,

3 H, SCH₃), 2.05–1.98 (m, 2 H, βCH₂-Met), 1.89 (s, 3 H, CH₃-

Thy), 1.88–1.61 (m, 8 H, 2 βCH₂-Lys and 2 δCH₂-Lys), 1.52–1.36

(m, 4 H, 2 γCH₂-Lys) ppm. ¹³C NMR (150 MHz, D₂O): δ = 175.0

(CO-Lys_N-Ter), 173.7 (CO-Lys_C-Ter), 172.9 (CO-Met), 166.4 (C-4),

164.0 (CHO), 157.3 (OCONH), 151.5 (C-2), 136.9 (C-6), 111.7 (C-

3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-L-Phe-OMe]-2Ј-deoxy-

thymidine-5Ј-monophosphate Triethylammonium Salt (34): Com-

pound 34 was obtained as a white solid (239 mg, 78%) according

to the general procedure, starting from a stirred solution of 26

(300 mg, 0.301 mmol), Et₃N (0.084 mL, 0.603 mmol), and Pd/C

1

(10%; Degussa; 240 mg, 80% w/w) in MeOH (25 mL). H NMR

(600 MHz, D₂O): δ = 11.29 (br. s, 1 H, NH-Thy), 8.43 (d, J =

7.1 Hz, 1 H, NH-Lys), 8.35 (d, J = 8.3 Hz, 1 H, NH-Met), 8.01 (s,

1 H, CHO), 7.90 (s, 1 H, 6-H), 7.34 (t, J = 5.5 Hz, εNH-Lys), 6.23

(dd, J = 9.0, 5.6 Hz, 1 H, 1Ј-H), 5.14–5.13 (m, 1 H, 3Ј-H), 4.49–

4.45 (m, 1 H, αH-Met), 4.21–4.17 (m, 1 H, αH-Lys), 4.05 (br. s, 1

H, 4Ј-H), 3.86 (br. s, 2 H, 5Ј-H and 5ЈЈ-H), 3.62 (s, 3 H, OCH₃),

2.99–2.96 (m, 2 H, εCH₂-Lys), 2.44 (t, J = 7.9 Hz, 2 H, γCH₂-

Met), 2.30–2.13 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.04 (s, 3 H, SCH₃),

1.93–1.77 (m, 5 H, βCH₂-Met and CH₃-Thy), 1.72–1.60 (m, 2 H,

βCH₂-Lys), 1.41–1.37 (m, 2 H, δCH₂-Lys), 1.32–1.27 (m, 2 H,

γCH₂-Lys) ppm. ¹³C NMR (150 MHz, D₂O): δ = 172.8 (CO-Lys),

172.5 (CO-Phe), 172.1 (CO-Met), 165.9 (C-4), 163.4 (CHO), 156.9

(OCONH), 151.1 (C-2), 136.5 (C-6), 135.6 (1C of Phe), 128.5 (Ar-

C), 128.0 (Ar-C), 126.5 (Ar-C), 111.3 (C-5), 84.2 (C-1Ј), 83.1 (d,

3

5), 84.7 (C-1Ј), 83.5 (d, J_C,P= 8.6 Hz, C-4Ј), 75.5 (C-3Ј), 64.9 (d,

²J_C,P= 4.0 Hz_,C-5Ј), 53.5 (αC-Lys_N-Ter), 52.6 (OCH₃), 52.2 (αC-

Lys_C-Ter), 51.3 (αC-Met), 39.7 (εC-Lys), 36.4 (C-2Ј), 30.4 (βC-

Lys_N-Ter), 30.2 (βC-Met), 29.6 (βC-Lys_N-Ter), 28.7 (γC-Met), 25.9

(2 δC-Lys), 21.9 (2 γC-Lys), 13.7 (SCH₃), 11.4 (CH₃-Thy) ppm. ³¹

P

NMR (202 MHz, D₂O): δ = –0.2 ppm. HRMS: calcd. for

C₅₂H₆₈N₇O₁₆PS [M – H]^–794.2801; found 794.2811.

3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)OMe]-2Ј-deoxythymidine-

5Ј-monophosphate Triethylammonium Salt (38): Compound 38 was

obtained as a white solid (217 mg, 79%) according to the general

procedure, starting from a stirred solution of 30 (240 mg,

0.316 mmol), Et₃N (0.088 mL, 0.632 mmol), and Pd/C (10%; De-

1

2

³J_C,P= 9.0 Hz, C-4Ј), 75.2 (C-3Ј), 64.4 (d, J_C,P= 4.0 Hz_,C-5Ј),

gussa; 120 mg, 50% w/w) in MeOH (25 mL). H NMR (500 MHz,

[D₆]DMSO): δ = 11.29 (br. s, 1 H, NH-Thy), 8.43 (d, J = 7.1 Hz,

1 H, NH-Lys), 8.35 (d, J = 8.3 Hz, 1 H, NH-Met), 8.01 (s, 1 H,

CHO), 7.90 (s, 1 H, 6-H), 7.34 (t, J = 5.5 Hz, εNH-Lys), 6.23 (dd,

J = 9.0, 5.6 Hz, 1 H, 1Ј-H), 5.14–5.13 (m, 1 H, 3Ј-H), 4.49–4.45

(m, 1 H, αH-Met), 4.21–4.17 (m, 1 H, αH-Lys), 4.05 (br. s, 1 H,

4Ј-H), 3.86 (br. s, 2 H, 5Ј-H and 5ЈЈ-H), 3.62 (s, 3 H, OCH₃), 2.99–

2.96 (m, 2 H, εCH₂-Lys), 2.44 (t, J = 7.9 Hz, 2 H, γCH₂-Met),

2.30–2.13 (m, 2 H, 2Ј-H and 2ЈЈ-H), 2.04 (s, 3 H, SCH₃), 1.93–1.77

(m, 5 H, βCH₂-Met and CH₃-Thy), 1.72–1.60 (m, 2 H, βCH₂-Lys),

1.41–1.37 (m, 2 H, δCH₂-Lys), 1.32–1.27 (m, 2 H, γCH₂-Lys) ppm.

¹³C NMR (125 MHz, [D₆]DMSO): δ = 172.5 (CO-Lys), 171.1 (CO-

Met), 163.8 (C-4), 161.0 (CHO), 155.4 (OCONH), 150.7 (C-2),

136.2 (C-6), 110.2 (C-5), 83.8 (C-1Ј and C-4Ј), 75.3 (C-3Ј), 64.3 (d,

²J_C,P= 5.2 Hz_,C-5Ј), 52.1 (αC-Lys), 51.9 (OCH₃), 50.2 (αC-Met),

39.6 (εC-Lys), 36.7 (C-2Ј), 32.3 (βC-Met), 30.3 (βC-Lys), 29.2 (γC-

Met), 28.9 (δC-Lys), 22.8 (γC-Lys), 14.7 (-SCH₃), 12.1 (CH₃-Thy)

ppm. ³¹P NMR (202 MHz, [D₆]DMSO): δ = –0.3 ppm. HRMS:

calcd. for C₂₄H₃₈N₅O₁₃PS [M – H]^–666.1851; found 666.1854.

53.4 (αC-Phe), 52.9 (αC-Lys), 52.2 (OCH₃), 50.6 (αC-Met), 39.3

(εC-Lys), 36.1 (C-2Ј), 35.9 (βC-Phe), 30.0 (βC-Lys), 29.7 (βC-Met),

28.3 (γC-Met), 27.5 (δC-Lys), 21.4 (γC-Lys), 13.4 (-SCH₃), 11.0

(CH₃-Thy) ppm. ³¹P NMR (202 MHz, D₂O): δ = –0.2 ppm.

HRMS: calcd. for C₃₃H₄₇N₆O₁₄PS [M – H]^–813.2536; found

813.2543.

3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-L-Ala-OMe]-2Ј-deoxy-

thymidine-5Ј-monophosphate Triethylammonium Salt (35): Com-

pound 35 was obtained as a white solid (148 mg, 76%) according

to the general procedure, starting from a stirred solution of 27

(190 mg, 0.206 mmol), Et₃N (0.058 mL, 0.413 mmol), and Pd/C

(10%; Degussa; 95 mg, 50% w/w) in MeOH (25 mL). ¹H NMR

(500 MHz, D₂O): δ = 8.21 (s, 1 H, CHO), 7.97 (s, 1 H, 6-H), 6.44

(dd, J = 8.6, 6.3 Hz, 1 H, 1Ј-H), 5.35–5.34 (m, 1 H, 3Ј-H), 4.63–

4.61 (m, 1 H, αH-Met), 4.50–4.45 (q, J = 7.2 Hz, 1 H, αH-Ala),

4.41–4.38 (m, 2 H, 4Ј-H and αH-Lys), 4.11 (br. s, 2 H, 5Ј-H and

5ЈЈ-H), 3.84 (s, 3 H, OCH₃), 3.24 (t, J = 6.4 Hz, 2 H, εCH₂-Lys),

2.69–2.64 (m, 2 H, γCH₂-Met), 2.58–2.50 (m, 2 H, 2Ј-H and 2ЈЈ-

H), 2.19 (s, 3 H, SCH₃), 2.18–2.07 (m, 2 H, βCH₂-Met), 2.02 (s, 3

H, CH₃-Thy), 1.94–1.80 (m, 2 H, βCH₂-Lys), 1.65–1.59 (m, 2 H,

δCH₂-Lys), 1.53–1.48 (m, 5 H, γCH₂-Lys and CH₃-Ala) ppm. ¹³C

NMR (125 MHz, D₂O): δ = 174.3 (CO-Ala), 173.0 (CO-Lys), 172.5

(CO-Met), 166.1 (C-4), 163.5 (CHO), 157.0 (OCONH), 151.3 (C-

2), 136.9 (C-6), 111.4 (C-5), 84.2 (C-1Ј), 83.5 (d, ³J_C,P= 9.2 Hz, C-

3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-L-Ala-L-Phe-OMe]-2Ј-de-

oxythymidine-5Ј-monophosphate Triethylammonium Salt (39): Com-

pound 39 was obtained as a white solid (232 mg, 81%) according

to the general procedure, starting from a stirred solution of 31

(280 mg, 0.263 mmol), Et₃N (0.073 mL, 0.525 mmol), and Pd/C

(10%; Degussa; 224 mg, 80% w/w) in MeOH (30 mL). ¹H NMR

(500 MHz, [D₆]DMSO): δ = 11.28 (br. s, 1 H, NH-Thy), 8.37 (d, J

= 7.8 Hz, 1 H, NH-Met), 8.27 (d, J = 7.5 Hz, 1 H, NH-Phe), 8.09

(d, J = 7.8 Hz, 1 H, αNH-Lys), 8.02 (s, 1 H, CHO), 7.95 (d, J =

7.7 Hz, 1 H, αNH-Ala), 7.89 (s, 2 H, 6-H), 7.31 (d, J = 5.4 Hz, 1

H, εNH-Lys), 7.28–7.19 (m, 5 H, Ar-H of Phe), 6.23 (dd, J = 8.7,

5.9 Hz, 1 H, 1Ј-H), 5.14–5.13 (m, 1 H, 3Ј-H), 4.47–4.40 (m, 2 H,

αH-Met and αH-Phe), 4.29–4.24 (m, 1 H, αH-Ala), 4.21–4.17 (m,

1 H, αH-Lys), 4.05 (br. s, 1 H, 4Ј-H), 3.89–3.86 (m, 2 H, 5Ј-H and

5ЈЈ-H), 3.57 (s, 3 H, OCH₃), 3.03–2.89 (m, 4 H, εCH₂-Lys and

2

4Ј), 75.5 (C-3Ј), 63.8 (d, J_C,P= 3.4 Hz_,C-5Ј), 53.0 (αC-Lys), 52.2

(OCH₃), 50.7 (αC-Met), 48.1 (αC-Ala), 39.4 (εC-Lys), 36.0 (C-2Ј),

29.9 (βC-Met and βC-Lys), 28.3 (γC-Met), 27.7 (δC-Lys), 21.4 (γC-

Lys), 15.2 (CH₃-Ala), 13.5 (SCH₃-Met), 11.1 (CH₃-Thy) ppm. ³¹P

NMR (202 MHz, D₂O): δ = 2.0 ppm. HRMS: calcd. for

C₂₇H₄₃N₆O₁₄PS [M – H]^–737.2222; found 737.2228.

