Towards nucleopeptides containing any trifunctional amino acid (II)

Tetrahedron 58 (2002) 6965–6978

Towards nucleopeptides containing any trifunctional

amino acid (II)

*

´ ´ `

Laurent Debethune, Vicente Marchan, Gemma Fabregas, Enrique Pedroso and Anna Grandas

´ ` ´ ´ `

Departament de Quımica Organica, Facultat de Quımica, Universitat de Barcelona, Martı i Franques 1-11, E-08028 Barcelona, Spain

Received 14 March 2002; revised 7 May 2002; accepted 18 June 2002

Abstract—Nucleopeptides with no restriction in the amino acid composition can be synthesized using stepwise solid-phase methodology.

Best conditions for the protection of arginine and cysteine, as well as for the ﬁnal deprotection treatment are established, and our conclusions

can be extended to the preparation of any peptide–oligonucleotide hybrid. The association between certain side reactions and nucleopeptide

1. Introduction

introduced in the hybrid molecule, which required to ﬁnd a

suitable synthetic strategy and an appropriate set of

permanent protecting groups. The stepwise solid-phase

methodology was more appropriate than a convergent solid-

phase approach.¹¹Although the latter was useful in some

cases,^10–20we have found problems associated with the

insolubility of protected peptides, in particular during the

phosphitylation and coupling steps, and the target nucleo-

peptide has not always been obtained. The insolubility of

some protected peptides may also render unsuitable the

solution phase convergent approaches used for the prep-

aration of certain peptide–oligonucleotide conjugates.^21–23

Stepwise oligonucleotide elongation (phosphite triester

approach) at the side chain of a hydroxylated amino acid

on a peptide-resin proceeds smoothly, whereas the sub-

sequent incorporation of amino acids onto linker-modiﬁed

oligonucleotide-resins does not give high, reproducible

yields.²⁴Regarding the protection scheme, nucleobases,

phosphate groups, amino acid side chains, and the C-

terminal carboxyl group (linkage to the solid support) were

permanently protected with base-labile groups, whereas the

temporary protection of 5⁰-hydroxyls and N ^a-amines was

achieved by acid-labile groups.

Phosphodiester linkages between the side chain of

hydroxylated amino acids and the ends of oligonucleotide

chains are established during the replication of the genetic

material of some viruses¹and the topoisomerase-mediated

relaxation of supercoiled DNA.²The small hybrid mol-

ecules that reproduce this particular type of amino acid–

nucleoside covalent union are referred to as nucleopeptides.

Nucleopeptides may be thus used as models of such

nucleoproteins and offer great potential for the development

of new antiviral and anticancer drugs.

The covalent attachment of a peptide to an oligonucleotide

chain has been exploited³to facilitate the oligonucleotide

transport through cell membranes and compartments, to

strengthen the hybridization between two polyanionic DNA

chains, to obtain artiﬁcial nucleases, and to introduce

reporter groups.⁴Peptide–oligonucleotide hybrids, either

with a phosphodiester or with other types of covalent

linkage, can also be used for the study of DNA–protein

interactions, to investigate the molecular requirements for

enzyme activity,⁵and to evaluate how metals or metal-

based anticancer drugs like cisplatin behave when DNA and

proteins are in close proximity.^6–8Finally, conjugation to

the 3⁰end delays oligonucleotide degradation by the most

ubiquitous 3⁰exonucleases.^9,10

In previous reports,^{11,24 – 26}we have described the

synthesis of nucleopeptides incorporating all deoxyribo-

nucleotides and, in addition to the linking residue,

amino acids with most side chain functional groups

(aspartic acid, histidine, methionine, serine, threonine,

tyrosine, lysine, arginine, and tryptophan). This manu-

script describes our latest results on the incorporation

and protection of arginine, asparagine (or glutamine),

and cysteine (Table 1, nucleopeptides 1 to 10). We also

discuss the compatibility of the ﬁnal basic deprotection

conditions with some nucleopeptide sequences. This

study completes our nucleopeptide synthesis project,

Our research group has been involved, over the last years, in

the development of a methodology for the preparation of

nucleopeptides. Our aim has been to set up a simple

procedure to allow any nucleoside or amino acid to be

Keywords: nucleopeptide; arginine; asparagine; cysteine; protection.

*

Corresponding author. Tel.: þ34-93-4021263; fax: þ34-93-3397878;

e-mail: agrandas@qo.ub.es

PII: S0040-4020(02)00793-7

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6966

Table 1. Nucleopeptides prepared in this study

0

1

2

3

4

5

6

9

10

11

12

13

14

Ac-Tyr(p³dTTTCAGAAAATCTAG)-Leu-Asp-Pro-Arg-Ile-Thr-Val-OH

0

Ac-Tyr(p³dTTTCAGAA)-Leu-Asp-Pro-Arg-Ile-Thr-Val-OH

0

Ac-Asn-Phe-Val-Ser(p³TTTT)-Gly-OH

0

Ac-Asn-Phe-Val-Ser(p³TTTT)-Gly-NH₂

0

H-Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-Tyr(p³dACGT)-Leu-Asp-Pro-OH

0

H-Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-Tyr(p³dTTTCAGAAAATCTAG)-Leu-Asp-Pro-OH

0

Ac-Cys-Gly-Tyr(p³dACTAGT)-Pro-OH

0

Ac-Cys-Gly-Ala-Hse(p³dGCATGC)-Ala-OH

0

Ac-Gly-Ser(p³T₅)-Leu-Ala-Val-OH

0

Ac-Gly-Thr(p³T₅)-Leu-Ala-Val-OH

0

Ac-Gly-Tyr(p³T₅)-Leu-Ala-Val-OH

0

Ac-Gly-Tyr(p⁵T₅)-Leu-Ala-Gly-OH

since the methodology for incorporating any amino acid

into nucleopeptides is now established.

namely Ac-Tyr-Leu-Asp-Pro-Orn(Ac-Tyr-Leu)-Ile-Thr-

Val-OH, as inferred from MALDI-TOF mass spectrometric

analysis.

All nucleopeptide crudes contained the target products, as

conﬁrmed by MALDI-TOF MS analysis, but products with

2. Results and discussion

0

m/z ratios corresponding to Ac-Tyr(p³dTTTCAGAA)-Leu-

2.1. Amino acids side chain protection

0

Asp-Pro-Arg(p³dTTTCAG)-Ile-Thr-Val-OH and Ac-

0

Tyr(p³dTTTCAGAA)-Leu-Asp-Pro-Arg(p³dTTTCAGA)-

Ile-Thr-Val-OH were also detected when nucleopeptide 2

was assembled on Ac-Tyr-Leu-Asp(OFm)-Pro-Arg(H^þ)-

Ile-Thr(Ac)-Val-resin. Both HPLC and PAGE analysis after

overnight deprotection with NH₃/dioxane 1:1 at 558C

showed that nucleopeptide crudes obtained using

Arg(Fmoc)₂were much more homogeneous than those

obtained using Arg(H^þ) (Fig. 2). Bands with an electro-

phoretic mobility lower than that of the target nucleopep-

tides, corresponding to products with approximately twice

as many nucleotide units, were found in the crudes obtained

from Arg(H^þ).

2.1.1. Arginine. Protection of the guanidine function with

two Fmoc^†groups was shown to be suitable for preparing

nucleopeptides, but N ^a-Boc-Arg(Fmoc)₂-OH was not

easily obtained.²⁶The arginine side chain was also protected

with two Fmoc groups for the preparation of peptide–

oligonucleotide hybrids.²⁷Protected arginines were

obtained by reaction of₀unprotected ornithine-containing

peptide-resins with N,N -di-Fmoc-guanidine-N⁰⁰-triﬂate in

this case.

To facilitate the preparation of peptide–oligonucleotide

hybrids, we evaluated the protection of the guanidine group

simply by protonation, which gives good results₀in peptide

synthesis.^28,29The nucleopeptides Ac-Tyr(p³dTTTCA-

Altogether, these results show that although protonation

keeps guanidine mostly unreactive during peptide assembly,

the use of Arg(Fmoc)₂derivative is recommended for the

stepwise solid-phase assembly of nucleopeptides, and, in

general, of peptide–oligonucleotide hybrids. Arginine

protection with two Fmoc groups was safer for the

construction of both the peptide and the oligonucleotide,

since it avoided some side reactions during the elongation of

the two chains. Using the protonated derivative, arginine

was partially transformed into ornithine³⁰and acylated by

some of the incoming amino acids, and nucleotide

incorporation onto the guanidine group occurred during

the subsequent nucleotide incorporation cycles.

