Citation
N- and O- Acylation of Peptides and Sugars in Partially Aqueous Media

Material Information

Title:
N- and O- Acylation of Peptides and Sugars in Partially Aqueous Media
Creator:
Cusido, Yanet
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (67 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Katritzky, Alan R.
Committee Members:
Castellano, Ronald K.
Hong, Sukwon
Graduation Date:
12/14/2007

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Carbon ( jstor )
Carboxylic acids ( jstor )
Esters ( jstor )
Microcrystals ( jstor )
Microwaves ( jstor )
Protons ( jstor )
Room temperature ( jstor )
Solvents ( jstor )
Sugars ( jstor )
Chemistry -- Dissertations, Academic -- UF
benzotriazole, carbohydrates, dipeptides, fluorescent, sugars, tripeptides
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, M.S.

Notes

Abstract:
The convenient preparation of N-(Fmoc- or Z-alpha-aminoacyl)benzotriazoles and N-protected peptidoylbenzotriazoles from aspartic and glutamic amino acids is discussed. Additionally, diverse N-protected di- and tripeptides are synthesized under mild reaction conditions in good to excellent yields by acylation with N-(Z- and Fmoc-alpha-aminoacyl)benzotriazoles of the amino groups of free aspartic and glutamic acids. Examples of peptide coupling utilizing free amino acids in partially aqueous solution are reported and the products are obtained without the use of chromatography. Evidence of maintained chirality was supported by NMR and HPLC. In addition, we present the suitable and efficient fluorescent labeling of sugars by O-acylation of diisopropylidene protected sugars and N-acylation of pivaloyl protected aminosugar with N-(coumarin-3-carbonyl)benzotriazole and benzotriazole derivatives of N-epsilon-coumarin-labeled N-alpha-protected-L-lysines under microwave irradiation or/and at room temperature. Monosaccharide containing Fmoc-lysine fluorescent building blocks can be useful as water soluble organic fluorophores for peptide labeling at the C-terminus in solid-phase peptide synthesis (SPPS). ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2007.
Local:
Adviser: Katritzky, Alan R.
Statement of Responsibility:
by Yanet Cusido.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Cusido, Yanet. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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(LS)-Benzyl-3-(((9H-fluoren-9-yl)methoxy)croymi)-(1123beztiol-

yl)-4-oxobutanoate (Fmoc-L-Asp(OBzl)-Bt, 2.8a): White microcrystals (87%); mp 91-92 oC,

[a]D23 = -26.5 (c 2.58, DMF). 1H NMR (DMSO-d6): 6 3.03 (dd, J= 16.8, 8.8 Hz, 1H), 3.30 (dd, J

= 16.8, 5.1 Hz, 1H), 4.24 (t, J= 6.3 Hz, 1H), 4.37 (d, J= 6.9 Hz, 2H), 5.14 (s, 2H), 5.87-5.92 (m,

1H), 7.29-7.44 (m, 9H), 7.65 (t, J= 7.5 Hz, 1H) 7.71 (d, J= 7.4 Hz, 2H), 7.81 (t, J= 7.5 Hz,

1H), 7.90 (d, J= 7.4 Hz, 2H), 8.23 (d, J= 8. 1 Hz, 1H), 8.30 (d, J= 8.2 Hz, 1H), 8.46 (d, J= 7. 1

Hz, 1H). 13C NMR (DMSO-d6): 6 35.3, 46.6, 51.0, 66.0, 66.2, 114.0, 120.2, 120.3, 125.2, 126.8,

127.1, 127.7, 128.0, 128.1, 128.4, 130.7, 131.2, 135.7, 140.8, 143.7, 145.4, 156.0, 169.5, 170.3.

Anal. called for C32H26N405: C, 70.32; H, 4.79; N, 10.25. Found: C, 70.08; H, 5.14; N, 9.47.

(LS)-Benzyl-4-(((9H-fluoren-9-yl)methoxy)croymi)-(1123beztiol-

yl)-5-oxopentanoate (Fmoc-L-Glu(OBzl)-Bt, 2.8b): White microcrystals (83%); mp 96-97 oC,

[a]D23 = -22.5 (c 2.08, DMF). 1H NMR (DMSO-d6): 6 2. 15-2.27 (m, 1H), 2.32-2.40 (m, 1H),

2.68 (t, J= 7. 1 Hz, 2H), 4.27 (t, J= 6.5 Hz, 1H), 4.37 (d, J= 6.9 Hz 2H), 5.03-5.13 (m, 2H),

5.54-5.63 (m, 1H), 7.30-7.47 (m, 9H), 7.66 (t, J= 7.7 Hz, 1H), 7.75 (d, J= 7.3 Hz, 2H), 7.84 (t, J

= 7.4 Hz, 1H), 7.92 (d, J= 7.3 Hz, 2H), 8.26 (d, J = 8.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.39

(d, J= 7.0 Hz, 1H). 13C NMR (DMSO-d6): 6 25.9, 29.9, 46.6, 53.6, 65.6, 65.9, 114.1i, 120.1i,

120.2, 125.2, 126.8, 127.1, 127.7, 127.9, 128.0, 128.4, 130.7, 131.1, 136.0, 140.8, 143.7, 145.4,

156.3, 171.5, 171.9. Anal. called for C33H28N405: C, 70.70; H, 5.03; N, 9.99. Found: C, 70.35; H,

5.09; N, 9.91.

Benzyl (S)-4-benzotriazol-1-yl-2-benzyloxycarbonyaio4oouaot (Z-L-Asp-

OBzl-Bt, 2.11a): White microcrystals (91%), mp 97-99 oC, [a]D23 = -22.5 (c 2.08, DMF). 1H

NMR (CDCl3): 6 4.01 (dd, J= 18.1i, 4.7 Hz, 1H), 4. 14 (dd, J= 18.1i, 4.6 Hz, 1H), 4.96-5.03 (m,

1H), 5.11 (s, 2H), 5.20 (s, 2H), 5.90 (d, J= 8.2 Hz, 1H), 7.21-7.32 (m, 10H), 7.52 (t, J= 7.5 Hz,










mL of CH2 12 WAS added, washed with 4N HCI soln (3 x 15 mL), sat. NaCl soln (10 mL) and

dried over MgSO4. After evaporation of solvent, the residue was recrystallized from CH2 12-

hexanes.

6-O-Coumarin-3-carbonyl-1,2:3,4-di-O-isopoyie--Dgltprnse 3.12:

White microcrystals (90%), mp 146.2 148.0 oC, 1H NMR (CDCl3): 6 1.34 (s, 3H), 1.36 (s, 3H),

1.48 (s, 3H), 1.54 (s, 3H), 4. 16 4.23 (m, 1H), 4.33 4.40 (m, 2H), 4.47 (dd, J= 11.4, 7.6 Hz,

1H), 4.55 (dd, J= 11.5, 4.9 Hz, 1H), 4.66 (dd, J= 7.8, 2.3 Hz, 1H), 5.56 (d, J= 4.9 Hz, 1H), 7.30

- 7.38 (m, 2H ), 7.58 7.70 (m, 2H), 8.53 (s, 1H). 13C NMR (CDCl3): 6 24.4, 24.9, 25.9, 26.0,

54.5, 64.4, 65.9, 70.6, 70.9, 96.2, 108.9, 109.6, 116.8, 117.8, 117.9, 124.8, 129.5, 134.4, 148.8,

155.1, 156.5, 162.6. Anal. called for C22H2409: C, 61.11; H, 5.59; N, 0.00. Found: C, 61.06; H,

5.71; N, 0.19.

3-O-Coumarin-3-carbonyl- 1,2: 5,6-Di-O-isopropylidene-a-D-glucose, 3.13: White

microcrystals (89%), mp 65.1 66.7 oC 1H NMR (CDCl3): 6 1.32 (s, 3H), 1.33 (s, 3H), 1.43 (s,

3H), 1.56 (s, 3H), 4.07 (dd, J= 8.8, 4.7 Hz, 1H), 4.15 (dd, J= 8.7, 5.9 Hz, 1H), 4.29 (dd, J =

8.5, 3.0 Hz, 1H), 4.45 4.51 (m, 1H), 4.68 (d, J= 3.8 Hz, 1H), 5.48 (d, J= 3.0 Hz, 1H), 5.97 (d,

J= 3.8 Hz, 1H), 7.32 7.40 (m, 2H), 7.60 7.72 (m, 2H), 8.53 (s, 1H). 13C NMR (CDCl3): 6

25.2, 26.1, 26.7, 26.9, 67.4, 72.4, 77.6, 79.9, 83.1, 105.1, 109.4, 112.3, 116.8, 117.6, 124.9,

129.6, 134.7, 149.3, 155.2, 156.2, 162.1i. Anal. called for C22H2409: C, 61.11; H, 5.59; N, 0.00.

Found: C, 60.91; H, 5.72; N, 0.03.

1-O-Coumarin-3 carbonyl-2,3:5,6-Di-O-isopropylidene-a-D-mnouaoe 3.14:

White microcrystals (65%), mp 158.2 160.0 oC, 1H NMR (CDCl3): 6 1.35 1.40 (m, 6H), 1.46

(s, 3H), 1.52 (s, 3H), 4.06 (dd, J= 9.07, 4.4 Hz, 1H), 4.08 4.15 (m, 1H), 4.19 (dd, J= 7.8, 3.4

Hz, 1H), 4.40 -4.48 (m, 1H), 4.89 -4.98 (m, 2H), 6.36 (s, 1H), 7.32 -7.40 (m, 2H), 7.62 -7.71










Hz, 1H), 4.00-4.10 (m, 1H), 4.25-4.45 (m, 2H), 4.94-5.08 (m, 4H), 7.06-7.35 (m, 15H), 7.48 (d, J

= 7.7 Hz, 1H), 7.92 (d, J= 7.7 Hz, 1H), 8. 17 (d, J= 7.7 Hz, 1H), 12.75 (br s, 1H). 13C NMR

(DMSO-d6): 8 18.1, 26.3, 30.1, 36.5, 49.8, 51.0, 53.5, 65.4, 65.5, 126.5, 127.8, 127.9, 128.0,

128.2, 128.3, 128.4, 128.5, 129.1, 136.2, 137.0, 137.4, 155.7, 172.2, 172.7, 172.8, 173.1. Anal.

called for C32H35N30s: C, 65.18; H, 5.98; N, 7.13. Found: C, 64.86; H, 6.03; N, 7.18.

Benzyl (S)-4-((LS)-2-benzyloxycarbonylaminopropnmio-(R)1crxy2

phenylethylamino)-5-oxopentanoate (Z-L-Ala-L-Glu(OBzl)-D-Phe-OH, 2.14a'): White

microcrystals (83%); mp 153-155 oC, [a]D23 = +5.5 (c 1.91, DMF.). 1H NMR (DMSO-d6): 8 1.15

(d, J= 6.6 Hz, 3H), 1.59-1.65 (m, 1H), 1.70-1.78 (m, 1H), 1.93-2.18 (m, 2H), 2.29-2.34 (m, 1H),

2.81 (dd, J= 13.3, 10.5 Hz, 1H) 4.04 (quintet, J= 7.4 Hz, 1H), 4.28-4.35 (m, 1H), 4.43-4.50 (m,

1H), 4.96 (d, J= 13.3 Hz, 1H, A part of AB system), 5.01 (d, J= 13.3 Hz, 1H, B part of AB

system), 5.06 (s, 2H), 7.06-7.08 (m, 1H), 7. 13-7.36 (m, 14H), 7.49 (d, J= 7.0 Hz, 1H), 7.84 (d, J

= 8.4 Hz, 1H), 8.31 (d, J= 8.4 Hz, 1H), 12.81 (br s, 1H). 13C NMR (DMSO-d6): 8 18.0, 27.6,

29.4, 36.9, 50.1, 51.2, 53.3, 65.4, 65.5, 126.4, 127.7, 127.8, 127.9, 128.0, 128.1, 128.4, 128.5,

129.1, 136.2, 137.0, 137.4, 155.7, 170.5, 172.1, 172.3, 172.8. Anal. called for C32H35N30s: C,

65.18; H, 5.98; N, 7. 13. Found: C, 64.88; H, 6. 11; N, 7.04.

Benzyl (S)-4-((LS)-2-benzyloxycarbonylamino-3-pheypoaaio--()1

carboxyethylamino)-5-oxopentanoate (Z-L-Phe-L-Glu(OBzl)-L-Ala-OH, 2.14b): White

microcrystals (95%); mp 163-165 oC, [a]D23 = -13.0 (c 2.33, DMF). 1H NMR (DMSO-d6): 8 1.15

(d, J= 7.0 Hz, 3H), 1.79-2.00 (m, 2H), 2.40-2.48 (m, 2H), 2.67-2.75 (m, 1H), 2.98 (d, J= 12.0

Hz, 1H), 4.91 (s, 2H), 5.09 (s, 2H), 7.06-7.35 (m, 15H), 7.52 (d, J= 8.4 Hz, 1H), 8. 14 (d, J= 7.0

Hz, 1H), 8.28 (d, J= 6.3 Hz, 1H), 12.51 (br s, 1H). 13C NMR (DMSO-d6): 8 17.0, 27.6, 29.8,

37.3, 47.6, 51.4, 56.1, 65.2, 65.5, 126.3, 127.5, 127.7, 127.9, 128.1, 128.3, 128.4, 128.5, 129.2,










and the mixture 2.5c+c' were obtained in 87-92% yields (Figure 2-3 and Table 2-2) and were

further recrystallized from CH2C 2/hexanes for further characterization.

The natural dipeptide 2.3b was obtained in higher yields than the unnatural dipeptide 2.5a.

For example, the reaction of 7-benzyl L-glutamate (2.2a) with Z-L-Phe-Bt (2.1b) gave dipeptide

2.3b in 97% yield, whereas the reaction of a-benzyl L-glutamate (2.4a) with Z-L-Phe-Bt (2.1b)

gave product 2.5a in 87% yield.

Z-O OH RN H O~P
R N--N Z'N N O P
I CH3CN /H20
N O H2NO Ph Et3N, r.t., 2h.
O O O
2.1b-d, 2.1d+d' 2.4a 2.5a-c, 2.5c+c'

Amino acids with R: Ala, Phe, Met, DL-Met.
Figure 2-3. Preparation of novel unnatural dipeptides 2.5a-c and diastereomeric mixture 2.5c+c'

Table 2-2. Preparation of novel unnatural dipeptides 2.5a-c and mixture 2.5c+c'
Entry N~-(Z-a-amninoacyl) Yield Rento
benzotriazoles Product a[a]2 D time Mp (oC)
(min)
Z-L-Phe-L-
1 Z-L-Phe-Bt (2.1b) 87 -10.7 6.4 140-141
Glu(OBzl)-OH (2.5a)
Z-D-Phe-L-
2 Z-D-Phe-Bt (2.1c) 91 -5.4 7.6 119-120
Glu(Obzl)-OH (2.5b)
Z-L-Met-L-
3 Z-L-Met-Bt (2.1d) 87 -7.1 6.3 98-99
Glu(Obzl)-OH (2.5c)
Z-DL-Met-L-
Z-DL-Met-Bt
4 Glu(OBzl) -OH 92 -5.3 6.3,7.5 72-73
(2.1d+d')
(2.5c+c')
alsolated yield



Dipeptides 2.5a-c were obtained with no detectable racemization evidenced by their NMR

analyses. The enantiopure LL- dipeptides (2.5a,c) gave doublets for the two -NH protons.

However, for the diastereomeric mixture (2.5c+c'), each of the two -NH protons appeared as two

pairs of equal doublets. 1H NMR spectrum of 2.5c displayed a singlet for the methyl protons










137.5, 155.9, 168.9, 171.4, 172.9. Anal. called for C28H28N207: C, 66.66; H, 5.59; N, 5.55.

Found: C, 66.67; H, 5.58; N, 5.48.

Benzyl (S)-2-benzyloxycarbonylamino-N-(1-carboxy2pelthlmn)4

oxobutanoate (Z-L-Asp-OBzl-DL-Phe-OH, 2.12b+b'): White microcrystals (91%); mp 116-

118 oC, [a]D23 = -18.6 (c 2.08, DMF). 1H NMR (DMSO-d6): 6 2.58-2.88 (m, 3H), 3.01 (dd, J =

17.4, 5.2 Hz, 1H), 4.47 (quintet, J = 6.9 Hz, 2H), 5.03 (d, J = 12.6 Hz, 1H, A part of AB system),

5.08 (d, J= 12.6 Hz, 1H, B part of AB system), 5.13 (s, 2H), 7. 17-7.35 (m, 15H), 7.60 (d, J = 8.1

Hz, 0.5H), 7.85 (d, J= 8.2 Hz, 0.5H), 8.35 (d, J= 8.0 Hz, 1H), 12.78 (br s, 1H). 13C NMR

(DMSO-d6): 8 30.7, 35,9, 36.6, 36.9, 50.6, 53.7, 65.6, 66.1, 66.3, 126.5, 127.6, 127.7, 127.8,

127.9, 128.0, 128.1, 128.2, 128.4, 135.8, 135.9, 136.9, 137.5, 155.9, 156.0, 168.8, 171.1, 171.4,

171.5, 172.9. Anal. called for C28H28N207: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.31; H, 5.53;

N, 5.58.

2.4.4 General Procedure for the Preparation of N"-Protected-Dipeptidoylbenzotriazoles
2.13a-c and 2.13a+a'

The preparation of 2.13a-c, 2.13a+a' was performed at -15 OC under similar conditions as

those described for 2.8 and 2.11.

(LS)-Benzyl 5-(1H-1,2,3-benzotriazol-1-yl)-4-((S)-2-

(benzyloxycarbonylamino)propanamido)-5-oxpnnot (Z-L-Ala-L-Glu(OBzl)-Bt,

2.13a): White microcrystals (93%); mp 133-135 oC, [a]D23 = -17.9 (c 2.08, DMF). 1H NMR

(DMSO-d6): 8 1.26 (d, J= 7.0 Hz, 3H), 1.83-2.42 (m, 2H), 2.47-2.52 (m, 1H), 2.68 (t, J= 7.4

Hz, 1H), 4. 17 (dt, J= 19.6, 7.4 Hz, 1H), 4.98-5.12 (m, 4H), 5.66-5.72 (m, 1H), 7.28-7.42 (m,

10H), 7.50-7.57 (m, 1H), 7.64 (t, J= 7.9 Hz, 1H), 7.80 (t, J= 7.5 Hz, 1H), 8.22 (d, J= 7.9 Hz,

1H), 8.29 (d, J= 8.2 Hz, 1H), 8.85 (d, J= 6.3 Hz, 1H). 13C NMR (DMSO-d6): 8 17.9, 25.8, 29.7,

49.6, 52.0, 65.4, 65.6, 114.1, 120.2, 126.7, 127.8, 127.9, 128.0, 128.3, 128.4, 128.5, 130.7, 131.0,










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White microcrystals (93%); mp 165-167 oC, [a]D23 = +11.5 (c 2. 16, DMF). 1H NMR (DMSO-

d6): 6 1.81-1.89 (m, 1H), 1.95-2.03 (m, 1H), 2.44 (t, J= 7.7 Hz, 2H), 3.10 (dd, J= 14.6, 8.0 Hz,

1H), 3.21 (dd, J= 14.7, 5.0 Hz, 1H), 4. 10-4.30 (m, 4H), 4.48-4.55 (m, 1H), 5.11 (s, 2H), 6.99 (t,

J= 7.5 Hz, 1H), 7.08 (t, J= 7. 1 Hz, 1H), 7.20 (s, 1H), 7.28-7.43 (m, 10H), 7.55 (d, J= 7.7 Hz,

1H), 7.60 (d, J= 8.2 Hz, 1H), 7.74 (t, J= 6.9 Hz, 2H), 7.88 (d, J= 7.4 Hz, 2H), 8.23 (d, J= 7.4

Hz, 1H), 10.90 (s, 1H), 12.75 (s, 1H). 13C NMR (DMSO-d6): 6 27.0, 27.5, 30.2, 46.7, 53.0, 53.6,

65.5, 65.7, 109.6, 111.4, 118.2, 118.4, 120.1, 121.0, 123.7, 125.4, 127.1, 127.3, 127.7, 128.0,

128.1, 128.5, 136.1, 136.3, 140.8, 143.8, 144.0, 155.9, 171.5, 172.3, 173.3. Anal. called for

C38H35N307: C, 70.68; H, 5.46; N, 6.51. Found: C, 70.43; H, 5.48; N, 6.47.

(LS)-2-((LS)-2-(((9H-Fluoren-9-yl)mI1ethoxy)carbonylam ino)-4-(benzyloxy)-4-

oxobutanamido)-3-(1H-indol-3-yl)propanoic acid (Fmoc-L-Asp(OBzl)-L-Trp-OH, 2.10b):

White microcrystals (94%); mp 136-137 oC, [a]D23 = -4.6 (c 2.16, DMF). 1H NMR (DMSO-d6):

6 2.66 (dd, J= 16.3, 9.6 Hz, 1H), 2.84 (dd, J= 16.5, 4.2 Hz, 1H), 3.10 (dd, J= 15.0, 8.1 Hz, 1H),

3.20 (dd, J= 14.8, 4.8 Hz, 1H), 4.21-4.30 (m, 3H), 4.45-4.57 (m, 2H), 5.11 (s, 2H), 6.99 (t, J=

7.0 Hz, 1H), 7.07 (t, J= 7. 1 Hz, 1H), 7. 18 (s, 1H), 7.28-7.45 (m, 10H), 7.54 (d, J= 7.7 Hz, 1H),

7.70-7.77 (m, 3H), 7.89 (d, J= 7.6 Hz, 2H), 8.18 (d, J= 7.4 H, 1H), 10.91 (s, 1H), 12.75 (s, 1H).

13C NMR (DMSO-d6): 6 26.9, 36.3, 46.6, 51.1, 53.1, 65.7, 65.8, 109.5, 111.4, 118.2, 118.5,

120.2, 121.0, 123.7, 125.4, 127.1, 127.2, 127.7, 127.9, 128.0, 128.4, 136.0, 136.1, 140.8, 143.9,

155.9, 170.1, 170.8, 173.2. Anal. called for C37H33N307: C, 70.35; H, 5.27; N, 6.65. Found: C,

70.02; H, 5.29; N, 6.65.

(S)-2- [(S)-4-Benzyl oxycarbo nyl-2-(9H-fluo ren-9-

ylm ethoxycarbonylam ino)butyrylam ino] -4-m ethyls ulfanylbutyric acid (Fm oc-L-

Glu(OBzl)-L-Met-OH, 2.10c): White microcrystals (91%); mp 96-97 oC, [a]D23 = -7.6 (c 2.16,










(benzopyranones), the largest class of laser dyes for the "blue-green" region, are highly

sensitive.8-9 They have provided the most commercially accepted categories of fluorescent

derivatives with the advantages of an extended spectral range, high emission quantum yield,

photostability, and good solubility in many solvents.

Primary amine-containing fluorescence-tags can be introduced by reductive amination at

the reducing end of sugar chains. The saccharide reacts with 3.2 containing 2-aminopyridine and

sodium cyanoborohydrate, or sodium borohydride to give glycamine 3.3 (Figure 3-1).76,96-100

However, using these methods cleave the cyclic structure at the reducing end changing the

properties of the sugar moieties.


OR Fluorescent

RO OFT NBH3C /OH FT
| RO NH
NHCOCH3 NH2
3.1 3.2 3.3

Figure 3-1. Fluorescent labeling of saccharides by reductive amination

Classical labeling procedures applied to proteins, based on acylation of -NH2 grOups are

not useful for many sugars because of the absence of the free amino groups in the saccharide

structure. However, mono- and disaccharides with amino groups have been labeled with

fluorescent mass tags as an alternative method for measuring a special class of enzymes that are

responsible for the synthesis of carbohydrates (glycosyltransferases).101-103 The coupling of 7-

hydroxycoumarin-3-carboxylic acid, a fluorescent tag, to glycosylamine has been performed

with HBTU/HOBT/DIEA in DMF.101 Also, amino-rich polysaccharides have been labeled with

the fluorescein derivative 5-([4, 6-dichlorotriazine-2-yl] amino)-fluorescein (DTAF).102,103

Fluorescence detection depends on the physical characteristics of the dyes employed.

Many dyes with high extinction coefficients and high quantum yields are of limited utility due to











83. Ehrhart, D. Curr. Opin. Plant Biol. 2003, 6, 622.

84. Faure, M. P.; Gaurdeau, P.; Shaw, I.; Cashman, N. R.; Beaudet, A. J. Histochent.
Cytochent. 1994, 42, 755.

85. Fernandez-Carneado, J.; Kogan, M. J.; Castel, S.; Giralt, E. Angew. Chens. Int. Ed. 2004,
43, 1811.

86. Ammar, H.; Fery-Forgues, S.; Gharbi, R. E. Dyes andPignzents 2003, 57, 259.

87. Gikas, E.; Parissi-Poulou, M.; Kazanis, M.; Vavagianis, A. Anal. China. Acta 2003, 489,
153.

88. Sastry, S. Biophys. Chent. 2001, 91, 191.

89. Malkar, N. B.; Fields, G. B. Lett. Pept. Sci. 2000, 7, 263.

90. Berthelot, T.; Lain, G.; Latxague, L.; Deleris, G. J. Fluoresc. 2004, 14, 671.

91. Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.; Lansley, A. B.; Lawrence,
M. J.; Marriott, C.; Bansal, S. S. Tetrahedron Lett. 1997, 38, 7449.

92. Wang, J.; Xie, J.; Schultz, P. G. J. Am. Chent. Soc. 2006, 128, 8738.

93. Esteves, A. P.; Rodrigues, L. M.; Silva, M. E.; Gupta, S.; Oliviera-Campos, A. M. F.;
Machalicky, O.; Mendonca, A. J. Tetrahed~rtrt~t~ ron~r~rtrt 2005, 61, 8625.

94. Heiner, S.; Detert, H.; Kuhn, A.; Kunz, H. Bioorg. M~ed. Chent. 2006, 14, 6149.

95. Malsch, R.; Guerrini, M.; Torri, G.; Lohr, G.; Casu, B.; Harenberg, J. Anal. Biochent.
1994, 217, 255.

96. Uozumi, N.; Teshima, T.; Yamamoto, T.; Nishikawa, A.; Gao, Y. E.; Miyoshi, E.; Gao,
C. X.; Noda, K.; Islam, K. N.; Ihara, Y.; Fujii, S.; Shiba, T.; Taniguchi, N. J. Biochent.
1996, 120, 385.

97. Hase, S.; Ibuki, T.; Ikenaka, T. J. Biochent. 1984, 95, 197.

98. Tomiya, N.; Kurono, M.; Ishihara, H.; Tejima, S.; Endo, S.; Arata, Y.; Takahashi, N.
Anal. Biochent. 1987, 163, 489.

99. Gross, H. J.; Sticher, U.; Brossmer, R. Anal. Biochent. 1990, 186, 127.

100. Taniguchi, N. Nishikawa, A.; Fujii, S.; Gu, J. Methods in Enzyntology 1989, 1 79, 397.










136.0, 137.0, 145.3, 155.7, 170.8, 171.9, 173.2. Anal. called for C29H29N5O6: C, 64.08; H, 5.38;

N, 12.88. Found: C, 63.80; H, 5.38; N, 12.29.

(LS)-Benzyl 5-(1H-1,2,3-benzotriazol-1-yl)-4-((LS)-2-bnyoyroylmo)3

phenylpropanamido)-5-oxopentanoate (Z-L-Phe-L-Glu(OBzl)-Bt, 2.13b): White

microcrystals (92%); mp 90-92 oC, [a]D23 = -24.7 (c 1.91, DMF). 1H NMR (DMSO-d6): 6 2.20-

2.40 (m, 2H), 2.63-2.80 (m, 3H), 3 .00-306 (m, 1H), 4.30-4.44 (m, 1H), 4.94 (s, 2H), 5.05 (d, J=

14.6 Hz, 1H, A part of AB system), 5.10 (d, J = 14.9 Hz, 1H, B part of AB system), 5.67-5.74

(m, 1H), 7. 17-7.40 (m, 15H), 7.54-7.67 (m, 2H), 7.82 (t, J= 7.5 Hz, 1H), 8.22 (d, J= 8.0 Hz,

1H), 8.30 (d, J= 8.2 Hz, 1H), 9.00 (d, J= 6.3 Hz, 1H). 13C NMR (DMSO-d6): 6 25.9, 29.8, 37.3,

52.1, 55.8, 65.3, 65.7, 114.1, 120.2, 126.3, 126.7, 127.5, 127.7, 128.0, 128.1, 128.3, 128.4, 129.2,

130.7, 131.1, 136.0, 136.9, 137.9, 145.4, 155.9, 170.8, 172.0, 172.4, 173.1. Anal. called for

C35H33N5O6: C, 67.84; H, 5.37; N, 11.30. Found: C, 67.72; H, 5.43; N, 11.09.

(LS)-Benzyl 4-(1H-1,2,3-benzotriazol-1-yl)-3-((LS)--bnylxcroylmn)3

phenylpropanamido)-4-oxobutanoate (Z-L-Phe-L-Asp(OBzl)-Bt, 2.13c): White microcrystals

(89%); mp 110-113 oC, [a]D23 = -20.7 (c 2.75, DMF). 1H NMR (DMSO-d6): 6 2.71-2.84 (m, 1H),

2.97-3.10 (m, 2H), 3.34 (dd, J= 17.0, 5.9 Hz, 1H), 4.32-4.40 (m, 1H), 4.90 (d, J= 6.7 Hz, 1H, A

part of AB system), 5.10 (d, J= 10.6 Hz, 1H, B part of AB system), 5.15 (s, 2H), 6.03 (dd, J=

13.5, 6.6 Hz, 1H), 7.14-7.39 (m, 15H), 7.56-7.67 (m, 2H), 7.82 (t, J= 8.1 Hz, 1H), 8.22 (d, J=

8.2 Hz, 1H), 8.30 (d, J= 8.2 Hz, 1H), 9.17 (d, J= 6.6 Hz, 1H). 13C NMR (DMSO-d6): 6 35.3,

37.4, 49.6, 55.9, 65.3, 66.3, 114.0, 120.3, 126.3, 126.8, 127.4, 127.5, 127.7, 128.0, 128.1, 128.2,

128.3, 128.4, 129.2, 130.7, 131.1, 135.6, 136.0, 137.0, 137.9, 145.4, 155.9, 169.5, 169.7, 172.1.

Anal. called for C34H31N5O6: C, 67.43; H, 5.16; N, 11.56. Found: C, 67.21; H, 5.16; N, 11.53.










170.3, 172.7, 173.1. Anal. called for C33H34N40s: C, 64.48; H, 5.58; N, 9.12. Found: C, 64.13; H,


5.70; N, 8.78.






















Yield

65
86
94
90
85


Mp
(oC)
121-123
154-156
119-120
69-71
179-181


[L25

+0.7
-12.8
-8.2
-24.0
-1.7


In addition, it was also found that the dipeptides 2.7a,b were obtained in lower yields as

compared to the natural dipeptides 2.3a,b and the unnatural dipeptides 2.5a. For example, the

reaction of L-glutamic acid (2.6a) with Z-L-Phe-Bt (2.1b) gave a dipeptide 2.7b in 86% yield,

whereas the reaction of 7-benzyl L-glutamate (2.2a) with Z-L-Phe-Bt (2.1b) gave compound 2.3b

in 97% yield and a-benzyl L-glutamate (2.4a) with L-Phe-Bt (2.1b) gave dipeptide 2.5a in 87%

yield. As expected, these three compounds gave different melting points: the dipeptide 2.7b

(with unprotected side chain) melted at 154-1560C, while the unnatural dipeptide 2.5a had

melting point 140-1410C and the natural dipeptide 2.3b had 139-1420C. Also, the compound

2.7a had a higher melting point (121-1230C) than the dipeptides 2.3a.

2.2.4 Peptide Chain Extension at the Alpha C-Terminus to Give Natural Dipeptides
2.10a-c.

Peptide coupling was carried out by reacting P or y-monobenzylesters (2.8a, Asp) and

(2.8b, Glu) of the N-(Fmoc-a-aminoacyl)b enzotriazoles with equimolar amounts of unprotected


I


OH
R O' .(CH2 n CH3CN / H20 ZN

,Ho O H2 Et3N, r.t., 2h.

2.1a,b,d,e 2.6a,b
n=1, 2.
Amino acids with R: Ala, Phe, Trp. Met
Figure 2-4. Preparation of novel dipeptides 2.7a-e

Table 2-3. Preparation of novel dipeptides 2.7a-e
Et N-(Z-an-amninoacyl) Prdc
benzotriazoles
1 Z-L-Ala-Bt (2.1a) Z-L-Ala-L-Glu-OH (2.7a)
2 Z-L-Phe-Bt (2.1b) Z-L-Phe-L-Glu-OH (2.7b)
3 Z-L-Met-Bt (2.1d) Z-L-Met-L-Glu-OH (2.7c)
4 Z-L-Trp-Bt (2.1e) Z-L-Trp-L-Glu-OH (2.7d)
5 Z-L-Phe-Bt (2.1b) Z-L-Phe-L-Asp-OH (2.7e)
a Isolated yield


OH
O (CH2)n
OH
2.7a-e










136.2, 137.0, 138.1, 155.9, 170.6, 171.5, 172.4, 174.1. Anal. called for C32H35N30s: C, 65.18; H,

5.78; N, 7.30. Found: C, 64.88; H, 6.26; N, 7.04.

Benzyl (S)-4-(2-benzyloxycarbonylaminopropanamido--()1croy2

phenylethylamino)-4-oxopentanoate (Z-DL-Ala-L-Glu(OBzl)-L-Phe-OH, 2.14a+a"): White

microcrystals (92%); mp 123-125 oC, [a]D23 = -5.5 (c 1.66, DMF). 1H NMR (DMSO-d6): 6 1.17

(m, 3H), 1.61-2. 10 (m, 2H), 2.29-2.45 (m, 2H), 2.82-2.95 (m, 1H), 3 .04-3.11 (m, 1H), 4.06

(quintet, J= 6.9 Hz, 1H), 4.20-4.31 (m, 1H), 4.39-4.42 (m, 1H), 4.94-5.07 (m, 4H), 7.06-7.51

(m, 15H), 7.45 (d, J= 7. 1 Hz, 0.5H), 7.50 (d, J= 6.3 Hz, 0.5H) 7.86-8.01 (m, 1H), 8.13-8.32 (m,

1H), 12.75 (br s, 1H). 13C NMR (DMSO-d6): 18.2, 18.3, 18.7, 26.4, 26.5, 27.6, 27.7, 29.9, 30.0.

30.1, 36.7, 50.0, 50.1, 50.3, 51.1, 51.4, 51.5, 53.6, 53.7, 65.5, 65.6, 126.6, 127.9, 128.1, 128.2,

128.2, 128.3, 128.5, 128.6, 129.2, 136.3, 137.1, 137.2, 137.5, 137.6, 155.8, 155.9, 171.0, 171.1,

172.2, 172.3, 172.4, 172.5, 172.6, 172.8, 172.9, 173.1, 173.2. Anal. called for C32H35N30s: C,

65.18; H, 5.98; N, 7.13. Found: C, 65.23; H, 6.14; N, 7.22.