3Ј-O-[N-For-L-Met-L-Lys(ε-carbamate)-L-Lys(ε-NH₂)-OMe]-2Ј-de-

oxythymidine-5Ј-monophosphate Triethylammonium Hydrochloride

2340

www.eurjoc.org

Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

βCH₂-Phe), 2.43 (t, J = 8.0 Hz, 2 H, γCH₂-Met), 2.29–2.11 (m, 2 dried repeatedly until constant mass to give 41 (258 mg, 71% over

H, 2Ј-H and 2ЈЈ-H), 2.02 (s, 3 H, SCH₃), 1.93–1.73 (m, 5 H, βCH₂- two steps) as a white solid. ¹H NMR (500 MHz, D₂O): δ = 7.75 (s,

Met and CH₃-Thy), 1.64–1.45 (m, 2 H, βCH₂-Lys), 1.40–1.34 (m,

1 H, 6-H), 6.31 (dd, J = 8.7, 6.0 Hz, 1 H, 1Ј-H), 5.21–5.20 (m, 1

2 H, δCH₂-Lys), 1.30–1.20 (m, 2 H, γCH₂-Lys), 1.20–1.16 (m, 3 H, H, 3Ј-H), 4.35–4.30 (m, 1 H, αH-Ala_C-Ter), 4.29 (br. s, 1 H, 4Ј-H),

CH₃-Ala) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 172.3 (CO- 4.25–4.22 (m, 1 H, αH-Lys), 4.09–4.05 (m, 2 H, 5Ј-H and 5ЈЈ-H),

Ala), 171.8 (CO-Phe), 171.1 (CO-Lys), 170.7 (CO-Met), 163.8 (C-

4.05–4.02 (m, 1 H, αH-Ala_N-Ter), 3.70 (s, 3 H, OCH₃), 3.12–3.10

4), 161.1 (CHO), 155.4 (OCONH), 150.6 (C-2), 137.1 (1C of Phe), (m, 2 H, εCH₂-Lys), 2.40–2.34 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.87 (s,

136.2 (C-6), 129.1 (Ar-C), 128.3 (Ar-C), 126.6 (Ar-C), 110.2 (C-5), 3 H, CH₃-Thy), 1.76–1.70 (m, 1 H, βCH₂-Lys), 1.52–1.44 (m, 5 H,

2

83.7 (C-1Ј and C-4Ј), 75.2 (C-3Ј), 64.3 (d, J_C,P= 5.0 Hz_,C-5Ј),

δCH₂-Lys and CH₃-Ala_N-Ter), 1.43–1.33 (m, 5 H, γCH₂-Lys and

53.6 (αC-Phe), 52.5 (αC-Lys), 51.9 (OCH₃), 50.5 (αC-Met), 47.9 CH₃-Ala_C-Ter) ppm. ¹³C NMR (125 MHz, D₂O): δ = 174.2 (CO-

(αC-Ala), 40.2 (εC-Lys), 36.7 (C-2Ј), 36.6 (βC-Phe), 32.1 (βC-Met), Ala_C-Ter), 173.1 (CO-Lys), 170.1 (CO-Ala_N-Ter), 165.9 (C-4), 157.0

31.5 (βC-Lys), 29.4 (γC-Met), 29.1 (δC-Lys), 22.7 (γC-Lys), 18.2 (OCONH), 151.2 (C-2), 136.5 (C-6), 111.3 (C-5), 84.3 (C-1Ј), 83.1

(CH₃-Ala), 14.6 (-SCH₃), 12.1 (CH₃-Thy) ppm. ³¹P NMR

(d, ³J_C,P= 9.0 Hz, C-4Ј), 75.2 (C-3Ј), 64.5 (d, ²J_C,P= 2.8 Hz_,C-5Ј),

(202 MHz, [D₆]DMSO): δ = –0.3 ppm. HRMS: calcd. for 53.1 (αC-Lys), 52.2 (OCH₃), 48.2 (αC-Ala_C-Ter), 48.1 (αC-Ala_N-Ter),

C₃₆H₅₂N₇O₁₅PS [M – H]^–884.2907; found 884.2920.

39.3 (εC-Lys), 36.0 (C-2Ј), 29.9 (βC-Lys), 27.8 (δC-Lys), 21.3 (γC-

Lys), 15.9 (CH₃-Ala_N-Ter), 15.1 (CH₃-Ala_C-Ter), 11.0 (CH₃-Thy)

ppm. ³¹P NMR (202 MHz, D₂O): δ = –0.3 ppm. HRMS: calcd. for

C₂₄H₃₉N₆O₁₃P [M – H]^–649.2240; found 649.2241.

3Ј-O-[N-For- -Met- -Lys(ε-carbamate)- -Ala- -Ala-L-Phe-OMe]-

L

2Ј-deoxythymidine-5Ј-monophosphate Triethylammonium Salt (40):

Compound 40 was obtained as a white solid (254 mg, 78%) accord-

ing to the general procedure, starting from a stirred solution of

32 (320 mg, 0.281 mmol), Et₃N (0.078 mL, 0.563 mmol), and Pd/C

3Ј-O-[H-L-Ala-L-Lys(ε-carbamate)-L-Ala-OH]-2Ј-deoxythymidine-

5Ј-monophosphate TFA Salt (42): LiOH (16.2 mg, 0.687 mmol) was

added to a stirred solution of 41 (150 mg, 0.196 mmol) in THF/

H₂O/MeOH, 1:1:1 (3 mL), and the resulting solution was stirred

at room temp. for 3 h. The reaction mixture was neutralized with

trifluoroacetic acid, and the volatiles were removed under reduced

pressure. The crude residue was purified by RP-Prep HPLC [TFA

(0.05%) in H₂O/MeCN, 98:2; and TFA (0.05%) in MeCN/H₂O,

98:2] to give 42 (109 mg, 74 %) as a white solid. ¹H NMR

(600 MHz, D₂O): δ = 7.77 (s, 1 H, 6-H), 6.33 (dd, J = 8.9, 5.9 Hz,

1 H, 1Ј-H), 5.22–5.21 (m, 1 H, 3Ј-H), 4.32–4.29 (m, 2 H, αH-

Ala_N-Terand 4Ј-H), 4.27–4.25 (m, 1 H, αH-Lys), 4.09–4.08 (m, 2

H, 5Ј-H and 5ЈЈ-H), 4.07–4.05 (m, 1 H, αH-Ala_C-Ter), 3.12–3.10 (m,

2 H, εCH₂-Lys), 2.42–2.36 (m, 2 H, 2Ј-H and 2ЈЈ-H), 1.88 (s, 3 H,

CH₃-Thy), 1.81–1.69 (m, 1 H, βCH₂-Lys), 1.53–1.46 (m, 5 H,

δCH₂-Lys and CH₃-Ala_C-Ter), 1.44–1.36 (m, 5 H, γCH₂-Lys and

CH₃-Ala_N-Ter) ppm. ¹³C NMR (150 MHz, D₂O): δ = 176.0 (CO-

Ala_C-Ter), 173.3 (CO-Lys), 170.4 (CO-Ala_N-Ter), 166.3 (C-4), 157.4

(OCONH), 151.5 (C-2), 136.9 (C-6), 111.7 (C-5), 84.7 (C-1Ј), 83.5

(d, ³J_C,P= 9.0 Hz, C-4Ј), 75.5 (C-3Ј), 64.9 (d, ²J_C,P= 3.4 Hz_,C-5Ј),

53.4 (αC-Lys), 48.5 (αC-Ala_C-Terand αC-Ala_N-Ter), 39.7 (εC-Lys),

36.4 (C-2Ј), 30.3 (βC-Lys), 28.1 (δC-Lys), 21.7 (γC-Lys), 16.3 (CH₃-

Ala_C-Ter), 15.7 (CH₃-Ala_N-Ter), 11.4 (CH₃-Thy) ppm. ³¹P NMR

(202 MHz, D₂O): δ = –0.3 ppm. HRMS: calcd. for C₂₃H₃₇N₆O₁₃P

[M – H]^–635.2083; found 635.2093.

1

(10%; Degussa; 256 mg, 80% w/w) in MeOH (30 mL). H NMR

(500 MHz, [D₆]DMSO): δ = 11.28 (br. s, 1 H, NH-Thy), 8.37 (d, J

= 8.2 Hz, 1 H, NH-Met), 8.28 (d, J = 7.5 Hz, 1 H, NH-Phe), 8.10

(d, J = 7.7 Hz, 1 H, αNH-Lys), 8.02 (s, 1 H, CHO), 7.99 (d, J =

7.4 Hz, 1 H, αNH-Ala), 7.89–7.86 (m, 2 H, 6-H and αNH-Ala),

7.31 (d, J = 5.5 Hz, 1 H, εNH-Lys), 7.28–7.19 (m, 5 H, Ar-H of

Phe), 6.23 (dd, J = 8.8, 5.8 Hz, 1 H, 1Ј-H), 5.15–5.14 (m, 1 H, 3Ј-

H), 4.47–4.41 (m, 2 H, αH-Met and αH-Phe), 4.30–4.25 (m, 1 H,

αH-Ala_C-ter), 4.25–4.23 (m, 1 H, αH-Ala_N-ter), 4.22–4.17 (m, 1 H,

αH-Lys), 4.05 (br. s, 1 H, 4Ј-H), 3.89–3.86 (m, 2 H, 5Ј-H and 5ЈЈ-

H), 3.57 (s, 3 H, OCH₃), 3.03–2.92 (m, 4 H, εCH₂-Lys and βCH₂-

Phe), 2.43 (t, J = 8.2 Hz, 2 H, γCH₂-Met), 2.30–2.12 (m, 2 H, 2Ј-

H and 2ЈЈ-H), 2.03 (s, 3 H, SCH₃), 1.94–1.74 (m, 5 H, βCH₂-Met

and CH₃-Thy), 1.68–1.47 (m, 2 H, βCH₂-Lys), 1.40–1.35 (m, 2 H,

δCH₂-Lys), 1.30–1.21 (m, 2 H, γCH₂-Lys), 1.20–1.16 (m, 6 H, 2

CH₃-Ala) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 172.2 (CO-

Ala_C-ter), 171.7 (CO-Phe), 171.5 (CO-Ala_N-ter), 171.2 (CO-Lys),

170.7 (CO-Met), 163.8 (C-4), 161.0 (CHO), 155.3 (OCONH), 150.4

(C-2), 137.0 (1C of Phe), 136.1 (C-6), 129.1 (Ar-C), 128.2 (Ar-C),

126.5 (Ar-C), 110.1 (C-5), 83.7 (C-1Ј and C-4Ј), 75.2 (C-3Ј), 64.2

2

(d, J_C,P= 4.6 Hz_,C-5Ј), 53.6 (αC-Phe), 52.6 (αC-Lys), 51.8

(OCH₃), 50.4 (αC-Met), 48.0 (αC-Ala_C-ter), 47.8 (αC-Ala_N-ter), 40.2

(εC-Lys), 36.7 (C-2Ј), 36.6 (βC-Phe), 32.1 (βC-Met), 31.4 (βC-Lys),

29.3 (γC-Met), 29.1 (δC-Lys), 22.7 (γC-Lys), 18.2 (CH₃-Ala), 17.9

(CH₃-Ala), 14.6 (-SCH₃), 12.0 (CH₃-Thy) ppm. ³¹P NMR

(202 MHz, [D₆]DMSO): δ = –0.3 ppm. HRMS: calcd. for

C₃₉H₅₇N₈O₁₆PS [M – H]^–955.3278; found 955.3293.

5Ј-O-(4-Monomethoxytrityl)-xylo-2Ј-deoxythymidine (43): Et₃N

(1.72 mL, 12.4 mmol), DMAP (0.13 g, 1.03 mmol), and then 4-

monomethoxytrityl chloride (3.66 g, 11.9 mmol) were added to a

stirred solution of 2Ј-deoxythymidine (2.50 g, 10.3 mmol) in pyr-

idine (45 mL) at room temp. The resulting mixture was stirred over-

night at room temp. The reaction mixture was cooled, and Et₃N

(1.72 mL, 12.4 mmol) and then MsCl (0.88 mL, 11.4 mmol) were

added. The mixture was stirred for 2 h at room temp., then it was

filtered, the solid was washed with ethyl acetate, and the filtrate

was concentrated in vacuo. The residue was dissolved in EtOH

(45 mL), and NaOH (1 m; 25 mL) was added. The mixture was

heated at reflux for 1.5 h, then it was cooled to room temp. and

neutralized with HCl (1 n; 10 mL). The ethanol was removed in

vacuo, and the residue was extracted with CH₂Cl₂(3ϫ 70 mL).