GAAAATCTAG)-Leu-Asp-Pro-Arg-Ile-Thr-Val-OH (1)⁵

0

and Ac-Tyr(p³dTTTCAGAA)-Leu-Asp-Pro-Arg-Ile-Thr-

Val-OH (2) were assembled (Fig. 1) using the two modes

of protection.

The amino acid analyses of the two peptide-resins, Ac-Tyr-

Leu-Asp(OFm)-Pro-Arg(H^þ)-Ile-Thr(Ac)-Val-resin and

Ac-Tyr-Leu-Asp(OFm)-Pro-Arg(Fmoc)₂-Ile-Thr(Ac)-Val-

resin, gave similar results, and the HPLC traces of the

peptide crudes obtained after ammonia deprotection (NH₃/

dioxane 1:1, 4 h, rt) were also similar (Fig. 2). However, in

the crude obtained from protonated arginine, the

target peptide was accompanied by a minor side product,

2.1.2. Asparagine and glutamine. Primary carboxamides

react with activated phosphines and phosphoramidites³¹

(Fig. 3A). This reaction was exploited for the prep-

aration of peptide–oligonucleotide conjugates,³¹nucleic

acid analogues³²and aminoacyl-adenylate³³with N-

acylphosphoramidate linkages. We inferred that nucleo-

peptide assembly without protection of the asparagine

and glutamine side chains may afford branched

molecules, and searched for a permanent protecting

group for these amino acid side chains that could be

removed in mild, basic conditions. We disregarded the

alternative of replacing asparagine (or glutamine) by the

phenyl ester derivative of aspartic (or glutamic) acid,

which may afford asparagine or glutamine by ammono-

lysis, because ammonia treatment may give rise to the

†

Abbreviations: amino acid and oligonucleotides nomenclature follows the

IUPAC-IUB recommendations (Eur. J. Biochem. 1984, 138, 9–37 and

Pure Appl. Chem. 1974, 279–290); Aa¼amino acid, Ac¼acetyl,

Acm¼acetamidomethyl, Boc¼t-butoxycarbonyl, Bz¼benzoyl, cHex¼

cyclohexyl, CNE¼2-cyanoethyl, DBU¼1,8-diazabicyclo[5.4.0]undece-

7-ene, DCC¼N,N⁰-dicyclohexylcarbodiimide, DCM¼dichloromethane,

DIEA¼N,N-diisopropylethylamine,

Dmf¼N,N-dimethylamino-

methylene, DMAP¼4-N,N-dimethylaminopyridine, DMT¼4,4⁰-

dimethoxytrityl, Dnp¼2,4-dinitrophenyl, DTT¼1,4-dithiothreitol,

For¼formyl, Fm¼9-ﬂuorenylmethyl, Fmoc¼9-ﬂuorenylmethoxy-

carbonyl, HMFS¼2-(9-oxymethylﬂuorenyl)succinyl, HOBt¼1-

hydroxybenzotriazole, iBu¼isobutyryl, IRAA¼internal reference amino

acid, MBHA¼4-methylbenzhydrylamine, N¼2⁰-deoxynucleoside,

NMI¼N-methylimidazole,

pr¼protected/protecting

group,

TBAF¼tetrabutylammonium ﬂuoride, TCA¼trichloroacetic acid,

TFA¼triﬂuoroacetic acid, Tfa¼triﬂuoroacetyl, THAP¼2,4,6-

trihydroxyacetophenone, Tos¼tosyl.

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L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6967

Figure 1. General nucleopeptide synthesis scheme.

formation of aspartimides to large extents.^34,35In

addition, both a- and b-peptide chains can be obtained

from the reaction of ammonia with the aspartimide ring

(Fig. 3B).

pyridine 1:1, at rt for 24 h; and reaction with 0.1 M TBAF in

anhydrous THF for 24 h at rt).

Attempts to oxidize –CH₂–NH– groups to –CO–NH–

functions following published methods^37,38also failed (Fig.

3C, b). This approach would have allowed protected 2,4-

diaminobutyric acid and ornithine to be transformed into

protected asparagine and glutamine, respectively. Efforts to

protect the CONH₂function with either benzoyl, diphenyl-

carbamoyl or Fmoc groups (Fig. 3C, c) were also

unsuccessful.

We ﬁrst considered the use of protecting groups removable

by b-elimination reactions, in particular the 2,4-dinitrophe-

nylethyl group.³⁶For preliminary evaluation, N-[b-(2,4-

dinitrophenyl)ethyl]acetamide was obtained from N-[(b-

phenyl)ethyl]acetamide by reaction with conc. H₂SO₄and

fuming HNO₃. We expected basic reagents to transform this

product into acetamide and 2,4-dinitrophenylstyrene (Fig.

3C, a), but the starting material remained unaltered after a

variety of treatments (overnight treatment with conc. aq.

NH₃/H₂O 1:1 at rt or at 558C; reaction with 0.5 M solutions

of DBU in dioxane, EtOH/dioxane 1:1, pyridine or MeOH/

After these negative results, we re-examined the extent of

carboxamide phosphitylation on peptide-resins. Boc-Gly-

Asn-Ala-Pro-MBHA, Boc-Gly-Gln-Ala-Pro-MBHA and

Boc-Asn-Ala-Val-O-HMFS-Leu-MBHA were submitted

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L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6968

the side products in the peptide crude was very low (less

than 3%).

As a ﬁnal test, four nucleotide (T) incorporation cycles were

carried out on a peptide-resin suitable for nucleopeptide

synthesis, Ac-Asn-Phe-Val-Ser-Gly-O-HMFS-Leu-MBHA,

which contained free hydroxyl and carboxamide groups.

HPLC Analysis showed that the target nucleopeptide was

the main product (75%) in the crude obtained after

ammonolysis at rt (a mixture of C-terminal acid, Ac-Asn-

0

Phe-Val-Ser(p³TTTT)-Gly-OH, 3, and carboxamide, Ac-

0

Asn-Phe-Val-Ser(p³TTTT)-Gly-NH₂, 4, was obtained, see

below). The only side product detected in substantial

amount (ca. 25%) was the phosphorylated tetranucleotide

TpTpTpTp, present in the mixture as the result of a b-

elimination process that cleaves the nucleopeptide (see

below).

Although there is a risk of obtaining branched products

when the side chains of asparagine and glutamine are

left unprotected, the extent of carboxamide

phosphitylation by tetrazole-activated phosphoramidites

seems to remain low and acceptable, unless stronger

activators are used.³³Hence, asparagine and glutamine

protection is not essential for the standard stepwise solid-

phase assembly of nucleopeptides (or of differently linked

peptide–oligonucleotide hybrids), as reported elsewhere

for phosphitylation of glutamine-containing peptide-

resins.³⁹

Nucleopeptides H-Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-

0

Tyr(p³dACGT)-Leu-Asp-Pro-OH (5)⁵and H-Ile-Ala-Leu-

0

Gly-Thr-Ser-Lys-Leu-Asn-Tyr(p³dTTTCAGAAAATC-

TAG)-Leu-Asp-Pro-OH (6)⁵were then prepared by step-

wise solid-phase assembly leaving unprotected the

asparagine side chain.

Before oligonucleotide elongation, an aliquot of peptide-

resin Fmoc-Ile-Ala-Leu-Gly-Thr(Ac)-Ser(Ac)-Lys(Tfa)-

Leu-Asn-Tyr-Leu-Asp(OFm)-Pro-O-HMFS-Phe-MBHA

was treated with the 1:1 conc. aq. NH₃/dioxane mixture at rt

(overnight reaction). The target peptide was the main

product of the crude, as inferred from MALDI-TOF MS and

HPLC analysis (Fig. 4A). The subsequent incorporation of

the required nucleoside derivatives (A^Bz, C^Bz, G^iBuand T)

onto this peptide-resin yielded 5 and 6 after overnight

deprotection and cleavage with conc. aq. NH₃/dioxane 1:1

at 558C.

Figure 2. HPLC proﬁles of arginine-containing peptide and nucleopeptide

crudes. Crudes obtained from protonated arginine: Ac-Tyr-Leu-Asp-Pro-

Arg-Ile-Thr-Val-OH (A), nucleopeptides 1 (C) and 2 (E). Crudes obtained

from bis-Fmoc-protected arginine: peptide (B), nucleopeptides 1 (D) and 2

(F).

to four or ﬁve consecutive nucleotide (T) incorporation

cycles, and the amount of DMT^þformed after each cycle

was quantitated. It appeared that some nucleoside incor-

poration had taken place during the ﬁrst cycle (4–10% with

respect to phosphitylation of all carboxamide groups on the

solid matrix), but the amount of DMT^þgroups found after

the subsequent synthesis cycles differed between assays.

