Bioactive pseudopeptidic analogues and cyclostereoisomers of osteogenic growth peptide C-terminal pentapeptide, OGP(10-14)

Article abstract of DOI:10.1021/jm010479l

The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic response to local bone marrow injury. When administered in vivo, OGP stimulates osteogenesis and hematopoiesis. The C-terminal pentapeptide OGP(10-14) is the minimal amino acid sequence that retains the full OGP-like activity. Apparently, it is also the physiologic active form of OGP. Residues Tyr¹⁰, Phe¹², Gly¹³, and Gly¹⁴ of OGP are essential for the OGP(10-14) activity. The present study explored the functional role of the peptide bonds, carboxyl and amino terminal groups, and conformational freedom in OGP(10-14). Transformations replacing the peptide bonds with surrogates such as ψ(CH₂NH), ψ(CONMe), and ψ(CH₂CH₂) demonstrated that amide bonds do not contribute significantly to OGP(10-14) bioactivity. End-to-end cyclization yielded the fully bioactive cyclic pentapeptide c(Tyr-Gly-Phe-Gly-Gly). The retroinverso analogue c(Gly-Gly-phe-Gly-tyr), a cyclostereoisomer of c(Tyr-Gly-Phe-Gly-Gly), is at least as potent as the parent cyclic pentapeptide. The unique structure-activity relations revealed in this study suggest that the spatial presentation of the Tyr and Phe side chains has a major role in the productive interaction of OGP(10-14) and its truncated and conformationally constrained analogues with their cognate cellular target.

Full text of DOI:10.1021/jm010479l

1624
J . Med. Chem. 2002, 45, 1624-1632
Bioa ctive P seu d op ep tid ic An a logu es a n d Cycloster eoisom er s of Osteogen ic
Gr ow th P ep tid e C-Ter m in a l P en ta p ep tid e, OGP (10-14)
Yu-Chen Chen,† Andras Muhlrad,‡ Arie Shteyer,§ Marina Vidson,† Itai Bab,*,† and Michael Chorev|
Bone Laboratory and Department of Oral Biology, Institute of Dental Sciences and Department of Oral and Maxillofacial
Surgery, Faculty of Dental Medicine, The Hebrew University of J erusalem, J erusalem 91120, Israel, Bone and Mineral
Metabolism Unit, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, Massachusetts 02215
Received October 17, 2001
The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic
response to local bone marrow injury. When administered in vivo, OGP stimulates osteogenesis
and hematopoiesis. The C-terminal pentapeptide OGP(10-14) is the minimal amino acid
sequence that retains the full OGP-like activity. Apparently, it is also the physiologic active
form of OGP. Residues Tyr10, Phe12, Gly13, and Gly14 of OGP are essential for the OGP(10-14)
activity. The present study explored the functional role of the peptide bonds, carboxyl and
amino terminal groups, and conformational freedom in OGP(10-14). Transformations replacing
the peptide bonds with surrogates such as Ψ(CH2NH), Ψ(CONMe), and Ψ(CH2CH2) demon-
strated that amide bonds do not contribute significantly to OGP(10-14) bioactivity. End-to-
end cyclization yielded the fully bioactive cyclic pentapeptide c(Tyr-Gly-Phe-Gly-Gly). The
retroinverso analogue c(Gly-Gly-phe-Gly-tyr), a cyclostereoisomer of c(Tyr-Gly-Phe-Gly-Gly),
is at least as potent as the parent cyclic pentapeptide. The unique structure-activity relations
revealed in this study suggest that the spatial presentation of the Tyr and Phe side chains has
a major role in the productive interaction of OGP(10-14) and its truncated and conformationally
constrained analogues with their cognate cellular target.
In tr od u ction
tivity, and matrix mineralization via an autoregulated
feedback mechanism.1,4,9 In vivo it also regulates the
expression of type I collagen and the receptor for basic
fibroblast growth factor.8 In addition, OGP and OGP-
(10-14) enhance hematopoiesis, including the stimula-
tion of bone marrow transplant engraftment and he-
matopoietic regeneration after ablative chemotherapy.10,11
Apparently, the hematopoietic effects of OGP and OGP-
(10-14) are secondary to their effect on the bone
marrow stroma.
The osteogenic growth peptide (OGP) is a naturally
occurring tetradecapeptide identical to the C-terminal
amino acid sequence 89-102 (H-Ala-Leu-Lys-Arg-Gln-
Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-OH) of histone
H4 (H4).1 The endogenous OGP is a proteolytic cleavage
product of PreOGP translated from H4 mRNA via
alternative translational initiation at a downstream
initiation codon.2 OGP in high abundance occurs physi-
ologically in human and rodent serum and in serum-
free medium of osteoblastic and fibroblastic cells. It is
mainly found as an inactive complex with R2-macroglo-
bulin (R2M). Following its dissociation from the complex
with R2M, the peptide is proteolytically cleaved, thus
generating the C-terminal pentapeptide H-Tyr-Gly-Phe-
Gly-Gly-OH [OGP(10-14)], which activates an intrac-
ellular Gi-protein-MAP kinase signaling pathway.1-6
Recently, OGP and OGP(10-14) have attracted con-
siderable clinical interest. They increase bone formation
and trabecular bone density and stimulate fracture
healing when administered to mice and rats.1,5,7,8 In
cultures of osteoblastic and other bone marrow stromal
cells derived from human and other mammalian species,
OGP regulates proliferation, alkaline phosphatase ac-
A primary structure-activity analysis carried out
recently has demonstrated that OGP(10-14) is the
minimal OGP-derived sequence that retains the full
OGP-like biological potency.5 Elimination of the amino
group at the N terminal in OGP(10-14) affords the
desaminoTyr10OGP(10-14), which retains ∼70% and
∼100% of the respective in vitro and in vivo potency of
the full-length OGP. Because of the simplified structure,
high in vivo potency, and the anticipated reduced
enzymatic susceptibility of the desaminoTyr10OGP(10-
14), we selected this analogue as our lead peptide for
detailed structural manipulations. These studies showed
that the integrity of pharmacophores such as the Tyr
and Phe side chains as well as the Gly residues at the
C terminus is important for the optimal bioactivity of
the OGP(10-14).5
Occasionally, a stepwise decrease in the peptidic
nature of bioactive peptides by the replacement of
peptide bonds with peptide bond surrogates or the
introduction of global conformational constraint by end-
to-end cyclization results in the enhancement of bio-
availability and metabolic stability. Therefore, the
present study explored the role of individual peptide
* To whom correspondence should be addressed. Address: Bone
Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine,
The Hebrew University of J erusalem, P.O. Box 12272, J erusalem
91120, Israel. Phone: 972-2-6758572. Fax: 972-2-6757623. E-mail:
babi@cc.huji.ac.il.