The combined organic extracts were washed with brine, dried with

Na₂SO₄, and concentrated under reduced pressure. The crude resi-

due was purified by column chromatography on silica gel (gradient

hexane/EtOAc, 2:1, v/v; 1:1, v/v; 1:2, v/v) to give 43 (2.54 g, 48%)

3Ј-O-[H-L-Ala-L-Lys(ε-carbamate)-L-Ala-OMe]-2Ј-deoxythymidine-

5Ј-monophosphate TFA Salt (41): According to the general pro-

cedure, a stirred solution of 29 (400 mg, 0.476 mmol) and Pd/C

(10%; Degussa; 40 mg, 10% w/w) in MeOH (15 mL) was hydroge-

nated to give crude 3Ј-O-[N-Boc-l-Ala-l-Lys-(ε-carbamate)-l-Ala-

OMe]-2Ј-deoxythymidine-5Ј-monophosphate 37 (357 mg, quantita-

tive) as a white solid, which was used in the next step without

further purification.

Thioanisole (0.056 mL, 0.476 mmol) and then trifluoroacetic acid

(1 mL) were added to a stirred solution of 37 (357 mg, 0.476 mmol)

in H₂O (3 mL) at 0 °C. The reaction mixture was stirred at room

temp. for 1.5 h, and then the volatiles were removed in vacuo. The

crude residue was coevaporated with toluene (3ϫ), and then puri-

fied by RP-Prep HPLC [TFA (0.05%) in H₂O/MeCN, 98:2; and

1

as a pale yellow foam. H NMR (300 MHz, CDCl₃): δ = 9.85 (br.

TFA (0.05%) in MeCN/H₂O, 98:2]. The collected eluate was freeze- s, 1 H, NH), 7.61 (s, 1 H, 6-H), 7.48–7.46 (m, 4 H, Ar-H), 7.37–

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2341

S. De, E. Groaz, P. Herdewijn

FULL PAPER

7.20 (m, 8 H, Ar-H), 6.86–6.83 (m, 2 H, Ar-H), 6.16 (dd, J = 7.8,

(s, 1 H, 6-H), 7.34 (s, 10 H, Ar-H), 6.28 (dd, J = 8.2, 5.8 Hz, 1 H,

1.7 Hz, 1 H, 1Ј-H), 4.42–4.40 (m, 1 H, 3Ј-H), 4.06–4.01 (m, 1 H, 1Ј-H), 5.46 (br. s, 2 H, ONH₂), 5.12–4.99 (m, 4 H, OCH₂Ph), 4.22–

4Ј-H), 3.78 (s, 3 H, OCH₃), 3.66–3.61 (m, 1 H, 5Ј-H), 3.61–3.46 4.19 (m, 4 H, 3Ј-H, 4Ј-H, 5Ј-H, and 5ЈЈ-H), 2.93–2.92 (m, 1 H, 2Ј-

(m, 1 H, 5ЈЈ-H), 2.56–2.479 (m, 1 H, 2Ј-H), 2.29–2.24 (m, 1 H, 2ЈЈ- H), 2.40–2.34 (m, 1 H, 2ЈЈ-H), 1.81 (s, 3 H, CH₃) ppm. ¹³C NMR

3

H), 1.72 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = (75 MHz, CDCl₃): δ = 164.1 (C-4), 150.6 (C-2), 135.4 (d, J_C,P

=

164.4, 158.8, 151.0, 144.1, 144.0, 137.4, 135.1, 130.4, 128.4, 128.3,

128.1, 127.2, 113.4, 109.8, 87.2, 85.6, 83.5, 62.2, 55.3, 41.4,

12.5 ppm. HRMS: calcd. for C₃₀H₃₀N₂O₆[M + Na]⁺537.1996;

found 537.1995.

6.3 Hz, 1C of OCH₂Ph), 135.2 (C-6), 128.8 (Ar-C), 128.6 (Ar-C),

3

128.0 (Ar-C), 111.3 (C-5), 84.8 (C-1Ј), 83.5 (C-3Ј), 81.5 (d, J_C,P

=

2

8.1 Hz, C-4Ј), 69.7–69.6 (2 d, J_C,P= 5.2 Hz, 2 OCH₂Ph), 67.9 (d,

²J_C,P= 5.6 Hz_,C-5Ј), 36.4 (C-2Ј), 12.2 (-CH₃) ppm. ³¹P NMR

(121 MHz, CDCl ₃) : δ = – 0 . 5 p p m . H R M S : c a l c d . for

C₃₁H₃₀N₃O₁₂P [M + Na]⁺540.1506; found 540.1501.

3Ј-O-Phthalimido-2Ј-deoxythymidine (44): DIAD (0.98 g,

4.85 mmol) was added dropwise to a stirred suspension of 43

(1.85 g, 3.59 mmol), N-hydroxyphthalimide (0.79 g, 4.85 mmol),

and PPh₃(1.25 g, 4.75 mmol) in toluene (35 mL) at 0 °C. The reac-

tion mixture was allowed to warm slowly to room temp. and stirred

for 2 h. After the reaction was complete, the mixture was concen-

trated in vacuo, and the residue was dissolved in AcOH (80 %;

50 mL). This solution was stirred for 3 h at room temp., then the

mixture was concentrated in vacuo, coevaporating with toluene

(3ϫ). The crude residue was purified by column chromatography

on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v; 98:2, v/v; 97:3, v/

v) to give 44 (0.42 g, 28% over two steps) as a white solid. ¹H NMR

(300 MHz, [D₆]DMSO): δ = 11.34 (br. s, 1 H, NH), 7.89 (s, 4 H,

Ar-H), 7.72 (s, 1 H, 6-H), 6.39 (dd, J = 8.9, 5.6 Hz, 1 H, 1Ј-H),

5.20–5.14 (m, 1 H, 3Ј-H), 4.97–4.95 (m, 1 H, OH), 4.23–4.22 (m, 1

H, 4Ј-H), 3.62–3.59 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.46–2.26 (m, 1 H,

2Ј-H and 2ЈЈ-H), 1.77 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz,

[D₆]DMSO): δ = 163.7 (C-4), 163.6 (CO-phthalimide), 150.5 (C-2),

135.9 (C-6), 134.9 (Ar-C), 128.6 (Ar-C), 123.4 (Ar-C), 109.7 (C-5),

88.1 (C-1Ј), 83.7 (app. C-3Ј), 82.9 (app. C-4Ј), 61.4 (C-5Ј), 35.4 (C-

2Ј), 12.3 (CH₃) ppm. HRMS: calcd. for C₁₈H₁₇N₃O₇[M + Na]⁺

410.0959; found 410.0958.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Glu(δ-carbox-

amide)-OMe]-2Ј-deoxythymidine (47): DCC (77.0 mg, 0.371 mmol)

was added to a stirred solution of 46 (120 mg, 0.232 mmol), dipept-

ide 2a (85.5 mg, 0.267 mmol), and DMAP (1.10 mg, 0.009 mmol)

in a mixture of dry DMF (1.0 mL) and dry CH₂Cl₂(5 mL) at 0 °C.

The reaction mixture was allowed to warm slowly to room temp.

and stirred for 24 h. All the volatiles were removed under reduced

pressure, and CH₂Cl₂(10 mL) was added to the residue. The solid

residue was filtered off and washed with CH₂Cl₂. The combined

organic layers were concentrated under reduced pressure, and the

crude residue was purified by column chromatography on silica gel

(gradient CH₂Cl₂/MeOH, 99:2, v/v; 97:4, v/v; 94:7, v/v) to give 47

(120 mg, 63%) as a colourless foam. ¹H NMR (300 MHz, CDCl₃):

δ = 10.65 (br. s, 1 H, NH), 10.34 (br. s, 1 H, NH), 8.20 (s, 1 H,

CHO), 7.80 (d, J = 7.9 Hz, 1 H, NH-Met), 7.74 (d, J = 7.1 Hz, 1

H, NH-Glu), 7.43 (s, 1 H, 6-H), 7.34–7.28 (m, 10 H, Ar-H of OBn),

6.24 (dd, J = 8.2, 5.7 Hz, 1 H, 1Ј-H), 5.08–5.00 (m, 4 H, 2

OCH₂Ph), 4.74–4.70 (m, 1 H, αH-Glu), 4.63–4.62 (m, 1 H, 3Ј-H),

4.57–4.53 (m, 1 H, αH-Met), 4.33 (br. s, 1 H, 4Ј-H), 4.25–4.17 (m,

2 H, 5Ј-H and 5ЈЈ-H), 3.70 (s, 3 H, OCH₃), 2.60–2.54 (m, 3 H, 2Ј-

H and γCH₂-Met), 2.32–2.04 (unresolved m, 7 H, γCH₂-Glu,

βCH₂-Met, and SCH₃), 2.00–1-91 (m, 2 H, βCH₂-Glu), 1.85–1.79

(m, 4 H, 2ЈЈ-H and CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

171.8 (CO₂Me-Glu), 171.7 (CO-Met), 170.5 (δCO-Glu), 164.1 (C-

4), 162.4 (CHO), 151.0 (C-2), 135.2 (C-6), 135.1 (d, ³J_C,P= 6.0 Hz,

1C of OCH₂Ph), 128.8 (Ar-C), 128.6 (Ar-C), 128.0 (Ar-C), 111.7

5Ј-O-(Dibenzylphosphate)-3Ј-O-phthalimido-2Ј-deoxythymidine

(45): Following a similar procedure to that used for the synthesis

of 22, compound 45 was obtained starting from 44 (0.40 g,

1.03 mmol), tetrazole (0.45 m in MeCN; 11.47 mL, 5.16 mmol), and

dibenzyldiisopropyl phosphoramidite (0.78 mL, 2.27 mmol) in dry

CH₂Cl₂(5 mL), and H₂O₂(30%; 0.44 mL, 5.16 mmol). The crude

residue was purified by column chromatography on silica gel (gra-

dient CH₂Cl₂/MeOH, 100:0, v/v; 99:1, v/v; 98:2, v/v) to give 45

3

(C-5), 84.9 (C-3Ј), 84.7 (C-1Ј), 81.0 (d, J_C,P= 6.7 Hz, C-4Ј), 69.8–

2

69.6 (2 d, J_C,P= 5.3 Hz, 2 -OCH₂Ph), 67.6 (d, J_C,P= 4.8 Hz_,C-

5Ј), 52.6 (OCH₃), 51.4 (αC-Met), 51.0 (αC-Glu), 36.0 (C-2Ј), 31.4

(βC-Met), 29.6 (γC-Met), 29.0 (γC-Glu), 27.2 (βC-Glu), 15.1

(SCH₃), 12.3 (CH₃) ppm. ³¹P NMR (121 MHz, CDCl₃): δ =

–0.8 ppm. HRMS: calcd. for C₃₆H₄₆N₅O₁₃PS [M – H]^–818.2477;

found 818.2446.

1

(0.62 g, 98%) as a colourless foam. H NMR (300 MHz, CDCl₃):

δ = 10.08 (br. s, 1 H, NH), 7.82–7.73 (m, 4 H, Ar-H), 7.37 (s, 1 H,

6-H), 7.31–7.30 (m, 10 H, Ar-H), 6.49 (dd, J = 8.2, 6.0 Hz, 1 H,

1Ј-H), 5.12–5.00 (m, 4 H, OCH₂Ph), 4.86–4.84 (m, 1 H, 3Ј-H),

4.53–4.52 (m, 1 H, 4Ј-H), 4.26–4.25 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.69–

2.63 (m, 1 H, 2Ј-H), 2.10–2.02 (m, 1 H, 2ЈЈ-H), 1.80 (s, 3 H, CH₃)

ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 164.1 (C-4), 163.6 (CO-

phthalimide), 150.3 (C-2), 135.3 (C-6), 135.2 (d, ³J_C,P= 6.1 Hz, 1C

of OCH₂Ph), 134.7 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C), 128.4 (Ar-

C), 127.9 (Ar-C), 127.8 (Ar-C), 123.6 (Ar-C), 111.2 (C-5), 87.8 (C-

3Ј-O-[N-For-L-Met-L-Glu(δ-carboxamide)-OMe]-2Ј-deoxy-

thymidine-5Ј-monophosphate Triethylammonium Salt (48): Com-

pound 48 was obtained as a white solid (96 mg, 78%) according to

the general procedure used for the synthesis of 19 and 33–40, start-

ing from a stirred solution of 47 (120 mg, 0.146 mmol), Et₃N

(0.041 mL, 0.293 mmol), and Pd/C (10%; Degussa; 60 mg, 50%

3

2

1Ј), 84.9 (C-3Ј), 81.0 (d, J_C,P= 8.3 Hz, C-4Ј), 69.6 (app t, J_C,P

=

1

w/w) in MeOH (15 mL). H NMR (500 MHz, D₂O): δ = 8.16 (s, 1

2

5.0 Hz, OCH₂Ph), 67.0 (d, J_C,P= 5.4 Hz_,C-5Ј), 36.4 (C-2Ј), 12.2

(CH₃) ppm. ³¹P NMR (121 MHz, CDCl₃): δ = –0.8 ppm. HRMS:

calcd. for C₃₁H₃₀N₃O₁₂P [M + H]⁺648.1742; found 648.1728.

