An efficient synthesis of the ribozyme-folate conjugate

Article abstract of DOI:10.1016/S0040-4039(02)00837-7

γ-Cysteamine modified folic acid was synthesized by reductive alkylation of N²-ⁱBu-6-formylpterin with suitably protected cysteaminyl-L-glutamyl-p-aminobenzoic acid, followed by deprotection. Following activation with 2,2′-dipyridyl disulfide, this synthon was conjugated to the 5′-end of 5′-thiol modified ribozyme to afford target ribozyme-folate conjugate in a good yield.

Full text of DOI:10.1016/S0040-4039(02)00837-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4439–4441
An efficient synthesis of the ribozyme–folate conjugate
Jasenka Matulic-Adamic, Mark Sanseverino and Leonid Beigelman*
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, CO 80301, USA
Received 20 March 2002; revised 26 April 2002; accepted 30 April 2002
Abstract—g-Cysteamine modified folic acid was synthesized by reductive alkylation of N2-iBu-6-formylpterin with suitably
protected cysteaminyl-L-glutamyl-p-aminobenzoic acid, followed by deprotection. Following activation with 2,2%-dipyridyl
disulfide, this synthon was conjugated to the 5%-end of 5%-thiol modified ribozyme to afford target ribozyme–folate conjugate in
a good yield. © 2002 Elsevier Science Ltd. All rights reserved.
The folic acid endocytosis pathway has been success-
fully exploited to deliver peptides, proteins, antisense
oligonucleotides, plasmids, liposomes and radiophar-
maceutical imaging agents into cells expressing folate
binding protein (FBP).1 The fact that FBP is vastly
overexpressed on certain malignant cells has permitted
the selective destruction of transformed cells without
modifying the behavior of normal cells in the same
culture.2 Electron microscopic analysis of the intracellu-
lar itinerary of folate–protein conjugates has demon-
strated that the conjugates enter receptor-bearing cells
primarily at uncoated pits, termed caveolae.2
experienced in attempts to scale up this synthesis to
obtain enough material for the in vivo animal studies.
Therefore, we decided to use post-synthetic conjugation
of folate to ribozyme through the cleavable disulfide
bond. It was shown that the nature of the linkage
between folate and the protein drug is crucial for the
activity: cleavable disulfide linked conjugate demon-
strated higher potency than amide or thioether linked
conjugate.8
The synthetic strategy of assembling folate molecule
through peptide coupling of pteroic and glutamic acid
is often plagued with low yields.9,10 On the other side,
reductive amination between 6-formylpterin and p-
aminobenzoic acid (PABA) segments seems less trou-
blesome.11 We successfully applied the latter approach
for the preparation of folate molecule in a high overall
yield.
In the development of ribozymes as potential anti-
cancer therapeutics, it would be desirable to achieve
high intracellular concentration of ribozyme at the
tumor site. An attractive approach to this problem is
conjugation of ribozyme to targeting ligands like folate
or peptides, which possess cell membrane translocation
and/or nuclear localization properties. Several groups
have recently conjugated folic acid to oligonucleotides,
but most methods are difficult to scale up and lack
regioselectivity.3,4 Recently, Bhat et al.5 reported the
synthesis of folic acid conjugated nucleoside building
blocks and their incorporation into oligonucleotides
using solid supported phosphoramidite chemistry. Their
synthetic approach allowed for the regioselective
attachment of folate through a- or g-carboxylic group.
It has been demonstrated that only those conjugates
that contain folate attached through the g-position
retain the ability to bind to cell surface folate receptors
with the same affinity as the free folic acid.6 We used a
similar approach for the conjugation of folate to the
ribozyme targeted against Erb2/Her.7 Difficulties were
Synthesis of the cysteamine modified folate 10 is pre-
sented in Fig. 1. Monomethoxytrityl cysteamine 1 was
prepared by selective tritylation of the thiol group of
cysteamine with 4-methoxytrityl alcohol in trifluoro-
acetic acid. Peptide coupling of 1 with Fmoc-Glu-
OtBu in the presence of PyBOP yielded 2 in a high
yield. N-Fmoc group was removed smoothly with pipe-
ridine to give 3. Condensation of 3 with p-(4-
methoxytrityl)aminobenzoic acid, prepared by reaction
of p-aminobenzoic acid with 4-methoxytrityl chloride in
pyridine, afforded the fully protected conjugate 4.
Selective cleavage of N-MMTr group with acetic acid
afforded 5 in a quantitative yield. Schiff base formation
between 5 and N2-iBu-6-formylpterin 6,9 followed by
reduction with borane–pyridine complex proceeded
with a good yield to give fully protected cysteamine–
folate adduct 7.12 The consecutive cleavage of protect-
* Corresponding author. E-mail: lnb@rpi.com
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00837-7