† Bone Laboratory, The Hebrew University of J erusalem.
‡ The Hebrew University of J erusalem.
§ Department of Oral and Maxillofacial Surgery, The Hebrew
University of J erusalem.
| Beth Israel Deaconess Medical Center and Harvard Medical School.
10.1021/jm010479l CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/15/2002

Pseudopeptidic Analogues and Cyclostereoisomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1625
bonds in the OGP(10-14) bioactivity, replacing them
with peptide bond surrogates such as N-methylated
(CONCH3) and reduced (CH2NH) amide bonds. In
addition, we developed N-to-C-terminal cyclic analogues
with reversed sequence and inverted chirality that
displayed improved in vivo and in vitro activity.
HPLC, and the reaction was generally carried out for
2-3 days until the disappearance of the linear starting
material. Under the above conditions only the cyclic
monomers 11-13, 15-20, 22, and 23 were obtained.
For the synthesis of the respective Aib- and Asp-
containing cyclic pentapeptides 23 and 24, we used
Kaiser’s oxime resin method.21 Recently, a convenient
method for the synthesis of cyclic peptides has been
developed by the use of the p-nitrobenzophenone oxime
resin.22 The peptidyl oxime ester linkage to this resin,
which has high acid stability, is highly reactive toward
primary amines. The free amino terminus of the fully
assembled oxime-resin-bound peptide interacts intramo-
lecularly with the oxime ester moiety, leading to cy-
clization with a concomitant cleavage of the cyclic
peptide from the resin. Cyclization on the resin takes
advantage of the self-diluting effect, which favors uni-
molecular reactions over the formation of oligomeric
byproducts via a bimolecular mechanism.23 The cyclic
OGP(10-14) analogues 23 and 24, synthesized via on
oxime resin cyclization, were obtained as the predomi-
nant products in the crude and were easily purified by
reversed-phase high-performance liquid chromatogra-
phy (RP-HPLC).
Syn th esis
The peptides reported herein were prepared by the
solid-phase peptide synthesis methodology using tert-
butyloxycarbonyl (Boc) chemistry and standard side
chain protection strategy.12 The N-terminal Boc group
was removed at the end of each cycle with 30% trifluo-
roacetic acid (TFA) in dichloromethane (DCM). After
neutralization with 10% N,N-diisopropylethylamine
(DIEA) in DCM, the extension of the resin-bound
peptide was carried out by the preformed symmetrical
anhydride generated by N,N′-dicyclohexylcarbodiimide
(DCC) in DCM.13 Similarly, the last coupling in the
desamino series, peptides 1-9, employed the sym-
metrical anhydride generated from the 3-(4-hydrox-
yphenyl)propionic acid. Completion of the coupling
reaction was monitored by the ninhydrin test.14 Treat-
ment of the fully assembled side chain protected, resin-
bound peptide with anhydrous hydrogen fluoride con-
taining 10% anisole as a cation scavenger simultaneously
removed the side chain protecting groups and cleaved
the peptide from the resin.15
Introduction of the Ψ(CH2NH) peptide bond isostere
into analogues 5-8 and 10 was accomplished in the
solid phase. The free N-terminal amino group of the
resin-bound peptide was reductively alkylated by the
requisite Boc-protected R-aminoaldehyde in the pres-
ence of sodium cyanoborohydride (NaBH3CN) in DMF
containing 1% AcOH. The aldehydes Boc-Tyr(OBzl)-H,
Boc-Phe-H, and Boc-Gly-H were prepared by LiAlH4
reduction of their corresponding N,O-dimethyl hydrox-
amates at 0-10 °C in dried THF.16 All components were
added at the same time to minimize racemization.17
Interestingly, in the early attempts to synthesize
analogue 9, we have obtained a hydrophobic product (k′
) 4.9) that in the FAB-MS showed a protonated mo-
lecular ion ([M + H]+) that is 338 atomic mass units
(amu) higher than the anticipated pseudopeptide 9 (data
not shown). This product corresponds to [desaminoTyr-
Gly-NHCH(CH2Φ)CH2]2Gly-Gly-OH, suggesting that
N-dialkylation of the nonhindered Gly by excess Boc-
Phe-H took place during the reductive alkylation step
and was elongated by the subsequent coupling cycles
to produce a branched product.18 Eventually, this reduc-
tive dialkylation was prevented by reducing the ratio
of Boc-Phe-H to the free amino-terminus resin-bound
Gly2 to 1.2:1. Under these conditions the anticipated
pseudopeptide 9 was obtained as the sole product. To
avoid the dialkylation of the free amino-terminus resin-
bound Gly during the reductive alkylation with Boc-Gly-
H, the pseudopeptide 8 was prepared by esterification
of Merrifield resin with the cesium salt of N-protected
pseudopeptide Boc-GlyΨ(CH2NZ)Gly-OH by the stan-
dard procedure.19
Purification of the synthetic peptides was accom-
plished in a single RP-HPLC step, yielding homoge-
neous products. The homogeneity of the peptides was
established by TLC and analytical HPLC. Their struc-
tural integrity was confirmed by mass spectrometry
and/or amino acid composition analysis (data not shown).
The physicochemical data of synthetic OGP(10-14)
analogues are detailed in Table 1.
Biologica l Eva lu a tion
Previously, it has been demonstrated that the endog-
enous OGP C-terminal pentapeptide OGP(10-14) is a
potent in vitro mitogen and in vivo stimulator of
osteogenesis and hematopoiesis.5,11 We also reported
that the elimination of the R-amino function from the
N-terminus is associated with a moderate reduction in
the in vitro potency but not the in vivo potency,5 which
can be attributed to the potentially greater metabolic
stability of the resultant desaminoTyr10OGP(10-14)
toward aminopeptidases.24 In this study we therefore
used the desaminoTyr10OGP(10-14) as a platform to
probe the contribution of the amide bonds to the
mitogenic activity of OGP(10-14).