H, CHO), 7.98 (s, 1 H, H-6), 6.43 (dd, J = 9.1, 5.7 Hz, 1 H, 1Ј-H),

4.76–4.75 (m, 1 H, 3Ј-H), 4.60–4.57 (m, 1 H, αH-Glu), 4.47–4.45

(m, 1 H, αH-Met), 4.38 (br. s, 1 H, 4Ј-H), 3.97–3.96 (m, 2 H, 5Ј-H

and 5ЈЈ-H), 3.77 (s, 3 H, OCH₃), 2.64–2.59 (m, 2 H, γCH₂-Met),

3Ј-O-Amino-5Ј-O-(dibenzylphosphate)-2Ј-deoxythymidine (46):

Methylamine (4% in H₂O; 2.73 mL, 3.52 mmol) was slowly added 2.53–2.49 (m, 1 H, 2Ј-H), 2.43–2.37 (m, 1 H, 2ЈЈ-H), 2.32–2.10 (un-

to a stirred solution of 45 (0.57 g, 0.88 mmol) in EtOH (15 mL) at resolved m, 7 H, γCH₂-Glu, βCH₂-Met, and SCH₃), 2.06–2.01 (m,

0 °C. The mixture was stirred at room temp. for 0.5 h, then it was

filtered, and the solid was washed with CH₂Cl₂. The filtrate was

concentrated in vacuo, and the crude residue was purified by col-

umn chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1,

v/v; 98:2, v/v; 97:3, v/v) to give 46 (0.37 g, 81%) as a colourless

2 H, βCH₂-Glu), 1.95 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz,

D₂O): δ = 172.9 (αCO-Glu), 172.7 (CO-Met), 171.3 (δCO-Glu),

166.4 (C-4), 163.7 (CHO), 151.4 (C-2), 137.3 (C-6), 111.4 (C-5),

85.7 (C-3Ј), 84.3 (C-1Ј), 82.1 (d, ³J_C,P= 8.7 Hz, C-4Ј), 63.7 (d, ²J_C,P

= 3.5 Hz_,C-5Ј), 52.5 (OCH₃), 51.5 (αC-Met), 50.8 (αC-Glu), 34.7

foam. ¹H NMR (300 MHz, CDCl₃): δ = 9.91 (br. s, 1 H, NH), 7.39 (C-2Ј), 30.0 (βC-Met), 28.4 (γC-Met), 28.1 (γC-Glu), 25.3 (βC-

2342

www.eurjoc.org

Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

Glu), 13.5 (SCH₃), 11.2 (CH₃) ppm. ³¹P NMR (202 MHz, [D₆]-

DMSO): δ = 3.4 ppm. HRMS: calcd. for C₂₂H₃₄N₅O₁₃PS

[M – H]^–638.1538; found 638.1542.

H), 5.16 (s, 2 H, OCH₂O), 5.11–5.09 (m, 3 H, 3Ј-H), 4.18 (br. s, 1

H, 4Ј-H), 4.14–4.00 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.56–2.50 (m, 1 H,

2Ј-H), 2.26–2.17 (m, 2 H, 2ЈЈ-H), 1.59 (s, 3 H, CH₃), 1.14 (s, 9 H,

CH₃-tBu) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 164.0 (C-4), 163.5

(CO-phthalimide), 150.6 (C-2), 135.7 (Ar-C), 135.4 (Ar-C), 135.3

(C-6), 134.6 (Ar-C), 133.2 (Ar-C), 132.4 (Ar-C), 130.2 (Ar-C),

130.1 (Ar-C), 129.0 (Ar-C), 128.1 (Ar-C), 128.0 (Ar-C), 123.7 (Ar-

C), 111.3 (C-5), 97.5 (OCH₂O), 85.3 (C-1Ј), 84.7 (C-4Ј), 78.6 (C-

3Ј), 64.6 (C-5Ј), 37.9 (C-2Ј), 27.1 [(CH₃)₃], 19.5 (CMe₃), 12.1 (CH₃)

ppm. HRMS: calcd. for C₃₅H₃₇N₃O₈Si [M + Na]⁺678.2242; found

678.2258.

5Ј-O-(tert-Butyldiphenylsilyl)-2Јdeoxythymidine (49):^[53]A solution

of imidazole (1.57 g, 23.08 mmol) and TBDPSCl (2.86 mL,

11.01 mmol) in dry DMF (15 mL) was added to a stirred solution

of 2Ј-deoxythymidine (2.54 g, 10.49 mmol) in dry DMF (20 mL) at

room temp. The mixture was stirred for 4 h at room temp., then it

was diluted with water (350 mL), and the aqueous layer was ex-

tracted with ethyl acetate (3ϫ 150 mL). The combined organic ex-

tracts were washed with brine, dried with Na₂SO₄, filtered and con-

centrated under reduced pressure. The crude residue was purified

by column chromatography on silica gel (gradient CH₂Cl₂/MeOH,

99:1, v/v; 98:2, v/v; 95:5, v/v) to give 49 (4.17 g, 83%) as a white

solid. HRMS: calcd. for C₂₆H₃₂N₂O₅Si [M + H]⁺481.2153; found

481.2156.

3Ј-O-(Phthalimidooxymethyl)-2Ј-deoxythymidine (52): Triethyl-

amine trihydrofluoride (1.72 mL, 10.55 mmol) was added to a

stirred solution of 51 (1.73 g, 2.64 mmol) in THF (25 mL) at room

temp. The reaction mixture was stirred at room temp. for 36 h, and

then it was concentrated under reduced pressure. The crude residue

was purified by column chromatography on silica gel (gradient

CH₂Cl₂/MeOH, 99:1, v/v; 98:2, v/v; 95:5, v/v) to give 52 (0.93 g,

84%) as a white solid. ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.30

(s, 1 H, NH), 7.88 (s, 4 H, Ar-H), 7.78 (d, J = 1.0 Hz, 1 H, 6-H),

6.12 (dd, J = 8.9, 5.6 Hz, 1 H, 1Ј-H), 5.26–5.19 (m, 3 H, OCH₂O

and 5Ј-OH), 4.80–4.78 (m, 1 H, 3Ј-H), 4.03–4.02 (m, 1 H, 4Ј-H),

3.78–3.63 (m, 2 H, 5Ј-H and 5ЈЈ-H), 2.38–2.32 (m, 1 H, 2Ј-H), 2.25–

2.16 (m, 2 H, 2ЈЈ-H), 1.79 (d, J = 0.9 Hz, 3 H, CH₃) ppm. ¹³C

NMR (75 MHz, [D₆]DMSO): δ = 163.7 (C-4), 163.2 (CO-phthal-

imide), 150.4 (C-2), 135.9 (C-6), 134.9 (Ar-C), 128.5 (Ar-C), 123.4

(Ar-C), 109.5 (C-5), 97.8 (OCH₂O), 84.9 (C-1Ј), 83.9 (C-4Ј), 78.7

(C-3Ј), 61.5 (C-5Ј), 36.6 (C-2Ј), 12.3 (CH₃) ppm. HRMS: calcd. for

C₁₉H₁₉N₃O₈[M + Na]⁺440.1064; found 440.1071.

5Ј-O-(tert-Butyldiphenylsilyl)-3Ј-O-(methylthiomethyl)-2Ј-deoxy-

thymidine (50):^[41]Acetic anhydride (19.4 mL) and acetic acid

(6.2 mL) were added to a stirred solution of 49 (4.08 g, 8.50 mmol)

in DMSO (27.5 mL). The reaction mixture was stirred at room

temp. for 48 h, then it was concentrated under reduced pressure.

The residue was neutralized with saturated aq. NaHCO₃(300mL),

and the mixture was extracted with ethyl acetate (3ϫ 180 mL). The

combined organic extracts were washed with brine, dried with

Na₂SO₄, filtered and concentrated under reduced pressure. The

crude residue was purified by column chromatography on silica gel

(gradient hexane/EtOAc, 4:1, v/v; 3:1, v/v; 2:1, v/v) to give 50 (3.3 g,

1

72%) as a white solid. H NMR (300 MHz, CDCl₃): δ = 9.54 (br.

s, 1 H, NH), 7.70–7.67 (m, 4 H, Ar-H), 7.47–7.38 (m, 7 H, 6-H

and Ar-H), 6.35 (dd, J = 8.5, 5.7 Hz, 1 H, 1Ј-H), 4.66–4.56 (m, 3

H, 3Ј-H and OCH₂S), 4.09–4.08 (m, 1 H, 4Ј-H), 4.00–3.82 (m, 2

H, 5Ј-H and 5ЈЈ-H), 2.49–2.43 (m, 1 H, 2Ј-H), 2.16–2.04 (m, 4 H,

SCH₃and 2ЈЈ-H), 1.66 (s, 3 H, CH₃), 1.10 (s, 9 H, CH₃-tBu) ppm.

¹³C NMR (75 MHz, CDCl₃): δ = 164.1 (C-4), 150.6 (C-2), 135.6

(Ar-C), 135.4 (Ar-C), 135.2 (C-6), 130.2 (Ar-C), 130.1 (Ar-C),

128.1 (Ar-C), 128.0 (Ar-C), 111.3 (C-5), 85.0 (C-1Ј), 84.8 (C-4Ј),

76.2 (C-3Ј), 73.7 (OCH₂S), 64.1 (C-5Ј), 37.9 (C-2Ј), 27.1

[(CH₃)₃], 19.4 (CMe₃), 13.9 (SCH₃), 12.2 (CH₃) ppm. HRMS:

calcd. for C₂₈H₃₆N₂O₅SSi [M + Na]⁺563.2006; found 563.2009.

5Ј-O-(Dibenzylphosphate)-3Ј-O-(phthalimidooxymethyl)-2Ј-deoxy-

thymidine (53): Following a similar procedure to that used for the

synthesis of 22, compound 53 was obtained starting from 52

(0.93 g, 2.23 mmol), tetrazole (0.45 m in MeCN; 24.75 mL,

11.14 mmol), and dibenzyldiisopropyl phosphoramidite (1.52 mL,

4.46 mmol) in dry CH₂Cl₂(10 mL), and H₂O₂(30 %; 0.96 mL,

11.14 mmol). The crude residue was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 100:0, v/v;

99:1, v/v; 98:2, v/v) to give 53 (1.39 g, 92%) as a colourless foam.

¹H NMR (300 MHz, CDCl₃): δ = 9.5 (app d, 1 H, NH), 7.84–7.74

(m, 4 H, phthamido Ar-H), 7.37 (d, J = 1.0 Hz, 1 H, 6-H), 7.38–

7.33 (m, 10 H, benzyl Ar-H), 6.49 (dd, J = 9.1, 5.3 Hz, 1 H, 1Ј-H),

5.11–5.04 (m, 6 H, OCH₂Ph and OCH₂O), 4.85–4.83 (m, 1 H, 3Ј-

H), 4.53–4.52 (m, 1 H, 4Ј-H), 4.55–4.49 (m, 1 H, 5Ј-H), 4.30–4.24

(m, 1 H, 5ЈЈ-H), 4.20–4.19 (m, 1 H, 4Ј-H), 2.37–2.30 (m, 1 H, 2Ј-

H), 1.90–1.80 (m, 4 H, 2ЈЈ-H and CH₃) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 163.9 (C-4), 163.5 (CO-phthalimide), 150.6 (C-2),

5Ј-O-(tert-Butyldiphenylsilyl)-3Ј-O-(phthalimidooxymethyl)-2Јdeoxy-

thymidine (51): Sulfuryl chloride (1 m solution in CH₂Cl₂; 3.32 mL,

3.32 mmol) was added to a stirred solution of 50 (1.50 g,

2.77 mmol) in dry CH₂Cl₂(20 mL) at 0 °C. The reaction mixture

was allowed to warm slowly to room temp. over 2 h, then it was

concentrated under reduced pressure to give a 3Ј-O-chlorometh-

yluridine derivative as a sticky mass, which was used without fur-

ther purification.