Nucleoside incorporation seemed to remain at ca. 1% in

each of the subsequent cycles. No differences were found

between asparagine and glutamine on the peptide-resin.

MALDI-TOF analysis of the crude obtained after ammonia

treatment of Boc-Asn-Ala-Val-O-HMFS-Leu-MBHA

Analysis of the composition of the crude obtained from

Fmoc-Ile-Ala-Leu-Gly-Thr(Ac)-Ser(Ac)-Lys(Tfa)-Leu-

0

Asn-Tyr(p³dACGT)^pr-Leu-Asp(OFm)-Pro-O-HMFS-Phe-

MBHA provided useful information. The target nucleopep-

tide, 5 (30%; MALDI-TOF MS, negative mode: m/z

0

2639.0), was accompanied by H-Tyr(p³dACGT)-Leu-

Asp-Pro-OH (7, 20%; MALDI-TOF MS, negative mode:

m/z 1740.6) and a second product (8, 30%) with an m/z ratio

virtually identical to that of 5 (m/z 2639.2, MALDI-TOF

MS, negative mode) (Fig. 4B). Compound 5 was also

identiﬁed by nucleoside and amino acid composition after

enzymatic digestion. Amino acid analysis after proteolytic

digestion conﬁrmed the presence of both asparagine and

aspartic acid. The digestion of 5 by a mixture of proteolytic

afforde₀d peaks with m/z values corresponding to Boc-

0

Asn(p³T)-Ala-Pro-OH, Boc-Asn(p³TT)-Ala-Pro-OH, Boc-

0

Asn(p³TTT)-Ala-Pro-OH and Boc-Asn(p³TTTT)-Ala-Pro-

OH in addition to tripeptide Boc-Asn-Ala-Pro-OH. How-

ever, the HPLC traces showed that the proportion of each of

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L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6969

O

C

A)

R

NH₂

O

P

i) oxidation

ii) deprotection

tetrazole

R

C NH

P

OR'

R

C NH

OR'

R'

O

P

NiPr₂

O-pr

O

O-pr

N-acylphosphoramidate

O

B) NH CH C NH

NH CH C NH

NH CH C NH₂

NH CH

H₂C

C

CH₂

NH₃

N

NH₃

+

C

O

(base)

C

OPh

C

NH₂

C

NH

O

-Asp(OPh)-

aspartimide

-Asn-

β-peptide

O₂N

O

C) a) H₃C C NH CH₂CH₂

base

H₃C C NH₂

NO₂

+

H₂C CH

NO₂

O

RuO₄

b) Ph CH₂CH₂NH

X

X = Fmoc, Boc

Ph CH₂C NH

X

O

Ph CH₂C NH

Ph-CO

Ph₂N-CO

Fmoc

Ph CH₂

C

NH₂

Y =

Y

c)

Figure 3. Side reactions associated with Asn (A) or protected aspartic acid (B), and summary of the essays carried out to obtain suitably protected carboxamide

derivatives (C).

enzymes was the expected behavior for an a-peptide chain

(see below).

for 8. Nevertheless, 8 was stable to treatment with NH₃at

558C, supporting the ﬁrst hypothesis. The primary carbox-

amide of the b-peptide may again react intramolecularly to

give the aspartimide, which, in turn, may yield an [a-þb-]

nucleopeptides mixture. In other words, if 8 were the b-

peptide, it might be partially transformed into 5 in these

reaction conditions, which did not happen.

Concerning the formation of the two side products, 7

seemed to result from the cleavage of the Asn-Tyr peptide

bond, as a result of the nucleophilic attack of the side chain

primary carboxamide to the asparagine carbonyl group of

the Asn-Tyr union. Peptide cleavage is unlikely,⁴⁰but

intramolecular acylations leading to the formation of

aspartimides are readily promoted by bases.

The main conclusion from this study concerns the ﬁnal

deprotection conditions. Peptide H-Ile-Ala-Leu-Gly-Thr-

Ser-Lys-Leu-Asn-Tyr-Leu-Asp-Pro-OH and nucleopep-

0

Side product 8 was formed from both the protected and the

free, pure nucleopeptide upon ammonia treatment at 558C,

and it was shown to remain stable in these reaction

conditions. An impurity with a mass virtually identical to

that of the target product was also found to accompany 6, as

inferred from the MALDI-TOF MS analysis of the crude.

tides Ac-Asn-Phe-Val-Ser(p³TTTT)-Gly-OH/NH₂(3þ4)

were the main products after ammonolysis at rt, whereas

ammonia treatment of the protected precursors of 5 and 6 at

558C afforded complex reaction mixtures. Moreover, pure 5

was partially transformed into the other two products after a

5 h treatment with conc. aq. NH₃/dioxane 1:1 at 558C.

Therefore, the use of the most base-labile set of permanent

protecting groups to allow the ﬁnal ammonia deprotection at

rt is preferable, especially when asparagine is present in the

peptide chain.

Compound 8 may be a nucleopeptide containing aspartic

acid instead of asparagine. The deamidation reaction is

enhanced by increased pH, ionic strength and temperature.⁴¹

Since carboxylic acids do not react with ammonia to give

amides, 8 cannot be transformed into 5, which is consistent

with its stability in these reaction conditions. Alternatively,

the origin of 8 may be the nucleophilic attack of the NH of

the Asn-Tyr amide bond to the side chain carboxamide, with

the concomitant formation of aspartimide, followed by basic

hydrolytic opening of the ring (Fig. 3C). Opening of the

aspartimide ring by reaction with ammonia may yield both

the target nucleopeptide and the b-peptide isomer. For-

mation of the latter was also consistent with the mass found

2.1.3. Cysteine. At this point, the only trifunctional amino

acid that remained to be introduced in nucleopeptides was

cysteine. However, we needed a protecting group able to

prevent sulfur oxidation (and nucleotide coupling onto the

thiol group) during nucleotide incorporation cycles. Only

three commercially available Boc-cysteine derivatives were

protected with groups removable in conditions compa-

tible with a nucleopeptide structure, Boc-Cys(Fm)-OH,

Boc-Cys(Acm)-OH and Boc-Cys(S–t Bu)-OH. The latter

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L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6970

Figure 4. HPLC traces of crude H-Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-Tyr-Leu-Asp-Pro-OH (A) and H-Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-

Tyr(p³dACGT)-Leu-Asp-Pro-OH (5) (B). Possible mechanism accounting for the formation of side product 7 (C).

0

was the only one stable in the phosphite to phosphate

oxidation conditions (both aqueous iodine and t-

butylhydroperoxide, TLC analysis).

the synthesis of 9, while 10 was prepared using Boc-Cys-

(S–t Bu)-OH; (ii) standard phosphoramidite derivatives

were used to obtain 9, but, as deduced from the synthesis

of 9 (see below), 10 was assembled using nucleoside

derivatives protected with more base-labile groups (A^Bz,

Thiol groups have been protected as disulﬁdes, with the S–

tert-butyl group, for the preparation of peptide–oligonu-

cleotide conjugates either containing a cysteine residue^42,43

or linked through N ⁴-mercaptoethylcytosine.⁴⁴Standard

oxidation conditions were used during oligonucleotide

elongation and the S–tert-butyl group was removed by

reaction with conc. aq. NH₃solutions containing DTT. The

S–tert-butyl group is not labile in the ammonia deprotection

conditions, but Schwope and co-workers⁴³reported that,

during this treatment, the SSt Bu group is lost by a b-

elimination reaction, giving rise to dehydroalanine at the

cysteine position. Dehydroalanine may be subsequently

transformed into serine (or 2,3-diaminopropionic acid) by

conjugate addition of OH²(or NH₃) during the concen-

trated aqueous ammonia treatment (Fig. 5A).

C

^Ac, G^Dmf).

Regarding the deprotection of peptide-resins (Fig. 5B, a),

the thiol-protected peptides (Ac-Cys(S–t Bu)-Gly-Tyr-Pro-

OH and Ac-Cys(S–t Bu)-Gly-Ala-Hse-Ala-OH) were the

main products of the respective crudes when deprotection

was carried out with NH₃/dioxane 1:1 at rt (overnight

treatment). The serine-containing peptide was the main

product when the overnight ammonolysis reaction was

carried out at 558C, and the free peptide was obtained after

cleavage and deprotection with the 1:1 mixture NH₃/[1 M

DTT in dioxane] for 4 h at rt.

