Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoammino acids

Article abstract of DOI:10.1016/S0960-894X(00)00121-9

Nδ-Fmoc protected nucleoamino acids of type I (base=T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00121-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 929±933
Synthesis and Hybridization Properties of Polyamide Based
Nucleic Acid Analogues Incorporating Pyrrolidine-derived
Nucleoamino Acids
Karl-Heinz Altmann, a,* Dieter Husken, c Bernard Cuenoud b
and Carlos Garcõa-Echeverrõa a
aNovartis Pharma AG, TA Oncology Research, WKL-136.4.21, CH-4002 Basel, Switzerland
bRespiratory Disease TA, Horsham, UK
cFunctional Genomics Area, Basel, Switzerland
Received 4 January 2000; accepted 17 February 2000
AbstractÐNd-Fmoc protected nucleoamino acids of type I (Base=T, C, A) have been synthesized and employed as building blocks
for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA
with signi®cant anity and in a sequence-speci®c fashion, while no binding was observed to complementary DNA. # 2000 Elsevier
Science Ltd. All rights reserved.
DNA analogues with modi®ed backbone structures have
attracted considerable attention as nuclease resistant
antisense and antigene agents.1,2 The majority of mod-
i®ed oligonucleotide analogues that have been investi-
gated in this context are characterized by an alternating
arrangement of natural phosphodiester and modi®ed
internucleoside linkages;2 however, as illustrated by the
discovery of peptide nucleic acids (PNA), which are
entirely based on an aminoethyl glycyl backbone, even
the complete replacement of the deoxyribose-phosphate
backbone in natural oligodeoxyribonucleotides can lead
to analogues with superior DNA- and RNA-binding
properties.3 A variety of other polyamide-type struc-
tures have subsequently been suggested to represent
useful DNA surrogates for possible antisense applica-
tions,4 some of which were reported to possess DNA-
and RNA-binding anities close to those of natural
DNA or even PNA.5
As part of a comprehensive program directed at the
identi®cation of genuinely new (i.e., not PNA-derived)
polyamide-based oligonucleotide analogues with RNA-
binding anities at least comparable to those of natural
oligodeoxyribonucleotides, we have recently described
the synthesis and RNA-binding properties of a series of
homopyrimidine 15-mers that were composed of rela-
tively ¯exible linear d-amino acids (Fig. 1, II±IV).6 As an
extension of this previous study we have now investigated
Figure 1.
*Corresponding author. Fax: +41-61-6966246; e-mail: karl-heinz.
altmann@pharma.novartis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00121-9

930
K.-H. Altmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 929±933
a conformationally more rigid type of polyamide-based
DNA analogue incorporating pyrrolidine-derived
monomer units I (Fig. 1).7
ester moiety by treatment with aqueous base, acid cata-
lyzed cleavage of the Nd-BOC protecting group, and
®nally reprotection of the free amino acid by reaction
with Fmoc-OSu11 to provide 8 in 35% overall yield
(based on 5).
In this communication we want to report on the synth-
esis of protected nucleoamino acids8 of type I as well as a
corresponding homo-pyrimidine oligomer and provide
some preliminary data on the RNA- and DNA-binding
properties of this novel type of oligonucleotide analogue.
Nd-Fmoc-N4-benzyloxycarbonyl-protected cytosine deri-
vative 13 (Scheme 2) was prepared via uracil 9 (obtained
from 5 and N3-Bz-uracil followed by cleavage of the N3-
Bz protecting group with aq ammonia/dioxane in 51%
overall yield).12 Conversion of 9 into triazolide 10 followed
by treatment of 10 with aq ammonia/dioxane6a,13 provided
cytosine derivative 11, whose exocyclic amino function
was protected with a benzyloxycarbonyl (Cbz) group by
reaction with Cbz-OBt.14 Concomitant cleavage of the
BOC- and tert-butyl ester moieties and reprotection of
the d-amino group gave 13 in 18% overall yield for the
®ve step sequence from 9.