2.4.6 General Procedure for the Preparation of Tripeptides 16 a,b.

N-Protected dipeptides 2.10a,b were treated with piperidine at room temperature for 2

hours to deprotect Fmoc group according to the reported procedures to provide the free

dipeptides 2.15a,b. Free dipeptides (without significant purification) (1 mmol) were dissolved in

a mixture of acetonitrile (10 mL), water (5 mL), and triethylamine (2.5 mmol). N-(Protected-a-

aminoacyl)benzotriazoles 2.1a,b (1 mmol) were added to the reaction mixture at -15oC and the

stirring continued for additional 2 hours. Resulting solution was acidified with (1 mL) 4N HCI

and acetonitrile was evaporated under reduced pressure at room temperature. The residue was

dissolved in EtOAc (50 mL) and was then washed 3 times with 4N HCI (3 x 15 mL) followed by

saturated NaCl (20 mL). The organic layer was dried over magnesium sulfate and the solvent









CHAPTER 1
GENERAL INTRODUCTION

Over the last 25 years, Katritzky and colleagues have studied the design of new synthetic

approaches that can produce scientifically attractive compounds in good quantities and with easy

purification methods. The development of better methods for the preparation of useful

compounds in our research group is based on the versatility of a well known synthetic auxiliary,

benzotriazole.

A useful synthetic auxiliary must possess several characteristics. First, benzotriazole can

be introduced readily at the beginning of a sequence. Second, benzotriazole is easily removed at

the end of the synthetic sequence so that it can be recovered and reused. Last, benzotriazole is

inexpensive and stable during various chemical reactions and, to some extent, activates groups

on other parts of the molecule that is attached to.'

1H Benzotriazole strongly exhibits all of the above characteristics. Benzotriazole (Bt)

offers many advantages as a resourceful synthetic auxiliary because it is soluble in many

solvents, such as benzene, toluene, chloroform, ethanol, tetrahydrofuran (THF), ethyl acetate

(EtOAc), diethyl ether, and dimethyl formamide (DIVF).2 Moreover, benzotriazole is partially

soluble in water, but extremely soluble in basic solutions because of its acidic pKa of 8.2.

N-Substituted derivates of benzotriazole with an a heteroatom (usually nitrogen, oxygen

and sulfur) attached to a benzotriazole nitrogen can ionize in two ways due to the electron

donating and electron accepting properties of benzotriazole.3 The benzotriazole anion and an

immonium, oxonium, or thionium cation 1.2 can be formed or it can ionize off the heteroatom

substituent to produce 1.3 (Figure 1-1). Generally, benzotriazole is considered to be comparable

with other activating groups because it shows good leaving ability 1.2 and activates the a-CH

toward proton loss 1.1. This type of activation to proton loss and leaving ability can be










and 2.14a+a" were obtained in 73-95% yields (Figure 2-8 and Table 2-7) and were further

recrystallized from CH2 2z/hexanes for NMR spectroscopy, elemental analysis, and ORP.

R' BN t R' ` O R"
Z' N CH3CN / H20 ZN N N CO
O (CH2)n H2N COOH H O (CH2) n
O~ Et3N, -150C, 2h. O~
O 2.9c,e ,f O
2.13a,b, 2.13a+a~hP

n = 2.2.14a,b,a',a+a"
Bt = benzotriazol-1-yl.
Amino acids with R': Ala, DL-Ala, Phe
Amino acids with R": Ala, Phe, D-Phe
Figure 2-8. Preparation of novel tripeptides 2.14a,b,a', and 2.14a+a"

Table 2-7. Preparation of novel tripeptides 2.14a,b,a' and mixture 2.14a+a"
Amino Yield 25~S M~C
Entry Acd Reactant Product o @ p(C
L-Phe Z-L-Al a-L-Glu(OBzl)- Z -L-Al a-L -Glu(OB zl)-
1 73 -2.9 74-76
(2.9c) Bt(2.13a) L-Phe-OH (2.14a)
D-Phe Z-L-Al a-L-Glu(OBzl)- Z -L-Al a-L -Glu(OB zl)-
2 83 +5.5 153-155
(2.9e) Bt (2.13a) D-Phe-OH (2.14a')
Z-L-Phe-L-Glu(OBzl)-
L-Ala Z-L-Phe-L-Glu(OBzl)- 9 1. 6-6
3 L-Ala-OH (2.14b) 9 1. 6-6
(2.9f) Bt (2.13b)
Z-DL-Ala-L-
L-Phe Z-DL-Ala-L-Glu(OBzl)-
4 Glu(OBzl)-Bt 92 -5.5 123-125
(2.9c) L-Phe-OH (2.14a+a")
(2.13a+a')
alsolated yield


NMR analysis showed no racemization for the tripeptides 2.14a and 2.14a' when the

reaction was carried out -15 OC, however when the same reaction was carried out at room

temperature, extensive racemization of the desired products was observed. H NMR showed a

clear doublet for all -NH protons and the methyl protons of the L-Ala fragment, while NMR

spectra of the mixture 2.14a+a" gave complicated multiplets for -NH groups. 13C NMR

displayed a singlet for each carbonyl carbon for compounds 2.14a and 2.14a' whereas the










(d, J = 7.7 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 12.41 (s, 2H). 13C NMR (DMSO-d6): 6 8.1, 26.4,

30.0, 49.8, 51.1, 65.4, 127.3, 127.8, 128.4, 137.0, 155.7, 172.7, 173.3, 173.8. Anal. called for

C16H20N207: C, 54.54; H, 5.72; N, 7.95. Found: C, 54.21; H, 5.69; N, 7.81.

(LS)-2-((LS)-2-Benzyloxycarbonylamino-3-phnlrpoyaino)pentanedioic acid (Z-

L-Phe-L-Glu-OH, 2.7b): White microcrystals (86%); mp 154-156 oC, [a]D23 = -12.8 (c 1.66,

DMF). 1H NMR (DMSO-d6): 6 1.76-1.88 (m, 1H), 1.99-2.06 (m, 1H), 2.28-2.36 (m, 2H), 2.68-

2.77 (m, 1H), 2.99-3.04 (m, 1H), 4.22-4.32 (m, 2 H), 4.94 (s, 2H), 7.05-7.32 (m, 10H), 7.52 (d, J

= 8.8 Hz, 1H), 8.33 (d, J= 7.7 Hz, 1H), 12.47 (s, 2H). 13C NMR (DMSO-d6): 6 26.6, 30.2, 37.6,

51.5, 56.2, 65.5, 126.5, 127.7, 127, 9, 128.3, 128.5, 129.4, 137.2, 138.3, 156.1, 172.1, 173.5,

174.1. Anal. called for C22H24N207: C, 61.67; H, 5.65; N, 6.54. Found: C, 61.76; H, 5.57; N, 6.56.

(LS)-2-((LS)-2-Benzyloxycarbonylam ino-4-methylsulfanylbutyrylamino)pentanedii

acid (Z-L-Met-L-Glu-OH, 2.7c): White microcrystals (94%); mp 119-120 oC, [a]D23 = -8.2 (c

1.66, DMF). 1H NMR (DMSO-d6): 6 1.75-1.99 (m, 4H), 2.02 (s, 3H), 2.30 (t, J= 7.2 Hz, 2H),

2.39-2.47 (m, 2H), 4.01-4.24 (m, 2H), 5.02 (s, 2H), 7.30-7.36 (m, 5H), 7.51 (d, J= 8.0, 1H), 8.20

(d, J= 7.7 Hz, 1H), 12.42 (br s, 2H). 13C NMR (DMSO-d6): 6 14.8, 26.3, 29.7, 30.1i, 31.9, 51.3,

53.8, 65.6, 127.9, 128.0, 128.5, 137.1, 156.1, 171.8, 173.3, 173.9. Anal. called for C18H24N207S:

C, 52.42; H, 5.86; N, 6.79. Found: C, 52.47; H, 5.89; N, 6.69.

(LS)-2-((LS)-2-Benzyloxycarbonylamino-3-(Hidl3ypriolmnoetndoi

acid (Z-L-Trp-L-Glu-OH, 2.7d): Yellow microcrystals (90%); mp 69-71 oC, [a]D23 = -24.0 (c

1.66, DMF). 1H NMR (DMSO-d6): 6 1.81-1.91 (m, 1H), 1.98-2.06 (m,1H), 2.33 (t, J= 7.1 Hz, 2

H), 2.87-2.95 (m, 1H), 3.10-3.15 (m, 1H), 4.26-4.38 (m, 2H), 5.01 (s, 2H), 7.00 (t, J= 7.4 Hz, 1

H), 7.07 (t, J= 7.4 Hz, 1H), 7.18-7.41 (m, 8H) 7.68 (d, J= 7.7 Hz, 1H), 8.35 (d, J= 7.4 Hz,

1H), 10.82 (s, 1H), 12.44 ( br s, 2H) 13C NMR (DMSO-d6): 6 26.4, 27.8, 30.1, 51.3, 55.3, 65.3,










3.2.3. Preparation of Coumarin-N-Tagged Monosaccharide: N-(Coumarin-3-
carbonyl)tetrapivaloyl Sugar 3.16.

2,3,4,6-tetra-O-pivaloyl-P-D-galactopyranslme 3.15 was coupled with N-(coumarin-

3-carbonyl) benzotriazole 3.4 in dry dichloromethane in the presence of 1 equivalent of DMAP

in 24 hours at 20oC. After silica-gel column chromatography using ethyl acetate/hexane (1:3) as

eluent, product 3.16 was isolated in 60% yield.


OOO



03.13 Bt

Oij~H 3.4OPiO
3.11 PivNH


X


Y


3.12


~~o o /


3 16


3.14 O u.I


Figure 3-3. Syntheses of O-(coumarin-3 -carbonyl)diisopropylidene sugars 3.12, 3.13, 3.14 and N-
(coumarin-3-carbonyl)tetrapivaloyl sugar 3.16.

3.2.4 Preparation of O- and N-(N"-Coumarin-3-Carbonyl-N"(Fmoc or Z-L-lys)protected
Sugars 3.17a,b, 3.18, 3.19, 3.20.

L-Lysine scaffold based coumarin labeled sugars 3.17a,b, 3.18, 3.19, 3.20 were

synthesized by O-acylation of the free -OH groups present in diacetonide protected sugars 3.9,

3.10, 3.11 and N-acylation of the amino group of 3.15 by AP-coumarin-3-carbonyl-N"-Z or

Fmoc-L-lysine benzotriazole 3.7, 3.8 (Figure 3-4).

Coupling reactions were carried out in dry DCM, in the presence of 1 eq. of DMAP, at

room temperature for 18-24 hrs. Under microwave irradiation at 600C, the preparation of










Ph Ph R 'H P

Fmoc Z' BtH R""~ O
HN Opiperidine H2N, O H21aO HN O
($-CH2)n .(C2)n > (CH2)
O ~r.t., 2h O~ CH3CN / H20O
R> NH R> NH Et3N, -150C, 2h. R't* NH
COOH COOH COOH

2.10a,b 2.15a,b 2.16a,b
n =1, 2.
Amino acids with R': Trp
Amino acids with R": Ala, Phe

Figure 2-9. Preparation of novel tripeptides 2.16a,b

Table 2-8. Preparation of novel tripeptides 2.16a,b containing Glu and Asp fragments
Product Yield (%) a 25D Mp (oC)
Z-L-Phe-L-Glu(OBzl)-L-Trp-OH (2.16a) 84 -3.2 109-111
Z-L-Ala-L-Asp(OBzl)-L-Trp-OH (2.16b) 83 -1.7 134-137
a Isolated yield


NMR analysis demonstrated no detectable racemization for the tripeptides 2.16a,b. No

signals arising from the diastereomers were observed in the NMR spectra of compounds 2.16a,b.

1H NMR showed a clear doublet for all -NH protons of 2.16a,b and for the methyl protons from

L-Ala fragment for compound 2.16b. 13C NMR also gave a singlet for each carbonyl carbon in

2.16a,b.

2.3 Conclusion

The convenient preparation under mild conditions of N-(Fmoc- or Z-oc-

aminoacyl)benzotriazoles, N-protected peptidoylbenzotriazoles and diastereomeric mixtures

prepared from aspartic and glutamic amino acid has been demonstrated. Additionally, the

preparation of di- and tri-peptides starting from C- and N-termini of glutamic and aspartic acids

has been demonstrated under mild reaction conditions. Products were obtained without the use

of column chromatography. Evidence of maintained chirality was supported by NMR and HPLC

analyses.









154.3, 156.3, 161.3, 162.1, 175.3. Anal. Called for C2 H24N207: C, 63.71; H, 5.35; N, 6.19.

Found: C, 63.82; H, 5.09; N, 6.04.

(LS)-2-(9H-Fluoren-9-ylmethoxycarbonylam in)6[(2-oxo-2H-chromene-3-carbo

nyl)amino]hexanoic acid (N"-Fmoc-N"-Coumoyl-L-Lys-OH, 3.6): White microcrystals (87%);

mp 110.0-111.0 oC, [a]23D = -1.62 (c 1.85, DMF), 1H NMR (DMSO-d6): 6 1.32-1.50 (m, 2H),

1.50-1.62 (m, 2H), 1.62-1.85 (m, 2H), 3.26-3.38 (m, 2H), 3.92-4.01(m, 1H), 4.17-4.36 (m,

3H), 7.22-7.54 (m, 6H), 7.60-7.80 (m, 4H), 7.87 (d, J= 7.4 Hz, 2H), 7.96 (d, J= 7.4 Hz, 1H),

8.73 (t, J= 5.5 Hz, 1H), 8.84 (s, 1H). 12.62 (s, 1H). 13C NMR (DMSO-d6): 23.2, 28.6, 30.5,

46.7, 53.8, 65.6, 116.1, 118.5, 119.0, 120.1, 125.1, 125.3, 127.1, 127.7, 130.2, 134.0, 140.7,

143.8, 147.3, 153.8, 156.2, 160.4, 161.0, 174.0. Anal. Called for C31H28N207: C, 68.88; H, 5.22;

N, 5.18. Found: C, 68.59; H, 5.11; N, 5.16.

3.4.3 General Procedure for the Preparation of Compound 3.7 and 3.8.

Thionyl chloride (1.2 mmol) was added to a solution of 1H-Benzotriazole (5 mmol) in

anhydrous CH2 12 (15 mL) at room temperature, and the reaction mixture was stirred for 20 min.

Either compound 3.7 or 3.8 (1 mmol) was added to the reaction mixture and stirred for 2 hours at

room temperature. The white precipitate formed during the reaction was filtered off, the filtrate

was diluted with additional CH2 12 (80 mL) and the solution was washed with sat. Na2CO3 SOln

(3x50mL), sat. NaCl soln (50mL), and dried over MgSO4. Removing solvent under reduced

pressure gave product in 79% or 85 % yields, which was recrystallized from CH2 2z-hexanes for

elemental analysis.

{(S)-1-(Benzotriazole-1-carbonyl)-5- [(2-oxo-2H-chromene-3-carbonyl)-amino]-

pentyl}-carbamic acid benzyl ester (N"-Z-N"-Coumoyl-L-Lys-Bt, 3.7): White microcrystals

(79%); mp 156-1570C, 1H NMR (CDCl3): 6 1.50-1.80 (m, 4H), 1.96-2,12 (m, 1H), 2.13-2.28





























To my family, friends and everyone who always believed in me











2.4.5 General Procedure for the Preparation of Tripeptides 2. 14a,b, 2. 14a' and
2.14a+a" .............. ... .. ......... .. .... .. .............4
2.4.6 General Procedure for the Preparation of Tripeptides 16 a,b. ...........................43

3 EFFICIENT LABELING OF SUGARS TO PROVIDE WATER SOLUBLE
F LUORE SCENT T AGS ................. ...............46.......... ......

3.1 Introducti on ................. ...............46........... ....
3.2 Results and Discussion ............... ....................... ... .........4
3.2.1 Preparation of F~-Coumarin-Labeled N"-Fmoc-L-lysine Benzotriazolide 3.8......49
3.2.2 Preparation of Coumarin-O-Tagged Monosaccharides: O-(Coumarin-3-
carbonyl)diisopropylidene Sugars 3.12, 3.13, 3.14. ............. .. ..... ...............4
3.2.3.Preparation of Coumarin-N-Tagged Monosaccharide: N-(Coumarin-3-
carbonyl)tetrapivaloyl Sugar 3.16............... ...... ............ ... .......5
3.2.4 Preparati on of O- and N-(N"-C oumarin-3 -C arb onyl-N"(Fmoc or Z -L-
lys)protected Sugars 3.17a,b, 3.18, 3.19, 3.20. ................... ...............5
3.2.5 Deprotection of the Diisopropylidene Groups of O-(Coumarin
Labeled)diisopropylidene Protected Sugars 3.13, 3.17b and 3.18.........................51
3.3 Conclusion ................. ...............52........ .....
3.4 Experimental Section................. ..... ..... .. .........5
3.4.1 General Procedure for the Preparation of Compound 3.4. ................. ...............53
3.4.2 General Procedure for the Preparation of Compounds 3.5 and 3.6. ................... ...54
3.4.3 General Procedure for the Preparation of Compound 3.7 and 3.8. .......................55
3.4.4 General Procedure for the Preparation of O-(Coumarin)diacetonide Sugars
3.12, 3.13, 3.14 Under Microwave Irradiation. ......................._. ........._.....56

LI ST OF REFERENCE S ............_ ..... ..__ ...............61...

BIOGRAPHICAL SKETCH .............. ...............67....










155.7, 155.9, 171.5, 171.8, 172.0, 172.1, 172.9, 173.0. Anal. called for C29H30N207: C, 67.17; H,

5.83; N, 5.40. Found: C, 66.86; H, 5.83; N, 5.21.

1-Benzyl (LS)-2-((S)-2-benzyloxycarbonylamino-4-

methylsulfanylbutyrylamino)pentanoate (Z-L-Met-L-Glu(OBzl)-OH, 2.5c): White

microcrystals (87%); mp 98-99 oC, [a]D23 = -7.1 (c 2.0, DMF). 1H NMR (CDCl3): 6 1.92-1.97

(m, 2H), 2.06 (s, 3H), 2.22-2.27 (m, 2H), 2.46-2.57 (m, 4H), 4.41 (q, J= 7.4 Hz, 1H), 4.54-4.58

(m, 1H), 5.07-5.13 (m, 4H), 5.64 (d, J= 7.9 Hz, 1H), 7. 17 (d, J= 7.4 Hz, 1H), 7.26-7.33 (m,

10H), 10.10 (br s, 1H). 13C NMR (CDCl3): 6 15.1, 26.5, 29.7, 30.2, 31.5, 51.8, 53.6, 66.7, 67.2,

128.0, 128.2, 128.3, 128.4, 128.5, 128.6, 135.5, 136.0, 156.2, 172.0, 173.0, 174.2. Anal. called for

C25H30N207S: C, 59.75; H, 6.02; N, 5.57. Found: C, 59.38; H, 6.02; N, 5.34.

1-Benzyl (LS)-2-(2-benzyloxycarbonylamino-4-

methylsulfanylbutyrylamino)pentanoate (Z-DL-Met-L-Glu(OBzl)-OH, 2.5c+c'): White

microcrystals (92%); mp 72-73 oC, [a]D23 = -5.3 (c 1.66, DMF). 1H NMR (CDCl3): 6 1.88-1.97

(m, 2H), 2.03 (s, 3H), 2.10-2.23 (m, 2H), 2.46-2.54 (m, 4H), 4.40-4.45 (m, 1H), 4.54-4.59 (m,

1H), 5.02-5.13 (m, 4H), 5.82 (d, J= 8.5 Hz, 0.5H), 5.95 (d, J= 8.5 Hz, 0.5H), 7.30-7.32 (m,

10H), 7.36 (d, J= 8.0 Hz, 1H), 8.40 (br s, 1H). 13C NMR (CDCl3): 6 14.9, 15.1, 26.4, 29.6, 29.7,

29.8, 30.1, 31.4, 31.7, 51.5, 52.8, 53.5, 53.7, 66.5, 66.9, 126.9, 127.8, 128.0, 128.1, 128.3, 128.4,

135.4, 135.9, 156.1, 156.2, 156.5, 172.1, 172.7, 172.8, 173.8, 174.9. Anal. called for

C25H30N207S: C, 59.75; H, 6.02; N, 5.57. Found: C, 59.36; H, 6.00; N, 5.19.

(LS)-2-((LS)-2-Benzyloxycarbonylaminopropoymioenndoi acid (Z-L-Ala-L-

Glu-OH, 2.7a): White microcrystals (65%); mp 121-123 oC, [a]D23 = +0.7 (c 1.66, DMF). 1H

NMR (DMSO-d6): 6 1.20 (d, J= 6.9 Hz, 3H), 1.71-1.84 (m, 1H), 1.95-2.01 (m, 1H), 2.30 (t, J=

7.6 Hz, 2H), 4.07 (q, J= 7.1 Hz, 1H), 4.17-4.25 (m, 1H), 5.01 (s, 2H), 7.30-7.36 (m, 5H), 7.45










DMF). 1H NMR (DMSO-d6): 6 1.87-2.10 (m, 7H), 2.47-2.53 (m, 4H), 4. 10-4.46 (m, 5H), 5.13

(s, 2H), 7.30-7.46 (m, 9H), 7.66 (d, J= 8. 1 Hz, 1H), 7.76 (t, J= 6.9 Hz, 1H), 7.89 (d, J= 7.4 Hz,

2H), 8.32 (d, J= 7.6 Hz, 1H), 12.76 (s, 1H). 13C NMR (DMSO-d6): 8 14.6, 27.4, 29.7, 30.2, 30.7,

46.8, 51.1, 53.7, 65.6, 65.8, 120.2, 125.4, 127.1, 127.7, 128.0, 128.1, 128.5, 136.3, 140.8, 143.8,

144.0, 156.0, 171.6, 172.4, 173.3. Anal. called for C32H34N207S: C, 65.07; H, 5.80; N, 4.74.

Found: C, 65.25; H, 5.80; N, 4.89.

Benzyl (S)-2-benzyloxycarbonylamino-N- [(S)-1-carboxy-2-(1H-indol-3-

yl)ethylamino]-4-oxobutanoate (Z-L-Asp-OBzl-L-Trp-OH, 2.12a): White microcrystals

(92%); mp 98-100 oC, [a]D23 = -7.1 (c 2.08, DMF). 1H NMR (DMSO-d6): 6 2.58-2.71 (m, 3H),

3.01 (dd, J= 18.1i, 8.1 Hz, 1H), 3.14 (dd, J= 18.1, 4.6 Hz, 1H), 4.42-4.54 (m, 2H), 5.03 (s, 2H),

5.10 (s, 2H), 6.98 (t, J= 7.4 Hz, 1H), 7.06 (t, J= 7.4 Hz, 1H), 7.14 (s, 1H), 7.26-7.38 (m, 10H),

7.52 (d, J= 7.8 Hz, 1H), 7.63 (d, J= 8.2 Hz, 1H), 8.33 (d, J= 7.7 Hz, 1H), 10.04 (s, 1H), 10.85

(br s, 1H). 13C NMR (DMSO-d6): 6 27.2, 36.6, 50.7, 53.2, 65.6, 66.1, 109.7, 111.4, 118.2, 118.4,

120.9, 123.6, 127.2, 127.7, 127.8, 127.9, 128.0, 128.3, 128.4, 135.9, 136.1, 136.8, 155.8, 168.8,

171.4, 173.3. Anal. called for C30H29N307: C, 66.29; H, 5.38; N, 7.73. Found: C, 65.96; H, 5.36;

N, 7.51.

Benzyl (S)-2-benzyloxycarbonylamino-N-((S)-1-caroy2pelthlmn)4

oxobutanoate (Z-L-Asp-OBzl-L-Phe-OH, 2.12b): White microcrystals (91%); mp 138-140 oC,

[a]D23 = +0.08 (c 2.08, DMF). 1H NMR (DMSO-d6): 6 2.58-2.90 (m, 3H), 3.01 (dd, J= 15.4, 4.6

Hz, 1H), 4.43- (quintet, J= 8.0 Hz, 2H), 5.03 (s, 2H), 5.11 (s, 2H), 7.16-7.34 (m, 15H), 7.60 (d, J

= 8.2 Hz, 1H), 8.33 (d, J= 8.0 Hz, 1H), 12.78 (br s, 1H). 13C NMR (DMSO-d6): 6 30.7, 36.8,

50.7, 53.7, 65.6, 66.1, 126.4, 127.7. 127.8, 127.9, 128.0, 128.2, 128.4, 128.5, 129.1, 135.9, 136.8,









ACKNOWLEDGMENTS

I thank my family, my teachers, and my colleagues. I am greatly indebted to Dr.

Encarnacion Lopez for my fascination with organic chemistry. I am grateful to Prof. Alan R.

Katritzky for giving me the opportunity to experience the j oys and the trials of being a true

scientist and to my committee members for their assistance and care. Very special thanks to

Valerie Rodriguez-Garcia for setting me on the right path. My thanks for the invaluable help and

suggestions in the preparation of this thesis go to Boris Grinkot, Adam Vincek, and Danniebelle

Haase. I also wish to thank all my friends and colleagues for their company, team spirit, and

international food!











RN N O Ph Z' \N COOH
CH3CNO / H20 H
Z'NH N ` ~ +(H) OHCN (CH2 O CH
HO j\Et3N, r.t., 2h. Oi
H2N COOH Oq
2.1a, b, 2.1a+a' 2.2a,b Ph

O$Gu22 2.3a-c, 2.3a+a'



n= 1, 2.
Amino acids with R: a, Ala; b, Phe; a+a', DL-Ala.
Figure 2-2. Preparation of novel dipeptides 2.3a-c and diastereomeric mixture 2.3a+a'

Table 2-1. Preparation of novel natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a'
Entry N-(Z-a-aminoacyl) Yield ,.25
benzotriazoles rlUCL(%)u U
Z -L -Al a-L -Glu(OB zl)-OH
1 Z-L-Ala-Bt (2.1a) 94 +1.0 89-91
(2.3a)
Z-L-Phe-L-Glu(OBzl)-OH
2 Z-L-Phe-Bt (2.1b) 97 -10.3 139-142
(2.3b)
Z-L-Phe-L-Asp(OBzl)-OH
3 Z-L-Phe-Bt (2.1b) 95 -14.0 102-105
(2.3c)
Z-DIL-Ala-Bt Z -DLL-Al a-L -Gl u(OB zl)-OH
S(2.1a+a') (2.3a+a') +. 18
a Isolated yield


The dipeptide 2.3a and the diastereomeric mixture 2.3a+a' were further subjected to HPLC

analysis using Chirobiotic T column (detection at 254 nm, flow rate 0.5 mL/min, and 100%

MeOH as solvent). As expected, HPLC analysis of the enantiopure LL- dipeptide (2.3a) showed

a single peak at 6.3 min. In contrast, two peaks of equal intensity at 6.3 and 6.7 min were

observed for the corresponding diastereomeric mixture 2.3a+a'.

2.2.2 Extensions of y-acids with ot-CO2H Protected.

The preparation of unnatural dipeptides 2.5a-c and mixture 2.5c+c' from a-benzyl L-

glutamate 2.4a and N-(a-aminoacyl)benzotriazoles 2.1b-d and 2.1d+d' were prepared using the

same procedure as mentioned for the natural dipeptides 2.3a-c. The two LL-dipeptides 2.5a,c










LIST OF TABLES


Table page

2-1. Preparation of novel natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a' ............18

2-2. Preparation of novel unnatural dipeptides 2.5a-c and mixture 2.5c+c' ................ ...............19

2-3. Preparation of novel dipeptides 2.7a-e .........__. .......... ...............21...

2-4. Preparation of novel dipeptides 2.10a-c ........._._. ......_.._ ....._ ...........2

2-5. Preparation of novel dipeptides 2.12a,b and the diastereomeric mixture 2.12b+b' .............23

2-6. Conversion of novel N"-Z-dipeptides 2.3a,b,d and the diastereomeric mixture 2.3a+a'
into N"-Z-dipeptidoylbenzotriazoles 2.13a-c, 2.13a+a' ........._._... ......__. ........._...25

2-7. Preparation of novel tripeptides 2.14a,b,a' and mixture 2.14a+a" .........__ .... .............. ..26

2-8. Preparation of novel tripeptides 2.16a,b containing Glu and Asp fragments .................. .....28










(m, 2H), 8.54 (s, 1H). 13C NMR (CDCl3): 6 24.6, 25.1, 25.9, 26.9, 66.8, 72.8, 79.2, 82.6, 85.0,

101.9, 109.4, 113.3, 116.8, 117.7, 124.9, 129.7, 134.8, 149.6, 155.3, 155.6, 162.0. Anal. called

for C22H2409: C, 61.11; H, 5.59; N, 0.00. Found: C, 61.02;H, 5.54; N, 0.03

2,2-Dimethyl-propionic acid (3S,5S,6R)-3,4,5-tris-(2,2-dimethyl-propioyox)6[(2-

oxo-2H-chromene-3-carbonyl)-amino] -tetrahydro-pyran-2-ylmethyl ester, 3.16: Clear solid

(60%), 1H NMR (CDCl3): 6 1.00 (s, 9H), 1.06 (s, 9H), 1.10 (s, 9H), 1.23 (s, 9H), 3.91 3.99 (m,

1H), 4.06 -4.16 (m, 2H), 5.20 5.35 (m, 2H), 7.30 -7.39 (m, 2H), 7.60 -7.68 (m, 2H), 8.83 (s,

1H), 9.29 (d, J=9.1Hz, 1H). 13C NMR (CDCl3): 6 26.7, 27.0, 27.1, 29.6, 38.6, 38.6, 38.7, 39.0,

60.7, 66.7, 67.7, 71.1, 72.7, 78.5, 116.7, 117.2, 118.3, 125.3, 130.0, 134.6, 149.5, 154.6, 160.6,

162.0, 176.8, 177.0, 177.1, 177.7. Anal. called for C22H2409: C, 62.87; H, 7.18; N, 2.04. Found:

C, 62.69; H, 7.68; N, 2.07.

(LS)-2-Benzyloxycarbonylamino-6- [(2-oxo-2H-chromene-3-carbonyl)

-amino]-hexanoic acid 5-(2,2- dimethyl-[1,3] dioxolan-4-yl)-2,2 -dimethyl-tetrahydro-

furo[2,3-d] [1,3]dioxol-6-yl ester, 3.17a: White microcrystals (82%), mp 123.0 124.0oC. 1H

NMR (CDCl3): 6 1.29 (s, 3H), 1.31 (s, 3H), 1.38 (s, 3), 1.41-1.48 (m, 2H), 1.51 (s, 3H), 1.58 -

1.98 (m, 4H), 3.38 3.58 (m, 2H), 3.97 (dd, J = 8.6, 4.2 Hz, 1H), 4.07 (dd, J = 8.6, 5.1 Hz, 1H),

4.1 4-4.25 (m, 2H), 4.29-4.40 (m, 1H), 4.48 (d, J = 3.4 Hz, 1H), 5.07(d. J = 12.2 Hz, 1H, B part

of AB system), 5.13 (d, J = 12.2 Hz, 1H, A part of AB system), 5.40-5.52 (m, 1H), 5.65 (d, J =

7.4 Hz, 1H), 5.82 (d, J = 3.4 Hz, 1H), 7.28-7.43 (m, 7H), 7.50 (d, J = 7.1 Hz, 1H), 7.62-7.72 (m,

1H), 8.82-8.96 (m, 2H). 13C NMR (CDCl3): 6 22.2, 25.2, 26.2, 26.7, 26.8, 29.1, 31.3, 38.7, 54.0,

66.9, 67.2, 72.4, 76.9, 79.7, 83.0, 105.0, 109.3, 112.4, 116.5, 116.5, 118.2, 118.6, 125.3, 128.0,

128.2, 128.5, 129.8, 134.1, 136.2, 148.6, 154.3, 156.0, 161.5, 161.9, 171.0. Anal. called for

C36H42N2012: C, 62.24; H, 6.09; N, 4.03. Found: C, 62.34; H, 6. 11; N, 4.09.









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

N- AND O- ACYLATION OF PEPTIDES AND SUGARS INT PARTIALLY AQUEOUS
MEDIA

By

Janet Cusido

December 2007

Chair: Alan R. Katritzky
Major: Chemistry

The convenient preparation of N-(Fmoc- or Z-a -aminoacyl)benzotriazoles and N-protected

peptidoylbenzotriazoles from aspartic and glutamic amino acids is discussed. Additionally,

diverse N-protected di- and tripeptides are synthesized under mild reaction conditions in good to

excellent yi elds by acyl ati on with N-(Z and Fmoc-a-aminoacyl)b enzotri azol es of the amino

groups of free aspartic and glutamic acids. Examples of peptide coupling utilizing free -amino

acids in partially aqueous solution are reported and the products are obtained without the use of

chromatography. Evidence of maintained chirality was supported by NMR and HPLC.

In addition, we present the suitable and efficient fluorescent labeling of sugars by O-

acylation of diisopropylidene protected sugars and N-acylation of pivaloyl protected aminosugar

with N-(coumarin-3 -carbonyl)benzotriazole and benzotriazole derivatives of N"-coumarin-

labeled N"-protected-L-lysines under microwave irradiation or/and at room temperature.

Monosaccharide containing Fmoc-lysine fluorescent building blocks can be useful as water

soluble organic fluorophores for peptide labeling at the C-terminus in solid-phase peptide

synthesis (SPPS).









110.2, 111.4, 118.2, 118.6, 120.9, 124.0, 127.3, 127.6, 127.7, 128.4, 136.1, 137.0, 155.9, 172.2,

173.3, 173.9 Anal. called for C2 H25N307: C, 61.66; H, 5.39; N, 8.99. Found: C, 61.72; H, 5.73;

N, 8.41.

(LS)-2-((LS)-2-Benzyloxycarbonylamino-3-phnlrpoyaioscii acid (Z-L-

Phe-L-Asp-OH, 2.7e): White microcrystals (85%); mp 179-181 oC, [a]D23 = -1.66 (c 1.66,

DMF). 1H NMR (DMSO-d6): 6 2.60-2.71 (m, 3H), 2.99-3.03 (m, 1H), 4.30 (apparent t, J= 7.6

Hz, 1H), 4.55-459 (m, 1H), 4.93 (s, 2H), 7.14-7.31 (m, 10 H), 7.53 (d, J= 8.5 Hz, 1H), 8.42 (d, J

= 8.0 Hz, 1H), 12.64 (br s, 2H). 13C NMR (DMSO-d6): 6 36.0, 37.5, 48.7, 56.0, 65.2, 126.3,

127.4, 127.7, 128.1, 128.3, 129.3, 137.0, 138.2, 155.9, 171.6, 171.7, 172.4. Anal. called for

C21H22N207: C, 60.86; H, 5.35; N, 6.76. Found: C, 61.07; H, 5.75; N, 6.53.

2.4.2 General Procedure for the Preparation ofN-(Z- and Fmoc-Aminoacyl)benzotriazoles
2.8 a,b and 2.11 a,b.

Thionyl chloride (5 mmol) was added to a solution of 1H-benzotriazole (20 mmol) in dry

CH2 12 (15 mL) at 20 oC, and the reaction mixture was stirred for 20 min at 40-50 oC. To the

reaction mixture at 0 oC, the N-protected amino acid (5 mmol) dissolved in dry CH2 12 (5 mL)

was added dropwise, and was then stirred for 2 hours at 20 oC. The white precipitate formed

during the reaction was filtered off, and the filtrate was concentrated under reduced pressure. The

residue was diluted with ethyl acetate (100 mL) and the solution was washed with 4N HCI

solution (50 mLx3) or saturated Na2CO3 Solution (50 mLx3), saturated NaCl solution (50 mL),

and dried over anhydrous MgSO4. Removing solvents under reduced pressure gave products

2.8a,b and 2.11a,b which were recrystallized from CHCl3/hexanes, unless specified otherwise.

Compounds 2.8a,b and 2.11a,b are novel and fully characterized by NMR and elemental

analy si s.