Most of the deprotection assays on nucleopeptide-resins

were carried out with the precursor of 9, and essentially

reproduced the results obtained with peptide-resins (Fig. 5B,

b). The target nucleopeptide was obtained as the main

product after ammonia/DTT treatments at rt or for 7 h at

558C. However, when the deprotection reaction was

prolonged overnight at 558C, the main product was a

Two cysteine-containing nucleopeptides were synthesized,

0

Ac-Cys-Gly-Tyr(p³dACTAGT)-Pro-OH (9) and Ac-Cys-

0

Gly-Ala-Hse(p³dGCATGC)-Ala-OH (10). Both were

assembled using standard stepwise solid-phase protocols,

with two differences: (i) Ac-Cys-(S–t Bu)-OH was used for

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6971

Figure 5. Side reactions associated with the deprotection of cysteine-containing peptides or nucleopeptides, and products obtained.

modiﬁed nucleopeptide, resulting from b-elimination and

DTT addition to dehydroalanine.

treatment with 1 M DTT (1 h), either in dioxane or in

dioxane/Tris HCl at pH¼8, and then to reaction with NH₃/

dioxane 1:1 at 558C.

In another experiment, we aimed to selectively remove the

cysteine protecting group from the nucleopeptide-resin,

since b-elimination does not occur if a cysteine-free

nucleopeptide is treated with ammonia. In addition, this

procedure was attractive because it avoided the presence of

DTT in the nucleopeptide crude. Unfortunately, this

In conclusion, the best choice is to assemble cysteine-

containing nucleopeptides using Cys(S–t Bu) in combi-

nation with the most base-labile nucleobase and amino acid

side chain permanent protecting groups, to allow the mildest

deprotection conditions (NH₃/[1 M DTT in dioxane] 1:1,

8 h to overnight treatment, rt).

experiment failed (Fig. 5B, c). Nucleopeptide Ac-Ser-Gly-

0

Tyr(p³dACTAGT)-Pro-OH was the main product, as

expected for a base-treated Cys(S–t Bu)-containing nucleo-

peptide, when the nucleopeptide-resin was ﬁrst submitted to

For the isolation of 9, the ﬁnal cleavage and deprotection

step was carried out by reaction with the NH₃/DTT mixture

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6972

for 7 h at 558C, searching for nucleobase deprotection

conditions compatible with the stability of the target

molecule. After oxidation with aqueous dimethylsulfox-

ide,⁴⁵we obtained the nucleopeptide dimer, with a disulﬁde

2.2. Stability of nucleopeptides in the ﬁnal basic

deprotection treatment conditions

The most delicate point of our stepwise solid-phase

synthesis scheme is, as stated elsewhere,²⁶the compatibility

between the basic conditions used for the removal of the

permanent protecting groups and the base-lability of serine-

and threonine–nucleoside phosphate diester linkages.⁴⁷

Unfortunately, not all serine–nucleoside linkages have the

same stability,¹¹since, for instance, the ﬁrst serine-

nucleopeptide that we₀synthesized and used as model

compound, Boc-Ser(p⁵dT)-NH-cHex, is the most base-

stable nucleopeptide that we have ever obtained. We have

found that: (i) some serine-nucleopeptides are extremely

labile to bases and cannot be obtained even using the

mildest deprotection conditions (1:1 NH₃/dioxane mixtures,

rt), (ii) the extent of the b-elimination reaction is rather high

in other cases, but (iii), it can be kept to acceptable limits

(10–25%) in most situations. Examples of the ﬁrst

bond

linking

the

two

units,

{Ac-Cys-Gly-

0

Tyr(p³dACTAGT)-Pro-OH}₂[S–S] (as conﬁrmed by

analytical ultracentrifugation).

Unexpectedly, the isolation of 10 was much more difﬁcult.

Although the optimal protection scheme and deprotection

conditions were used, a complex crude was obtained. As

stated above, the same deprotection treatment at the level of

peptide-resin had afforded the free peptide Ac-Cys-Gly-

Ala-Hse-Ala-OH as the main product. As expected, the

peptide cleanly dimerized to the disulﬁde form.

HPLC and MALDI-TOF MS analysis of the nucleopeptide

crude showed that some of the contaminants were

nucleopeptides lacking the Ac-Cys, Ac-Cys-Gly and Ac-

Cys-Gly-Ala fragments. Moreover, pure 10, in the reduced

form, decomposed affording the same mixture of products

nucleopeptides (quantitative degradation after 2–6 h ₀NH₃

0

treatments) are H-Ser(p⁵dG)-His-OH⁴⁴and H-Ser(p⁵dG)-

and dimerized very slowly. The isolated nucleopeptide

Met-OH.²⁴Up to 60–70% degradation was found in the

0

disulﬁde dimer {Ac-Cys-Gly-Ala-Hse(p³dGCATGC)-Ala-

OH}₂[S–S] was stable. We cannot explain how the

covalent linkage of an oligonucleotide to the homoserine

side chain can give rise to a readily decomposing

nucleopeptide, whereas peptide Ac-Cys-Gly-Ala-Hse-Ala-

OH is stable.

synthesis of Ac-Gly-Ala-Ser(p³dACTAGT)-His-Val-OH,⁴⁴

but the extent of the side reaction was lower for Ac-Gly-

0

Ala-Ser(p³dACTAGT)-Lys-Val-OH (23%), which only

differed from the previous molecule in that histidine was

replaced by lysine.⁴⁸So far, we cannot reach any conclusion

relating the base-lability of nucleopeptides with their

sequence. The -Ser-His- sequence may be very prone to

undergo the b-elimination reaction, but no association

between size and stability can be established.

We would also like to highlight that, in both nucleopeptide

crudes, some m/z values corresponded to increases of 53

mass units with respect to the expected product. Alkylation

of the free thiol groups by the acrylonitrile generated upon

deprotection of the phosphates accounts for the formation of

these [–S–CH₂–CH₂–CN]-modiﬁed side products. In

agreement with other authors,⁴⁶the proportion of these

impurities was found to decrease when ﬁnal deprotection

was carried out in higher solvent volumes.

Threonine-nucleopeptides are more stable to basic reagents

than serine-nucleopeptides,⁴⁷in agreement with our results.

The extent of the b-elimination reaction after treatment of

0

nucleopeptide-resin Ac-Gly-Ser(p³T₅)^pr-Leu-Ala-Val-O-

HMFS-resin with a 1:1 mixture of NH₃/dioxane at rt was

32% after 6 h (as deduced, upon HPLC analysis, from the

relative areas of the peaks corresponding to the target

nucleopeptide, Ac-Gly-Ser(p³T₅)-Leu-Ala-Val-OH, 11,

0

To gain more insight into the synthesis of cysteine-

containing nucleopeptides, we checked the peptide stability

in the oligonucleotide assembly conditions. A sample of Ac-

Cys(S–t Bu)-Gly-Ala-Hse-Ala-resin was submitted to a

nucleotide incorporation cycle, with no phosphoramidite

delivery to the column to prevent coupling onto the

homoserine side chain, and two aliquots were subsequently

treated with either the NH₃/DTT mixture or with NH₃(rt),

respectively. The NH₃/DTT treatment afforded the free

peptide. The NH₃treatment gave rise to a complex mixture

in which the expected thiol-protected peptide was a minor

product, and which contained, among others, products with

m/z ratios corresponding to oxidized peptides (the m/z ratios

found indicated transformation of the S–S–tBu moiety into

SO₂–S–tBu, and even to SO²₃groups). On one hand, this

experiment showed that, in contrast with the results of our

initial tests, the S–S–t Bu moiety was not stable to

oxidation with t BuOOH. These oxidized cysteines probably

undergo b-elimination more easily than the unoxidized

residues. On the other hand, the expected peptide was the

main product when deprotection was carried out with the

NH₃/DDT mixture, indicating that most, if not all, of these

oxidized products were reduced by reaction with DTT

during the deprotection process.

and the phosphorylated tetranucleotide, TpTpTpTp). In

0

the case of nucleopeptide Ac-Gly-Thr(p³T₅)-Leu-Ala-Val-

OH, 12, 23% degradation was detected after the same

treatment. Other authors have described that threonine-

nucleopeptides undergo the b-elimination reaction.⁴⁹

As expected, with both linking amino acids, most of the side

reaction occurs at the beginning of the basic treatment, before

phosphate groups are fully deprotected, since phosphate

diesters are much more efﬁcient leaving groups than

monoesters, and the prolonged reaction time required to

fully remove the nucleobase permanent protecting groups

does not excessively affectthe target product. Protection ofthe

phosphates and the nucleobases with allyl and allyloxycarbo-

nyl groups, which can be removed in neutral conditions, has

been claimed to be the best alternative to obtain serine-

nucleopeptides, since only a short ammonia treatment is

required to cleave the oligonucleotide–resin bond.^18,19The

disadvantage of this protection scheme is that nucleoside

phosphoramidite derivatives are not commercially available.