3

135.6 (d, J_C,P= 6.1 Hz, 1C of OCH₂Ph), 135.2 (C-6), 134.7 (Ar-

C), 128.9 (Ar-C), 128.8 (Ar-C), 128.7 (Ar-C), 128.2 (Ar-C), 128.1

(Ar-C), 127.8 (Ar-C), 123.7 (Ar-C), 111.6 (C-5), 97.5 (OCH₂O),

In a separate round-bottomed flask, N-hydroxyphthalimide (1.81 g,

11.08 mmol) was suspended in dry CH₂Cl₂(30 mL), and DBU

(1.45 mL, 9.69 mmol) was then added. After 10 min, the red-col-

oured solution was added to the crude 3Ј-O-chloromethyluridine

derivative, and the reaction mixture was kept stirring at room temp.

for 24 h. The mixture was then diluted with CH₂Cl₂(100 mL), and

acetic acid (1 m aq.; 30 mL) was added with vigorous stirring. The

aqueous layer was discarded, and the organic layer was washed

with saturated aq. NaHCO₃(2 ϫ 50 mL) and brine, dried with

Na₂SO₄, filtered, and concentrated under reduced pressure. The

crude residue was purified by column chromatography on silica gel

(gradient CH₂Cl₂/MeOH, 99:1, v/v; 98:2, v/v; 97:3, v/v) to give 51

3

84.7 (C-1Ј), 83.1 (d, J_C,P= 8.3 Hz, C-4Ј), 78.3 (C-3Ј), 69.8–69.7 (2

2

d, J_C,P= 5.4 Hz, 2 OCH₂Ph), 67.5 (d, J_C,P= 5.7 Hz_,C-5Ј), 37.2

(C-2Ј), 12.4 (-CH₃) ppm. ³¹P NMR (121 MHz, CDCl₃): δ =

–0.5 ppm. HRMS: calcd. for C₃₁H₃₀N₃O₁₂P [M + Na]⁺570.1612;

found 570.1615.

3Ј-O-(Aminooxymethyl)-5Ј-O-(dibenzylphosphate)-2Ј-deoxy-

thymidine (54): Following a similar procedure to that used for the

synthesis of 46, compound 54 was obtained starting from 53 (1.3 g,

1.92 mmol), methylamine (4% in H₂O; 5.96 mL, 3.52 mmol), and

EtOH (30 mL). The crude residue was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v;

1

(1.73 g, 95%) as a white solid. H NMR (300 MHz, CDCl₃): δ =

9.35 (br. s, 1 H, NH), 7.84–7.69 (m, 8 H, Ar-H), 7.53 (s, 1 H, 6- 98:2, v/v; 96:4, v/v) to give 54 (0.86 g, 82%) as a colourless foam.

H), 7.45–7.37 (m, 6 H, Ar-H), 6.34 (dd, J = 9.2, 5.1 Hz, 1 H, 1Ј-

¹H NMR (300 MHz, CDCl₃): δ = 9.75 (br. s, 1 H, NH), 7.34–7.33

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2343

S. De, E. Groaz, P. Herdewijn

86.3 (C-1Ј), 84.3 (d, J_C,P= 7.8 Hz, C-4Ј), 79.2 (C-3Ј), 71.1–71.0 (2

FULL PAPER

(m, 11 H, 6-H and Ar-H), 6.25 (app t, J = 6.6 Hz, 1 H, 1Ј-H), 5.64

(br. s, 2 H, NH₂), 5.12–4.99 (m, 4 H, 2 OCH₂Ph), 4.78–4.72 (m, 2

3

2

d, J_C,P= 5.6 Hz, 2 OCH₂Ph), 68.7 (d, J_C,P= 5.6 Hz_,C-5Ј), 53.6

H, OCH₂O), 4.31–4.26 (m, 1 H, 3Ј-H), 4.22–4.12 (m, 3 H, 4Ј-H, (αC-Met), 52.8 (αC-Glu), 38.4 (C-2Ј), 32.6 (βC-Met), 30.9 (γC-

5Ј-H, and 5ЈЈ-H), 2.41–2.33 (m, 1 H, 2Ј-H), 2.03–1.98 (m, 1 H, 2ЈЈ- Met), 29.9 (γC-Glu), 28.7 (βC-Glu), 15.3 (SCH₃), 12.5 (CH₃) ppm.

H), 1.81 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 164.0

³¹P NMR (121 MHz, [D₄]methanol): δ = –1.1 ppm. HRMS: calcd.

(C-4), 150.5 (C-2), 135.4 (d, ³J_C,P= 6.3 Hz, 1C of OCH₂Ph), 135.3 for C₃₆H₄₇N₆O₁₃PS [M – H]^–833.2586; found 833.2567.

(C-6), 128.9 (Ar-C), 128.7 (Ar-C), 128.1 (Ar-C), 111.2 (C-5), 98.3

3Ј-O-[N-For-L-Met-L-Glu-(methyloxy-δ-carboxamide)-OMe]-2Ј-

3

(OCH₂O), 84.8 (C-1Ј), 82.9 (d, J_C,P= 7.9 Hz, C-4Ј), 76.4 (C-3Ј),

deoxythymidine-5Ј-monophosphate Triethylammonium Salt (57):

Compound 57 was obtained as a white solid (154 mg, 75%) accord-

ing to the general procedure used for the synthesis of 19 and 33–

40, starting from a stirred solution of 55 (200 mg, 0.235 mmol),

Et₃N (0.065 mL, 0.471 mmol), and Pd/C (10%; Degussa; 100 mg,

50% w/w) in MeOH (20 mL). ¹H NMR (500 MHz, [D₆]DMSO): δ

= 12.51 (br. s, 1 H, NH), 11.24 (br. s, 1 H, NH), 9.03 (d, J = 8.0 Hz,

1 H, NH-Met), 8.94 (d, J = 6.4 Hz, 1 H, NH-Glu), 8.04 (s, 1 H,

CHO), 7.80 (s, 1 H, 6-H), 6.11 (app t, J = 5.8 Hz, 1 H, 1Ј-H), 4.88–

4.77 (m, 2 H, OCH₂O), 4.67–4.64 (m, 1 H, 3Ј-H), 4.49–4.45 (m, 1

H, αH-Glu), 4.17–4.13 (m, 1 H, αH-Met), 4.06–4.02 (m, 1 H, 5Ј-

H), 3.99 (br. s, 1 H, 4Ј-H), 3.92–3.90 (m, 2 H, 5ЈЈ-H), 3.60 (s, 3 H,

OCH₃), 2.46 (t, J = 7.9 Hz, 2 H, γCH₂-Met), 2.34–2.29 (m, 1 H,

2Ј-H), 2.26–2.06 (m, 3 H, 2ЈЈ-H and γCH₂-Glu), 2.04 (s, 3 H,

SCH₃), 2.03–1.86 (m, 4 H, βCH₂-Glu and βCH₂-Met), 1.81 (s, 3

H, CH₃) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 172.1 (αCO-

Glu), 171.6 (CO-Met), 168.7 (δCO-Glu), 164.0 (C-4), 161.2 (CHO),

150.5 (C-2), 136.2 (C-6), 109.8 (C-5), 96.2 (OCH₂O), 83.6 (C-1Ј),

2

69.8–69.7 (2 d, ²J_C,P= 5.5 Hz, 2 OCH₂Ph), 66.5 (d, J_C,P= 5.5 Hz_,

C-5Ј), 38.2 (C-2Ј), 12.4 (-CH₃) ppm. ³¹P NMR (121 MHz, CDCl₃):

δ = –0.4 ppm. HRMS: calcd. for C₂₅H₃₀N₃O₉P [M + H]⁺548.1792;

found 548.1812.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Glu-(methyloxy-

δ-carboxamide)-OMe]-2Ј-deoxythymidine (55): Following a similar

procedure to that used for the synthesis of 47, compound 55 was

obtained starting from 54 (200 mg, 0.365 mmol), dipeptide 2a

(134.6 mg, 0.420 mmol), DCC (120.6 mg, 0.584 mmol), and

DMAP (1.8 mg, 0.0146 mmol) in a mixture of dry DMF (2.0 mL)

and dry CH₂Cl₂(10 mL). The crude residue was purified by col-

umn chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1,

v/v; 97:3, v/v; 94:6, v/v) to give 55 (211.0 mg, 68%) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ = 10.52 (br. s, 1 H, NH), 10.20 (br.

s, 1 H, NH), 8.18 (s, 1 H, CHO), 7.90 (d, J = 7.0 Hz, 1 H, NH-

Met), 7.74 (d, J = 6.7 Hz, 1 H, NH-Glu), 7.34–7.33 (m, 11 H, H-

6 and Ar-H of OBn), 6.24 (app t, J = 6.5 Hz, 1 H, 1Ј-H), 5.13–

5.00 (m, 4 H, 2 OCH₂Ph), 4.92–4.86 (m, 2 H, OCH₂O), 4.79–4.72

(m, 1 H, 3Ј-H), 4.54–4.52 (m, 1 H, αH-Glu), 4.42 (br. s, 1 H, αH-

Met), 4.34–4.30 (m, 1 H, 5Ј-H), 4.22–4.16 (m, 2 H, 4Ј-H and 5ЈЈ-

H), 3.71 (s, 3 H, OCH₃), 2.56 (t, J = 7.2 Hz, 2 H, γCH₂-Met), 2.44–

2.42 (m, 1 H, 2Ј-H), 2.35–2.15 (m, 3 H, 2ЈЈ-H and γCH₂-Glu),

2.14–1.85 (unresolved m, 7 H, βCH₂-Met, SCH₃, and βCH₂-Glu),

1.79 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.9

(αCO-Glu), 171.8 (CO-Met), 170.0 (δCO-Glu), 164.1 (C-4), 162.1

(CHO), 150.8 (C-2), 135.3 (C-6), 135.2 (1C of OCH₂Ph), 128.9 (Ar-

C), 128.7 (Ar-C), 128.0 (Ar-C), 111.4 (C-5), 98.0 (OCH₂O), 84.8

3

2

83.2 (d, J_C,P= 5.8 Hz, C-4Ј), 74.1 (C-3Ј), 62.5 (d, J_C,P= 4.8 Hz_,

C-5Ј), 52.1 (αC-Met), 52.0 (OCH₃), 50.7 (αC-Glu), 36.9 (C-2Ј), 32.4

(βC-Met), 29.5 (γC-Met), 29.4 (γC-Glu), 27.0 (βC-Glu), 14.8

(SCH₃), 12.3 (CH₃) ppm. ³¹P NMR (202 MHz, [D₆]DMSO): δ =

0.0 ppm. HRMS: calcd. for C₂₃H₃₆N₅O₁₄PS [M – H]^–668.1644;

found 668.1656.