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78. Kolter, K.; Sandhoff, K. Angew.Chens. Int. Ed. 1999, 38, 1532.

79. a) Dell, A.; Morris, H. R.; Science 2001, 291, 2351. b) Shriver, Z.; Raguram, S.;
Sasisekharan, R. Nat. Rev. Drug Discovely 2004, 3, 863. c) Morelle, W.; Michalski, J. C.
Curr. Anal. Chent. 2005, 1, 29.

80. Mechrev, Y.; Novotny, M. Chens. Rev. 2002, 102, 321.

81. Tinnefeld, P.; Sauer, Markus. Angew. Chenz. hIt. Ed. 2005, 44, 2642.

82. Swedlow, J. R.; Platani, M. Cell Sruct. Funct. 2002, 27, 335.









BIOGRAPHICAL SKETCH

Janet Cusido grew up in Miami, Florida, with her parents Ramon and Oneida, and brother

Ramon Alej andro. In high school, she developed a love for the sciences, especially for math and

chemistry. She graduated from Coral Gables Senior High and enrolled at the University of

Florida in order to pursue a degree in chemical engineering. During her junior year at the

University of Florida, she took organic chemistry lab led by teaching assistant Valerie

Rodriguez-Garcia, where Janet discovered her passion for laboratory experimentation. Thanks to

Valerie, Janet decided to switch maj ors from chemical engineering to chemistry. Janet started to

perform research with Professor Alan R. Katritzky under the supervision of Valerie and learned

how important organic chemistry was in everyday life. Janet graduated with High Honors from

the University of Florida in April 2005 and then enrolled in the graduate program at UF,

continuing her research with Dr. Katritzky in the field of Benzotriazole Chemistry. After Janet

graduates from the University of Florida in December 2007, she plans to continue her graduate

studies at another institution to expand her knowledge and experience.










was removed under reduced pressure affording the tripeptides 2.16a,b. Further purification was

performed by recrystallization from ether-hexanes for elemental analysis.

Benzyl (S)-4-((LS)-2-benzyloxycarbonylamino-3-pheypoanmd)4[(S)-1-

carb oxy-2-( 1H- in dol-3-yl)ethyl amin o]-5- oxo pentano ate (Z-L-P he-L-Gl u(OBzl)-L-T rp- OH,

2.16a): White microcrystals (84%); mp 109-111 oC, [a]D23 = -3.2 (c 1.66, DMF). 1H NMR

(DMSO-d6): 8 1.80-2.10 (m, 2H), 2.44 (t, J= 8. 1 Hz, 2H), 2.66-2.83 (m, 2H), 2.95-3.12 (m, 2H),

3.21 (dd, J= 14.7, 4.9 Hz, 1H), 4.30-4.51 (m, 3H), 4.93 (s, 2H), 5.10 (s, 2H), 6.96-7.10 (m, 2H),

6.99 (t, J= 7.6 Hz, 1H), 7.07 (t, J= 6.9 Hz, 1H), 7. 16-7.36 (m, 15H), 7.54 (t, J= 7.2 Hz, 2H),

8. 17 (d, J= 7.7 Hz, 1H), 8.28 (d, J= 7. 1 Hz, 1H), 10.89 (br s, 1H). 13C NMR (DMSO-d6): 8

26.9, 27.7, 29.9, 37.3, 51.6, 53.1, 56.1, 65.3, 65.6, 109.6, 111.4, 118.2, 118.5, 121.0, 123.7,

126.3, 127.3, 127.5, 127.6, 127.7 128.0, 128.1, 128.3, 128.5, 129.3, 136.1, 136.3, 137.0, 138.1,

155.9, 171.0, 171.5, 172.4, 173.3. Anal. called for C40H40N40s: C, 68.17; H, 5.72; N, 7.95.

Found: C, 67.84; H, 5.79; N, 7.68.

Benzyl (S)-3-((,S)-2-benzyloxycarbonylam inopropionylam ino)-N-[(S)-1 -carboxy-2-

(1H-indol-3-yl)ethyl] butanoate (Z-L-Ala-L-Asp(OBzl)-L-Trp-OH, 2.16b): White

microcrystals (83%); mp 134-137 oC, [a]D23 = -1.7 (c 1.66, DMF). 1H NMR (DMSO-d6): 8 1.15

(d, J= 7. 1 Hz, 3H), 2.60-2.71 (m, 1H), 2.82 (dd, J= 14.7, 5.2 Hz, 1H), 3.07-3.14 (m, 2H), 4.03

(quintet, J= 7. 1 Hz, 1H), 4.45 (q, J= 6.3 Hz, 1H), 4.70 (q, J= 6.3 Hz, 1H), 4.92-5.10 (m, 4H),

6.97 (t, J= 7.1 Hz, 1H), 7.06 (t, J= 8.0 Hz, 1H), 7.15 (apparent s, 1H), 7.31-7.35 (m, 11H), 7.48-

7.54 (m, 2H), 7.94 (d, J= 7.4 Hz, 1H), 8.26 (d, J= 7.0 Hz, 1H), 10.85 (s, 1H), 12.85 (br s, 1H).

13C NMR (DMSO-d6): 8 18.2, 27.1, 36.3, 49.4, 50.3, 53.2, 65.6, 65.9, 109.6, 111.5, 118.3, 118.6,

121.1, 123.9, 127.7, 127.9, 128.0, 128.1, 128.2, 128.5, 128.6, 136.1, 136.2, 137.1, 155.9, 170.2,



































O 2007 Janet Cusido









(SMe) from Z-L-Met fragment. 13C NMR displayed a singlet for each carbonyl carbon for

compounds 2.5a,c whereas for the diastereomeric mixture 2.5c+c', most of the aliphatic and

carbonyl carbons were observed as doublets, but no significant changes were observed for the

aromatic carbons. In addition, the formation of rotamers was observed for the DL-dipeptide

(2.5b), showing complicated 1H and 13C NMR spectra, e.g. 13C analysis gave a second set of

signals for all aliphatic, carbonyl and aromatic carbons.

The enantiopurity of the dipeptides 2.5a-c was further confirmed by the HPLC analysis

using a Chirobiotic T column (detection at 254 nm, flow rate 0.5mL/min, and MeOH as solvent).

As expected, HPLC analysis of the enantiopure LL (2.5a,c) and DL (2.5b) dipeptides gave a

single peak for each compound (Table 2-2).

2.2.3 Preparation of Dipeptides 2.7a-e from Unprotected Glutamic or Aspartic Acids and
N-(a-Aminoacyl)benzotriazoles 2.1a,b,d and e.

Dipeptides 2.7a-e were obtained from unprotected glutamic (2.6a) and aspartic acids

(2.6b) (1 equiv.), N-(Z-a-aminoacyl)benzotriazoles 2.1a,b,d and e (1 equiv.), and triethylamine

(2 equiv.) in acetonitrile-water mixture (2:1 by volume) at room temperature for 2 hours (Figure

2-4 and Table 2-3). The crude products were washed with 4N HCI to remove the by-product,

BtH. Compounds 2.7a-e were obtained in 65-94% yields and were further recrystallized from

CH2 2z/hexanes for further characterization.

NMR analysis of 2.7a-e showed no detectable racemization: each compound revealed two

doublets for each of the two -NH protons in the range of 7.40-8.50 ppm. The methyl protons

from the Z-L-Ala fragment showed a clear doublet in the 1H NMR of 2.7a. The 1H NMR spectra

also displayed a singlet for the methyl protons (SMe) from Z-L-Met fragment for compounds

2.7c. In addition, 13C NMR displayed a singlet for each carbonyl carbon for dipeptides 2.7a-e.









NMR analysis of the enantiopure LL-dipeptides 2.12a,b revealed no detectable

racemization. Signals arising from diastereomers were not observed in the NMR spectra of

compounds 2.12a,b. Thus, each dipeptide revealed two sets of doublets for the two -NH proton

signals. However, for the diastereomeric mixture 2.12b+b' one of the two -NH protons

appeared as two pairs of equal doublets. 13C NMR displayed a singlet for each carbonyl carbon

for compounds 2.12a,b whereas for the diastereomeric mixture 2.12b+b', most of the aliphatic

and carbonyl and aromatic carbons were observed as doublets.

The dipeptides 2.12a,b were further characterized by HPLC analysis using a Chirobiotic T

column (detection at 254nm, flow rate 0.5mL/min, and MeOH as solvent). As expected, HPLC

analysis of enantiopure LL (2.12a,b) dipeptides showed a single peak for each compound. In

contrast, two peaks were observed for the corresponding diastereomeric mixture 2.12b+b'.

2.2.6 Preparation of F-Protected-Dipeptidoylbenzotriazoles 2.13a-c and 2.13a+a' from N"-
Protected Dipeptides 2.3a,b,d and 2.3a+a'

N"-Z-Dipeptides 2.3a,b,d and 2.3a+a' were successfully converted into the corresponding

benzotriazole derivatives 2.13a-c and 2.13a+a' (Figure 2-7 and Table 2-6). The reactions were

carried out at -15 oC following the same simple procedure as in references 62 and 63. The

reaction was continued until the starting materials 2.3a,b,d and 2.3a+a' were completely

consumed as observed under TLC and NMR analyses. Compounds 2.13a-c, 2.13a+a' were

isolated after acid (4N HC1) workup in good yields (89-93%) and were recrystallized using

CH2 2z/hexanes for 1H and 13C NMR spectroscopy, elemental analysis, and ORP.

Compounds 2.13a-c were obtained with no detectable racemization as evidenced by NMR.

Compounds 2.13a-c gave two doublets for the two -NH protons. 1H NMR spectrum of 2.13a

indicated doublet for the methyl proton of L-Ala fragment. In contrast, the same signal of the

methyl group showed two sets of doublets for the diastereomeric mixture 2.13a+a'. 13C NMR









Glutamic and aspartic acids are important elements for the biological activity of diverse

naturally occurring and synthetic peptides and their analogs.14 The side-chain carboxylic acid

group of both of these amino acids enables specific recognition by various receptors through

ionic interactions; hence numerous biologically active peptidomimetics incorporate glutamic and

aspartic acid fragments in their structures.26

Glutamic acid plays a pivotal important role as the main excitatory neurotransmitter of the

central nervous system (CNS), operating through four different classes of receptors. Therefore,

glutamic acid has received much attention in the design of glutamate receptor ligands for drugs.27

Numerous small peptides and peptidomimetics, containing Asp and Glu residues have been

suggested as prodrugs to enhance CNS effects.27-31

In peptide coupling incorporating aspartic and glutamic acids could involve either of the

two carboxylic acids.35 Especially in the case of Asp, the two isomeric forms frequently

interconvert during coupling or subsequently. Procedures developed previously for peptide

coupling incorporating Asp and Glu include: (i) carbodiimides in combination with additives

such as 1 -hydroxybenzotriazole (HOBt),36,37 1 -hydroxy-7-azabenzotriazole (HOAt) and

analogs38 Or N-hydroxysuccinimide (HOSu),39 (ii) phosphonium,40,41 and uronium saltS42,43 Of

HOBt or HOAt; (iii) N-acylazoles such as 1,1'-carbonylbis(1H-imidazole) (CDI);44 (iv) mixed

anhydrides;45 Or (V) carboxylic acid fluorides.46,47 The most common procedures for the

preparation of peptides containing glutamic and aspartic acids are based on solid-phase

methodology .28,32-34

A commonly encountered problem in peptide synthesis is epimerization of the amino acid

chiral center during activation of the carboxylic acid group. Many of the coupling reagents

require prior protection and subsequent deprotection of various amino acid functional groups.48









compared to other activating groups such as cyano, phenylsulfonyl and halogen analogues;

however, Bt offers intermediates that are more stable, nonvolatile and less physiologically

hazardous to prepare.




N N



R H
1.2 acidic proton 1.3
prone to deprotonation
typically for typically for
X= NR2, OR, SR 1.1 X= Halogen, OH

Figure 1-1. Properties of N-Substituted benzotriazoles as electron donors or electron acceptors

Benzotriazole methodology has become a fundamental synthetic tool for many chemical

processes, such as multi step preparations of drugs, biologically active compounds and synthetic

analogs of natural products. Our group has focused some of its research on the preparation of N-

acylbenzotriazoles. Two methods were utilized in our laboratory to prepare N-

acylbenzotriazoles directly from carboxylic acids. The first method employs

sulfonylbenzotriazoles as a counter attack reagent.4,5 In the presence of triethylamine, carboxylic

acids were converted into the desired acylbenzotriazoles, probably through intermediate

formation of the mixed carboxylic sulfonic anhydride and benzotriazole anion, which was then

acylated by the mixed anhydride (Figure 1-2).



.jiOHBtSO2R2
R OH Et3N R O-S M2 + Bt + Et3NHR B



Figure 1-2. N-Acylbenzotriazoles from sulfonylbenzotriazoles









2.4 Experimental Section

Melting points were determined on a capillary point apparatus equipped with a digital

thermometer. NMR spectra were recorded in CDCl3 Or DMSO-d6 with TMS for 1H (300 MHz)

and 13C (75 MHz) as an internal reference. N-Z- and Fmoc-amino acids were purchased from

Fluka and Acros and were used without further purification. Elemental analyses were performed

on a Carlo Erba-1 106 instrument. Optical rotation values were measured with the use of the

sodium D line. HPLC analyses were performed on Beckman system gold programmable solvent

module 126, using Chirobiotic T column (4.6 x 250 mm), detection at 254 nm, flow rate of 0.5

mL/min and 100% MeOH as eluting solvent.

2.4.1 General Procedure for the Preparation of Dipeptides 2.3a-c, 2.3a+a', 2.5a-c, 2.5c+c'
and 2.7a-e

Benzyl protected (2.2a,b and 2.4a) or unprotected (2.6a,b) aspartic or glutamic acids (5

mmol) were added to a solution of Et3N (10 mmol) in CH3CN (15 mL) and H20 (7 mL) at 25 oC,

and the reaction mixture was stirred for 15 min at 25 oC. N-(Protected-a-

aminoacyl)benzotriazoles (2.1a-e and 2.1a+a') (for characterization and preparation refer to

references 51, 61, and 63) (5 mmol) were added to the mixture with continued stirring for 2 h at

25 oC. About 4N HCI (5 mL) was added to the reaction mixture and CH3CN was evaporated

under reduced pressure. The residue was dissolved in EtOAc (50 mL), the organic extract was

then washed with 4 N HCI (3x15 mL), saturated NaCl (20 mL), and dried over MgSO4.

Evaporation of the solvent gave the desired products (2.3a-c, 2.3a+a', 2.5a-c, 2.5c+c', 2.7a-e),

which were further recrystallized from CH2C 2-hexane, unless specified otherwise.

5-Benzyl (LS)-2-((S)-2-benzyloxycarbonylamino-propoymioennot (Z-L-Ala-

L-Glu(OBzl)-OH, 2.3a): White microcrystals (94%); mp 89-91 oC, [a]D23 = +1.0 (c 1.66, DMF).

1HNMR (DMSO-d6): 6 1.26 (d, J = 7.1 Hz, 3H), 1.80-1.96 (m, 1H), 2.00-2.18 (m, 1H), 2.30-










LIST OF REFERENCES

1. Katritzky, A. R.; Rogovoy, Boris V. Chem. Eur. J. 2003, 9, 4586.

2. Katritzky, A. R.; Lan, X.; Yang, J.; Denisko, O. Chem. Rev. 1998, 98, 409.

3. Katritzky, A. R.; Belyakov, S. Aldchim. Act. 1998, 31, 35.

4. Katritzky, A. R.; Shobana, N.; Pernak, J.; Afridi, A. S.; Fan, W. Q. Tetr~rt~rt~raheron~rt~t~rt 1992, 48,
7817.

5. Katritzky, A. R.; He, H.-Y.; Suzuki, K. J. Org. Chem. 2000, 65, 8210.

6. Katritzky, A. R.; Zhang, Y.; Singh, S. K. Synthesis 2003, 2795.

7. Katritzky, A. R., Suzuki, K.; Singh, S.K. Syinhesis\i 2004, 2645.

8. Katritzky, A. R.; Angrish, P.; Hiir, D.; Suzuki, K. Syinhesis\i 2005, 3, 397.

9. Katritzky, A. R.; Angrish, P.; Suzuki, K. Sy inhes~i\ 2006, 3, 411.

10. Katritzky, A. R.; Todadze, E.; Cusido, J. Angrish, P.; Shestopalov A. Chem. Biol.
Drug Des. 2006, 68, 42.

11. Katritzky, A. R.; Todadze, E.; Shestopalov A.; Cusido, J. Angrish, P. Chem. Biol. Drug
Des. 2006, 68, 37.

12. Katritzky, A. R.; Narindoshvili, T, Angrish, P. Manuscript submitted to Synlett

13. Katritzky, A. R.; Cusido, J.; Narindoshvili, T. Manuscript in progress

14. Gill, I.; L6pez-Fandifio, R.; Jorba, X.; Vulfson, E. N. Enzym. M~icro. Tech. 1996, 18, 162.

15. Nishimura, T.; Kato, H. FoodRev Int. 1988, 4, 39.

16. Sturtevant, Frank M. J. Environ. Sci. Health Part A Environ. Sci. Eng. 1985, 20, 863.

17. Grenby, T. H. Trends Food Sci. Technol. 1991, 2, 2.

18. Nosho, Y.; Seki, T.; Kondo, M.; Ohfuji, T.; Tamura, M.; Okai, H. J. Agric. Biol. Chem.
1990, 38, 1368.

19. Arai, S. A. Anal. Control Less Desirable Flavor Foods Beverages, (Proc Symp).
Academic, New York, 1980, 133.

20. Matoba, T.; Hayashi, R.; Hata, T. Agric. Biol. Chem. 1988, 34, 1235.










Benzyl (S)-5-Benzotriazol-1-yl-4-(2-benzyloxycaroymipopn id)5

oxopentanoate (Z-DL-Ala-L-Glu(OBzl)-Bt, 2.13a+a'): White microcrystals (91%); mp 79-81

oC, [a]D23 = -22.4 (c 1.66, DMF). 1H NMR (CDCl3): 6 1.40 (d, J= 6.9 Hz, 3H), 2.27-2.34 (m,

1H), 2.47-2.68 (m, 3 H), 4.30-4.40 (m, 1H), 5.07 (s, 2H), 5.12 (s, 2H), 5.31 (d, J= 6.6 Hz, 1H)

5.93 (br s, 1H), 7.25-7.42 (m, 11H), 7.52 (t, J= 7.6 Hz, 1H), 7.65 (t, J= 6.9 Hz, 1H), 8.13 (d, J=

8.2 Hz, 1H), 8.19-8.24 (m, 1H). 13C NMR (CDCl3): 6 18.4, 18.6, 26.7, 27.0, 30.2, 30.3, 50.3,

51.6, 52.7, 66.5, 66.8, 67.1, 114.3, 120.3, 125.8, 126.5, 127.9, 128.0, 128.1, 128.2, 128.3, 128.5,

128.6, 128.7, 130.7, 131.0, 135.3, 135.5, 136.0, 145.9, 156.0, 156.1, 170.3, 170.4, 172.8, 173.6,

173.7. Anal. called for C29H29N5O6: C, 64.08; H, 5.38; N, 12.88. Found: C, 64.37; H, 5.28; N,

12.49.

2.4.5 General Procedure for the Preparation of Tripeptides 2.14a,b, 2.14a' and 2.14a+a"

Unprotected amino acids (2.9c,e and f; 1 mmol) were dissolved in a mixture of acetonitrile

(10 mL), water (5 mL), and triethylamine (2.5 mmol). N"-protected-dipeptidoylbenzotriazoles

2.13a,b, 2.13a+a' (1 mmol) were added to the reaction mixture at -15oC and the stirring

continued for additional 2 hours. Resulting solution was acidified with (1 mL) 4N HCI and

acetonitrile was removed under reduced pressure at room temperature. The residue was dissolved

in EtOAc (50 mL) and was then washed 3 times with 4N HCI (3 x 15 mL) followed by saturated

NaCl (20 mL). The organic layer was dried over magnesium sulfate and the solvent was removed

under reduced pressure yielding the tripeptides 2.14a,b, 2.14a' and 2.14a+a". Further

purification was performed by recrystallization from CH2 2z-hexanes for elemental analysis.

Benzyl (S)-4-((S)-2-benzyloxycarbonylaminopropanaio--()1croy2

phenylethylamino)-5-oxopentanoate (Z-L-Ala-Glu(OBzl)-L-Phe-OH, 2.14a): White

microcrystals (73%); mp 74-76 oC, [a]D23 = -2.9 (c 1.91, DMF.). 1H NMR (DMSO-d6): 6 1.15 (d,

J= 6.3 Hz, 3H), 1.70-2.00 (m, 2H), 2.32-2.45 (m, 2H), 2.86-2.93 (m, 1H), 3.05 (dd, J= 13.4, 4.2









2.2 Results and Discussion

2.2.1 Extensions of ot-Amino Acids with P and y-CO2H PrOtected.

The preparation of natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a' from 7-

benzyl-L-glutamate 2.2a or P-benzyl-L-aspartate 2.2b and N-(Z-a-aminoacyl)benzotriazoles

2.1a,b and 2.1a+a' was carried out by using as one component 2.1a or 2.1b and as the second

component equimolar amounts of L-Glu(OBzl)-OH (2.2a) or L-Asp(OBzl)-OH (2.2b). Each

coupling took place in acetonitrile-water mixture (2: 1 in volume) in the presence of triethylamine

(2 equiv.) at room temperature for 2 hours. The crude products were washed with 4N HCI to

remove the by-product (BtH) to yield the three LL-dipeptides 2.3a-c (94-97%) (Figure 2-2 and

Table 2-1). Diastereomeric mixture 2.3a+a' was prepared from N-(Z-DL-Ala)acylbenzotriazole

(2.1a+a') and y-benzyl-L-glutamate 2.2a in 94% yield using the same procedure as mentioned

for the enantiopure dipeptides 2.3a-c.


NMR analysis showed no detectable racemization for the three enantiopure LL-dipeptides

2.3a-c. No signals from their corresponding diastereomers were observed in the NMR spectra of

2.3a-c suggesting the enantiopurity of the N-protected dipeptides. Each dipeptide revealed two

sets of doublets for the two -NH protons ranging from 7.40 to 8.60 ppm. However, for the

diastereomeric mixture 2.3a+a' one of the two -NH protons appeared as two pairs of equal

doublets. In addition, the 1H NMR spectrum of 2.3a gave a clear doublet for the methyl protons

present in compound at 1.26 ppm (DMSO-d6), whereas a multiple was observed for the

corresponding diastereomeric mixture 2.3a+a' ranging from 1.34 to 1.38 ppm (CDCl3). The 13C

NMR spectrum displayed a singlet for each carbonyl carbon for compound 2.3a. However, for

2.3a+a' NMR analysis displayed doublets for most of the aliphatic and carbonyl carbons,

although no significant difference was observed for the aromatic carbons.









CHAPTER 2
SELECTIVE PEPTIDE CHAIN EXTENSION AT THE C- AND N-TERMINI OF ASPARTIC
AND GLUTAMIC ACIDS UTILIZINGN-PROTECTED (ALPHA-AMINOACYL)
BENZOTRIAZOLES

2.1 Introduction


Recently many short, long, and cyclic biologically active peptides have been isolated from

bacterial, fungal, plant, animal, and other sources. Peptides play pivotal roles as taste additives,

neuroactive or enzyme regulators, and as antibiotics. They also influence cell-cell

communication upon interaction with receptors and are involved in a number of biochemical

processes such as metabolism, pain, reproduction and immune response; such diverse roles have

driven intensive peptide research.14-2 Among the 20 naturally occurring peptide amino acids,

glutamic and aspartic acids are the representatives of dicarboxylic acids (Figure 2-1), often found

in peptides with sensory properties including sweetness, bitterness, bitterness-masking, and

flavor enhancements.14-25 The dipeptides aspartame (L-Asp-L-Phe-OMe) and alitame (L-Asp-D-

Ala-NH2) exemplify non calorific sweeteners and are used worldwide.14,17 Small peptides are

important biomolecules and many have therapeutic value. Unlike large peptides that are

commonly isolated from natural sources or produced through recombinant techniques, small

peptides are usually prepared using organic synthetic methods.

HO ,





OH
Aspartic Acid Glutamic Acid

Figure 2-1. Structures of Aspartic (Asp) and Glutamic (Glu) Amino Acids









diastereomeric mixture 2.14a+a" showed most of the aliphatic and carbonyl and aromatic

carbons as doublets.

To confirm the ab sence of racemizati on, the compounds Z -L-Al a-L-Glu(OBzl)-L-Phe-OH

(2.14a), Z-L-Ala-L-Glu(OBzl)-D-Phe-OH (2.14a') and the mixture Z-DL-Al a-L-Glu(OBzl)-L-

Phe-OH (2.14a+a") were analyzed by HPLC. Single peaks were obtained for tripeptides 2.14a

at 6.4 min and 2.14a' at 7.3 min whereas the diastereomeric mixture 2.14a+a" gave two peaks at

6.4 and 7.3 min.

2.2.8 Preparation of Novel Tripeptides 2.16a,b Containing Glu and Asp Fragments.

The general applicability of our coupling method was demonstrated by the preparation of

tripeptides 2.16a,b containing glutamate or aspartate moieties. This was achieved by the

elongation of Asp and Glu fragments from the N-terminus, utilizing the free amino group of

unprotected dipeptides 2.15a,b and N-(Z-a-aminoacyl)benzotriazoles 2.1a,b.

Synthesis of tripeptides 2.16a,b was achieved in two steps without purification of the

dipeptides intermediates 2.15a,b. First, the Fmoc- group in compounds 2.10a,b was removed

with piperidine utilizing a literature method;65 thereafter the remaining piperidine was removed,

the dipeptides obtained 2.15a,b were then coupled with N-(Z-a-aminoacyl)benzotriazoles 2.1a,b

yielding the desired tripeptides 2.16a,b. The procedure does not require purification of

compounds 2.15a,b; however, care must be taken to remove all remaining piperidine, as it can

compete with free amino acids and peptides in their reactions with N-protected(a-

aminoacyl)benzotriazoles. Crystalline tripeptides 2.16a,b were purified by washing with diethyl

ether-hexanes mixture giving desired product in the yields of 83-84% (Table 2-8). Novel

compounds 2.16a,b were characterized by 1H and 13C MR Spectroscopy, elemental and ORP

analyses.










present the convenient and efficient fluorescent labeling by O-acylation, of diisopropylidene

protected sugars 3.9-3.11, and N-acylation of pivaloyl protected aminosugar 3.15 with (i) N-

(coumarin-3-carbonyl)benzotriazole 3.4 and (ii) the benzotriazole derivatives 3.7, 3.8 of F-

coumarin-labeled N"-protected-L-lysines 3.5, 3.6 under microwave irradiation or at room

temperature. Monosaccharide containing Fmoc-lysine fluorescent building blocks can be useful

as water soluble organic fluorophores for peptide labeling at the C-terminus in solid-phase

peptide synthesis (SPPS).

3.2 Results and Discussion

3.2.1 Preparation of M-Coumarin-Labeled N"-Fmoc-L-lysine Benzotriazolide 3.8.

N"-Coumarin-3 -carbonyl-N"-Fmoc-L-lysine benzotriazole 3.8 (Figure 3-3) was prepared

(87%) from coumarin-labeled N-Fmoc-protected lysine 3.6 utilizing benzotriazole methodology

optimized in our laboratories, by reacting 1H-benzotriazole with thionyl chloride in CH2 12 at

200C for 2 hourS.10,11,49,61-63

3.2.2 Preparation of Coumarin-O-Tagged Monosaccharides: O-(Coumarin-3-
carbonyl)diisopropylidene Sugars 3.12, 3.13, 3.14.

O-Coumarin labeled diisopropylidene sugars 3.12, 3.13, 3.14 were prepared by coupling of

3.4 with the 6-OH of 1 ,2:3,4-Di-O-isopropylidene-a-D-galactopyrns 3.9, 3-OH of 1,2:5,6-Di-

O-i sopropylidene-a-D-glucose 3.10, and 1-OH of 2,3:5,6-Di-O-i sopropylidene-a-D-

mannofuranose 3.11, respectively in dichloromethane, utilizing equivalent of 4-

Dimethylaminopyridine (DMAP) under 100W microwave irradiation at 500C for 45 min (Figure

3-3). Products were isolated after simple acid work up without chromatography in 60-90%

yields.










Coupling reactions with such reagents are frequently moisture sensitive. Furthermore, isolation

and purification processes often involve column chromatography due to the formation of by-

products from the coupling reagents.

In Katritzky's group, researchers have applied N-acylbenzotriazoles for N-acylation,49-52 C

acyl ati on53-58 and O-acyl ati on.59,60 Recently, the synthesi s of N-(Z -aminoacyl)b enzotri azole s was

achieved in our laboratory and such were found to be efficient coupling reagents for N- and O-

aminoacylation. N-(Z-aminoacyl)benzotriazoles compounds are isolable, stable and storable at

room temperature for months, and easy to handle without special procedures to exclude air or

moisture. The reagents used in the preparation of aminoacylbenzotriazoles are inexpensive,

thereby offering at the same time an overall cost effective methodology. Additionally, these N-

(Z-aminoacyl)b enzotri azoles can b e used in aqueous soluti ons to effi ciently allow the coupling

of several non-derivatized amino acids. Thus, these coupling reagents enable fast synthesis of

peptides and peptoids in high yields, and purity under mild conditions with full retention of the

original chirality.

We now rep ort (i) the prep arati on of N-(Z or Fm oc -a-ami noacyl)b enz otri azol es derived

from L-Asp and L-Glu amino aci ds, and (ii) pepti de coupling of the se aminoacylb enzotri azol es

with unprotected a-amino acids and dipeptides yielding the corresponding natural and unnatural

di- and tri-peptides. Additionally, a new and convenient procedure for the selective stepwise

elaboration of peptide chains incorporating aspartic and glutamic acids by utilizing N-(protected

aminoacyl)benzotriazoles as coupling reagents is described. We show components of Asp and

Glu are elongated from the N-terminus by acylation at the free amino group of glutamic or

aspartic acids in which the -CO2H groups are unprotected or partially protected.










LIST OF FIGURES


Figure page

1-1. Properties of N-Substituted benzotriazoles as electron donors or electron acceptors ...........1 1

1-2. N-Acylbenzotriazoles from sulfonylbenzotriazoles ................. .............. ......... .....11

1-3. N-Acylbenzotriazoles from carboxylic acids, excess BtH and thionyl chloride ................... 12

1-4. Structure of N-(coumarin-3-carbonyl) benzotriazole .............. ...............13....

2-1. Structures of Aspartic (Asp) and Glutamic (Glu) Amino Acids ................. ............... .....14

2-2. Preparation of novel dipeptides 2.3a-c and diastereomeric mixture 2.3a+a'............._._... .....18

2-3. Preparation of novel unnatural dipeptides 2.5a-c and diastereomeric mixture 2.5c+c' ........19

2-4. Preparation of novel dipeptides 2.7a-e ........._._.. .. ...............21_._.. ....

2-5. Preparation of novel dipeptides 2.10a-c .........._.... ...............22.._._.. ...

2-6. Preparation of novel dipeptides 2.12a,b and diastereomeric mixture 2.12b+b' ..................23

2-7. Preparation of novel dipeptidoylbenzotriazoles 2.13a-c and diastereomeric mixture
2.13a+a' ............. ...............25.....

2-8. Preparation of novel tripeptides 2.14a,b,a', and 2.14a+a" ........._... ........_..........._26

2-9. Preparation of novel tripeptides 2.16a,b .............. ...............28....

3-1. Fluorescent labeling of saccharides by reductive amination ................. .......................47

3-2. Structures of N-(coumarin-3 -carbonyl) benzotriazole and N"-coumarin-labeled N"
protected-L-lysines............... ..........4

3-3. Syntheses of O-(coumarin-3-carbonyl)diisopropylidene sugars 3.12, 3.13, 3.14 and N-
(coumarin-3-carbonyl)tetrapivaloyl sugar 3.16. ............. ...............50.....

3 -4. Preparati on of O- and N-(N"-coumarin-3 -carb onyl-N"(Fmoc or Z -L-ly s)protected
sugars 3.17a,b, 3.18, 3.19, 3.20. ............. ...............51.....

3-5. Deprotection of diacetonide groups for compound 3.13 ................... ...............5

3-6. Deprotection of diacetonide groups for 3.17b............... ...............52.

3-7. Deprotection of diacetonide groups for 3.18 ........_........_...__ ........__ .........5










solution was washed with sat. Na2CO3 Soln. (3 x 50mL), sat. NaCl soln. (50mL), and dried over

MgSO4. Removal of the solvent under reduced pressure gave 3 -(Benzotriazole-1 -carbonyl)-

chromen-2-one 3.4, which was recrystallized from CH2 2z-hexanes for elemental analysis.

3-(Benzotriazole-1-carbonyl)chromen-2-one (Coum-Bt, 3.4): White microcrystals

(87%); mp 186-187 OC, 1H NMR (CDCl3): 6 7.41 (t, J= 7.6 Hz, 1H), 7.46 (d, J= 8.2 Hz, 1H),

7.58 (t, J= 8.2 Hz, 1H), 7.64-7.80 (m, 3H), 8. 16 (d, J= 8.2 Hz, 1H), 8.34 (s, 1H), 8.36 (d, J=

8.4 Hz, 1H). 13C NMR (CDCl3): 114.4, 117.2, 117.6, 120.5, 121.9, 125.3, 126.8, 129.6, 130.9,

131.2, 134.5, 146.2, 147.0, 154.9, 157.4, 162.6. Anal. Called for C16H9N303: C, 65.98; H, 3.11;

N, 14.43. Found: C, 65.67; H, 3.10; N, 14.22.

3.4.2 General Procedure for the Preparation of Compounds 3.5 and 3.6.

3-(Benzotriazole-1 -carbonyl)chromen-2-one 3.4 (1 mmol) was added to a solution of 1

mmol of N"-Fmoc- or Z-L-lysine in MeCN-H20 (10mL/5mL) mixture, in the presence of Et3N

(1 mmol). The reaction mixture was stirred at 20 OC for about 1h (until TLC shows absence of

3.4). Aqueous 4N HCI (1mL) was then added and MeCN was removed under reduced pressure.

The residue obtained was dissolved in EtOAc (150 mL), and washed with 4N HCI soln. (3 x 50

mL), sat. NaCl soln. (50 mL) and dried over MgSO4. After evaporation of solvent, the residue

was recrystallized from EtOAc-hexanes or CH2 2z-hexanes.