The synthesis of nucleoamino acids of type I was based
on alcohol 5 as the central common intermediate, which
can be obtained in a four step sequence from known d-
hydroxyproline derivative 19 in 69% overall yield (Scheme
1). The conversion of 5 into fully protected nucleoamino
acids was achieved through alkylation of appropriately
protected nucleic acid base derivatives (N3-Bz-thymine or
-uracil) or nucleic acid base precursors (6-chloropurine)
under Mitsunobu conditions.6a,10 These alkylation pro-
ducts were subsequently elaborated into Nd-9-¯uor-
enylmethoxycarbonyl-(Fmoc-)protected nucleoamino
acids through base conversion (where required) fol-
lowed by appropriate protecting group manipulations.
In the case of fully protected thymine derivative 6
(Scheme 1) this involved simultaneous removal of the
base protecting group and hydrolysis of the tert-butyl
Preparation of Nd-Fmoc-N6-Cbz-protected adenine deri-
vative 15 initially involved reaction of 5 with 6-chloro-
purine as an adenine surrogate (Scheme 3).10 Although
the resulting N9-alkylated 6-chloropurine could not be
obtained in entirely pure form (contamination by residual
N,N0-diethoxycarbonyl hydrazine) treatment of this
material with aq ammonia at 60 ꢀC provided adenine
Scheme 1. (i) TBS-Cl (1.5 equiv), imidazole (2.2 equiv), Et3N, DMF, rt, 2 h, quant. (ii) LiBH4 (3.5 equiv), THF, 0ꢀ, 2.5 h, 86%. (iii) BrCH2COOBut,
(2.5 equiv), Bu4NHSO4 (0.25 equiv), benzene/50% NaOH 1/1, 2 h, 88%. (iv) TBAF (2.5 equiv), THF, 0ꢀ, 1 h, 91%. (v) N3-Bz-thymine (2 equiv),
DEAD (2.5 equiv), Ph3P (2.5 equiv), THF, 0ꢀ, 7 h, 63%. (vi) 2N NaOH/MeOH/DMF 1.2/3/4 (4 equiv OH ), rt, 3.5 h, 77%. (vii) CF3COOH, rt, 45
min. (viii) Fmoc-OSu (1.15 equiv), Na2CO3 (1.6 equiv), dioxane/H2O 1/1, 73% (2 steps).
Scheme 2. (i) POCl3 (3.5 equiv), triazole (22.5 equiv), Et3N (23 equiv), CH3CN, rt, 43%. (ii) Concd NH3/dioxane 1/3, rt, 93%. (iii) Cbz-OBt (1.3
equiv), Et3N (1.3 equiv), CH2Cl2, rt, 82%. (iv) a) CF3COOH, rt; b) Fmoc-OSu (1.2 equiv), Na2CO3 (2.5 equiv), dioxane/H2O 6/4, rt, 55% (2 steps).

K.-H. Altmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 929±933
931
Scheme 3. (i) DEAD (2.5 equiv), Ph3P (2.5 equiv), 6-Cl-purine (2 equiv), THF, 0 ꢀC, 1 h, rt, 48 h. (ii) Concd NH3/dioxane 1/1, 60ꢀ, 24 h, 75% (2
steps). (iii)
(4 equiv), rt, 16h, 70%. (iv) CF3COOH, rt, 3 h, 60%. (v) Fmoc-OSu (1.15 equiv), Na2CO3 (2.3 equiv), dioxane/H2O
1/1, rt, 48 h, 62%.
derivative 14 in a satisfactory 75% overall yield based
on 5. 14 was then elaborated into 15 by the same
sequence of reactions as described above for the pre-
paration of cytosine derivative 13. It should be noted,
however, that in contrast to 11 (Scheme 2) protection of
the exocyclic amino group of 14 with a Cbz-group could
only be achieved by means of the Rapaport reagent,15
whereas neither Cbz-OBt nor Cbz-Cl, even in large
excess, resulted in any of the desired acylation product.
removed in solution at rt (TFMSA/TFA/DMS/m-cresol
(1/10/6/2, v/v/v/v),22 3.5 h). The crude compounds were
subjected to analysis and puri®cation on a standard C18
reversed-phase column eluting with an acetonitrile±
water gradient. The purity of the oligomer was veri®ed
by C18 reversed-phase analytical HPLC and was found
to be >95%. In addition, the identity of the ®nal pro-
duct was assessed by mass spectral (matrix-assisted laser
desorption ionization time-of-¯ight mass spectrometry,
MALDI-TOF) analysis, which gave the expected mole-
cular weight (4179.4 versus 4178.4 (calc)).