The activity was screened in osteoblastic MC3T3 E1
and fibroblastic NIH 3T3 cell cultures. In these cell
systems, OGP and all of its active analogues so far
tested (previously and herein), including those with low
mitogenic activity, show a bell-shaped dose-response
curve, which in the MC3T3 E1 and NIH 3T3 typically
peaks at 10-13 and 10-11 M analogue concentration,
respectively.1,3-5 In addition, as previously reported,5
the relative potencies of the different analogues showed
a very high correlation between the osteoblastic and
fibroblastic cell systems (Figure 1). We therefore con-
sider the loss of activity of the presently reported, poorly
potent analogues as authentic rather than representing
a shift of the dose-response curve. Table 2 summarizes
the activity of OGP(10-14) analogues with peptide bond
surrogates. N-methylation [Ψ(CO-NMe)] of either Gly13
or Gly14 resulted in a dramatic loss of activity (analogues
The cyclization of linear peptides was carried out
under optimized conditions (high dilution, 0 °C) with
diphenylphosphoryl azide as the coupling agent.20
Progress of the cyclization reaction was followed by

1626 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Ta ble 1. Physicochemical Characteristics of OGP(10-14) Analogues
Chen et al.
TLCb
HPLC (K′),a
Rf1,
Rf2,
Rf3,
FAB-MS (MH+),
analogue no.
analogue
min
min
min
min
m/z
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
desaminoTyr10,Sar11OGP(10-14)
desaminoTyr10,N(Me)Phe12OGP(10-14)
desaminoTyr10,Sar13OGP(10-14)
desaminoTyr10,Sar14OGP(10-14)
desaminoTyr10Ψ(CH2NH)Gly11OGP(10-14)
desaminoTyr10,Gly11Ψ(CH2NH)Phe12OGP(10-14)
desaminoTyr10,Phe12Ψ(CH2NH)Gly13OGP(10-14)
desaminoTyr10Gly13Ψ(CH2NH)Gly14OGP(10-14)
desaminoTyr10Gly13Ψ(CH2CH2)Gly14OGP(10-14)
Tyr10(CH2NH)Gly11OGP(10-14)
c(Tyr-Gly-Phe-Gly-Gly)
c(Gly-Gly-Phe-Gly-Tyr)
c(tyr-Gly-phe-Gly-Gly)
retro-inverso OGP(10-14)
c(Gly-Gly-phe-Gly-tyr)
c(Tyr-Gly-Phe-Gly)
c(Gly-Tyr-Gly-Phe-Gly-Gly)
c(âAla-Tyr-Gly-Phe-Gly-Gly)
3.9 (A)
4.1 (A)
3.5 (B)
3.4 (C)
3.6 (A)
4.0 (A)
3.6 (A)
4.5 (A)
2.4 (A)
4.5 (D)
3.4 (A)
4.8 (A)
4.5 (A)
2.8 (A)
4.8 (A)
3.0 (A)
3.8 (A)
4.0 (A)
4.8 (A)
5.0 (A)
2.8 (A)
4.6 (A)
4.9 (C)
3.5 (A)
0.71
0.67
0.71
0.89
0.77
0.55
0.82
0.81
0.60
0.76
0.87
0.68
0.75
0.66
0.60
0.53
0.75
0.64
0.73
0.36
0.40
0.79
0.83
0.75
0.60
0.54
0.60
0.73
0.75
0.74
0.57
0.76
0.81
0.72
0.64
0.42
0.80
0.49
0.35
0.52
0.65
0.54
0.73
0.42
0.53
0.75
0.79
0.69
0.58
0.51
0.66
0.71
0.66
0.80
0.77
0.79
0.80
0.58
0.78
0.52
0.66
0.10
0.24
0.48
0.86
0.66
0.78
0.90
0.55
0.55
0.66
0.36
499.4
499.3
499.4
471.4
471.6
471.5
486.0
482.3
482.5
482.5
500.0
482.5
553.2
567.5
581.2
c(γAbu-Tyr-Gly-Phe-Gly-Gly)
c(δAva-Tyr-Gly-Phe-Gly-Gly)
[Pro11]OGP(10-14)
c(Tyr-Pro-Phe-Gly-Gly)
c(Tyr-Aib-Phe-Gly-Gly)
522.0
540.5
c(Tyr-Gly-Phe-Gly-Asp)-OH
a
b
Linear acetonitrile gradient (30 min) used: A, 15-40%; B, 5-95%; C, 5-25%; D, 5-35%. Solvent system used (v/v): (1) 1-BuOH/
AcOH/H2O (4:1:1); (2) 1-BuOH/AcOH/EtOAc/H2O (5:1:3:1); (3) CHCl3/MeOH/AcOH (9:3:1).
Ta ble 2. Proliferative Activity of Pseudopeptide OGP(10-14) Analogues with Peptide Bond Surrogates
relative potency (95% confidence limit)
analogue no.
analogue
MC 3T3 E1 cells
NH3T3 cells
OGP(10-14)
1.00 (reference)
0.31 (0.25-0.37)
0.52 (0.46-0.58)
0.15 (0.07-0.23)
0.16 (0.10-0.22)
0.81 (0.71-0.91)
0.61 (0.53-0.69)
0.70 (0.65-0.75)
0.78 (0.73-0.83)
0.78 (0.73-0.83)
0.92 (0.83-1.01)
1.00 (reference)
0.39 (0.26-0.52)
0.67 (0.55-0.70)
0.11 (0.05-0.17)
0.14 (0.09-0.19)
0.79 (0.67-0.91)
0.67 (0.60-0.74)
0.88 (0.76-1.00)
0.80 (0.67-0.93)
0.88 (0.79-0.97)
0.87 (0.83-0.91)
1
2
3
4
5
6
7
8
9
desaminoTyr10,Sar11OGP(10-14)
desaminoTyr10,N(Me)Phe12OGP(10-14)
desaminoTyr10,Sar13OGP(10-14)
desaminoTyr10,Sar14OGP(10-14)
desaminoTyr10Ψ(CH2NH)Gly11OGP(10-14)
desaminoTyr10,Gly11Ψ(CH2NH)Phe12OGP(10-14)
desaminoTyr10,Phe12Ψ(CH2NH)Gly13OGP(10-14)
desaminoTyr10Gly13Ψ(CH2NH)Gly14OGP(10-14)
desaminoTyr10Gly13Ψ(CH2CH2)Gly14OGP(10-14)
Tyr10Ψ(CH2NH)Gly11OGP(10-14)
10
more potent than the corresponding desamino derivative
(cf. analogues 10 and 5). Apparently, the similar potency
of analogues 8 and 9, containing a Ψ(CH2NH) and a
Ψ(CH2CH2) between Gly13 and Gly14, suggests that the
chemical nature of this bond is of minor significance.