3Ј-O-[N-For-L-Met-L-Glu-(methyloxy-δ-carboxamide)-NH₂]-2Ј-

deoxythymidine-5Ј-monophosphate Triethylammonium Salt (58):

Compound 58 was obtained as a white solid (135 mg, 73%) accord-

ing to the general procedure used for the synthesis of 19 and 33–

40, starting from a stirred solution of 56 (180.0 mg, 0.216 mmol),

Et₃N (0.06 mL, 0.431 mmol) and Pd/C (10%; Degussa; 90.0 mg,

50% w/w) in a mixture of EtOH (18 mL) and H₂O (2 mL). ¹H

NMR (600 MHz, [D₆]DMSO): δ = 12.26 (br. s, 1 H, NH), 11.28

(br. s, 1 H, NH), 9.26 (d, J = 7.7 Hz, 1 H, NH-Met), 8.37 (d, J =

7.7 Hz, 1 H, NH-Glu), 8.07 (s, 1 H, CHO), 7.80 (s, 1 H, 6-H), 7.34

(s, 1 H, CONH₂-Glu), 7.04 (s, 1 H, CONH₂-Glu), 6.13 (app t, J =

6.1 Hz, 1 H, 1Ј-H), 4.87–4.79 (m, 2 H, OCH₂O), 4.65–4.62 (m, 1

H, 3Ј-H), 4.40–4.36 (m, 1 H, αH-Glu), 4.12–4.09 (m, 1 H, αH-

Met), 4.02–4.00 (m, 2 H, 4Ј-H and 5Ј-H), 3.92–3.89 (m, 1 H, 5ЈЈ-

H), 2.48 (t, J = 8.0 Hz, 2 H, γCH₂-Met), 2.33–2.29 (m, 1 H, 2Ј-H),

2.26–2.22 (m, 1 H, 2ЈЈ-H), 2.13–2.00 (unresolved m, 5 H, γCH₂-

Glu and SCH₃), 1.98–1.82 (unresolved m, 7 H, βCH₂-Glu, βCH₂-

Met, and CH₃) ppm. ¹³C NMR (150 MHz, [D₆]DMSO): δ = 173.2

(αCO-Glu), 171.2 (CO-Met), 169.0 (δCO-Glu), 163.9 (C-4), 161.6

(CHO), 150.4 (C-2), 136.1 (C-6), 109.7 (C-5), 96.0 (OCH₂O), 83.6

3

(C-1Ј), 82.9 (d, J_C,P= 6.8 Hz, C-4Ј), 77.1 (C-3Ј; merged with

2

CDCl₃), 68.9–69.8 (2 d, J_C,P= 5.6 Hz, 2 OCH₂Ph), 66.7 (d, J_C,P

= 5.1 Hz_,C-5Ј), 52.5 (OCH₃), 51.8 (αC-Met), 51.1 (αC-Glu), 37.7

(C-2Ј), 31.7 (βC-Met), 29.8 (γC-Met), 29.1 (γC-Glu), 27.3 (βC-

Glu), 15.1 (SCH₃), 12.3 (CH₃) ppm. ³¹P NMR (121 MHz, CDCl₃):

δ = –0.6 ppm. HRMS: calcd. for C₃₇H₄₈N₅O₁₄PS [M – H]^–

848.2583; found 848.2560.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Glu-(methyloxy-

δ-carboxamide)-NH₂]-2Ј-deoxythymidine (56): Following a similar

procedure to that used for the synthesis of 47, compound 56 was

obtained starting from 54 (200 mg, 0.365 mmol), dipeptide 2b

(128.3 mg, 0.420 mmol), DCC (120.6 mg, 0.584 mmol), and

DMAP (1.8 mg, 0.0146 mmol) in a mixture of dry DMF (2.0 mL)

and dry CH₂Cl₂(10 mL). The crude residue was purified by col-

umn chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1,

v/v; 97:3, v/v; 92:8, v/v) to give 56 (186.0 mg, 61%) as a white solid.

¹H NMR (300 MHz, [D₄]methanol): δ = 8.09 (s, 1 H, CHO), 7.42

(s, 1 H, 6-H), 7.31(br. s, 10 H, Ar-H of OBn), 6.17 (app t, J =

6.9 Hz, 1 H, 1Ј-H), 5.06–5.03 (m, 4 H, 2 OCH₂Ph), 4.86 (s, 2 H,

OCH₂O), 4.52–4.48 (m, 2 H, 3Ј-H and αH-Glu), 4.39–4.35 (m, 1

H, αH-Met), 4.27–4.24 (m, 1 H, 4Ј-H and 5Ј-H), 4.19–4.15 (m, 2

H, 5ЈЈ-H), 2.58–2.50 (m, 2 H, γCH₂-Met), 2.47–2.34 (m, 1 H, 2Ј-H),

2.23–2.07 (m, 3 H, 2ЈЈ-H and γCH₂-Glu), 2.04–1.84 (unresolved m,

7 H, βCH₂-Met, SCH₃, and βCH₂-Glu), 1.71 (s, 3 H, CH₃) ppm.

¹³C NMR (75 MHz, [D₄]methanol): δ = 175.8 (αCO-Glu), 173.4

(CO-Met), 172.0 (δCO-Glu), 166.1 (C-4), 164.0 (CHO), 152.2 (C-

3

2

(C-1Ј), 83.1 (d, J_C,P= 6.0 Hz, C-4Ј), 74.3 (C-3Ј), 62.6 (d, J_C,P

=

4.1 Hz_,C-5Ј), 52.5 (αC-Met), 51.2 (αC-Glu), 36.7 (C-2Ј), 31.7 (βC-

Met), 29.6 (γC-Met), 29.5 (γC-Glu), 28.2 (βC-Glu), 14.7 (SCH₃),

12.2 (CH₃) ppm. ³¹P NMR (202 MHz, [D₆]DMSO): δ = 0.0 ppm.

HRMS: calcd. for C₂₂H₃₅N₆O₁₃PS [M – H]^–653.1647; found

653.1644.

5Ј-O-(tert-Butyldiphenylsilyl)-3Ј-O-[N-For-Gly-

L-Glu(γ-methyl-

oxy ester)- -Ala-OMe]-2Ј-deoxythymidine (59): Following a similar

L

procedure to that used for the synthesis of 51, compound 59 was

obtained in two steps starting from 50 (1.69 g, 3.12 mmol) and

SO₂Cl₂(1 m in CH₂Cl₂; 3.75 mL, 3.75 mmol), followed by reaction

3

2), 137.3 (C-6), 137.0 (d, J_C,P= 6.2 Hz, 1C of OCH₂Ph), 129.9

(Ar-C), 129.7 (Ar-C), 129.2 (Ar-C), 112.0 (C-5), 99.1 (OCH₂O),

2344

www.eurjoc.org

Eur. J. Org. Chem. 2014, 2322–2348

Tailoring Peptide–Nucleotide Conjugates

with tripeptide 5a (1.24 g, 3.91 mmol) and DBU (0.61 mL, βCH₂-Glu), 1.51 (d, J = 0.9 Hz, 3 H, CH₃-Thy), 1.28 (d, J =

4.06 mmol) in dry CH₂Cl₂(50 mL). The crude residue was purified

by column chromatography on silica gel (gradient CH₂Cl₂/MeOH,

99:1, v/v; 98:2, v/v; 96:4, v/v) to give 59 (1.87 g, 74%) as a colour-

less foam. ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.36 (s, 1 H,

NH-Thy), 8.43 (d, J = 6.7 Hz, 1 H, NH-Ala), 8.21 (t, J = 5.5 Hz,

7.3 Hz, 3 H, CH₃-Ala) ppm. ¹³C NMR (75 MHz, [D₆]DMSO): δ

= 172.8 (CO-Ala), 171.9 (δCO-Glu), 170.7 (αCO-Glu), 168.3 (CO-

3

Gly), 163.6 (C-4), 161.4 (CHO), 150.3 (C-2), 135.9 (d, J_C,P

=

6.8 Hz, 1C of OCH₂Ph), 135.7 (C-6), 128.5 (Ar-C), 128.4 (Ar-C),

127.8 (Ar-C), 109.9 (C-5), 87.8 (OCH₂O), 84.0 (C-1Ј), 82.0 (d, ³J_C,P

2

1 H, NH-Gly), 8.11 (d, J = 8.3 Hz, 1 H, NH-Glu), 8.05 (s, 1 H, = 7.4 Hz, C-4Ј), 78.8 (C-3Ј), 68.7 (d, J_C,P= 5.4 Hz, 2 OCH₂Ph),

CHO), 7.65–7.41 (m, 11 H, 6-H and Ar-H of TBDPS), 6.14 (app

2

66.8 (d, J_C,P= 4.8 Hz_,C-5Ј), 51.8 (OCH₃), 51.1 (αC-Glu), 47.6

t, J = 7.0 Hz, 1 H, 1Ј-H), 5.32 (dd, J = 19.9, 6.6 Hz, 2 H, OCH₂O), (αC-Ala), 40.5 (αC-Gly), 36.5 (C-2Ј), 29.7 (γC-Glu), 27.2 (βC-Glu),

4.50–4.48 (m, 1 H, 3Ј-H), 4.40–4.32 (m, 1 H, αH-Glu), 4.29–4.20 16.6 (CH₃-Ala), 12.0 (CH₃-Thy) ppm. ³¹P NMR (121 MHz,

(m, 1 H, αH-Ala), 4.04–4.01 (m, 1 H, 4Ј-H), 3.93–3.82 (m, 2 H, 5Ј-

[D₆]DMSO): δ = –0.9 ppm. HRMS: calcd. for C₃₇H₄₆N₅O₁₅P [M

H and 5ЈЈ-H), 3.76 (d, J = 5.8 Hz, 2 H, αCH₂-Gly), 3.57 (s, 3 H, + H]⁺832.2800; found 832.2803.

OCH₃), 2.39 (t, J = 8.1 Hz, 2 H, γCH₂-Glu), 2.34–2.25 (m, 1 H,

3Ј-O-[N-For-Gly-L-Glu(γ-methyloxy ester)-L-Ala-OMe]-2Ј-deoxy-

2Ј-H and 2ЈЈ-H), 1.98–1.74 (m, 2 H, βCH₂-Glu), 1.51 (s, 3 H, CH₃-

Thy), 1.28 (d, J = 7.3 Hz, 3 H, CH₃-Ala), 1.02 (s, 9 H, tBu) ppm.

¹³C NMR (75 MHz, [D₆]DMSO): δ = 172.8 (CO-Ala), 172.0 (δCO-

Glu), 170.7 (αCO-Glu), 168.2 (CO-Gly), 163.5 (C-4), 161.4 (CHO),

150.3 (C-2), 135.4 (C-6), 135.1 (1C of Ph), 134.9 (1C of Ph), 132.7

(Ar-C), 132.3 (Ar-C), 130.1 (Ar-C), 130.0 (Ar-C), 128.0 (Ar-C),

127.9 (Ar-C), 109.7 (C-5), 87.7 (OCH₂O), 83.9 (C-4Ј), 83.6 (C-1Ј),

79.1 (C-3Ј), 63.8 (C-5Ј), 51.7 (OCH₃), 51.1 (αC-Glu), 47.6 (αC-

Ala), 40.5 (αC-Gly), 36.8 (C-2Ј), 29.7 (γC-Glu), 27.2 (βC-Glu), 26.6

(tBu), 18.8 (1C of tBu), 16.6 (CH₃-Ala), 11.8 (CH₃-Thy) ppm.

HRMS: calcd. for C₃₉H₅₁N₅O₁₂Si [M – H]^–808.3230; found

808.3233.

thymidine-5Ј-monophosphate Triethylammonium Salt (62): Com-

pound 62 was obtained as a white solid (63.6 mg, 62%) according

to the general procedure used for the synthesis of 19 and 33–40,

starting from a stirred solution of 61 (100 mg, 0.120 mmol),

NaHCO₃(20.2 mg, 0.240 mmol), and 20 % Pd(OH)₂/C (20 %;

10.0 mg, 10 % w/w) in EtOH/H₂O, 9:1 (10 mL). ¹H NMR

(500 MHz, D₂O): δ = 8.14 (s, 1 H, CHO), 7.81 (s, 1 H, 6-H), 6.26

(app t, J = 7.2 Hz, 1 H, 1Ј-H), 5.38 (dd, J = 44.5, 6.9 Hz, 2 H,

OCH₂O), 4.62–4.61 (m, 1 H, 3Ј-H), 4.39–4.34 (m, 2 H, αH-Glu

and αH-Ala), 4.26–4.25 (m, 1 H, 4Ј-H), 3.98–3.94 (m, 4 H, αCH₂-

Gly, 5Ј-H, and 5ЈЈ-H), 3.69 (s, 3 H, OCH₃), 2.56 (t, J = 7.4 Hz, 2

H, γCH₂-Glu), 2.46–2.34 (m, 1 H, 2Ј-H and 2ЈЈ-H), 2.17–1.95 (m,

2 H, βCH₂-Glu), 1.88 (d, J = 0.9 Hz, 3 H, CH₃-Thy), 1.38 (d, J =

7.3 Hz, 3 H, CH₃-Ala) ppm. ¹³C NMR (125 MHz, D₂O): δ = 174.5

(CO-Ala), 174.0 (δCO-Glu), 172.8 (αCO-Glu), 170.7 (CO-Gly),

166.4 (C-4), 164.6 (CHO), 151.5 (C-2), 137.4 (C-6), 111.5 (C-5),

3Ј-O-[N-For-Gly-L-Glu(γ-methyloxy ester)-L-Ala-OMe]-2Ј-deoxy-

thymidine (60): Following a similar procedure to that used for the

synthesis of 52, compound 60 was obtained starting from 59

(0.60 g, 0.74 mmol) and Et₃N·3HF (0.483 mL, 2.96 mmol) in THF

(10 mL). The crude residue was purified by column chromatog-

raphy on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v; 96:4, v/v;

3

88.3 (OCH₂O), 84.6 (C-1Ј), 83.9 (d, J_C,P= 9.0 Hz, C-4Ј), 80.3 (C-

3Ј), 64.0 (d, ²J_C,P= 4.4 Hz, C-5Ј), 52.6 (OCH₃), 52.3 (αC-Glu), 48.5

(αC-Ala), 40.7 (αC-Gly), 36.8 (C-2Ј), 29.5 (γC-Glu), 25.6 (βC-Glu),

15.5 (CH₃-Ala), 11.4 (CH₃-Thy) ppm. ³¹P NMR (202 MHz, D₂O):

δ = 1.8 ppm. HRMS: calcd. for C₂₃H₃₄N₅O₁₅P [M + H]⁺650.1716;

found 650.1718.