Tyrosine gives rise to the most base-stable amino

acid–nucleoside linkages, which are fully resistant to

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L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6973

NH₃/dioxane treatments.²⁶No lability to tetrabutylammo-

nium ﬂuoride in aqueous solvents has been described,^50,51

but their stability to this reagent in anhydrous solutions has

showed that C-terminal amino acids with more sterically

hindered side chains give rise, as expected, to considerably

lower or undetectable amounts of carboxamide product.

Nevertheless, the relative proportion of products probably

varies depending on the structure (length, sequence) of the

immobilized molecule, as in the case of our nucleopeptides

with glycine at the C-terminus, since ammonia treatment of

a peptide-HMFS-resin with a leucine residue at the C-

terminal position has been described to yield a 3:1 –OH/–

NH₂mixture.³⁹

0

not been proved. The nucleopeptides Ac-Gly-Tyr(p³T₅)-

0

Leu-Ala-Val-OH (13) and Ac-Gly-Tyr(p⁵T₅)-Leu-Ala-Gly-

OH (14) were obtained by deprotection and cleavage of the

nucleopeptide–resin bond with 0.05 M TBAF in anh. THF

(30 min, rt), and no pentanucleotide was found upon HPLC

analysis of the crudes. We thus conclude that tyrosine–

nucleoside phosphodiester linkages are resistant to treat-

ment with ﬂuorides in anhydrous media.

Finally, we did not detect the formation of asparagine after

ammonia deprotection of -Asp(OFm)- derivatives, which

does not agree with the results found for C-terminal glycine

nucleopeptides, but may be due to the proximity of the solid

support. If the acidic proton of the ﬂuorenylmethyl ester is

less accessible, thus delaying the b-elimination reaction,

nucleophilic attack of ammonia to the carbonyl of the ester

may occur, obviously more easily if bulky groups are not

close. Fluorenylmethyl groups on amino acid side chains are

more accessible and may be smoothly removed, releasing

the carboxyl group, whereas the solid matrix may hinder the

elimination of the acidic proton of the ﬂuorenylmethyl

esters of C-terminal amino acids, thus enhancing the

formation of acid/amide nucleopeptide mixtures.

2.3. C-Terminal acids vs C-terminal carboxamides

The NH₃/dioxane treatment used for the ﬁnal deprotection

of nucleopeptides is known to afford a mixture of

nucleopeptides with the C-terminal amino acid either as

carboxylic acid or as carboxamide derivative.^26,39As

expected, the relative proportion of these two products

depends on the nature of the C-terminal amino acid (Table

2).

A C-terminal glycine residue always gives rise to acid/

carboxamide mixtures, in a relative proportion which varies

between nucleopeptides. In any case, b-elimination seems

to take place faster than the ammonia nucleophilic attack to

the carbonyl of the ester, since the acid is always obtained in

higher yields than the amide. Alkaline hydrolysis of the

ester may also account for the formation of nucleopeptide-

acids.

3. Conclusion

Nucleopeptides, as well as other peptide–oligonucleotide

conjugates, can be easily obtained by stepwise solid-phase

synthesis. It is recommended to ﬁrst assemble the peptide

moiety, and then to elongate the oligonucleotide chain at the

hydroxyl group of an amino acid side chain.

0

Nucleopeptide Phac-Phe-Val-Ser(p³dACTAGT)-Gly was

also assembled using a TentaGel solid support instead of the

polystyrene-1%-divinylbenzene copolymer. After cleavage

and deprotection using the same conditions as described in

Table 2, HPLC analysis of the crude revealed a 1.7:1 –OH/

NH₂mixture.⁵²Should we rely on a single result to reach

conclusions, this would indicate that the nucleophilic attack

of ammonia to the carbonyl of the ester (and thus

carboxamide formation) is slightly more favored with

polystyrene resins, which have worse swelling properties

in polar solvents than polystyrene–polyethyleneglycol

copolymers.

As often stated, the most delicate step is the choice of

protecting groups. The use of base-labile permanent

protecting groups is not devoid of problems, but, in our

opinion, it is advantageous with respect to other alternatives

because it allows the use of many commercially available

amino acid and nucleoside synthons. Boc-Thr(Ac)-OH,

Boc-Arg(Fmoc₂)-OH and Boc-Cys(S-t Bu)-OH are the only

amino acid derivatives that cannot be purchased from

commercial sources. It may also be necessary to prepare the

Analysis of the last three nucleopeptide crudes (Table 2)

5⁰-phosphoramidites of dC^Ac(or dC^iBu) and dG^{Dmf 54–56}

, or

Table 2. Relative proportion nucleopeptide-OH/nucleopeptide-NH₂after deprotection with ammonia

Nucleopeptide sequence

Deprotection conditions (NH₃/dioxane 1:1)

–OH/–NH₂

0

Phac-Phe-Val-Ser(p³dACTAGT)-Gly⁵²

17 h, rt

24 h, rt

17 h, 558C

6 h, rt

1.3:1

1.7:1

1.4:1

2:1

3:1

0

Ac-Ser(p⁵dG)-Gly⁴⁷

0

Phac-Ser(p⁵dG)-Gly⁴⁷

0

H-Trp-Val-Hse(p³dACTAGT)-Gly²⁶

0

Phac-Hse(p³dACTAGT)-Ala-Thr-Ser-Val-Tyr-Gly²⁶

0

Ac-Gly-Tyr(p³TTTTT)-Leu-Ala-Gly

6 h, 558C

17 h, 558C

6 h, rt

4:1

0

Ac-Lys-Trp-Lys-Hse(p³₀dGCATCG)-Ala⁵³

.93:7

.99:1

Ac-Ala-Trp-Ala-Hse(p³dGCATCG)-Ala⁵³

Ac-Gly-Tyr(p³TTTTT)-Leu-Ala-Val

0

6 h, 558C

The relative proportion nucleopeptide-OH/nucleopeptide-NH₂was deduced from the peak areas at the HPLC trace of each crude (the HFMS handle was used

in all cases; the acid always eluted before the amide). The isolated products were characterized as the C-terminal acid and the C-terminal amide, respectively,

by electrospray mass spectrometric analysis.

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6974

those in which nucleobases are protected with tert-

signiﬁcant differences between the base-lability of 3⁰and

5⁰-unions.

butylphenoxyacetyl groups,⁵⁷to obtain nucleopeptides

0

with serine-₀or threonine–⁵oligonucleotide linkages, since

the only 5 -phosphoramidite derivatives now available

require deprotection with ammonia at 558C.

4. Experimental

We have obtained the best results by protecting the side

chain of arginine with two Fmoc groups rather than by

protonation. Guanidine protonation is insufﬁcient to wholly

prevent the formation of branched products resulting from

nucleotide incorporation at this group, and less homo-

geneous crudes are obtained.

Fmoc-^L-amino acids, Boc-L-amino acids and resins for

peptide synthesis (MBHA resin and Boc-Val-Pam-resin)

were obtained from Bachem and Novabiochem. The 3⁰-

phosphoramidite derivatives of DMT-dA^Bz, DMT-dC^Bz,

5⁰

DMT-dG^iBuand DMT-T, and the

phosphoramidite

derivative of T-DMT were purchased from Glen

Research Corporation. Solid-phase syntheses were

performed manually in a polypropylene syringe ﬁtted

with a polyethylene disc (peptides), or on the 380B

Applied Biosystems synthesizer (oligonucleotide assem-

bly in nucleopeptides).

Protection of the primary carboxamides of asparagine and

glutamine side chains is not mandatory, since the extent of

phosphitylation of these groups is kept to minimal,

acceptable levels during oligonucleotide elongation cycles.

Ammonia deprotection at rt is required to avoid side

reactions in asparagine-containing nucleopeptides.

HPLC analysis (Nucleosil C-18, 250£4.6 mm, 10 mm,

1 mL/min, detection wavelength 260 nm): A¼0.01 M

ammonium acetate, B¼acetonitrile/H₂O 1:1 (v/v). Unless

otherwise indicated, puriﬁcations at the semi-preparative

scale were carried out on a Kromasil column (Phase

Separations, 250£10 mm, 10 mm, 3 mL/min) using basi-

cally the same solvent system (A¼0.05 M ammonium

acetate).

The cysteine thiol can be blocked with the tert-butylsulfenyl

group, which can be removed simultaneously to all the

other permanent protecting groups by adding DTT to the

ﬁnal ammonia deprotection solution. We have found that,

even in the presence of DTT, b-elimination of HS–St Bu

occurs when the ammonia treatment is carried out at

558C, and that the extent of this side reaction grows with

prolonged reaction times. Although no mention is made

to this side reaction in some reports,^9,58we recommend rt

conditions for the ﬁnal ammonia deprotection reaction.