(LS)-2-Benzyloxycarbonylamino-6- [(2-oxo-2H-chromene-3-carbonyl)amino] hexa noic

acid (N"-Z-N"-Coumoyl-L-Lys-OH, 3.5): White microcrystals (89%); mp 144-145 OC, [a]23D

-8.54 (c 1.68, DMF). 1H NMR (CDCl3): 6 1.37-1.56 (m, 2H), 1.58-1.74 (m, 2H), 1.75-2.40 (m,

2H), 3.33-3.58 (m, 2H), 4.34-4.45 (m, 1H), 5.09 (s, 2H), 5.75 (d, J= 8.0 Hz, 1H), 7.27-7.42 (m,

7H), 7.52-7.72 (m, 2H), 8.92 (s, 1H), 8.95-9.04 (m, 1H). 13C NMR (CDCl3): 22.3, 28.9, 31.5,

39.3, 53.6, 66.9, 116.5, 117.9, 118.5, 125.3, 128.0, 128.1, 128.4, 130.0, 134.1, 136.2, 148.8,









poor photostability and crucially poor aqueous solubility. Solubility characteristics affects the

degree of self interaction in solution of chromophores conjugated to substrates and therefore

light absorption and emission propertieS.104 Bright fluorescent reagents, with good aqueous

solubility and low non-specific staining, are needed. Incorporating sugar units to fluorescent

reagents confers useful water solubility to organic fluorophores without significant change in

absorption and fluorescent propertieS.105,106

Researchers in the Katritzky group have recently uncovered N-(coumarin-3 -carbonyl)

benzotriazole 3.4,12 as a useful starting material, for convenient and reliable fluorescent labeling

of amino acids and dipeptides. Such coumarin-labeled lysines, including N"-coumarin-labeled

N"-protected-L-lysines 3.5, 3.6 are of considerable interest for the design and synthesis of

fluorogenic substrates to analyze matrix metalloproteinases (MMP).107-110 Their successful

labeling of amino acids and peptides in solution utilized 3.7, and a benzotriazole activated 3.5

(Figure 3-2).12
















Figure 3 -2. Structures of N-(coumarin-3 -carbonyl) benzotriazole and N"-coumarin-labeled N"-
protected-L-lysines

They also recently reported efficient O-acylation of diacetonide protected sugars with

readily available N-(Z-a-aminoacyl)benzotriazoles under microwave irradiation.97 We now











TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....

LIST OF TABLES ........._.___..... .__. ...............7....

LIST OF FIGURES .............. ...............8.....

AB S TRAC T ..... ._ ................. ............_........9

CHAPTER

1 GENERAL INTRODUCTION .............. ...............10....

2 SELECTIVE PEPTIDE CHAIN EXTENSION AT THE C- AND N-TERMINI OF
ASPARTIC AND GLUTAMIC ACIDS UTILIZING N-PROTECTED (ALPHA-
AMINOACY L) BENZ OTRIAZOLE S .............. ............... 14....

2.1 Introducti on .........._...... ...............14......__......
2.2 Results and Discussion .........._....._ ... .. ....... .. ......._...._ ... ...........1
2.2. 1 Extensions of a-Amino Acids with P and y-CO2H Protected.............__ ............ 17
2.2.2 Extensions of y-acids with a-CO2H Protected. ............... ....... ...............1
2.2.3 Preparation of Dipeptides 2.7a-e from Unprotected Glutamic or Aspartic
Acids and N-(a-Aminoacyl)benzotriazoles 2.1la,b,d and e ................... ................20
2.2.4 Peptide Chain Extension at the Alpha C-Terminus to Give Natural Dipeptides
2. 10a-c .................. .............. .. ........ ... .......................2
2.2.5 Peptide Chain Extension at the P or y C-Terminus to Give Unnatural
Dipeptides 2. 12a-d ................... .. ............. ... ......... .... .. .. .........2
2.2.6 Preparation of N"-Protected-Dipeptidoylbenzotriazoles 2. 13a-c and 2. 13a+a'
from N"-Protected Dipeptides 2.3 a,b,d and 2.3 a+a' ................. ......................24
2.2.7 Preparation of F-Protected Tripeptides 2.14a,b,a' and Diastereomeric
Mixture 2.14a+a" from Dipeptidoylbenzotriazoles 2.13a,b, 2.13a+a' and Free
Amino Acids 2.9c,e and f ................ ...... ... .. ...... ...............2
2.2.8 Preparation of Novel Tripeptides 2.16a,b Containing Glu and Asp Fragments....27
2.3 Conclusion ............ _...... ._ ...............28....
2.4 Experim ental Section................ .. ... ... ... .. .. .......................2
2.4.1 General Procedure for the Preparation of Dipeptides 2.3a-c, 2.3a+a', 2.5a-c,
2.5c+c' and 2.7a-e ................ ....... ........... .. .......2
2.4.2 General Procedure for the Preparation ofN-(Z- and Fmoc-
Aminoacyl)benzotriazoles 2.8 a,b and 2.11 a,b. .............. ... .......... ............3
2.4.3 General Procedure for the Preparation of Dipeptides 2.10a-c, 2.12a,b and
2.12b+b'....... .. ... .. ... .................... ...............3
2.4.4 General Procedure for the Preparation offa-Protected-
Dipeptidoylbenzotriazoles 2.13a-c and 2.13a+a' .............. ....................3










analysis displayed a singlet for each carbonyl carbon for compounds 2.13a-c, whereas in the

mixture 2.13a+a' most of the carbonyl, aliphatic and aromatic carbons appeared as doublets.


Z' N COOH Z N, O H22 N O
O1 Cl '`CI -1 50C O


Ph Ph

2.3a,b,d, 2.3a+a' 2.13a-c, 2.13a+a'

n= 1, 2.
Bt =benzotriazol-1 -yl
Figure 2-7. Preparation of novel dipeptidoylbenzotriazoles 2.13a-c and diastereomeric mixture
2.13a+a'

Table 2-6. Conversion of novel N"-Z-dipeptides 2.3a,b,d and the diastereomeric mixture
2.3a+a' into Na-Z -dipepti doylb enzotri azoles 2.13a-c, 2.13a+a'
Yield [a25oM(C
Entry Reagent Product (o/)a[]DMp(C
Z-L-Ala-L- Z-L-Ala-L-
1 93 -17 9 133-135
Glu(OBzl)-OH (2.3a) Glu(OBzl)-Bt (2.13a)
Z-L-Phe-L- Z-L-Phe-L-
2 92 -24.7 90-92
Glu(OBzl)-OH (2.3b) Glu(OBzl)-Bt (2.13b)
Z- L-Phe-L- Z-L-Phe-L-
3 89 -20.7 110-113
Asp(OBzl)-OH (2.3d) Asp(OBzl)-Bt (2.13c)
Z -DL -Al a-L- Glu(OB zl) Z -DL -Al a-L -Glu(OB zl)
4 91 -22.4 79-81
-OH (2.3a+a') -Bt (2.13a+a')
alsolated yield


2.2.7 Preparation of N"-Protected Tripeptides 2.14a,b,a' and Diastereomeric Mixture
2.14a+a" from Dipeptidoylbenzotriazoles 2.13a,b, 2.13a+a' and Free Amino Acids
2.9c,e and f

Tripeptides 2.14a,b and a' were prepared by peptide coupling reactions between N"-

protected-dipeptidoylbenzotriazoles 2.13a,b (1 equiv.) with unprotected amino acids 2.9c,e,f (1

equiv.) in acetonitrile-water mixture (2: 1 v/v) in the presence of triethylamine (2 equiv.) at -15

oC for 2 hours. The mixture 2.14a+a" was prepared using the same procedure. The crude

products were washed with 4N HCI to remove the by-product, BtH. Compounds 2.14a,b, 2.14a'










2.2.5 Peptide Chain Extension at the (3 or y C-Terminus to Give Unnatural Dipeptides
2.12a-d

Peptide coupling reactions were carried out using equimolar amounts of the ot-monobenzyl

ester N-(Z-a-aminoacyl)b enzotri azoles (Asp) 2.11a and (Glu) 2.11b and unprotected amino aci ds

(L-Trp, L-Phe, DL-Phe,) 2.9a,c,d in partially aqueous solution (CH3CN/H20, 2:1 v/v) in the

presence of triethylamine (2 equiv.) at room temperature for 2 hours (Figure 2-6). The crude

products were washed with 4N HCI to remove the by-product, BtH. The dipeptides 2.12a,b were

obtained in 91-92% yields (Table 2-5) and were further recrystallized from CH2 2z/hexane for

NMR, elemental analysis, and ORP. Diastereomeric mixture 2.12b+b' was prepared from N-(Z-

L-Asp)benzotriazole and DL-Phe-OH using the same procedure as for the enantiopure dipeptides

2.10a-c. Dipeptide mixture 2.12b+b' was obtained in 95% yield and was recrystallized from

diethyl ether/hexanes.

Btz O Oh NZ~OH E,~rth COOH

CH3CN / H20 R
H2 COO
HO HOPh
2.11a,b 2.9a,c,d
Bt = benzotriazol-1-yl. 21abbb
Amino acids with R: Trp, Phe, DL-Phe

Figure 2-6. Preparation of novel dipeptides 2.12a,b and diastereomeric mixture 2.12b+b'

Table 2-5. Preparation of novel dipeptides 2.12a,b and the diastereomeric mixture 2.12b+b'
Yie
Retention Melting
Amino Id [(25 tm on
Entry Product []D tm on
Acid (%).
a (min) (oC

L-Trp Z-L-Asp-OBzl-L-Trp-OH
1 92 -7.1 7.5 98-100
(2.9a) (2.12a)
L-Phe Z-L-Asp-OBzl-L-Phe-OH
2 91 +0.07 7.6 138-140
(2.9c) (2.12b)
DL-Phe Z-L-Asp-OBzl-DL-Phe-OH 9 1. .,1. 1-1
(2.9d) (2.12b+b')
alsolated yield









2.54 (m, 2H), 4.09-4.19 (m, 1H), 4.27-4.34 (m, 1H), 5.00-5.10 (m, 2H), 5.14 (s, 2H), 7.30-7.41

(m, 10H), 7.52 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 12.70 (s, 1H). 13C NMR (DMSO-

d6): 6 18.1, 26.3, 30.0, 49.9, 51.0, 65.4, 65.6, 127.8, 128.0, 128.1, 128.4, 128.5, 136.2, 137.1,

149.7 155.7, 172.2, 172.8, 173.2. Anal. called for C23H26N207: C, 62.43; H, 5.92; N, 6.33. Found:

C, 62.05; H, 5.95; N, 6.01.

5-Benzyl (LS)-2-((S)-2-benzyloxycarbonylamino-3-phnlrpoymioetnoe

(Z-L-Phe-L-Glu(OBzl)-OH, 2.3b): White microcrystals (97%); mp 139-142 oC, [a]D23 = -10.3

(c 1.66, DMF). 1H NMR (DMSO-d6): 6 1.88-1.95 (m, 1H), 2.08-2. 14 (m, 1H), 2.32-2.55 (m,

2H), 2.72-2.82 (m, 1H), 2.99-3.09 (m, 1H), 4.30-4.37 (m, 2H), 4.95 (s, 2H), 5.13 (s, 2H),

7. 16-7.41 (m, 15H), 7.57 (d, J= 8.7 Hz, 1H), 8.39 (d, J= 7.8 Hz, 1H), 12.73 (s, 1H). 13C NMR

(DMSO-d6): 6 26.3, 30.1, 37.4, 51.2, 56.1, 65.3, 65.6, 126.3, 127.5, 127.7, 128.0, 128.1, 128.2,

128.3, 128.5, 129.3, 136.2, 137.0, 138.2, 155.9, 171.9, 172.2, 173.1. Anal. called for C29H30N207:

C, 67.17; H, 5.83; N, 5.40. Found: C, 66.88; H, 5.87; N, 5.31.

4-Benzyl (LS)-2-((S)-2-benzyloxycarbonylamino-3-pheypoinlmn~uaot

(Z-L-Phe-L-Asp(OBzl)-OH, 2.3c): White microcrystals (95%); mp 102-105 oC, [a]D23 = -14.0

(c 1.66, DMF). 1H NMR (DMSO-d6): 6 2.65-3.07 (m, 4H), 4.29-4.35 (m, 1H), 4.65-4.73 (m,

1H), 4.89-4.98 (m, 2H), 5.13 (s, 2H), 7. 14-7.37 (m, 15H), 7.56 (d, J= 8.4 Hz, 1H), 8.52 (d, J=

7.7 Hz, 1H), 12.9 (s, 1H). 13C NMR (DMSO-d6): 6 36.0, 37.4, 48.6, 56.0, 65.2, 65.9, 126.3,

127.4, 127.7, 127.9, 128.0, 128.3, 128.4, 128.5, 129.2, 135.9, 137.0, 138.1, 155.8, 170.1, 171.6,

172.5. Anal. called for C28H28N207: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.33; H, 5.68; N, 5.60.

5-Benzyl (LS)-2-(2-benzyloxycarbonylaminopropionylaiopnaot (Z-DL-Ala-L-

Glu(OBzl)-OH, 2.3a+a'): White microcrystals (94%); mp 81-83 oC, [a]D23 = +0.6 (c 1.66,

DMF). 1H NMR (CDCl3): 6 1.34-1.38 (m, 3H), 2.04-2. 11 (m, 1H), 2.27-2.30 (m, 1H), 2.30-2.50










(m, 1H), 3.36-3.48 (m, 1H), 3.48-3.64 (m, 1H), 5.13 (s, 2H), 5.69-5.83 (m, 1H), 6.12 (d, J= 7.8

Hz, 1H), 7.26-7.47 (m, 7H), 7.52 (t, J= 7.6 Hz, 1H), 7.60-7.74 (m, 2H), 8. 13 (d, J= 8.2 Hz,

1H), 8.27 (d, J= 8.2 Hz, 1H), 8.86 (s, 1H), 8.82-8.97 (m, 1H). 13C NMR (CDCl3): 22.3, 28.9,

31.6, 38.5, 54.6, 67.1, 114.4, 116.5, 118.2, 118.6, 120.3, 125.2, 126.4, 128.0, 128.1, 128.5, 129.8,

130.6, 131.1, 134.0, 136.2, 145.9, 148.6, 154.3, 156.2, 161.4, 162.0, 171.7. Anal. Called for

C30H27N5O6: C, 65.09; H, 4.92; N, 12.65. Found: C, 64.91; H, 4.76; N, 12.59.


{(S)-1-(Benzotriazole-1-carbonyl)-5-[(2-ox-Hcrmn--abnl-mn]

pentyl}-carbamic acid 9H-fluoren-9-ylmethyl ester acid (N"-Fmoc-Ne-Coumoyl-L-Lys-Bt,

3.8): White microcrystals from CH2 2z-hexanes (85%); mp 113.0 115.0 oC. 1H NMR (CDCl3):

6 1.40 -1.90 (m, 4H), 1.95 -2.15 (m, 1H), 2.15 -2.23 (m, 1H), 3.40 -3.68 (m, 2H), 4.20 -4.35

(m, 2H), 4.36 4.48 (m, 1H), 6.20 (d, J = 7.7 Hz, 1H), 7.20 7.45 (m, 7H), 7.50 7.80 (m, 7H),

8.13 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.20 8.97 (m, 2H). 13C NMR (CDCl3): 6 22.4,

28.9, 31.6, 38.5, 47.1, 54.6, 67.1, 114.4, 116.4, 118.0, 118.5, 119.9, 120.2, 125.2, 126.4, 127.0,

127.6, 129.7, 130.6, 131.1, 134.0, 141.2, 143.6, 143.9, 146.0, 148.6, 154.3, 156.2, 161.4, 162.1,

171.7. Anal. called for C37H31N5O6: C, 69.26; H, 6.87; N, 10.91. Found: C, 69.01; H, 4.76; N,

11.03.

3.4.4 General Procedure for the Preparation of O-(Coumarin)diacetonide Sugars 3.12,
3.13, 3.14 Under Microwave Irradiation.

A dried heavy walled Pyrex tube containing a small stir bar was charged with 3-

(Benzotriazole-1 -carbonyl)chromen-2-one 3.4 (1.0 mmol), sugars 3.9, 3.10, 3.11 (1 mmol),

DMAP (1 mmol), and CH2 12 (1 mL). The reaction mixture was exposed to microwave

irradiation (100 W) for 45 minutes to obtain 3.12, 3.13 and 3.14 at a temperature of 600C. After

the irradiation, the reaction mixture was allowed to cool through an inbuilt system in the

instrument until the temperature had fallen below 300C (ca. 10 min). To the reaction mixture 20










(m, 2H), 4.30-4.43 (m, 1H), 4.63-4.65 (m, 1H), 5.05-5.13 (m, 4H), 5.97 (d, J= 7.4 Hz, 0.5H),

6.06 (d, J= 7.5 Hz, 0.5H), 7.26-7.46 (m, 11H), 10.27 (s, 1H). 13C NMR (CDCl3): 6 18.0, 18.3,

26.5, 30.0, 50.2, 51.3, 66.3, 66.8, 127.7, 127.8, 127.8, 128.0. 128.2, 128.3, 135.3, 135.8, 156.1,

156.2, 172.7, 172.8, 173.4, 173.7. Anal. called for C23H26N207: C, 62.43; H, 5.92; N, 6.33.

Found: C, 62.12; H, 5.93; N, 6.23.

1-Benzyl (LS)-2-((S)-2-benzyloxycarbonylamino-3-phnlrpoymioetnoe

(Z-L-Phe-L-Glu(OBzl)-OH, 2.5a): White microcrystals (87%); mp 140-141 oC, [a]D23 = -10.7

(c 2.00, DMF). 1H NMR (CDCl3): 6 1.99 (quintet, J = 7. 1 Hz, 1H), 2. 15-2.20 (m, 1H), 2.35-2.49

(m, 2H), 3.05 (d, J = 6.3 Hz, 2H), 4.48-4.55 (m, 2H), 5.02 (d, J= 12.9 Hz, 1H, A part of AB

system), 5.06 (d, J = 12.6 Hz, 1H, B part of AB system), 5.08 (s, 2H), 5.47 (d, J = 8.0 Hz, 1H),

6.97 (d, J= 6.9 Hz, 1H), 7. 12-7.36 (m, 15H), 12.85 (br s, 1H). 13C NMR (CDCl3): 6 26.6, 30.1i,

38.3, 51.8, 56.0, 66.6, 67.1, 127.0, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 129.3,

135.5, 136.0, 156.2, 171.7, 173.0, 174.0. Anal. called for C29H30N207: C, 67.17; H, 5.83; N, 5.40.

Found: C, 66.83; H, 5.81; N, 5.22.

1-Benzyl (,S)-2-((R)-2-benzyloxycarbonylamino-3-phnlrpoymioetnoe

(Z-D-Phe-L-Glu(OBzl)-OH, 2.5b): White microcrystals (91%); mp 119-120 oC, [a]D23 = -5.4 (c

1.66, DMF). 1H NMR (DMSO-d6) (two rotameric forms): 6 1.82-1.91 (m, 1H), 1.96-2.09 (m,

1H), 2.30 (t, J= 7.2 Hz, 1H), 2.43-2.46 (m, 1H), 2.69-2.79 (m, 1H), 2.92-3.04 (m, 1H), 4.26-4.35

(m, 2H), 4.99 (d, J = 12.4 Hz, 1H, A part of AB system), 5.02 (d, J= 12.6 Hz, 1H, B part of AB

system), 5.08 (d, J = 12.4 Hz, 1H, A part of AB system), 5.11 (d, J = 12.4 Hz, 1H, B part of AB

system), 7.12-7.36 (m, 15H), 7.51 (d, J= 8.8 Hz, 1H), 8.31-8.37 (m, 1H), 12.83 (br s, 1H). 13C

NMR (CDCl3) (two rotameric forms): 6 26.3, 26.4, 29.8, 30.0, 37.3, 51.0, 51.1, 56.0, 65.2, 65.5,

126.3, 127.4, 127.5, 127.7, 127.9, 128.0, 128.3, 128.4, 129.2, 136.1, 136.9, 137.0, 137.9, 138.1,









CHAPTER 3
EFFICIENT LABELING OF SUGARS TO PROVIDE WATER SOLUBLE FLUORESCENT
TAGS

3.1 Introduction

Carbohydrate moieties have pivotal roles in numerous biological processes including cell-

cell communication,66-68 cell adhesion,69,70 fertilization, protein folding, and microbial

infections.7-7 Glycosylation is the process of adding saccharides to proteins and lipids. Over

50% of all protein sequences in eukaryotic systems sequences are glycosylated.77 Glycosylated

lipids constitute up to 5-10 % of the membrane content in animal cells, and are involved in a

wide array of pathological disorders.'

Oligosaccharides are present in the form of glycoconjugates glycoproteinss and

glycolipids) in all cell walls mediating a variety of events such as inflammation, immunological

response, and metastasis. Separation of glycoproteins has been achieved with modern

chromatographic and electrophoretic methodologies. However, glycoproteins, once isolated are

difficult to study structurally because glycoproteins usually destroy when analyzed with X-ray

crystallography.79 Also, the amount of glycoproteins obtained from biological materials is too

small to study their structures. Therefore, glycoproteins are often cleaved to smaller fragments

such as their glycopeptides or glycans, which are easier to analyze.so Glycopeptides and glycans

are frequently labeled to increase detection sensitivity.8

Fluorescent tagging with organic fluorophores82 Or green fluorescent protein is the

visualization tool most commonly used for analyzing carbohydrate structures in biological

systems.83 Highly sensitive fluorescence derivatization techniques are gaining an increasing

share of the analytical world market, becoming competitive with, for example,

radioimmunoassay. Derivatives of rhodamin, fluorescein, and coumarin are widely used as

fluorescent markers for peptides and other biomolecules.84,85 Derivates of coumarins










(LS)-2-(9H-Fluoren-9-ylmethoxycarbonylam in)6[(2-oxo-2H-chromene-3-

carbonyl)-amino]-hexanoic acid 2,2,7,7-tetramethyl-tetrahydro-bis [1,3]dioxolo [4,5-b; 4',5'-

d]pyran-5-ylmethyl ester, 3.17b: White microcrystals (85%), mp 122.3 124.1 oC. 1H NMR

(CDCl3): 6 1.33 (s, 6H), 1.46 (s, 3H), 1.52 (s, 3H), 1.63 -2.30 (m, 4H), 3.40 -3.57 (m, 2H), 4.01

- 4.08 (m, 1H), 4.20 4.29 (m, 3H), 4.30 4.46 (m, 6H), 4.62 (dd, J = 7.8, 2.3 Hz, 1H), 5.54 (d, J

= 4.9Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 7.29 7.43 (m, 7H), 7.54 (d, J = 7.7Hz, 1H), 7.60 7.67

(m, 3H), 7.72 7.78 (m, 2H), 8.82 8.88 (m, 1H), 8.90 (s, 1H). 13C NMR (CDCl3): 6 22.3, 24.4,

24.9, 25.9, 26.0, 29.0, 31.9, 37.3, 39.2, 47.1, 53.8, 64.2, 66.0, 67.0, 68.8, 70.3, 70.7, 70.9, 96.2,

108.8, 109.7, 116.5, 118.3, 118.6, 119.9, 125.2, 127.0, 127.6, 129.7, 133.9, 141.2, 143.8, 148.3,

154.3, 155.9, 161.4, 161.7, 172.3. Anal. called for C43H46N2012: C, 65.97; H, 5.92; N, 3.58.

Found: C, 65.63; H, 6.04; N, 3.62.

(LS)-2-(9H-Fluoren-9-ylmethoxycarbonylam in)6[(2-oxo-2H-chromene-3-

carbonyl)-amino]-hexanoic acid 5-(2,2-dimethyl-[1 ,3] dioxolan-4-yl)-2,2-dimethyl-

tetrahydro-furo [2,3-dj [1,3]dioxol-6-yl ester, 3.18: White microcrystals (67%), mp 118.2 -

120.4. 1H NMR (CDCl3): 6 1.29 (s,3H), 1.31 (s, 3H), 1.38 (s, 3H), 1.51 (s, 3H), 1.60 2.00 (m,

4H), 3.40 -3.52 (m, 2H), 3.97 -4.1 I(m, 3H), 4. 19 -4.27 (m, 3H), 4.30 -4.45 (m, 3H), 4.51 (d,

J= 3.4 Hz, 1H), 5.30 (s, 1H), 5.77 (d, J = 7.6 Hz, 1H), 5.86 (d, J = 3.6 Hz, 1H), 7.25 7.46 (m,

8H), 7.60 7.67 (m, 3H), 7.72 7.79 (m, 2H), 8.84 8.96 (m, 2H). 13C NMR (CDCl3): 6 22.2,

25.1, 26.2, 26.7, 26.8, 29.0, 31.3, 38.7, 47.1, 53.9, 60.4, 67.0, 67.2, 72.3, 79.7, 83.1, 105.0,

109.4, 112.4, 116.5, 118.1, 118.5, 120.0, 125.0, 125.1, 125.2, 127.1, 127.7, 129.7, 134.0, 141.2,

143.6, 143.8, 148.6, 154.3, 156.0, 161.5, 162.0, 171.1. Anal. called for C43H46N2012: C, 65.97;

H, 5.92; N, 3.58. Found: C, 65.68; H, 6.07; N, 3.60.










compounds 3.17a,b, 3.18, 3.19 needed 45 minutes. After washing with 4N HC1, products

3.17a,b, 3.18 were obtained without chromatography in 85-89% yields. The crude products were

estimated to be >95% pure. Compounds 3.19 and 3.20 were isolated using column

chromatography in 74% and 40% yields respectively.


HN-Pg \WFo--


N O O .1


SO OO NH 8~



3.18
Pg =Cbz 3.7, 3.17a 31
Pg =Fmoc 3.8, 3.17b HN


OHN PivOO HNN O O7

O O OPiv NH-Fmoc

X~o;Y O NH-Fmoc
O 3.20

3.19

Figure 3 -4.Preparation of O- and N-(AP-coumarin-3 -carbonyl-N"(Fmoc or Z-L-lys)protected
sugars 3.17a,b, 3.18, 3.19, 3.20.

3.2.5 Deprotection of the Diisopropylidene Groups of O-(Coumarin
Labeled)diisopropylidene Protected Sugars 3.13, 3.17b and 3.18.

Deprotection of diacetonide groups of 3.13, 3.17b and 3.18 were performed by TFA/H20

(9:1, v/v; 5mL) mixture at 20 oC for 3-5 minutes. The unprotected coumarin-sugar conjugate

3.21 (Figure 3-5) and coumarin-L-lysine-free sugar conjugates 3.22 and 3.23 (Figures 3-6, 3-7)

were obtained in quantitative yields and characterized by 1H NMR and 13C NMR spectroscopy,

elemental analysis, melting point, Mass Spec. and ORP.









derivative 1.4 was coupled with Fmoc-protected lysine to give lf-coumarin-labeled N"-

protected-L-lysines. The Lysines were coupled with protected sugars by O- and N-acylation at

free OH and NH2 grOups to yield water soluble fluorescent markers in 40-90% yields.


Figure 1-4. Structure of N-(coumarin-3-carbonyl) benzotriazole










(LS)-2-(9H-Fluoren-9-ylmethoxycarbonylam in)6[(2-oxo-2H-chromene-3-

carbonyl)-amino]-hexanoic acid 6-(2,2-dimethyl- [1,3] dioxolan-4-yl)-2,2-dimethyl-

tetrahydro-furo [3,4-d] [1,3]dioxol-4-yl ester, 3.19: White microcrystals (74%), mp 87.0-88.0

1H NMR (CDCl3): 6 1.22-1.30 (m, 2H), 1.33 (s, 3H), 1.37 (s, 3H), 1.43 (s, 3H), 1.48 (s, 3H),

1.52-1.98 (m, 4H), 3.46-3.60 (m, 2H), 3.98-4.12 (m, 3H), 4.22 (t, J= 7.1 Hz, 1H), 4.30-4.48 (m,

3H), 4.73 (d, J = 5.9 Hz, 1H), 4.80-4.90 (m, 1H), 5.65 (d, J = 7.8 Hz, 1H), 6.17 (s, 1H), 7.25-7.42

(m, 7H), 7.50 (d, J = 7.8 Hz, 1H), 7.58-6.67 (m, 3H), 7.74 (dd, J = 7.3, 2.8,Hz, 2H), 8.82-8.88

(m, 2H). 13C NMR (CDCl3): 6 22.3, 22.6, 24.6, 25.1, 25.9, 26.9, 29.0, 31.5, 38.9, 47.1, 53.7,

66.8, 67.0, 72.7, 79.2, 82.5, 85.0, 101.6, 109.3, 113.3, 116.5, 118.2, 118.5, 119.9, 125.2, 127.0,

127.6, 129.7, 134.0, 141.2, 143.7, 143.8, 148.4, 154.3, 155.8, 161.4, 161.8, 171.1. Anal. called

for C43H46N2012: C, 65.97; H, 5.92; N, 3.58. Found: C, 65.57; H, 6.06; N, 3.40.

{(S)-1l-Formyl-5- [(2-oxo-2H-chrom ene-3-carbonyl)-am ino] -pentyl}-carbam ic acid

9H-fluoren-9-ylmethyl ester; compound with 2,2-dimethyl-propionic acid (3S,5S,6R)-3,4,5-

tris-(2,2-dimethyl-propionyloxy)-6-methylmn-erhdopr-2yetl ester, 3.20:

2-Oxo-2H-chromene-3-carboxylic acid (3S,5R)-2,3,5-trihydroxy-6-hydroxymethyl-

tetrahydro-pyran-4-yl ester, 3.21:

(LS)-2-(9H-Fluoren-9-ylmethoxycarbonylam in)6[(2-oxo-2H-chromene-3-

carbonyl)-amino]-hexanoic acid 3,4,5,6-tetrahydroxy-tetrahydro-pyran-2-ymtl ester,

3.22:

(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino-6[(2-oxo-2H-chromene-3-

carbonyl)-amino]-hexanoic acid (3S,5R)-2,3,5-trihydroxy-6-hydroxymethyl-erhdo

pyran-4-yl ester, 3.23:













O H





,- 3.21


TFA-H20
9:1i, 5mL


Figure 3-5. Deprotection of diacetonide groups for compound 3.13


TFA-H20

9:1, 5mL


'L OH
OH


3.17b


Figure 3-6. Deprotection of diacetonide groups for 3.17b


3.22






OH


HHO OH
,N OH
Fmoc O


TFA-H20

9:1i, 5mL


3.18 3.23


Figure 3-7. Deprotection of diacetonide groups for 3.18

3.3 Conclusion

In conclusion, we have demonstrated a convenient and efficient O-fluorescence labeling of

diisopropylidene protected sugars 3.12-3.14 and N-labeling of pivaloyl protected aminosugar

3.15 in yields of 55-87%. Fmoc and Z-protected L-Lysine scaffold based coumarin labeled









amino acids Trp 2.9a and Met 2.9b in acetonitrile-water mixture (2:1 v/v) in the presence of

triethylamine (2 equiv.) at room temperature for 2 hours (Figure 2-5 and Table 2-4). The crude

products were washed with 4N HCI to remove the by-product BtH, yielding compounds 2.10a-c

in 91-94%, which were further recrystallized from CH2 2z/hexanes before 1H and 13C M

spectroscopy, elemental analysis, and optical rotatory power (ORP).

The Katritzky group researchers have previously found that coupling reactions utilizing N-

(Z or Fmoc-a-aminoacyl)benzotriazoles gave dipeptide products with no detectable racemization

as demonstrated by their 1H NMR and HPLC analyses.61-63 Likewise, NMR analysis of the

enantiopure LL-dipeptides 2.10a-c showed no detectable racemization: each compound revealed

two doublets for each of the two -NH protons in the range of 7.80 8.40 ppm. The NMR

spectra also displayed a singlet for the methyl protons (SMe) for compounds 2.10c. In addition,

13C NMR displayed a singlet for each carbonyl carbon for the dipeptides 2.10a-c.

O O

Ph `O. (CH2)n CH3CN /H2,O Ph O (CH2)nH
Fmoc, 2 CO Bt Et3N, r.t., 2h. Fmoc, rN ~COOH
N N
HO HO R
2.8a,b 2.9a, R =-CH2(3-indolyl NH) (Trp) 2.10a-c
a: n= 1; b: n =2 2.9b, R= -(CH2)2-S-CH3 (Met)
Bt = benzotriazol-1-yl







Figure 2-5. Preparation of novel dipeptides 2.10a-c

Table 2-4. Preparation of novel dipeptides 2.10a-c
Amino Yield [a]25D Mp (oC)
Entry Acid Product (%)a
1 L-Trp (2.9a) Fmoc-L-Glu(OBzl)-L-Trp-OH (2.10a) 93 +11.5 165-167
2 L-Trp (2.9a) Fmoc-L-Asp(OBzl)-L-Trp-OH (2.10b) 94 -4.6 136-137
3 L-Met (2.9b) Fmoc-L-Glu(OBzl)-L-Met-OH (2.10c) 91 -7.6 96-97
alsolated yield










21. Mojarro-Guerra, S.H.; Amado, R.; Arrigoni, E.; Solms, J. J. FoodSci. 1991, 56, 943.

22. Kirimura, J.; Shimizu, A.; Kimizura, A.; Ninomiya, T.; Katsuya, N. J. Agric. Food Chem.
1969, 17, 689.

23. Ohyama, S.; Ishibashi, N.; Tamura, M.; Nishizaki, H.; Okai, H. Agric. Biol. Chem. 1988,
52, 871.

24. Tada, M.; Shinoda, I.; Okai, H. JAgric. Food Chem. 1984, 32, 992.

25. Kawasaki, Y.; Seki, T.; Tamura, M.; Kikuchi, E.; Tada, M.; Okai, H. Agric. Biol. Chem.
1988, 52, 2679.

26. Stefanic, P.; Dolenc, M. S. Current Med. Chem. 2004, 11, 945.

27. Braiuner-Osborne, H.; Egebj erg, J.; Nielsen, E. O.; Madsen, U.; Krogsgaard-Larsen, P. J.
M~ed. Chem. 2000, 43, 2609.

28. Prokai-Tatrai, K.; Nguyen, V.; Zharikova, A. D.; Braddy, A. C.; Stevens, S. M. Jr.;
Prokai, L. Biorg. M~ed. Chem. Lett. 2003, 13, 1011.

29. Mann, E.; Kessler, H. Org. Lett. 2003, 5, 4567.

30. Dauban, P.; De Saint-Fuscien, C.; Acher, F.; Prezeau, L.; Brabet, I.; Pin, J. -P.; Dodd, R.
H. Biorg. M~ed. Chem. Lett. 2000, 10, 129.

31. Bessis, A. S.; Bolte, J.; Pin, J. P.; Acher, F. Biorg. M~ed. Chem. Lett. 2001, 11, 1569.

32. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.

33. Schnolzer, M., Alewood, D.; Kent, S. B.1Int. J. Peptide andProtein Research, 1992, 40,
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34. Dorwald, F.Z. Organ2ic Sywhelrlri\ in Solid Support. Wiley-VCH, Weinheim, Germany,
2000.

35. Capecchi, J. T.; Miller, M. J.; Loudon, G. M. J. Org. Chem. 1983, 48, 2014.

36. Konda-Yamada, Y.; Okada, C.; Yoshida, K.; Umeda, Y.; Arima, S.; Sato, N.; Kai, T.;
Takayanagi, H.; Harigaya, Y. Tetrahedron 2002, 58, 7851.

37. Baek, B. -H.; Lee, M. -R.; Kim, K. -Y.; Cho, U. -I.; Boo, D. W.; Shin, I. Org. Lett. 2003,
5, 97 1.

38. Carpino, L. A.; Ferrer, F. J. Org. Lett. 2001; 3, 2793.










1H), 7.65 (t, J= 7.7 Hz, 1H), 8.12 (d, J= 8.2 Hz, 1H), 8. 16 (d, J= 8.2 Hz, 1H). 13C M

(CDCl3): 6 38.3, 50.2, 67.3, 67.9, 114.2, 120.4, 126.5, 128.2, 128.3, 128.5, 128.5, 128.6, 130.6,

130.7, 134.8, 135.9, 146.1, 155.9, 169.8, 170.3. Anal. called for C25H22N405: C, 65.49; H, 4.84;

N, 12.12. Found: C, 65.71; H, 4.80; N, 12.15.