While N-methylation of the amide bond reduces the
barrier for rotation around the modified bond and
therefore increases the population of the cis vs trans
isomer,25 the reduction and “carba” modifications of the
amide bond increase local flexibility at the site of the
peptide bond surrogate.26 Thus, the lower potency of the
N-methylated amide bond series compared to the parent
peptide and reduced amide bond series may be related
to the steric hindrance introduced by the N-CH3 group.
At this stage we cannot determine whether this modi-
fication perturbs the bioactive conformation or the fully
productive interaction of the bioactive conformation with
the putative receptor.
F igu r e 1. Regression analysis of proliferative potency of
pseudopeptide and cyclic OGP(10-14) analogues between
osteoblastic MC3T3 E1 and fibroblastic NIH 3T3 cell
lines. Values are in vitro potencies relative to activity of OGP-
(10-14).
3 and 4). This modification is more acceptable at
position 12 (analogue 2) than position 11 (analogue 1),
with the former being almost 2-fold more potent than
the latter. Interestingly, all amide bonds are equally
more tolerant to amide bond reduction [Ψ(CH2NH)]
than to N-methylation (cf. analogues 1-4 and 5-8,
Table 2). As in the case of the parent pentapeptide, the
Tyr10Ψ(CH2NH)Gly11-containing pentapseudopeptide is
Structural rigidification provides the means to delin-
eate the conformational space and offers insights into
the biologically relevant conformations. In general, end-
to-end cyclization, especially when carried out on small
peptides, is an established mode for introducing global

Pseudopeptidic Analogues and Cyclostereoisomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1627
Ta ble 3. Proliferative Activity of Cyclostereoisomers of OGP(10-14)
relative potency (95% confidence limit)
analogue no.
analogue
OGP(10-14)
c(Tyr-Gly-Phe-Gly-Gly)
c(Gly-Gly-Phe-Gly-Tyr)
c(D-Tyr-Gly-D-Phe-Gly-Gly)
retro-inverso OGP(10-14)
c(Gly-Gly-phe-Gly tyr)
MC 3T3 E1 cells
NH3T3 cells
1.00 (reference)
0.79 (0.72-0.86)
0.95 (0.89-1.01)
0.69 (0.62-0.76)
0.71 (0.52-0.90)
1.03 (0.95-1.11)
1.00 (reference)
1.12 (1.06-1.17)
1.02 (0.93-1.11)
0.84 (0.80-0.88)
0.82 (0.75-0.89)
1.16 (1.10-1.22)
11
12
31
14
15
F igu r e 2. Cyclostereoisomers of OGP(10-14) retain full OGP-
like, dose-dependent proliferative activity in osteoblastic MC3T3
E1 cell culture: (O) OGP(10-14); (b) c(Tyr-Gly-Phe-Gly-Gly);
(0) c(Gly-Gly-Phe-Gly-Tyr); (9) c(Gly-Gly-phe-Gly-tyr). Data
are mean ( SE obtained in triplicate culture wells per
condition.
rigidification.27 Remarkably, all four cyclostereoisomers
of the C-terminal OGP-derived pentapeptide, the all-L-
isomer (11), the retro-isomer (12), the all-D-isomer (13),
and the retro-inverso isomer (15), were highly mitogenic
(Table 3), demonstrating a dose/response relationship
identical to that of the parent OGP(10-14) (Figure 2).
The cyclic retro-inverso pentapeptide 15 was the most
active cyclostereoisomer, significantly more active than
the parent linear peptide OGP(10-14) and substantially
more potent than the linear retro-inverso pentapeptide
14 (Table 3). These results suggest that the charged
amino and carboxyl end groups and the nonpalindro-
micity of the backbone do not have an important role
in the interaction with the putative OGP receptor.
Moreover, global conformational constraint is not det-
rimental to bioactivity. Comparison of the potencies of
the enantiomeric pairs 11 and 13, and 12 and 15
indicates opposite effects of chain reversal and chirality
switch. The all-L-isomer 11 is more potent than its
enantiomer 13 (same chain direction but opposite
chirality), and the retro-inverso isomer 15 (reversed
chain direction and opposite chirality) is more active
than the retro-isomer 12 (reversed chain direction and
the same chirality). Formally, the retro-inverso analogue
15, the most reactive cyclostereoisomer, is topologically
equivalent to the all-L-isomer 11.
F igu r e 3. c(Tyr-Gly-Phe-Gly-Gly) rescues CFU-Obl derived
from femoral and tibial bone marrow of OVX rats. (A) Shown
is the percent CFU-Obl of total CFU-f in sham-OVX, untreated
control rats, and OVX rats treated with either PBS vehicle or
indicated peptides. Data are mean ( SE obtained in five rats
per condition. (B, C) double ALP/ARS staining of CFU-f derived
from OVX rats treated with OGP(10-14) (B) or PBS (C). Note
the absence of single-positive ARS colonies.
in line with our previous demonstration of anabolic
activity in the bone of the full-length OGP, OGP(10-
5
14), and desaminoTyr10OGP(10-14).1, The CFU-Obl
stain positively for both alkaline phosphatase (ALP) and
alizarin-red-S (ARS, staining of mineral deposits). Be-
cause mineralization is the final stage of osteogenic
differentiation preceded by the expression and activity
of ALP,29 ARS positive deposits typically occur only in
the center of ALP positive colonies (Figure 3B,C). In this
context it should be pointed out that the OGP(10-14)
and its all-L-cycloisomer affected only the number of the
double-positive ALP/ARS colonies and not the number
of total or single-positive ALP CFUs (data not shown).
It is therefore suggested that in the bone marrow
system, OGP(10-14) and its cyclic analogues preferen-
In vivo, even the all-L-cycloisomer (11) was more
effective than the less rigid linear OGP(10-14) in
reversing the ovariectomy (OVX)-induced decrease in
colony-forming units, osteoblastic (CFU-Obl) (Figure 3).
Ex vivo CFU-Obl is a surrogate of the in vivo number
of preosteoblastic cells capable of terminal osteoblastic
differentiation and bone formation.28 The stimulation
of CFU-Obl by OGP(10-14) and its all-L-cycloisomer is

1628 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Chen et al.