1

92:8, v/v) to give 60 (0.34 g, 80%) as a colourless foam. H NMR

(300 MHz, [D₆]DMSO): δ = 11.31 (s, 1 H, NH-Thy), 8.44 (d, J =

6.7 Hz, 1 H, NH-Ala), 8.21 (t, J = 5.0 Hz, 1 H, NH-Gly), 8.10 (d,

J = 8.0 Hz, 1 H, NH-Glu), 8.06 (s, 1 H, CHO), 7.68 (s, 1 H, 6-H),

6.11 (app t, J = 7.0 Hz, 1 H, 1Ј-H), 5.31 (dd, J = 11.6, 6.7 Hz, 2

H, OCH₂O), 4.39–4.35 (m, 2 H, 3Ј-H and αH-Glu), 4.27–4.23 (m,

1 H, αH-Ala), 3.93–3.90 (m, 1 H, 4Ј-H), 3.77 (d, J = 6.8 Hz, 2 H,

αCH₂-Gly), 3.61–3.58 (m, 5 H, 5Ј-H, 5ЈЈ-H, and OCH₃), 2.40 (t, J

= 8.0 Hz, 2 H, γCH₂-Glu), 2.25–2.19 (m, 1 H, 2Ј-H and 2ЈЈ-H),

2.00–1.80 (m, 2 H, βCH₂-Glu), 1.78 (s, 3 H, CH₃-Thy), 1.29 (d, J

= 7.3 Hz, 3 H, CH₃-Ala) ppm. ¹³C NMR (75 MHz, [D₆]DMSO):

δ = 172.8 (CO-Ala), 172.0 (δCO-Glu), 170.7 (αCO-Glu), 168.3

(CO-Gly), 163.6 (C-4), 161.4 (CHO), 150.4 (C-2), 135.9 (C-6),

109.5 (C-5), 87.6 (OCH₂O), 84.7 (C-4Ј), 83.6 (C-1Ј), 79.3 (C-3Ј),

61.1 (C-5Ј), 51.8 (OCH₃), 51.1 (αC-Glu), 47.6 (αC-Ala), 40.5 (αC-

Gly), 36.9 (C-2Ј), 29.8 (γC-Glu), 27.2 (βC-Glu), 16.6 (CH₃-Ala),

12.2 (CH₃-Thy) ppm. HRMS: calcd. for C₂₃H₃₃N₅O₁₂[M – H]^–

570.2053; found 570.2054.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-L-Met-L-Lys(Boc)-L-Lys-

(Boc)-ester]-2Ј-deoxythymidine (63): Following a similar procedure

to that used for the synthesis of 47, compound 63 was obtained

starting from 23 (250 mg, 0.498 mmol), tripeptide 4h (378.4 mg,

0.597 mmol), DCC (164.3 mg, 0.796 mmol), and DMAP (6.1 mg,

0.050 mmol) in a mixture of dry DMF (3 mL) and dry CH₂Cl₂

(15 mL). The crude residue was purified by column chromatog-

raphy on silica gel (gradient CH₂Cl₂/MeOH 99:1, v/v; 97:2, v/v;

94:4, v/v) to give 63 (423 mg, 76%) as a colourless foam. ¹H NMR

(300 MHz, CDCl₃): δ = 9.51 (br. s, 1 H, NH), 9.31 (br. s, 1 H, NH),

8.22 (s, 1 H, CHO), 7.41 (s, 1 H, 6-H), 7.35–7.34 (m, 10 H, Ar-H

of OBn), 6.33 (dd, J = 9.2, 5.4 Hz, 1 H, 1Ј-H), 5.18–5.12 (m, 1 H,

3Ј-H), 5.10–4.95 (m, 4 H, OCH₂Ph), 4.89–4.77 (m, 1 H, αH-Met),

4.73–4.34 (m, 2 H, 2 αH-Lys), 4.22–4.10 (m, 3 H, 4Ј-H, 5Ј-H, and

5ЈЈ-H), 3.15–3.04 (m, 4 H, 2 εCH₂-Lys), 2.56 (t, J = 6.7 Hz, 2 H,

γCH₂-Met), 2.35–2.22 (m, 1 H, 2Ј-H), 2.13–2.07 (m, 4 H, 2ЈЈ-H

and SCH₃), 1.96–1.87 (m, 2 H, βCH₂-Met), 1.82 (s, 3 H, CH₃-Thy),

1.77–1.64 (m, 4 H, 2 βCH₂-Lys), 1.54–1.31 (m, 26 H, 2 δCH₂-Lys,

2 γCH₂-Lys, and 2 CH₃-tBu) ppm. ¹³C NMR (75 MHz, CDCl₃): δ

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-Gly-

L-Glu(γ-methyloxy-

ester)- -Ala-OMe]-2Ј-deoxythymidine (61): Following a similar pro-

L

cedure to that used for the synthesis of 22, compound 61 (383 mg,

86%) was obtained starting from 60 (306 mg, 0.535 mmol) as a

1

colourless foam. H NMR (300 MHz, [D₆]DMSO): δ = 11.33 (s, 1

H, NH-Thy), 8.43 (d, J = 6.7 Hz, 1 H, NH-Ala), 8.20 (t, J = 5.7 Hz, = 171.9 (CO-Lys_N-Ter), 171.7 (CO-Met), 171.3 (CO-Lys_C-Ter), 163.7

1 H, NH-Gly), 8.10 (d, J = 8.1 Hz, 1 H, NH-Glu), 8.06 (s, 1 H, (C-4), 161.9 (CHO), 156.4 (2 OCONH), 150.9 (C-2), 135.5 (d, ³J_C,P

CHO), 7.46 (d, J = 0.9 Hz, 1 H, 6-H), 7.36–7.35 (m, 10 H, Ar-H = 6.0 Hz, 1C of OCH₂Ph), 135.1 (C-6), 129.1 (Ar-C), 128.9 (Ar-

3

of OBn), 6.12 (app t, J = 7.1 Hz, 1 H, 1Ј-H), 5.29 (dd, J = 15.7,

6.6 Hz, 2 H, OCH₂O), 5.05 (d, J = 8.1 Hz, 4 H, 2 OCH₂Ph), 4.37–

C), 128.2 (Ar-C), 112.1 (C-5), 84.5 (C-1Ј), 82.6 (d, J_C,P= 8.9 Hz,

2

C-4Ј), 79.4 (2 C of tBu), 75.7 (C-3Ј), 70.0 (app t, J_C,P= 5.6 Hz, 2

4.29 (m, 2 H, 3Ј-H and αH-Glu), 4.27–4.19 (m, 3 H, αH-Ala, 5Ј- OCH₂Ph), 67.3 (d, ²J_C,P= 5.8 Hz_,C-5Ј), 53.5 (2 αC-Lys), 52.8 (αC-

H, and 5ЈЈ-H), 4.10–4.07 (m, 1 H, 4Ј-H), 3.77 (d, J = 5.8 Hz, 2 H,

αCH₂-Gly), 3.58 (s, 3 H, OCH₃), 2.41 (t, J = 7.8 Hz, 2 H, γCH₂-

Met), 40.1 (2 εC-Lys), 37.0 (C-2Ј), 31.8 (βC-Met), 31.0 (2 βC-Lys),

30.1 (γC-Met), 29.5 (2 δC-Lys), 28.6 (2 CH₃-tBu), 22.9 (2 γC-Lys),

Glu), 2.26–2.13 (m, 1 H, 2Ј-H and 2ЈЈ-H), 1.99–1.74 (m, 2 H, 15.4 (SCH₃), 12.5 (CH₃-Thy) ppm. ³¹P NMR (121 MHz, CDCl₃):

Eur. J. Org. Chem. 2014, 2322–2348

www.eurjoc.org

2345

S. De, E. Groaz, P. Herdewijn

FULL PAPER

δ = –0.7 ppm. HRMS: calcd. for C₅₂H₇₆N₇O₁₆PS [M + H]⁺

1118.4879; found 1118.4878.

2.29–2.27 (m, 1 H, 2Ј-H), 2.22–2.18 (m, 1 H, 2ЈЈ-H), 1.85 (s, 3 H,

CH₃-Thy), 0.85 [s, 9 H, C(CH₃)₃], 0.05 (s, 6 H, CH₃) ppm. ¹³C

NMR (75 MHz, CDCl₃): δ = 164.1 (C-4), 150.4 (C-2), 137.0 (C-6),

110.8 (C-5), 87.5 (C-4Ј), 86.5 (C-1Ј), 71.5 (C-3Ј), 61.8 (C-5Ј), 40.5

(C-2Ј), 25.6 (tBu), 17.9 (1C of tBu), 12.4 (CH₃-Thy), –4.8 (CH₃),

–4.9 (CH₃) ppm. HRMS: calcd. for C₁₆H₂₉N₂O₅Si [M + H]⁺

357.1840; found 357.1841.

3Ј-O-[N-For-L-Met-L-Lys-L-Lys-ester]-2Ј-deoxythymidine-5Ј-

monophosphate TFA Salt (65): Following a similar procedure to

that used for the synthesis of 41, compound 65 was obtained as a

white solid in two steps (214 mg, 62%) starting from 63 (400 mg,

0.358 mmol) and Pd/C (10 %; Degussa; 200 mg, 50 % w/w) in

EtOH/H₂O, 10:1 (30 mL), to give crude 3Ј-O-[N-For-Met-l-Lys-

(Boc)-l-Lys(Boc)-ester]-2Ј-deoxythymidine-5Ј-monophosphate (64)

(335.5 mg, quantitative) as a white solid, which was immediately

deprotected by treatment with H₂O (4.5 mL), thioanisole

(0.056 mL, 0.476 mmol), and trifluoroacetic acid (1.5 mL). ¹H

NMR (500 MHz, D₂O): δ = 8.11 (s, 1 H, CHO), 7.80 (s, 1 H, 6-

H), 6.38–6.33 (m, 1 H, 1Ј-H), 5.47–5.44 (m, 1 H, 3Ј-H), 4.52–4.48

(m, 1 H, αH-Met), 4.46–4.33 (m, 3 H, 2 αH-Lys and 4Ј-H), 4.14–

4.09 (m, 2 H, 5Ј-H and 5ЈЈ-H), 3.00–2.98 (m, 4 H, 2 εCH₂-Lys),

2.61–2.51 (m, 2 H, γCH₂-Met), 2.48–2.42 (m, 1 H, 2Ј-H and 2ЈЈ-

H), 2.09 (m, 3 H, SCH₃), 2.05–1.97 (m, 2 H, βCH₂-Met), 1.91 (s,

3 H, CH₃-Thy), 1.85–1.78 (m, 4 H, 2 βCH₂-Lys), 1.72–1.66 (m, 4

H, 2 δCH₂-Lys), 1.52–1.41 (m, 4 H, 2 γCH₂-Lys) ppm. ¹³C NMR

(125 MHz, D₂O): δ = 173.3 (CO-Lys_N-Ter), 172.7 (CO-Met), 171.7

(CO-Lys_C-Ter), 166.0 (C-4), 163.7 (CHO), 151.2 (C-2), 136.5 (C-6),

5Ј-O-(Dibenzylphosphate)-3Ј-O-(tert-butyldimethylsilyl)-2Ј-deoxy-

thymidine (68): Following a similar procedure to that used for the

synthesis of 22, compound 68 was obtained starting from 67

(0.47 g, 1.32 mmol), tetrazole (0.45 m in MeCN; 15 mL,

6.57 mmol), and dibenzyldiisopropyl phosphoramidite (0.95 mL,

2.87 mmol) in dry CH₂Cl₂(10 mL), and H₂O₂(35 %; 0.57 mL,

7.01 mmol). The crude residue was purified by column chromatog-

raphy on silica gel (gradient hexane/EtOAc, 90:10, v/v; 70:30, v/v;