Large volumes of deprotecting solution are also helpful to

minimize the extent of cysteine alkylation by

acrylonitrile.

Amino acid analyses were performed on a Beckman

System 6300 analyzer. Acid hydrolyses of peptide-resins,

peptides and nucleopeptides were carried out in 12 M

HCl/propionic acid (1:1) either at 1508C for 60–90 min or

at 1108C for 24 h (homoserine, serine and threonine are to

some extent degraded in these conditions). Enzymatic

hydrolyses of the peptide moiety of nucleopeptides were

carried out as follows (the three enzymes were purchased

from Sigma): to a solution of 3–5 nmol of product in

15 mL of 0.1 M ammonium acetate (pH¼5.3) was added

1 mL of a 1:32 (v/v) 2-mercaptoethanol/H₂O mixture and

3 mL of a 20 mg/mL papaine (EC 3.4.22.2) solution

(286 U/mL). The mixture was incubated at 378C for 2 h,

and, after addition of 1 drop of acetic acid, lyophilised.

The sample was dissolved in 15 mL of 0.2 M ammonium

acetate (pH¼8.3) and 1 mL of the 2-mercaptoethanol/H₂O

mixture. 3 mL of microsomal leucine aminopeptidase (EC

3.4.11.2) solution (3 mg/mL, 72 U/mL) was added, and

the mixture was incubated at 378C for 3 h. After a second

addition of 3 mL of the enzyme solution, incubation was

prolonged for 12 h (or overnight), 1 drop of acetic acid

was added and the sample was lyophilized. Finally, 3 mL

of a prolidase (EC 3.4.13.9) solution (5 mg/mL,

875 U/mL) was added to the amino acid/nucleopeptide

mixture dissolved in 15 mL of 0.1 M ammonium acetate

and 0.025 M MnCl₂(pH¼8.3). The mixture was

incubated for 3 h at 378C, after which 1 drop of acetic

acid was added and the sample was lyophilized.

The base-lability of serine- and threonine–nucleoside

linkages is one of the major points of concern, but ﬁnal

deprotection with ammonia at rt allows most nucleopeptides

to be obtained in fairly good yields. Since mild deprotection

conditions are also convenient to minimize or circumvent

side reactions associated with the presence of asparagine

and -Cys(S-t Bu), the use of the most base-labile nucleobase

protecting groups is required for the stepwise synthesis of

both nucleopeptides and any peptide–oligonucleotide

conjugate incorporating cysteine or asparagine.

In summary, the most convenient scenario for nucleopeptide

synthesis is that in which glycine is not the C-terminal

amino acid, and either tyrosine or the natural, non-

proteinogenic homoserine residue is the linking residue.⁵⁹

Anchoring of glycine to the solid support is racemization-

free, but cleavage of the nucleopeptide–resin bond with

ammonia yields acid/amide mixtures in unpredictable

proportions. Although the mixture of the two products can

be used in some biological experiments, the presence of two

compounds renders puriﬁcation and characterization more

difﬁcult. The only alternative to obtain a single product (the

acid) is to detach the nucleopeptide from the solid matrix by

reaction with tetrabutylammonium ﬂuoride in anhydrous

conditions, but this rather strong basic treatment may not be

compatible with all nucleopeptide sequences. Regarding the

base-stability of serine- and threonine–nucleoside phos-

phodiester linkages, on the basis of the few data available

predictions cannot be made, and we have not found

The nucleoside content in nucleopeptides was determined

after reversed-phase HPLC analysis of the mixtures

obtained after digestion with snake venom phosphodiester-

ase (EC 3.1.4.1, Sigma) and alkaline phosphatase (EC

3.1.3.1, Sigma).

Mass spectrometric analysis was carried out using

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6975

VG-Quattro (ES) or Applied Biosystems Voyager-DERP

(MALDI-TOF) instruments.

treatment with a 1:1 conc. aq. ammonia/dioxane solution

(dioxane was ﬁrst added to swell the polystyrene resin).

Overall synthesis, deprotection and puriﬁcation yields

ranged between the following values: nucleopeptides 13

and 14: 25–30%; nucleopeptides 1 and 2: 15–20%;

nucleopeptides 3, 4, 11 and 12: 10–15%; nucleopeptides

5, 6, 9 and 10: 1–5%.

4.1. Nucleopeptide synthesis

Incorporation of the internal reference amino acid onto p-

methylbenzhydrylamine-resin ( f¼0.5–0.6 mmol/g) was

carried out by reaction with 0.5 equiv. of both the

corresponding Fmoc-amino acid derivative and DCC

(15 min) in order to achieve partial acylation of polymeric

amine groups. The substitution degree was determined on

an aliquot by UV spectrophotometric quantitation of the

amount of N-(9-ﬂuorenylmethyl)piperidine formed upon

deprotection with piperidine, and, if suitable for oligonu-

cleotide synthesis,⁶⁰unreacted amine groups were capped

by reaction with Ac₂O and DIEA (substitution degree

values ranged between 0.13 and 0.25 mmol/g). The Fmoc

group was eliminated and the bifunctional linker N-2-(9-

hydroxymethylﬂuorenyl)succinamic acid, H-HMFS-OH⁶¹

coupled in the presence of DCC (3 equiv. of each reagent,

2–4 h). Hydroxyl groups were esteriﬁed by reaction with

the C-terminal Boc-amino acid and DCC (10 equiv.) in the

presence of 0.5 equiv. of DMAP for 2 h. The coupling yield

was determined from the relative proportion [C-terminal

residue]/[internal reference amino acid] after acid hydroly-

sis and amino acid analysis (typical values: 0.93–0.97, 0.79

in the case of 9), and unreacted hydroxyl groups were

capped by reaction with benzoyl chloride (or acetic

anhydride) and pyridine.

0

4.2. Ac-Tyr(p³dTTTCAGAAAATCTAG)-Leu-Asp-

Pro-Arg-Ile-Thr-Val-OH (1)

Peptide-resins: Ac-Tyr-Leu-Asp(OFm)-Pro-Arg(X)-Ile-

Thr(Ac)-Val-O-HMFS-Phe-MBHA-PS (X¼H/Fmoc₂,

0.14–0.15 mmol/g). Amino acid analysis after acid

hydrolysis: X¼H: Asp 0.97, Thr 0.36, Val 1.66, Ile 0.78,

Leu 1.01, Tyr 1.00, Arg 1.01, Pro 1.00; X¼Fmoc₂: Asp

1.02, Thr 0.39, Val 1.68, Ile 0.84, Leu 1.03, Tyr 1.02, Arg

1.08, Pro 0.86. Crude nucleopeptide was obtained by

overnight ammonia treatment of the two nucleopeptide-

resins at 558C. The product obtained from X¼Fmoc₂was

puriﬁed by semi-preparative reversed-phase HPLC (10–

40% of B in 30 min).

Amino acid composition after acid hydrolysis: Asp 0.41,

Thr 0.44, Pro 1.28, Val 1.17, Ile 0.82, Leu 1.33, Tyr 1.47,

Arg 1.09. Nucleoside composition after enzymatic diges-

tion: dC/dG/T/dA: 2.12/2.05/4.95/5.92. Compound 1:

C₁₉₅H₂₆₀N₆₇O₁₀₃P₁₅, calcd mass: 5655.19 (average).

MALDI-TOF MS (2,4,6-THAP, negative mode, linear),

m/z: 5651.6 [M2H]²; ES MS (negative mode), m/z: 1412.5

[M24H]⁴², 1130.2 [M25H]⁵², 941.6 [M26H]⁶², 807.2

[M27H]⁷², mass found: 5652.

The remaining amino acids of the peptide sequence were

incorporated following the standard procedures for solid-

phase peptide assembly (3 equiv. of Boc-amino acid and

DCC and 1–1.5 h reaction time were used for the coupling

step), with the addition of an equimolar amount of HOBt in

the incorporation of the homoserine⁵⁹and Arg(Fmoc)₂

derivatives. When arginine was protected by protonation, a

two-fold excess of HOBt was added during the coupling

step, and, after the incorporation of this residue, the TFA-

mediated removal of the Boc group was followed by

neutralization with a 5% DIEA solution in DCM and

treatment with a 0.5 M solution of HOBt in DMF

(4£1 min). The N ^a-Boc derivative of the linking amino

acid was incorporated with the hydroxyl group unprotected.

