Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques

372 Pages·1994·20.174 MB·English

by  Sudhir Agrawal

Protecting Groups
in Oligonucleotide Synthesis
Etienne Sonveaux
1. Introduction
A biopolymer is synthesized by assembling monomeric or oligo-
merit blocks. Each block features at least a nucleophilic and an elec-
trophilic function, i.e., the a-amino and the carboxylic functions for
peptides, the S-OH and the 3’-function (phosphate, phosphoramidite,
or phosphonate), for n&leotides. The nucleophilic and electrophilic
sites are linked together at the coupling step. Protection is a necessity.
It guarantees the chemoselectivity of coupling and the solubility of
synthons in organic solvents.
There are two classes of protecting groups: persistent and transient.
The persistent protections remain on the biopolymer during all the
synthesis. They are cleaved at the very end. They cap the functions of
the aglycone residue of nucleotides, or of the side chains of amino
acids in peptide synthesis. They also cap the phosphate oxygen of
oligonucleotides. The transient protections block the functions to be
coupled at a given time of the synthesis. They are specifically cleaved
before each coupling.
When the synthesis is performed on a solid support, the first mono-
mer of a hundred-mer has to survive to a hundred cleavages of a tran-
sient protecting group. The yield of successful removal is thus as
limiting ast he coupling yield. This is also true for the final deprotection.
If each monomeric unit is only 90% deprotected, the yield of a dimer
of correct structure is g2%, of a trimer g3/10%, and of a n-mer 9V10”-2%.
From Methods m Molecular Biology, Vol. 26 Protocols for O//gonuc/eot/de Conpgates
Edited by. S. Agrawal CopyrIght 01994 Humana Press Inc., Totowa, NJ
1
2 Sonveaux
Yields drop dramatically with length. Paradoxically, the crucial prob-
lem of protection is thus deprotection. Good results obtained with a
protection strategy on small sequencesd o not guaranteet hat the method
is viable. The discriminating test is the successi n obtaining high yields
of long oligomers.
In oligonucleotide synthesis, the academic research is nowadays
moving from DNA synthesis to the synthesis of RNA and modified
DNA/RNA structures. As functions and types of aglycone residues
entering oligonucleotide synthesis diversify, protection strategies have
to be adapted. That is why this review may be useful.
Its content is as follows. The persistent protections of the nucleic
basesa nd of the 2’-OH of ribonucleotides are considered first. Both are
usually introduced at the beginning of the synthesis. The transient
protection of the S-OH is then discussed. This function is indeed
capped before the phosphorylation or phosphitylation of the synthons.
The last paragraph describes the protections of phosphorus. This last
topic is much related to coupling strategies. The reader is thus invited
to consult the other parts of this book to embrace the whole field.
2. Protection of the Heterocyclic Bases
and Protocols for Oligonucleotides
and Analogs
The protecting groups that have been proposed for the aglycone
residues are presented in Tables 1-5. The most useful protections are
briefly described in the following paragraphs.
2.1, Thymidine and Uridine
It is possible to synthesize oligo DNA or RNA by one of the three
classical methods (phosphotriester, phosphoramidite, phosphonate
stategies) without protecting these residues. However, the N-3 nitro-
gen being acidic (T, pK, = 9.79) (I), a certain amount of the anionic
form is present in basic media. In these conditions, the thymine and
uracil residues react with electrophiles like alkylating agents (2,3)
(inter alia, the methyl phosphate of the internucleotidic bond in one of
thephosphoramidite strategies( 3-5), carbodiimides (6), activatedphos-
phates (7-14) and sulfonates (15,16), bis(diisopropylamino) meth-
oxyphosphine (I7), trimethylsilyl chloride (18), and acid chlorides.
Protecting Groups in Synthesis 3
The instable O-4 phosphorylated products undergo a nucleophilic
attack on C-4 by nucleophiles usually present in the medium, like 1,2,4-
triazole, 3-nitro-1,2,4-triazole, l-hydroxybenzotriazole, N-methylim-
idazole or pyridine. In the case of acylation, the N-3 acylated derivative
is usually obtained (19). It is the thermodynamic product. The O-4
acylated derivative, accessible by PTC, spontaneously rearranges to
the N-3 acylated isomer (16).
