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New Hydrophobic Protecting Groups for the Chemical Synthesis of Oligonucleotides.

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of the complexes (21, and their M-M and M-E bond
lengths show the same trend as in (2a) and (2b).
The intrinsic novelty of the clusters (2) is, therefore, their
systematic preparation. Since numerous functional dinuclear complexes having the butterfly structure shown in
(la) and (lb) and suitable simple carbonylmetals for reaction exist, the route described here to planar tetranuclear
clusters should not be merely limited to (2a) and (2b).
MgBr
8
l2a -el
[la-el
Thymidine
Procedure
Equimolar amounts (1-2 mmol) of (la) o r (Ib) and
C O ~ ( C Oare
) ~ added to 50 ml of hexane. For the formation
of (2a) the mixture is stored in the dark for 20 d at room
temperature, then chromatographed on silica gel, and finally the product obtained from the third fraction is crystallized from hexane at - 30 “C. The complex (2b) precipitates from the reaction mixture in analytically pure form
after 12 h at 0°C. The yields of(2a) (m.p. 134”C, dec.) and
(2b) (dec. at 100-200°C without recognizable m.p.) are
20-25%.
Received: October 27, 1980 [Z 821 b IE]
German version: Angew. Chem. 93. 715 (1981)
[ I ] D . Seyferth, Adv. Organomet. Chem. 14, 97 (1976).
[2] H . Vahrenkamp, Angew. Chem. 87, 363 (1975); Angew. Chem. Int. Ed.
Engl. 14. 322 (1975).
[3] F. Richter. H . Beurich, H . Vahrenkamp. J. Organomet. Chem. 166, C 5
(1979).
[4] 6 . K . Teo. M . B. Hall, R. F. Fenske, L. F. Dahl, Inorg. Chem. 14. 3103
(1975).
[S] (2aj: triclinic, Pi, 2 = 2 , a=962.7(1), b = 1567.8(2), c=960.2(2) pm,
a=91.04(1), B= 109.05(1), y=84.83(1)”; 3190 reflections, R=0.059.(26): monoclinic, P2,/c, Z = 4 , a = 1615.4(5), b=655.6(2), c = 1872.2(5)
pm, B= 115.06(2)”; 2946 reflections, R=0.051.
161 P. Chin;, B T. Heaton. Top. Cum. Chem. 71. 1 (1977).
171 R. C Ryan. L. F. Dahl, J. Am. Chem. SOC.97, 6904 (1975).
HO
As expected, the R,-values of the 5’-0-(4-alkoxytrity1)thymidines (3a-e) on silica gel are somewhat higher
and o n RP-2 somewhat lower than the R,-values of 5 ’ 0
(4,4’-dimethoxytrity1)thymidine. These changes reflect the
reduction in total polarity. The behavior of (3a-e) upon
R P (reversed phase) chromatography on a C,,-phase is,
however, different: here, an apparently specific interaction
1
New Hydrophobic Protecting Groups for the
Chemical Synthesis of Oligonucleotides‘**]
8
10
12
IL
16
n
Fig. 1. Dependence of the Rf-values on the alkyl chain length n of compounds (3) [n=8, 10, 12, 14, 16 = (3a, b, e. d, e)]. 0: silica gel 60 (Merck),
chloroform/methanol 9 : 1; 0 : silica gel RP-2 (Merck), acetone/water
75 : 2 5 ; A : silica gel K C , B(Whatman), acetone/water 75 :25.
By Hans-Helmut Gortz and Hartmut Seliger[*’
In the chemical synthesis of polynucleotide fragments
between the alkyl chains of the sorbent and sorbate leads
by the triester procedure”.*’, chromatographic methods are
to a dramatic reduction of the R,-values with increasing
important for the purification of protected intermediates
chain length upon thin layer chromatography (Fig. 1 ) and
and for the isolation of the unprotected final p r o d ~ c t [ ~ . ~ ~ .
increasing retention times in HPLC (Fig. 2).