Ta ble 4. Proliferative Activity of Variable Ring Size Cyclic OGP(10-14) Analogues
relative potency (95% confidence limit)
MC 3T3 E1 cells NH3T3 cells
analogue no.
analogue
OGP(10-14)
1.00 (reference)
0.35 (0.30-0.40)
0.26 (0.19-0.33)
0.36 (0.30-0.42)
0.20 (0.16-0.24)
0.14 (0.09-0.19)
1.00 (reference)
0.43 (0.40-0.46)
0.20 (0.17-0.23)
0.37 (0.31-0.43)
0.22 (0.19-0.25)
0.18 (0.13-0.23)
16
17
81
19
20
c(Tyr-Gly-Phe-Gly)
c(Gly-Tyr-Gly-Phe-Gly-Gly)
c(âAla-Tyr-Gly-Phe-Gly-Gly)
c(γAbu-Tyr-Gly-Phe-Gly-Gly)
c(δAva-Tyr-Gly-Phe-Gly-Gly)
Ta ble 5. Proliferative Activity of Miscellaneous Cyclic OGP(10-14) Analogues
relative potency (95% confidence limit)
analogue no.
analogue
MC 3T3 E1 cells
NIH3T3 cells
OGP(10-14)
1.00 (reference)
0.89 (0.80-0.98)
0.99 (0.79-1.19)
0.19 (0.14-0.24)
0.14 (0.09-0.19)
1.00 (reference)
0.96 (0.87-1.05)
0.90 (0.77-1.03)
0.31 (0.29-0.33)
0.11 (0.07-0.15)
21
22
23
24
[Pro11]OGP(10-14)
c(Tyr-Pro-Phe-Gly-Gly)
c(Tyr-Aib-Phe-Gly-Gly)
c(Tyr-Gly-Phe-Gly-Asp)-OH
tially stimulate cells in their late stage of osteoblastic
differentiation.
this function was “sacrificed” during the end-to-end
cyclization. To this end, we prepared the cyclic pen-
tapeptide 24 in which Gly14 is replaced with Asp, thus
yielding a carboxyl-bearing side chain, which mimics the
original C-terminal carboxyl in the linear parent pen-
tapeptide (Table 5). The 5- to 10-fold decrease in potency
of analogue 24 compared to the parent cyclopentapep-
tide 11 suggests that the presentation of the carboxyl
in the form of Asp14 is compromising the interaction
with the putative receptor. It cannot be ruled out that
introduction of Asp14 presents the carboxyl in an
unfavorable spatial orientation for taking advantage of
a putative complementary charge within the receptor.
Contraction (analogue 16, a 12-member ring) or
expansion (analogues 17-20, 18- to 21-member rings,
respectively) of the ring size of the cyclopentapeptide
11 (a 15-member ring) leads to substantial loss in
proliferative potency (Table 4). Evidently, over-rigidi-
fication, as in the case of the cyclotetrapeptide 16,
results in 2- to 3-fold reduction in potency compared to
the cyclopentapeptide 11. It may suggest that the
contraction in ring size eliminates some degree of
conformational freedom, which is apparently important
for the formation of the putative bioactive conformation.
An even greater loss of activity results from increasing
the degree of conformational freedom by adding, be-
tween the ring-forming end groups, a Gly residue or
homologues thereof as spacers, thus generating homolo-
gous cyclohexapeptides that are 2- to 7-fold less potent
than the linear parent pentapeptide and the cyclopen-
tapeptide 11. These results suggest that the cyclo-
hexapeptides 17-20, which are conformationally con-
strained in comparison to the flexible linear pentapeptide,
not only do not favor the putative bioactive conformation
but may also discriminate against it.
Con clu sion
Our studies of the OGP structure-activity relation-
ship led to the identification of the C-terminal pen-
tapeptide, H-Tyr-Gly-Phe-Gly-Gly-OH, as the minimal
sequence required for full biological activity. In this
sequence, the Tyr and Phe side chains act as the critical
pharmacophores. The promiscuity of the peptidic and
nonpalindromic nature of the backbone allowed for the
development of the retro-inverso cyclic pentapeptide,
c(Gly-Gly-phe-Gly-tyr), a fully potent cyclostereoisomer
of the c(Tyr-Gly-Phe-Gly-Gly), which is equipotent to the
linear OGP(10-14). These findings suggest that the
molecular topology in general and the spatial disposition
of the pharmacophores in particular play a key role in
the recognition and activation of the putative macro-
molecular OGP receptor.
Local rigidification between Tyr10 and Phe13 was
achieved by replacing Gly12 with either Pro or Aib (Table
5). The former represents an intraresidue side-chain-
to-backbone rigidification, while the latter represents
a rigidification that originates from steric hindrance
contributed by the disubstituted CR carbon. Interest-
ingly, position 12 accommodates substitution by Pro,
both when introduced to the linear pentapeptide 21 or
to the cyclopentapeptide 22. However, introduction of
Aib11 results in a 4- to 10-fold loss of potency. It may be
speculated that the different conformational biases of
Pro and Aib may lead to differential effects on the
bioactive conformation. For example, Aib, which is
known to promote helical conformation,30 is not toler-
ated, while Pro, which is a helix breaker,31 is well
tolerated.
Exp er im en ta l Section
Gen er a l. Boc-amino acids32 were purchased from either
Bachem California or prepared with di-tert-butyl dicarbonate
by a conventional procedure.33 All chemicals were purchased
from either Aldrich Chemica Co., Fluka Chemie AG Switzer-
land, or Pierce Chemical Co. and were used without further
purification. Where appropriate, solvents were freshly distilled
from proper drying materials before use. The tyrosine side
chain was protected as an O-benzyl ether, namely, Boc-Tyr-
(O-Bzl)-OH. Peptides were treated with liquid HF in an all-
Teflon apparatus (Protein Research Foundation, Osaka, J a-
pan). Thin-layer chromatography (TLC) was performed on
precoated silica gel plates 60F-254 (E. Merck, Darmstadt,
FRG) in the following solvent systems (all v/v): (1) 1-BuOH/
AcOH/H2O (4:1:1); (2) 1-BuOH/AcOH/EtOAc/H2O (5:1:3:1); (3)
CHCl3/MeOH/AcOH (9:3:1). Peptides were visualized by one
Earlier studies carried out in our laboratories sug-
gested that the C-terminal free carboxyl is crucial for
mitogenic activity. Amidation, esterification, and reduc-
tion of this carboxyl led to a 2- to 30-fold loss in potency.5
We revisited the role of the carboxyl function in the
context of the cyclic C-terminal pentapeptide, in which

Pseudopeptidic Analogues and Cyclostereoisomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1629
or two of the following procedures: (1) UV; (2) ninhydrin spray
reagent. Analytical and semipreparative HPLC separations
were performed on a Merck Hitachi 655A-11 liquid chromato-
graph, equipped with a model 655A variable wavelength and
model L-5000 LC controller, model D-2000 chromatointegrator,
and a model AS-2000 autosampler injector. Absorptions were
measured at 220 nm. For all analytical applications, a Lichro-
spher 100 RP-18 column was used. The crude peptides were
purified on a µBondapak C-18, 19 cm × 150 cm column or a
Vydac Protein & Peptide C-18 column. HPLC was carried out
using a gradient made from two solvents: (A) 0.1% TFA in
water and (B) 0.1% TFA in acetonitrile. The gradient condi-
tions used for purification were determined by analytical
HPLC. The flow rates used for the analytical and semi-
preparative columns were 1 and 6 mL/min, respectively. The
analytical column was protected with a C-18 guard cartridge.