Acylation of the N-terminal amine group was carried out by

reaction with acetic anhydride. The homoserine derivative

Boc-Hse(DMT)-O²HTEA^þwas prepared as previously

described.⁵⁹

0

4.3. Ac-Tyr(p³dTTTCAGAA)-Leu-Asp-Pro-Arg-Ile-

Thr-Val-OH (2)

Peptide-resins: the same as above. Ammonia ﬁnal deprotec-

tion treatment: overnight, 558C. The product obtained from

X¼Fmoc₂was puriﬁed by semi-preparative reversed-phase

HPLC (10–40% of B in 30 min).

Amino acid composition after acid hydrolysis: Asp 0.57,

Thr 0.66, Pro 1.83, Val 1.21, Ile 0.71, Leu 0.80, Tyr 1.31,

Arg 0.91. Nucleoside composition after enzymatic diges-

tion: dC/dG/T/dA: 1.06/1.02/2.91/3.05. Compound 2:

C₁₂₆H₁₇₄N₄₀O₆₂P₈, calcd mass: 3488.78 (average).

MALDI-TOF MS (2,4,6-THAP, negative mode, linear),

m/z: 3487.0 [M2H]²; ES MS (negative mode), m/z: 1743.1

[M22H]²², 1162.0 [M23H]³², 871.3 [M24H]⁴², 697.0

[M25H]⁵², mass found: 3487.

Oligonucleotide elongation was carried out (5–10 mmol-

scale) following standard phosphite triester procedures with

the minor modiﬁcations that render this methodology

compatible with the use of polystyrene supports.⁶₀⁰

0.15–0.2 M solutions of commercially available 3 -

phosphoramidite derivatives (5⁰-DMT-N-P(OCNE)NiPr₂,

N¼T, A^Bz, C^Bz, G^iBu) in anhydrous DCM and 0.7–0.8 M

solutions of tetrazole in anhydrous THF were used for the

coupling step (30 min coupling time for the ﬁrst nucleo-

side and 15 min for the others). 1 M t-BuOOH was

always used for the oxidation of phosphites to phosphates

during the oligonucleotide elongation. The 5⁰-DMT group

was removed from the nucleopeptide-resin. Nucleo-

peptides were detached from the solid support by

0

4.4. Ac-Asn-Phe-Val-Ser(p³TTTT)-Gly-OH (3) and Ac-

0

Asn-Phe-Val-Ser(p³TTTT)-Gly-NH₂(4)

Peptide-resin: Ac-Asn-Phe-Val-Ser-Gly-O-HMFS-Leu-

MBHA-resin (0.21 mmol/g). Amino acid analysis after

acid hydrolysis: Asp 1.11, Ser 0.47, Gly 1.01, Val 1.03, Phe

0.86. Ammonia ﬁnal deprotection treatment: 6 h, rt.

Puriﬁcation: MPLC (glass column, 22£2 cm, Vydac

C-18), gradient from 0 to 30% of B, 600 mL of each

solvent. Isolated products: TpTpTpTp (C₄₀H₅₄N₈O₂₉P₄,

calcd mass: 1234.8 (average), 1234.2 (monoisotopic):

MALDI-TOF MS (2,4,6-THAP, negative mode, reﬂectron),

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6976

0

m/z: 1233.5 [M2H]². Ac-Asn-Phe-Val-Ser(p³dTTTT)-

Gly-OH (3, C₆₅H₈₈N₁₄O₃₇P₄, calcd mass: 1781.4

(average)): MALDI-TOF MS (2,4,6-THAP, negative

mode,₀reﬂectron), m/z: 1779.2 [M2H]². Ac-Asn-Phe-Val-

Ser(p³dTTTT)-Gly-NH₂(4, C₆₅H₈₉N₁₅O₃₆P₄, calcd mass:

1780.4 (average)): MALDI-TOF MS (2,4,6-THAP, nega-

tive mode, reﬂectron), m/z: 1778.1 [M2H]².

4.7. Ac-Cys-Gly-Tyr(p³dACTAGT)-Pro-OH (9)

Cysteine derivative used: Ac-Cys(S–t Bu)-OH. Peptide-

resin: Ac-Cys(S–t Bu)-Gly-Tyr-Pro-O-HMFS-Phe-MBHA-

resin (0.19 mmol/g). Final deprotection conditions: conc.

aq. ammonia/[1 M DTT in dioxane] 1:1, 7 h, 558C.

Puriﬁcation: (i) gel ﬁltration (Sephadex G-15, 0.05 M

ammonium acetate) to remove the excess of DTT; (ii)

semi-preparative HPLC on a Hamilton PRP-1 column

4.5. H-₀Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-

Tyr(p³dACGT)-Leu-Asp-Pro-OH (5)

(305£7 mm,

10 mm,

1.5 mL/min),

A¼0.01 M

ammonium acetate, B¼acetonitrile/H₂O 1:1, linear

gradient from 5 to 35% of B in 30 min. The two

main products isolated (t_R¼16.2 and 17.3 min) were the

two conformational isomers of the Tyr-Pro peptide

bond in 9. A third minor product was also isolated

(t_R¼19.5 min). Treatment of 9 with a 10% aqueous

DMSO solution afforded, almost quantitatively, the

product eluting at 19.5 min, which was characterized

as the disulﬁde dimer form of 9. This product was

puriﬁed using the same conditions as for the nucleo-

peptide monomer.

Peptide-resin: Fmoc-Ile-Ala-Leu-Gly-Thr(Ac)-Ser(Ac)-

Lys(Tfa)-Leu-Asn-Tyr-Leu-Asp(OFm)-Pro-O-HMFS-Phe-

MBHA-resin (0.13 mmol/g). To prevent diketopiperazine

formation from H-Asp-Pro-resin, the Boc group of Boc-

Asp(OFm)-Pro-resin was removed as usual, and Boc-

leucine (5 equiv.) was coupled to the dipeptide-resin

(triﬂuoroacetate salt) by reaction with PyAOP (7-azabenzo-

triazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexaﬂuoro-

phosphate) and DIEA (10 equiv.). The N-terminal amino

acid was incorporated as Fmoc-derivative. Amino acid

analysis after acid hydrolysis: Asp 2.15, Thr 0.65, Ser 0.33,

Gly 0.87, Ala 0.95, Ile 0.60, Leu 1.97, Tyr 1.09, Lys 1.02,

Pro 1.04. HPLC analysis of crude nucleopeptide after

overnight ammonia deprotection at 558C (10–40% of B in

30 min): t_R¼11.7 (<20%, impurity 1, 7), 24.5 (<30%, 5)

and 25.5 min (<30%, impurity 2, 8). Puriﬁcation: HPLC

(10–40% of B in 30 min).

Compound 9: calcd mass for C₈₀H₁₀₂N₂₆O₄₃P₆S: 2333.7

(average). Nucleoside composition after enzymatic

digestion: dC/dG7T7dA: 1.31/1.10/2.10/1.49. MALDI-

TOF MS (2,4,6-THAP, negative mode, reﬂectron): m/z

2332.3 [M2H]²; electrospray MS: m/z 776.9 [M23H]³²

1165.3 [M22H]²², calcd mass: 2333.24^0.58.

,

0

Compound 5: calcd mass for C₁₀₃H₁₅₄N₃₀O₄₄P₄: 2640.4

(average). Amino acid composition after enzymatic diges-

tion: Asp 0.88, Thr 0.73, Ser 1.05, Asn 0.79, Pro 1.72, Gly

1.75, Ala 1.51, Ile 1.11, Leu 1.52, Tyr 1.09, Lys 0.90.

Nucleoside composition after enzymatic digestion:

dC/dG/T/dA: 1.25/1.19/1.06/0.51. MALDI-TOF MS

(2,4,6-THAP, negative mode, reﬂectron): m/z: 2639.1

[M2H]².

4.8. {Ac-Cys-Gly-Tyr(p³dACTAGT)-Pro-OH}₂[S–S]

Disulﬁde dimer of 9: calcd mass for C₁₆₀H₂₀₂N₅₂O₈₆P₁₂S₂:

4665.4 (average). MALDI-TOF MS (2,4,6-THAP, negative

mode, linear): m/z 4670.5 [M2H]²; electrospray MS: m/z

663.9 [M27H]⁷², 776.7 [M26H]⁶², 1166.1 [M24H]⁴²

calcd mass: 4663.8^5.4.