In the conditions of a normal oligonucleotide synthesis, the contact
times with electrophiles are short and these side reactions are limited
(20). Thyrnidine is less sensitive than uridine (15), and is usually not
protected (21). Some side reactions are reversed (17), either during syn-
thesis, or by an adequate final deprotection (15,22) (e.g., oximate). The
side reactions of uracil in phosphotriester synthesis may be a serious
nuisance (22). They have been carefully studied by Reese( 7,15,23,24).
Two protecting groups are well established: the N-3 anisoyl and the
4-(2,4-dimethylphenyloxy) derivatives I and 4 (Table 1).
The N-3 anisoyl compound is a little more resistant to nucleophilic
attack than the N-3 benzoyl(2526). It is introduced, by PTC (16) or
in homogeneous conditions (27) (See also ref. 28), by selective N-3
acylation of 3’,5’-0-( 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine,
followed by the protection of the 2’-OH and desilylation. The 2’-O-
(tetrahydropyran-2-yl)-3’,5’-O-( 1,1,3,3-tetraisopropyldisiloxane- 1,3-
diyl)uridine is also easily anisoylable on N-3 (29). The selective N-3
acylation is possible by the so called Jones’ method (in situ protection
of the 5’,3’, and 2’-0 by trimethylsilyl chloride during acylation, fol-
lowed by the hydrolysis of the silyl ethers in the workup) (26,28,30).
Protection 4 is introduced by reaction of 2’-0-(4-methoxytetrahy-
dropyran-4-yl)-3’,5’-0-dimethoxyacetyluridine with diphenylphos-
phochloridate and 3-nitro- 1,2,4-triazole in pyridine, followed by a
treatment with a phenol and triethylamine. The labile 3’ and 5’-0 acyl
groups are cleaved by 8M ammonia in methanol (23). Care is needed
because this type of uracil protection may be substituted by ammonia
to give cytosine (31,32).
2.2. Cytidine and Deoxycytidine
These nucleosides are usually protected by acylation on N-4. Cyto-
sine is the most basic of the aglycone residues (dC, pK, = 4.25). It is
also the most nucleophilic. The rate of acylation decreasesi n the order:
C(N-4) > OH > A(N-6) > G(N-2) (52).
4 Sonveaux
Table 1
Protections of Thymine and Uracil Residuesa
Properties of the tabulated protecting groups
(solvent mixtures are expressed in volumic proportions)
1. Resists to K&O3 0.2M (short time); TBAF lM/THF; chromatography on silica
(CHCl&HsOH); NEts/pyridine. It 1s cleaved by NH, cc in CH,OH/H,O;
n-butylamine 0 04MICHsOH; TBAF/pyridine/HzO (8*1:1). See refs.
25,26,28,29,33-35.
2. Resists to CHsCOOH 80% (2 h); TBAF/THF; ZnBrz lM, pyndinelt-butylamine/
Hz0 (8:l:l) (24 h). It is cleaved by cont. NI-Is in CHsOH (9:l) (T,., 3 h). See refs.
36,37.
3. Resists to t-butylamine 0.14WCHsOH (20 min). It is cleaved by Znlpyndine.
See refs 16,38,39.
4. Resists to K&JO3 0.2M, morpholme 0 05M, NH3 8MICHsOH (~15 mm). It is
cleaved by oximate. See refs. 23,24,40.
5. Is cleaved by oximate. See refs. 31,41.
6. Resists to NHs/CHsOH. It is cleaved by DBU OSMlpyridine. See refs 42,43,45
For possible side reactions involving the 0-Calkylthymme residues, see ref. 44.
7. Resists to CHsCOOH 80%, cont. NHs/CHsOH. It is cleaved by I, O.lM/THF/
collidine&O. See refs 16,46.
8. Resists to CHsCOOH 80%. It is cleaved by pyridine/H,O; TBAF 0 2MTHF
See ref. 47.
9. Resists to CHsCOOH 80%; HCl pH 2; oximate; NaOH 0.2M. It is cleaved by
NH3 cc/pyridine. (50°C) (C6Hs),C+ BFJ-Does not cleave well. See refs. 28,48,49.
10 Resists to NH2-NH2 1M in pyridine/CH$OOH (3: 1). It is cleaved by cont. NH,
See ref. 50.
11. Resists to cont. NH,; DBU OSWpyridine (1 h); TBAF 1M: THF. It is cleaved
by cont. NHs, 50°C (2.5 h) See refs. 21,51.
?Yeeo pposite page for corresponding structure.