In order to assess the hybridization anity of oligomers
of type I to complementary RNA and DNA, oligonu-
cleotide analogue A was prepared as a simple model
system, employing Fmoc-protected nucleoamino acids 8
and 13 as building blocks:16
The interactions of oligomer A with complementary
RNA and DNA were investigated in UV-melting
experiments and the results of these studies are sum-
marized in Table 1. Slow heating of a 1/1 mixture of A
and its antiparallel23 RNA complement immediately
after mixing produced a biphasic melting curve with two
cooperative transitions around 33 and 68 ꢀC, respec-
tively. Upon subsequent cooling of the mixture only the
low temperature transition was still observed (33.2 ꢀC);
likewise, re-heating resulted in a cooperative melting
curve with only a single transition at 33.4 ꢀC. Although
the origin of the high-temperature transition in the
initial heating cycle is currently unresolved, the coop-
erative nature of the melting curve as well as its full
reversibility with regard to the low-temperature transi-
tion clearly indicate that A is able to bind to its anti-
parallel RNA complement, r[(AG)5A5], in a cooperative
fashion. The presence of a single mismatched base in the
RNA target sequence (r[(AG)4ACA5]) resulted in a
decrease in melting temperature of ꢁ10 ꢀC (Table 1)
which con®rms that binding is (Watson±Crick)
sequence-speci®c. In addition, the results of titration
experiments indicate that complex formation between A
and r[(AG)5A5] occurs with 1/1 stoichiometry.24 Based
on the di€erence in Tm-values between the A/r[(AG)5A5]
complex and the corresponding natural DNA/RNA
H-Lys-ttt ttc tct ctc tct-Lys-NH2 (A) t,c=I-T, I-C
Lysine residues were attached to the N- as well as the C-
terminus of the actual base sequence in order to ensure
adequate solubility in UV-melting experiments. Oligomer
synthesis was performed on a 4-(20,40-dimethoxyphenyl-
aminomethyl)-phenoxy resin (copoly(styrene)-1% DVB;
fꢁ0.24 mmol/g),17 with lysine residues being incorporated
into the growing polyamide chain as the Na-Fmoc-NE-tert-
butoxycarbonyl derivative (Fmoc-Lys(BOC)-OH). Chain
elongation was achieved by 2-(2-oxo-1(2H)-pyridyl))-
1,1,3,3-tetramethyluronium tetra¯uoroborate (TPTU)18-
mediated single couplings (2- or 3-fold excess of nucleo-
amino acid, similar excess of TPTU) in the presence
of ethyl-diisopropyl-amine (3.3 equiv) in N-methyl-
pyrrolidinone, followed by capping of unreacted amino
groups with acetic anhydride (Ac2O/DMA/pyridine 1/8/1).
The coupling time was set to 90 min, including 30 min at
40 ꢀC.19 In general, coupling eciencies were >95%, as
determined by recording the UV absorption of the ful-
vene-piperidine adduct formed upon removal of the Nd-
Fmoc protecting group with 20% piperidine/DMF
(l=299.8 nm, E=7800 M Âcm 1).20 In order to
duplex (ÁTm
ꢁ
19 ꢀC, Table 1), it is clear that A binds
1
ensure complete deprotection of the terminal amino
group, on-line monitoring of the Fmoc-cleavage step
proved to be of crucial importance, as cleavage rates
varied as a function of sequence position. The 20%
piperidine/DMF treatment interval was thus adjusted in
each deprotection step according to the observed lability
of the protecting group. After cleavage of the protected
oligomer from the solid support with TFA/H2O (95/5,
v/v; 3.5 h), the Cbz-protecting groups on cytosine were
to complementary antiparallel RNA with lower anity
than the corresponding oligodeoxyribonucleotide,
d[(T)5(CT)5]; however, the resulting ÁTm-value of
between 1.2 and 1.3ꢀ/residue should be compared to
similar or signi®cantly more negative values that have
been reported for a variety of di€erently modi®ed DNA
analogues, which are more closely related to the struc-
ture of natural DNA than oligomer A.1,2 It should also
be noted that the complex between A and r[(AG)5A5] is

932
K.-H. Altmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 929±933
Table 1. Melting temperatures (Tm's) of complexes between oligomer A and complementary RNA and DNAa
RNA/DNA complement
r[(AG)5A5]
r[A5(GA)5]
d[(AG)5A5]
d[A5(GA)5]
r[(AG)4ACA5]
Tm (ꢀC):
TmWT (ꢀC):b
34.0c (33.2d)
52.4
34.5c (34.9d)
53.3
n.c.e
42.8
n.c.e
43.2
23.3
42.9
aTm's were determined in 10 mM phosphate bu€er, pH7, 100 mM Na+, 0.1 mM EDTA at 4 mM strand concentrations (for details cf. ref 21).