,

0

4.9. Ac-Cys-Gly-Ala-Hse(p³dGCATGC)-Ala-OH (10)

0

Compound 7: H-Tyr(p³dACGT)-Leu-Asp-Pro-OH. Calcd

mass C₆₃H₈₃N₁₉O₃₂P₄: 1742.4 (average). MALDI-TOF MS

Cysteine derivative used: Boc-Cys(S–t Bu)-OH. The

N-terminal was acetylated by reaction with AcOH

(5 equiv.) and DCC (5 equiv.) for 30 min. Peptide-resin:

Ac-Cys(S–t Bu)-Gly-Ala-Hse-Ala-O-HMFS-Leu-MBHA-

resin (0.20 mmol/g). The oligonucleotide chain was

assembled using the standard 3⁰-phosphoramidite

derivatives of T, A^Bz, C^Acand G^Dmf(Glen Res. products,

Eurogentec, BE). Deprotection and cleavage: treatment

with conc. aq. ammonia/[1 M DTT in dioxane] 1:1, 6 h,

rt. DTT was removed by gel ﬁltration (Sephadex G-15,

0.05 M ammonium acetate) and pure 10 was isolated

after MPLC puriﬁcation (glass column, 22£2 cm, Vydac

C-18, gradient from 0 to 30% of B, 600 mL of each

solvent). 10 was dissolved (ca. 50 mM) in 60 mM

triethylammonium acetate buffer, pH¼8, and air-oxidized

by vigorous magnetic stirring for 2 days at rt. The

disulﬁde dimer of 10 was puriﬁed by semi-preparative

HPLC (5–25% of B in 40 min) followed by several runs

of analytical HPLC (5–25% of B in 40 min, analytical

Kromasil column).

(negative mode, reﬂectron), m/z: 1740.6 [M2H]².

Compound 8: MALDI-TOF MS (2,4,6-THAP, negative

mode, reﬂectron), m/z: 2639.2 [M2H]².

4.6. H-₀Ile-Ala-Leu-Gly-Thr-Ser-Lys-Leu-Asn-

Tyr(p³dTTTCAGAAAATCTAG)-Leu-Asp-Pro-OH (6)

Peptide-resin: the same as above. Final ammonia deprotec-

tion: overnight, 558C. Puriﬁcation: preparative PAGE,

followed by HPLC separation (10–40% of B in 30 min)

of the two products contained in the main electrophoretic

band (t_R¼13.6 and 14.2 min).

Compound 6: calcd mass for C₂₁₂H₂₉₀N₇₁O₁₀₉P₁₅: 6041.6

(average). Amino acid analysis after enzymatic

digestion: Asp 0.65, Thr 0.71, Ser 1.09, Asn 1.13, Pro

1.92, Gly 2.00, Ala 1.36, Ile 0.78, Leu 1.06, Tyr 1.45, Lys

0.85. Nucleoside composition after enzymatic digestion:

dC/dG/T/dA: 3.05/2.04/4.23/5.77. MALDI-TOF MS (2,4,6-

THAP, negative mode): m/z: 6036.3 [M2H]². Impurity:

MALDI-TOF MS (2,4,6-THAP, negative mode, linear):

m/z: 6036.9.

Compound 10: calcd mass for C₇₅H₁₀₂N₂₈O₄₄P₆S: 2317.7

(average). MALDI-TOF MS (2,4,6-THAP, negative mode,

reﬂectron): m/z 2315.5 [M2H]².

´

L. Debethune et al. / Tetrahedron 58 (2002) 6965–6978

6977

4.10. Ac-Cys(S–t Bu)-Gly-Ala-Hse-Ala-OH

phase obtained after extraction of the reaction mixture with

0.1 M HCl (2£) and that obtained from reextraction of the

aqueous phase with methylene chloride was dried over

sodium sulfate, ﬁltered and evaporated to dryness. Pure Ac-

Cys(S–t Bu)-OH was obtained after recrystallization from

Obtained from cleavage of an aliquot of peptide-resin with

conc. aq. ammonia/dioxane 1:1, 4 h, rt): calcd mass for

C₂₁H₃₇N₅O₈S₂: 551.7 (average). MALDI-TOF MS (2,5-

dihydroxybenzoic acid, negative mode, reﬂectron): m/z

550.6 [M2H]².

1

hexanes/dichloromethane (76% yield): mp 176–1778C; H

NMR (CDCl₃, 200 MHz): d 1.9 (s, 9H), 2.1 (s, 3H), 3.1 (m,

CH₂), 3.7 (m, CH). Calcd mass for C₉H₁₇NO₃S₂: 251.4

(average), CI MS (NH₃, positive mode): m/z 269 [Mþ18]^þ,

286 [Mþ35]^þ.

4.11. Ac-Cys-Gly-Ala-Hse-Ala-OH

Obtained from cleavage of an aliquot of peptide-resin with

conc. aq. ammonia/[1 M DTT in dioxane] 1:1, 5 h, rt, and

removal of DTT by extraction with ethyl acetate): calcd

mass for C₁₇H₂₉N₅O₈S: 463.5 (average). MALDI-TOF MS

(2,5-dihydroxybenzoic acid, positive mode, reﬂectron): m/z

465.2 [MþH]^þ, 487.3 [MþNa]^þ, 503.3 [MþK]^þ.

4.16. Boc-Cys(S–tBu)-OH

H-Cys(S–t Bu)-OH (Novabiochem, 0.5 g, 2.4 mmol) was

suspended in 15 mL of a 2:1 dioxane/H₂O mixture, and the

pH was adjusted to 9 by addition of 1 M NaOH. After

addition of 0.60 g of di-tert-butyldicarbonate (Boc₂O,

2.74 mmol), the reaction was kept overnight at rt (magnetic

stirring). After extraction with hexanes (2£50 mL), the

aqueous phase was chilled and acidiﬁed to pH 2–3 by

addition of 1 M HCl. The mixture was extracted with ethyl

acetate (3£50 mL), and the combined organic phases were

washed with H₂O (2£25 mL), dried over sodium sulfate,

ﬁltered and evaporated to dryness. Pure Boc-Cys(S–t Bu)-

OH was obtained (80% yield) without further puriﬁcation:

mp 119–1208C; ¹H NMR (CDCl₃, 200 MHz): d 5.4 (d, 1H),

4.6 (m, 1H), 3.2 (m, 2H), 1.5 (s, 3H), 1.3 (s, 3H). Calcd mass

for C₁₂H₂₃NO₄S₂: 309.5 (average), FAB MS (4-nitrobenzyl

alcohol, positive mode): m/z 309.4 [MþH]^þ, 332.4

[MþNa]^þ.

0

4.12. {Ac-Cys-Gly-Ala-Hse(p³dGCATGC)-Ala-OH}₂

[S–S]

Disulﬁde dimer of 10: calcd mass for C₁₅₀H₂₀₂N₅₆O₈₈P₁₂S₂:

4633.4 (average). Nucleoside composition after enzymatic

digestion: dC 2.2, dG 0.7, T 2.2, dA 1.0.MALDI-TOF MS

(2,4,6-THAP, negative mode, linear): m/z 4628.9 [M2H]²;

2316.5 [M22H]²²

.

0

4.13. Ac-Gly-Ser(p³T₅)-Leu-Ala-Val-OH, 11, and Ac-

0

Gly-Thr(p³T₅)-Leu-Ala-Val-OH (12)

Peptide-resins: Ac-Gly-Xxx-Leu-Ala-Val-O-HMFS-Phe-

resin, Xxx¼Ser, Thr (0.19 mmol/g). Final ammonia depro-

tection treatment: 2 h, rt. Puriﬁcation: anal HPLC, linear

gradient from 5 to 35% of B in 30 min.

Acknowledgments

Compound 11: calcd mass for C₇₁H₁₀₂N₁₅O₄₃P₅2008.53

(average). MALDI-TOF MS (2,4,6-THAP, negative mode,

reﬂectron) m/z 2006.50 [M2H]².

´

Laurent Debethune was a recipient fellow of the Generalitat

de Catalunya, and Vicente Marchan of the Universitat de

Barcelona. This work was supported by funds from the

Spanish MEC (grants PB97-0941-C02-01 and BQU 3693-

C02-01) and the Generalitat de Catalunya (2000SGR18,

´

Compound 12: calcd mass for C₇₂H₁₀₄N₁₅O₄₃P₅2022.56

(average). MALDI-TOF MS (2,4,6-THAP, negative mode,

reﬂectron) m/z 2020.50 [M2H]².

`

2001SGR49 and Centre de Referencia de Biotecnologia).

0

4.14. Ac-Gly-Tyr(p³T₅)-Leu-Ala-Val-OH (13), and Ac-

0

Gly-Tyr(p⁵T₅)-Leu-Ala-Gly-OH (14)

References

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35% of B in 30 min.

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Article Doi

Towards nucleopeptides containing any trifunctional amino acid (II)

DOI: 10.1016/S0040-4020(02)00793-7

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Article abstract of DOI:10.1016/S0040-4020(02)00793-7

Full text of DOI:10.1016/S0040-4020(02)00793-7

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