Benzyl (S)-5-Benzotriazol-1-yl-2-benzyloxycarbonlmo-oxptnoe (Z-L-

Glu-OBzl-Bt, 2.11b): White microcrystals (81%), mp 50.0-52.0 oC, [a]D23 = -20.7 (c 1.66,

DMF). 1H NMR (CDCl3): 6 2.23-2.30 (m, 1H), 2.45-2.50 (m, 1H), 3.37-3.60 (m, 2H), 4.60-4.70

(m, 1H), 5.01-5.20 (m, 4H), 5.79 (d, J= 8.0 Hz, 1H), 7.22-7.31 (m, 10 H), 7.46 (t, J= 7.9 Hz,

1H), 7.60 (t, J= 7.6 Hz, 1H), 8.06 (d, J= 8.2 Hz, 1H), 8.20 (d, J= 8. 1 Hz, 1H). 13C M

(CDCl3): 21.7, 31.0, 58.8, 67.5, 68.3, 114.3, 120.2, 126.2, 128.2, 128.3, 128.4, 128.5, 128.6,

128.7, 130.5, 131.0, 135.0, 136.1, 146.1, 156.1, 171.4, 171.7. Anal. called for C26H24N405: C,

66.09; H, 5.12; N, 11.86. Found: C, 65.99; H, 5.14; N, 11.65.

2.4.3 General Procedure for the Preparation of Dipeptides 2.10a-c, 2.12a,b and 2.12b+b'

Free amino acids (2.9a-f) (5 mmol) were added to a solution of Et3N (10 mmol) in CH3CN

(15 mL) and H20 (7 mL) at 25 oC, and the reaction mixture was stirred for 15 min at 25 oC. N-

(Protected-a-aminoacyl)benzotriazoles (2.8a,b and 2.11a,b; 5 mmol) were added to the mixture

with continued stirring for 2 h at 25 oC. About 4N HCI (5 mL) was added to the reaction mixture

and CH3CN was removed under reduced pressure. The residue was dissolved in EtOAc (50 mL),

and the organic extract was washed with 4N HCI (3x15 mL), saturated NaCl (20 mL) and dried

over MgSO4. Evaporation of the solvent gave the desired products (2.10a-c, 2.12a,b, 2.12b+b'),

which were further recrystallized from CH2 2z-hexanes (2.10a-c) and ether-hexanes (2.12a,b,

2.12b+b').

(LS)-2-((LS)-2-(((9H-Fluoren-9-yl)mI1ethoxy)carbonylam ino)-5-(benzyloxy)-5-

oxopentanamido)-3-(1H-indol-3-yl)propanoi acid (Fmoc-L-Glu(OBzl)-L-Trp-OH, 2.10a):









The second method involves the treatment of a carboxylic acid with thionyl chloride in the

presence of excess of benzotriazole (Figure 1-3). This method was further applied in the field of

peptides to prepare N-protected aminoacylbenzotriazoles from N-(Z- and Fmoc-a) amino acids.

These N-protected aminoacylbenzotriazoles are stable enough to participate in amino acid

coupling at ambient temperature under mild conditions to produce di- and tripeptides with

complete retention of chirality.6-1


O O O O
S3 BtH II and/or RI 'O
/S aS S
Cl Cl Bt Cl Bt BtRt

Figure 1 -3. N-Acylb enzotriazoles from carboxylic acids, excess BtH and thionyl chloride

Chapter 2 describes the preparation of N-(Z- or Fmoc-a-aminoacyl)benzotriazoles derived

from L-Asp and L-Glu amino acid, as well as peptide coupling of these acylbenzotriazoles with

unprotected a-amino acids/dipeptides yielding the corresponding natural and unnatural di- and

tripeptides. Such reactions were carried out with diverse N-(protected aminoacyl)benzotriazoles

on Asp and Glu residues, in which the CO2H groups were free or partially protected.l0.11

Recently, our group reported the straightforward syntheses of coumarin-labeled amino

acids and dipeptides which afforded enantiomerically pure fluorescent building blocks suitable

for solid phase peptide synthesis (SPPS). The two-step synthetic route converted coumarin-3-

carboxylic acid into its active benzotriazolide 1.4 (Figure 1-4), which was coupled with Z- and

Fmoc-N-protected lysines. The N-terminus of free amino acids, as well as dipeptides, provided

diverse optically pure fluorescent probes in good to excellent yields.12

Helpful water soluble fluorescent building blocks for peptide labeling at the C-terminus in

solid-phase peptide synthesis (SPPS) are described in Chapter 3.13 The same benzotriazole









protected sugars 3.17a,b, 3.18, 3.19, and 3.20 were obtained from 3.7 and 3.8. Deprotection of

diisopropylidene groups from 3.13, 3.17b and 3.18 provided water soluble conjugates 3.21-3.23

in quantitative yields. Fluorescent building blocks 3.17a,b, and 3.18 can be considered to be

useful markers for labeling C-terminus of peptides in solid phase peptide synthesis (SPPS) and

after deprotection of diisopropylidene groups, the free sugar will provide the water solubility of

organic fluorophores for coumarin labeled protein molecules.

3.4 Experimental Section

Melting points were determined on a capillary point apparatus equipped with a digital

thermometer. NMR spectra were recorded in CDCl3 Or DMSO-d6 with TMS for 1H (300 MHz)

and 13C (75 MHz) as an internal reference. Coumarin-3 carboxylic acid was purchased from

Acros. Sugars and N-Fmoc-amino acids were purchased from Fluka, Acros and Aldrich and were

used without further purification. Most of the reactions were carried out under microwave

irradiation with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC)

producing a continuous irradiation at 2450 MHz. Elemental analyses were performed on a Carlo

Erba-1106 instrument. Optical rotation values were measured with the use of sodium D line.

Column chromatography was performed on silica gel (200-425 mesh). HPLC analyses were

performed on Beckman system gold programmable solvent module 126 using Chirobiotic T

column (4.6 x 250 mm), detection at 254 nm, flow rate 1.0 mL/min, and methanol as solvent.

3.4.1 General Procedure for the Preparation of Compound 3.4.

Thionyl chloride (7.5 mmol) was added to a solution of 1H-benzotriazole (25 mmol) in dry

CH2C 2 Or THF (30 mL) at room temperature, and the reaction mixture was stirred for 20 min.

To the reaction mixture, coumarin-3-carboxylic acid (5 mmol) was added and stirred for 4 h at

250C. The white precipitate formed during the reaction was filtered off, and the filtrate was

concentrated under reduced pressure. The residue was diluted with EtOAc (150 mL) and the









N- AND O- ACYLATION OF PEPTIDES AND SUGARS IN PARTIALLY AQUEOUS
MEDIA



















By

JANET CUSIDO


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007










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Full Text

PAGE 1

1 NAND OACYLATION OF PEPTIDES AND SUGARS IN PARTIALLY AQUEOUS MEDIA By JANET CUSIDO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Janet Cusido

PAGE 3

3 To my family, friends and everyone who always believed in me

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4 ACKNOWLEDGMENTS I thank my family, my teachers, and my co lleagues. I am greatly indebted to Dr. Encarnacion Lopez for my fascination with organi c chemistry. I am grateful to Prof. Alan R. Katritzky for giving me the opportunity to experi ence the joys and the trials of being a true scientist and to my committee members for their a ssistance and care. Very special thanks to Valerie Rodriguez-Garcia for setting me on the right path. My thanks for the invaluable help and suggestions in the preparation of this thesis go to Boris Grinkot, Adam Vincek, and Danniebelle Haase. I also wish to thank all my friends and colleagues for their company, team spirit, and international food!

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 GENERAL INTRODUCTION..............................................................................................10 2 SELECTIVE PEPTIDE CHAIN EXTENSION AT THE C AND N -TERMINI OF ASPARTIC AND GLUTAMIC ACIDS UTILIZING N -PROTECTED (ALPHAAMINOACYL) BE NZOTRIAZOLES..................................................................................14 2.1 Introduction.............................................................................................................. ........14 2.2 Results and Discussion.....................................................................................................17 2.2.1 Extensions of -Amino Acids with and -CO2H Protected...............................17 2.2.2 Extensions of -acids with -CO2H Protected......................................................18 2.2.3 Preparation of Dipeptides 2.7a-e from Unprotected Glutamic or Aspartic Acids and N-( -Aminoacyl)benzotriazoles 2.1a,b,d and e....................................20 2.2.4 Peptide Chain Extension at the Alpha C-Te rminus to Give Natural Dipeptides 2.10a-c....................................................................................................................21 2.2.5 Peptide Chain Extension at the or C-Terminus to Give Unnatural Dipeptides 2.12a-d.................................................................................................23 2.2.6 Preparation of N-Protected-Dipeptidoylbenzot riazoles 2.13a-c and 2.13a+a' from N-Protected Dipeptides 2.3a,b,d and 2.3a+a'...............................................24 2.2.7 Preparation of N-Protected Tripeptides 2.14a,b,a' and Diastereomeric Mixture 2.14a+a'' from Dipeptidoylb enzotriazoles 2.13a,b, 2.13a+a' and Free Amino Acids 2.9c,e and f......................................................................................25 2.2.8 Preparation of Novel Tripeptides 2.16a ,b Containing Glu and Asp Fragments....27 2.3 Conclusion................................................................................................................. .......28 2.4 Experimental Section....................................................................................................... .29 2.4.1 General Procedure for the Preparation of Dipeptides 2.3a-c, 2.3a+a', 2.5a-c, 2.5c+c' and 2.7a-e..................................................................................................29 2.4.2 General Procedure for the Preparation of N -(Zand FmocAminoacyl)benzotriazoles 2.8 a,b and 2.11 a,b....................................................34 2.4.3 General Procedure for the Prepara tion of Dipeptides 2.10a-c, 2.12a,b and 2.12b+b'..................................................................................................................36 2.4.4 General Procedure for the Preparation of N-ProtectedDipeptidoylbenzotriazo les 2.13a-c and 2.13a+a'...................................................39

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62.4.5 General Procedure for the Prepara tion of Tripeptides 2.14a,b, 2.14a' and 2.14a+a''.................................................................................................................41 2.4.6 General Procedure for the Prepar ation of Tripeptides 16 a,b............................43 3 EFFICIENT LABELING OF SUGARS TO PROVIDE WATER SOLUBLE FLUORESCENT TAGS.........................................................................................................46 3.1 Introduction.............................................................................................................. ........46 3.2 Results and Discussion....................................................................................................49 3.2.1 Preparation of N-Coumarin-Labeled N-FmocL -lysine Benzotriazolide 3.8......49 3.2.2 Preparation of Coumarin-O-Tagged Monosaccharides : O-(Coumarin-3carbonyl)diisopropylidene Sugars 3.12, 3.13, 3.14...............................................49 3.2.3. Preparation of CoumarinN -Tagged Monosaccharide: N -(Coumarin-3carbonyl)tetrapivaloyl Sugar 3.16..........................................................................50 3.2.4 Preparation of O and N -( N-Coumarin-3-CarbonylN(Fmoc or ZL lys)protected Sugars 3.17a,b, 3.18, 3.19, 3.20.......................................................50 3.2.5 Deprotection of the D iisopropylidene Groups of O -(Coumarin Labeled)diisopropylid ene Protected Sugars 3.13, 3.17b and 3.18.........................51 3.3 Conclusion................................................................................................................. .......52 3.4 Experimental Section...................................................................................................... .53 3.4.1 General Procedure for the Preparation of Compound 3.4.....................................53 3.4.2 General Procedure for the Preparation of Compounds 3.5 and 3.6.......................54 3.4.3 General Procedure for the Pr eparation of Compound 3.7 and 3.8........................55 3.4.4 General Procedure for the Preparation of O -(Coumarin)diacetonide Sugars 3.12, 3.13, 3.14 Under Microwave Irradiation......................................................56 LIST OF REFERENCES............................................................................................................. ..61 BIOGRAPHICAL SKETCH.........................................................................................................67

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7 LIST OF TABLES Table page 2-1. Preparation of novel natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a' ............18 2-2. Preparation of nov el unnatural dipeptides 2.5a-c and mixture 2.5c+c' .................................19 2-3. Preparation of novel dipeptides 2.7a-e ..................................................................................21 2-4. Preparation of novel dipeptides 2.10a-c ................................................................................22 2-5. Preparation of novel dipeptides 2.12a,b and the diastereomeric mixture 2.12b+b' .............23 2-6. Conversion of novel N-Z-dipeptides 2.3a,b,d and the diastereomeric mixture 2.3a+a' into N-Z-dipeptidoylbenzotriazoles 2.13a-c, 2.13a+a' ....................................................25 2-7. Preparation of novel tripeptides 2.14a,b,a' and mixture 2.14a+a'' .......................................26 2-8. Preparation of novel tripeptides 2.16a,b containing Glu a nd Asp fragments.......................28

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8 LIST OF FIGURES Figure page 1-1. Properties of N -Substituted benzotriazoles as electr on donors or electron acceptors...........11 1-2. N -Acylbenzotriazoles from sulfonylbenzotriazoles..............................................................11 1-3. N -Acylbenzotriazoles from carboxylic acid s, excess BtH and thionyl chloride...................12 1-4. Structure of N -(coumarin-3-carbonyl) benzotriazole............................................................13 2-1. Structures of Aspartic (Asp ) and Glutamic (Glu) Amino Acids...........................................14 2-2. Preparation of novel dipeptides 2 3a-c and diastereomeric mixture 2 3a+a' ........................18 2-3. Preparation of nov el unnatural dipeptides 2.5a-c and diastereomeric mixture 2.5c+c' ........19 2-4. Preparation of novel dipeptides 2.7a-e ..................................................................................21 2-5. Preparation of novel dipeptides 2.10a-c ................................................................................22 2-6. Preparation of novel dipeptides 2.12a,b and diastereomeric mixture 2.12b+b' ...................23 2-7. Preparation of novel dipeptidoylbenzotriazoles 2.13a-c and diastereomeric mixture 2.13a + a' .............................................................................................................................25 2-8. Preparation of novel tripeptides 2.14a,b a', and 2.14a+a'' ...................................................26 2-9. Preparation of novel tripeptides 2.16a,b ...............................................................................28 3-1. Fluorescent labeling of saccharides by reductive amination.................................................47 3-2. Structures of N -(coumarin-3-carbonyl) benzotriazole and N-coumarin-labeled N protectedL -lysines.............................................................................................................48 3-3. Syntheses of O -(coumarin-3-carbonyl)diisopropylidene sugars 3.12, 3.13, 3.14 and N (coumarin-3-carbonyl)te trapivaloyl sugar 3.16 .................................................................50 3-4. Preparation of O and N -( N-coumarin-3-carbonylN(Fmoc or ZL -lys)protected sugars 3.17a,b, 3.18, 3.19, 3.20 .........................................................................................51 3-5. Deprotection of diacetonide groups for compound 3.13 .......................................................52 3-6. Deprotection of diacetonide groups for 3.17b .......................................................................52 3-7. Deprotection of diacetonide groups for 3.18 .........................................................................52

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NAND OACYLATION OF PEPTIDES AND SUGARS IN PARTIALLY AQUEOUS MEDIA By Janet Cusido December 2007 Chair: Alan R. Katritzky Major: Chemistry The convenient preparation of N -(Fmocor Z-aminoacyl)benzotriazoles and N -protected peptidoylbenzotriazoles from aspartic and glut amic amino acids is discussed. Additionally, diverse N -protected diand tripeptides are synthesi zed under mild reaction conditions in good to excellent yields by acylation with N(Zand Fmoc-aminoacyl)benzotriazoles of the amino groups of free aspartic and glutamic acids. Exam ples of peptide coupling utilizing free -amino acids in partially aqueous solution are reported and the products are obtai ned without the use of chromatography. Evidence of maintained chirality was supported by NMR and HPLC. In addition, we present the suitable and e fficient fluorescent la beling of sugars by O acylation of diisopropylidene protected sugars and N -acylation of pivaloyl protected aminosugar with N -(coumarin-3-carbonyl)benzotriazole a nd benzotriazole derivatives of N-coumarinlabeled N-protectedL -lysines under microwave irradia tion or/and at room temperature Monosaccharide containing Fmoc-lysine fluorescent building blocks can be useful as water soluble organic fluorophores for peptide labeling at the C -terminus in solid-phase peptide synthesis (SPPS).

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10 CHAPTER 1 GENERAL INTRODUCTION Over the last 25 years, Katritzky and colleague s have studied the design of new synthetic approaches that can produce scientifically attr active compounds in good quantities and with easy purification methods. The development of be tter methods for the preparation of useful compounds in our research group is based on the versatility of a well known synthetic auxiliary, benzotriazole. A useful synthetic auxiliary must possess severa l characteristics. First, benzotriazole can be introduced readily at the beginni ng of a sequence. Second, benz otriazole is easily removed at the end of the synthetic sequence so that it can be recovered and reused. Last, benzotriazole is inexpensive and stable during various chemical r eactions and, to some extent, activates groups on other parts of the molecu le that is attached to.1 1 H Benzotriazole strongly exhib its all of the above characteristics. Benzotriazole (Bt) offers many advantages as a resourceful synt hetic auxiliary because it is soluble in many solvents, such as benzene, toluene, chloroform ethanol, tetrahydrofuran (THF), ethyl acetate (EtOAc), diethyl ether, and dimethyl formamide (DMF).2 Moreover, benzotri azole is partially soluble in water, but extremely soluble in basic solutions because of its acidic p Ka of 8.2. N -Substituted derivates of benzotriazole with an heteroatom (usua lly nitrogen, oxygen and sulfur) attached to a benz otriazole nitrogen can ionize in two ways due to the electron donating and electron accepting pr operties of benzotriazole.3 The benzotriazole anion and an immonium, oxonium, or thionium cation 1.2 can be formed or it can ionize off the heteroatom substituent to produce 1.3 (Figure 1-1). Generally, benzotriazol e is considered to be comparable with other activating groups becau se it shows good leaving ability 1.2 and activates the -CH toward proton loss 1.1. This type of activation to proton loss and leaving ability can be

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11 compared to other activating groups such as cyano, phenylsulfonyl and halogen analogues; however, Bt offers intermediates that are mo re stable, nonvolatile and less physiologically hazardous to prepare. N N N H R X N N N H R N N N X H R X typicallyfor X=NR2,OR,SR1.2typicallyfor X=Halogen,OH1.3 1.1acidicproton pronetodeprotonation Figure 1-1. Properties of N -Substituted benzotriazoles as electron donors or electron acceptors Benzotriazole methodology has become a fundame ntal synthetic tool for many chemical processes, such as multi step preparations of drugs, biologically active compounds and synthetic analogs of natural products. Our group has focuse d some of its research on the preparation of N acylbenzotriazoles. Two methods were utilized in our laboratory to prepare Nacylbenzotriazoles directly from carboxy lic acids. The first method employs sulfonylbenzotriazoles as a counter attack reagent.4,5 In the presence of triethylamine, carboxylic acids were converted into the desired acylben zotriazoles, probably through intermediate formation of the mixed carboxylic sulfonic anhydride and benzot riazole anion, which was then acylated by the mixed anhydride (Figure 1-2). R1OH O Et3N R1O O S O O R2 Bt Et3NH R1Bt O BtSO2R2 Figure 1-2. N -Acylbenzotriazoles from sulfonylbenzotriazoles

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12 The second method involves the tr eatment of a carboxylic acid w ith thionyl chloride in the presence of excess of benzotriazole (Figure 1-3). This method was further applied in the field of peptides to prepare Nprotected aminoacylbenzotriazoles from N -(Zand Fmoc) amino acids. These N -protected aminoacylbenzotr iazoles are stable enough to participate in amino acid coupling at ambient temperature under mild c onditions to produce diand tripeptides with complete retention of chirality.6-11 Cl S Cl O 3BtH Bt S Cl O Bt S Bt O R1OH O R1Bt O and/or Figure 1-3. N -Acylbenzotriazoles from carboxylic acids, excess BtH and thionyl chloride Chapter 2 describes the preparation of N -(Zor Fmoc-aminoacyl)benzotriazoles derived from L -Asp and L -Glu amino acid, as well as peptide coup ling of these acylbenzotriazoles with unprotected -amino acids/dipeptides yielding the corr esponding natural and unnatural diand tripeptides. Such reactions we re carried out with diverse N -(protected aminoacyl)benzotriazoles on Asp and Glu residues, in which the CO2H groups were free or partially protected.10,11 Recently, our group reported the straightforw ard syntheses of coumarin-labeled amino acids and dipeptides which afforded enantiomeri cally pure fluorescent building blocks suitable for solid phase peptide synthesis (SPPS). The tw o-step synthetic route converted coumarin-3carboxylic acid into its actived benzotriazolide 1.4 (Figure 1-4), which was coupled with Zand FmocNprotected lysines. The N -terminus of free amino acids, as well as dipeptides, provided diverse optically pure fluorescent probes in good to excellent yields.12 Helpful water soluble fluorescent building blocks for peptide labeling at the Cterminus in solid-phase peptide synthesis ( SPPS) are described in Chapter 3.13 The same benzotriazole

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13 derivative 1.4 was coupled with Fmoc-protected lysine to give N-coumarin-labeled NprotectedL -lysines. The Lysines were c oupled with protected sugars by O and N -acylation at free OH and NH2 groups to yield water soluble fl uorescent markers in 40-90% yields. OO N N N O 1.4 Figure 1-4. Structure of N -(coumarin-3-carbonyl) benzotriazole

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14 CHAPTER 2 SELECTIVE PEPTIDE CHAIN EXTENSION AT THE C AND N -TERMINI OF ASPARTIC AND GLUTAMIC ACIDS UTILIZING N -PROTECTED (ALPHA-AMINOACYL) BENZOTRIAZOLES 2.1 Introduction Recently many short, long, and cyclic biologically active peptides have been isolated from bacterial, fungal, plant, animal, a nd other sources. Peptides play pivotal roles as taste additives, neuroactive or enzyme regulators, and as an tibiotics. They also influence cell-cell communication upon interaction with receptors and are involved in a number of biochemical processes such as metabolism, pain, reproduction and immune response; such diverse roles have driven intensive peptide research.14-25 Among the 20 naturally occurring peptide amino acids, glutamic and aspartic acids are the representatives of dicarboxylic acids (Figure 2-1), often found in peptides with sensory properties including sweetness, bitterness, bitterness-masking, and flavor enhancements.14-25 The dipeptides aspartame ( L -AspL -Phe-OMe) and alitame ( L -AspD Ala-NH2) exemplify non calorif ic sweeteners and are used worldwide.14,17 Small peptides are important biomolecules and many have therapeu tic value. Unlike large peptides that are commonly isolated from natural sources or produced through recombinant techniques, small peptides are usually prepared using organic synthetic methods. Figure 2-1. Structures of Aspartic (Asp) and Glutamic (Glu) Amino Acids

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15 Glutamic and aspartic acids are important el ements for the biological activity of diverse naturally occurring and syntheti c peptides and their analogs.14 The side-chain carboxylic acid group of both of these amino acids enables sp ecific recognition by various receptors through ionic interactions; hence numer ous biologically active peptidomimetics incorporate glutamic and aspartic acid fragments in their structures.26 Glutamic acid plays a pivotal important role as the main excitatory neurotransmitter of the central nervous system (CNS), operating through f our different classes of receptors. Therefore, glutamic acid has received much attention in th e design of glutamate rece ptor ligands for drugs.27 Numerous small peptides and peptidomimetics, containing Asp and Glu residues have been suggested as prodrugs to enhance CNS effects.27-31 In peptide coupling inco rporating aspartic and glutamic ac ids could involve either of the two carboxylic acids.35 Especially in the case of Asp, the two isomeric forms frequently interconvert during coupling or s ubsequently. Procedures developed previously for peptide coupling incorporating Asp and Glu include: (i) carbodiimides in combination with additives such as 1-hydroxybenzotriazole (HOBt),36,37 1-hydroxy-7-azabenzotriazole (HOAt) and analogs38 or N -hydroxysuccinimide (HOSu),39 (ii) phosphonium,40,41 and uronium salts42,43 of HOBt or HOAt; (iii) N -acylazoles such as 1,1'-carbonylbis(1 H -imidazole) (CDI);44 (iv) mixed anhydrides;45 or (v) carboxylic acid fluorides.46,47 The most common procedures for the preparation of peptides containing glutamic and aspartic acids are based on solid-phase methodology.28,32-34 A commonly encountered problem in peptide synt hesis is epimerizati on of the amino acid chiral center during activation of the carboxylic acid group. Ma ny of the coupling reagents require prior protection and s ubsequent deprotection of vari ous amino acid functional groups.48

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16 Coupling reactions with such reagents are frequen tly moisture sensitive. Furthermore, isolation and purification processes ofte n involve column chromatography due to the formation of byproducts from the coupling reagents. In Katritzkys group, res earchers have applied N -acylbenzotriazoles for N -acylation,49-52 C acylation53-58 and O -acylation.59,60 Recently, the synthesis of N -(Z-aminoacyl)benzotriazoles was achieved in our laboratory and such were found to be efficient coupling reagents for N and O aminoacylation. N -(Z-aminoacyl)benzotriazoles compounds are isolable, stable and storable at room temperature for months, and easy to handle without special procedures to exclude air or moisture. The reagents used in the preparati on of aminoacylbenzotriazoles are inexpensive, thereby offering at the same time an overall cost effective methodology. Additionally, these N (Z-aminoacyl)benzotriazoles can be used in aque ous solutions to efficiently allow the coupling of several non-derivatized amino acids. Thus, th ese coupling reagents enable fast synthesis of peptides and peptoids in high yi elds, and purity under mild conditions with full retention of the original chirality. We now report (i) the preparation of N -(Zor Fmoc-aminoacyl)benzotriazoles derived from L -Asp and L -Glu amino acids, and (ii) peptide coupl ing of these aminoacylbenzotriazoles with unprotected -amino acids and dipeptides yielding the corresponding na tural and unnatural diand tri-peptides. Additionally, a new and c onvenient procedure for the selective stepwise elaboration of peptide chains incorporating aspartic and glutamic acids by utilizing N -(protected aminoacyl)benzotriazoles as coupl ing reagents is described. We show components of Asp and Glu are elongated from the N -terminus by acylation at the free amino group of glutamic or aspartic acids in which the -CO2H groups are unprotected or partially protected.

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17 2.2 Results and Discussion 2.2.1 Extensions of -Amino Acids with and -CO2H Protected. The preparation of natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a' from benzylL -glutamate 2.2a or -benzylL -aspartate 2.2b and N -(Z-aminoacyl)benzotriazoles 2.1a,b and 2.1a+a' was carried out by using as one component 2.1a or 2.1b and as the second component equimolar amounts of L -Glu(OBzl)-OH ( 2.2a ) or L -Asp(OBzl)-OH ( 2.2b ). Each coupling took place in ac etonitrile-water mixture (2:1 in volum e) in the presence of triethylamine (2 equiv.) at room temperature for 2 hours. Th e crude products were washed with 4N HCl to remove the by-product (BtH) to yield the three LL -dipeptides 2.3a-c (94 97%) (Figure 2-2 and Table 2-1). Diastereomeric mixture 2.3a+a was prepared from N -(ZDL -Ala)acylbenzotriazole ( 2.1a+a' ) and -benzylL -glutamate 2.2a in 94% yield using the same procedure as mentioned for the enantiopure dipeptides 2.3a-c NMR analysis showed no detectable racemization for the three enantiopure LL -dipeptides 2.3a-c No signals from their corresponding diastere omers were observed in the NMR spectra of 2.3a-c suggesting the enantiopurity of the N -protected dipeptides. E ach dipeptide revealed two sets of doublets for the two NH protons rang ing from 7.40 to 8.60 ppm. However, for the diastereomeric mixture 2.3a+a' one of the two NH protons a ppeared as two pairs of equal doublets. In addition, the 1H NMR spectrum of 2.3a gave a clear doublet for the methyl protons present in compound at 1.26 ppm (DMSOd6), whereas a multiplet was observed for the corresponding diastereomeric mixture 2.3a+a' ranging from 1.34 to 1.38 ppm (CDCl3). The 13C NMR spectrum displayed a singlet for each carbonyl carbon for compound 2.3a However, for 2.3a+a' NMR analysis displayed doublets for most of the al iphatic and carbonyl carbons, although no significant difference was obs erved for the aromatic carbons.

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18 Figure 2-2. Preparati on of novel dipeptides 2 3a-c and diastereomeric mixture 2 3a+a' Table 2-1. Preparation of novel natural dipeptides 2.3a-c and diastereomeric mixture 2.3a+a' Entry N -(Z-aminoacyl) benzotriazoles Product Yield (%)a [ ]25 D Mp (oC) 1 ZL -Ala-Bt ( 2.1a ) ZL -AlaL -Glu(OBzl)-OH ( 2.3a ) 94 +1.0 89-91 2 ZL -Phe-Bt ( 2.1b ) ZL -PheL -Glu(OBzl)-OH ( 2.3b ) 97 -10.3 139-142 3 ZL -Phe-Bt ( 2.1b ) ZL -PheL -Asp(OBzl)-OH ( 2.3c ) 95 -14.0 102-105 4 ZDL -Ala-Bt ( 2.1a+a' ) ZDL -AlaL -Glu(OBzl)-OH ( 2.3a+a' ) 94 +0.6 81-83 a Isolated yield The dipeptide 2.3a and the diastereomeric mixture 2.3a+a' were further subjected to HPLC analysis using Chirobiotic T column (detec tion at 254 nm, flow rate 0.5 mL/min, and 100% MeOH as solvent). As expected, HPLC analysis of the enantiopure LLdipeptide ( 2.3a ) showed a single peak at 6.3 min. In contrast, two p eaks of equal intensity at 6.3 and 6.7 min were observed for the corresponding diastereomeric mixture 2.3a+a' 2.2.2 Extensions of -acids with -CO2H Protected. The preparation of unnatural dipeptides 2.5a-c and mixture 2.5c+c' from -benzyl L glutamate 2.4a and N -( -aminoacyl)benzotriazoles 2.1b-d and 2.1d+d' were prepared using the same procedure as mentioned for the natural dipeptides 2.3a-c. The two LL -dipeptides 2.5a,c N H Z R N O N N H2NCOOH (CH2)nO O Ph N H Z R H N O COOH (CH2)nO O Ph 2.1a,b,2.1a+a' CH3CN/H2O Et3N,r.t.,2h. 2.2a,b n=1,2. AminoacidswithR: a ,Ala; b ,Phe; a+a' DL -Ala. 2.3a-c,2.3a+a' Z= O O Glu= 2.2a Asp= 2.2b

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19 and the mixture 2.5c+c' were obtained in 87-92% yields (Fi gure 2-3 and Table 2-2) and were further recrystallized from CH2Cl2/hexanes for further characterization. The natural dipeptide 2.3b was obtained in higher yields than the unnatural dipeptide 2.5a For example, the reaction of -benzyl L -glutamate ( 2.2a ) with ZL -Phe-Bt ( 2.1b ) gave dipeptide 2.3b in 97% yield, whereas the reaction of -benzyl L -glutamate ( 2.4a ) with Z L -Phe-Bt ( 2.1b ) gave product 2.5a in 87% yield. N H Z R N O N N H2N O O Ph OH O N H Z R H N O O O Ph OH O 2.1b-d, 2.1d+d' CH3CN / H2O Et3N, r.t., 2h. 2.5a-c, 2.5c+c' Amino acids with R: Ala, Phe, Met, DL -Met. 2.4a Figure 2-3. Preparation of novel unnatural dipeptides 2.5a-c and diastereomeric mixture 2.5c+c' Table 2-2. Preparation of novel unnatural dipeptides 2.5a-c and mixture 2.5c+c' Entry N -(Z-aminoacyl) benzotriazoles Product Yield (%)a [ ]25 DRetention time (min) Mp (oC) 1 ZL -Phe-Bt ( 2.1b ) ZL -PheL Glu(OBzl)-OH ( 2.5a ) 87 -10.7 6.4 140-141 2 ZD -Phe-Bt ( 2.1c ) ZD -PheL Glu(Obzl)-OH ( 2.5b ) 91 -5.4 7.6 119-120 3 ZL -Met-Bt ( 2.1d ) ZL -MetL Glu(Obzl)-OH ( 2.5c ) 87 -7.1 6.3 98-99 4 ZDL -Met-Bt ( 2.1d+d' ) ZDL -MetL Glu(OBzl) -OH ( 2.5c+c' ) 92 -5.3 6.3,7.5 72-73 aIsolated yield Dipeptides 2.5a-c were obtained with no detectable racemization evidenced by their NMR analyses. The enantiopure LLdipeptides ( 2.5a,c ) gave doublets for the two NH protons. However, for the diastereomeric mixture ( 2.5c+c' ), each of the two NH protons appeared as two pairs of equal doublets. 1H NMR spectrum of 2.5c displayed a singlet for the methyl protons

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20 (SMe) from ZL -Met fragment. 13C NMR displayed a singlet for each carbonyl carbon for compounds 2.5a,c whereas for the diastereomeric mixture 2.5c+c' most of the aliphatic and carbonyl carbons were observed as doublets, but no significant changes were observed for the aromatic carbons. In addition, the formation of rotamers was observed for the DL -dipeptide ( 2.5b ), showing complicated 1H and 13C NMR spectra, e.g. 13C analysis gave a second set of signals for all aliphatic, car bonyl and aromatic carbons. The enantiopurity of the dipeptides 2.5a-c was further confirmed by the HPLC analysis using a Chirobiotic T column (detection at 254 nm flow rate 0.5mL/min, and MeOH as solvent). As expected, HPLC analysis of the enantiopure LL ( 2.5a,c) and DL ( 2.5b ) dipeptides gave a single peak for each compound (Table 2-2). 2.2.3 Preparation of Dipeptides 2.7a-e from U nprotected Glutamic or Aspartic Acids and N-( -Aminoacyl)benzotriazoles 2.1a,b,d and e. Dipeptides 2.7a-e were obtained from unprotected glutamic ( 2.6a ) and aspartic acids ( 2.6b ) (1 equiv.), N -(Z-aminoacyl)benzotriazoles 2.1a,b,d and e (1 equiv.), and triethylamine (2 equiv.) in acetonitrile-water mixture (2:1 by volume) at room temperature for 2 hours (Figure 2-4 and Table 2-3). The crude products were wa shed with 4N HCl to remove the by-product, BtH. Compounds 2.7a-e were obtained in 65-94% yields and were further recrystallized from CH2Cl2/hexanes for further characterization. NMR analysis of 2.7a-e showed no detectable racemizati on: each compound revealed two doublets for each of the two NH protons in th e range of 7.40-8.50 ppm. The methyl protons from the ZL -Ala fragment showed a clear doublet in the 1H NMR of 2.7a The 1H NMR spectra also displayed a singlet for th e methyl protons (SMe) from ZL -Met fragment for compounds 2.7c In addition, 13C NMR displayed a singlet for each carbonyl carbon for dipeptides 2.7a-e