bTm's for the corresponding natural (antiparallel) DNA/RNA and DNA/DNA duplexes.
cTransition-temperature obtained upon slow heating (0.5 ꢀC/min) of a 1/1 mixture of oligomer A and the corresponding RNA complement from
5
to 95ꢀ after initial slow heating and subsequent slow cooling over the same temperature range. In the case of A/r[(AG)5A5] two cooperative transi-
tions were observed at 33.5 and 68.4 ꢀC, respectively, upon immediate slow heating of a freshly prepared 1/1 mixture. The latter transition was not
observed upon subsequent cooling or re-heating of the same sample (cf text).
dTransition-temperature obtained upon slow cooling of a 1/1 mixture of oligomer A and the corresponding RNA complement from 95 to 5 ꢀC
after initial slow heating over the same temperature range.
eNo cooperative transition observed.
signi®cantly more stable than the corresponding com-
plexes incorporating oligonucleotide analogues of types
Acknowledgements
II±IV (cf. Fig. 1).6b On the other hand, Lowe et al.5a
have recently reported that oligonucleotide analogues
We thank A. Garnier, R. Wille, V. von Arx, and M.-L.
Piccolotto for technical assistance.
related to those of type I by the replacement of the
-CH2O- unit with an amide group -C(O)NH- supposedly
bind to natural RNA as well as DNA with similar a-
nities as PNA (and thus more tightly than natural DNA).
References and Notes
The di€erence between Lowe's system and our structure I
1. For recent reviews on antisense technology and the
could conceivably be caused by an increase in conforma-
importance of structural modi®cations of oligonucleotides
tional ¯exibility in the latter, which would entropically
for antisense applications cf., e.g.: a Crooke, S. T. Med. Res.
disfavor hybrid formation (cf., however, ref 25).
Rev. 1996, 16, 319. (b) Matteucci, M. D. Persp. Drug Disc.
Des. 1996, 4, 1. (c) Altmann K.-H.; Cuenoud, B.; von Matt,
P. In Applied Antisense Oligonucleotide Technology; Stein, C.
A.; Krieg, A. M., Eds.; Wiley-Lyss: New York, 1997; pp 73±
107.
2. For recent reviews on backbone modi®ed oligonucleotides
cf., e.g.: (a) De Mesmaeker, A.; Haner, R.; Martin, P.; Moser,
H. E. Acc. Chem. Res. 1995, 28, 366. (b) De Mesmaeker, A.;
Altmann, K.-H.; Waldner, A.; Wendeborn, S. Curr. Opinion
Struct. Biol. 1995, 5, 343.
3. For recent reviews on PNA cf., e.g.: (a) Nielsen, P. Curr.
Opin. Struct. Biol. 1999, 9, 353. (b) Hyrup; B.; Nielsen, P. E.
Bioorg. Med. Chem. 1996, 4, 5.
4. Cf., e.g.: (a) Schah, V. J.; Cerpa, R.; Kuntz, I. D.; Kenyon,
G. L. Bioorg. Chem. 1996, 24, 201. (b) Petersen, K. H.;
Buchardt, O.; Nielsen, P. E. Bioorg. Med. Chem. Lett. 1996,
6, 793. (c) Lenzi, A.; Reginato, G.; Taddei, M. Tetrahedron
Lett. 1995, 36, 1713; Lenzi, A.; Reginato, G.; Taddei, M.;
Tri®lie€, E. Tetrahedron Lett. 1995, 36, 1717. (d) Garner, P.;
Yoo, J. U. Tetrahedron Lett. 1993, 34, 1275. (e) Lewis, I.