The chromatographic behavior of the oligomers can be
The 4-hexadecyloxytrityl (cl6Tr-) derivatives [(6). (8).
varied within certain limits by protecting groups, generally
(11). (13) and (16)] among the oligonucleotide phosphoproducing an unspecific change in the total polarity of the
triesters (4)-(16) (Table 1) exhibit the same characteristic
molecule. We have introduced 4-alkoxytrityl groups as a
behavior as the monomers (3) upon RP-18 chromatogranovel type of hydrophobic protection for the 5’-end of
phy. Of prime interest for preparative application is the
o l i g ~ n u c l e o t i d e s For
~ ~ ~ this
.
purpose, the 4-alkoxytritanols
magnitude of the R,-difference between the Ci6Tr-deriva(2a-e) were prepared by Grignard reaction from (la-e)
tives and the corresponding compounds having free 5’-OH
and the trityl chlorides reacted with thymidine to produce
groups, which indicates an easy and complete HPLC sepa(3a-e).
ration of both types of components irrespective of the chain
[*] Prof. Dr. H. Seliger, Dr. H.-H. Gortz
[**I
Sektion Polymere der Universitat
Oberer Eselsberg, D-7900 Ulm (Germany)
Syntheses with Nucleic Acid Constituents, Part 9.-Par1 8: H. Seliger. B.
Haas. M . Holupirek. T. Knable. G. Todling. M . Philipp. Nucleic Acids
Res. Symp. Ser. 7, 191 (1980).
Angew. Chem In,. Ed. Engl 20 ( I Y U I )
No. 8
length and sequence. This is in contrast to separations
of the 4-methoxy- or 4,4‘-dimethoxytrityl (DMTr) derivatives used so far, in which the starting components with 3’terminal phosphate charge can readily be separated from
the reaction mixture arising from nucleotide condensations, however, good separation of the oligonucleotides
0 Verlag Chemie GmbH. 6940 Weinheim, I981
0510-0833/81/0808-0681 $02.50/0
681
ded to a solution of 0.2 mol sodium ethoxide in 200 mL
ethanol. After refluxing the mixture for 5 h, the ethanol is
removed in uacuu and the residue is taken u p in 100 mL
CH2C12/l N NaOH. The aqueous phase is separated, extracted twice with CH2CI2,the combined organic phases
washed with a little water, dried over sodium sulfate and
the solvent removed. Vacuum distillation of the residue
(air condenser!) gives 45.6 g (54%) of a light beige liquid,
b.p. =255 "C/1 torr, which solidify on cooling
(m. p. = 73 OC)['I.
(2e): A solution of ( l e ) (42.2 g , 0.1 mol) in 50 mL tetrahydrofuran (THF) is dropped into a solution of 0.125 mol
phenylmagnesium bromide in 50 mL ether over 30 min.
After the addition, the mixture is boiled for 1 h and upon
cooling poured onto an ice/hydrochloric acid slush. The
organic phase is separated and the aqueous phase extracted with T H F (2 x 50 mL). After being washed with water, NaHCO, solution and water again, the combined organic phases are dried over sodium sulfate and the solvent
removed. The oily residue is taken u p in pentane and recrystallized at - 2 0 ° C ; yield 37.6 g (75%), m.p.=5658 oC[6J.
(3e): A solution of (2e) (750 mg, 1.5 mmol) in 10 mL benzene and 3 mL acetyl chloride are heated for 1 h under reflux, concentrated and evaporated to dryness three times
with benzene (3 x 10 mL). The residual oil is taken u p in 3
mL anhydrous pyridine, and thymidine (242 mg, I mmol)
and 2 mg 4-dimethylaminopyridine are added. At the end
of the reaction (ca. 2 h, monitored by thin layer chromatography) and after addition of 10 mL 5% NH,HCO, solution, the mixture is extracted twice (2 x 10 mL) with chloroform. The dried organic phase is concentrated, succes-
with free 5'-OH groups requires a complicated after-treatmerit['). For purposes of clarification, the R,-values of the
nucleotide sequences having a terminal CI6Tr-[(8), (13),
(16)] or DMTr-group [(7), (12), (IS)] shown in Table 1,
should be compared with their precursors (4). (9) and
(14).
C
1
- l
I
t
Cminl
o
Fig. 2. HPLC separation of compounds (3a-e). Column: p-Bondapak C l X
(Waters), eluent: 2-propanol/methanol/water6 :2 :2, flow rate: I mL/min,
pressure: 130 bar, detection: UV2s4.
Table I . R,-values of compounds (4)-(16) [a] in systems A, B and C [bl
(4)
(5)
(6)
(71
(8)
(9)
Cpd.
A
B
C
dTpTpTtl.