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21 N H Z R N O N N H2N (CH2)nOH O N H Z R H N O(CH2)nOH O 2.1a,b,d,e CH3CN/H2O Et3N,r.t.,2h. 2.7a-e n=1,2. AminoacidswithR:Ala,Phe,Trp.Met OH O OH O 2.6a,b Figure 2-4. Preparati on of novel dipeptides 2.7a-e Table 2-3. Preparati on of novel dipeptides 2.7a-e Entry N -(Z-aminoacyl) benzotriazoles Product Yield (%)a [ ]25 D Mp (oC) 1 ZL -Ala-Bt ( 2.1a ) ZL -AlaL -Glu-OH ( 2.7a ) 65 +0.7 121 123 2 ZL -Phe-Bt ( 2.1b ) ZL -PheL -Glu-OH ( 2.7b ) 86 -12.8 154 156 3 ZL -Met-Bt ( 2.1d ) ZL -MetL -Glu-OH ( 2.7c ) 94 -8.2 119-120 4 ZL -Trp-Bt ( 2.1e ) ZL -TrpL -Glu-OH ( 2.7d ) 90 -24.0 69 71 5 ZL -Phe-Bt ( 2.1b ) ZL -PheL -Asp-OH ( 2.7e ) 85 -1.7 179 181 a Isolated yield In addition, it was also f ound that the dipeptides 2.7a,b were obtained in lower yields as compared to the natural dipeptides 2.3a,b and the unnatural dipeptides 2.5a For example, the reaction of L -glutamic acid ( 2.6a ) with ZL -Phe-Bt ( 2.1b ) gave a dipeptide 2.7b in 86% yield, whereas the reaction of -benzyl L -glutamate ( 2.2a ) with ZL -Phe-Bt ( 2.1b ) gave compound 2.3b in 97% yield and -benzyl L -glutamate ( 2.4a ) with L -Phe-Bt ( 2.1b ) gave dipeptide 2.5a in 87% yield. As expected, these th ree compounds gave different me lting points: the dipeptide 2.7b (with unprotected side chain) melted at 154-156 C, while the unnatural dipeptide 2.5a had melting point 140-141 C and the natural dipeptide 2.3b had 139-142 C. Also, the compound 2.7a had a higher melting point (121-123 C) than the dipeptides 2.3a 2.2.4 Peptide Chain Extension at the Alpha CTerminus to Give Natural Dipeptides 2.10a-c. Peptide coupling was carried out by reacting or -monobenzylesters ( 2.8a Asp) and ( 2.8b Glu) of the N -(Fmoc-aminoacyl)benzotriazoles with equimolar amounts of unprotected

PAGE 22

22 amino acids Trp 2.9a and Met 2.9b in acetonitrile-water mixture (2:1 v/v) in the presence of triethylamine (2 equiv.) at room temperature for 2 hours (Figure 2-5 and Table 2-4). The crude products were washed with 4N HCl to re move the by-product BtH, yielding compounds 2.10a-c in 91-94%, which were furthe r recrystallized from CH2Cl2/hexanes before 1H and 13C NMR spectroscopy, elemental analysis, and optical rotatory power (ORP). The Katritzky group researchers have previous ly found that coupling reactions utilizing N (Z or Fmoc-aminoacyl)benzotriazoles gave dipeptide products with no detectable racemization as demonstrated by their 1H NMR and HPLC analyses.61-63 Likewise, NMR analysis of the enantiopure LL -dipeptides 2.10a-c showed no detectable racemization : each compound revealed two doublets for each of the two NH protons in the range of 7.80 8.40 ppm. The NMR spectra also displayed a singlet for th e methyl protons (SMe) for compounds 2.10c. In addition, 13C NMR displayed a singlet for each carbonyl carbon for the dipeptides 2.10a-c (CH2)nN H FmocBt O O O Ph H2NCOOH R O O (CH2)nN H Fmoc O O O Ph H N COOH R 2.10a-c a : n = 1; b : n = 2 Bt = benzotriazol-1-yl 2.8a,b CH3CN / H2O Et3N, r.t., 2h. 2.9a R = -CH2(3-indolyl NH) (Trp) 2.9b R = -(CH2)2-S-CH3 (Met) Fmoc = Figure 2-5. Preparati on of novel dipeptides 2.10a-c Table 2-4. Preparati on of novel dipeptides 2.10a-c Entry Amino Acid Product Yield (%)a [ ]25 D Mp (oC) 1 L -Trp (2.9a) FmocL -Glu(OBzl)L -Trp-OH ( 2.10a ) 93 +11.5 165-167 2 L -Trp (2.9a) FmocL -Asp(OBzl)L -Trp-OH ( 2.10b )94 -4.6 136-137 3 L -Met (2.9b) FmocL -Glu(OBzl)L -Met-OH ( 2.10c ) 91 -7.6 96-97 aIsolated yield

PAGE 23

23 2.2.5 Peptide Chain Extension at the or C-Terminus to Give Unnatural Dipeptides 2.12a-d Peptide coupling reactions were carried out using equimolar amounts of the -monobenzyl ester N -(Z-aminoacyl)benzotriazoles (Asp) 2.11a and (Glu) 2.11b and unprotected amino acids ( L -Trp, L -Phe, DL -Phe,) 2.9a c,d in partially aqueous solution (CH3CN/H2O, 2:1 v/v) in the presence of triethylamine (2 equiv.) at room temperature for 2 hours (Figure 2-6). The crude products were washed with 4N HCl to remove the by-product, BtH. The dipeptides 2.12a,b were obtained in 91-92% yields (T able 2-5) and were furthe r recrystallized from CH2Cl2/hexane for NMR, elemental analysis, and OR P. Diastereomeric mixture 2.12b+b' was prepared from N -(ZL -Asp)benzotriazole and DL -Phe-OH using the same procedure as for the enantiopure dipeptides 2.10a-c Dipeptide mixture 2.12b+b' was obtained in 95% yield a nd was recrystallized from diethyl ether/hexanes. N H ZO O Ph BtO H2NCOOH R N H ZO OPh H N OCOOH R 2.12a,b,b+b' Bt=benzotriazol-1-yl. AminoacidswithR:Trp,Phe, DL -Phe 2.11a,b CH3CN/H2O Et3N,r.t.,2h. 2.9a,c,d Figure 2-6. Preparati on of novel dipeptides 2.12a,b and diastereomeric mixture 2.12b+b' Table 2-5. Preparati on of novel dipeptides 2.12a,b and the diastereomeric mixture 2.12b+b' Entry Amino Acid Product Yie ld (%)a [ ]25 D Retention time (min) Melting point (oC) 1 L -Trp (2.9a) ZL -Asp-OBzlL -Trp-OH (2.12a) 92 -7.1 7.5 98-100 2 L -Phe (2.9c) ZL -Asp-OBzlL -Phe-OH (2.12b) 91 +0.07 7.6 138-140 3 DL -Phe (2.9d) ZL -Asp-OBzlDL -Phe-OH (2.12b+b') 95 -18.6 7.6, 10.1 116-118 aIsolated yield

PAGE 24

24 NMR analysis of the enantiopure LL -dipeptides 2.12a b revealed no detectable racemization. Signals arising from diastereom ers were not observed in the NMR spectra of compounds 2.12a,b. Thus, each dipeptide revealed two se ts of doublets for the two NH proton signals. However, for the diastereomeric mixture 2.12b+b' one of the two NH protons appeared as two pairs of equal doublets. 13C NMR displayed a singlet for each carbonyl carbon for compounds 2.12a,b whereas for the diastereomeric mixture 2.12b+b' most of the aliphatic and carbonyl and aromatic carbons were observed as doublets. The dipeptides 2.12a,b were further characterized by HPLC analysis using a Chirobiotic T column (detection at 254nm, flow rate 0.5mL/mi n, and MeOH as solvent). As expected, HPLC analysis of enantiopure LL ( 2.12a,b ) dipeptides showed a single peak for each compound. In contrast, two peaks were observed for the corresponding diastereomeric mixture 2.12b+b' 2.2.6 Preparation of N-Protected-Dipeptidoylbenzotriazoles 2.13a-c and 2.13a+a' from NProtected Dipeptides 2.3a,b,d and 2.3a+a' N-Z-Dipeptides 2.3a,b,d and 2.3a+a' were successfully converted into the corresponding benzotriazole derivatives 2.13a-c and 2.13a+a' (Figure 2-7 and Table 26). The reactions were carried out at C following the same simple procedure as in references 62 and 63. The reaction was continued until the starting materials 2.3a,b,d and 2.3a+a' were completely consumed as observed under TLC and NMR analyses. Compounds 2.13a-c 2.13a+a' were isolated after acid (4N HCl) workup in good yields (89-93%) and were recrystallized using CH2Cl2/hexanes for 1H and 13C NMR spectroscopy, elemental analysis, and ORP. Compounds 2.13a-c were obtained with no detectable racemization as evidenced by NMR. Compounds 2.13a-c gave two doublets for the two NH protons. 1H NMR spectrum of 2.13a indicated doublet for the methyl proton of L -Ala fragment. In contrast, the same signal of the methyl group showed two sets of doubl ets for the diastereomeric mixture 2.13a + a' 13C NMR

PAGE 25

25 analysis displayed a singlet fo r each carbonyl carbon for compounds 2.13a-c whereas in the mixture 2.13a + a' most of the carbonyl, aliphatic and ar omatic carbons appeared as doublets. N H Z R H N O COOH (CH2)nO O Ph N H N N Cl S Cl O CH2Cl2N H Z R H N O(CH2)nO O Ph O Bt -150C 2.3a,b,d,2.3a+a' 2.13a-c,2.13a+a' n=1,2. Bt=benzotriazol-1-yl Figure 2-7. Preparation of nove l dipeptidoylbenzotriazoles 2.13a-c and diastereomeric mixture 2.13a + a' Table 2-6. Conversion of novel N-Z-dipeptides 2.3a,b,d and the diastereomeric mixture 2.3a+a' into N-Z-dipeptidoylbenzotriazoles 2.13a-c, 2.13a+a' Entry Reagent Product Yield (%)a [ ]25 D Mp (oC) 1 ZL -AlaL Glu(OBzl)-OH ( 2.3a ) ZL -AlaL Glu(OBzl)-Bt ( 2.13a ) 93 -17.9 133-135 2 ZL -PheL Glu(OBzl)-OH ( 2.3b ) ZL -PheL Glu(OBzl)-Bt ( 2.13b ) 92 -24.7 90-92 3 ZL -PheL Asp(OBzl)-OH ( 2.3d ) ZL -PheL Asp(OBzl)-Bt ( 2.13c ) 89 -20.7 110-113 4 ZDL -AlaL -Glu(OBzl) -OH ( 2.3a+a' ) ZDL -AlaL -Glu(OBzl) Bt ( 2.13a+a' ) 91 -22.4 79-81 aIsolated yield 2.2.7 Preparation of N-Protected Tripeptides 2.14a,b,a' and Diastereomeric Mixture 2.14a+a'' from Dipeptidoylbenzotriazoles 2.13a,b, 2.13a+a' and Free Amino Acids 2.9c,e and f Tripeptides 2.14a,b and a' were prepared by peptide coupling reactions between Nprotected-dipeptidoylbenzotriazoles 2.13a,b (1 equiv.) with unprotected amino acids 2.9c e f (1 equiv.) in acetonitrile-water mixture (2 :1 v/v) in the presence of tr iethylamine (2 equiv.) at C for 2 hours. The mixture 2.14a+a'' was prepared using the same procedure. The crude products were washed with 4N HCl to remove the by-product, BtH. Compounds 2.14a,b 2.14a'

PAGE 26

26 and 2.14a+a'' were obtained in 73-95% yields (Figur e 2-8 and Table 2-7) and were further recrystallized from CH2Cl2/hexanes for NMR spectroscopy, el emental analysis, and ORP. N H Z R' H N O(CH2)nO O Ph O Bt H2NCOOH R'' N H Z R' H N O(CH2)nO O Ph N H O 2.14a,b,a',a+a'' n=1,2. Bt=benzotriazol-1-yl. AminoacidswithR':Ala,DL-Ala,Phe AminoacidswithR'':Ala,Phe,D-Phe 2.13a,b,2.13a+a' CH3CN/H2O Et3N,-150C,2h. 2.9c,e,f COOH R'' Figure 2-8. Preparati on of novel tripeptides 2.14a,b a', and 2.14a+a'' Table 2-7. Preparati on of novel tripeptides 2.14a,b,a' and mixture 2.14a+a'' Entry Amino Acid Reactant Product Yield (%)a [ ]25 D Mp (oC) 1 L -Phe (2.9c) ZL -AlaL -Glu(OBzl)Bt (2.13a) ZL -AlaL -Glu(OBzl)L -Phe-OH ( 2.14a ) 73 -2.9 74-76 2 D -Phe (2.9e) ZL -AlaL -Glu(OBzl)Bt (2.13a) ZL -AlaL -Glu(OBzl)D -Phe-OH ( 2.14a' ) 83 +5.5 153-155 3 L -Ala (2.9f) ZL -PheL -Glu(OBzl)Bt (2.13b) ZL -PheLGlu(OBzl)L -Ala-OH ( 2.14b ) 95 -13.0 163-165 4 L -Phe (2.9c) ZDL -AlaL Glu(OBzl)-Bt (2.13a+a') ZDL -AlaLGlu(OBzl)L -Phe-OH (2.14a+a'') 92 -5.5 123-125 aIsolated yield NMR analysis showed no racemization for the tripeptides 2.14a and 2.14a' when the reaction was carried out -15 0C, however when the same reaction was carried out at room temperature, extensive racemization of the desired products was observed. 1 H NMR showed a clear doublet for all NH protons and the methyl protons of the L -Ala fragment, while NMR spectra of the mixture 2.14a+a'' gave complicated multiplets for -NH groups. 13C NMR displayed a singlet for e ach carbonyl carbon for compounds 2.14a and 2.14a' whereas the

PAGE 27

27 diastereomeric mixture 2.14a+a'' showed most of the aliphatic and carbonyl and aromatic carbons as doublets. To confirm the absence of racemization, the compounds ZL -AlaL -Glu(OBzl)L -Phe-OH ( 2.14a ) ZL -AlaL -Glu(OBzl)D -Phe-OH ( 2.14a' ) and the mixture ZDL -AlaLGlu(OBzl)L Phe-OH ( 2.14a+a'' ) were analyzed by HPLC. Single peaks were obtained for tripeptides 2.14a at 6.4 min and 2.14a' at 7.3 min whereas the diastereomeric mixture 2.14a+a'' gave two peaks at 6.4 and 7.3 min. 2.2.8 Preparation of Novel Tripeptides 2.16a,b Containing Glu and Asp Fragments. The general applicabil ity of our coupling method was dem onstrated by the preparation of tripeptides 2.16a,b containing glutamate or aspartate moieties. This was achieved by the elongation of Asp and Glu fragments from the N -terminus, utilizing the free amino group of unprotected dipeptides 2.15a,b and N -(Z-aminoacyl)benzotriazoles 2.1a b Synthesis of tripeptides 2.16a,b was achieved in two steps wi thout purification of the dipeptides intermediates 2.15a,b. First, the Fmocgroup in compounds 2.10a,b was removed with piperidine utilizi ng a literature method;65 thereafter the remaining piperidine was removed, the dipeptides obtained 2.15a,b were then coupled with N -(Z-aminoacyl)benzotriazoles 2.1a b yielding the desired tripeptides 2.16a,b The procedure does not require purification of compounds 2.15a,b ; however, care must be taken to remove all remaining piperidine, as it can compete with free amino acids and peptides in their reactions with N -protected( aminoacyl)benzotriazoles. Crystalline tripeptides 2.16a,b were purified by washing with diethyl ether-hexanes mixture giving de sired product in the yields of 83-84% (Table 2-8). Novel compounds 2.16a,b were characterized by 1H and 13C NMR spectroscopy, elemental and ORP analyses.

PAGE 28

28 (CH2)nHN O O O Ph NH COOH R' Fmoc (CH2)nH2N O O O Ph NH COOH R' N H Z R'' Bt O (CH2)nHN O O O Ph NH COOH R' O NH Z R'' n=1,2. AminoacidswithR':Trp AminoacidswithR'':Ala,Phe 2.1a,b 2.10a,b piperidine r.t.,2h CH3CN/H2O Et3N,-150C,2h. 2.15a,b2.16a,b Figure 2-9. Preparati on of novel tripeptides 2.16a,b Table 2-8. Preparati on of novel tripeptides 2.16a,b containing Glu and Asp fragments Product Yield (%)a[ ]25 D Mp (oC) ZL -PheL -Glu(OBzl)L -Trp-OH ( 2.16a ) 84 -3.2 109 111 ZL -AlaL -Asp(OBzl)L -Trp-OH ( 2.16b ) 83 -1.7 134-137 a Isolated yield NMR analysis demonstrated no detectable racemization for the tripeptides 2.16a,b No signals arising from the diastereomers were observed in the NMR spectra of compounds 2.16a,b 1H NMR showed a clear doublet for all NH protons of 2.16a,b and for the methyl protons from L -Ala fragment for compound 2.16b 13C NMR also gave a singlet for each carbonyl carbon in 2.16a,b 2.3 Conclusion The convenient preparation under mild conditions of N -(Fmocor Zaminoacyl)benzotriazoles, N -protected peptidoylbenzotriazole s and diastereomeric mixtures prepared from aspartic and glutamic amino acid has been demonstrat ed. Additionally, the preparation of diand tr i-peptides starting from Cand N -termini of glutamic and aspartic acids has been demonstrated under mild reaction conditi ons. Products were obtained without the use of column chromatography. Evidence of main tained chirality was supported by NMR and HPLC analyses.

PAGE 29

29 2.4 Experimental Section Melting points were determined on a capillary point apparatus equi pped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSOd6 with TMS for 1H (300 MHz) and 13C (75 MHz) as an internal reference. N -Zand Fmoc-amino acids were purchased from Fluka and Acros and were used without further purification. Elemental an alyses were performed on a Carlo Erba-1106 instrument. Optical rotation values were measured with the use of the sodium D line. HPLC analyses were performe d on Beckman system gold programmable solvent module 126, using Chirobiotic T column (4.6 250 mm ), detection at 254 nm, flow rate of 0.5 mL/min and 100% MeOH as eluting solvent. 2.4.1 General Procedure for the Preparation of Dipeptides 2.3a-c, 2.3a+a', 2.5a-c, 2.5c+c' and 2.7a-e Benzyl protected ( 2.2a,b and 2.4a ) or unprotected ( 2.6a,b ) aspartic or glutamic acids (5 mmol) were added to a solution of Et3N (10 mmol) in CH3CN (15 mL) and H2O (7 mL) at 25 oC, and the reaction mixture was stirred for 15 min at 25 oC. N -(Protectedaminoacyl)benzotriazoles ( 2.1a e and 2.1a+a' ) (for characterization and preparation refer to references 51, 61, and 63) (5 mmol) were added to the mixture with continued stirring for 2 h at 25 oC. About 4N HCl (5 mL) was added to the reaction mixture and CH3CN was evaporated under reduced pressure. The residu e was dissolved in EtOAc (50 mL), the organic extract was then washed with 4 N HCl (3x15 mL), saturated NaCl (20 mL), and dried over MgSO4. Evaporation of the solvent ga ve the desired products ( 2.3a c 2.3a+a' 2.5a c, 2.5c+c' 2.7a-e ), which were further recrystallized from CH2Cl2hexane, unless specified otherwise. 5-Benzyl ( S )-2-(( S )-2-benzyloxycarbonylamino-pro pionylamino)pentanoate (ZL -AlaL -Glu(OBzl)-OH, 2.3a): White microcrystals (94%); mp 89-91 oC, [ ]D 23 = +1.0 (c 1.66, DMF). 1H NMR (DMSOd6): 1.26 (d, J = 7.1 Hz, 3H), 1.80-1.96 (m, 1H), 2.00-2.18 (m, 1H), 2.30-

PAGE 30

30 2.54 (m, 2H), 4.09-4.19 (m, 1H), 4.27-4.34 (m, 1H ), 5.00-5.10 (m, 2H), 5.14 (s, 2H), 7.30-7.41 (m, 10H), 7.52 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 12.70 (s, 1H). 13C NMR (DMSOd6): 18.1, 26.3, 30.0, 49.9, 51.0, 65.4, 65.6, 127.8, 128.0, 128.1, 128.4, 128.5, 136.2, 137.1, 149.7 155.7, 172.2, 172.8, 173.2. Anal. calcd for C23H26N2O7: C, 62.43; H, 5.92; N, 6.33. Found: C, 62.05; H, 5.95; N, 6.01. 5-Benzyl ( S )-2-(( S )-2-benzyloxycarbonylamino-3-phe nylpropionylamino)pentanoate (ZL -PheL -Glu(OBzl)-OH, 2.3b): White microcrystals (97%); mp 139-142 oC, [ ]D 23 = -10.3 (c 1.66, DMF). 1H NMR (DMSOd6): 1.88 1.95 (m, 1H), 2.08 2.14 (m, 1H), 2.32 2.55 (m, 2H), 2.72 2.82 (m, 1H), 2.99 3.09 (m, 1H), 4.30 4.37 (m, 2H), 4.95 (s, 2H), 5.13 (s, 2H), 7.16 7.41 (m, 15H), 7.57 (d, J = 8.7 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 12.73 (s, 1H). 13C NMR (DMSOd6): 26.3, 30.1, 37.4, 51.2, 56.1, 65.3, 65.6, 126.3, 127.5, 127.7, 128.0, 128.1, 128.2, 128.3, 128.5, 129.3, 136.2, 137.0, 138.2, 155.9, 171.9, 172.2, 173.1. Anal. calcd for C29H30N2O7: C, 67.17; H, 5.83; N, 5.40. Found: C, 66.88; H, 5.87; N, 5.31. 4-Benzyl ( S )-2-(( S )-2-benzyloxycarbonylamino-3phenylpropionylamino)butanoate (ZL -PheL -Asp(OBzl)-OH, 2.3c): White microcrystals (95%); mp 102-105 oC, [ ]D 23 = -14.0 (c 1.66, DMF). 1H NMR (DMSOd6): 2.65 3.07 (m, 4H), 4.29 4.35 (m, 1H), 4.65 4.73 (m, 1H), 4.89 4.98 (m, 2H), 5.13 (s, 2H), 7.14 7.37 (m, 15H), 7.56 (d, J = 8.4 Hz, 1H), 8.52 (d, J = 7.7 Hz, 1H), 12.9 (s, 1H). 13C NMR (DMSOd6): 36.0, 37.4, 48.6, 56.0, 65.2, 65.9, 126.3, 127.4, 127.7, 127.9, 128.0, 128.3, 128.4, 128.5, 129.2, 135.9, 137.0, 138.1, 155.8, 170.1, 171.6, 172.5. Anal. calcd for C28H28N2O7: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.33; H, 5.68; N, 5.60. 5-Benzyl ( S )-2-(2-benzyloxycarbonylaminopr opionylamino)pentanoate (ZDL -AlaL Glu(OBzl)-OH, 2.3a+a'): White microcrystals (94%); mp 81-83 oC, [ ]D 23 = +0.6 (c 1.66, DMF). 1H NMR (CDCl3): 1.34-1.38 (m, 3H), 2.04-2.11 (m, 1H), 2.27-2.30 (m, 1H), 2.30-2.50

PAGE 31

31 (m, 2H), 4.30-4.43 (m, 1H), 4.63-4.65 (m, 1H), 5.05-5.13 (m, 4H), 5.97 (d, J = 7.4 Hz, 0.5H), 6.06 (d, J = 7.5 Hz, 0.5H), 7.26-7.46 (m, 11H), 10.27 (s, 1H). 13C NMR (CDCl3): 18.0, 18.3, 26.5, 30.0, 50.2, 51.3, 66.3, 66.8, 127.7, 127.8, 127.8, 128.0. 128.2, 128.3, 135.3, 135.8, 156.1, 156.2, 172.7, 172.8, 173.4, 173.7. Anal. calcd for C23H26N2O7: C, 62.43; H, 5.92; N, 6.33. Found: C, 62.12; H, 5.93; N, 6.23. 1-Benzyl ( S )-2-(( S )-2-benzyloxycarbonylamino-3-phe nylpropionylamino)pentanoate (ZL -PheL -Glu(OBzl)-OH, 2.5a): White microcrystals (87%); mp 140-141 oC, [ ]D 23 = -10.7 (c 2.00, DMF). 1H NMR (CDCl3): 1.99 (quintet, J = 7.1 Hz, 1H), 2.15-2.20 (m, 1H), 2.35-2.49 (m, 2H), 3.05 (d, J = 6.3 Hz, 2H), 4.48-4.55 (m, 2H), 5.02 (d, J = 12.9 Hz, 1H, A part of AB system), 5.06 (d, J = 12.6 Hz, 1H, B part of AB system), 5.08 (s, 2H), 5.47 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 6.9 Hz, 1H), 7.12-7.36 (m, 15H), 12.85 (br s, 1H). 13C NMR (CDCl3): 26.6, 30.1, 38.3, 51.8, 56.0, 66.6, 67.1, 127.0, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 129.3, 135.5, 136.0, 156.2, 171.7, 173.0, 174.0. Anal. calcd for C29H30N2O7: C, 67.17; H, 5.83; N, 5.40. Found: C, 66.83; H, 5.81; N, 5.22. 1-Benzyl ( S )-2-(( R )-2-benzyloxycarbonylamino-3-phe nylpropionylamino)pentanoate (ZD -PheL -Glu(OBzl)-OH, 2.5b): White microcrystals (91%); mp 119-120 oC, [ ]D 23 = -5.4 (c 1.66, DMF). 1H NMR (DMSOd6) (two rotameric forms): 1.82-1.91 (m, 1H), 1.96-2.09 (m, 1H), 2.30 (t, J = 7.2 Hz, 1H), 2.43-2.46 (m, 1H), 2.69-2.79 (m, 1H), 2.92-3.04 (m, 1H), 4.26-4.35 (m, 2H), 4.99 (d, J = 12.4 Hz, 1H, A part of AB system), 5.02 (d, J = 12.6 Hz, 1H, B part of AB system), 5.08 (d, J = 12.4 Hz, 1H, A part of AB system), 5.11 (d, J = 12.4 Hz, 1H, B part of AB system), 7.12-7.36 (m, 15H), 7.51 (d, J = 8.8 Hz, 1H), 8.31-8.37 (m, 1H), 12.83 (br s, 1H). 13C NMR (CDCl3) (two rotameric forms): 26.3, 26.4, 29.8, 30.0, 37.3, 51.0, 51.1, 56.0, 65.2, 65.5, 126.3, 127.4, 127.5, 127.7, 127.9, 128.0, 128.3, 128.4, 129.2, 136.1, 136.9, 137.0, 137.9, 138.1,

PAGE 32

32 155.7, 155.9, 171.5, 171.8, 172.0, 172.1, 172.9, 173.0. Anal. calcd for C29H30N2O7: C, 67.17; H, 5.83; N, 5.40. Found: C, 66.86; H, 5.83; N, 5.21. 1-Benzyl ( S )-2-(( S )-2-benzyloxycarbonylamino-4methylsulfanylbutyrylamino)pentanoate (ZL -MetL -Glu(OBzl)-OH, 2.5c): White microcrystals (87%); mp 98-99 oC, [ ]D 23 = -7.1 (c 2.0, DMF). 1H NMR (CDCl3): 1.92-1.97 (m, 2H), 2.06 (s, 3H), 2.22-2.27 (m 2H), 2.46-2.57 (m, 4H), 4.41 (q, J = 7.4 Hz, 1H), 4.54-4.58 (m, 1H), 5.07-5.13 (m, 4H), 5.64 (d, J = 7.9 Hz, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.26-7.33 (m, 10H), 10.10 (br s, 1H). 13C NMR (CDCl3): 15.1, 26.5, 29.7, 30.2, 31.5, 51.8, 53.6, 66.7, 67.2, 128.0, 128.2, 128.3, 128.4, 128.5, 128.6, 135.5, 136.0, 156.2, 172.0, 173.0, 174.2. Anal. calcd for C25H30N2O7S: C, 59.75; H, 6.02; N, 5.57. Found: C, 59.38; H, 6.02; N, 5.34. 1-Benzyl ( S )-2-(2-benzyloxycarbonylamino-4methylsulfanylbutyrylamino)pentanoate (ZDL -MetL -Glu(OBzl)-OH, 2.5c+c ): White microcrystals (92%); mp 72-73 oC, [ ]D 23 = -5.3 (c 1.66, DMF). 1H NMR (CDCl3): 1.88-1.97 (m, 2H), 2.03 (s, 3H), 2.10.23 (m, 2H), 2.462.54 (m, 4H), 4.40-4.45 (m, 1H), 4.54-4.59 (m, 1H), 5.02-5.13 (m, 4H), 5.82 (d, J = 8.5 Hz, 0.5H), 5.95 (d, J = 8.5 Hz, 0.5H), 7.30-7.32 (m, 10H), 7.36 (d, J = 8.0 Hz, 1H), 8.40 (br s, 1H). 13C NMR (CDCl3): 14.9, 15.1, 26.4, 29.6, 29.7, 29.8, 30.1, 31.4, 31.7, 51.5, 52.8, 53.5, 53.7, 66.5, 66.9, 126.9, 127.8, 128.0, 128.1, 128.3, 128.4, 135.4, 135.9, 156.1, 156.2, 156.5, 172.1, 172.7, 172.8, 173.8, 174.9. Anal. calcd for C25H30N2O7S: C, 59.75; H, 6.02; N, 5.57. Found: C, 59.36; H, 6.00; N, 5.19. ( S )-2-(( S )-2-Benzyloxycarbonylaminopropionylamino)pentanedioic acid (ZL -AlaL Glu-OH, 2.7a): White microcrystals (65%); mp 121-123 oC, [ ]D 23 = +0.7 (c 1.66, DMF). 1H NMR (DMSOd6): 1.20 (d, J = 6.9 Hz, 3H), 1.71-1.84 (m, 1H), 1.95-2.01 (m, 1H), 2.30 (t, J = 7.6 Hz, 2H), 4.07 (q, J = 7.1 Hz, 1H), 4.17-4.25 (m, 1H), 5.01 (s, 2H), 7.30-7.36 (m, 5H), 7.45

PAGE 33

33 (d, J = 7.7 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 12.41 (s, 2H). 13C NMR (DMSOd6): 8.1, 26.4, 30.0, 49.8, 51.1, 65.4, 127.3, 127.8, 128.4, 137.0, 155.7, 172.7, 173.3, 173.8. Anal. calcd for C16H20N2O7: C, 54.54; H, 5.72; N, 7.95. Found: C, 54.21; H, 5.69; N, 7.81. ( S )-2-(( S )-2-Benzyloxycarbonylamino-3-phenylpro pionylamino)pentanedioic acid (ZL -PheL -Glu-OH, 2.7b): White microcrystals (86%); mp 154-156 oC, [ ]D 23 = -12.8 (c 1.66, DMF). 1H NMR (DMSOd6): 1.76-1.88 (m, 1H), 1.99-2.06 (m, 1H), 2.28-2.36 (m, 2H), 2.682.77 (m, 1H), 2.99-3.04 (m, 1H), 4.22-4.32 (m, 2 H), 4.94 (s, 2H), 7.05-7.32 (m, 10H), 7.52 (d, J = 8.8 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H), 12.47 (s, 2H). 13C NMR (DMSOd6): 26.6, 30.2, 37.6, 51.5, 56.2, 65.5, 126.5, 127.7, 127, 9, 128.3, 128.5, 129.4, 137.2, 138.3, 156.1, 172.1, 173.5, 174.1. Anal. calcd for C22H24N2O7: C, 61.67; H, 5.65; N, 6.54. Found: C, 61.76; H, 5.57; N, 6.56. ( S )-2-(( S )-2-Benzyloxycarbonylamino-4-methylsu lfanylbutyrylamino)pentanedioic acid (ZL -MetL -Glu-OH, 2.7c): White microcrystals (94%); mp 119-120 oC, [ ]D 23 = -8.2 (c 1.66, DMF). 1H NMR (DMSOd6): 1.75-1.99 (m, 4H), 2.02 (s, 3H), 2.30 (t, J = 7.2 Hz, 2H), 2.39-2.47 (m, 2H), 4.01-4.24 (m, 2H), 5.02 (s, 2H), 7.30-7.36 (m, 5H), 7.51 (d, J = 8.0, 1H), 8.20 (d, J = 7.7 Hz, 1H), 12.42 (br s, 2H). 13C NMR (DMSOd6): 14.8, 26.3, 29.7, 30.1, 31.9, 51.3, 53.8, 65.6, 127.9, 128.0, 128.5, 137.1, 156.1, 171.8, 173.3, 173.9. Anal. calcd for C18H24N2O7S: C, 52.42; H, 5.86; N, 6.79. Found: C, 52.47; H, 5.89; N, 6.69. ( S )-2-(( S )-2-Benzyloxycarbonylamino-3-(1 H -indol-3-yl)propionylamino)pentanedioic acid (ZL -TrpL -Glu-OH, 2.7d): Yellow microcrystals (90%); mp 69-71 oC, [ ]D 23 = -24.0 (c 1.66, DMF). 1H NMR (DMSOd6): 1.81-1.91 (m, 1H), 1.98-2.06 (m,1H), 2.33 (t, J = 7.1 Hz, 2 H), 2.87-2.95 (m, 1H), 3.10-3.15 (m, 1H), 4.26-4.38 (m, 2H), 5.01 (s, 2H), 7.00 (t, J = 7.4 Hz, 1 H), 7.07 (t, J = 7.4 Hz, 1H), 7.18-7.41 (m, 8H) 7.68 (d, J = 7.7 Hz, 1H), 8.35 (d, J = 7.4 Hz, 1H), 10.82 (s, 1H), 12.44 ( br s, 2H) 13C NMR (DMSOd6): 26.4, 27.8, 30.1, 51.3, 55.3, 65.3,

PAGE 34

34 110.2, 111.4, 118.2, 118.6, 120.9, 124.0, 127.3, 127.6, 127.7, 128.4, 136.1, 137.0, 155.9, 172.2, 173.3, 173.9 Anal. calcd for C24H25N3O7: C, 61.66; H, 5.39; N, 8.99. Found: C, 61.72; H, 5.73; N, 8.41. ( S )-2-(( S )-2-Benzyloxycarbonylamino-3-phenylpropionylamino)succinic acid (ZL PheL -Asp-OH, 2.7e): White microcrystals (85%); mp 179-181 oC, [ ]D 23 = -1.66 (c 1.66, DMF). 1H NMR (DMSOd6): 2.60-2.71 (m, 3H), 2.99-3.03 (m, 1H), 4.30 (apparent t, J = 7.6 Hz, 1H), 4.55-459 (m, 1H), 4.93 (s, 2H), 7.14-7.31 (m, 10 H), 7.53 (d, J = 8.5 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 12.64 (br s, 2H). 13C NMR (DMSOd6): 36.0, 37.5, 48.7, 56.0, 65.2, 126.3, 127.4, 127.7, 128.1, 128.3, 129.3, 137.0, 138.2, 155.9, 171.6, 171.7, 172.4. Anal. calcd for C21H22N2O7: C, 60.86; H, 5.35; N, 6.76. Found: C, 61.07; H, 5.75; N, 6.53. 2.4.2 General Procedure for the Preparation of N -(Zand Fmoc-Aminoacyl)benzotriazoles 2.8 a,b and 2.11 a,b. Thionyl chloride (5 mmol) was added to a solution of 1 H -benzotriazole (20 mmol) in dry CH2Cl2 (15 mL) at 20 oC, and the reaction mixture was stirred for 20 min at 40 50 oC. To the reaction mixture at 0 oC, the N -protected amino acid (5 mmol) dissolved in dry CH2Cl2 (5 mL) was added dropwise, and was then stirred for 2 hours at 20 oC. The white precipitate formed during the reaction was filtered off, and the filtrate was concentrated under reduced pressure. The residue was diluted with ethyl acetate (100 mL) and the solution was washed with 4N HCl solution (50 mL 3) or saturated Na2CO3 solution (50 mL 3), saturated NaCl solution (50 mL), and dried over anhydrous MgSO4. Removing solvents under reduced pressure gave products 2.8a,b and 2.11a,b which were recrystallized from CHCl3/hexanes, unless specified otherwise. Compounds 2.8a,b and 2.11a,b are novel and fully characterized by NMR and elemental analysis.