It is possible to specifically acylate the exocyclic amino function of
cytidine or deoxycytidine, under controlled conditions: activated esters
(53,54), acid anhydrides (55,56), some acid chlorides (57), mixed anhy-
drides (58), l-alkyloxycarbonylbenzotriazoles (43,59), carboxylic acids
activated by EEDQ(60) and thioacetic acid (61,62) have been used for
this purpose.
The rate of N-deacylation by ammonia or sodium hydroxyde decreases
in the order C > A > G (63-69) (acylated adeninea nd guanine residues of
nucleosides may loose a proton in basic media, rendering them more
resistant to nucleophilic attack). It is to be noticed that nucleophiles may
attack on C-4, displacing the acylated nitrogen. The N-4 acylcytosine
residuei s deaminatedt o thec orrespondingu racil by hot acetic acid (70,71).
Similarly, n-butylamine gives the N-4-butyl derivative (72).
Protecting Groups in Synthesis 5
Table 1
Structures
R=H,CH,
Ar=-(=J, -@b
R=H,CH3
r@m3
R=H,CH,
u
0
a K! NO
I
R=H,CH,
6 Sonveaux
As a result of easy deacylation under acidic or basic catalysis, the
protection of cytidine and deoxycytidine need some tuning. The halo-
acetyl groups are useless, being to labiles (73). The N-4 acetyl protec-
tion is fragile(68,73,74), (HCl 0. lM, tii2 = 2 h; KOH O.OlM, tiiZ = 6 h;
NaOH 0.2M/CH30H (1: l), RT, tu2 = 0.2 min; cont. NH,/C,HSOH
(1: l), RT, t1,2 = 10 min). It is cleaved by boiling ethanol (61). The p-
methoxybenzoyl group, more resistant to nucleophilic attack, was
formerly used with the phosphodiester method (75). The most resistent
acyl group is o,p-dimethylbenzoyl (68). The benzoyl group is rou-
tinely used nowadays, although it is less robust (70) (e.g., deacylation
of the 2’, 3’ or 5’ 0-silylated derivatives by methanol; refs. 76,77);
deacylation by hydrazine in pyridine/acetic acid (78). It is cleaved
without problems by ammonia at room temperature (66) (NH3 9M, t,,
= 16 min (70); NH3 5iWdioxane (1: l), complete cleavage in 6.5
&63); NH, 29%/pyridine (80:20), tu2 = 2 h (64,6.5).
On a preparative point of view, the acylation method of Jones-Sung-
Narang (in situ protection of the alcohol functions by trimethylsilyl
chloride) (I#, 79) is particularly practical. It is also possible to ZVO, -
peracylate the nucleoside and to selectively cleave the ester functions
afterwards (23,71). Deoxycytidine has been selectively ZV-4-benzoyl-
ated on a multikilogram scale by simply shaking the nucleoside with
one equivalent of benzoic anhydride in DMF (56). If a more easily
cleavable protection is necessary, one would prefer theZV-4-isobutyryl
group (64,65) (cleaved by ammonia 28%, RT, 5 h) (80,81)
2.3. Adenosine and Deoxyadenosine
The protection of these nucleotides requires a special comment,
because the chemical stability of the nucleoside is altered by the pro-
tection of the exocyclic amino function of the nucleic base. The dis-
cussion thus starts with an account of the encountered problems.
2.3.1. Stability Toward Acids
Purine nucleosides can loose their purine residue in acidic condi-
tions, leaving an unsubstituted sugar (apurinic site). This reaction is a
nuisance because oligonucleotides are repeatedly submitted to acid
detritylation during routine synthesis on a solid support.
The mechanism of acid depurination involves a rapid preequilibrium
of protonation (or deprotonation) of the purine residue, followed by
Protecting Groups in Synthesis 7
Table 2
Protections on N-4 of the Cytosine Residuea
List of methods of cleavage and relevant references
(solvent mixtures are expressed in volumlc proportions)
1. Cont. NHs, TY Refs. 56,60,74.
2. Cont. NH,, TV Refs 64,65,80.
3a. Cont. NH,, Tp Refs 14,56,60,71,79. This protection is selectively removed by
NH2-NH2 in pyridine/acetic acid (4.1) Refs. 150-1.52
3b. Cont. NHs, T, Ref. 23.
3c. Cone NH,, TV Refs. 60,71,82.