DMTrdTpTpTp(CE)
C i,TrdTpTpTp(CE)
DMTrdTpTpTpTpTpT,,
0.3 1
0.41
0.48
0.35
0.45
0.19/0.30 [c]
0.57/0.65 [cl
0.6310.69 [c]
0.40
0.48
0.73
0.56
0.72
0.s 1
0.72
0.52
0.69
0.52
0.92
0.81/0.83 [c]
0.36/0.39 [c]
0.76
0.40
0.89
0.86
0.33
0.68
0.50
0.19
C ,,TrdTpTpTpTpTpTs.
(14)
dG'""pA"p(CE)
DMTrdG'h"pAhLp(CE)
C ,,TrdG'""pA"p(CE)
DMTrdG'h"pAhzpG'""pAbzp(CE)
C ,bTrdG'hUpAh'pC'h""pAh'p(CE)
dTpCh'pTpC&
(15)
(16)
C 16TrdC'h""pAb'pC'hYpA
"pTpC "pTpC:
(10)
(11)
(12)
(13)
DMTrdG'"""pAh'pG'"""pAh7pTpCh'pTpC~
0.44
0.18
0.97
0.89/0.91 [c]
0.24/0.29 [cl
0.81
0.28
0.96
0.87
0.21
[a] p =p-chlorophenylphosphoryl; CE = B-cyanoethyl: DMTr=4,4'-dimethoxytrityl, CloTr= 4-hexadecyloxytrityl. [bl A: silica gel 60 (Merck); chloroform/methano1 9 : I : B: silica gel RP-8 (Merck); acetondwater 85 : 15; C: silica gel RP-18(Merck); acetone/water 85 : 15. [c] Diastereomeric pairs.
Bearing in mind that the advantage of the triester
method rests mainly upon the rapid and simple removal of
the reaction precursors, the possibility of also separating
the hydroxy component in a selective way should substantially increase the efficiency of the process. In this context
the analogy of the C16Tr-to the 4-methoxy-group with respect to the introduction and cleavage conditions requires
no change in the synthetic strategy.
Procedure
(le): 4-Hydroxybenzophenone (39.6 g, 0.2 mol), l-bromohexadecane (61.1 g, 0.2 mol) and a trace of KI are ad-
682
0 Verlug Chemie CmbH, 6940 Weinheim. 1981
sively twice evaporated to dryness with toluene, ethanol
and chloroform, dissolved in a little chloroform and chromatographed on silica gel (Merck 7733, 1.5 x 7 cm column,
chloroform eluent). The product fractions are concentrated
and solid (3c) obtained by concentrating twice with pentane; yield 545 mg (75%)[61.
Received: December 17, 1980 [Z 807a IE]
German version: Angew. Chem. 93, 708 (1981)
[I] H . Seliger, T. C. Bach. E. Happ. M. Holupirek. E. H . Teufel, Hoppe-Seylers 2. Physiol. Chem. 360. 1044 (1979).
121 S. A . Narang. R . Brousseau. H. M . Hsiung, J. J . Michniewicz. Methods
Enzymol. 65, 610 (1980); S. A . Narang. H. M. Hsiung. R . Brousseau. rbid.
68,90 ( 1979).
0570-0833/81/0808-0682 $02.50/0
Angew. Chem. Int. Ed. Engl. 20 (1981) N o 8
[3] H. Seliger. M . Holupirek. T. C. Each. E . Happ. Proceedings, Konigsteiner
Chromatographie-Tage, Bad Homburg, 1979, 132.
141 H. Seliger, H.-H. Gortz, Proceedings, Konigsteiner ChromatographieTage, Bad Homburg, 1979,304.
[5] J. Stawinski. T. Hozumi. S . A . Narang, C. P. Bahl, R . Wu.Nucleic Acids
Res. 4, 353 (1977).
161 (le). IR: 2960, 2850, 1640, 1600, 1575, 1500, 1250 cm-’; ’H-NMR
(CDCIdTMS): 6eO.88 (t. 3H), 1.28 (s, 26H), 1.79 (t, 2H), 4.00 (t, 2H),
6.85-7.85 (m. 9H); ( 2 K ) : IR: 3580, 3480, 3060, 2920, 2850, 1610, 1580,
1510, 1245 c m - ’ ; (3e): IR: 3400,3050, 2920, 2850, 1700, 1600, 1500, 1250
cm ’.
A
9
o=c
0
__z
*=b
@L
~
i2a.bl
I1a.bJ
Specific Separation of Products in Supported
Oligonucleotide Syntheses Using the Triester
Method [**’
By Hartrnut Seliger and Hans-Helmut Gortz‘.]