The crude peptides were dissolved in appropriate solvents and
then filtered through a PTFE filter (0.2 mm) and directly
injected. In some cases, a repeated purification step was
required to obtain high purity. Fractions containing the
purified peptides were pooled and lyophilized.
P ep tid e Syn th esis. (A) Syn th esis of Lin ea r P ep tid es,
Gen er a l. The OGP analogues were synthesized on a Milligen
504 synthesizer (manual) or a 430A Applied Biosystem peptide
synthesizer unless otherwise indicated. Boc-amino acids were
assembled on a PAM resin or Merrifield resin. The PAM resin
was purchased with the first Boc-amino acid already attached.
The Boc-amino acid-resin was subjected to the required
number of coupling and deprotection cycles according to the
SPPS method of choice. In each cycle, resins were treated with
25% TFA in the presence of 1% dimethyl sulfide (DMS) in
DCM to remove Boc groups. After neutralization with 10%
DIEA in DCM, the resin was treated with a 4-fold excess of
the appropriate DCC-mediated preactivation of the Boc-amino
acids in DCM. Where possible, completion of each coupling step
was monitored by the Kaiser test. The fully assembled peptides
were removed from the resin either by aminolysis (in the case
of Merrifield resin-bound peptide) or by treatment with
anhydrous hydrogen fluoride (HF, 18 mL/g resin-bound pep-
tide) containing anisole (2 mL/g resin-bound peptide) as a
cation scavenger at -5 °C for 1 h. After the removal of HF in
vacuo, the residues were precipitated with diethyl ether and
extracted from the resin with 30% aqueous acetic acid. The
aqueous fractions were combined and then lyophilized.
The peptides were assayed for purity using analytical HPLC
(Vydac C-18 column) and were shown to be >95% pure.
Peptides were hydrolyzed for amino acid analysis in 6 N HCl
for 24 h at 110 °C in vacuum-sealed tubes. The samples were
analyzed after precolumn derivatization as Fmoc-amino acid,
performed at the Institute of Life Sciences, The Hebrew
University of J erusalem, J erusalem, Israel. Molecular weights
of the peptides were determined by FAB-MS at the Faculty of
Chemistry, Technion, Haifa, Israel.
Boc-N-Me-P h e-OH. The preparation followed a previ-
ously reported procedure.34 Briefly, Boc-Phe-OH was dissolved
in dry THF and treated with MeI (8 equiv). NaH (60%
dispersion in oil, 3 equiv) was added portionwise to the reaction
mixture, and the reaction was monitored by HPLC. The
solvent was removed in vacuo, yielding a crude product that
was purified by flash column chromatography eluted with
EtOAc/petroleum ether to yield the pure product.
Desam in oTyr 10Ψ[CH2NH]Gly11-P h e-Gly-Gly-OH (5). 3-(4-
hydroxyphenyl)propionic acid (1.33 g, 8.0 mmol) was dissolved
in dry DCM (50 mL), and the salt of N,O-dimethylhydroxy-
lamine (0.9 g, 9.2 mmol), Et3N (1.22 mL, 8.8 mmol), DCC (1.65
g, 8.0 mmol), and p-(dimethylamino)pyridine (DMAP) (0.0486
g, 3.9 mmol) were added. The solution was stirred overnight
at room temperature, and then the DCCU was filtered off and
the solvents were evaporated to dryness. The residue was
dissolved in EtOAc and was washed with a saturated aqueous
solution of NaCl (2 × 20 mL), a saturated aqueous solution of
NaHCO3 (2 × 20 mL), a 10% aqueous solution of citric acid (2
× 20 mL), and finally a saturated aqueous solution of NaCl.
The organic layer was dried over anhydrous MgSO4 and was
evaporated to dryness. The crude product was purified by
column chromatography on silica gel eluted with EtOAc/
petroleum ether, obtaining 1.03 g (62%) of HO-C6H5CH2CH2-
CON(OMe)Me as an oil.
HO-C6H5CH2CH2CON(OMe)Me (0.24 g, 1.44 mmol) was
dissolved in anhydrous diethyl ether, and the solution was
cooled to 0 °C, and then 0.069 g (1.8 mmol) of LiAlH4 was
added in small portions. After 3 h, 3 mL of a 10% aqueous
solution of KHSO4 was added dropwise and the aqueous layer
was extracted with diethyl ether (3 × 30 mL). The organic
layer was collected and washed with 3 N HCl (2 × 20 mL), a
saturated solution of NaHCO3 (2 × 20 mL), and a saturated
solution of NaCl (2 × 20 mL). The solution was then dried
over anhydrous MgSO4 and was evaporated to dryness to
obtain 0.17 g (77%) of 4-HO-C6H5CH2CH2CHO, which was
used without further purification.
3-(4-Hydroxyphenyl)propional (50 mg, 0.33 mmol) was dis-
solved in dry DMF (10 mL) containing 1% AcOH, and the
solution was added to H-Gly-Phe-Gly-Gly-PAM resin (0.3
mmol, prepared by SPPS) followed by the addition of NaC-
NBH3 (0.4 mmol) in several portions. The reaction mixture
was allowed to shake overnight at room temperature followed
by removal of the N-terminal protecting group with 25% TFA
in DCM and a liquid HF/anisole cleavage step. The crude
peptide (84 mg) was purified on preparative HPLC to yield
the pure peptide (8 mg).
Desam in oTyr -Gly11Ψ(CH2NH)P h e12-Gly-Gly-OH (6). The
N,O-dimethyl hydroxamate of Boc-Gly-OH was obtained as a
white solid from the DCC coupling of Boc-Gly-OH with N,O-
dimethylhydroxylamine in the presence of DMAP and then
was converted to Boc-glycinal by LiAlH4 reduction. The peptide
resin Phe-Gly-Gly-PAM was reductively alkylated with Boc-
glycinal, as above, and then elongated by coupling with 3(4-
hydroxyphenyl)propionic acid followed by cleavage of the
peptide from the resin with HF/anisole. The crude peptide was
purified using semipreparative HPLC with 0-60% acetonitrile
in 120 min, thus affording the pure peptide.