Tetrahedron Lett. 1993, 36, 5697. (f) Hudziak, R. M.; Bar-
ofsky, E.; Douglas, F.; Weller, D. L.; Huang, S.-B.; Weller,
D. D. Antisense Nucleic Acid Drug Dev. 1996, 6, 267. For a
review on earlier attempts to construct polyamide-based
nucleic acid analogues see: Jones, S. A. Int. J. Biol. Macro-
molecules 1979, 1, 194.
Cooperative binding of A to its parallel23 RNA comple-
ment (r[A5(GA)5]) was also observed and occurs with
comparable anity as for complementary RNA with an
antiparallel alignment (Table 1). Most notably, only a
single transition was observed under all experimental
conditions and the binding curve was fully reversible upon
heating and cooling without any indication of hysterisis. It
is not clear, however, whether the interaction between A
and r[A5(GA)5] in fact involves formation of a parallel A/
RNA duplex, as binding of A to r[A5(GA)5] could also
occur through the formation of an 11-base pair anti-
parallel duplex based on Watson±Crick base pairs
between residues 5 to 15 of A and 15 to 5 of r[A5(GA)5].26
In contrast to complementary RNA, cooperative bind-
ing of A to complementary DNA, either in an antiparallel
or parallel fashion, was not observed. This is contrary to
the behaviour of the corresponding oligomers of type II±
IV (Fig. 1), for which complexes with complementary
DNA appear to be of comparable stability as those with
RNA.6b
In summary, we have achieved the synthesis of a new
polyamide based oligonucleotide analogue incorporat-
ing thymine- and cytosine-derived building blocks of
type I. Homo-pyrimidine oligomer A exhibits sequence-
speci®c binding to complementary RNA, but not DNA.
As nucleoamino acids of type I easily lend themselves to
further chemical modi®cation (e.g., through introduction
of additional substituents on the pyrrolidine ring or at
the position a to the carboxy group; cf. also ref 6b),
these building blocks could represent an appropriate
template for the design of DNA analogues exhibiting
further improved RNA-binding properties.
5. (a) Lowe, G.; Vilaivan, T.; Westwell, M. S. Bioorg. Chem.
1997, 25, 321. (b) Jordan, S.; Schwemler, C.; Kosch, W.;
Kretschmer, A.; Stropp, U.; Schwenner, E.; Mielke, B. Bioorg.
Med. Chem. Lett. 1997, 7, 687.
6. (a) Altmann, K.-H.; Schmit Chiesi, C.; Garcõa-Echeverrõa,
C. Bioorg. Med. Chem. Lett. 1997, 7, 1119. (b) Garcõa-Eche-
verrõa, C.; Schmit Chiesi, C.; Husken, D.; Altmann, K.-H.
Bioorg. Med. Chem. Lett. 1997, 7, 1123.
7. Related structures have recently been described (cf. ref. 5).
8. The term nucleoamino acid is meant to signify amino acids
with side chains bearing nucleic acid bases.
9. Baker, G. L.; Fritschel, S. J.; Stille, J. R.; Stille, J. K. J. Org.
Chem. 1981, 46, 2954.

K.-H. Altmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 929±933
933
10. Jenny, T. F.; Horlacher, J.; Previsiani, N.; Benner, S. A.
Helv. Chim. Acta. 1993, 76, 248.
23. The terms `antiparallel' and `parallel' RNA/DNA comple-
ment refer to the Watson±Crick alignment of oligomer A
in the N!C direction with the RNA (DNA) in the 30!50
(`antiparallel') and 50!30 (`parallel') direction, respectively.