3d Cont. NH,, 50% Ref 68.
3e. Cone NHs, 50°C Ref. 57.
4 Cone NH,, 50°C Ref. 83.
5 TBAF/pyridine/H,O (1: 1) Ref 84.
6. K&O:, O.OSM/CH,OH, T, or cont. NHs, T, Ref 85
7. NH2-NH2 0 SM/pyridine/CH&OOH (4: 1) Ref. 86
8. Cone NH,, T, Ref 78.
9 NEtdpyridine or oxrmate. Refs 63,87-90.
10 DBU Refs. 43JP
11. Co(I)/phthalocyanin anion Refs. 39,9X
12. Pd/C/1,3-cyclohexadiene/EtOAc/EtOH. Ref. 92
13. Pd(0)/P(C6Hs)s/n-butylamine/HCOOH/THF. Ref 93.
14. p-Methylthiophenolate/pyridine, Tr Ref 94.
15 NHs, 50°C or NH40Ac/conc. NHs, 50°C. Refs. 9.5,96.
16. NaOH O.SM/pyridine (1: 1) Ref. 97.
17. HCl O.OlM. Refs. 50,98-100.
YSeep ages 8 and 9 for correspondmg structures.
the cleavage of the glycosidic linkage, that is the rate-determining step
(101-107). The unstable species are the N-7 protonated derivatives
(Scheme 1). Both purine nucleosides (or deoxynucleosides) depurinate
at about the same rate (104). A carbocation being generated at C- 1’ in
the rate-determining step, electron withdrawing substituents on the
sugar reduce the tendency to depurinate. Accordingly, adenosine depur-
inates 1200 times more slowly than deoxyadenosine (108), and deoxy-
adenosine 3’,5’-diphosphate (in the middle of an oligonucleotide
sequence) depurinates less easily than the nucleotide (109). That is
why people usually avoid starting a sequence with deoxyadenosine
directly attached to the solid support with the 3’-succinate link (110).
Depurination is not a problem in the ribo series (except for hyper-
modified residues like wyosine [111]). In the deoxyribo series, the
8 Sonveaux
Table 2
Structures
1
1 0
0 P
h \ / c-
-sQ - f+3
4
R2 R3
H H
;;HH
c CH30 H H
d H CH,
e ;' c1H30 H
most sensitive residue is N-6-acylated deoxyadenosine. The site of
first protonation of N-6-acylated deoxyadenosine is N-7 (pK, = 1.75)
(I12,113). This quickly depurinating form is thus readily accessible.
On the contrary, for deoxyadenosine itself, the site of first protonation
is N- 1(114), and the N- 1, N-7 diprotonated form is accessible only at
low pH (pKa1 = - 1.48 (115) pKa2= 3.65 (I, 112).T he acidic depurination
of deoxyadenosine is thus limited by the access to the N-7 protonated
form, but this is not the case for N-6-acylated derivatives. N-6 benzoyl
Protecting Groups in Synthesis 9
Table 2
f%uctures CcontlnuedJ
14
Ll
-
s-
/
4
NO2
R1=H, R2=-0-W
0 RI =R2 = -o-cl+j
dA depurinates about ten times more rapidly than N-2-isobutyryl dG
(109,116). A diacylation on N-6 corrects this effect by lowering the
pK, (pK, << 1.4) (112,117). A N-6 amidine protection (95,118,119)
presumably does not shift the first protonation site of deoxyadenosine
from N- 1 to N-7, but this protection is cleaved in aqueous acidic con-
ditions at a rate similar to the depurination rate (112).
Sonveaux
-
OH OH
OH
y
HO
OH
0
+H20,-H
HO
OH
Scheme 1. Mechanism of acidic depurination.
When a partially depurinated oligomer is exposed to the strongly
basic conditions of final deprotection, the chain is cleaved at the apurinic
site by p-elimination (120,121). The 5’-dimethoxytrityl oligonucleo-
tides usually obtained by an automatic DNA synthesis are thus con-
taminated by 5’-DMTr truncated sequences difficult to remove by
reversed phase chromatography. That this is really the case has been
proved by Horn et al. (122,123), who found a method to cleave apurinic
sites directly on the solid support, without detaching much of the full
length oligonucleotide (1M lysine.HCl, pH 9, 6O”C, 90 min). Their
protocol gave long oligonucleotides of high purity (98-l 18-mers).