4440
J. Matulic-Adamic et al. / Tetrahedron Letters 43 (2002) 4439–4441
O
O
C
R
i
ii
+
FmocHN
COOH
H2N
FmocHN
C
SMmt
H2N
SMmt
SMmt
N
H
N
H
COOtBu
COOtBu
COOtBu
1
2
3
O
COOtBu
O
COOtBu
MmtHN
COOH
H
N
H
N
iv
N
H
SMmt
N
H
SMmt
O
O
MmtHN
H2N
iii
4
5
O
O
O
COOtBu
N
O
COOtBu
H
N
H
N
HN
H
O
N
H
SMmt
O
N
N
H
SMmt
vi
6
BuHiN
N
N
N
N
O
N
N
O
HN
N
H
HN
N
H
v
H2N
N
BuHiN
8
7
O
COO-TEA+
O
COOH
H
N
H
N
S
N
viii
O
N
N
H
S
O
N
H
SH
vii
N
O
N
N
O
HN
N
H
HN
H2N
N
H
H2N
N
N
9
10
Figure 1. Synthesis of 2-dithiopyridyl activated folic acid. Reagents and conditions: (i) PyBOP, DIPEA, DMF, 98%; (ii) piperidine,
DMF, 84%; (iii) PyBOP, DIPEA, DMF, 65%; (iv) 80% CH3COOH, quant.; (v) CH3COOH, pyridine·BH3, CH3OH, 70%; (vi) 40%
CH3NH2, DMF, quant.; (vii) TFA:CH2Cl2:iPr3SiH 7.5:2:0.5, 90%; (viii) 2,2%-dipyridyl disulfide, DMSO, 45°C, 18 h, TEA+HCO3
,
−
51%.
ing groups of 7 with base and acid yielded thiol deriva-
tive 9. The thiol exchange reaction of 9 with 2,2-
dipyridyl disulfide afforded the desired S-pyridyl
activated synthon 10 as a yellow powder in a moderate
yield.13 It is worth noting that the isolation of 10 as its
TEA+ or Na+ salt made it soluble in DMSO and/or
water, which is an important requirement for its use in
conjugation reactions.
Oligonucleotide synthesis, deprotection and purification
was performed as described previously.14 5%-Thiol-
Modifier C6 (Glen Research, Sterling, Virginia) was
coupled as the last phosphoramidite to the 5%-end of a
growing oligonucleotide chain. After cleavage from the
solid support and base deprotection, the disulfide
modified ribozyme 11 (Fig. 2) was purified using ion
exchange chromatography. The thiol group was
i
10
O
O
O
S
O
SH
S
OH
p
p
5'-Ribozyme
O
5'-Ribozyme
O
O-
O-
12
ii
11
HOOC
O
H
N
O
O
S
N
H
O
N
S
p
5'-Ribozyme
O
O-
O
N
N
N
H
NH
NH2
13
Figure 2. Preparation of ribozyme–folate conjugate. Reagents and conditions: (i) DTT, ethanol, TEA, H2O; (ii) 50 mM NH4OAc,
pH 7.