PAGE 35

35 ( S )-Benzyl-3-(((9 H -fluoren-9-yl)methoxy) carbonylamino)-4-(1 H -1,2,3-benzotriazol-1yl)-4-oxobutanoate (FmocL -Asp(OBzl)-Bt, 2.8a): White microcrystals (87%); mp 91-92 oC, [ ]D 23 = -26.5 (c 2.58, DMF). 1H NMR (DMSOd6): 3.03 (dd, J = 16.8, 8.8 Hz, 1H), 3.30 (dd, J = 16.8, 5.1 Hz, 1H), 4.24 (t, J = 6.3 Hz, 1H), 4.37 (d, J = 6.9 Hz, 2H), 5.14 (s, 2H), 5.87-5.92 (m, 1H), 7.29-7.44 (m, 9H), 7.65 (t, J = 7.5 Hz, 1H) 7.71 (d, J = 7.4 Hz, 2H), 7.81 (t, J = 7.5 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H), 8.23 (d, J = 8.1 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.46 (d, J = 7.1 Hz, 1H). 13C NMR (DMSOd6): 35.3, 46.6, 51.0, 66.0, 66.2, 114.0, 120.2, 120.3, 125.2, 126.8, 127.1, 127.7, 128.0, 128.1, 128.4, 130.7, 131.2, 135.7, 140.8, 143.7, 145.4, 156.0, 169.5, 170.3. Anal. calcd for C32H26N4O5: C, 70.32; H, 4.79; N, 10.25. Found: C, 70.08; H, 5.14; N, 9.47. ( S )-Benzyl-4-(((9 H -fluoren-9-yl)methoxy) carbonylamino)-5-(1 H -1,2,3-benzotriazol-1yl)-5-oxopentanoate (FmocL -Glu(OBzl)-Bt, 2.8b): White microcrystals (83%); mp 96-97 oC, [ ]D 23 = -22.5 (c 2.08, DMF). 1H NMR (DMSOd6): 2.15-2.27 (m, 1H), 2.32-2.40 (m, 1H), 2.68 (t, J = 7.1 Hz, 2H), 4.27 (t, J = 6.5 Hz, 1H), 4.37 (d, J = 6.9 Hz 2H), 5.03-5.13 (m, 2H), 5.54-5.63 (m, 1H), 7.30-7.47 (m, 9H), 7.66 (t, J = 7.7 Hz, 1H), 7.75 (d, J = 7.3 Hz, 2H), 7.84 (t, J = 7.4 Hz, 1H), 7.92 (d, J = 7.3 Hz, 2H), 8.26 (d, J = 8.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.39 (d, J = 7.0 Hz, 1H). 13C NMR (DMSOd6): 25.9, 29.9, 46.6, 53.6, 65.6, 65.9, 114.1, 120.1, 120.2, 125.2, 126.8, 127.1, 127.7, 127.9, 128.0, 128.4, 130.7, 131.1, 136.0, 140.8, 143.7, 145.4, 156.3, 171.5, 171.9. Anal. calcd for C33H28N4O5: C, 70.70; H, 5.03; N, 9.99. Found: C, 70.35; H, 5.09; N, 9.91. Benzyl ( S )-4-benzotriazol-1-yl-2-benzyloxyc arbonylamino-4-oxobutanoate (ZL -AspOBzl-Bt, 2.11a): White microcrystals (91%), mp 97-99 oC, [ ]D 23 = -22.5 (c 2.08, DMF). 1H NMR (CDCl3): 4.01 (dd, J = 18.1, 4.7 Hz, 1H), 4.14 (dd, J = 18.1, 4.6 Hz, 1H), 4.96-5.03 (m, 1H), 5.11 (s, 2H), 5.20 (s, 2H), 5.90 (d, J = 8.2 Hz, 1H), 7.21-7.32 (m, 10H), 7.52 (t, J = 7.5 Hz,

PAGE 36

36 1H), 7.65 (t, J = 7.7 Hz, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3): 38.3, 50.2, 67.3, 67.9, 114.2, 120.4, 126.5, 128.2, 128.3, 128.5, 128.5, 128.6, 130.6, 130.7, 134.8, 135.9, 146.1, 155.9, 169.8, 170.3. Anal. calcd for C25H22N4O5: C, 65.49; H, 4.84; N, 12.12. Found: C, 65.71; H, 4.80; N, 12.15. Benzyl ( S )-5-Benzotriazol-1-yl-2-benzyloxyc arbonylamino-5-oxopentanoate (ZL Glu-OBzl-Bt, 2.11b): White microcrystals (81%), mp 50.0-52.0 oC, [ ]D 23 = -20.7 (c 1.66, DMF). 1H NMR (CDCl3): 2.23-2.30 (m, 1H), 2.45-2.50 (m, 1H), 3.37-3.60 (m, 2H), 4.60-4.70 (m, 1H), 5.01-5.20 (m, 4H), 5.79 (d, J = 8.0 Hz, 1H), 7.22-7.31 (m, 10 H), 7.46 (t, J = 7.9 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H). 13C NMR (CDCl3): 21.7, 31.0, 58.8, 67.5, 68.3, 114.3, 120.2, 126.2, 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 130.5, 131.0, 135.0, 136.1, 146.1, 156.1, 171.4, 171.7. Anal. calcd for C26H24N4O5: C, 66.09; H, 5.12; N, 11.86. Found: C, 65.99; H, 5.14; N, 11.65. 2.4.3 General Procedure for the Preparation of Dipeptides 2.10a-c, 2.12a,b and 2.12b+b' Free amino acids ( 2.9a-f ) (5 mmol) were added to a solution of Et3N (10 mmol) in CH3CN (15 mL) and H2O (7 mL) at 25 oC, and the reaction mixture was stirred for 15 min at 25 oC. N (Protected-aminoacyl)benzotriazoles ( 2.8a,b and 2.11a,b ; 5 mmol) were added to the mixture with continued stirring for 2 h at 25 oC. About 4N HCl (5 mL) was added to the reaction mixture and CH3CN was removed under reduced pressure. The residue was dissolved in EtOAc (50 mL), and the organic extract was washed with 4N HC l (3x15 mL), saturated NaCl (20 mL) and dried over MgSO4. Evaporation of the solvent gave the desired products ( 2.10a c 2.12a,b, 2.12b+b' ), which were further recrystallized from CH2Cl2hexanes ( 2.10a-c ) and ether-hexanes ( 2.12a,b, 2.12b+b' ). ( S )-2-(( S )-2-(((9 H -Fluoren-9-yl)methoxy)carbon ylamino)-5-(benzyloxy)-5oxopentanamido)-3-(1 H -indol-3-yl)propanoic acid (FmocL -Glu(OBzl)L -Trp-OH, 2.10a):

PAGE 37

37 White microcrystals (93%); mp 165-167 oC, [ ]D 23 = +11.5 (c 2.16, DMF). 1H NMR (DMSOd6): 1.81-1.89 (m, 1H), 1.95-2.03 (m, 1H), 2.44 (t, J = 7.7 Hz, 2H), 3.10 (dd, J = 14.6, 8.0 Hz, 1H), 3.21 (dd, J = 14.7, 5.0 Hz, 1H), 4.10-4.30 (m, 4H), 4.48-4.55 (m, 1H), 5.11 (s, 2H), 6.99 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.1 Hz, 1H), 7.20 (s, 1H), 7.28-7.43 (m, 10H), 7.55 (d, J = 7.7 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.74 (t, J = 6.9 Hz, 2H), 7.88 (d, J = 7.4 Hz, 2H), 8.23 (d, J = 7.4 Hz, 1H), 10.90 (s, 1H), 12.75 (s, 1H). 13C NMR (DMSOd6): 27.0, 27.5, 30.2, 46.7, 53.0, 53.6, 65.5, 65.7, 109.6, 111.4, 118.2, 118.4, 120.1, 121.0, 123.7, 125.4, 127.1, 127.3, 127.7, 128.0, 128.1, 128.5, 136.1, 136.3, 140.8, 143.8, 144.0, 155.9, 171.5, 172.3, 173.3. Anal. calcd for C38H35N3O7: C, 70.68; H, 5.46; N, 6.51. Found: C, 70.43; H, 5.48; N, 6.47. ( S )-2-(( S )-2-(((9 H -Fluoren-9-yl)methoxy)carbon ylamino)-4-(benzyloxy)-4oxobutanamido)-3-(1 H -indol-3-yl)propanoic acid (FmocL -Asp(OBzl)L -Trp-OH, 2.10b): White microcrystals (94%); mp 136-137 oC, [ ]D 23 = -4.6 (c 2.16, DMF). 1H NMR (DMSOd6): 2.66 (dd, J = 16.3, 9.6 Hz, 1H), 2.84 (dd, J = 16.5, 4.2 Hz, 1H), 3.10 (dd, J = 15.0, 8.1 Hz, 1H), 3.20 (dd, J = 14.8, 4.8 Hz, 1H), 4.21-4.30 (m, 3H), 4.454.57 (m, 2H), 5.11 (s, 2H), 6.99 (t, J = 7.0 Hz, 1H), 7.07 (t, J = 7.1 Hz, 1H), 7.18 (s, 1H), 7.28-7.45 (m, 10H), 7.54 (d, J = 7.7 Hz, 1H), 7.70-7.77 (m, 3H), 7.89 (d, J = 7.6 Hz, 2H), 8.18 (d, J = 7.4 H, 1H), 10.91 (s, 1H), 12.75 (s, 1H). 13C NMR (DMSOd6): 26.9, 36.3, 46.6, 51.1, 53.1, 65.7, 65.8, 109.5, 111.4, 118.2, 118.5, 120.2, 121.0, 123.7, 125.4, 127.1, 127.2, 127.7, 127.9, 128.0, 128.4, 136.0, 136.1, 140.8, 143.9, 155.9, 170.1, 170.8, 173.2. Anal. calcd for C37H33N3O7: C, 70.35; H, 5.27; N, 6.65. Found: C, 70.02; H, 5.29; N, 6.65. ( S )-2-[( S )-4-Benzyloxycarbonyl-2-(9 H -fluoren-9ylmethoxycarbonylamino)butyrylamino]-4 -methylsulfanylbutyric acid (FmocL Glu(OBzl)L -Met-OH, 2.10c): White microcrystals (91%); mp 96-97 oC, [ ]D 23 = -7.6 (c 2.16,

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38 DMF). 1H NMR (DMSOd6): 1.87-2.10 (m, 7H), 2.47-2.53 (m, 4H), 4.10-4.46 (m, 5H), 5.13 (s, 2H), 7.30-7.46 (m, 9H), 7.66 (d, J = 8.1 Hz, 1H), 7.76 (t, J = 6.9 Hz, 1H), 7.89 (d, J = 7.4 Hz, 2H), 8.32 (d, J = 7.6 Hz, 1H), 12.76 (s, 1H). 13C NMR (DMSOd6): 14.6, 27.4, 29.7, 30.2, 30.7, 46.8, 51.1, 53.7, 65.6, 65.8, 120.2, 125.4, 127.1, 127.7, 128.0, 128.1, 128.5, 136.3, 140.8, 143.8, 144.0, 156.0, 171.6, 172.4, 173.3. Anal. calcd for C32H34N2O7S: C, 65.07; H, 5.80; N, 4.74. Found: C, 65.25; H, 5.80; N, 4.89. Benzyl ( S )-2-benzyloxycarbonylaminoN -[( S )-1-carboxy-2-(1 H -indol-3yl)ethylamino]-4-oxobutanoate (ZL -Asp-OBzlL -Trp-OH, 2.12a) : White microcrystals (92%); mp 98-100 oC, [ ]D 23 = -7.1 (c 2.08, DMF). 1H NMR (DMSOd6): 2.58-2.71 (m, 3H), 3.01 (dd, J = 18.1, 8.1 Hz, 1H), 3.14 (dd, J = 18.1, 4.6 Hz, 1H), 4.42-4.54 (m, 2H), 5.03 (s, 2H), 5.10 (s, 2H), 6.98 (t, J = 7.4 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 7.14 (s, 1H), 7.26-7.38 (m, 10H), 7.52 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H), 10.04 (s, 1H), 10.85 (br s, 1H). 13C NMR (DMSOd6): 27.2, 36.6, 50.7, 53.2, 65.6, 66.1, 109.7, 111.4, 118.2, 118.4, 120.9, 123.6, 127.2, 127.7, 127.8, 127.9, 128.0, 128.3, 128.4, 135.9, 136.1, 136.8, 155.8, 168.8, 171.4, 173.3. Anal. calcd for C30H29N3O7: C, 66.29; H, 5.38; N, 7.73. Found: C, 65.96; H, 5.36; N, 7.51. Benzyl ( S )-2-benzyloxycarbonylaminoN -(( S )-1-carboxy-2-pheny lethylamino)-4oxobutanoate (ZL -Asp-OBzlL -Phe-OH, 2.12b) : White microcrystals (91%); mp 138-140 oC, [ ]D 23 = +0.08 (c 2.08, DMF). 1H NMR (DMSOd6): 2.58-2.90 (m, 3H), 3.01 (dd, J = 15.4, 4.6 Hz, 1H), 4.43(quintet, J = 8.0 Hz, 2H), 5.03 (s, 2H), 5.11 (s, 2H), 7.16-7.34 (m, 15H), 7.60 (d, J = 8.2 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 12.78 (br s, 1H). 13C NMR (DMSOd6): 30.7, 36.8, 50.7, 53.7, 65.6, 66.1, 126.4, 127.7. 127.8, 127.9, 128.0, 128.2, 128.4, 128.5, 129.1, 135.9, 136.8,

PAGE 39

39 137.5, 155.9, 168.9, 171.4, 172.9. Anal. calcd for C28H28N2O7: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.67; H, 5.58; N, 5.48. Benzyl ( S )-2-benzyloxycarbonylaminoN -(1-carboxy-2-phenylethylamino)-4oxobutanoate (ZL -Asp-OBzlDL -Phe-OH, 2.12b+b') : White microcrystals (91%); mp 116118 oC, [ ]D 23 = -18.6 (c 2.08, DMF). 1H NMR (DMSOd6): 2.58-2.88 (m, 3H), 3.01 (dd, J = 17.4, 5.2 Hz, 1H), 4.47 (quintet, J = 6.9 Hz, 2H), 5.03 (d, J = 12.6 Hz, 1H, A part of AB system), 5.08 (d, J = 12.6 Hz, 1H, B part of AB system), 5.13 (s, 2H), 7.17-7.35 (m, 15H), 7.60 (d, J = 8.1 Hz, 0.5H), 7.85 (d, J = 8.2 Hz, 0.5H), 8.35 (d, J = 8.0 Hz, 1H), 12.78 (br s, 1H). 13C NMR (DMSOd6): 30.7, 35,9, 36.6, 36.9, 50.6, 53.7, 65.6, 66.1, 66.3, 126.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.4, 135.8, 135.9, 136.9, 137.5, 155.9, 156.0, 168.8, 171.1, 171.4, 171.5, 172.9. Anal. calcd for C28H28N2O7: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.31; H, 5.53; N, 5.58. 2.4.4 General Procedure for the Preparation of N-Protected-Dipeptidoylbenzotriazoles 2.13a-c and 2.13a+a' The preparation of 2.13a-c 2.13a+a' was performed at -15 0C under similar conditions as those described for 2.8 and 2.11 ( S )-Benzyl 5-(1 H -1,2,3-benzotriaz ol-1-yl)-4-(( S )-2(benzyloxycarbonylamino)propanamido)-5-oxopentanoate (ZL -AlaL -Glu(OBzl)-Bt, 2.13a): White microcrystals (93%); mp 133-135 oC, [ ]D 23 = -17.9 (c 2.08, DMF). 1H NMR (DMSOd6): 1.26 (d, J = 7.0 Hz, 3H), 1.83-2.42 (m, 2H), 2.47-2.52 (m, 1H), 2.68 (t, J = 7.4 Hz, 1H), 4.17 (dt, J = 19.6, 7.4 Hz, 1H), 4.98-5.12 (m, 4H), 5.66-5.72 (m, 1H), 7.28-7.42 (m, 10H), 7.50-7.57 (m, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.85 (d, J = 6.3 Hz, 1H). 13C NMR (DMSOd6): 17.9, 25.8, 29.7, 49.6, 52.0, 65.4, 65.6, 114.1, 120.2, 126.7, 127.8, 127.9, 128.0, 128.3, 128.4, 128.5, 130.7, 131.0,

PAGE 40

40 136.0, 137.0, 145.3, 155.7, 170.8, 171.9, 173.2. Anal. calcd for C29H29N5O6: C, 64.08; H, 5.38; N, 12.88. Found: C, 63.80; H, 5.38; N, 12.29. ( S )-Benzyl 5-(1 H -1,2,3-benzotriaz ol-1-yl)-4-(( S )-2-(benzyloxycarbonylamino)-3phenylpropanamido)-5-oxopentanoate (ZL -PheL -Glu(OBzl)-Bt, 2.13b): White microcrystals (92%); mp 90-92 oC, [ ]D 23 = -24.7 (c 1.91, DMF). 1H NMR (DMSOd6): 2.202.40 (m, 2H), 2.63-2.80 (m, 3H), 3.00-306 (m, 1H ), 4.30-4.44 (m, 1H), 4.94 (s, 2H), 5.05 (d, J = 14.6 Hz, 1H, A part of AB system), 5.10 (d, J = 14.9 Hz, 1H, B part of AB system), 5.67-5.74 (m, 1H), 7.17-7.40 (m, 15H), 7.54-7.67 (m, 2H), 7.82 (t, J = 7.5 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 9.00 (d, J = 6.3 Hz, 1H). 13C NMR (DMSOd6): 25.9, 29.8, 37.3, 52.1, 55.8, 65.3, 65.7, 114.1, 120.2, 126.3, 126.7, 127.5, 127.7, 128.0, 128.1, 128.3, 128.4, 129.2, 130.7, 131.1, 136.0, 136.9, 137.9, 145.4, 155.9, 170.8, 172.0, 172.4, 173.1. Anal. calcd for C35H33N5O6: C, 67.84; H, 5.37; N, 11.30. Found: C, 67.72; H, 5.43; N, 11.09. ( S )-Benzyl 4-(1 H -1,2,3-benzotriaz ol-1-yl)-3-(( S )-2-(benzyloxycarbonylamino)-3phenylpropanamido)-4-oxobutanoate (ZL -PheL -Asp(OBzl)-Bt, 2.13c): White microcrystals (89%); mp 110-113 oC, [ ]D 23 = -20.7 (c 2.75, DMF). 1H NMR (DMSOd6): 2.71-2.84 (m, 1H), 2.97-3.10 (m, 2H), 3.34 (dd, J = 17.0, 5.9 Hz, 1H), 4.32-4.40 (m, 1H), 4.90 (d, J = 6.7 Hz, 1H, A part of AB system), 5.10 (d, J = 10.6 Hz, 1H, B part of AB system), 5.15 (s, 2H), 6.03 (dd, J = 13.5, 6.6 Hz, 1H), 7.14-7.39 (m, 15H), 7.56-7.67 (m, 2H), 7.82 (t, J = 8.1 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 9.17 (d, J = 6.6 Hz, 1H). 13C NMR (DMSOd6): 35.3, 37.4, 49.6, 55.9, 65.3, 66.3, 114.0, 120.3, 126.3, 126.8, 127.4, 127.5, 127.7, 128.0, 128.1, 128.2, 128.3, 128.4, 129.2, 130.7, 131.1, 135.6, 136.0, 137.0, 137.9, 145.4, 155.9, 169.5, 169.7, 172.1. Anal. calcd for C34H31N5O6: C, 67.43; H, 5.16; N, 11.56. Found: C, 67.21; H, 5.16; N, 11.53.

PAGE 41

41 Benzyl (S)-5-Benzotriazol-1-yl-4-(2benzyloxycarbonylaminopropanamido)-5oxopentanoate (ZDL -AlaL -Glu(OBzl)-Bt, 2.13a+a'): White microcrystals (91%); mp 79-81 oC, [ ]D 23 = -22.4 (c 1.66, DMF). 1H NMR (CDCl3): 1.40 (d, J = 6.9 Hz, 3H), 2.27-2.34 (m, 1H), 2.47-2.68 (m, 3 H), 4.30-4.40 (m, 1H), 5.07 (s, 2H), 5.12 (s, 2H), 5.31 (d, J = 6.6 Hz, 1H) 5.93 (br s, 1H), 7.25-7.42 (m, 11H), 7.52 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 6.9 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.19-8.24 (m, 1H). 13C NMR (CDCl3): 18.4, 18.6, 26.7, 27.0, 30.2, 30.3, 50.3, 51.6, 52.7, 66.5, 66.8, 67.1, 114.3, 120.3, 125.8, 126.5, 127.9, 128.0, 128.1, 128.2, 128.3, 128.5, 128.6, 128.7, 130.7, 131.0, 135.3, 135.5, 136.0, 145.9, 156.0, 156.1, 170.3, 170.4, 172.8, 173.6, 173.7. Anal. calcd for C29H29N5O6: C, 64.08; H, 5.38; N, 12.88. Found: C, 64.37; H, 5.28; N, 12.49. 2.4.5 General Procedure for the Preparation of Tripeptides 2.14a,b, 2.14a' and 2.14a+a'' Unprotected amino acids ( 2.9c,e and f ; 1 mmol) were dissolved in a mixture of acetonitrile (10 mL), water (5 mL), and triethylamine (2.5 mmol). N-protected-dipepti doylbenzotriazoles 2.13a,b, 2.13a+a' (1 mmol) were added to the reacti on mixture at -15C and the stirring continued for additional 2 hours. Resulting solu tion was acidified with (1 mL) 4N HCl and acetonitrile was removed under reduced pressure at room temperature. The residue was dissolved in EtOAc (50 mL) and was then washed 3 times with 4N HCl (3 mL) followed by saturated NaCl (20 mL). The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure yi elding the tripeptides 2.14a,b 2.14a' and 2.14a+a'' Further purification was performed by recrystallization from CH2Cl2-hexanes for elemental analysis. Benzyl (S)-4-((S)-2-benzyloxycarbonyla minopropanamido)-4-((S)-1-carboxy-2phenylethylamino)-5 -oxopentanoate (ZL -Ala-Glu(OBzl)L -Phe-OH, 2.14a): White microcrystals (73%); mp 74-76 oC, [ ]D 23 = -2.9 (c 1.91, DMF.). 1H NMR (DMSOd6): 1.15 (d, J = 6.3 Hz, 3H), 1.70-2.00 (m, 2H), 2.32-2.45 (m, 2H), 2.86-2.93 (m, 1H), 3.05 (dd, J = 13.4, 4.2

PAGE 42

42 Hz, 1H), 4.00-4.10 (m, 1H), 4.25-4.45 (m, 2H), 4.94-5.08 (m, 4H), 7.06-7.35 (m, 15H), 7.48 (d, J = 7.7 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 8.17 (d, J = 7.7 Hz, 1H), 12.75 (br s, 1H). 13C NMR (DMSOd6): 18.1, 26.3, 30.1, 36.5, 49.8, 51.0, 53.5, 65.4, 65.5, 126.5, 127.8, 127.9, 128.0, 128.2, 128.3, 128.4, 128.5, 129.1, 136.2, 137.0, 137.4, 155.7, 172.2, 172.7, 172.8, 173.1. Anal. calcd for C32H35N3O8: C, 65.18; H, 5.98; N, 7.13. Found: C, 64.86; H, 6.03; N, 7.18. Benzyl ( S )-4-(( S )-2-benzyloxycarbonylaminopropanamido)-4-(( R )-1-carboxy-2phenylethylamino)-5 -oxopentanoate (ZL -AlaL -Glu(OBzl)D -Phe-OH, 2.14a'): White microcrystals (83%); mp 153-155 oC, [ ]D 23 = +5.5 (c 1.91, DMF.). 1H NMR (DMSOd6): 1.15 (d, J = 6.6 Hz, 3H), 1.59-1.65 (m, 1H), 1.70-1.78 (m, 1H), 1.93-2.18 (m, 2H), 2.29-2.34 (m, 1H), 2.81 (dd, J = 13.3, 10.5 Hz, 1H) 4.04 (quintet, J = 7.4 Hz, 1H), 4.28-4.35 (m, 1H), 4.43-4.50 (m, 1H), 4.96 (d, J = 13.3 Hz, 1H, A part of AB system), 5.01 (d, J = 13.3 Hz, 1H, B part of AB system), 5.06 (s, 2H), 7.06-7.08 (m, 1H), 7.13-7.36 (m, 14H), 7.49 (d, J = 7.0 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 12.81 (br s, 1H). 13C NMR (DMSOd6): 18.0, 27.6, 29.4, 36.9, 50.1, 51.2, 53.3, 65.4, 65.5, 126.4, 127.7, 127.8, 127.9, 128.0, 128.1, 128.4, 128.5, 129.1, 136.2, 137.0, 137.4, 155.7, 170.5, 172.1, 172.3, 172.8. Anal. calcd for C32H35N3O8: C, 65.18; H, 5.98; N, 7.13. Found: C, 64.88; H, 6.11; N, 7.04. Benzyl ( S )-4-(( S )-2-benzyloxycarbonylamino-3 -phenylpropanamido)-4-(( S )-1carboxyethylamino)-5-oxopentanoate (ZL -PheL -Glu(OBzl)L -Ala-OH, 2.14b): White microcrystals (95%); mp 163-165 oC, [ ]D 23 = -13.0 (c 2.33, DMF). 1H NMR (DMSOd6): 1.15 (d, J = 7.0 Hz, 3H), 1.79-2.00 (m, 2H), 2.40-2.48 (m, 2H), 2.67-2.75 (m, 1H), 2.98 (d, J = 12.0 Hz, 1H), 4.91 (s, 2H), 5.09 (s, 2H), 7.06-7.35 (m, 15H), 7.52 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 8.28 (d, J = 6.3 Hz, 1H), 12.51 (br s, 1H). 13C NMR (DMSOd6): 17.0, 27.6, 29.8, 37.3, 47.6, 51.4, 56.1, 65.2, 65.5, 126.3, 127.5, 127.7, 127.9, 128.1, 128.3, 128.4, 128.5, 129.2,

PAGE 43

43 136.2, 137.0, 138.1, 155.9, 170.6, 171.5, 172.4, 174.1. Anal. calcd for C32H35N3O8: C, 65.18; H, 5.78; N, 7.30. Found: C, 64.88; H, 6.26; N, 7.04. Benzyl ( S )-4-(2-benzyloxycarbonylaminopropanamido)-4-(( S )-1-carboxy-2phenylethylamino)-4 -oxopentanoate (ZDL -AlaL -Glu(OBzl)L -Phe-OH, 2.14a+a''): White microcrystals (92%); mp 123-125 oC, [ ]D 23 = -5.5 (c 1.66, DMF). 1H NMR (DMSOd6): 1.17 (m, 3H), 1.61-2.10 (m, 2H), 2.29-2.45 (m, 2H), 2.82-2.95 (m, 1H), 3.04-3.11 (m, 1H), 4.06 (quintet, J = 6.9 Hz, 1H), 4.20-4.31 (m, 1H), 4.39-4.42 (m, 1H), 4.94-5.07 (m, 4H), 7.06-7.51 (m, 15H), 7.45 (d, J = 7.1 Hz, 0.5H), 7.50 (d, J = 6.3 Hz, 0.5H) 7.86-8.01 (m, 1H), 8.13-8.32 (m, 1H), 12.75 (br s, 1H). 13C NMR (DMSOd6): 18.2, 18.3, 18.7, 26.4, 26.5, 27.6, 27.7, 29.9, 30.0. 30.1, 36.7, 50.0, 50.1, 50.3, 51.1, 51.4, 51.5, 53.6, 53.7, 65.5, 65.6, 126.6, 127.9, 128.1, 128.2, 128.2, 128.3, 128.5, 128.6, 129.2, 136.3, 137.1, 137.2, 137.5, 137.6, 155.8, 155.9, 171.0, 171.1, 172.2, 172.3, 172.4, 172.5, 172.6, 172.8, 172.9, 173.1, 173.2. Anal. calcd for C32H35N3O8: C, 65.18; H, 5.98; N, 7.13. Found: C, 65.23; H, 6.14; N, 7.22. 2.4.6 General Procedure for the Prep aration of Tripeptides 16 a,b. N -Protected dipeptides 2.10a,b were treated with piperidine at room temperature for 2 hours to deprotect Fmoc group according to the reported procedure65 to provide the free dipeptides 2.15a,b. Free dipeptides (without significant purification) (1 mmol) were dissolved in a mixture of acetonitrile (10 mL), water (5 mL), and triethylamine (2.5 mmol). N -(Protectedaminoacyl)benzotriazoles 2.1a b (1 mmol) were added to the reaction mixture at -15C and the stirring continued for additional 2 hours. Result ing solution was acidified with (1 mL) 4N HCl and acetonitrile was evaporated under reduced pr essure at room temperature. The residue was dissolved in EtOAc (50 mL) and was then wash ed 3 times with 4N HCl (3 mL) followed by saturated NaCl (20 mL). The organic layer wa s dried over magnesium sulfate and the solvent

PAGE 44

44 was removed under reduced pressure affording the tripeptides 2.16a,b Further purification was performed by recrystallization from et her-hexanes for elemental analysis. Benzyl ( S )-4-(( S )-2-benzyloxycarbonylamino-3 -phenylpropanamido)-4-[( S )-1carboxy-2-(1 H -indol-3-yl)ethylamino]-5-oxopentanoate (ZL -PheL -Glu(OBzl)L -Trp-OH, 2.16a): White microcrystals (84%); mp 109-111 oC, [ ]D 23 = -3.2 (c 1.66, DMF). 1H NMR (DMSOd6): 1.80-2.10 (m, 2H), 2.44 (t, J = 8.1 Hz, 2H), 2.66-2.83 (m, 2H), 2.95-3.12 (m, 2H), 3.21 (dd, J = 14.7, 4.9 Hz, 1H), 4.30-4.51 (m, 3H), 4.93 (s, 2H), 5.10 (s, 2H), 6.96-7.10 (m, 2H), 6.99 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 6.9 Hz, 1H), 7.16-7.36 (m, 15H), 7.54 (t, J = 7.2 Hz, 2H), 8.17 (d, J = 7.7 Hz, 1H), 8.28 (d, J = 7.1 Hz, 1H), 10.89 (br s, 1H). 13C NMR (DMSOd6): 26.9, 27.7, 29.9, 37.3, 51.6, 53.1, 56.1, 65.3, 65.6, 109.6, 111.4, 118.2, 118.5, 121.0, 123.7, 126.3, 127.3, 127.5, 127.6, 127.7 128.0, 128.1, 128.3, 128.5, 129.3, 136.1, 136.3, 137.0, 138.1, 155.9, 171.0, 171.5, 172.4, 173.3. Anal. calcd for C40H40N4O8: C, 68.17; H, 5.72; N, 7.95. Found: C, 67.84; H, 5.79; N, 7.68. Benzyl ( S )-3-(( S )-2-benzyloxycarbonylaminopropionylamino)N -[( S )-1-carboxy-2(1 H -indol-3-yl)ethyl]butanoate (ZL -AlaL -Asp(OBzl)L -Trp-OH, 2.16b): White microcrystals (83%); mp 134-137 oC, [ ]D 23 = -1.7 (c 1.66, DMF). 1H NMR (DMSOd6): 1.15 (d, J = 7.1 Hz, 3H), 2.60-2.71 (m, 1H), 2.82 (dd, J = 14.7, 5.2 Hz, 1H), 3.07-3.14 (m, 2H), 4.03 (quintet, J = 7.1 Hz, 1H), 4.45 (q, J = 6.3 Hz, 1H), 4.70 (q, J = 6.3 Hz, 1H), 4.92-5.10 (m, 4H), 6.97 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.15 (apparent s, 1H), 7.31-7.35 (m, 11H), 7.487.54 (m, 2H), 7.94 (d, J = 7.4 Hz, 1H), 8.26 (d, J = 7.0 Hz, 1H), 10.85 (s, 1H), 12.85 (br s, 1H). 13C NMR (DMSOd6): 18.2, 27.1, 36.3, 49.4, 50.3, 53.2, 65.6, 65.9, 109.6, 111.5, 118.3, 118.6, 121.1, 123.9, 127.7, 127.9, 128.0, 128.1, 128.2, 128.5, 128.6, 136.1, 136.2, 137.1, 155.9, 170.2,

PAGE 45

45 170.3, 172.7, 173.1. Anal. calcd for C33H34N4O8: C, 64.48; H, 5.58; N, 9.12. Found: C, 64.13; H, 5.70; N, 8.78.

PAGE 46

46 CHAPTER 3 EFFICIENT LABELING OF SUGARS TO PROVIDE WATER SOLU BLE FLUORESCENT TAGS 3.1 Introduction Carbohydrate moieties have pivotal roles in num erous biological processes including cellcell communication,66-68 cell adhesion,69,70 fertilization, protein folding, and microbial infections.72-76 Glycosylation is the process of adding saccharides to proteins and lipids. Over 50% of all protein sequences in eukaryotic systems sequences are glycosylated.77 Glycosylated lipids constitute up to 5 % of the membrane c ontent in animal cells, and are involved in a wide array of pathological disorders.78 Oligosaccharides are present in the form of glycoconjugates (glycoproteins and glycolipids) in all cell walls me diating a variety of events su ch as inflammation, immunological response, and metastasis. Separation of gl ycoproteins has been achieved with modern chromatographic and electrophoret ic methodologies. However, gl ycoproteins, once isolated are difficult to study structurally because glycoprotei ns usually destroy when analyzed with X-ray crystallography.79 Also, the amount of glycoproteins obt ained from biological materials is too small to study their structures. Therefore, glycoproteins are of ten cleaved to smaller fragments such as their glycopeptides or gl ycans, which are easier to analyze.80 Glycopeptides and glycans are frequently labeled to in crease detection sensitivity.81 Fluorescent tagging with organic fluorophores82 or green fluorescent protein is the visualization tool most commonly used for anal yzing carbohydrate struct ures in biological systems.83 Highly sensitive fluorescence derivatizati on techniques are gain ing an increasing share of the analytical world market, becoming competitive with, for example, radioimmunoassay. Derivatives of rhodamin, fluorescein, and coumarin are widely used as fluorescent markers for peptides and other biomolecules.84,85 Derivates of coumarins

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47 (benzopyranones), the largest cl ass of laser dyes for the "b lue-green" region, are highly sensitive.86-94 They have provided the most commerci ally accepted categories of fluorescent derivatives with the advantages of an exte nded spectral range, high emission quantum yield, photostability, and good solubility in many solvents. Primary amine-containing fluorescence-tags can be introduced by reductive amination at the reducing end of sugar chains. The saccharide reacts with 3.2 containing 2-aminopyridine and sodium cyanoborohydrate, or sodium borohydride to give glycamine 3.3 (Figure 3-1).76,96-100 However, using these methods cleave the cyclic structure at the reduc ing end changing the properties of the sugar moieties. Figure 3-1. Fluorescent labeling of s accharides by reductive amination Classical labeling procedures applied to proteins, based on acylation of -NH2 groups are not useful for many sugars because of the abse nce of the free amino groups in the saccharide structure. However, monoand disaccharides with amino groups have been labeled with fluorescent mass tags as an alternative method for measuring a special class of enzymes that are responsible for the synthesis of car bohydrates (glycosyltransferases).101-103 The coupling of 7hydroxycoumarin-3-carboxylic acid, a fluorescent tag, to glycosylamine has been performed with HBTU/HOBT/DIEA in DMF.101 Also, amino-rich polysaccharides have been labeled with the fluorescein derivative 5-([4, 6-dichloro triazine-2-yl] amino )-fluorescein (DTAF).102,103 Fluorescence detection depends on the physical characteristics of the dyes employed. Many dyes with high extinction coef ficients and high quantum yields are of limited utility due to O RO RO NHCOCH3OR OH OH RO RO NHCOCH3OR NH NH2NaBH3CNFluorescent tag+3.1 3.2 3.3 FT FT

PAGE 48

48 poor photostability and crucially poor aqueous solubi lity. Solubility char acteristics affects the degree of self interaction in solution of chrom ophores conjugated to s ubstrates and therefore light absorption and emission properties.104 Bright fluorescent reagents, with good aqueous solubility and low non-specific st aining, are needed. Incorporati ng sugar units to fluorescent reagents confers useful water solubility to organic fluorophores without significant change in absorption and fluorescent properties.105,106 Researchers in the Katritzky group have recently uncovered N -(coumarin-3-carbonyl) benzotriazole 3.4 ,12 as a useful starting material, for c onvenient and reliable fluorescent labeling of amino acids and dipeptides. Such coumarin-labeled lysines, including N-coumarin-labeled N-protectedL -lysines 3.5 3.6 are of considerable interest for the design and synthesis of fluorogenic substrates to analyze matrix metalloproteinases (MMP).107-110 Their successful labeling of amino acids and peptides in solution utilized 3.7 and a benzotriazole activated 3 5 (Figure 3-2).12 Figure 3-2. Structures of N -(coumarin-3-carbonyl ) benzotriazole and N-coumarin-labeled NprotectedL -lysines They also recently reported efficient O -acylation of diacetonide protected sugars with readily available N -(Z-aminoacyl)benzotriazoles under microwave irradiation.97 We now

PAGE 49

49 present the convenient and e fficient fluorescent labeling by O -acylation, of diisopropylidene protected sugars 3.9-3.11, and N -acylation of pivaloyl protected aminosugar 3.15 with (i) N (coumarin-3-carbonyl)benzotriazole 3.4 and (ii) the benzotriazole derivatives 3.7, 3.8 of Ncoumarin-labeled N-protectedL -lysines 3.5, 3.6 under microwave irradiation or at room temperature Monosaccharide containing Fmoc-lysine fluor escent building blocks can be useful as water soluble organic fluorophor es for peptide labeling at the C -terminus in solid-phase peptide synthesis (SPPS). 3.2 Results and Discussion 3.2.1 Preparation of N-Coumarin-Labeled N-FmocL -lysine Benzotriazolide 3.8. N-Coumarin-3-carbonylN-FmocL -lysine benzotriazole 3.8 (Figure 3-3) was prepared (87%) from coumarin-labeled N -Fmoc-protected lysine 3.6 utilizing benzotriazole methodology optimized in our laboratories, by reacting 1 H -benzotriazole with thionyl chloride in CH2Cl2 at 20C for 2 hours.10,11,49,61-63 3.2.2 Preparation of Coumarin-O-Tagged Monosaccharides : O-(Coumarin-3carbonyl)diisopropylidene Sugars 3.12, 3.13, 3.14. O -Coumarin labeled diisopropylidene sugars 3.12, 3.13, 3.14 were prepared by coupling of 3.4 with the 6-OH of 1,2:3,4-DiO -isopropylidene-D-galactopyranose 3.9, 3-OH of 1,2:5,6-DiO -isopropylidene-D-glucose 3.10, and 1-OH of 2,3:5,6-DiO -isopropylidene-Dmannofuranose 3.11 respectively in dichloromethane, utilizing 1equivalent of 4Dimethylaminopyridine (DMAP) under 100W microwave irradiation at 50C for 45 min (Figure 3-3). Products were isolated after simple acid work up without chromatography in 60-90% yields.