In the search for simplified syntheses of oligonucleotides having a specific sequence, methods involving polymeric supports are of considerable interest. However, incomplete lengthening of the chains immobilized on the
carrier leads to formation of a mixture of homologous sequence fragments from which the desired product can only
be obtained in sufficient purity with considerable expenditure of effort. Earlier we described procedures to separate
the product chain, i. e. generally the longest sequence, from
all by-products by selective affinity labellingc’]. We have
now applied this scheme to supported oligonucleotide syntheses using the triester method12].This aspect has not been
considered in other triester carrier ~yntheses‘~’.
As new support materials we prepared “popcorn” copol y m e r ~ from
[ ~ ~ styrene and the 5’-tritylated deoxynucleoside-3’pvinyl benzoates (1) [styrene :(I)G nucleoside
loading of support material = 0.09 mmol/g (2a) and 0.02
mmol/g (Zb)](Scheme 1).
With this support a chain-lengthening step was performed in which, I) the 5‘-OH protecting group was removed by treatment with acid, 2) the product was condensed with ca. a threefold excess of 5’-0-(4,4‘-dimethoxytrityl)thymidine-3’-p-chlorophenylphosphate and 3) the
unreacted chain-ends were blocked by reaction with acetic
anhydride in pyridine. This chain-lengthening step was repeated. Subsequently, the chains were further lengthened
by block condensation with a trithymidylate moiety, which
was protected at the 5’-end by a hexadecyloxytrityl
groupc5].Following ammoniacal cleavage from the support,
the product mixture was separated by HPLC. By this
means the hexanucleotide C,,TrdT(pT), was specifically
separated (Fig. 1, fraction P); after cleavage of the C,,Trgroup it proved to be chromatographically homogeneous
and identical with authentic material synthesized by a support-free methodc5].The yields, relative to the immediate
precursor, were determined to be 51, 57 and 58% respectively, following VIS spectroscopic investigation of the
acid-treated carrier samples. By means of the supportmethod described here, the advantages of the triester method-generally increased yields at shortened reaction times
and with only a slight excess of one reactant-can be used
in solid phase synthetic procedures.
I*]
Prof. Dr. H. Seliger, Dr. H.-H. Gdrtz
Sektion Polymere der Universitat
Oberer Eselsberg, D-7900 Ulm (Germany)
I**] Polymer supported synthesis, Part 9.-Part 8: see [I].
Angew. Chem. Int. Ed. Engl 20 (1981) No. 8
(3al
[&I
R=
-
0
c
C,6TrdTpITplLl-C ‘I-@
0
dTfpTI,
1
. .
Thymin-I-yl
I
14al
N‘-Eenzoylcytosin-I-y(
Scheme 1. Synthesis of dT(pT), on support. A : 1) “Popcorn” copolymerization, 2) H + ;B: 1) DMTrTp-, mesitylenesulfonyl tetrazolide, 2) acetic anhydride, pyridine, 3) H e , 4) Repeat of 1)-3); C: C,,Trd(Tp):, mesitylenesulfonyl tetrazolide; D: 1) aqueous NHJpyridine, 2) HPLC on C,,-phase, 3)
H +.p=p-chlorophenylphosphoryl, DMTr= 4,4’-dimethoxytrityl, C,,Tr= 4hexadecyloxytrityl.
Fig. I. Separation of the sequence C,,TrdT(pT), (P) from the by-products of
the supported synthesis by HPLC. Column: pBondapak CCa(Waters),
eluent: 2-propanol/methanol/water50 : 15 :35, flow rate: 2 ML/min, pressure: 110 bar, detection: UVz54.
Procedure
(la): 5’-O-(4-Hexadecyloxytrityl)thymidinecs1
(725 mg, 1
mmol) is dissolved in 5 mL anhydrous pyridine and the solution mixed with 4-vinylbenzoyl chloridec6](500 mg, 3
mmol). After stirring for 30 min, 10 mL of a 5% NH4HC03
solution is added to the mixture which is then extracted
with chloroform (2 x 15 mL). After drying over sodium sulfate, the organic phase is separated, the pyridine removed
by azeotropic distillation with toluene, and the residue
chromatographed (Merck 7734, column 1.5 x 7 cm, eluent
chloroform). Approximately 700 mg (80%)of a light yellow
oil, Rf = 0.73 (educt: 0.43; silica gel, chloroform/methanol
9 :1); UV: A,,, =265 nm.-(Zb) is synthesized analogously.
0 Verlag Chemie GmbH, 6940 Weinheim, 1981
0570-0833/81/0808-0683 $02.50/0
683
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