J. Matulic-Adamic et al. / Tetrahedron Letters 43 (2002) 4439–4441
4441
Figure 3. HPLC of ribozyme–folate conjugation reaction. HPLC conditions: Column: C18–Transgenomic, Temp. 80°C, Buffer A:
100 mM TEAA, Buffer B: 100 mM TEAA/50% ACN, Gradient: 22%B–40%B in 35 min. 1: starting ribozyme, 2: ribozyme–folate
conjugate.
unmasked by reduction with dithiothreitol (DTT) to
afford 12 which was purified by gel filtration and
immediately conjugated with 10. The resulting conju-
gate 13 was separated from the excess folate by gel
filtration and then purified by RP HPLC using gradient
of acetonitrile in 100 mM triethylammonium acetate
(TEAA) (Fig. 3).
References
1. Reddy, J. A.; Low, P. S. Crit. Rev. Ther. Drug Carrier Syst.
1998, 15, 587–627 and references cited therein.
2. Leamon, C. P.; Low, P. S. Biochem. J. 1993, 291, 855–860.
3. Habus, I.; Xie, J.; Iyer, R. P.; Zhou, W.-Q.; Shen, L. X.;
Agarwal, S. Bioconjugate Chem. 1998, 9, 283–291.
4. Harrison, J. G.; Balasubramanian, S. Bioorg. Med. Chem.
Lett. 1997, 7, 1041–1046.
5. Bhat, B.; Balow, G.; Guzaev, A.; Cook, D. P.; Manoharan,
M. Nucleosides Nucleotides 1999, 18, 1471–1472.
6. Wang, S.; Mathias, C. J.; Green, M. A.; Low, P. S.
Bioconjugate Chem. 1996, 8, 673–679.
Desalting was performed by RP HPLC. Reactions
were conducted on 400 mg of disulfide modified
ribozyme 11 (5%-LgscsasgsuggccgaaggCgagUgaGGu-
CuagcucaB, where g,c,a,u=2%-O-Me G,C,A,U; G=ribo
G; C=2%-amino-C, U=2%-C-allyl-U; L=Spacer 6 from
Glen Research, S=phosphorotioate linkage and B=
inverted abazic) to afford 200–250 mg (50–60% yield)
of conjugate 13. MALDI TOF MS confirmed the struc-
ture: MW calcd. 12083.82, found 12084.74.
7. Matulic-Adamic, J. et al., Bioconjugate Chem. 2002, in
press.
8. Lamon, C. P.; Pastan, I.; Low, P. S. J. Biol. Chem. 1993,
268, 24847–24854.
9. Manoharan, M.; Bhat, B.; Cook, P. D.; Guzaev, A. P. 1999,
WO 99/66063.
10. Nomura, M.; Shuto, S.; Matsuda, A. J. Org. Chem. 2000,
65, 5016–5021.
In conclusion, folate–cysteamine adduct is prepared by
a scaleable solution phase synthesis in a good overall
yield. Disulfide conjugation of this novel targeting lig-
and to the thiol-modified oligonucleotide is suitable for
the multigram scale synthesis. The 9-atom spacer pro-
vides an essential spatial separation between folate and
attached ribozyme cargo. Importantly, conjugation of
folate to ribozyme through a disulfide bond should
permit intermolecular separation which was suggested
to be required for the functional cytosolic entry of a
protein drug.2 Tissue localization and animal efficacy
studies using folate–ribozyme conjugates are underway
and will be reported in due course.
11. Plante, L. T. J. Org. Chem. 1971, 36, 860–861.
12. 1H NMR spectrum for 7 in DMSO-d6-D2O: l 8.92 (s, 1H,
H-7), 7.70(d, J=8.8, 2H, PABA), 7.39–6.92(m, 14H, trityl),
4.66 (s, 2H, 6-CH2), 4.25 (m, 1H, Glu), 3.79 (s, 3H, OCH3),
i
3.02 (m, 2H, cysteamine), 2.84 (m, 1H, Bu), 2.22 (m, 4H,
cysteamine, Glu), 2.10–1.85 (m, 2H, Glu), 1.43 (s, 9H, tBu),
1.20 (s, 3H, iBu), 1.19 (s, 3H, iBu). MS/ESI+ m/z 899.3
[M+H]+.
13. Isolated as a TEA+ salt: 1H NMR spectrum for 10 in D2O:
l 8.68 (s, 1H, H-7), 8.10 (d, J=3.6, 1H, pyr), 7.61 (d, J=8.8,
2H, PABA), 7.43 (m, 1H, pyr), 7.04 (d, J=7.6, 1H, pyr),
6.93 (m, 1H, pyr), 6.82 (d, J=8.8, 1H, PABA), 4.60 (s, 2H,
6-CH2), 4.28 (m, 1H, Glu), 3.30–3.08 (m, 2H, cysteamine),
3.05 (m, 6H, TEA), 2.37 (m, 2H, cysteamine), 2.10 (m, 4H,
Glu), 1.20 (m, 9H, TEA). MS/ESI− m/z 608.02 [M−H]−.
14. Wincott, F. E.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz,
D.; Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe,
S.; Usman, N. Nucleic Acids Res. 1995, 23, 2677–2684.
Acknowledgements
We thank the Process Chemistry Department for the
large scale synthesis of ribozymes and Professor Alex
Azhayev for helpful advice.