Desam in oTyr -Gly-P h e12Ψ(CH2NH)Gly13-Gly-OH (7). The
N,O-dimethyl hydroxamate of Boc-Phe-OH was obtained as a
colorless oil from the BOP-mediated coupling of Boc-Phe-OH
with N,O-dimethylhydroxylamine in the presence of triethy-
lamine. Reduction of the N,O-dimethyl hydroxate of Boc-Phe-
OH with LiAlH4 afforded Boc-phenylalaninal (Boc-Phe-H) as
a white solid, which was used without further purification. A
total of 1.25 equiv of the crude aldehyde was dissolved in dry
DMF (15 mL) containing 1% AcOH, and the solution was
added to H-Gly-Gly-PAM resin (0.4 mmol), which had been
prepared by the SPPS procedure described above. This was
followed by the addition of NaCNBH3 (0.5 mmol) in several
portions. The reaction mixture was allowed to shake overnight,
and then Z-Cl and TEA (5 equiv) were added to protect the
secondary amine between Phe and Gly. The reaction was
carried out overnight and the N-terminal Boc group was
removed by 25% TFA in DCM. The peptide was further
elongated by coupling sequentially with Boc-Gly and 3-(4-
hydroxyphenyl)propionic acid, followed by cleavage from the
resin with liquid HF/anisole. The crude peptide was purified
using semipreparative HPLC with 0-60% acetonitrile in 120
min, which afforded the desired peptide.
Desa m in oTyr -Gly-P h e-Gly13Ψ(CH 2NH )Gly14-OH (8).
Boc-NHCH CH2NH2 (Boc-EDA). A solution of di-tert-butyl
dicarbonate 2(4.3 g, 19.7 mmol) in DCM (70 mL) was added
dropwise (5 mL/min) to a solution of 1,2-diaminoethane (13
mL, 195 mmol) in DCM (70 mL). The reaction mixture was
then left to stir for 24 h at room temperature, followed by
extraction with cold 1 N KHSO4 (3 × 20 mL). The aqueous
phase was pooled together, and solid NaOH was added until
pH 10-11 was reached. The alkaline aqueous solution was
extracted with DCM (3 × 20 mL), which was pooled together
and dried over MgSO4. Evaporation under reduced pressure
yielded Boc-EDA as an oil (1.52 g, 48.3%).
Boc-GlyΨ(CH2NH)Gly-OH. A solution of chloroacetic acid
(2.3 g, 24.3 mmol) in aqueous NaOH (1 N, 24.4 mL) was added
dropwise to a solution of Boc-EDA (2.46 g, 15.3 mmol) in

1630 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Chen et al.
ethanol (95%, 6 mL) followed by addition of 1 N NaOH (12.2
mL). The reaction mixture was stirred overnight at room
temperature, and the ethanol was removed in vacuo. The
residue was extracted with diethyl ether (3 × 20 mL). The
aqueous phase was cooled to 0 °C, and benzyloxycarbonyl
chloride (1.9 g) was added dropwise, maintaining pH 9-10
with 1 N NaOH. After being stirred for 5 h at room temper-
ature, the reaction mixture was extracted with diethyl ether,
cooled to 0 °C, acidified with cold citric acid solution (30%),
and washed with EtOAc. Evaporation of the washed and dried
EtOAc extract resulted in an oil that was recrystallized on
trituration with petroleum ether to yield 2.3 g (42.6%).
The Boc-protected pseudodipeptide (0.7 g, 2 mmol) was
attached to Merrifield resin using the cesium salt. The resin-
bound peptide was treated with 25% TFA in DCM, neutralized
with DIEA, and then elongated with Boc-Phe-OH, Boc-Gly-
OH and 3-(4-hydroxyphenyl)propionic acid. After cleavage from
the resin, the crude peptide was purified by preparative HPLC
to yield the pure product.
(B) Cyclic An a logu es. Meth od 1. Solu tion Cycliza tion .
The crude linear peptides were purified on preparative HPLC,
or the nonpurified peptide was used directly for cyclization.
Solid NaHCO3 (5 equiv) was added to a cooled (0 °C) solution
of the linear peptide in an anhydrous, amine-free DMF (0.008
M), and the suspension was stirred for 5 min. After addition
of diphenylphosphoryl azide (DPPA) (1.5 equiv), the reaction
mixture was stirred at 0-5 °C for 2-4 days and HPLC-
monitored. Upon completion of the reaction, the solvent was
evaporated to dryness in vacuo (30 °C bath). The residues were
purified on preparative HPLC to yield the pure cyclic peptides.
In Vitr o P r olifer a tion Assa y. This assay was carried out
as reported recently.5 Briefly, osteoblastic MC3T3 E1 and
fibroblastic NIH 3T3 cells derived from confluent maintenance
cultures grown in minimal essential medium (RMEM) supple-
mented with 10% fetal calf serum (FCS) were seeded in 16
mm multiwell dishes at 2 × 104 cells per well and incubated
in the same medium at 37 °C in CO2/air. For the initial 46 h
the cells were incubated in the same FCS-containing medium.
The cultures were then washed and kept for an additional 2 h
period under serum-free conditions. This was followed by
replacement of the serum-free RMEM with a medium contain-
ing 4% fatty acid free bovine serum albumin (BSA) (Sigma
Chemical Co., St. Louis, MO; catalog no. A-7030) with or
without OGP(10-14) (positive control) or its analogue prepa-
rations. The analogue-containing culture medium was pre-
pared as follows: the corresponding OGP(10-14) analogue [or
OGP(10-14)] was freshly weighed, dissolved in the BSA-
supplemented medium at 10-6 M, and further diluted to the
final concentration with the same medium. Prior to use in the
cell culture, the BSA-supplemented RMEM with the final
analogue concentration was preincubated for 30 min at 37 °C.
Unless otherwise specified, the analogue concentration was
10-13 and 10-11 M in the MC3T3 E1 and NIH 3T3 cell culture,
respectively. By use of a variety of OGP and OGP(10-14)
analogues, these concentrations were shown previously to
induce maximal proliferative activity in the corresponding cell
cultures.3 To verify the relevance of these concentrations for
the present study, some of the analogues were subjected to a
dose-response analysis (Figure 2). Cell counts were carried out
using a hemocytometer 48 h after addition of the BSA
supplemented, analogue-containing RMEM.