50:50, v/v) to give 68 (0.75 g, 88%) as a colourless foam. ¹H NMR

(300 MHz, CDCl₃): δ = 9.11 (br. s, 1 H, NH), 7.33–7.32 (m, 11 H,

Ar-H and 6-H), 6.29 (dd, J = 7.5, 6.1 Hz, 1 H, 1Ј-H), 5.11–4.98

(m, 4 H, OCH₂Ph), 4.32–4.28 (m, 1 H, 3Ј-H), 4.15–4.09 (m, 2 H,

5Ј-H and 5ЈЈ-H), 3.95–3.93 (m, 1 H, 4Ј-H), 2.15 (ddd, J = 13.4, 6.0,

3.1 Hz, 1 H, 2Ј-H), 1.93–1.84 (m, 1 H, 2ЈЈ-H), 1.82 (d, J = 0.9 Hz,

3 H, CH₃-Thy), 0.86 [s, 9 H, C(CH₃)₃], 0.04 (s, 3 H, CH₃), 0.03 (s,

3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 163.5 (C-4),

150.0 (C-2), 135.2 (1C of OCH₂Ph), 135.0 (C-6), 128.5 (Ar-C),

128.4 (Ar-C), 127.7 (Ar-C), 110.9 (C-5), 85.0 (d, ³J_C,P= 8.0 Hz, C-

4Ј), 84.5 (C-1Ј), 71.5 (C-3Ј), 69.4 (2 d, ²J_C,P= 5.4 Hz, OCH₂), 66.2

3

111.4 (C-5), 84.2 (C-1Ј), 82.7 (d, J_C,P= 8.9 Hz, C-4Ј), 76.1 (C-3Ј),

2

64.5 (d, J_C,P= 4.8 Hz_,C-5Ј), 52.9 (αC-Lys), 52.0 (αC-Lys), 50.8

(αC-Met), 38.5 (2 εC-Lys), 35.7 (C-2Ј), 29.8 (βC-Met), 28.8 (2 βC-

Lys), 28.4 (γC-Met), 25.5 (2 δC-Lys), 21.5 (2 γC-Lys), 13.5 (SCH₃),

11.0 (CH₃-Thy) ppm. ³¹P NMR (202 MHz, D₂O): δ = –0.2 ppm.

HRMS: calcd. for C₂₈H₄₈N₇O₁₂PS [M – H]^–736.2746; found

736.2742.

2

(d, J_C,P= 5.8 Hz_,C-5Ј), 40.4 (C-2Ј), 25.3 (tBu), 17.6 (1C of tBu),

12.0 (CH₃-Thy), –5.0 (CH₃), –5.2 (CH₃) ppm. ³¹P NMR (121 MHz,

CDCl₃): δ = –0.5 ppm. HRMS: calcd. for C₃₀H₄₂N₂O₈PSi

[M + H]⁺617.2442; found 617.2447.

3Ј,5Ј-O-Di(tert-butyldimethylsilyl)-2Ј-deoxythymidine (66):^[51]Imid-

azole (3.37 g, 49.5 mmol) and then TBDMSCl (3.73 g, 24.8 mmol)

were added to a stirred solution of thymidine (2.00 g, 8.26 mmol)

in dry DMF, and the solution was stirred overnight at room temp.

The mixture was then diluted with CH₂Cl₂, washed with water,

dried with Na₂SO₄, and filtered, and the solvents were evaporated

in vacuo. The crude residue was purified by column chromatog-

raphy on silica gel (gradient hexane/EtOAc, 90:10, v/v; 80:20, v/v;

70:30, v/v) to give 66 (3.03 g, 78%) as a white foam. ¹H NMR

(300 MHz, CDCl₃): δ = 9.23 (br. s, 1 H, NH), 7.45 (s, 1 H, 6-H),

6.34–4.30 (m, 1 H, 1Ј-H), 4.39–4.37 (m, 1 H, 3Ј-H), 3.91–3.83 (m,

2 H, 5Ј-H and 4Ј-H), 3.76–3.71 (m, 1 H, 5ЈЈ-H), 2.27–2.20 (m, 1

H, 2Ј-H), 2.02–1.96 (m, 1 H, 2ЈЈ-H), 1.89 (s, 3 H, CH₃-Thy), 0.91

[s, 9 H, C(CH₃)₃], 0.97 [s, 9 H, C(CH₃)₃], 0.09 (s, 6 H, CH₃), 0.06

(s, 6 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 163.6 (C-4),

150.1 (C-2), 135.1 (C-6), 110.5 (C-5), 87.5 (C-4Ј), 84.5 (C-1Ј), 71.9

(C-3Ј), 62.6 (C-5Ј), 41.5 (C-2Ј), 25.6 (tBu), 25.4 (tBu), 18.1 (1C of

tBu), 17.7 (1C of tBu), 12.2 (CH₃-Thy), –5.0 (CH₃), –5.2 (CH₃),

–5.7 (CH₃), –5.8 (CH₃) ppm. HRMS: calcd. for C₂₂H₄₃N₂O₅Si₂[M

+ H]⁺471.2705; found 471.2706.

5Ј-O-(Dibenzylphosphate)-2Ј-deoxythymidine (23)

Method 1: TBAF (1 m in THF; 0.92 mL, 0.92 mmol) was added to

a solution of 68 (570 mg, 0.92 mmol) in THF (10 mL) at 0 °C. The

reaction mixture was stirred for 45 min at 0 °C, and for 15 min at

room temp. The mixture was partially evaporated and then purified

by column chromatography on silica gel (gradient CH₂Cl₂/MeOH,

99:1, v/v; 98:2, v/v; 96:4, v/v) to give 23 (227 mg, 49%) as a colour-

less foam.

Method 2: Et₃N·3HF (0.53 mL, 3.24 mmol) was added to a solu-

tion of 68 (0.80 g, 1.30 mmol) in THF (10 mL), and the solution

was stirred at room temp. for 24 h. All the volatiles were removed,

and the residue was redissolved in CH₂Cl₂and washed with

NaHCO₃(10% aq. solution). The organic layer was then dried with

Na₂SO₄and filtered, and the solvents were evaporated under re-

duced pressure. The crude residue was purified by column

chromatography on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v;

98:2, v/v; 96:4, v/v) to give 23 (0.58 g, 89%) as a colourless foam.

5Ј-O-(Dibenzylphosphate)-3Ј-O-[N-For-Gly-L-Glu(δ-ester)-L-Phe-

OMe]-2Ј-deoxythymidine (69): Following a similar procedure to

that used for the synthesis of 47, compound 69 was obtained start-

ing from 23 (230 mg, 0.458 mmol), tripeptide 5b (200.0 mg,

0.508 mmol), DCC (110.0 mg, 0.533 mmol), and DMAP (1.1 mg,

0.090 mmol) in a mixture of dry DMF (4 mL) and dry CH₂Cl₂

(4 mL). The crude residue was purified by column chromatography

on silica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v; 95:5, v/v; 93:7,

v/v) to give 69 (230 mg, 57%) as a white foam. ¹H NMR (300 MHz,

[D₄]methanol): δ = 8.18 (s, 1 H, CHO), 7.46 (s, 1 H, 6-H), 7.36 (br.

s, 10 H, Ar-H of OBn), 7.29–7.19 (m, 5 H, Ar-H of Phe), 6.25 (dd,

J = 8.6, 5.7 Hz, 1 H, 1Ј-H), 5.21–5.20 (m, 1 H, 3Ј-H), 5.13–5.09

(m, 4 H, OCH₂Ph), 4.68 (dd, J = 8.5, 5.7 Hz, 1 H, αCH-Phe), 4.68

3Ј-O-(tert-Butyldimethylsilyl)-2Ј-deoxythymidine (67):^[51]Com-

pound 66 (2.39 g, 5.08 mmol) was dissolved in CH₂Cl₂(28 mL),

and the solution was cooled to 0 °C. A cooled TFA/H₂O mixture

(8:1; 2.8 mL) was then added dropwise, and the reaction mixture

was stirred for 4 h at 0 °C. The solution was then diluted with co-

oled CH₂Cl₂, washed with ice-cold water and brine, dried with

Na₂SO₄, and filtered, and the solvents were evaporated in vacuo.

The crude residue was purified by column chromatography on sil-

ica gel (gradient CH₂Cl₂/MeOH, 99:1, v/v; 95:5, v/v) to give 67

1

(1.07 g, 59%) as a white foam. H NMR (300 MHz, CDCl₃): δ =

9.40 (br. s, 1 H, NH), 7.38 (d, J = 1.08 Hz, 1 H, 6-H), 6.13 (t, J =

6.7 Hz, 1 H, 1Ј-H), 4.48–4.43 (m, 1 H, 3Ј-H), 3.89–3.86 (m, 2 H, (dd, J = 8.3, 5.7 Hz, 1 H, αCH-Glu), 4.28–4.26 (m, 2 H, 5Ј-H and

5Ј-H and 5ЈЈ-H), 3.73–3.72 (m, 1 H, 4Ј-H), 3.07 (br. s, 1 H, OH), 5ЈЈ-H), 4.19–4.18 (m, 1 H, 4Ј-H), 3.93 (br. s, 2 H, CH₂-Gly), 3.68

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Tailoring Peptide–Nucleotide Conjugates

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3

(C-6), 135.3 (d, J_C,P= 6.1 Hz, 1C of OCH₂Ph), 128.6 (Ar-C),

128.2 (Ar-C), 128.1 (Ar-C), 127.8 (Ar-C), 127.5 (Ar-C), 126.2 (Ar-

C), 110.4 (C-5), 84.5 (C-1Ј), 82.2 (d, ³J_C,P= 7.8 Hz, C-4Ј), 74.1 (C-

2

3Ј), 69.4 (d, J_C,P= 5.7 Hz, 2 OCH₂Ph), 66.9 (d, J_C,P= 5.8 Hz,

C-5Ј), 53.5 (αC-Phe), 51.6 (αC-Glu), 51.1 (OCH₃), 40.3 (αC-Gly),

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(CH₃-Thy) ppm. ³¹P NMR (121 MHz, [D₄]methanol): δ =

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found 878.3006.

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3Ј-O-[N-For-L-Gly-L-Glu(δ-ester)-L-Phe-OMe]-2Ј-deoxythymidine-

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obtained as a white solid (160 mg, 66%) according to the general

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stirred solution of 69 (190 mg, 0.216 mmol) and Pd/C (10%; De-

gussa; 120 mg, 50% w/w) in THF (20 mL). ¹H NMR (300 MHz,

D₂O): δ = 8.15 (s, 1 H, CHO), 7.70 (s, 1 H, 6-H), 7.34–7.20 (m, 5

H, Ar-H of Phe), 6.36 (dd, J = 9.1, 5.5 Hz, 1 H, 1Ј-H), 5.35 (d, J

= 5.2 Hz, 1 H, 3Ј-H), 4.68 (dd, J = 9.5, 5.5 Hz, 1 H, αCH-Phe),

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[16]

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[18]

[19]

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3

[25]

111.5 (C-5), 85.6 (d, J_C,P= 8.0 Hz, C-4Ј), 84.5 (C-1Ј), 71.0 (C-3Ј),

2

63.4 (d, J_C,P= 4.06 Hz_,C-5Ј), 53.7 (αC-Phe), 55.8 (αC-Glu), 53.5

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Supporting Information (see footnote on the first page of this arti-

1

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Acknowledgments

The authors thank P. Marliere for helpful discussions. The research

leading to these results has received funding from the European

Research Council under the European Union’s Seventh Framework

Programme (FP7/2007-2013), ERC Grant agreement number ERC-

2012-ADG_20120216/ 320683.

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Received: November 29, 2013

Published Online: February 12, 2014

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Article Doi

Tailoring peptide-nucleotide conjugates (PNCs) for nucleotide delivery in bacterial cells

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