24. Titration experiments were performed at 260 nm and 280
nm by mixing 4 mM solutions of r[(AG)5A5] and A at molar
fractions of A ranging from 1 to 0 in steps of 0.1 (i.e., molar
fractions of A were 1, 0.9, 0.8, etc.). Absorbance was measured
at rt either directly 15 min after mixing or after prior heating
to 85 ꢀC for 5 min. At 280 nm, no signi®cant di€erences in
absorbance were found between heated and unheated samples,
with a trough in absorbance being observed for a 1/1 ratio of
r[(AG)5A5] and A in both cases. At 260 nm absorbance versus
molar fraction A curves were biphasic; the slope of the curves
increased signi®cantly for ratios of r[(AG)5A5]/A <0.5 (0.29ꢀ
!0.56ꢀ for the unheated and 0.11!0.68 ꢀC for the heated
samples). These data strongly suggest that the complex
between A and r[(AG)5A5] is of 1/1 stoichiometry.
25. Independent of Lowe's work (ref 5a) we have also pre-
pared an analogue of A with amide linkages -C(O)NH- sub-
stituting for all ether linkages -CH2O- in A. In our hands,
under the experimental conditions referred to in Table 1, a 1/1
mixture of this oligomer with r[(AG)5A5] upon initial heating
gave a reasonably cooperative melting curve with an apparent
Tm of 30 ꢀC. However, no cooperative transition was observed
upon cooling the sample. Furthermore, subsequent re-heating
did not reproduce the original transition curve, but merely
resulted in a virtually monotonous increase in absorption.
26. One could argue that the similar stabilities of the com-
plexes of A with r[(AG)5A5] (15 Watson±Crick base pairs) and
r[A5(GA)5], respectively, render the existence of an 11-base
pair duplex between A and its parallel RNA complement a
rather unlikely possibility. However, it should be kept in mind
that no signi®cant stability di€erence exists between the
duplexes of d[T5(CT)5] with r[(AG)5A5] and r[A5(GA)5] (Tm-
values of 52 and 48ꢀ, respectively).
11. Sigler, G. F.; Fuller, W. D.; Chaturverdi, N. C.;
Goodman, M.; Verlander, M. Biopolymers 1983, 22, 2157.
12. In related alcohol substrates, direct Mitsunobu reaction
with cytosine or N4-isobutyryl cytosine predominantly resulted
in O-2 alkylation (Altmann, K.-H., unpublished results).
13. Divakar, K. J.; Reese, C. B. J. Chem. Soc. Perkin Trans. 1
1982, 1171
14. Wunsch, E.; Graf, W.; Keller, O.; Keller, W.; Wersin, G.
Synthesis 1986, 958.
15. Watkins, B. E.; Kiely, J. S.; Rapaport, H. J. Org. Chem.
1982, 104, 5702. For use of this reagent in the synthesis of
PNA building blocks cf.: Egholm, M.; Behrens, C.; Chris-
tensen, L.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Chem.
Soc., Chem. Commun. 1993, 800.
16. Using the oligomerization/puri®cation protocol described
in this work, we have also prepared an oligomer H-Lys-cau
aau cau caa ucu-Lys-NH2 (u=I-U), which incorporates pyr-
imidine- as well as purine-derived building blocks.
17. Rink, H. Tetrahedron Lett. 1987, 28, 3787. Syntheses were
carried out on a semi-automated peptide synthesizer using
140±190 mg of resin.
18. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillesen, D. Tet-
rahedron Lett. 1989, 30, 1927.
19. Ecient incorporation of the N-terminal lysine residue in
general required double-coupling.
20. Atherton, E.; Sheppard, R. C. In Solid Phase Peptide
Synthesis: A Practical Approach; IRL Press, Oxford, 1989.
21. Concentrations of oligomer A were determined using the
corresponding nucleotide extinction coecients (E260) for thy-
mine and cytosine derivatives, respectively (cf. Puglisi, J. D.
Methods Enzymol. 1989, 180, 304). For further details regarding
Tm measurements cf.: Lesnik, E. A.; Guinosso, C. J.; Kawasaki,
A. M.; Sasmor, H.; Zounes, M.; Cummins, L. L.; Ecker, D. J.;
Cook, P. D.; Freier, S. M. Biochemistry 1993, 32, 7832.
22. Tam, J. P.; Heath, W. F.; Merri®eld, R. B. J. Am. Chem.
Soc. 1986, 108, 5242.