PAGE 50

50 3.2.3. Preparation of CoumarinN -Tagged Monosaccharide: N -(Coumarin-3carbonyl)tetrapivaloyl Sugar 3.16. 2,3,4,6-tetraO -pivaloyl-D-galactopyranosylamine 3.15 was coupled with N -(coumarin3-carbonyl) benzotriazole 3.4 in dry dichloromethane in the pres ence of 1 equivalent of DMAP in 24 hours at 20C. Afte r silica-gel column chromatography us ing ethyl acetate/hexane (1:3) as eluent, product 3.16 was isolated in 60% yield. O PivO PivO OPiv NH2OPiv O O O O O OH O O O O O O O O O OO O Bt PivO OPiv H N OPiv O O O PivO O O O O O O O O O O O O O OH O O 3.10 3.133 1 63.4 3.14 3.9 3.12 3.11 3.15 OH O O O O O O O O O O O O O O Figure 3-3. Syntheses of O -(coumarin-3-carbonyl)diisopropylidene sugars 3.12, 3.13, 3.14 and N (coumarin-3-carbonyl)te trapivaloyl sugar 3.16 3.2.4 Preparation of O and N -( N-Coumarin-3-CarbonylN(Fmoc or ZL -lys)protected Sugars 3.17a,b, 3.18, 3.19, 3.20. L -Lysine scaffold based coumarin labeled sugars 3.17a,b, 3.18, 3.19, 3.20 were synthesized by O -acylation of the free -OH groups pres ent in diacetonide protected sugars 3.9, 3.10 3.11 and N -acylation of the amino group of 3.15 by N-coumarin-3-carbonylN-Z or FmocL -lysine benzotriazole 3.7 3.8 (Figure 3-4). Coupling reactions were carried out in dry DC M, in the presence of 1 eq. of DMAP, at room temperature for 18-24 hrs. Under microwave irradiation at 60 C, the preparation of

PAGE 51

51 compounds 3.17a,b 3.18, 3.19 needed 45 minutes. After washing with 4N HCl, products 3.17a,b 3.18 were obtained without chromatography in 85-89% yields. The crude products were estimated to be >95% pure. Compounds 3.19 and 3.20 were isolated using column chromatography in 74% and 40% yields respectively. O OO O O O O NH O O O HN Fmoc O O O HN O N H Pg O O O O O O O O O O O O O O O O NH Fmoc NH PivO OPiv H N OPiv O OPiv Pg = Cbz 3.7, 3.17a Pg = Fmoc 3.8, 3.17b3 2 03.7, 3.8 3.9 3.10 3.11 3.17a, 3.17b 3.18 3.19 3.15 O NH O O O HN Fmoc Figure 3-4. Preparation of O and N -( N-coumarin-3-carbonylN(Fmoc or ZL -lys)protected sugars 3.17a,b, 3.18, 3.19, 3.20 3.2.5 Deprotection of the Diisopropylidene Groups of O -(Coumarin Labeled)diisopropylidene Protected Sugars 3.13, 3.17b and 3.18. Deprotection of diacetonide groups of 3.13 3.17b and 3.18 were performed by TFA/H2O (9:1, v/v; 5mL) mixture at 20 C for 3-5 minutes. The unprotected coumarin-sugar conjugate 3.21 (Figure 3-5) and coumarinL -lysine-free sugar conjugates 3.22 and 3.23 (Figures 3-6, 3-7) were obtained in quantitative yields and characterized by 1H NMR and 13C NMR spectroscopy, elemental analysis, melting point, Mass Spec. and ORP.

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52 O O O O O O O O O 3.13 TFA-H2O 9:1,5mL O HO O OH OH OH O O O 3.21 Figure 3-5. Deprotection of diacetonide groups for compound 3.13 O O O HN O N H Fmoc O O O O O O 3.17bO O O HN O N H Fmoc O HO HO OH O 3.22TFA-H2O 9:1,5mL OH Figure 3-6. Deprotection of diacetonide groups for 3.17b O O O O O O O O O O NH Fmoc NH 3.18O HO O OH OH OH 3.23 O O O H N Fmoc NH O TFA-H2O 9:1,5mL Figure 3-7. Deprotection of diacetonide groups for 3.18 3.3 Conclusion In conclusion, we have demonstr ated a convenient and efficient O -fluorescence labeling of diisopropylidene protected sugars 3 12-3.14 and N -labeling of pivaloyl protected aminosugar 3.15 in yields of 55-87%. Fmoc and Z-protected L -Lysine scaffold based coumarin labeled

PAGE 53

53 protected sugars 3 17a,b, 3.18, 3.19, and 3.20 were obtained from 3.7 and 3.8. Deprotection of diisopropylidene groups from 3.13, 3.17b and 3 18 provided water soluble conjugates 3.21-3.23 in quantitative yields. Fluorescent building blocks 3 17a,b, and 3 18 can be considered to be useful markers for labeling C -terminus of peptides in solid phase peptide synthesis (SPPS) and after deprotection of diisopropylidene groups, the fr ee sugar will provide the water solubility of organic fluorophores for coumar in labeled protein molecules. 3.4 Experimental Section Melting points were determined on a capilla ry point apparatus e quipped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSOd6 with TMS for 1H (300 MHz) and 13C (75 MHz) as an internal reference. Coum arin-3 carboxylic acid was purchased from Acros. Sugars and N -Fmoc-amino acids were purchased from Fluka, Acros and Aldrich and were used without further purificati on. Most of the reactions were carried out under microwave irradiation with a sing le mode cavity Discover Microwave Synthesizer (CEM Corporation, NC) producing a continuous irradiati on at 2450 MHz. Elemental analyses were performed on a Carlo Erba-1106 instrument. Optical rota tion values were measured with the use of sodium D line. Column chromatography was performed on sili ca gel (200-425 mesh). HPLC analyses were performed on Beckman system gold programma ble solvent module 126 using Chirobiotic T column (4.6 x 250 mm), detection at 254 nm, flow rate 1.0 mL/min, and methanol as solvent. 3.4.1 General Procedure for the Preparation of Compound 3.4. Thionyl chloride (7.5 mmol) was added to a solution of 1 H -benzotriazole (25 mmol) in dry CH2Cl2 or THF (30 mL) at room temperature, an d the reaction mixture was stirred for 20 min. To the reaction mixture, coumarin-3-carboxylic ac id (5 mmol) was added and stirred for 4 h at 25 C. The white precipitate formed during the reac tion was filtered off, and the filtrate was concentrated under reduced pre ssure. The residue was diluted with EtOAc (150 mL) and the

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54 solution was washed with sat. Na2CO3 soln. (3 50mL), sat. NaCl soln. (50mL), and dried over MgSO4. Removal of the solvent under reduced pr essure gave 3-(Benzotriazole-1-carbonyl)chromen-2-one 3.4 which was recrystallized from CH2Cl2-hexanes for elemental analysis. 3-(Benzotriazole-1-carbonyl) chromen-2-one (Coum-Bt, 3.4) : White microcrystals (87%); mp 186 187 C, 1H NMR (CDCl3): 7.41 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.58 (t, J = 8.2 Hz, 1H), 7.64 7.80 (m, 3H), 8.16 (d, J = 8.2 Hz, 1H), 8.34 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3): 114.4, 117.2, 117.6, 120.5, 121.9, 125.3, 126.8, 129.6, 130.9, 131.2, 134.5, 146.2, 147.0, 154.9, 157.4, 162.6. Anal. Calcd for C16H9N3O3: C, 65.98; H, 3.11; N, 14.43. Found: C, 65.67; H, 3.10; N, 14.22. 3.4.2 General Procedure for the P reparation of Compounds 3.5 and 3.6. 3-(Benzotriazole-1-carbonyl)chromen-2-one 3.4 (1 mmol) was added to a solution of 1 mmol of N-Fmocor ZL -lysine in MeCN H2O (10mL/5mL) mixture, in the presence of Et3N (1 mmol). The reaction mixture was stirred at 20 C for about 1h (until TLC shows absence of 3.4 ). Aqueous 4N HCl (1mL) was then added an d MeCN was removed under reduced pressure. The residue obtained was dissolved in EtOAc ( 150 mL), and washed with 4N HCl soln. (3 50 mL), sat. NaCl soln. (50 mL) and dried over MgSO4. After evaporation of solvent, the residue was recrystallized from EtOAc-hexanes or CH2Cl2-hexanes. ( S )-2-Benzyloxycarbonylamino-6-[(2-oxo-2 H -chromene-3-carbonyl)amino]hexa noic acid ( N-ZN-CoumoylL -Lys-OH, 3.5): White microcrystals (89%); mp 144 145 C, [ ]23 D = 8.54 (c 1.68, DMF). 1H NMR (CDCl3): 1.37 1.56 (m, 2H), 1.58 1.74 (m, 2H), 1.75 2.40 (m, 2H), 3.33 3.58 (m, 2H), 4.34 4.45 (m, 1H), 5.09 (s, 2H), 5.75 (d, J = 8.0 Hz, 1H), 7.27 7.42 (m, 7H), 7.52 7.72 (m, 2H), 8.92 (s, 1H), 8.95 9.04 (m, 1H). 13C NMR (CDCl3): 22.3, 28.9, 31.5, 39.3, 53.6, 66.9, 116.5, 117.9, 118.5, 125.3, 128.0, 128.1, 128.4, 130.0, 134.1, 136.2, 148.8,

PAGE 55

55 154.3, 156.3, 161.3, 162.1, 175.3. Anal. Calcd for C24H24N2O7: C, 63.71; H, 5.35; N, 6.19. Found: C, 63.82; H, 5.09; N, 6.04. ( S )-2-(9 H -Fluoren-9-ylmethoxycar bonylamino)-6-[(2-oxo-2 H -chromene-3-carbo nyl)amino]hexanoic acid ( N-FmocN-CoumoylL -Lys-OH, 3.6): White microcrystals (87%); mp 110.0 111.0 C, [ ]23 D = 1.62 (c 1.85, DMF), 1H NMR (DMSO-d6): 1.32 1.50 (m, 2H), 1.50 1.62 (m, 2H), 1.62 1.85 (m, 2H), 3.26 3.38 (m, 2H), 3.92 4.01(m, 1H), 4.17 4.36 (m, 3H), 7.22 7.54 (m, 6H), 7.60 7.80 (m, 4H), 7.87 (d, J = 7.4 Hz, 2H), 7.96 (d, J = 7.4 Hz, 1H), 8.73 (t, J = 5.5 Hz, 1H), 8.84 (s, 1H). 12.62 (s, 1H). 13C NMR (DMSOd6): 23.2, 28.6, 30.5, 46.7, 53.8, 65.6, 116.1, 118.5, 119.0, 120.1, 125.1, 125.3, 127.1, 127.7, 130.2, 134.0, 140.7, 143.8, 147.3, 153.8, 156.2, 160.4, 161.0, 174.0. Anal. Calcd for C31H28N2O7: C, 68.88; H, 5.22; N, 5.18. Found: C, 68.59; H, 5.11; N, 5.16. 3.4.3 General Procedure for the P reparation of Compound 3.7 and 3.8. Thionyl chloride (1.2 mmol) was added to a solution of 1H-Benzotr iazole (5 mmol) in anhydrous CH2Cl2 (15 mL) at room temperature, and the reaction mixture was stirred for 20 min. Either compound 3.7 or 3.8 (1 mmol) was added to the reaction mixture and stirred for 2 hours at room temperature. The white precipitate formed during the reaction was filtered off, the filtrate was diluted with additional CH2Cl2 (80 mL) and the solution was washed with sat. Na2CO3 soln (3x50mL), sat. NaCl soln (50mL), and drie d over MgSO4. Removing solvent under reduced pressure gave product in 79% or 85 % yields, which was recrystallized from CH2Cl2-hexanes for elemental analysis. {( S )-1-(Benzotriazole-1-carbonyl)-5-[(2-oxo-2 H -chromene-3-carbonyl)-amino]pentyl}-carbamic acid benzyl ester ( N-ZN-CoumoylL -Lys-Bt, 3.7) : White microcrystals (79%); mp 156 157C, 1H NMR (CDCl3): 1.50 1.80 (m, 4H), 1.96 2,12 (m, 1H), 2.13 2.28

PAGE 56

56 (m, 1H), 3.36 3.48 (m, 1H), 3.48 3.64 (m, 1H), 5.13 (s, 2H), 5.69 5.83 (m, 1H), 6.12 (d, J = 7.8 Hz, 1H), 7.26 7.47 (m, 7H), 7.52 (t, J = 7.6 Hz, 1H), 7.60 7.74 (m, 2H), 8.13 (d, J = 8.2 Hz, 1H), 8.27 (d, J = 8.2 Hz, 1H), 8.86 (s, 1H), 8.82 8.97 (m, 1H). 13C NMR (CDCl3): 22.3, 28.9, 31.6, 38.5, 54.6, 67.1, 114.4, 116.5, 118.2, 118.6, 120.3, 125.2, 126.4, 128.0, 128.1, 128.5, 129.8, 130.6, 131.1, 134.0, 136.2, 145.9, 148.6, 154.3, 156.2, 161.4, 162.0, 171.7. Anal. Calcd for C30H27N5O6: C, 65.09; H, 4.92; N, 12.65. Found: C, 64.91; H, 4.76; N, 12.59. {(S)-1-(Benzotriazole-1-carbonyl)-5-[(2-oxo-2H-chromene-3-carbonyl)-amino]pentyl}-carbamic acid 9H-flu oren-9-ylmethyl ester acid ( N-FmocN-CoumoylL -Lys-Bt, 3.8): White microcrystals from CH2Cl2-hexanes (85%); mp 113.0 115.0 oC. 1H NMR (CDCl3): 1.40 1.90 (m, 4H), 1.95 2.15 (m, 1H), 2.15 2.23 (m, 1H), 3.40 3.68 (m, 2H), 4.20 4.35 (m, 2H), 4.36 4.48 (m, 1H), 6.20 (d, J = 7.7 Hz, 1H), 7.20 7.45 (m, 7H), 7.50 7.80 (m, 7H), 8.13 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.20 8.97 (m, 2H). 13C NMR (CDCl3): 22.4, 28.9, 31.6, 38.5, 47.1, 54.6, 67.1, 114.4, 116.4, 118.0, 118.5, 119.9, 120.2, 125.2, 126.4, 127.0, 127.6, 129.7, 130.6, 131.1, 134.0, 141.2, 143.6, 143.9, 146.0, 148.6, 154.3, 156.2, 161.4, 162.1, 171.7. Anal. calcd for C37H31N5O6: C, 69.26; H, 6.87; N, 10.91. Found: C, 69.01; H, 4.76; N, 11.03. 3.4.4 General Procedure for the Preparation of O -(Coumarin)diacetonide Sugars 3.12, 3.13, 3.14 Under Microwave Irradiation. A dried heavy walled Pyrex t ube containing a small stir bar was charged with 3(Benzotriazole-1-carbonyl)chromen-2-one 3.4 (1.0 mmol), sugars 3.9 3.10 3.11 (1 mmol), DMAP (1 mmol), and CH2Cl2 (1 mL). The reaction mixture was exposed to microwave irradiation (100 W) fo r 45 minutes to obtain 3.12, 3.13 and 3.14 at a temperature of 60 C. After the irradiation, the reaction mixture was allowe d to cool through an inbuilt system in the instrument until the temperature had fallen below 30 C (ca. 10 min). To the reaction mixture 20

PAGE 57

57 mL of CH2Cl2 was added, washed with 4N HCl soln (3 15 mL), sat. NaCl soln (10 mL) and dried over MgSO4. After evaporation of solvent, the residue was recrystallized from CH2Cl2hexanes. 6O -Coumarin-3-carbonyl-1,2:3,4-di-O-isopropylidene-D-galactopyranose, 3.12: White microcrystals (90%), mp 146.2 148.0 oC, 1H NMR (CDCl3): 1.34 (s, 3H), 1.36 (s, 3H), 1.48 (s, 3H), 1.54 (s, 3H), 4.16 4.23 (m, 1H), 4.33 4.40 (m, 2H), 4.47 (dd, J = 11.4, 7.6 Hz, 1H), 4.55 (dd, J = 11.5, 4.9 Hz, 1H), 4.66 (dd, J = 7.8, 2.3 Hz, 1H), 5.56 (d, J = 4.9 Hz, 1H), 7.30 7.38 (m, 2H ), 7.58 7.70 (m, 2H), 8.53 (s, 1H). 13C NMR (CDCl3): 24.4, 24.9, 25.9, 26.0, 54.5, 64.4, 65.9, 70.6, 70.9, 96.2, 108.9, 109.6, 116.8, 117.8, 117.9, 124.8, 129.5, 134.4, 148.8, 155.1, 156.5, 162.6. Anal. calcd for C22H24O9: C, 61.11; H, 5.59; N, 0.00. Found: C, 61.06; H, 5.71; N, 0.19. 3O -Coumarin-3-carbonyl-1,2:5,6-Di-O-isopropylidene-D-glucose, 3.13: White microcrystals (89%), mp 65.1 66.7 oC 1H NMR (CDCl3): 1.32 (s, 3H), 1.33 (s, 3H), 1.43 (s, 3H), 1.56 (s, 3H), 4.07 (dd, J = 8.8, 4.7 Hz, 1H), 4.15 (dd, J = 8.7, 5.9 Hz, 1H), 4.29 (dd, J = 8.5, 3.0 Hz, 1H), 4.45 4.51 (m, 1H), 4.68 (d, J = 3.8 Hz, 1H), 5.48 (d, J = 3.0 Hz, 1H), 5.97 (d, J = 3.8 Hz, 1H), 7.32 7.40 (m, 2H), 7.60 7.72 (m, 2H), 8.53 (s, 1H). 13C NMR (CDCl3): 25.2, 26.1, 26.7, 26.9, 67.4, 72.4, 77.6, 79.9, 83.1, 105.1, 109.4, 112.3, 116.8, 117.6, 124.9, 129.6, 134.7, 149.3, 155.2, 156.2, 162.1. Anal. calcd for C22H24O9: C, 61.11; H, 5.59; N, 0.00. Found: C, 60.91; H, 5.72; N, 0.03. 1O -Coumarin-3 carbonyl-2,3:5,6-Di-O-isopropylidene-D-mannofuranose, 3.14: White microcrystals (65%), mp 158.2 160.0 oC, 1H NMR (CDCl3): 1.35 1.40 (m, 6H), 1.46 (s, 3H), 1.52 (s, 3H), 4.06 (dd, J = 9.07, 4.4 Hz, 1H), 4.08 4.15 (m, 1H), 4.19 (dd, J = 7.8, 3.4 Hz, 1H), 4.40 4.48 (m, 1H), 4.89 4.98 (m, 2H), 6.36 (s, 1H), 7.32 7.40 (m, 2H), 7.62 7.71

PAGE 58

58 (m, 2H), 8.54 (s, 1H). 13C NMR (CDCl3): 24.6, 25.1, 25.9, 26.9, 66.8, 72.8, 79.2, 82.6, 85.0, 101.9, 109.4, 113.3, 116.8, 117.7, 124.9, 129.7, 134.8, 149.6, 155.3, 155.6, 162.0. Anal. calcd for C22H24O9: C, 61.11; H, 5.59; N, 0.00. Found: C, 61.02;H, 5.54; N, 0.03 2,2-Dimethyl-propionic acid (3 S ,5 S ,6 R )-3,4,5-tris-(2,2-dimethyl-propionyloxy)-6-[(2oxo-2H-chromene-3-carbonyl)-amino]-tetrah ydro-pyran-2-ylmethyl ester, 3.16: Clear solid (60%), 1H NMR (CDCl3): 1.00 (s, 9H), 1.06 (s, 9H), 1.10 (s, 9H), 1.23 (s, 9H), 3.91 3.99 (m, 1H), 4.06 4.16 (m, 2H), 5.20 5.35 (m, 2H), 7.30 7.39 (m, 2H), 7.60 7.68 (m, 2H), 8.83 (s, 1H), 9.29 (d, J=9.1Hz, 1H). 13C NMR (CDCl3): 26.7, 27.0, 27.1, 29.6, 38.6, 38.6, 38.7, 39.0, 60.7, 66.7, 67.7, 71.1, 72.7, 78.5, 116.7, 117.2, 118.3, 125.3, 130.0, 134.6, 149.5, 154.6, 160.6, 162.0, 176.8, 177.0, 177.1, 177.7. Anal. calcd for C22H24O9: C, 62.87; H, 7.18; N, 2.04. Found: C, 62.69; H, 7.68; N, 2.07. ( S )-2-Benzyloxycarbonylamino-6-[(2 -oxo-2H-chromene-3-carbonyl) -amino]-hexanoic acid 5-(2,2dimethyl-[1,3 ] dioxolan-4-yl)-2,2 -dimethyl-tetrahydrofuro[2,3-d][1,3]dioxol-6 -yl ester, 3.17a: White microcrystals (82%), mp 123.0 124.0oC. 1H NMR (CDCl3): 1.29 (s, 3H), 1.31 (s, 3H), 1.38 (s, 3), 1.41-1.48 (m, 2H), 1.51 (s, 3H), 1.58 1.98 (m, 4H), 3.38 3.58 (m, 2H), 3.97 (dd, J = 8.6, 4.2 Hz, 1H), 4.07 (dd, J = 8.6, 5.1 Hz, 1H), 4.1 4-4.25 (m, 2H), 4.29-4.40 (m, 1H), 4.48 (d, J = 3.4 Hz, 1H), 5.07(d. J = 12.2 Hz, 1H, B part of AB system), 5.13 (d, J = 12.2 Hz, 1H, A part of AB system), 5.40-5.52 (m, 1H), 5.65 (d, J = 7.4 Hz, 1H), 5.82 (d, J = 3.4 Hz, 1H), 7.28-7.43 (m 7H), 7.50 (d, J = 7.1 Hz, 1H), 7.62-7.72 (m, 1H), 8.82-8.96 (m, 2H). 13C NMR (CDCl3): 22.2, 25.2, 26.2, 26.7, 26.8, 29.1, 31.3, 38.7, 54.0, 66.9, 67.2, 72.4, 76.9, 79.7, 83.0, 105.0, 109.3, 112.4, 116.5, 116.5, 118.2, 118.6, 125.3, 128.0, 128.2, 128.5, 129.8, 134.1, 136.2, 148.6, 154.3, 156.0, 161.5, 161.9, 171.0. Anal. calcd for C36H42N2O12: C, 62.24; H, 6.09; N, 4.03. Found: C, 62.34; H, 6.11; N, 4.09.

PAGE 59

59 ( S )-2-(9 H -Fluoren-9-ylmethoxycar bonylamino)-6-[(2-oxo-2 H -chromene-3carbonyl)-amino]-hexanoic acid 2,2,7,7-te tramethyl-tetrahydro-bis[1,3]dioxolo[4,5b ;4',5'd ]pyran-5-ylmethyl ester, 3.17b : White microcrystals (85%), mp 122.3 124.1 oC. 1H NMR (CDCl3): 1.33 (s, 6H), 1.46 (s, 3H), 1.52 (s, 3H), 1.63 2.30 (m, 4H), 3.40 3.57 (m, 2H), 4.01 4.08 (m, 1H), 4.20 4.29 (m, 3H), 4.30 4.46 (m, 6H), 4.62 (dd, J = 7.8, 2.3 Hz, 1H), 5.54 (d, J = 4.9Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 7.29 7.43 (m, 7H), 7.54 (d, J = 7.7Hz, 1H), 7.60 7.67 (m, 3H), 7.72 7.78 (m, 2H), 8.82 8.88 (m, 1H), 8.90 (s, 1H). 13C NMR (CDCl3): 22.3, 24.4, 24.9, 25.9, 26.0, 29.0, 31.9, 37.3, 39.2, 47.1, 53.8, 64.2, 66.0, 67.0, 68.8, 70.3, 70.7, 70.9, 96.2, 108.8, 109.7, 116.5, 118.3, 118.6, 119.9, 125.2, 127.0, 127.6, 129.7, 133.9, 141.2, 143.8, 148.3, 154.3, 155.9, 161.4, 161.7, 172.3. Anal. calcd for C43H46N2O12: C, 65.97; H, 5.92; N, 3.58. Found: C, 65.63; H, 6.04; N, 3.62. ( S )-2-(9 H -Fluoren-9-ylmethoxycar bonylamino)-6-[(2-oxo-2 H -chromene-3carbonyl)-amino]-hexanoic acid 5-(2,2-di methyl-[1,3]dioxolan4-yl)-2,2-dimethyltetrahydro-furo[2,3d ][1,3]dioxol-6-yl ester, 3.18: White microcrystals (67%), mp 118.2 120.4. 1H NMR (CDCl3): 1.29 (s,3H), 1.31 (s, 3H), 1.38 (s, 3H), 1.51 (s, 3H), 1.60 2.00 (m, 4H), 3.40 3.52 (m, 2H), 3.97 4.11 (m, 3H), 4.19 4.27 (m, 3H), 4.30 4.45 (m, 3H), 4.51 (d, J= 3.4 Hz, 1H), 5.30 (s, 1H), 5.77 (d, J = 7.6 Hz, 1H), 5.86 (d, J = 3.6 Hz, 1H), 7.25 7.46 (m, 8H), 7.60 7.67 (m, 3H), 7.72 7.79 (m, 2H), 8.84 8.96 (m, 2H). 13C NMR (CDCl3): 22.2, 25.1, 26.2, 26.7, 26.8, 29.0, 31.3, 38.7, 47.1, 53.9, 60.4, 67.0, 67.2, 72.3, 79.7, 83.1, 105.0, 109.4, 112.4, 116.5, 118.1, 118.5, 120.0, 125.0, 125.1, 125.2, 127.1, 127.7, 129.7, 134.0, 141.2, 143.6, 143.8, 148.6, 154.3, 156.0, 161.5, 162.0, 171.1. Anal. calcd for C43H46N2O12: C, 65.97; H, 5.92; N, 3.58. Found: C, 65.68; H, 6.07; N, 3.60.

PAGE 60

60 ( S )-2-(9H-Fluoren-9-ylmethoxycarbony lamino)-6-[(2-oxo-2H-chromene-3carbonyl)-amino]-hexanoic acid 6-(2,2-dime thyl[1,3]dioxolan4-yl)-2,2-dimethyltetrahydro-furo[3,4-d][1,3] dioxol-4-yl ester, 3.19: White microcrystals (74%), mp 87.0-88.0 1H NMR (CDCl3): 1.22-1.30 (m, 2H), 1.33 (s, 3H), 1.37 (s 3H), 1.43 (s, 3H), 1.48 (s, 3H), 1.52-1.98 (m, 4H), 3.46-3.60 (m, 2H), 3.98-4.12 (m, 3H ), 4.22 (t, J = 7.1 Hz, 1H), 4.30-4.48 (m, 3H), 4.73 (d, J = 5.9 Hz, 1H), 4.80-4.90 (m, 1H), 5.65 (d, J = 7.8 Hz, 1H), 6.17 (s, 1H), 7.25-7.42 (m, 7H), 7.50 (d, J = 7.8 Hz, 1H), 7.58-6.67 (m, 3H), 7.74 (dd, J = 7.3, 2.8,Hz, 2H), 8.82-8.88 (m, 2H). 13C NMR (CDCl3): 22.3, 22.6, 24.6, 25.1, 25.9, 26.9, 29.0, 31.5, 38.9, 47.1, 53.7, 66.8, 67.0, 72.7, 79.2, 82.5, 85.0, 101.6, 109.3, 113.3, 116.5, 118.2, 118.5, 119.9, 125.2, 127.0, 127.6, 129.7, 134.0, 141.2, 143.7, 143.8, 148.4, 154.3, 155.8, 161.4, 161.8, 171.1. Anal. calcd for C43H46N2O12: C, 65.97; H, 5.92; N, 3.58. Found: C, 65.57; H, 6.06; N, 3.40. {( S )-1-Formyl-5-[(2-oxo-2 H -chromene-3-carbonyl)-amino]-pentyl}-carbamic acid 9 H -fluoren-9-ylmethyl ester; compound with 2,2-dimethyl-propionic acid (3S,5S,6R)-3,4,5tris-(2,2-dimethyl-propionyloxy) -6-methylamino-tetrahydro-pyr an-2-ylmethyl ester, 3.20: 2-Oxo-2 H -chromene-3-carboxylic acid (3S ,5R)-2,3,5-trihydroxy-6-hydroxymethyltetrahydro-pyran-4-yl ester, 3.21: ( S )-2-(9 H -Fluoren-9-ylmethoxycar bonylamino)-6-[(2-oxo-2 H -chromene-3carbonyl)-amino]-hexanoic acid 3,4,5,6-tetrahyd roxy-tetrahydro-pyran-2-ylmethyl ester, 3.22: ( R )-2-(9 H -Fluoren-9-ylmethoxycar bonylamino)-6-[(2-oxo-2 H -chromene-3carbonyl)-amino]-hexanoic ac id (3S,5R)-2,3,5-trihydroxy-6hydroxymethyl-tetrahydropyran-4-yl ester, 3.23:

PAGE 61

61 LIST OF REFERENCES 1. Katritzky, A. R.; Rogovoy, Boris V. Chem. Eur. J. 2003 9 4586. 2. Katritzky, A. R.; Lan, X.; Yang, J.; Denisko, O. Chem. Rev. 1998 98 409. 3. Katritzky, A. R.; Belyakov, S. Aldchim. Act. 1998 31 35. 4. Katritzky, A. R.; Shobana, N.; Pernak, J.; Afridi, A. S.; Fan, W. Q. Tetrahedron 1992 48 7817. 5. Katritzky, A. R.; He, H.-Y.; Suzuki, K. J. Org. Chem. 2000 65 8210. 6. Katritzky, A. R.; Zhang, Y.; Singh, S. K. Synthesis 2003 2795. 7. Katritzky, A. R., Suzuki, K.; Singh, S.K. Synthesis 2004 2645. 8. Katritzky, A. R.; Angrish, P.; Hr, D.; Suzuki, K. Synthesis 2005 3, 397. 9. Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis 2006 3, 411. 10. Katritzky, A. R.; Todadze, E.; Cusido, J. Angrish, P.; Shestopalov A. Chem. Biol. Drug Des. 2006 68 42. 11. Katritzky, A. R.; Todadze, E.; Shestopalov A.; Cusido, J. Angrish, P. Chem. Biol. Drug Des. 2006 68 37. 12. Katritzky, A. R.; Narindoshvili, T, Angrish, P. Manuscript submitted to Synlett 13. Katritzky, A. R.; Cusido, J.; Narindoshvili, T. Manuscript in progress 14. Gill, I.; Lpez-Fandio, R.; Jorba, X.; Vulfson, E. N. Enzym. Micro. Tech. 1996, 18 162. 15. Nishimura, T.; Kato, H. Food Rev Int. 1988, 4, 39. 16. Sturtevant, Frank M. J. Environ. Sci. Health Part A Environ. Sci. Eng 1985 20, 863. 17. Grenby, T. H. Trends Food Sci. Technol. 1991 2 2. 18. Nosho, Y.; Seki, T.; Kondo, M.; Ohfuji, T.; Tamura, M.; Okai, H. J. Agric. Biol. Chem 1990 38 1368. 19. Arai, S. A. Anal. Control Less Desirable Flavor Foods Beverages (Proc Symp). Academic, New York, 1980 133. 20. Matoba, T.; Hayashi, R.; Hata, T. Agric. Biol. Chem 1988 34 1235.

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67 BIOGRAPHICAL SKETCH Janet Cusido grew up in Miami, Florida, w ith her parents Ramon and Oneida, and brother Ramon Alejandro. In high school, she developed a l ove for the sciences, especially for math and chemistry. She graduated from Coral Gables Seni or High and enrolled at the University of Florida in order to pursue a de gree in chemical engineering. During her junior year at the University of Florida, she took organic ch emistry lab led by teaching assistant Valerie Rodriguez-Garcia, where Janet discovered her passi on for laboratory experimentation. Thanks to Valerie, Janet decided to switch majors from chem ical engineering to chemistry. Janet started to perform research with Professor Alan R. Katritz ky under the supervision of Valerie and learned how important organic chemistry was in everyday life. Janet graduated with High Honors from the University of Florida in April 2005 and th en enrolled in the gr aduate program at UF, continuing her research with Dr. Katritzky in th e field of Benzotriazole Chemistry. After Janet graduates from the University of Florida in D ecember 2007, she plans to continue her graduate studies at another institution to expand her knowledge and experience.