Meth od 2. On -Resin Cycliza tion Em p loyin g Oxim e
Resin . Boc-amino acids and DCC (1 equiv) were added to a
suspension of the oxime resin (2 equiv) in DCM in a shaking
reaction vessel. After 15 h, the solvent was drained and the
resin was washed with DCM, DCM/EtOH (1:1), and DCM.
Excess oxime functional groups were capped with AcOH (1.5
equiv) and DIEA (1.5 equiv) in DCM. The Boc-amino acyl-
oxime resin was obtained after washing with DCM, EtOH/
DCM (1:1), EtOH, and DCM and was dried in vacuo. The level
of resin substitution was determined with picric acid. Single
couplings were performed with 3 equiv of a suitable protected
amino acid activated by BOP. A typical cycle for the coupling
of an individual amino acid was as follows: (i) activation of
the next amino acid to be coupled with BOP; (ii) deprotection
of the amino acid on the resin with 25% TFA/DCM + 1% DMS
as a scavenger; (iii) washing with DCM and neutralization
with 10% DIEA/DCM; (iv) mixing the BOP-activated amino
acid with the neutralized resin-bound peptide. The efficiency
of each coupling was monitored by the Kaiser test.
After final removal of the Boc protecting group from the N
terminus, the amino group was liberated from its TFA salt by
addition of DIEA (1.5 equiv). Intrachain aminolysis by the free
amino group cleaved the peptide from the oxime resin,
generating concomitantly the cyclic peptide. These cleavage/
cyclization reactions were carried out in DCM at room tem-
perature and catalyzed by the presence of 1.5 equiv of AcOH.35
After 24 h of reaction time, the cyclic peptides were obtained
from the solution phase by filtration and purified by prepara-
tive HPLC to yield a pure cyclic peptide.
Dep r otection P r oced u r e. Catalytic hydrogenation was
carried out in a solution mixture of 10% AcOH/MeOH under
atmospheric pressure at room temperature in the presence of
5% Pd black using a peptide-to-catalyst ratio of 3:1. After
complete removal of the benzyl-type protecting groups as
monitored by analytical HPLC, the solution was filtered, the
filtrate concentrated in vacuo, and the residue subjected to
purification on preparative HPLC.
Peptide purification was evaluated using analytical HPLC
(Vydac C-18 column) and was shown to be >95% in all cases.
Peptides were hydrolyzed for amino acid analysis in 6 N HCl
for 24 h at 110 °C in deaerated tubes. The samples were
analyzed after precolumn derivatization as the Fmoc-amino
acid. Molecular weights of the peptides were determined by
FAB-MS.
The results were initially expressed as the mean cell number
in triplicate culture wells per condition. The value obtained
was then transformed into peptide-treated over peptide-free
control ratios (T/C ratio) and then presented as relative
potency using results obtained in cultures challenged with
OGP(10-14) as reference. As in previous studies of the same
nature,36 the relative potencies of individual analogues in each
of the cell lines were expressed as the mean and 95%
confidence limit obtained in at least four repetitive experi-
ments.
R ever sa l of OVX-In d u ced R ed u ct ion in CF U-Ob l.
Fifteen female Sabra rats (The Hebrew University animal
facility, J erusalem), each weighing 250 g, were subjected to
bilateral OVX. Additional five control animals underwent
sham OVX. All animals were left untreated for 30 days. Then
the OVX animals were divided into three groups of five rats
each that were treated daily for 8 weeks by subcutaneous
injections of 100 ng/day/rat of either OGP(10-14) or c(Tyr-
Gly-Phe-Gly-Gly) or the peptide-free PBS vehicle. The animals
were sacrificed 1 day after treatment termination by an
anesthetic overdose (Avertin, 400 mg/Kg). Prior to death, bone
marrow from the two femoral and two tibial diaphyses of each
animal was flushed into tissue culture medium (RMEM) and
pooled. Determination of stromal colony forming units, fibro-
blastic (CFU-f, which include the CFU-Obl), was set as
described previously.28 Briefly, a suspension of single cells was
prepared by withdrawing and expelling the bone marrow
through graded syringe needles. The cells were seeded in 35
mm dishes at 106 cells/dish, 10 dishes per animal using RMEM
supplemented with 50 mg/mL ascorbic acid, 10 mM â-glyc-
erophosphate, and 10% fetal calf serum; these supplements
promote osteoblastic differentiation and formation of mineral-
ized nodules.29 The medium was changed twice a week.
On day 21 in culture, the CFU-f were counted and then
washed with PBS, fixed with citrate/acetone/formalin, and
double-stained for ALP (Sigma 86-R kit) and mineral deposits
(1% aqueous ARS). Counts of ALP positive colonies and double-
positive ALP/ARS colonies (representing CFU-Obl) then fol-
lowed. A colony was defined as a cluster of cells containing 16
or more members. The scores from each 10 dishes were
averaged for the individual animals. Results were expressed
as percent ALP positive CFU or percent CFU-Obl of the total
CFU-f.

Pseudopeptidic Analogues and Cyclostereoisomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1631
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Active R-(t-Butoxycarbonylamino)-Aldehydes from R-Amino Ac-
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Phase Synthesis of Peptides Containing the CH2NH Peptide
Bond Isostere. Peptides 1987, 8, 119-121. (c) Coy, D. H.; Hocart,
S. J .; Sasaki, Y. Solid Phase Reductive Alkylation Techniques
in Analogue Peptide Bond and Side-Chain Modification. Tetra-
hedron Lett. 1988, 44, 835-841.
Ack n ow led gm en t. This study was supported by
Grant No. 91-270 from the United States-Israel Bina-
tional Science Foundation and Grant No. 6305194 from
the Ministry of Science and The Arts, The Government
of Israel. Y.C. was a postdoctoral research fellow of The
Hebrew University of J erusalem.
(17) Ho, P. T.; Chang, D.; Zhong, J . W. X.; Musso, G. F. An Improved
Low Racemization Solid-Phase Methods for the Synthesis of
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Biosynthesis of Osteogenic Growth Peptide Via Alternative
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phosphatase; ARS, alizarin-red-S.; BH, Bolton-Hunter reagent;
BSA, bovine serum albumin; CFU-f, colony-forming units fibro-
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propionic acid; desaminoPhe, 3-phenylpropionic acid; desami-
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J M010479L